A new, efficient route was used in the synthesis of [2.2]-paracyclophane-2-carboxylic acid. The acid as then resolved and the Birch reduction performed yielding one enantiomer of tetrahydro-[2.2]-paracyclophane-2-carboxylic acid. The ultraviolet spectrum of tetrahydro-[2.2]-paracyclophane- 2-carboxylic acid in isopenthane shows one absorption at 206 nm (Emax = 5,271). There are three bands observed in the circular dichroism spectrum in isopentane at 236 nm ([] = 1.8 X 104), 201 nm ([0] = -16 X 104) and a positive band indicated below 180 nm but not observed. The bands were assigned and possible reasons for the occurrence of a mr'r* transition at unexpectedly long wavelengths are discussed.

<p> A series of derivatives of thiazolidine-4-carboxylic acid was prepared from two sulfur containing aminoacids, cysteine and D-penicillamine. Cysteine is very easily oxidized to a disulfide, while penicillamine is less susceptible to oxidation of the sulfhydryl group because of greater steric hindrance. Sulfhydryl aminoacids react readily with aldehydes and ketones to give thiazolidine rings with varying degrees of substitution.</p> <p> Many of the compounds prepared were studied in solution and in the solid state by means of nmr, vibrational spectroscopy, mass spectra and X-ray crystallographic data. Thiazolidine-4-carboxylic acids contain a free carboxyl group and a secondary amino group, both of which can be ionized. Infrared spectra and X-ray crystallography are useful in detecting ionization and hydrogen bonds. An example is given of a compound that can exist in both the zwitterion and the non ionized state depending on the solvent of recrystallization.</p> <p> From previous work in our group we were predicting that increasing steric crowding in the thiazolidine ring in close proximity to the ionizable groups would decrease the tendency of these molecules to ionize. No such simple relationship could be found.</p> / Thesis / Master of Science (MSc)

Biotin is an essential vitamin in plants and mammals functioning as the carbon dioxide carrier within central lipid metabolism. Biotin is composed of a fused bicylic ring system and a five carbon, carboxylic acid chain. Biotin biosynthesis in bacteria is catalysed by a series of enzymes that use fatty acid, amino acid and sulfur-containing substrates. In Bacillus subtilis, pimeloyl-CoA synthetase (PCAS, EC 6.2.1.14, UNIPROT code: P53559, 29.6 kDa) is the first enzyme in the biotin biosynthetic pathway and acts as a highly specific substrate selection gate ensuring the integrity of the carbon chain in biotin synthesis. PCAS catalyses the synthesis of the key acyl-thioester, pimeloyl-CoA in two steps; the first involves activation of pimelic acid (C7 dicarboxylic acid) using ATP to give an acyl-adenylate, enzyme-bound intermediate and pyrophosphate (PPi), and in the second step, this pimeloyl-adenylate reacts with coenzyme A (CoASH) to form the pimeloyl-CoA thioester. This thesis describes the results of biochemical, structural and mechanistic studies of B. subtilis PCAS. Recombinant PCAS was prepared by expressing the B. subtilis BioW gene in E. coli in various hexa-histidine affinity-tagged forms and the enzyme purified in high purity and yield. Enzyme activity and kinetic constants were measured using reverse-phase HPLC and enzyme coupled spectroscopic assays. These revealed the enzyme to have a strict carboxylic acid specificity. In collaboration with colleagues at the University of St. Andrews various commercial and in-house screens were used to obtain diffraction-quality crystals suitable for X-ray crystallography. This also included the generation of seleno-methionine (SeMet) labelled PCAS, as well as heavy-metal derivatives. Structures of B. subtilis PCAS in complex with the substrate pimelic acid and the pimeloyl-adenylate intermediate and product PPi were determined at 2.04 Å and 2.34 Å resolution respectively. The B. subtilis PCAS displays a novel 3D fold and defines a new class (Class IV) in the ANL superfamily of adenylate forming enzymes. The enzyme is a homodimer composed of two domains, a short N-terminus and a large C-terminal domain and the ligand-bound structures revealed the residues potentially involved in substrate specificity and enzyme catalysis. The enzyme uses an internal ruler composed of a number of conserved arginine residues (Arg213, Arg227 and Arg170) to select the correct dicarboxylic acid substrate. The X-ray structures guided the production of a number of site directed mutants to identify residues involved in the catalytic mechanism and stabilising the acyl-adenylate intermediate. This also allowed rational engineering of the PCAS active site to generate mutants with altered substrate specificity. Mutant PCAS Y211F was shown to synthesise both heptanoyl (C7) and octanoyl (C8) mono carboxylic acid-CoA and C8 dicarboxylic-CoA thioester products, highlighting the synthetic potential of PCAS. The PCAS pimeloyl-CoA product is the substrate for the next enzyme in the biotin pathway, a pyridoxal 5'phosphate (PLP)-dependent 8-amino 7-oxononanoate synthase (AONS). AONS catalyses the condensation of pimeloyl-CoA with L-alanine to give AON which is converted to biotin by the action of three other enzymes. We used genome mining to identify a putative ~66 kDa, bi-functional PCAS/AONS enzyme with an N-terminal PCAS domain fused to C-terminal AONS domain in the organism Corynebacterium amycolatum. A recombinant C. amycolatum PCAS/AONS fusion protein was expressed and purified from E. coli and initial studies suggest that it forms a functional, fused, dimeric enzyme.

Oil sand deposits in the Athabasca Basin in Alberta represent one of the largest global oil reserves. The bitumen contents of oil sand shallow deposits are recovered by surface mining using modified version of the Clark hot water process. Extraction of bitumen results in extremely large volumes of process water, which are contaminated with naphthenic acids. Various ex-situ treatment techniques including ozonation, advanced oxidation, adsorption, and bioremediation have been evaluated for the treatment of these waters. Previous studies conducted by Paslawski et al. (2009) investigated aerobic biodegradation of naphthenic acids in properly designed and carefully operated bioreactors. In the current work, anaerobic biodegradation of naphthenic acids under denitrifying condition was examined as a potential approach to eliminate the aeration cost in ex-situ treatment and as an alternative for application of in-situ treatment of oil sand process water in stabilization ponds was examined. Using trans-4-methyl-1-cyclohexane carboxylic acid (trans-4MCHCA), a microbial mixed culture developed in earlier works (Paslawski et al., 2009), and nitrate as an electron acceptor, anaerobic biodegradation of trans-4MCHCA were studied in batch and continuous bioreactors: continuous stirred tank reactor (CSTR) and biofilm system. Effects of naphthenic acid concentration, temperature, and loading rate on biodegradation process were investigated. The batch studies showed that initial concentration of trans-4MCHCA influenced the biodegradation rate where the increase in initial concentration of trans-4MCHCA from 100 to 250 mg L-1 led to a higher rate but further increase in concentration did not have a marked effect. Moreover, batch experiments at temperatures ranging from 10° to 35°C demonstrated that the optimum temperature was in the range of 20 - 24°C. Continuous anaerobic biodegradation in the CSTR showed that increase in loading rate of trans-4MCHCA caused an increase in removal rate of both trans-4MCHCA and nitrate. Rates were decreased as the system approached the cell washout. The maximum biodegradation rate and nitrate removal rate, achieved at trans-4MCHCA loading rate of 157.8 mg L-1 h-1, were 105.4 mg L-1 h-1 and 144.5 mg L-1 h-1, respectively. A similar dependency between the loading and removal rates was also observed in the biofilm reactor. The maximum removal rate of trans-4MCHCA and nitrate in the biofilm reactor, operated at room temperature (24 ± 2ºC) were 2,028.1 mg L-1 h-1 and 3,164.7 mg L-1 h-1, respectively and obtained at trans-4MCHCA loading rate of 2,607.9 mg L-1 h-1. Comparison of the results from aerobic batch systems obtained by Paslawski et al. (2009) and the current results showed similar profile where increase in initial concentration of naphthenic acid increased the biodegradation rate of trans-4MCHCA. As far as the effect of temperature is concerned, room temperature (20 - 24ºC) was identified as optimum temperature regardless of mode of biodegradation. Under continuous mode of operation (CSTR and biofilm reactors), anaerobic biodegradation was much faster than its aerobic counterpart. For instance the maximum anaerobic removal rate of trans-4MCHCA in the CSTR was 105.4 mg L-1 h-1, while the highest removal rate achieved in the aerobic CSTR was 9.6 mg L-1 h-1. Similarly, anaerobic biofilm reactor achieved a higher maximum removal rate of 2,028.1 mg L-1 h-1 compared to a 924.4 mg L-1 h-1 removal rate in the aerobic biofilm reactor. The overall finding indicated that biodegradation of trans-4MCHCA can be achieved effectively under anaerobic condition with the rates markedly higher than those for aerobic system.

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Thesis (Ph.D.); Submitted to Department of Chemical Engineering, Colorado School of Mines, Golden, CO (US); 1 Sep 2004. / Published through the Information Bridge: DOE Scientific and Technical Information. "LA-14178-T" R.L. Ames. 09/01/2004. Report is also available in paper and microfiche from NTIS.

Biocatalysis is a field rapidly expanding to meet a demand for green and sustainable chemical processes. As the use of enzymes for synthetic chemistry becomes more common, the construction of multistep enzyme reactions is likely to become more prominent providing excellent cost and productivity benefits. However, the design and optimisation of multistep reactions can be challenging. An enzyme toolbox of well-characterised enzyme parts is critical for the design of novel multistep reactions. Furthermore, while whole-cell biocatalysis offers an excellent platform for multistep reactions, we are limited to the use of mesophilic host organisms such as Escherichia coli. The development of a thermophilic host organism would offer a powerful tool allowing whole-cell biocatalysis at elevated temperatures. This study aimed to investigate the construction of a multistep enzyme reaction from well-characterised enzyme parts, consisting of an esterase, a carboxylic acid reductase and an alcohol dehydrogenase. A novel thermostable esterase Af-Est2 was characterised both biochemically and structurally. The enzyme shows exceptional stability making it attractive for industrial biocatalysis, and features what is likely a structural or regulatory CoA molecule tightly bound near the active site. Five carboxylic acid reductases (CARs) taken from across the known CAR family were thoroughly characterised. Kinetic analysis of these enzymes with various substrates shows they have a broad but similar substrate specificity and that electron rich acids are favoured. The characterisation of these CARs seeks to provide specifications for their use as a biocatalyst. The use of isolated enzymes was investigated as an alternative to whole-cell biocatalysis for the multistep reaction. Additional enzymes for the regeneration of cofactors and removal of by-products were included, resulting in a seven enzyme reaction. Using characterised enzyme parts, a mechanistic mathematical model was constructed to aid in the understanding and optimisation of the reaction, demonstrating the power of this approach. Thermus thermophilus was identified as a promising candidate for use as a thermophilic host organism for whole-cell biocatalysis. Synthetic biology parts including a BioBricks vector, custom ribosome binding sites and characterised promoters were developed for this purpose. The expression of enzymes to complete the multistep enzyme reaction in T. thermophilus was successful, but native T. thermophilus enzymes prevented the biotransformation from being completed. In summary, this work makes a number of contributions to the enzyme toolbox of well-characterised enzymes, and investigates their combination into a multistep enzyme reaction both in vitro and in vivo using a novel thermophilic host organism.

To shift from a petroleum-based to a biomass-based economy will require the development not only of biofuels, but also of biorenewable replacements for petroleum-derived chemicals. In this regard, environmentally friendly biomass-derived esters may serve as alternatives to fossil-derived chemicals such as toxic halogenated solvents and glycol ethers. Therefore, esterification of various carboxylic acids that find significant applications in the chemical, pharmaceutical, petrochemical, food, and cosmetic industries has been initiated by the chemical industry. At atmospheric condition, esterification is a reversible reaction limited by the low equilibrium conversion and slow reaction rate, and has recently been performed with excess alcohol to shift the equilibrium conversion. Heterogeneous or homogeneous acid catalysts are used to achieve acceptable reaction rates. The conventional acid-catalyzed process has been extensively developed; but it suffers from problems associated with the generation of side reactions, corrosion of equipment, expensive purification procedures, long reaction times and discharge of acidic wastes. Various attempts on esterification of carboxylic acids with ethanol have previously addressed important issues concerning product distribution, catalyst activity, and kinetics of acid-catalyzed esterification at lower reaction temperatures, but kinetics of uncatalyzed esterification at elevated reaction temperatures are still very limited. It is thus of great interest from a practical viewpoint that more information such as kinetic and thermodynamic parameters are required to develop a possible esterification process without using any catalyst. In this work, therefore, a fundamental study on the uncatalyzed esterification of different aliphatic carboxylic acids with stoichiometric amounts of ethanol was undertaken to examine the possibility of converting the biomass-derived carboxylic acids to ethyl esters and to determine the kinetic and thermodynamic parameters for the uncatalyzed esterification. Experiments were conducted with isothermal batch reactors at temperatures ranging from 298 K to 623 K. A 2nd-order reversible kinetics rate expression was used to fit the experimental data. The thermodynamic and kinetic values estimated were found to vary for different esterification systems studied. The dependence of Keq on temperature for esterification of short-chain and long-chain carboxylic acids varied. Despite the nonlinearity of the Van't Hoff plot for esterification of linoleic acid, the Arrhenius and Eyring plots were linear. Two thermodynamic paths were developed for estimating the equilibrium conversions, and the theoretical values compared well with the experimental results reported in this study. Additional experiments performed to assess the corrosive and catalytic influences of metallic materials on esterification reaction indicated Inconel 625 alloy, nickel wire and stainless steel materials have potential corrosion problems on the uncatalyzed esterification reaction at elevated reaction. However, tantalum and grade 5 titanium materials showed acceptable level of compatibility for similar reaction conditions, and this can encourage the design of a flow reactor system.

(ENGLISH) Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Candidate: Sarah Basem Bouz Supervisor: PharmDr. Jan Zitko, Ph.D. Title of diploma thesis: Derivatives of quinoxaline-2-carboxylic acid as potential antimicrobial compounds Despite the presence of well-established treatment plan, tuberculosis remains the number one killer of infections according to WHO. One of the reasons behind this failure in eradicating this infection is drug resistance. This fact potentiates worldwide efforts to develop new antituberculars. As part of our long-term research on pyrazine derivatives, we prepared a series of N-substituted quinoxaline-2-carboxamides, refer to fig. below. Quinoxaline-2-carboxylic acid was activated by oxalyl chloride and reacted with different anilines or benzylamines in the presence of pyridine at room temperature, overnight with stirring, and then obtained crudes were purified with flash chromatography. Final products were evaluated for in vitro antimicrobial activities against six mycobacterial strains, eight fungal stems, along with four gram positive and four gram negative bacteria of clinical importance. The most promising compound among all with broad spectrum of antimycobacterial activity (MICMtbH37Ra = 3.91...

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