abstract: Hydrothermal environments are important locales for carbon cycling on Earth and elsewhere in the Universe. Below its maximum temperature (~73 °C), microbial photosynthesis drives primary productivity in terrestrial hydrothermal ecosystems, which is thought to be performed by bacterial phototrophs in alkaline systems and eukaryotic algae in acidic systems, yet has received little attention at pH values intermediate to these extremes. Sequencing of 16S and 18S rRNA genes was performed at 12 hot springs with pH values 2.9-5.6 and revealed that cyanobacteria affiliated with the genus Chlorogloeopsis and algae of the order Cyanidiales coexisted at 10 of the sites. Cyanobacteria were present at pH values as low as 2.9, which challenges the paradigm of cyanobacteria being excluded below pH 4. Presence of the carotenoid β-cryptoxanthin in only 2 sites and quantitative PCR data suggest that algae were inactive at many of the sites when sampled. Spatial, but perhaps not temporal, overlap in the habitat ranges of bacterial and eukaryal microbial phototrophs indicates that the notion of a sharp transition between these lineages with respect to pH is untenable. In sedimentary basins, biosphere-derived organic carbon is subjected to abiotic transformations under hydrothermal conditions. Benzaldehyde was experimentally evaluated as a model to assess the chemistry of aldehydes under these conditions. It was first demonstrated that gold, a traditional vessel material for hydrothermal experiments, caused catalysis of benzaldehyde degradation. Experiments in silica tubes were performed at 250, 300, and 350 °C yielding time-dependent data at several starting concentrations, which confirmed second-order kinetics. Therefore, disproportionation was expected as a major reaction pathway, but unequal yields of benzoic acid and benzyl alcohol were inconsistent with that mechanism. Consideration of other products led to development of a putative reaction scheme and the time dependencies of these products were subjected to kinetic modeling. The model was able to reproduce the observed yields of benzoic acid and benzyl alcohol, indicating that secondary reactions were responsible for the observed ratios of these products. Aldehyde disproportionation could be an unappreciated step in the formation of carboxylic acids, which along with hydrocarbons are the most common organic compounds present in natural systems. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2016

Chapter 1: An assessment of the mechanism of the benzoin condensation. The reaction under non-hydroxylic conditions. The generally accepted mechanism for the well known benzion condensation is unviable for the following reasons: 1) No experimental evidence for formation of carbanion from oxyanion. 2) No experimental reports on pKa of ‘C-H” in intermediate oxyanion. 3) From previous reports, these types of carbanion are stable only at -78° C. 4) Carbanions possessing nucleofuges generally form carbenes. It was observed that the O- protected benzaldehyde cyanohydrin (1) and O-MOM protected ethyl mandelate (2) are possibly less acidic than benzyl cyanide (3). Attempts to affect hydrogen-deuterium exchange in the O-methyl ether of benzaldehyde cyanohydrin (1) did not yield clear-cut results; neither could the analogous carbanion from O-MOM protected ethyl mandelate be formed, under analogous conditions. O-Protected benzaldehyde cyanohydrin and O-MOM protected ethyl mandelate (2) did not condense with electrophiles like benzaldehyde (4); however benzyl cyanide (3) was condensed with benzaldehyde to form the stilbene cyanide (5) under similar conditions to the benzoin condensation. Scheme 1: Reactions under benzoin conditions. All these evidences indicate that carbanions derived from 1 and 2 are not formed under the conditions of the benzoin reaction. An alternative mechanism via intramolecular participation in the cyanohydrin oxyanion leads initially to an imino-oxirane intermediate; electrophilic capture of this in the key step finally leads to benzoin (Scheme 2). This is an attractive possibility that avoids many of the problems of the earlier mechanism, and is also not incompatible with most of the available experimental evidence. Further experimental and theoretical work is indicated before an acceptable mechanism for the benzoin condensation finally emerges. Scheme 2: A possible alternative mechanism of benzoin condensation Scheme 2: A possible alternative mechanism of benzoin condensation From the studies on mechanism of benzoin condensation, it seems possible to form imino-oxirane from oxyanion of benzaldehyde cyanohydrin instead of carbanion by participation of cyanide ion. To prove the cyanide ion participation in mechanism of benzoin condensation and to avoid ambiguities from O-H peak, the reaction was performed in 1,4 dioxane solvent with benzaldehyde (4) and cyanide with the phase transfer catalyst 18-crown-6, instead of EtOH and H2O as solvent. After mixing the IR spectra for the crude product, indicated the formation of benzoin (8), which was confirmed upon work up. This led to the developing of a novel method for the benzion under anhydrous conditions. Scheme 3: Formation of benzoin under anhydrous conditions Chapter 2: Stereochemical stability of benzion. Generally α-hydroxy ketones undergo tautomerism to the enediol form, which are stabilized by intramolecular hydrogen bonding. Because of this, they undergo racemisation. Benzoin is also a α-hydroxy ketone, but it can be resolved into its enantiomeric forms. It indicates enediol form of benzion is unstable possibly due to steric interference. It was observed from the crystal structure of the carbonate analog of ene-diol form of benzoin that there is steric interference between the two phenyl rings. These were twisted out of the plane of the carbonate moiety by 19.92° and -47.32°. (The crystal structure of 4,5-diphenyl-1,3-dioxol-2-one (9) was reported first time.) This structure also indicated the existence of atropisomerism in the crystalline lattice. Chapter 3: Polymerisation of benzaldehyde. α-Hydroxy esters can be viewed as surrogates of cyanohydrins. To prove the cyanohydrin anion intermediate in classical benzoin condensation mechanism is very difficult. An interesting alternative is to employ α-hydroxy esters instead. It was observed that methyl 2-phenylglyoxylate and methyl 2-(methoxymethyl)-2-phenylglyoxylate (2) failed to react with benzaldehyde in aqueous methanol with sodium carbonate as base. IR spectra indicated that the carbonyl peak of benzaldehyde has disappeared, but NMR spectra showed a mixture of methyl 2-(methoxymethyl)-2-phenylglyoxylate (2) and benzaldehyde (4). This seems to indicate the polymerization of benzaldehyde (10) (Scheme 4). However, the product was not stable enough to be isolated and purified. Scheme 4: Reaction between benzaldehyde and MOM-protected methyl mandalate (For structural formula pl see the abstract file.)

Ce travail traite de la synthèse et de la caractérisation structurale de nanoparticules d'oxyde de zirconium (ZrO2) ainsi que de leurs relations taille/structure. Nous avons élaboré des nanoparticules de taille inférieure à 5 nm avec une distribution de taille étroite en utilisant une voie sol-gel non hydrolytique solvothermale. La diffraction des rayons X classique a été utilisée pour évaluer la taille des cristallites via des affinements de Rietveld et la diffusion totale des rayons X pour extraire les fonctions de distribution des paires (PDF) des échantillons et effectuer leur analyse structurale.Nous avons réussi à synthétiser des nanoparticules cristallines de ZrO2 d'une taille d'environ 3 nm et quasi mono-disperses. Nous avons montré qu'une addition d'hydroxyde de sodium dans le mélange réactionnel était pertinente pour obtenir des nanoparticules bien cristallisées et monophasées présentant une structure moyenne soit monoclinique soit tétragonale en fonction du solvant utilisé, respectivement dans le benzaldéhyde ou dans l'alcool benzylique. Enfin, la dilution de l'alcool benzylique par l'anisole, un solvant inerte, conduit au contrôle fin de la taille moyenne des nanoparticules de 3,2 nm à 2 nm selon l'étude MET.Ces trois échantillons principaux préparés dans le benzaldéhyde alcalinisé, l'alcool benzylique ou l'anisole ont été soumis à une analyse structurale complète. Selon leur analyse PDF, ces trois échantillons offrent un aperçu de la compréhension des propriétés structurales de ces petites particules.La PDF expérimental de l'échantillon préparé avec du benzaldéhyde coïncide avec la PDF affinée d'un modèle structural monoclinique. Cependant, la PDF expérimentale de l'échantillon préparé avec de l'alcool benzylique n'est conforme à aucun des polymorphes connus de ZrO2 ayant une structure dérivée de la fluorine, mais présente plutôt une structure tétragonale moyenne avec des distorsions monocliniques à l'ordre local. Enfin, la PDF expérimentale de l'échantillon préparé avec de l'anisole présente de fortes distorsions structurales dans le domaine de l'ordre à moyenne distance, mais aussi une structure de type monoclinique à l'ordre local.Ensuite, l'échantillon préparé avec l'anisole a été soumis à un vieillissement assisté thermiquement (3 à 24 jours) et on a ajouté des quantités variables d'eau ex-situ (teneur en volume de 0,3 à 5%). Nous avons observé que les nanoparticules évoluaient d'une phase contenant un désordre élevé (3 jours) à une structure mieux cristallisée (24 jours) correspondant à une structure tétragonale moyenne tout en maintenant une distorsion monoclinique à l'ordre local. De manière similaire, l'addition d'eau ex-situ sur le mélange réactionnel favorise une cristallisation plus rapide et favorise l'apparition de pics typiques de la structure monoclinique. Cependant, la formation de phase monoclinique a été inhibée par la présence d'hydroxyde de sodium, mais limitée à environ 2% en volume d'eau, et une valeur plus élevée que cette quantité conduit à la formation de grandes particules de structure monoclinique. / This work deals with the synthesis and the structural characterization of zirconium oxide nanoparticles (ZrO2) as well as their size/structure relationships. We elaborated nanoparticles with a size inferior to 5 nm and a narrow size distribution using a non-hydrolytic solvothermal sol-gel route. Classical X-ray diffraction was used to evaluate the crystallite size via Rietveld refinement and X-ray total scattering to extract the pair distribution functions (PDF) of the samples for structural analysis.We succeeded in synthesizing crystalline nanoparticles of ZrO2 with a size of about 3 nm and quasi mono-dispersed. We showed that an adjusted addition of sodium hydroxide into the reaction mixture was pertinent to obtain a well crystallized and single-phase nanoparticles exhibiting an average monoclinic or tetragonal structure in benzaldehyde or in benzyl alcohol, respectively. Finally, the dilution of benzyl alcohol by anisole, an inert solvent, lead to the fine tuning of average crystallite size of the nanoparticles from 3.2 nm to 2 nm according to the TEM investigation.Those three major samples prepared in alkalinized benzaldehyde, benzyl alcohol, or anisole were subjected to complete structural analysis. According to their PDF analysis, these three samples offer an insight to the understanding of structural properties of such small particles.The experimental PDF of the benzaldehyde-prepared sample coincide with the refined PDF of a monoclinic structural model. However, the experimental PDF of the benzyl alcohol-prepared sample does not conform to any of the known polymorphs of ZrO2 having a fluorite-derived structure but rather it has an average tetragonal structure and a monoclinic distortion at the short-range order. Lastly, the experimental PDF of the anisole-prepared sample has large structural distortion at the medium-range order but has monoclinic structural features at the short-range order. We then subjected the anisole-prepared sample by thermally assisted ageing (3 to 24 days) and addition of varied amounts ex-situ water (0.3 to 5% volume content). We observed that the nanoparticles evolve from a phase containing high disorder (3 days) to a state of higher structural order (24 days) that fits with the average tetragonal structure while maintaining a monoclinic distortion at the short-range order. Similarly, the addition of ex-situ water onto the reaction mixture promoted a faster crystallization and promotes the onset of peaks that aligns with the monoclinic structure. However, the formation of monoclinic phases was inhibited by the presence of sodium hydroxide but limited to about 2% volume water content and higher than this amount lead to the formation of large particles with monoclinic structure.

Yes / Rhodium nanoparticles or rhodium organometallic complexes are mainly used in catalysis for reduction or hydroformylation reactions. In this work instead, we explored the capabilities of Rh nanoparticles as an oxidation catalyst, applied to the oxidation of benzyl alcohol to benzaldehyde under very mild conditions (100 °C, and atmospheric pressure) as a model reaction. Here we report the preparation of novel Rh/C catalysts by using an impregnation protocol, with particular emphasis on the pre-treatment of the carbon supports by using HNO3 and HCl, as well as the characterization of these materials by using an array of methods involving TEM, XPS and XRPD. Our preparation method led to a wide Rh particle size distribution ranging from 20 to 100 nm, and we estimate an upper limit diameter of Rh nanoparticles for their activity towards benzyl alcohol oxidation to be ca. 30 nm. Furthermore, a HNO3 pre-treatment of the activated carbon support was able to induce a smaller and narrower particle size distribution of Rh nanoparticles, whereas a HCl pre-treatment had no effect or sintered the Rh nanoparticles. We rationalise these results by HNO3 as an acid able to create new nucleation sites for Rh on the carbon surface, with the final effect of smaller nanoparticles, whereas for HCl the effect of sintering was most likely due to site blocking of the nucleation sites over the carbon surface. The roles of acid centres on the carbon surfaces for the oxidation reaction was also investigated, and the larger their amounts the larger the amounts of by-products. However, by treatment with HNO3 we were able to convert neutral or basic carbons into supports capable to enhance the catalytic activity of Rh, and yet minimised detrimental effects on the selectivity of the oxidation to benzaldehyde.

This thesis deals with the design and synthesis of chiralenantiopure nitrogencontaining ligands and the use of theseligands in asymmetric catalysis. A modular synthetic approachto enantiopure nitrogen-containing ligands was developed. Thesynthetic method is based on the ring-opening of activatedchiral aziridines by nitrogen nucleophiles. The aziridines areconveniently prepared from amino alcohols. The structure oftheaziridine and of the nucleophile can be extensively varied andlibraries of ligands are easily prepared. The use of primaryamines affords C2-symmetric bis(sulfonamides), whereas the use ofammonia affords C3-symmetric tris(sulfonamides) that can beelaborated into the corresponding tetra-amines. The C2- and C3-symmetric ligands were used in the asymmetrictitaniummediated addition of diethylzinc to benzaldehyderesulting in modest enantioselection, 76% ee. A thoroughinvestigation of the reaction conditions revealed that theamount of Ti(OiPr)4has a decisive effect on the reaction rate and thestereochemical outcome of the reaction. The reaction timedecreased from about 90 hours to 15 minutes and theenantioselectivity changed from 26% of the (R)- enantiomer to72% of the (S)-enantiomer when the Ti(OiPr)4:benzaldehyde ratio was increased from 0.125:1 to1.48:1. Moreover, the titanium-mediated addition of diethylzincto benzaldehyde was studied in the presence of chiraladditives. The bis(sulfonamides) were also used in thecyclopropanation of cinnamyl alcohol. However, only lowenantioselection was observed, 27% ee. The C3-symmetric tetra-amines were reacted to formazaphosphatranes. These weak acids were only partiallydeprotonated by the strong base KOtBu to form the correspondingproazaphosphatranes. The unexpectedly strong basicity of theproazaphosphatranes was believed to be due to steric effects assuggested by DFT calculations. The tetra-amines and thesulfonamides were used for the preparation of metal complexesof Lewis acidic metals such as titanium(IV) andzirconium(IV). <b>Keywords:</b>asymmetric catalysis, aziridine, benzaldehyde,diethylzinc, enantioselective, ligand, proazaphosphatrane,ring-opening, sulfonamide, symmetry, titanium, zirconium

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In this study, a methodology for separation of chiral molecules, by using enhanced ultrafiltration system was developed. Benzoin was the model chiral molecule studied. In the scope of developing this methodology, some parameters were investigated in the preliminary ultrafiltration experiments in order to set the operation conditions for enhanced ultrafiltration experiments. Due to the slight solubility of benzoin in pure water, 15% (v/v) Polyethylene glycol (PEG 400) and 30 % (v/v) Dimethyl sulfoxide (DMSO) were selected as cosolvents. Because of the high retention capacity of RC-10000 Da membranes for benzoin, a membrane saturation strategy was developed. In polymer enhanced ultrafiltration (PEUF) experiments bovine serum albumin (BSA) was used as ligand. Effects of ligand concentration and pH on total benzoin retention and on enantiomeric excess (ee %) were investigated. Benzoin concentration was almost kept constant at ~10 ppm and ~50 ppm for 15% (v/v) PEG 400 and 30 % (v/v) DMSO cosolvents, respectively. It was observed that the increase either in pH or in BSA concentration yielded an increase in total benzoin retention. In 15% (v/v) PEG 400-water, with BSA concentration of 10000 ppm, at pH 10, total benzoin retention reached to 48.7%. For this cosolvent, at different pH values and at different BSA concentrations, all ee % values were about or less than 10%. When 50000 ppm BSA was dissolved in 30 % (v/v) DMSO-water, total benzoin retention increased to 41.3% at pH 10 and ee % reached 16.7 % at pH 11. In enzyme enhanced ultrafiltration (EEUF) experiments, specific to benzoin, apo form of Benzaldehyde Lyase (BAL, E.C. 4.1.2.38) was used as ligand. These experiments were performed with constant ~ 10 ppm benzoin concentration in only 15% (v/v) PEG 400 &ndash / water solvent. Effect of BAL concentration on total benzoin retention and ee% was investigated. It was found that / for all the studied BAL concentrations in the range of 650- 1936 ppm total benzoin retention and ee % were kept almost constant at ~75% and ~60%, respectively.

Benzaldehyde lyase (BAL, EC 4.1.2.38) from Pseudomonas fluorescens Biovar I, a thiamine pyrophosphate (ThDP) dependent enzyme, catalyzes the enzymatic kinetic resolution of racemates by C-C bond cleavage and concomitant C-C bond formation. In this study, benzaldehyde lyase gene from Pseudomonas fluorescens Biovar I was cloned into Pichia pastoris, with the aim of the extracellular production of the enzyme. For this purpose, firstly, PCR amplified bal gene was cloned into an integration vector pPICZalphaA. Thereafter the recombinant plasmid pPICZalphaA::bal was transformed into P.pastoris. Extracellular benzaldehyde lyase enzyme was expressed under the control of the strong AOX promoter and the secretion of the enzyme in the fermentation medium was achieved by means of S. cerevisiae alpha factor signal sequence. The recombinant cells were grown for 48-72 hours in solid medium then the cells inoculated in glycerol containing medium. After being separated by centrifugation cells were transferred into methanol containing production medium. In methanol containing medium cells were grown for 72 h. Starting from t=24 h methanol was added to medium as an inducer of AOX promoter and the carbon source in order to produce BAL in every 24 hour. SDS-PAGE analyses illustrated that extracellular benzaldehyde lyase enzyme produced by the recombinant P.pastoris strain had the size of 59 kDa, which is the size of benzaldehyde lyase monomer. FPLC analysis showed that concentration of the tetrameric form of benzaldehyde lyase enzyme, active form, was much less than the monomeric form of the enzyme indicating that the enzyme produced by recombinant P.pastoris mostly could not fold into multimeric form in the fermentation medium.

In this study, the effects of oxygen transfer conditions on the synthesis of the enzyme benzaldehyde lyase as intracellular in recombinant E. coli BL21 (DE3) pLysS was investigated sistematically and a comprehensive model was developed to determine benzaldehyde lyase activity. For this purpose, the research program was carried out in mainly two parts. In the first part of study, the effects of oxygen transfer together with the mass transfer coefficient (KLa), enhancement factor E (=KLa/KLao), volumetric oxygen transfer rate, volumetric and specific oxygen uptake rates, mass transfer and biochemical reaction resistances / moreover, the variation in product and by-product distribution, specific substrate uptake rates, yield and maintenance coefficient were investigated in the pilot scale batch bioreactor at QO/VR = 0.5 vvm and agitation rates of N= 250, 500, 625, and 750 min-1, and dissolved oxygen levels DO= 20%, 40% conditions, while medium components were CGlucose= 8.0 kg m-3, C(NH4)2HPO4= 5.0 kg m-3 and salt solution at controlled pHc=7.2. The highest cell concentration and benzaldehyde lyase activity were obtained at DO=40% condition as 3.0 kg m-3 and A=1095 Ucm-3, respectively. v Then a mathematical model was proposed to estimate benzaldehyde lyase activity as function of time, agitation rate, cell concentration, dissolved oxygen concentration, and by-product concentration with reasonable accuracy.

This study focuses on the molasses based complex medium design for benzaldehyde lyase production by recombinant E. coli BL21 and development of a feeding strategy based on the designed complex medium. For this purpose, firstly, the effects of molasses were investigated in laboratory scale bioreactors. As E. coli BL21 was not able to utilize sucrose, molasses was pretreated and hydrolyzed to fructose and glucose. Thereafter, effect of pretreated molasses concentration was investigated in the range of 16 to 56 kg m-3 by batch-bioreactor experiments / and the highest cell concentration and benzaldehyde lyase activity were obtained as CX=5.3 kg m-3 and A=1617 U cm-3, respectively, in the medium containing 7.5 kg m-3 glucose and 7.5 kg m-3 fructose. Then, different feeding strategies were developed to produce efficient cells with high concentration and BAL activity. In the first strategy, after 10 hours of batch-cultivation with molasses based medium having 7.5 kg m-3 glucose and 7.5 kg m-3 fructose concentration, based on the airflow rate, pretreated molasses was fed to the system. When air flow rate decreased considerably, fed was given to the system that results in increase in glucose and fructose concentration in the medium to 2.5 kg m-3. At the end of the process, the highest cell concentration obtained was CX=7.4 kg m-3. The maximum activity was reached at 20th hour as A=2360 U cm-3. On the other hand, as air flow variation only demonstrated the absence of glucose not fructose, a second strategy, based on the detection of the fructose and glucose concentrations during the process, was applied. In this strategy when glucose and fructose were depleted, fed was given to the system that results in increase in glucose and fructose concentration in the medium to 2.5 kg m-3 / and the highest BAL activity was obtained as 2370 U cm-3 at t= 26 h where the cell concentration was 7.5 kg m-3. At the last strategy, when glucose and fructose were depleted, fed was given to the system that results in increase in CGlucose=1.5 kg m-3 and CFructose=1.5 kg m-3 in the production medium to decrease the accumulation of acetic acid. By this strategy highest cell concentration was obtained as 8.04 kg m-3 at t=24 h and the highest BAL activity was 2315 U cm-3. These strategies could be accepted having the same BAL activity with little distinctions. However, cell concentration of the last one was higher than others and also the lowest amount of carbon source was used. Thus, last one could be chosen as the most favorable strategy.