Thesis (Ph. D.)--University of Hong Kong, 2005.

M.Sc. (Biochemistry) / Manganese (Mn) is an essential trace element. Although it is vital for the normal development of mammals, too much Mn can be harmful. Most reported cases of toxicity have been found in occupational settings, such as welding, mining and ferro-manganese (FeMn) production plants. Long-term overexposure to Mn can result in lung epithelial necrosis and the development of a neurological disease, manganism. Even though evidence of Mn-associated diseases exists, some epidemiological studies have found no association between occupational exposure levels and possible indicators of neurotoxic effects. It is, therefore, important to establish Mn toxicity and the mechanisms involved in this toxicity, for a possible identification of biomarkers of exposure and effect. The hypothesis formulated states that, FeMn particulate matter consists of nano and micro sized particles that, upon inhalation, may cause injury to the lungs and translocate to the brain. Since Mn-induced injury to the brain and lungs is a possibility, this study aimed to investigate the effects of FeMn dust, which was collected from a FeMn smelter works, on primary rat astrocytes and human bronchial epithelial (BEAS-2B) cells. This was achieved by first characterizing the physicochemical properties of the particles by using a scanning mobility particle sizer (SMPS) and aerodynamic particle sizer (APS) for size distribution, Brunauer-Emmett-Teller (BET) for surface area determination and inductively coupled plasma atomic emission spectroscopy (ICP-AES) for elemental composition analysis. Cells were treated with 5, 10, 25 μg/cm2 FeMn, and particle uptake, by astrocytes and BEAS-2B cells, was confirmed using dark field microscopy e.g. Cytoviva® hyperspectral imaging system. The viability and toxicity of FeMn was studied using the conventional toxicity assay systems, including 3-bis [2-Methoxy-4- nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide salt (XTT), adenosine triphosphate (ATP) and lactate dehydrogenase (LDH) assays. It was, however, established that FeMn particles interfere with the final read-out produced by some of these assay systems. Therefore, a rare application of the xCELLigence real time cell analysis (RTCA) system was implemented, as a better option, in the assessment of the toxicity and viability of cells in the presence of FeMn particles. The ability of FeMn particles to cause deoxyribonucleic acid (DNA) damage in both cell types was also determined using the alkaline comet assay. Finally, the nuclear translocation of the antioxidant transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) and inflammatory transcription factor nuclear factor kappa B (Nf-κB), was studied using Western blotting. The results showed that FeMn, in a dose dependent manner, could enter the cell, decrease the viability, induce DNA damage, and initiate nuclear transport of the studied transcription factors. The same methodologies were implemented to determine the physicochemical properties of Min- U-Sil 5 crystalline silica, used as a positive control, to assess its toxicity and effect on cellular viability. As well as its ability to induce DNA damage and initiate nuclear translocation of the two transcription factors, in astrocytes and BEAS-2B cells. Similar to FeMn particles, crystalline silica also enters the cells with subsequent reduction in cellular viability. It results in increased DNA damage and increased nuclear translocation of the studied transcription factors. The effects of crystalline silica on these cellular effects were, however, always higher than those produced by FeMn particles. To conclude, these results indicate that depending on the size distribution of particles in the work environment, they may enter different regions of the lungs. However, for those particles in the nano size region, direct access to the brain is a possibility. These results also indicate that after deposition in the target organ, these particles will produce cellular changes through oxidative stress. This would lead to inflammation, decreased cellular viability and increased toxicity.

Yes / In the past decade, libraries of iridium organometallic arene compounds have expanded rapidly, with the majority of their applications aimed towards effective catalysts and potential anti-cancer drug candidates. Researchers have begun to adapt the traditional “piano-stool” structures to include different bidentate ligands, ancillary ligands and extend the aromaticity and functionality of the arene substituent, all in the hope to optimize their activities and allow the determination of structure activity relationships. Many of the complexes incorporate N- and O-donor ligands, but more recently, these structures have been expanded to include C-donor ligands such as cyclometalated bidentate ligands and N-heterocyclic carbenes. This mini-review highlights the recent and ongoing research in C-donor iridium arene complexes, and discusses their importance as potential anticancer drugs.

My research primarily focuses on biomimetics of the active sites of the [FeFe]- and [Fe]-hydrogenases (H2ase) and is classified into three parts. Part A: The one-electron oxidation of asymmetrically disubstituted FeIFeI models of the active site of the [FeFe]-H2ase, (mu-pdt)[Fe(CO)2PMe3][Fe(CO)2NHC] (pdt = 1,3- propanedithiolate, NHC = N-heterocyclic carbene) generates mixed valent FeIIFeI models of the Hox state of [FeFe]-hydrogenase. The spectroscopic properties, structures, reactivities and relative stabilities of the one-electron oxidized mixed valent complexes, (mu-pdt)(mu-CO)[FeII(CO)2PMe3][FeI(CO)NHC]+ are discussed in the context of experimental and theoretical data and biological relevance. Part B: DFT computations find the Fe-Fe bond in the FeIFeI diiron models ((mu- pdt)[Fe(CO)2L][Fe(CO)2L'] ( L, L' = CO, PPh3, or PMe3) is thermodynamically favored to produce the mu-oxo or oxidative addition product, FeII-O-FeII, nevertheless the sulfurbased HOMO-1 accounts for the experimentally observed mono- and bis-O-atom adducts at sulfur. The FeII(mu-H)FeII diiron model, (mu-pdt)(mu-H)[Fe(CO)2PMe3]2 (IV-5), for which the HOMO is largely of sulfur character, exclusively yields S-oxygenation. Deoxygenation with reclamation of the mu-pdt parent complexes occurs in a proton/electron coupled process. The possible biological relevance of oxygenation and deoxygenation studies is discussed. Comprehensive investigations of intramolecular CO site change and intermolecular CO/L (L = PMe3 or CN-) exchange of (mu-pst)[Fe(CO)3]2 (IV-1-O), (mu-pdt)[Fe(CO)3]2 (V-1), and their mono-CN-/PMe3 substituted derivatives indicated that the factors influencing the rate of the CO/L exchange reaction of such diiron carbonyls are intramolecular structural rearrangement (or fluxionality) and nucleophilic attack by the incoming ligand. Part C: X-ray diffraction and spectroscopic studies of a series of mono- and disubstituted complexes, FeI2(CO)xL4-x, x = 2 or 3, showed them to be rudimentary structural models of the [Fe]-H2ase active site in native (FeII(CO)2) or CO-inhibited (FeII(CO)3) states. Full characterization of the advanced model complexes ((NS)FeI(CO)2P, NS = 2-amidophenothiolate; P = phosphine) including x-ray diffraction, DFT computations, and Mossbauer studies revealed the interesting "noninnocent" character of these complexes due to the NS ligand. Ligand-based protonation with a strong acid, HBF4Et2O, interrupted the pi-delocalization over Fe and ligand of complex VII-1 and switched on CO uptake (1 bar) and 12CO/ 13CO exchange of VII-1. The intermediate, VII-1-H+, capable of CO uptake, was defined by DFT calculations.

abstract: Oxidoreductases catalyze transformations important in both bioenergetics and microbial technologies. Nonetheless, questions remain about how to tune them to modulate properties such as preference for catalysis in the oxidative or reductive direction, the potential range of activity, or coupling of multiple reactions. Using protein film electrochemistry, the features that control these properties are defined by comparing the activities of five [FeFe]-hydrogenases and two thiosulfate reductases. Although [FeFe]-hydrogenases are largely described as hydrogen evolution catalysts, the catalytic bias of [FeFe]-hydrogenases, i.e. the ratio of maximal reductive to oxidative activities, spans more than six orders of magnitude. At one extreme, two [FeFe]-hdyrogenases, Clostridium pasteuriaunum HydAII and Clostridium symbiosum HydY, are far more active for hydrogen oxidation than hydrogen evolution. On the other extreme, Clostridium pasteurianum HydAI and Clostridium acetobutylicum HydA1 have a neutral bias, in which both proton reduction and hydrogen oxidation are efficient. By investigating a collection of site-directed mutants, it is shown that the catalytic bias of [FeFe]-hydrogenases is not trivially correlated with the identities of residues in the primary or secondary coordination sphere. On the other hand, the catalytic bias of Clostridium acetobutylicum HydAI can be modulated via mutation of an amino acid residue coordinating the terminal [FeS] cluster. Simulations suggest that this change in catalytic bias may be linked to the reduction potential of the cluster. Two of the enzymes examined in this work, Clostridium pasteurianum HydAIII and Clostridium symbiosum HydY, display novel catalytic properties. HydY is exclusively a hydrogen oxidizing catalyst, and it couples this activity to peroxide reduction activity at a rubrerythrin center in the same enzyme. On the other hand, CpIII operates only in a narrow potential window, inactivating at oxidizing potentials. This suggests it plays a novel physiological role that has not yet been identified. Finally, the electrocatalytic properties of Pyrobaculum aerophilum thiosulfate reductase with either Mo or W in the active site are compared. In both cases, the onset of catalysis corresponds to reduction of the active site. Overall, the Mo enzyme is more active, and reduces thiosulfate with less overpotential. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2020

<p>The ability to manipulate the coordination chemistry of metal ions has significant ramifications for the study and treatment of metal-related health concerns, including iron overload, UV skin damage, and microbial infection among many other conditions. To address this concern, chelating agents that change their metal binding characteristics in response to external stimuli have been synthesized and characterized by several spectroscopic and chromatographic analytical methods. The primary stimuli of interest for this work are light and hydrogen peroxide.</p><p>Herein we report the previously unrecognized photochemistry of aroylhydrazone metal chelator ((E)-N&#8242;-[1-(2-hydroxyphenyl)ethyliden]isonicotinoylhydrazide) (HAPI) and its relation to HAPI metal binding properties. Based on promising initial results, a series of HAPI analogues was prepared to probe the structure-function relationships of aroylhydrazone photochemistry. These efforts elucidate the tunable nature of several aroylhydrazone photoswitching properties.</p><p>Ongoing efforts in this laboratory seek to develop compounds called prochelators that exhibit a switch from low to high metal binding affinity upon activation by a stimulus of interest. In this context, we present new strategies to install multiple desired functions into a single structure. The prochelator 2-((E)-1-(2-isonicotinoylhydrazono)ethyl)phenyl (E)-3-(2,4-dihydroxyphenyl)acrylate (PC-HAPI) is masked with a photolabile trans-cinnamic acid protecting group that releases umbelliferone, a UV-absorbing, antioxidant coumarin along with a chelating agent upon UV irradiation. In addition to the antioxidant effects of the coumarin, the released chelator (HAPI) inhibits metal-catalyzed production of damaging reactive oxygen species. Finally a peroxide-sensitive prochelator quinolin-8-yl (Z)-3-(4-hydroxy-2-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)phenyl)acrylate (BCQ) has been prepared using a novel synthetic route for functionalized cis-cinnamate esters. BCQ uses a novel masking strategy to trigger a 90-fold increase in fluorescence emission, along with the release of a desired chelator, in the presence of hydrogen peroxide.</p> / Dissertation

No description available.

PhD / A new family of cationic N-heterocyclic ligand derivatives was prepared and characterised. Among these compounds are halide salts of the dications [Y(spacer)Y]2+, each of which comprise two N heterocyclic donor groups (Y = 4,4′-bipy, pyz, apyz, apym) linked by a conformationally flexible spacer such as (CH2)n, α,α′-xylylene, 2,6-lutidylene or thiabicyclo[3.3.1]nonane-2,6 diyl. The diquaternary halide salts were converted to NO3- and PF6- salts, and interaction of these bridging ligands with labile palladium(II) and platinum(II) precursors afforded several multinuclear complexes. Bis(4,4′-bipyridinium) dications were incorporated into the dinuclear macrocycles [M2(2,2′ bipy)2{4,4′ bipy(CH2)n4,4′-bipy}2]8+ (M = Pd, Pt; n = 4, 6), cis [Pd2Cl4{4,4′ bipy(CH2)34,4′-bipy}2]4+, [Pt2(dppp)2{4,4′-bipy(1,2-xylylene)4,4′-bipy}2]8+ and cis-[Pt2Cl4{4,4′-bipy(1,2-xylylene)4,4′-bipy}2]4+. While bis(pyrazinium) analogues were unreactive towards the palladium(II) and platinum(II) precursors, the doubly deprotonated bis(3 aminopyrazinium) and bis(2 aminopyrimidinium) derivatives served as charge-neutral quadruply-bridging ligands in the complexes [Pt4(2,2′ bipy)4{apyz(CH2)6apyz–2H}2]8+ and [Pt4(2,2′ bipy)4{apym(CH2)5apym–2H}2]8+, both of which feature Pt(II). Pt(II) interactions. Larger species formed when the diamine O,O′-bis(2-aminoethyl)octadeca(ethylene glycol) (PEGda) was treated with cis dinitratopalladium(II) and platinum(II) precursors. The resulting complexes [M(N,N)(PEGda)]2+ (M = Pd, Pt; N,N = 2,2′-bipy, en, tmeda) possessed great size (62 membered chelate rings) and aqueous solubility. DNA-binding studies were conducted with selected complexes in order to investigate the types of interactions these species might participate in. Equimolar mixtures containing either the 16mer duplex DNA D2 or the single strand D2a and palladium(II)/platinum(II) complexes were prepared and analysed by negative-ion ESI MS. Studies of D2/Pd(II) mixtures suggested extensive fragmentation was occuring, and the use of [Pd(tmeda)(PEGda)]2+ and [Pd2(2,2′-bipy)2{4,4′-bipy(CH2)44,4′-bipy}2]8+ resulted in D2 adducts of [Pd(tmeda)]2+ and [4,4′-bipy(CH2)44,4′-bipy]2+, respectively. Decomposition also occurred when D2a was used, although 1 : 1 adducts were observed with [Pd(tmeda)(PEGda)]2+, [Pd(2,2′ bipy)(PEGda)]2+ and [Pd2(2,2′-bipy)2{4,4′-bipy(CH2)44,4′-bipy}2]8+. The low intensities of these adducts indicated that they are unstable towards ESI MS. Analogous ESI-MS experiments using platinum(II) derivatives were performed and, in contrast to those with palladium(II), indicated that the complexes remained largely intact. ESI-MS analysis of D2/Pt(II) mixtures allowed for the detection of 1 : 1 D2 adducts of [Pt(en)(PEGda)]2+, [Pt(tmeda)(PEGda)]2+ and [Pt2(2,2′-bipy)2{4,4′-bipy(CH2)44,4′-bipy}2]8+. Intensities of the adduct ions suggested the greater charge and aryl surface area allow the dinuclear species to bind D2 most strongly. Both [Pt(2,2′-bipy)(Mebipy)2]4+ and [Pt(2,2′ bipy)(NH3)2]2+ gave rise to 1 : 2 adducts of D2, although the latter was found to be a weaker binder, perhaps owing to its lower charge. Data obtained using 1 : 5 (D2 : complex) mixtures were consistent with the results above and suggested that D2 can bind more molecules of daunomycin than any of the platinum(II) species. Analyses of D2a/Pt(II) mixtures gave results similar to those obtained with D2, although fragmentation was more pronounced, indicating that the nucleobases in D2a play more significant roles in mediating decomposition than those in D2, in which they are paired in a complementary manner. Investigations into the effects of selected platinum(II) complexes on the thermal denaturation of calf-thymus DNA (CT-DNA) in solution were conducted. Both [Pt2(2,2′ bipy)2{4,4′-bipy(CH2)64,4′-bipy}2]8+ and [Pt(2,2′-bipy)(Mebipy)2]4+ greatly stabilised CT-DNA, most likely by intercalation. In contrast, [Pt(tmeda)(PEGda)]2+ and [Pt(en)(PEGda)]2+ (as well as PEGda) caused negligible changes in melting temperature (∆Tm), suggesting that these interact weakly with CT-DNA. Data for [Pt(2,2′ bipy)(PEGda)]2+ and [Pt(2,2′-bipy)(NH3)2]2+ indicated that these species perhaps intercalate CT-DNA, with similar ∆Tm values for both complexes implying that PEGda does not play a major role in binding. While findings from ESI-MS experiments were similar to those from the thermal denaturation experiments, discrepancies between results from the two methods could be found. In particular, fragmentation of cyclic species during ESI-MS caused the binding strength of the species to be underestimated when this method was employed.

Ph.D. / Biochemistry and Molecular Biology / The human copper chaperone to superoxide dismutase (hCCS) is a zinc and copper containing protein that delivers copper to the active site of the cytoplasmic protein superoxide dismutase (SOD). hCCS is a three domain protein with three possible copper binding sites: Domain I is called the Atx-like domain and contains the copper binding motif MXCXXC; Domain II is the SOD-like domain and includes the slightly altered histidine rich copper binding site seen in SOD; and Domain III is a short C-terminal tail that has the copper binding motif of CXC. Studies of the WT protein using EXAFS showed that the protein contained a binuclear copper-sulfur cluster. Initially, it was unknown whether this cluster was formed between domain I and domain II of the protein or formed intermolecularly between two hCCS monomers. Further studies, on the cysteine to serine mutants of the residues in the Domain I and Domain II motifs, showed that while the Domain I motif is capable of binding Cu(I), it is Domain III that is the site of the copper cluster and the cluster is formed between two hCCS monomers. Additional studies with cysteine to alanine mutants of the residues in the copper binding motifs of Domain I and Domain III showed that Domain III is not only the site of the copper cluster but also the site of transfer of copper from hCCS to SOD and also a imerization interface for hCCS. While Domain I can bind copper and may play a role in regulation of activity, it is Domain III that contains the activity of hCCS.

The A-cluster active site in acetyl coA synthase exploits a Ni(CGC)2- metallopeptide as a bidentate ligand to chelate the catalytically active square-planar nickel center used to produce acetyl coA. As Nature utilizes polypeptides to isolate and stabilize the active sites, we have set out to immobilize biomimetic complexes to polyethylene-glycol (PEG) rich polystyrene polymer beads (TentaGel). The PEG rich resin-beads serve to imitate the peptidic superstructure of enzyme active sites as well as to protect the resin-bound models from O2 decomposition. As a model of the NiN2S2 ligand observed in the A-cluster of acetyl coA synthase, the CGC tripeptide was constructed on resins using Merrifield solid phase peptide synthesis and then metallated with NiII to produce bright orange beads. Derivatization with M(CO)x (M = Rh, W) provided qualitative identification of Ο-Ni(CGC)M(CO)x n- via ATR-FTIR. Additionally, Neutron Activation Analysis (NAA) and UV-vis studies have determined the concentration of Ni and CGC, and qualitatively identify Ο-Ni(CGC)2-. Furthermore, infrared studies and NAA experiments have been used to identify and quantify Ο- Ni(CGC)Rh(CO)2 1-. The S-based reactivity of Ni(ema)2-, a good model of Ni(CGC)2-, toward oxygenation and alkylation has been pursued and compared to neutral NiN2S2 complexes. The spectroscopic, electrochemical and structural effects of these modifications will be discussed and supported using DFT computations and electrostatic potential maps of the resulting Ni(ema)*O2 2- and Ni(ema)*(CH2)3 complexes. Having firmly established the synthesis, characterization and reactivity of NiN2S2 2- systems in solution and resin-bound, CuIIN2S2 analogues were explored. The synthesis and identification of solution complexes, Cu(ema)2-, Cu(emi)2-, and Cu(CGC)2- via UV-Vis, EPR, and –ESI-MS will be discussed in addition to their S-based reactivity with Rh(CO)2 + . Furthermore, the resin-bound Cu(CGC)2- complex has been produced and characterized by EPR and its Rh(CO)2 adduct identified by ATR-FTIR and compared to the analogous NiN2S2 2- systems. As the active site of [FeFe] Hydrogenase utilizes a unique peptide-bound propane dithiolate bridge to support the FeFe organometallic unit, [FeFe]Hydrogenase models have been covalently anchored to the resin-beads via similar carboxylic acid functionalities. The characterization (ATR-FTIR, EPR, Neutron Activation Analysis), stability and reactivity of the immobilized models complexes are discussed as well as work toward establishing the microenvironment of resin-bound complexes.