Stopped-flow Technique Research Articles

Kinetic studies on the reaction of NO with iron(ii) complexes using low temperature stopped-flow techniques.

Low temperature stopped-flow techniques were used to investigate the reaction of three different iron(ii) complexes with nitrogen monoxide. The kinetic studies allowed calculation of the activation parameters from the corresponding Eyring plots for all three systems. The reaction of iron(ii) chloride with NO leading to the formation of MNIC (mononitrosyl-iron-complex) and DNIC (dinitrosyl-iron-complex) led to activation parameters of ΔH‡ = 55.4 ± 0.4 kJ mol-1 and ΔS‡ = 13 ± 2 J K-1 mol-1 for MNIC and ΔH‡ = 32 ± 6 kJ mol-1 and ΔS‡ = -193 ± 21 J K-1 mol-1 for DNIC. Formation of MNIC turned out to be much faster in comparison with DNIC. In contrast, activation parameters for the formation of monoculear [Fe(bztpen)(NO)](OTf)2 (bztpen = N-benzyl-N,N',N'-tris(2-pyridylmethyl)-ethylenediamine) ΔH‡ = 17.8 ± 0.8 kJ mol-1 and ΔS‡ = -181 ± 3 J K-1 mol-1 supported an associative mechanism. Interestingly, [Fe(bztpen)(CH3CN)](OTf)2 does not react with dioxygen at all. Furthermore, activation parameters of ΔH‡ = 37.7 ± 0.7 kJ mol-1 and ΔS‡ = -66 ± 3 J K-1 mol-1 were obtained for the reaction of NO with the dinuclear iron(ii) H-HPTB complex (H-HPTB = N,N,N',N'-tetrakis(2-benzimidazolylmethyl)-2-hydroxy-1,3-diaminopropane), [Fe2(H-HPTB)(Cl)3]. The kinetic data allowed postulation of the mechanisms for all of these reactions.

Role of Ionizing Amino Acid Residues in the Process of DNA Binding by Human AP Endonuclease 1 and in Its Catalysis

In the repair of the damage to bases, human apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is a key participant via the DNA base excision repair pathway. APE1 cleaves AP sites in DNA, which are potentially cytotoxic and highly mutagenic if left unrepaired. According to existing structural data, this enzyme's active site contains many polar amino acid residues, which form extensive contacts with a DNA substrate. A few alternative catalytic mechanisms of the phosphodiester bond hydrolysis by APE1 have been reported. Here, the kinetics of conformational changes of the enzyme and of DNA substrate molecules were studied during the recognition and cleavage of the abasic site in the pH range from 5.5 to 9.0 using stopped-flow fluorescence techniques. The activity of APE1 increased with an increase in pH because of acceleration of the rates of catalytic complex formation and of the catalytic reaction. Molecular dynamics simulation uncovered a significant increase in the pKa of His-309 located in the active site of the enzyme. This finding revealed that the observed enhancement of enzymatic activity with pH could be associated with deprotonation of not only Tyr-171 but also His-309. The obtained data allowed us to hypothesize that the ionized state of these residues could be a molecular switch between the alternative catalytic mechanisms, which involve different functionalities of these residues throughout the reaction.

Kinetics and new Brønsted correlations study of CO2 absorption into primary and secondary alkanolamine with and without steric-hindrance

To figure out the steric hindrance and its magnitude effects on CO2 absorption performance and Brønsted correlations of primary and secondary alkanolamines, the pKa values and kinetics data (second order reaction rate, k2) were investigated for nine alkanolamines at 293–313 K using pH meter and stopped-flow technique. The tested amines include monoethanolamine (MEA), diethanolamine (DEA), 2-methyl-ethynolamine (MAE), 2-ethyl-ethynolamine (EAE), 1-amino-2-propanol (1-AP), 3-amino-1-propanol (3-AP), 2-amino-2-methyl-1-propanol (AMP), 2-amino-1-propanol (2-AP) and 3-Amino-1,2-propanediol (3-APD). In addition, the equilibrium solubility of MEA, MAE, EAE, 2-AP and AMP were tested for evaluation of steric hindrance effect on carbamate’s stability. The comprehensive analysis of kinetics, solubility and Brønsted correlations indicated that the steric hindrance caused by alkyl group(s) attached to α-C atom has much significant effect on k2 and solubility than that caused by alkyl group connected to amino group. Moreover, the k2 and pKa values for sterically unhindered and hindered amines were separately fitted at 293–313 K, giving out an AARD of 5.2% and 16.5% between predicted and experimental k2, respectively. Finally, based on molecular structures and experimental results, mechanism of steric hindrance effect on reaction kinetics was proposed.

Synthesis, crystal structures and reactivity towards dioxygen of copper(I) complexes with tripodal aliphatic amine ligands

Copper(I) complexes of the tripodal, tetradentate, aliphatic ligands Me3tren (Me3tren = tris-(2-methylaminoethyl)amine), Isoprop3tren (Isoprop3tren = tris-(2-isopropylaminoethyl)amine) and Me3isoprop3tren (Me3isoprop3tren = tris-(2-isopropylmethylaminoethyl)amine) of the Tren-family (Tren = tris(2-aminoethyl)amine) were prepared and structurally characterized. Furthermore, they were reacted in solution at low temperatures with dioxygen. All complexes formed a short-lived superoxido intermediate complex that could be observed using low temperature stopped-flow techniques prior to consecutive reactions. However, the superoxido complexes were too reactive to allow isolation and full characterization in contrast to previous work.

A Rapid Fenton treatment of bio-treated dyeing and finishing wastewater at second-scale intervals: kinetics by stopped-flow technique and application in a full-scale plant

A rapid Fenton treatment at second-scale intervals was investigated for further removal of organic compounds in the effluent of bio-treated dyeing and finishing wastewater (BDFW). The decolorization kinetics was studied using a stopped-flow spectrophotometer (SFS) at second-scale intervals. A combined first-order model was found to fit well for the decrease of both methylene blue and rhodamine B in SFS as well as SCOD (soluble chemical oxygen demand) and DOC (dissolved organic carbon) in real BDFW in batch test during Fenton oxidation. A full-scale plant with treatment capacity of 400,000 m3·d−1 was designed and has been run continuously based on the results of the stopped-flow study to treat the effluent of BDFW using Fenton oxidation in 16 pipeline reactors, each with a volume of 6.9 m3 and 24 s of reaction time since 2014. The COD, SCOD and DOC decreased from 140, 110 and 35 mg·L−1 to 77, 71 and 26 mg·L−1 respectively, which can meet the latest strict discharge limitations. The natural fluorescent substances detected in the BDFW were completely removed. The main organic pollutants in the BDFW can be significantly reduced using both gas chromatography-mass spectrometry and ultrahigh-performance liquid chromatography-hybrid quadrupole time-of-flight mass spectrometry. The rapid Fenton reaction applied in pipeline reactors at second intervals has several advantages over the conventional Fenton’s process such as much shorter reaction time at second scale intervals, no need to build extra pH adjustment or reaction tanks, simple operation, low capital cost, etc.

Spectroscopic, kinetic, and theoretical analyses of oxidation of dl-ethionine by Pt(IV) anticancer model compounds.

Ethionine is an S-ethyl analog of methionine (Met) having a small change in structure. But it is a chemical carcinogen and an antagonist of Met, thus displaying a disparate biological profile. The oxidations of ethionine by biologically important oxidants have not been exploited. Oxidations of dl-ethionine by Pt(IV) anticancer model complexes trans-[PtX2(CN4)]2- (X = Cl or Br) were thus analyzed by time-resolved and stopped-flow spectral techniques. Overall second-order kinetics was established, being first-order in [Pt(IV)] and [Ethionine]tot (the total concentration of ethionine); the observed second-order rate constant k' versus pH profiles were obtained. A stoichiometry of Δ[Pt(IV)]:Δ[Ethionine]tot = 1:1 was unraveled, indicating that ethionine was oxidized to ethionine-sulfoxide which was confirmed by NMR spectroscopic and high-resolution mass spectral analyses. In the proposed reaction mechanism which is similar to that for the oxidation of Met by the same Pt(IV) compounds, the rate-determining steps are rationalized in terms of a bridge formation between one of the coordinated halides in [PtX2(CN4)]2- and the sulfur atom in ethionine, followed by an X+ transfer. Moreover, a large rate enhancement for the reaction of ethionine with [PtBr2(CN4)]2- compared with [PtCl2(CN4)]2- strongly supports an X+ transfer mechanism. Furthermore, a combined quantum-mechanical/molecular-mechanical (QM/MM) method was utilized to simulate a Cl+ transfer mechanism from trans-[PtCl2(CN)4]2- to ethionine. The simulations unraveled the energetically stable structures of reactants and products, which favor the Cl+ transfer process. Rate constants of the rate-determining steps have been derived. Ratios of k (ethionine)/k (Met) are between 2.2 and 2.6 obtained for the three protolytic species of ethionine and Met; the enhanced reactivity might be partially responsible for the disparate biological profiles.

Development of Large-Scale Stopped-Flow Technique and its Application in Elucidation of Initial Ziegler-Natta Olefin Polymerization Kinetics.

The stopped-flow (SF) technique has been extensively applied to study Ziegler–Natta (ZN) olefin polymerization kinetics within an extremely short period (typically <0.2 s) for understanding the nature of the active sites as well as the polymerization mechanisms through microstructure analyses of obtained polymers. In spite of its great applicability, a small amount of polymer that is yielded in a short-time polymerization has been a major bottleneck for polymer characterizations. In order to overcome this limitation, a large-scale SF (LSF) system has been developed, which offers stable and scalable polymerization over an expanded time range from a few tens milliseconds to several seconds. The scalability of the LSF technique has been further improved by introducing a new quenching protocol. With these advantages, the LSF technique has been effectively applied to address several unknown issues in ZN catalysis, such as the role of physical and chemical transformations of a catalyst on the initial polymerization kinetics, and regiochemistry of ZN propylene polymerization. Here, we review the development of the LSF technique and recent efforts for understanding heterogeneous ZN olefin polymerization catalysis with this new system.

The impact of single-nucleotide polymorphisms of human apurinic/apyrimidinic endonuclease 1 on specific DNA binding and catalysis

Human apurinic/apyrimidinic (AP) endonuclease APE1 is a crucial enzyme of the base excision repair (BER) pathway, which is in charge of recognition and initiation of removal of AP-sites in DNA. It is known that some single-nucleotide polymorphism (SNP) variants of APE1 have a reduced activity as compared to wild-type APE1. It has been hypothesized that genetic variation in APE1 might be responsible for an increased risk of some types of cancer. In the present work, analysis of SNPs of the APE1 gene was performed to select the set of variants having substitutions of amino acid residues on the surface of the enzyme globule and in the DNA-binding site, thereby affecting protein–protein interactions or the catalytic reaction, respectively. For seven APE1 variants (R221C, N222H, R237A, G241R, M270T, R274Q, and P311S), conformational dynamics and catalytic activities were examined. The conformational changes in the molecules of APE1 variants and in a DNA substrate were recorded as fluorescence changes of Trp and 2-aminopurine residues, respectively, using the stopped-flow technique. The results made it possible to determine the kinetic mechanism underlying the interactions of the APE1 variants with DNA substrates, to calculate the rate constants of the elementary stages, and to identify the stages of the process affected by mutation.

Design, DNA binding and kinetic studies, antibacterial and cytotoxic activities of stable dithiophenolato titanium(IV)-chitosan Nanocomposite

Highly thermal stable dithiophenolato Ti(IV)-chitosan nanocomposite (DBT-CS) was formed from dithiophenolato Ti(IV)-complex (DBT) and chitosan (CS) nanoparticles. As DNA-drugs binding is the key reaction step responsible for the anticancer activity, in vitro DNA binding studies to DBT and DBT-CS were investigated and showed a strong interaction to DNA helix via groove binding. Kinetic stability, affinity and association constants of compounds-DNA were studied for the first time using a stopped-flow technique and showed that the formulation of nanostructure DBT-CS does not only improve the rate of reaction through coordination affinity but also change the binding mechanism. Additionally, the antibacterial investigation showed that DBT and DBT-CS exhibited good antibacterial activity against both Gram-positive and Gram-negative bacteria. They also show high cytotoxicity against HepG2 human liver cancer cell. Although DBT-DNA complex is kinetically more stable than that of DBT-CS, the latter showed better antibacterial and cytotoxic activities which in agreement with the binding constants.

Rapid mixing of colliding picoliter liquid droplets delivered through-space from piezoelectric-actuated pipettes characterized by time-resolved fluorescence monitoring.

Rapid mixing of aqueous solutions is a crucial first step to study the kinetics of fast biochemical reactions with high temporal resolution. Remarkable progress toward this goal has been made through the development of advanced stopped-flow mixing techniques resulting in reduced dead times, and thereby extending reaction monitoring capabilities to numerous biochemical systems. Concurrently, piezoelectric actuators for through-space liquid droplet sample delivery have also been applied in several experimental systems, providing discrete picoliter sample volume delivery and precision sample deposition onto a surface, free of confinement within microfluidic devices, tubing, or other physical constraints. Here, we characterize the inertial mixing kinetics of two aqueous droplets (130 pl) produced by piezoelectric-actuated pipettes, following droplet collision in free space and deposition on a surface in a proof of principle experiment. A time-resolved fluorescence system was developed to monitor the mixing and fluorescence quenching of 5-carboxytetramethylrhodamine (5-Tamra) and N-Bromosuccinimide, which we show to occur in less than 10 ms. In this respect, this methodology is unique in that it offers millisecond mixing capabilities for very small quantities of discrete sample volumes. Furthermore, the use of discrete droplets for sample delivery and mixing in free space provides potential advantages, including the elimination of the requirement for a physical construction as with microfluidic systems, and thereby makes possible and extends the experimental capabilities of many systems.

The comparative kinetics study of CO2 absorption into non-aqueous DEEA/MEA and DMEA/MEA blended systems solution by using stopped-flow technique

The kinetics of the absorption of CO2 into solutions of (monoethanolamine) MEA-Ethanol, (N, N-diethylethanolamine) DEEA-MEA-Ethanol and (N, N-dimethylethanolamine) DMEA-MEA-Ethanol have been studied by applying a stopped-flow apparatus. In this study, the concentration range of both tertiary amine and MEA were chosen to be 0.2–0.4 kmol/m3 with a temperature range of 293–313 K under non-aqueous conditions. The model based on the termolecular reaction mechanism is proposed to interpret the experimental kinetic data of the reaction occurred in the non-aqueous blended amine systems, and the results indicate that the model is well fitted to the experimental data with an AARD of 5.70% for DEEA-MEA-Ethanol system and 6.53% for DMEA-MEA-Ethanol system. Blended amines show many advantages in kinetics because of the existence of the interaction between the tertiary amine and MEA. The kinetics behaviors of CO2 absorbed in the two non-aqueous blended amine systems are discussed in brief according to the molecular structures of the two tertiary amines. Different from the traditional aqueous systems, this study proved that tertiary amine enhanced the ability of primary amine to absorb CO2 which is due to tertiary amine didn’t react with CO2 directly in a non-aqueous system. The result in the non-aqueous system also shows that DMEA is more easily participate in the termolecular reaction between MEA and CO2 compared with DEEA, which indicate the length of alkyl chain on nitrogen atom also plays an important role in the enhancement effect. Additionally, after comparing the kinetic data of the two blended amine systems under aqueous and non-aqueous conditions, it can be found that tertiary amine can participate in the termolecular reaction between MEA and CO2, and that the protonated MEA can be reconverted to free MEA by the tertiary amine, making it easier for MEA to participate in its own termolecular reactions, so that the overall reaction rate of the blended amine system is accelerated. It can be summarized that there are interactions between the tertiary amine and the primary amine in the non-aqueous blended amine system that positively promote the kinetics.

Reaction Kinetics of Carbon Dioxide in Aqueous Blends of N-Methyldiethanolamine and L-Arginine Using the Stopped-Flow Technique

: Reduction of carbon dioxide emission from natural and industrial flue gases is paramount to help mitigate its effect on global warming. Efforts are continuously deployed worldwide to develop efficient technologies for CO2 capture. The use of environment friendly amino acids as rate promoters in the present amine systems has attracted the attention of many researchers recently. In this work, the reaction kinetics of carbon dioxide with blends of N-methyldiethanolamine and L-Arginine was investigated using stopped flow technique. The experiments were performed over a temperature range of 293 to 313 K and solution concentration up to one molar of different amino acid/amine ratios. The overall reaction rate constant (kov) was found to increase with increasing temperature and amine concentration as well as with increased proportion of L-Arginine concentration in the mixture. The experimental data were fitted to the zwitterion and termolecular mechanisms using a nonlinear regression technique with an average absolute deviation (AAD) of 7.6% and 8.0%, respectively. A comparative study of the promoting effect of L-Arginine with that of the effect of Glycine and DEA in MDEA blends showed that MDEA-Arginine blend exhibits faster reaction rate with CO2 with respect to MDEA-DEA blend, while the case was converse when compared to the MDEA-Glycine blend.

Dioxygen Reduction to Hydrogen Peroxide by a Molecular Mn Complex: Mechanistic Divergence between Homogeneous and Heterogeneous Reductants.

The selective electrocatalytic reduction of dioxygen (O2) to hydrogen peroxide (H2O2) could be an alternative to the anthraquinone process used industrially, as well as enable the on-demand production of a useful chemical oxidant, obviating the need for long-term storage. There are challenges associated with this, since the two-proton/two-electron reduction of H2O2 to two equivalents of water (H2O) or disproportionation to O2 and H2O can be competing reactions. Recently, we reported a Mn(III) Schiff base-type complex, Mn(tbudhbpy)Cl, where 6,6'-di(3,5-di- tert-butyl-2-phenolate)-2,2'-bipyridine = [tbudhbpy]2-, which is active for the electrocatalytic reduction of O2 to H2O2 (ca. 80% selectivity). The less-than-quantitative selectivity could be attributed in part to a thermal disproportionation reaction of H2O2 to O2 and H2O. To understand the mechanism in greater detail, spectrochemical stopped-flow and electrochemical techniques were employed to examine the catalytic rate law and kinetic reaction parameters. Under electrochemical conditions, the catalyst produces H2O2 by an ECCEC mechanism with appreciable rates down to overpotentials of 20 mV and exhibits a catalytic response with a strong dependence on proton donor p Ka. Mechanistic studies suggest that under spectrochemical conditions, where the homogeneous reductant decamethylferrocene (Cp*2Fe) is used, H2O2 is instead produced via a disproportionation pathway, which does not show a strong acid dependence. These results demonstrate that differences in mechanistic pathways can occur for homogeneous catalysts in redox processes, dependent on whether an electrode or homogeneous reductant is used.

One-Pot Multicomponent Synthesis of 2H-Chromene Derivative, Kinetics, and Thermodynamic Studies Using a Stopped-Flow Technique in Combination with Theoretical Computation Methods

The diethyl 6-bromo-2H-chromene-2,3-dicarboxylate (4) was successfully synthesized by the reaction between triphenylphosphine (1), diethyl acetylenedicarboxylate (DEAD, 2) and 5-bromo-2-hydroxybenzaldehyde acid (3) in the presence of dichloromethane. The 2H-chromene’s compound were characterized using IR, 1H, 13C, and 31P NMR spectroscopy. The kinetics and mechanism of the reaction were experimentally investigated by stopped-flow technique and UV–vis spectrophotometry. The first step in the proposed five-step mechanism, the reaction between 1 and 2 for the formation of intermediate 1 (I1), was fast. The reaction of I1 and 3 was slow and followed first-order kinetics. The experimental kinetic data proved that the step4 of the proposed mechanism is a rate-determining step. To make a further understanding about the proposed mechanism, theoretical investigations were carried out at the B3LYP/6-31++g(d,p) level of theory. The calculations showed that the step4 (Δ 82.1 kJ/mol) is in terms of energy undesirable and difficult in line with experimental results, and is an endothermic process. It also cleared that why I3 with diethyl groups in step4 tends to convert to the I4 very slowly through a four-member ring formation in comparison with other steps and ultimately it becomes impossible process (step4) when t-But groups are replaced instead of diethyl groups within the structure of I3. The experimental and theoretical data indicated that the overall reaction rate follows second-order kinetics and depends on the concentrations of compounds 1 and 2.

Generalization of the anthocyanins kinetics and thermodynamics multistate to 2,6-bis(2-hydroxybenzylidene)cyclohexanones

The thermodynamic and kinetic behavior of 4-(2,4-dihydroxybenzylidene)-6-hydroxy-1,2,3,4-tetrahydroxanthylium chloride was studied in detail. Similarly to anthocyanins, this compound generates a multistate of species that are reversibly interconverted by pH stimuli. The multistate system was studied by NMR and UV–visible (conventional spectroscopy and stopped-flow techniques). A theoretical approach adapted from the one reported for anthocyanins and related compounds was used to rationalize the complex multistate. In particular, the thermodynamic diagram where all the species are positioned was extended to the ionized species. Cis and trans isomers of flavylium cation, quinoidal bases, and ionized chalcone at extremely high pH values were identified. The multistate is dominated in the range 5 < pH < 8 by the spiro form in equilibrium with a small fraction of quinoidal bases. The spiro opening rates follow a different route and pH dependence in very acidic or moderately acidic pHs.

Kinetics of CO2 reaction with N-methyldiethanolamine and aminobutanol using stopped flow technique

Removal of carbon dioxide (CO2) from natural gas streams is mandatory to avoid pipeline corrosion, increase the heating value of the gas and reduce the gas volume in case of liquefied natural gas (LNG). The use of N-Methyldiethanolamine (MDEA) solution combined with other rate promoters such as Piperazine (PZ) is a common practice in Gas treatment technology. In this work, the use of Aminobutanol (AB) mixed with MDEA for the removal of (CO2) from natural gas streams is presented. The reaction kinetics of (CO2) with mixtures of MDEA and AB was investigated using stopped flow technique. The experiments were performed over a temperature range of 293 to 313 K and solution concentration of 0.5 and 1 moles/l in different MDEA/AB proportions. Obtained kinetics data were interpreted using zwitterion mechanisms for the primary amine AB. The individual rate constants of the participating reactions were regressed and their corresponding activation energies were estimated.

Metal-Ion Displacement Approach for Optical Recognition of Thorium: Application of a Molybdenum(VI) Complex for Nanomolar Determination and Enrichment of Th(IV).

An azine-based molybdenum (Mo(VI)) complex (M1) is exploited for selective detection of thorium (Th(IV)) ions through a metal-ion displacement protocol. Th(IV) displaces Mo(VI) from M1 instantly leading to the formation of the Th(IV) complex, having orange-red emission. Consequently, a red shift of the emission wavelength along with 41-fold fluorescence enhancement is observed. This unique method allows detection of Th(IV) as low as 1.5 × 10–9 M. The displacement of Mo(VI) from M1 by Th(IV) is established by spectroscopic studies and kinetically followed by the stopped-flow technique. The displacement binding constant for Th(IV) is notably strong, 4.59 × 106 M–1. Extraction of Th(IV) from aqueous solution to the ethyl acetate medium using M1 has been achieved. The silica-immobilized M1 efficiently enriches Th(IV) from its reservoir through solid-phase extraction. Computational studies (density functional theory) support experimental findings.

Isothermal titration calorimetry and stopped flow circular dichroism investigations of the interaction between lomefloxacin and human serum albumin in the presence of amino acids

The present study was designed to investigate the influence of two indispensable and two dispensable amino acids, including methionine, histidine, cysteine and proline, on the binding interaction between human serum albumin (HSA) and an antibiotic agent lomefloxacin (LMF). The fluorescence quenching experiments showed that the intrinsic emission of HSA was considerably quenched following binding to LMF in all the systems. Furthermore, in all the interactions the maximum wavelength of HSA was slightly decreased. The spectral changes observed in the binding systems we e all attributed to the alteration of the micro-environment around the tryptophan and tyrosine residues of HSA. The Kb values o HSA-LMF complex in the absence and presence of histidine, methionine, cysteine and proline have been obtained 6.02 × 105, 4.83 × 105, 5.05 × 105, 4.94 × 105 and 6.20 × 105 M−1 respectively. The various kind of Kb values showed the different interaction behavior between HSA and LMF in the absence and presence of amino acids mentioned. The data gathered by isothermal titration calorimetry (ITC) studies revealed that although all the binding interactions were exothermic, the amount of the heat exchanged during the HSA-LMF interaction increased in the presence of the amino acids especially cysteine. In the present study, the binding kinetics and affinity of LMF to HSA in the absence and presence of the amino acids were studies using stopped-flow circular dichroism and ITC techniques respectively. The results of these two techniques revealed that the bindig affinity and binding rate of the LMF-HSA interaction decreased in the presence of histidine, methionine and cysteine. In the presence of proline, the binding process of LMF-HSA was sped up and the affinity of LMF to HSA slightly increased. All the experimental results were then supported by the data collected from molecular modeling studies using density functional theory.Communicated by Ramaswamy H. Sarma

Synthesis and characterization of copper complexes with a series of tripodal amine ligands

A series of copper complexes with tripodal amine ligands was synthesized and structurally characterized. The copper(I) complexes of this series were investigated in regard to their reactivity towards dioxygen using stopped-flow techniques. For most complexes no “dioxygen adduct” complexes as intermediates could be detected. During the course of our investigations it was observed that the copper(II) complex with the ligand 3,3′-dimethylaminopropyl-(2-methylenpyridyl)-amine (Me4-p33) crystallized as a dinuclear complex, [Cu(Me4-p3H3)OH]2(ClO4)4, that could be structurally characterized. Interestingly, it turned out that in crystalline form the two bridging hydroxide ions were intact while two of the amine arms of the two ligands were protonated. DFT calculations were performed to get a better understanding of the behavior of this complex system in solution. Further protonation of two more amine ligand arms in solution was possible, still keeping the bridging hydroxide ions in place in the solid state. A related copper(II) complex system could be crystallized that contained a coordinated carbonate anion together with a protonated amine arm.

83 - Interrogating the cytoglobin binding pocket and thiol redox status with point mutations and chemical probes

Cytoglobin (Cygb) is a highly conserved, hexacoordinate heme protein expressed ubiquitously in mammalian tissues. Unlike other commonly found globins (i.e., hemoglobin and myoglobin), Cygb has a bound - but dissociable - proximal histidine that hinders ligand binding and, unlike the common globins, Cygb is not regarded to be closely associated with oxygen transport. The physiological functions of Cygb are still unclear but are thought to include ROS detoxification, lipid peroxidation, and vascular nitric oxide metabolism via hypoxic nitrite reduction and O2-dependent nitric oxide dioxygenation. All of these processes first require dissociation of the distal histidine to generate an active penta-coordinate heme. Further, these processes are likely further modulated by two conserved cysteine residues (Cys38 and Cys83), which can readily generate intermolecular dimeric and intramolecular monomeric disulfide bonds. For instance, formation of the intramolecular monomer greatly increases the nitrite reductase (NiR) activity, suggesting a change in pocket shape resulting in an increase in the rate and frequency the distal histidine dissociates. Predominantly using stopped-flow and kinetic techniques, we are assessing the effect of the redox states of these cysteines via small molecule binding/reaction characterization in both the ferrous and ferric form of the protein, elucidating differences in the binding pocket conformation in various redox environments. These methodologies consequently allow for the assessment of the thiol oxidation state, as different preparations of recombinant Cygbs vary and result in formation of differing cysteine redox states. Chemical modification and point mutations have been used to generate a number of modified cytoglobins to develop a more complete picture of the effect of the redox states of these cysteines.