Tesis sobre el tema "Copper monooxygenases"

Les monooxygénases polysaccharidiques lytiques (LPMO) cassent des polysaccharides et sont étudiés dans la valorisation de la biomasse. Ces enzymes contiennent un centre mononucléaire à cuivre, étudié grâce à ses propriétés magnétiques. Le mécanisme et les intermédiaires mis en jeu demeurent inconnus. On a utilisé un ensemble de complexes de la littérature pour développer un protocole pour prédire les propriétés magnétiques de systèmes à cuivre grâce aux calculs théoriques. Ce protocole a été utilisé sur des modèles de LPMO et sur un complexe tripeptidique afin de comprendre leurs propriétés. De plus, on a développé une série de complexes pour isoler des intermédiaires de haute-valence des LPMO. La réactivité d’un complexe en particulier a été étudié pour sa capacité à produire du formate dans des alcools. Le formate a été produit avec un rendement de 120%. De futures générations de complexes ont été synthétisés et seront étudiés pour mieux comprendre les chemins réactionnels des LPMOs
Lytic polysaccharide monooxygenases (LPMO) break down polysaccharides and are greatly studied in the context of biomass conversion. They contain a mononuclear copper center which is studied by its magnetic properties. The mechanism of reaction and its intermediates are still unknown. We use a large set of well-known complexes to develop a protocol to predict the magnetic properties of copper systems using computational calculations. This protocol was applied to LPMO models and to an LPMO-inspired tripeptidic complex to elucidate their structural and spectroscopic properties. In addition, we produced a series of complexes to capture potential high-valent reaction intermediates of LPMO. Eventually, the reactivity of one specific complex was studied for its capacity to produce formate in alcohol solvents. Formate was produced at around 120% conversion. Future generations of ligands and complexes were also synthesized and envisioned to understand the reaction pathways of LPMO

Les lytic polysaccharide monooxygenases (LPMO) sont des métalloenzymes à cuivre impliquées dans la dégradation des polysaccharides récalcitrants, tels que la cellulose et la chitine. L'ion cuivre du site actif est lié par un motif de coordination très inhabituel, composé de deux histidines entièrement conservées, dont l'une située à l'extrémité N-terminale qui est liée au cuivre à la fois par l'imidazole de la chaîne latérale et l’amine terminale. Ce motif de coordination, appelé «histidine brace», permet de catalyser le clivage oxydatif de liaisons glycosidiques dans des polysaccharides récalcitrants, en présence de dioxygène ou de peroxyde d'hydrogène et d'un donneur d'électrons. Ce travail a été réalisé par une approche interdisciplinaire avec des outils et des concepts fondamentaux allant de la biologie à la chimie. Nous nous sommes tout d'abord intéressés aux propriétés natives du site actif dans des LPMOs bactériennes appartenant à la sous-famille AA10. Notamment, la variabilité d'un résidu d'alanine en deuxième sphère de coordination a été étudiée, conduisant à la découverte de nouvelles enzymes avec des caractéristiques inhabituelles. Le rôle de l'alanine a ensuite été sondé en remplaçant ce résidu dans l'enzyme modèle SmAA10, par d’autres résidus naturellement présents chez certaines AA10s. Cette même enzyme modèle a ensuite été utilisée pour étudier l'influence du motif histidine brace sur les propriétés du site actif. Variants du site actif de SmAA10 nous ont permis de créer des sites de coordination originaux dans la perspective de catalyser de nouvelles réactions abiologiques dépendantes d’un ion métallique
Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes involved in the breakdown of recalcitrant polysaccharides, such as cellulose and chitin. The active site copper ion is coordinated by a very unusual coordination motif, consisting of two fully conserved histidines, one of which is located at the N-terminus and binds copper by both its the side chain nitrogen and the free amino terminal group. Such organization, known as “histidine brace” motif allows the oxidative cleavage of glycosidic bonds in recalcitrant polysaccharides, in the presence of dioxygen or hydrogen peroxide and an electron donor. The objectives of the present work have been pursued via an interdisciplinary approach, using tools and fundamental concepts that span from biology to chemistry. The focus was firstly addressed on the natural properties of the active site in the bacterial LPMOs belonging to the AA10 subfamily. Notably, the variability of a second coordination sphere alanine residue (~ 4 Å from the copper ion) was firstly investigated, leading to the discovery of new enzymes with unusual active-site features. The role of active site alanine was then probed by exploring the mutational effect induced by the other occurring residues on both the activity and the physico-chemical properties on the model enzyme SmAA10. Our results emphasize that this unusual coordination motif imparts unique structural and functional features to the copper centre. Furthermore, the SmAA10 active site variants allowed us to create original metal binding sites with the perspective of exploring new abiological metal-based biocatalytic reactions

Dissertation (Ph.D.)--University of Toledo, 2008.
"In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Title from title page of PDF document. Bibliography: p. 126-146.

Une attention croissante est portée aux cascades multi enzymatiques pour l’élaboration de procédés de synthèse plus efficaces. Cependant, le contrôle de l’expression hétérologue de plusieurs gènes dans un même hôte s’avère difficile et peut mener à un déséquilibre du flux réactionnel. Pour exploiter au mieux les avantages d’une cascade in vivo, il est nécessaire d’ajuster les activités de chaque étape, et de construire des catalyseurs cellulaires capables de programmer la stœchiométrie des enzymes. Nous avons développé dans ce projet une approche originale pour moduler le ratio de deux enzymes in cellulo en jouant sur le nombre de copies de plasmides par cellule (PCN). Nous avons choisi comme modèle un système autosuffisant associant une Alcool Déshydrogénase (ADH) et une Baeyer-Villiger MonoOxygenase (BVMO), NADP(H)-dépendantes. Plusieurs plasmides recombinants portant les deux gènes ont été conçus et combinés dans E. coli. Les souches de co-expression construites ont été comparées en termes de PCN, de production d’enzymes et d’activité. Nous avons montré l’importance d’un choix judicieux de la combinaison de plasmides ainsi que l’existence d’une corrélation entre ratios d’enzymes et activité. Nos biocatalyseurs s’étendent sur une gamme allant du système inactif à un système affichant un TTN d’environ 6000. Ce système a permis la synthèse de lactones d’intérêt industriel, la dihydrocoumarine et la caprolactone, à partir d’indanol et de cyclohexanol. Enfin, sur ce modèle de combinaison de plasmides, trois nouveaux biocatalyseurs cellulaires, associant l’ADH à diverses BVMOs, ont été créés afin d’élargir la gamme d’esters et de lactones synthétisables à partir d’alcools
Growing attention is paid to multienzymatic cascades to develop more efficient synthetic processes. However, in in cellulo process, the control of the simultaneous heterologous expression of several genes in the same host is often difficult and can lead to imbalances in the reaction flow. To exploit the benefits of cascades, activities of each step have to be adjusted and thus, cellular biocatalysts capable of programming enzymes stoichiometry have to be constructed. In this work, to modulate the stoichiometry of two enzymes in vivo, we developed an original approach based on the copy number per cell of plasmids (PCN) used as vectors. The PCN is regulated in bacteria by three main mechanisms leading, according to the replicon, to low, medium or high PCN. As proof of concept, we chose a self-sufficient system combining an Alcohol Dehydrogenase (ADH) and a Baeyer-Villiger MonoOxygenase (BVMO), both NADP(H)-dependent. Several recombinant plasmids harboring both genes were designed and combined in E. coli. Coexpression strains constructed were compared in terms of PCN, enzyme production and activity. We showed the importance of a judicious choice of plasmids combination and the existence of a correlation between enzymes ratios and activity. Our biocatalysts ranged from an inactive system to a system with a TTN of about 6000. This system allowed the synthesis of lactones of industrial interest, dihydrocoumarin and caprolactone, via double oxidation of indanol and cyclohexanol. Finally, based on this plasmids combination model, three new cellular biocatalysts combining ADH with various BVMOs were designed to broaden the range of esters and lactones synthesizable from alcohols

Une attention croissante est portée aux cascades multi enzymatiques pour l’élaboration de procédés de synthèse plus efficaces. Cependant, le contrôle de l’expression hétérologue de plusieurs gènes dans un même hôte s’avère difficile et peut mener à un déséquilibre du flux réactionnel. Pour exploiter au mieux les avantages d’une cascade in vivo, il est nécessaire d’ajuster les activités de chaque étape, et de construire des catalyseurs cellulaires capables de programmer la stœchiométrie des enzymes. Nous avons développé dans ce projet une approche originale pour moduler le ratio de deux enzymes in cellulo en jouant sur le nombre de copies de plasmides par cellule (PCN). Nous avons choisi comme modèle un système autosuffisant associant une Alcool Déshydrogénase (ADH) et une Baeyer-Villiger MonoOxygenase (BVMO), NADP(H)-dépendantes. Plusieurs plasmides recombinants portant les deux gènes ont été conçus et combinés dans E. coli. Les souches de co-expression construites ont été comparées en termes de PCN, de production d’enzymes et d’activité. Nous avons montré l’importance d’un choix judicieux de la combinaison de plasmides ainsi que l’existence d’une corrélation entre ratios d’enzymes et activité. Nos biocatalyseurs s’étendent sur une gamme allant du système inactif à un système affichant un TTN d’environ 6000. Ce système a permis la synthèse de lactones d’intérêt industriel, la dihydrocoumarine et la caprolactone, à partir d’indanol et de cyclohexanol. Enfin, sur ce modèle de combinaison de plasmides, trois nouveaux biocatalyseurs cellulaires, associant l’ADH à diverses BVMOs, ont été créés afin d’élargir la gamme d’esters et de lactones synthétisables à partir d’alcools
Growing attention is paid to multienzymatic cascades to develop more efficient synthetic processes. However, in in cellulo process, the control of the simultaneous heterologous expression of several genes in the same host is often difficult and can lead to imbalances in the reaction flow. To exploit the benefits of cascades, activities of each step have to be adjusted and thus, cellular biocatalysts capable of programming enzymes stoichiometry have to be constructed. In this work, to modulate the stoichiometry of two enzymes in vivo, we developed an original approach based on the copy number per cell of plasmids (PCN) used as vectors. The PCN is regulated in bacteria by three main mechanisms leading, according to the replicon, to low, medium or high PCN. As proof of concept, we chose a self-sufficient system combining an Alcohol Dehydrogenase (ADH) and a Baeyer-Villiger MonoOxygenase (BVMO), both NADP(H)-dependent. Several recombinant plasmids harboring both genes were designed and combined in E. coli. Coexpression strains constructed were compared in terms of PCN, enzyme production and activity. We showed the importance of a judicious choice of plasmids combination and the existence of a correlation between enzymes ratios and activity. Our biocatalysts ranged from an inactive system to a system with a TTN of about 6000. This system allowed the synthesis of lactones of industrial interest, dihydrocoumarin and caprolactone, via double oxidation of indanol and cyclohexanol. Finally, based on this plasmids combination model, three new cellular biocatalysts combining ADH with various BVMOs were designed to broaden the range of esters and lactones synthesizable from alcohols

Lignin and cellulose comprise a large portion of the renewable biomass on Earth. However, substantially due to laborious course of processing, the conversion efficiency of these biomaterials to accessible biofuel is very low. Therefore, effective depolymerization and utilization of these biopolymers are requirements for environmentally friendly and sustainable energy development. In the hope of finding solutions to these biomass utilization challenges, there have been growing interests in using biodegrading metalloenzymes as active biocatalysts. However, there still remain many questions regarding mechanistic details of enzyme catalysis and effective application of these enzymes. This thesis focuses on investigating the redox chemistry involved in the catalytic mechanisms of two main lignin- and cellulose- degrading copper enzymes: multicopper oxidases (MCOs) and lytic polysaccharide monooxygenases (LPMOs).

MCOs are capable of aerobic oxidation of lignin as their primary function, but the nature of their substrate variability also allows the oxidation of not only diverse high potential organic and inorganic complexes, but also earth abundant divalent metal ions such as manganese. LPMOs, on the other hand, enable the cleavage of glycosidic bonds in recalcitrant insoluble cellulosic substances, which are not degradable by other hydrolytic enzymes such as endoglucanases and cellulobiohydrolases.

It is remarkable that nature has created such versatile enzymes with specific active site metals and redox-active amino acids involved in electron transfer, which contribute to substrate oxidation as well as enzyme survival against oxidative damage during catalysis. By gaining a deeper understanding of how these enzymes work, we could greatly enhance current usage efficiencies and develop more energy-efficient biocatalysts.

Chapter I gives an introduction to biological coppers, two groups of bio-degrading copper enzymes: multicopper oxidases (MCOs) and lytic polysaccharide monooxygenases (LPMOs), and the role of redox-active amino acids in electron transfer and enzyme catalysis. For the MCO work, a thermophilic laccase (Tth-lac) from Thermus thermophilus HB27 and a CotA laccase (CotA-lac) from Bacillus Subtilis were studied. For the LPMO work, two cellulose active LPMOs (ScLPMO10B and ScLPMO10C) and a chitin active LPMO (BlLPMO10A) were studied.

Chapter II describes thermodynamic aspects of Tth-lac catalysis. The temperature dependence of the formal potential of type I copper (Cu_T1) in Tth-lac is reported, and the interplay between many competing dynamic and thermodynamic factors which results in thermostability and activity of Tth-lac is discussed.

Chapter III reports the electron transfer (ET) kinetics data obtained with Tth-lac using the transient absorption spectroscopy. The results of photochemical electron/hole transfer studies indicate that the chains of Trp and Tyr can participate in electron transfer through Tth-lac, which could potentially have a role in enzyme catalysis as well.

Chapter IV discusses the protective role of a Trp/Tyr pair positioned close to the trinuclear copper cluster (TNC) in Tth-lac. It is indeed remarkable that laccases are capable of utilizing the power of oxygen to catalyze the oxidation of diverse high-potential substrates. But, as a tradeoff, the utilization of dioxygen can make the enzyme highly susceptible to oxidative damage. Chapter IV provides supporting evidence that led us to conclude that the TNC-proximal Trp/Tyr pair functions as an internal antioxidant for prolonging the enzyme lifetime.

Chapter V describes investigations on the factors that affect MCO catalysis, which include the potentials of the active site coppers, possible reactive intermediates, and common structural motifs. Based on the structural homology between Tth-lac and CotA-lac, some preliminary work done on CotA-lac is also reported.

Chapter VI outlines the work on LPMOs. After the successful expression and purification of ScLPMO10B, ScLPMO10B and BlLPMO10A, standard activity assays were done with insoluble cellulose and chitin substrates to confirm the enzyme activity. The results are compared with that from the photo-degradation experiments to investigate if the photochemically generated Cu(III) species are active intermediates in LPMO catalysis.

Chapter VII reports the results on bioinformatics analysis on the distribution of vicinal amino acids in different enzyme classes. This study was to examine the biological significance of amino acid pairs and clusters existing in many different enzyme classes, with vicinal surface tyrosines in CotA-lac as an underlying motivation behind the work.

This thesis demonstrates that MCOs and LPMOs are truly versatile enzymes which can oxidize such diverse refractory substrates, and there could be multiple pathways that the enzymes achieve this task. As shown so far, not only the active site metals but also the chain of redox-active amino acids as well as metal coordinating residues can contribute to enzyme catalysis.

碩士
國立臺灣師範大學
化學系
100
In this study, two new ligands 7-Impy and 7-MeImpy have been synthesized, and they can coordinate with 3 equivalents of CuI ions to form a trinuclear copper complex [CuICuICuI(7-Impy)](BF4) and [CuICuICuI(7-MeImpy)](BF4). These ligands are similar to our previous developed 7-Dipy ligand in the scaffold to trap three CuI ions. It is known that the hydroxylation of alkane molecules catalyzed by trinuclear copper complexes through the “oxene” insertion mechanism. The active “oxene” with a “1D” spin state will have lower reaction energy barrier when it is tuned by three copper ions. In this study, these new catalysts, [CuICuICuI(7-ImPy)](BF4) and [CuICuICuI(7-MeImPy)](BF4), are also able to catalyze the oxidation of cyclohexane converting to cyclohexanol and cyclohexanone. Compared to our previous developed 7-Dipy ligand, the catalytic reaction is more favorable for the product of cyclohexanol. In this study, we replaced traditional heating by microwave for the ligand synthesis. Microwave can enhance internal heating efficiently, which significantly reduce the reaction time, increase the purity, and reduce the byproducts. In addition, for proving the reaction catalyzed by our trinuclear copper cluster via the O-atom insertion mechanism or radical mechanism, we designed a series of experiments using 5,5-Dimethyl-Pyrroline-N-Oxide (DMPO), which is very sensitive to radical. We added DMPO in catalytic reaction, and measured EPR while in the reaction. We found that there are no radical EPR peaks related to DMPO derivatives, similar to the case of 7-Dipy.

博士
國立清華大學
化學系
92
Copper ions play an essential role in particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath). The particulate methane monooxygenase (pMMO) contains 15 reduced copper ions which are arranged in five trinuclear clusters. Two of these clusters were subsequently found to participate in dioxygen chemistry and hydrocarbon hydroxylation chemistry, called C-clusters. The remaining copper ions were in the reduced d10 state, and were thought to be responsible for channeling electrons to the C-clusters from NADH, called E-clusters. The low temperature EPR spectrum of as-isolated pMMO was deconvoluted into a type 2 Cu(II) signal and a broad, but nearly isotropic EPR signal centered at g ~ 2.1. Earlier magnetization and magnetic susceptibility measurements have suggested that the latter EPR signal, which is not sensitive to microwave power saturation, arise from a ferromagnetically exchange-coupled trinuclear Cu(II) cluster with J = 15�{20 cm–1 and with a zero-field splitting D of +0.018cm-1 (175 G) and E value of 0.005 cm-1 (50 G). By combining EPR spectroscopy and rapid cryogenically trap, we successfully observed the different oxidative phases of the turnover cycle and practically proved the catalytic mechanisms of pMMO. Processing cell growth in a fermentor adapted with a hollow-fiber bioreactor, we successfully prepared the (Cu, Zn)-pMMO. The bulk of the copper ions of the E-clusters have been replaced by divalent Zn ions in (Cu, Zn)-pMMO. The Cu and Zn contents in the (Zn, Cu)-pMMO were determined by both ICP-MS and x-ray absorption K-edge spectroscopy. Further characterization of the (Zn, Cu)-pMMO was provided by low temperature electron paramagnetic spectroscopy during reductive titration and hydrocarbon hydroxylation. These studies indicate that the (Zn, Cu)-pMMO is still capable of supporting the activation of dioxygen, but that the replacement of the E-cluster copper ions has compromised the ability of the protein to mediate the transfer of reducing equivalents to the C-clusters. These observations provide strong support for the electron transfer and catalytic roles that we have previously proposed for the E-cluster and C-cluster copper ions, respectively.

碩士
國立中央大學
化學研究所
94
Recent DFT electronic calculations of a trinuclear copper cluster bis(��3-oxo)trinuclear copper(II, II, III) complex have showed that this structure harnesses a singlet “oxene”, mimicking one of the C-clusters in the particulate methane monooxygenase (pMMO), an important membrane enzyme that mediate the facile hydroxylation of methane and other small hydrocarbons. A series of supporting ligands that are capable to trap three copper ions toward developing a model compound to mimic this chemistry, we have designed and synthesized. Oxygenation of their corresponding [Cu3(I, I, I)L](X) complexes of these ligands leads to the formation of [Cu3(II, II, II)L(O)](X)2 (L = 7-Et, 7-Me, 6-Et, 6-Me; X = ClO4- and BF4-) through mass spectrometry analysis. Only one oxygen atom is locked in the trinuclear copper(II, II, II) complex. When the [Cu3(II, II, II)L(O)](X)2 complex is treated with three equivalent amounts of benzoin and triethylamine in CH3CN, and the solution purged by dioxygen, the benzoin is oxidized to benzil, which in turn is cleaved by further oxidation and hydrolyzes to 2 benzoic acid molecules. This chemistry is mediated by efficient oxo-transfer from the bis(μ3-oxo)trinuclear copper(II, II, III) complex to the benzil, as verified by 18O2 isotope labeling experiments and subsequent GC-MS analysis. We propose a mechanism involving intermolecular oxo-insertion across the C-C bond of the benzil by the bis(μ3-oxo)Cu(II)Cu(II)Cu(III) trinuclear copper intermediate.

碩士
國立中興大學
化學系所
101
Recently, we have successfully developed ligands for capturing three copper ions such as 7-Dipy and 7-N3Et. The 7-Dipy ligand can react with Cu(I) to form tricopper complex [CuICuICuI(7-Dipy)](X) (X = ClO4- or BF4-), which had been confirmed as a good catalyst for C-H bond oxygenation. For simulating the pMMO hydrophobic packet’s environment, we have made another ligand 7-N3Et. Although 7-N3Et system doesn’t have good catalytic efficiency, it has chances to observe intramolecular oxygenation to it’s ethyl substituents. Attempts to understand tricopper cluster, we study tricopper complex of 7-N3Et system, especially its Cu(I)Cu(I)Cu(I) species. When three equivalent of [Cu(CH3CN)4]+ was added to the 7-N3Et ligand to form copper(I, I, I) complex, this complex was instantly injected directly to ESI-MS in anaerobic environment. We first find the spectrum of tricopper complex signals with respect to {[CuICuICuI(7-N3Et)](ClO4)2}+ in ESI-MS. In addition, we successfully obtained several tricopper cluster crystals {[CuIICuII(μ-O)(μ-H2O)CuII(7-N3Et)]+2(H3O+)}(ClO4-)3 and {[CuII CuII(μ-Cl)2CuII(7-N3Et)(H+)]+3(ClO4-)3}, by using cation exchange column chromatography or directly addition of 3M NaClO4(aq) solution. The [CuICuICuI(7-N3Et)]+ was activated by TBHP to oxygenate itself and followed by generation of the acetaldehyde product through intramolecular or intermolecular reaction evidenced by GC-MS.

碩士
國立中興大學
化學系所
101
One-step methane oxidation is extremely difficult, because the hydrocarbon bond-dissociation energy of methane is high to 104 kcal / mol. This is very difficult process in industry, however, there is a methanotrophic bacteria and it can use the particulate methane monooxygenase (pMMO) itself to form methanol as its food by oxidation of methane at room temperature under atmospheric pressure. With the more understanding of the crystal structure of particulate methane monooxygenase (pMMO), we thought that it is the trinuclear copper structure in the active center. In our previous study, a ligand 7-Dipy has been first synthesized, and it is capable of trapping tree equivalents of copper ions to form a trinuclear copper cluster as an catalyst model of pMMO. In this study, catalyst is try to be activated by a variety of reducing agents and use the oxygen from the air to oxidize the alkane substrates at room temperature under atmospheric pressure. We found that when sodium ascorbate was employed as reducing agent, [CuIICuII(μ-O) CuII(7-Dipy)]2+ (2) can be fully reduced in 20 minutes. Currently, the oxidation of C-H bonds of cyclohexane can be achieved with reacting with oxygen and its TON can reach to 25 with conversion is 42.4% and TOF is 0.18 min-1, and the 50 equivalents sodium ascorbate could be consumed completely about three to four hours. There are no other byproducts in this catalyst, and the whole process is simple and facile. In other ways, we used Dipy as ligand to prepare (CuII -Dipy)2+ (6) and found that we can use the same method to do catalytic oxidation reaction of cyclohexane like [CuIICuII(μ-O)CuII(7-Dipy)]2+ (2). They both have great power of catalytic oxidation with cyclohexane. However, in comparison with their mechanisms, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was added in the catalytic process to trap if any free radicals form in reactions and observed by electron paramagnetic resonance spectroscopy (EPR). For tricopper cluster, there is no any radical formed during time course observation, but obvious ‧OH radical formation was present in monocoper case.

碩士
國立臺灣師範大學
化學系
100
In previous study, a ligand 7-Dipy has been synthesized, and it can coordinate with tree equivalents of [CuI(CH3CN)4](BF4) to form a trinuclear copper complex [CuICuICuI(7-Dipy)](BF4) (1). This trinuclear copper complex [CuICuICuI(7-Dipy)](BF4) (1) catalyst is able to oxidize the C－H bonds of cyclohexane (C－H BDE = 99.3 kcal/mol) to cyclohexanol and cyclohexanone with high turnover frequencies in the presence of H2O2 in acetonitrile under ambient conditions. The oxygenation of [CuICuICuI(7-Dipy)](BF4) (1) either by dioxygen or two equivalents of H2O2 will obtain a stable [CuIICuII(µ-O)CuII(7-Dipy)](BF4)2 (2). In my study, 400 MHz 1H-NMR and ESI-MS spectra demonstrate that the 7-Dipy add tree equivalents of [CuI(CH3CN)4](BF4) will obtain [CuICuICuI(7-Dipy)](BF4) (1) complex. In the same catalytic conditions, we found that mononuclear copper complexes [CuI(CH3CN)4](BF4) (7) and [CuI(bispicolylamine)](BF4) (8) compared to trinuclear copper complex [CuICuICuI(7-Dipy)](BF4) (1) a significantly lower level of cyclohexane is oxidized. A time-course study indicates that the H2O2 used to turn over the trinuclear copper complex [CuICuICuI(7-Dipy)](BF4) (1) catalyst for substrate oxidation is already exhausted within 45 min. To further understand the trinuclear copper complex [CuICuICuI(7-Dipy)](BF4) (1) carrying out catalytic reaction are free radical mechanism or direct oxene insertion mechanism, therefore, we designed a series of experiments using very sensitive to free radicals DMPO, to detect the trinuclear copper complex [CuICuICuI(7-Dipy)](BF4) (1) by adding H2O2 for the catalytic reaction whether involvement free radical mechanism. EPR spectra demonstrate that trinuclear copper complex [CuICuICuI(7-Dipy)](BF4) (1) catalytic reaction can rule out the involvement of free radical mechanism. Key Word:Particulate Methane Monooxygenase、Trinuclear Copper Clusters、Catalysis

碩士
國立中興大學
化學系所
103
The ligand diNpy (1,3-bis((pyridin-2-ylmethyl)amino)propan-2-ol), had been known to be capable of forming either mononuclear and dinuclear copper complexes by mediating the equivalent of metal ions. According to pMMO’s crystal structure, and EPR data, the mononuclear complex Cu(diNpy)(ClO4)2 (1) and the dinuclear complex [Cu2(diNpy)(O2P(OPh)2)(CH3OH)(ClO4)]ClO4 (2) can probably act as the pMMO models for the oxygenation of the C-H bond of alkanes. In the comparisons of the oxygen atom transfers (OAT) into cyclohexane under mild condition, however, the catalytic ability of the tricopper complexes [CuICuICuI(7-py)]+ and [CuICuICuI(7-Dipy)]+ are proved to be much superior than mono- or dimer-copper diNpy complexes. Based on the coordination chemistry of diNpy, the corresponding mono- or dinuclear iron(II) complexes were synthesized in air atmosphere, nevertheless, a new trinuclear iron(III), [FeIIIFeIII(μ-O)FeIII(diNpy)2((2-py)COO)2](ClO4)3 (5-2) was obtained. Clearly, these iron(II) diNpy complexes are very reactive with oxygen, and can proceed the OAT to the C-N or C-Cl bonds. In the treatment of two equiv. of TBHP, a characteristic EPR similar to the typical low-spin III (S=1/2) [FeIII(TPA)(OOH)(CH3CN)2], which was known as the high-valent FeIV=O or FeV=O precursor, was obtained. The mechanism for the catalytic reaction of iron(II or III) diNpy relative complexes were proposed on the basis how the oxygen atoms activated by sMMO.

碩士
國立臺灣師範大學
化學系
99
It is known that the hydroxylation of alkane molecules catalyzed by trinuclear copper complexes through the “oxene” insertion mechanism. The active “oxene” with a “1D” spin state will have lower reaction energy barrier when it is tuned by three copper ions. In my first study, for understanding the role of the copper ion in the apex position in the scaffold of isosceles tricopper cluster, we modified a hydroxyl group on the 7-membered amino ring to obtain two whole new 7-OH-Me and 7-OH-Et ligands. By means of the modified hydroxyl group, it is capable of mediating the electron densities on the vertex of copper triad. According to the ESI-MS spectra of oxygenated [CuICuICuI(7-OH-Me)] and [CuICuICuI(7-OH-Et)], which are synthesized by mixing one equiv. of ligand and three equiv. of [CuI(CH3CN)4](BF4 or ClO4), the cluster signals which are associated with [CuIICuII(-O)CuII(L)](X) (L = 7-OH-Me or 7-OH-Et; X = BF4 or ClO4) only appear. Interestingly, a minor signal with m/z = 14 molecular weight lower than [CuIICuII(-O)CuII(7-OH-Me)](X) are suspected to encounter N-demethylation, and the main signal of oxygenated [CuICuICuI(7-OH-Me)] present a [CuIICuII(-O)CuII(7-OH-Me)](X) tricopper complex plus a m/z = 34 amu. However, the main signal of oxygenated [CuICuICuI(7-OH-Et)] refers to a [CuIICuII(-O)CuII(7-OH-Me)](X) tricopper complex plus a m/z = 57 amu. These two ESI-MS spectra are not seen in the corresponding oxygenated [CuICuICuI(7-Me)]+ and [CuICuICuI(7- Et)]+. In my second study, based on our previous studies about efficient oxidation of cyclohexane catalyzed by [CuICuICuI(7-Dipy)]+ in the bulk of H2O2, we modified the ligand 7-Dipy to a 7-OH-Dipy as well. However, the ESI-MS of oxygenated [CuICuICuI(7-OH-Dipy)] present a main cluster, [CuIICuII(-O)CuII(7-OH-Dipy)](BF4 or ClO4)2, which shows the modified hydroxyl group is not linked to the copper ion completely different from the 7-OH-Me and 7-OH-Et ligands. Nevertheless, the hydroxyl group on the 7-membered ring might be replaced by a thiol group (-SH) to anchor the active [CuICuICuI(7-Dipy)]+ tricopper on the gold thin film for a series of electrochemical studies.

碩士
國立臺灣師範大學
化學系
99
In this study, a new ligand 7-N3Et has been synthesized, and it can coordinate with 3 equivalents of CuI ions to form a trinuclear copper complex [CuICuICuI(7-N3Et)](X) (X = ClO4 or BF4) (2). This 7-N3Et ligand is similar to our previous developed 7-Dipy ligand in the scaffold to trap three CuI ions. They all have a pair of symmetric N3 coordination which consists of N1-(2-(diethylamino)ethyl) -N2,N2-diethylethane-1,2-diamine in 7-N3Et instead of 2,2-bis(pyridylmethyl)amine group for 7-Dipy. According to the efficient oxidation of cyclohexane catalyzed by [CuICuICuI(7-Dipy)]+, the [CuICuICuI(7-N3Et)]+ was adopted for comparing the catalytic effects arisen from substitution groups. The ESI-MS spectra of oxygenated [CuICuICuI(7-N3Et)]+ by dioxygen molecules, two equivalents of H2O2, and two equivalents of TBHP present a exactly same cluster signal associated with a [CuIICuII(-O)CuII(7-N3Et)]2+ trinuclear copper complex. However, with more than two equivalents of TBHP, it will encounter N-deethylation to loss a molecular weight in m/z of 28 amu and 56 amu. When one equiv. of [CuICuICuI(7-Dipy)]+ is added in the presence of 10, 25, 50 and 75 equiv. of TBHP , respectively, as oxidant with external substrates such as cyclohexane (C-H BDE : 99.3 kcal/mol) and toluene (C-H BDE : 90 kcal/mol) in the solvent of CH3CN for two hours, substrates can be oxygenated with lower TON because of competing reactions between intra-N-deethylation and inter-oxygenation. In contrast, no external substrate was added in the above condition, and the acetalaldehyde was detected by GC-MS when 50 equiv. of TBHP was used. The production of acetalaldehyde is postulated from first hydroxylation on the -CH2- of the amine group, and then disassociation by proton migration of hydroxyl group to the nitrogen atom of amine.

碩士
國立臺灣師範大學
化學系
99
In first study, a new modified trinuclear copper complex, [CuICuICuI(7-dipy)](BF4) (2), was first employed as a catalyst to oxidize the CH bonds of cyclohexane (CH BDE is 99.3 kcal mol-1). ESI-MS spectra demonstrate that the oxygenation of [CuICuICuI(7-dipy)](BF4) (2)either by dioxygen will obtain a stable [CuIICuII(-O)CuII(7-dipy)](BF4 )2(3) complex. The catalysis of CH bond oxygenation of cyclohexane was carried out under room temperature in the presence of 50 equivalents of oxidant, and a product mixture of cyclohexanol and cyclohexanone were observed with 34% conversion according to the consuming of the oxidant by the quantitative GC-MS analysis. However, there is nearly no reaction by employing [CuIICuII(-O)CuII(7-dipy)](BF4)2 (3) complex instead of [CuICuICuI(7-dipy)](BF4) (2). This tricopper complex is a quite robust catalyst because most the remainders after the catalytic reaction are in the form of [CuIICuII(-O)CuII(7-dipy)](BF4)2 (3)evidenced by ESI-MS spectra. In second study, the same 7-dipy ligand was also adopted in the synthesis of trinuclear manganese complex. A first trimanganese complex [MnII(OAc)2MnII(-OAc)2MnII(7-dipy)] (2) was first synthesized as a precursor for the high-valent manganese species. Further oxidation of [MnII(OAc)2MnII(-OAc)2MnII(7-dipy)] (2) by treating two equivalents of TBHP (tert-butylhydroperoxide) is able to obtain a 16-line characteristic EPR spectrum with gx= 2.006, gy= 1.998, gz= 1.985, AIII xx=-16.3 mT, AIII yy= -11.7 mT, AIII zz= -16.2 mT, AIV xx= 8.2 mT, AIV yy= 8.0 mT, AIV zz= 7.4 mT acquired by simulation, which is postulated as a active intermediate, [MnIIIMnIII(-O)2MnIV(7-dipy)]4+ (3). While excess of TBHP up to 15 equivalents were added, and the 16-line EPR spectra still remain unchanged. [MnIIIMnIII(-O)2MnIV(7-dipy)]4+ (3) is able to catalyze the oxidation of CH bonds of cyclohexane (CH BDE is 99.3 kcal mol-1) to a mixture of cyclohexanol and cyclohexanone, CH bonds of n-hexane in the C-2 and C-3 position (CH BDE is 98 kcal mol-1 and 99.1 kcal mol-1, respectively) to a mixture of 2-hexanol, 3-hexanol, 2-hexanone and 3-hexanone. Except the CH bond oxidation in the secondary carbon atom position, ethane molecule which merely has primary CH bonds (CH BDE is 101 kcal mol-1) was applied as the substrate, and the suspected acetic acid product involving 6 oxidation equivalents was found. Ethanol molecule (CH BDE is 95.6 kcal mol-1) as the substrate was also oxidized in the same catalysis to form the acetic acid product, providing the support for the oxidation of ethane molecule.

碩士
國立臺灣大學
化學研究所
93
Potentiometric titrations of the copper centers in the particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath) are described. The reduction potentials of the various copper centers were measured by poising the enzyme in pMMO-enriched membranes in the presence of different redox mediators in an electrochemical cell, and quickly freezing the sample to record their low temperature EPR signals. Measurements of the low temperature EPR intensities of the type 2 sites and trinuclear Cu(II) centers yielded [Cu(I)]/[Cu(II)] for the various copper centers at the various different potentials, and the mid-point potentials were determined from the Nernst equation. The measurements were performed on two different forms of the enzyme in pMMO-enriched membranes: (i) the enzyme as isolated aerobically, but in the absence of hydrocarbon substrate; and (ii) the same preparation oxidized under air in the presence of acetylene or other hydrocarbon substrates. While the E-cluster copper sites exhibited essentially the same high reduction potential of +490 mV, substantially different results were obtained for the redox behavior of the C-cluster copper ions between the two forms. The reduction potentials of the oxidized C-cluster copper sites observed in the EPR were found to be substantially lower compared to that of the E-clusters. Accordingly, the redox potential of the oxidized bis(μ–oxo)dicopper(III) and bis(μ–peroxo)dicopper (II) dimer associated with the isolated type 2 site of the same cluster must be considerably higher than +490 mV, and there must be a kinetic barrier for transfer of electrons from the E-clusters to this site. This “splitting” of the redox potential of the trinuclear copper cluster is artifactual of the non-physiological state of enzyme created from the oxidation of the six copper ions by eight oxidizing equivalents from two dioxygen molecules. When the enzyme is turning over in the presence of hydrocarbon substrate, the dioxygen chemistry is tightly linked to the hydroxylation chemistry to achieve kinetic competence, and the redox potentials of the C-cluster copper ions were found to be substantially higher. The results of the present study add a significant chapter to our understanding of the structure and function of pMMO. First, the potentiometric titrations confirmed without any further question the existence of C-cluster and E-cluster copper ions, the number of distinct C-clusters, as well as the functional role they play in the methane hydroxylation chemistry, as the various copper centers are distinguished by distinct redox behaviors in the EPR, as manifested by their different appearance in the EPR spectrum at different potentials. Second, the different redox behavior of the C-clusters copper ions between the “as-isolated” enzyme and during turnover in the presence of hydrocarbon substrate has led to important insights into the details of how electron transport, dioxygen chemistry and hydrocarbon oxidation are linked in this complex system.

碩士
國立成功大學
化學系碩博士班
93
Methylococcus capsulatus (Bath) is one of methanotrophic bacteria.Differential expression of either sMMO or pMMO is regulated by the concentration of copper ions available to the cells. During the past decade, the experiments from either electron paramagnetic resonance (EPR) or X-ray absorption spectroscopy conclude that pMMO is a multicopper protein. The copper ions in the pMMO were classified into C-clusters and E-clusters. The E-clusters found to be associated with water-exposed domains of the 45 kDa subunit which were responsible for channeling electrons to the C-cluster. It would be crucial to shed light on the copper binding affinity on even coordination feature in this aqueous domain of PmoB subunit to show how the electrons transfered from E-Cluster to C-cluster where the methane oxidation take place. Therefore, over-expressed PmoB subunit, a water-soluble domain of of pMMO in E.coli. system was conducted by using gene engineering technique. Thus, whether partially expressed proteins can be folded properly or the copper ions exhibt capability to sequester can be possilbly deduced. Furthermore, the copper concentration was quantitated with atomic absorption spectroscopy, and the copper binding constant was examessed by Scatchard plot. The results indicated that the copper ion played at least two important roles in both coordinating with protein and stablized the protein folding structure. The maximum number of protein binding copper is about 16.70. In the future, we will express other domains of water-exposed protein to study which sequence has stronger copper binding effect, and resolve their structures and copper coordinations via X-ray crystallography.

碩士
國立中興大學
化學系所
105
This study demonstrated that the methane/ethane oxidation is able to be facilely mediated by the reaction of [CuICuICuI(7-Dipy)](ClO4)2 with hydrogen peroxide or oxygen. By means of combining ESI-MS (Electrospray Ionization Mass Spectrometry) with RFQ (Rapid Freeze Quench), we first successfully detected the highly acitve species [CuIICuII(μ-O)2CuII]4+ or [CuIICuIII(μ-O)2CuIII]4+, which are proposed to closely correlate with the active intermediate [CuIICuII(μ-O)2CuIII]3+ in pMMO hydroxylation. Moreover, for precisely quantifying the employed H2O2 in the catalytic reaction carried out by tricopper cluster, hydrogen peroxide (H2O2) concentration has been first determined by using absorption spectral titration of the CH2Cl2 solution containing Co(II)(OETTP) and imidazole with the ratio (1:2). In such reaction, H2O2 acts as one-electron oxidant. When Co(II)(OETTP) underwent one-electron oxidation, imidazole will rapidly coordinate to the Co(III) ion to give rise to clear changes of the absorption.

碩士
國立清華大學
化學系
91
The catalytic sites in the particulate methane monooxygenase from Methylococcus capsulatus (Bath) involve two sets of trinuclear Cu(II) clusters. It has been proposed that when the trinuclear copper clusters of pMMO are fully oxidized, each of the oxidased clusters consists of three type 2 copper centers that are mutually weakly ferromagnetically coupled (J ~ 20cm-1) and D ≦ 0.05 cm-1. Toward elucidating these results, several trinuclear Cu(II) model complexes are synthesized and characterized. The complexes, [Cu3C18H42N6O3(μ3-Br)2](Br)4 and [Cu3C18H42N6O3(μ3-Cl)2](Cl)4, are ferromagnetically coupled with J = 430 cm-1 and 470 cm-1, respectively. Both cases show the isotropic EPR signal at g = 2.092 and g = 2.104 with D ≦ 0.009 cm-1. Density functional calculations reproduce well these geometric, electronic and magnetic properties of these tricopper complexes, and provide insights into the spin coupling interactions mediated by the bridging ligands. Furthermore, they reasonably explain that the anisotropic EPR signal abserved of [Cu3C18H42N6O3(μ3-OH)2](Cl)4 might arise from the antiferromagnetic interactions among the three copper(II) ions.