Novel biocatalysts for the enantioselective reduction of imines and reductive amination of a broad range of carbonyl compounds have been developed. Unlike other imine reductases (IREDs), the IRED from Amycolaptosis orientalis (AoIRED) features an aprotic "catalytic" residue Asn171 and as such became an interesting candidate for detailed mechanistic, specificity and stereoselectivity studies. AoIRED has been shown to be an efficient catalyst for the enantioselective reduction of imines and iminium ions to yield the corresponding chiral amines in high conversions and good to excellent enantioselectivity. The enzyme exhibits unusual stereoselective properties, displaying a selectivity switch for structurally similar substrates and in certain cases for the same substrate depending on the age of the enzyme. Mutagenesis studies have highlighted important residues that may play key roles in the substrate specificity and stereoselectivity of the enzyme. The reductive aminase from Aspergillus oryzae (AspRedAm) is a multifunctional catalyst that efficiently catalyses i) the reductive coupling of carbonyl compounds and amine nucleophiles, ii) the enantioselective reduction of prochiral cyclic and preformed imines or iii) the oxidative deamination of amines towards kinetic resolution of racemic amines. Detailed kinetic studies have led to the construction of a kinetic model/mechanism and based on structure guided investigation of conserved active site residues, a putative catalytic mechanism has been proposed. It has also been possible to engineer wild-type AspRedAm for improved stereoselectivity as well as to invert the enzyme's enantioselectivity towards a range of substrates. Using AspRedAm as a catalyst, efficient systems have been developed that allow the kinetic resolution of several racemic amines. This thesis has been organised into separate chapters each addressing a specific theme. Chapter 1 gives an overview of recent advances in the field of amine biocatalysis with emphasis on biocatalytic imine reduction and reductive amination; it also outlines the objectives of this project. Chapter 2 describes methods and materials used in these studies while Chapters 3-7 present and discuss results from different projects that constitute the work in this thesis. Initial discovery and characterisation studies of IREDs are described in Chapter 3. Chapter 4 describes detailed characterisation of AoIRED with particular emphasis on stereoselectivity and synthetic applicability while Chapter 5 presents and discusses results from the study of the reductive aminase (AspRedAm) from Aspergillus oryzae. Chapters 6 and 7 respectively describe the engineering of AoIRED and AspRedAm, and the application of AspRedAm in kinetic resolution of racemic amines. The results from these chapters have been summarised and discussed in Chapter 8 and recommendations for future directions in this field have been offered.

A terapia de quelação tem sido descrita na literatura como uma ferramenta importante no combate de processos neurodegenerativos como a doença de Alzheimer (AD). Esta doença é caracterizada pela agregação de peptídeos β-amiloides, formando fibrilas, que parece ser induzida ou facilitada em presença de íons metálicos como Cu2+, Zn2+ ou Fe3+. Vários compostos já foram testados e descritos como ligantes competitivos para coordenar e retirar estes íons dos agregados proteicos em condições patológicas, na chamada hipótese da cascata amiloide. O presente projeto visou investigar a reatividade de ligantes imínicos, derivados de oxindóis, na quelação de cobre(II) e zinco(II), numa tentativa de inibir ou evitar a formação de agregados relacionados à AD. Foram sintetizados seis compostos imínicos, sendo duas hidrazonas inéditas: isahim e isahpy e quatro bases de Schiff: isapn, misapn, isaen e misaen, as três últimas também inéditas. Os compostos foram caracterizados por espectroscopia FTIR, RMN, UV/VIS e por análise elementar CHN e espectrometria de massas ESI-MS/MS. Os valores das constantes de estabilidade (log β 2,7 - 5,1) para a formação dos complexos [ML]2+ e do aduto [(Aβ1-16)2•isahim] em solução aquosa foram determinadas por espectroscopia UV/VIS. As formas de interação dos compostos isapn, misapn e isahim com o peptídeo Aβ1-16 foram analisadas por espectroscopia 1H RMN, observando-se uma forte interação com as histidinas His6, His13 e His14 e com os carboxilatos do peptídeo. A eficácia dos ligantes foi testada frente ao processo de inibição da agregação do peptídeo Aβ1-40 na presença e ausência de íons Cu2+ ou Zn2+ por turbidimetria. Estudos de docking e dinâmica molecular suportam que a interação dos ligantes imínicos com o peptídeo Aβ1-16 ocorre nos mesmos sítios de coordenação dos íons metálicos. Os resultados indicam que os compostos aqui estudados podem atuar como eficientes inibidores de agregação dos peptídeos amiloides implicados na AD. / Chelation therapy has been considered in the literature an important tool in neurodegenerative processes like Alzheimer disease (AD). This disease is characterized by aggregation of β-amyloid peptides that seems to be improved in the presence of metal ions, particularly copper, zinc and iron. Several compounds have been tested and reported as competitive ligands to withdrawal these metal ions from protein aggregates in pathologic conditions, in the \"amyloid cascade hypothesis\". The present project aims to investigate the reactivity of imine ligands, particularly those derived from oxindoles, in the chelation of copper(II) and zinc(II) ions trying to inhibit or avoid aggregate formation related to AD. Six iminic compounds were synthesized, being two of them hydrazones: isahim and isahpy and four Schiff bases: isapn, misapn, isaen, and misaen. The compounds were characterized by spectroscopic analysis (FTIR, NMR, UV/VIS), elemental analysis CHN and mass spectrometry ESI-MS/MS. The corresponding stability constants (log β 2,7-5,1) for each complex formation [ML]2+ as well as for the adduct [(Aβ1-16)2•isahim] in aqueous solution were determined by UV/VIS spectroscopy. Interactions of compounds isapn, misapn and isahim with the Aβ1-16 peptide were detected and analyzed by 1H NMR spectroscopy, indicating a strong interaction among the compounds and the histidines His6, His13 e His14 as well as the carboxylate residues in the peptides. The ligands efficiency toward the inhibition aggregation process for the Aβ1-40 peptide were evaluated in the presence, and in absence of Cu2+ or Zn2+ ions by turbidimetry. Computational calculations (docking and molecular dynamics) indicated that the interaction of the imine ligand with the Aβ1-16 peptide occurs in the metal coordination sites. The results indicate that these imine compounds studied may act as efficient inhibitors of amyloid peptides implicated in AD.

<p>In this thesis, I present the electrochemical studies of three dendrimeric polypropylene imine (PPI) nanomaterials and their applications as a platform in the development of a novel label free DNA nanobiosensor based on electrochemical impedance spectroscopy. Cyclic voltammetry (CV), differentia pulse voltammetry (DPV), square wave voltammetry (SWV) and electrochemical impedance spectroscopy (EIS) techniques were used to study and model the electrochemical reactivities of the nanomaterials on glassy carbon electrode (GCE) as the working electrode.</p>

The following study investigates the intramolecular and intermolecular interactions responsible for invoking stereoselectivity and stereospecificity in the synthesis of a chiral original species and amino acid receptor. The former commences with a brief overview of the nature, scope and applications of helical chirality, and culminates in the formation of a permanent helix via the synthesis of a novel chiral lactone. The latter is discussed as an extension of a naturally occurring cofactor whose identity is modulated to furnish a tailored receptor selective to the binding of amino-acid enantiomers. The study and analysis is executed via both synthetic and computational methods.

The following study investigates the intramolecular and intermolecular interactions responsible for invoking stereoselectivity and stereospecificity in the synthesis of a chiral original species and amino acid receptor. The former commences with a brief overview of the nature, scope and applications of helical chirality, and culminates in the formation of a permanent helix via the synthesis of a novel chiral lactone. The latter is discussed as an extension of a naturally occurring cofactor whose identity is modulated to furnish a tailored receptor selective to the binding of amino-acid enantiomers. The study and analysis is executed via both synthetic and computational methods.

Abstract The Copper Catalyzed Azide-Alkyne Cycloaddition (CuAAC) is a paradigm of ¡§click¡¨ chemistry which has been applied in different fields. To understand the interaction between organic azides and a copper surface, we use benzyl azide (Bn¡ÐN£\¡ÐN£]¡ÝN£^) as an adsorbate on Cu(111) under ultrahigh vacuum conditions. The thermal reaction process was explored by a combination of temperature-programmed desorption (TPD), reflection absorption infrared spectroscopy (RAIRS), and X-ray photoemission spectroscopy (XPS) techniques. The TPD profiles show a multilayer desorption peak at 190K, two peaks for N2 , and H2 from 270K to 390K. At 345K, peak of desorption product (m/z=103) represents phenyl cyanide (PhCN) or phenyl isocyanide (PhNC). RAIR and XP spectra demonstrate that at 190K benzyl azide on Cu(111) readily adopt the imine intermediate formalism involving N£\¡ÐN£] scission and phenyl group shift from carbon to nitrogen. The mechanism is analogous to the organic reaction of Schmidt rearrangement. To heat the surface to 250K, the CH2 group of the imine intermediate undergoes C¡ÐH bond scission to produce a surface isocyanide intermediate, (M=C=N¡ÐPh). Therefore the final desorption product is phenyl isocyanide at ~350K. Intriguingly, the thermal chemistry of benzyl azide involves both imine and isocyanide intermediacy, despite the fact that azido species usually generate nitrene or imido complexes under thermal conditions.

none

Quinolines have been studied for over a century as an important class of heterocyclic compounds and continue to attract considerable interest due to the broad range of biological activities they possess. The incorporation of the essential structural features of quinolines with a ferrocene moiety could provide new derivatives with unexpected and/or enhanced biological activities since several ferrocene derivatives have already been shown to be active against a number of tumors. For this reason, we investigated the synthesis of ferrocenyl-substituted quinolines, such as 2-ferrocenylquinoline, by employing the molecular iodine catalyzed reaction between enolizable aldehydes and ferrocenyl imines, which were prepared by the condensation reactions of ferrocenecarboxaldehyde with aniline derivatives. As anticipated, these reactions produced 2-ferrocenylquinoline derivatives. By employing this ethodology, we synthesized 2-ferrocenylquinoline, 6-chloro-2-ferrocenylquinoline, 6-bromo-2-ferrocenyl-quinoline, 2-ferrocenyl-7-methylquinoline and 2-ferrocenyl-3,7-dimethylquinoline. Due to the ready availability of ferrocenylimines and aldehydes, this practical onepot method represents a versatile synthesis of ferrocenyl-substituted quinolines.

Chiral 1,2-amino alcohols were synthesized via newly developed &ldquo / intramolecular unsaturation transfer&rdquo / using cyclohexanone, propargyl bromide, and various chiral amino alcohols as starting components. These amino alcohols can be potential chiral ligands for many asymmetric transformation reactions. Therefore, their effectiveness as chiral ligands in diethylzinc addition to benzaldehyde and N-diphenylphosphinoyl imines were tested. Various parameters including temperature, solvent, ligand amount etc. were screened for the synthesized chiral ligands. In diethylzinc addition to benzaldehyde high enantioselectivity could not be obtained. When N-diphenylphosphinoyl imines were used as substrate good ee values up to 80% were achieved.

Cyanide-catalyzed aldimine coupling was employed to synthesize compounds with 1,2-ene-diamine and α-imine-amine structural motifs: 1,2,N,N'- tetraphenyletheylene-1,2-diamine (13) and (+/-)-2,3-di-(2-hydroxyphenyl)-1,2- dihydroquinoxaline (17), respectively. Single crystal X-ray diffraction provided solidstate structures and density functional theory calculations were used to probe isomeric preferences within this and the related hydroxy-ketone/ene-diol system. The enediamine and imine-amine core structures were calculated to be essentially identical in energy. However, additional effects-such as π conjugation-in 13 render an enediamine structure that is slightly more stable than the imine-amine tautomer (14). In contrast, the intramolecular hydrogen bonding present in 17 significantly favors the imine-amine isomer over the ene-diamine tautomer (18). Aldimine coupling (AIC) is the nitrogen analogue of the benzoin condensation and has been applied to dialdimines, providing the first examples of cyclizations effected by cyanide-catalyzed AIC. Sodium cyanide promoted the facile, intramolecular cyclization of several dialdimines in N,N-dimethylformamide, methanol, or dichloromethane/water (phase-transfer conditions) yielding a variety of six-membered heterocycles. Under aerobic conditions, an oxidative cyclization occurs to provide the diimine heterocycle. Cyanide-catalyzed aldimine coupling was employed as a new synthetic method for oligomerization. Nine rigidly spaced dialdimines were oxidatively coupled under aerobic conditions to yield conjugated oligoketimines and polyketimines with unprecedented structure and molecular weight (DP = 2 - 23, ~700 -8200 g/mol). The α- diimine linkage was established based on IR spectroscopy, NMR spectroscopy, size exclusion chromatography, and X-ray crystallographic characterization of the model oxidized dimer of N-benzylidene-(p-phenoxy)-aniline. Cyclic voltammetry indicates ptype electrical conductivity, suggesting they are promising candidates for plastic electronic devices. The cyanide-catalyzed benzoin condensation reaction of 4-substituted benzaldehydes followed by oxidation to the diketone, and the Schiff Base condensation of two equivalents of o-aminophenol provides 2,3-(4-X-phenyl)2-1,4-(2- hydroxyphenyl)2-1,4-diazabutadiene. The ligand is given the moniker X-dabphol. These ligands are readily metallated to form M-X-dabphol complexes. The copper complexes catalytically fix CO2 with propylene oxide to yield propylene carbonate. DFT studies along with a comparison with Hammet parameters help validate and elaborate on the catalytic cycle and the catalytic results obtained. The nickel complex is competent for olefin epoxidation. Synthesis, characterization, X-ray structure, DFT analysis, and catalytic activity of the parent nickel dabphol complex are reported.