Efficient Reusable Gold-Mesoporous Silica Nanocatalysts for Aromatic Nitro Reduction: Role of Phenolic Chelating Ligands on Immobilizing Gold Nanoparticles and Catalytic Activity

Developing heterogeneous metal nanocatalysts is highly desirable since the catalyst can be easily separated and reused for several times. In this manuscript, we have immobilized gold nanoparticles (AuNPs) on the surface of mesoporous silica (SiO[Formula: see text] using simple amino acid-based phenolic chelating molecules and utilized as highly reusable catalyst for nitroarene reduction. The synthesized nanocomposites (Au@SiO2-1 and Au@SiO2-2) have been unambiguously confirmed using powder X-ray diffraction (PXRD), Fourier transform infrared (FT-IR), high resolution-transmission electron microscopy (HR-TEM) and X-ray photoelectron spectroscopy (XPS). Interestingly, Au@SiO2-1 exhibited highly enhanced 4-nitrophenol reduction that was studied using absorption spectroscopy. Further catalytic activity of Au@SiO2-1 was also explored for 2-nitroaninline and 4-nitroaniline. The reusable studies demonstrated that the catalyst did not show significant change in the activity up to ten cycles. After catalytic reactions studies confirmed the strong attachment of AuNPs on the SiO2 matrix.

Directed Evolution and Discovery of Nitroreductase Enzymes for Targeted Cell Ablation

<p>Nitroreductase enzymes are a superfamily of bacterial flavoproteins that can catalyze the reduction of aromatic nitro groups. The reduction of an aromatic nitro group, a highly electronegative functionality, causes a large electronic shift that can profoundly affect the activity of other substituents on the aromatic ring. For example, upon nitroreduction, initially non-toxic compounds known as prodrugs can be converted into a cytotoxic form. The ability of nitroreductases to alter the activity of compounds has lead to their development as tools for multiple biotechnological applications. Of particular note is the use of nitroreductase enzymes in combination with a nitroaromatic prodrug to study the role of specific cell populations in zebrafish (Danio rerio). Zebrafish are used as model organisms to study processes such as embryonic development and tissue regeneration. By expressing a nitroreductase enzyme in a specific tissue of a zebrafish, it is possible to selectively ablate that tissue upon administration of a prodrug. The subsequent phenotypic change induced by the ablation can provide information on the physiological role of the ablated tissue, or of the regenerative processes that can be recruited to repair the damage. The goal of this thesis was to engineer or discover new nitroreductase enzymes that could expand the capabilities of cell ablation studies in zebrafish. In particular, this work sought to develop a system that would enable the dual, or multiplexed, ablation of two tissues independently within the same organism. Control over the ablation of two distinct tissues could be useful for studying tissue interactions during developmental or regenerative processes. For this to be achievable, two different nitroreductase enzymes, each possessing distinct and non-overlapping prodrug selectivities would be required. Previous studies in the Ackerley lab had identified NfsA from Escherichia coli (NfsA_Ec) and NfsA from Pseudomonas putida (NfsA_Pp) as nitroreductase enzymes that were slightly more selective for the prodrug tinidazole compared than metronidazole. In contrast the NfsB nitroreductase from Vibrio vulnificus (NfsB_Vv) was substantially more selective for metronidazole than tinidazole. To further improve the tinidazole selectivity of the NfsA enzymes, directed evolution was employed as a tool to further enhance the substrate selectivity of each enzyme. The primary outcome of this work was the evolution of an NfsA_Ec mutant that was 12 fold more selective for tinidazole over metronidazole than wild type NfsA_Ec. In addition to engineering new enzymes for cell ablation experiments, this work also sought to discover new nitroreductase enzymes from unculturable bacteria, a previously unplumbed source. The genes and gene products of unculturable bacteria can be identified and studied by expressing fragments of their DNA in a readily culturable host such as E. coli. A variety of different screening methodologies were tested for identifying nitroreductase enzymes from eDNA inserts. The compound 4-nitroimidazole was found to be capable of detecting nitroreductase expression at the level of a single colony. While no novel nitroreductase enzymes were discovered in the scope of this work, the preliminary results are encouraging that a screening strategy centred on 4-nitroimidazole in particular could successfully do so in the near future.</p>

Directed Evolution and Discovery of Nitroreductase Enzymes for Targeted Cell Ablation

<p>Nitroreductase enzymes are a superfamily of bacterial flavoproteins that can catalyze the reduction of aromatic nitro groups. The reduction of an aromatic nitro group, a highly electronegative functionality, causes a large electronic shift that can profoundly affect the activity of other substituents on the aromatic ring. For example, upon nitroreduction, initially non-toxic compounds known as prodrugs can be converted into a cytotoxic form. The ability of nitroreductases to alter the activity of compounds has lead to their development as tools for multiple biotechnological applications. Of particular note is the use of nitroreductase enzymes in combination with a nitroaromatic prodrug to study the role of specific cell populations in zebrafish (Danio rerio). Zebrafish are used as model organisms to study processes such as embryonic development and tissue regeneration. By expressing a nitroreductase enzyme in a specific tissue of a zebrafish, it is possible to selectively ablate that tissue upon administration of a prodrug. The subsequent phenotypic change induced by the ablation can provide information on the physiological role of the ablated tissue, or of the regenerative processes that can be recruited to repair the damage. The goal of this thesis was to engineer or discover new nitroreductase enzymes that could expand the capabilities of cell ablation studies in zebrafish. In particular, this work sought to develop a system that would enable the dual, or multiplexed, ablation of two tissues independently within the same organism. Control over the ablation of two distinct tissues could be useful for studying tissue interactions during developmental or regenerative processes. For this to be achievable, two different nitroreductase enzymes, each possessing distinct and non-overlapping prodrug selectivities would be required. Previous studies in the Ackerley lab had identified NfsA from Escherichia coli (NfsA_Ec) and NfsA from Pseudomonas putida (NfsA_Pp) as nitroreductase enzymes that were slightly more selective for the prodrug tinidazole compared than metronidazole. In contrast the NfsB nitroreductase from Vibrio vulnificus (NfsB_Vv) was substantially more selective for metronidazole than tinidazole. To further improve the tinidazole selectivity of the NfsA enzymes, directed evolution was employed as a tool to further enhance the substrate selectivity of each enzyme. The primary outcome of this work was the evolution of an NfsA_Ec mutant that was 12 fold more selective for tinidazole over metronidazole than wild type NfsA_Ec. In addition to engineering new enzymes for cell ablation experiments, this work also sought to discover new nitroreductase enzymes from unculturable bacteria, a previously unplumbed source. The genes and gene products of unculturable bacteria can be identified and studied by expressing fragments of their DNA in a readily culturable host such as E. coli. A variety of different screening methodologies were tested for identifying nitroreductase enzymes from eDNA inserts. The compound 4-nitroimidazole was found to be capable of detecting nitroreductase expression at the level of a single colony. While no novel nitroreductase enzymes were discovered in the scope of this work, the preliminary results are encouraging that a screening strategy centred on 4-nitroimidazole in particular could successfully do so in the near future.</p>

Adsorption and reduction of carcinogenic organics by ordered semi-crystalline poly-m-chloroaniline

Poly-m-chloroaniline has been synthesized by chemical oxidative polymerization of m-chloroaniline and confirmed by spectroscopic studies. Powder X-ray diffraction and FESEM studies confirmed ordered arrangement in poly-m-chloroaniline. The peak at lower angle such as 7.09° having d spacing of 12.53 Å shows ordered structure of PmClA which was further confirmed by layered morphology in the FESEM images. FTIR spectra confirm the stretching and vibration mode of polymeric m-chloroaniline ring. BET surface area and pore diameter of PmClA were found to be 121 m2/g and 15.184 nm. Poly-m-chloroaniline was found to have better adsorption capacity toward anionic dyes over cationic dyes. The anionic dye (IC and EY) was 98 and 99% removed in just 25 min, whereas the cationic dye (MG and Rh6G) was 87 and 83% removed in 30 min. Langmuir adsorption isotherm model and pseudo second-order kinetic equation fitted the data best. Mechanism of adsorption has also been proposed. This is suggested that polymeric materials can be used for purification of water. The reduction of aromatic nitro compounds to amino compounds using organocatalyst has been done for the first time.