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Chiral amines are important chemical building blocks found in a wide range of bioactive pharmaceutical compounds and sustainable routes for their synthesis is highly desirable. In recent years, the use of biocatalysts has emerged as a popular choice for amine synthesis as it has many advantages over traditional chemical synthesis, such as the ability to catalyse a broad range of highly selective reactions under mild aqueous conditions as well as an improved atom economy, reducing waste. As such, the aim of this thesis was to develop multienzyme cascades towards the synthesis of chiral primary and secondary amines. With improved enzyme discovery and faster gene synthesis, it is increasingly possible to combine multiple enzymes in one-pot to perform sequential biocatalytic reactions for the generation of complex intermediates. These de novo enzymatic cascades take advantage of enzymes preferred and often compatible reaction conditions and can be implemented in vitro, in vivo or as hybrid cascades. Through computer-aided-synthesis-planning (CASP), several multi-enzyme cascades were established for the preparation of chiral and non-chiral, primary and secondary amines, utilising a range of galactose oxidase (GOase), imine reductase (IRED), D-fructose-6-phosphate aldolase (FSA), choline oxidase (AcO) and ω-transaminase (TA) enzymes. In particular, an in vitro GOase-IRED one-pot cascade for the synthesis of Cbz- protected 3-aminopiperidine homologs, a drug precursor, using starting materials derived from bio renewable amino acids ornithine and lysine. Another cascade using FSAs and IREDs in a one-pot, two-step approach was developed for the synthesis of chiral polyhydroxylated amines, making products with three chiral centres from two reaction steps with high diastereomeric ratios. Enzyme compatibility remains a key issue for some cascades, with reactants sometimes inhibiting one enzyme or causing side reactions. Through the use of enzyme immobilisation a 'plug-and-play' concept was developed for the synthesis of primary and secondary amines in continuous flow, utilising various oxidase and amine biocatalysts.

The Morita-Baylis-Hillman (MBH) reaction is a carbon-carbon (C-C) bond forming reaction between an activated alkene and an aldehyde. It is a synthetically useful reaction due to the high atom economy and retention of multiple functional groups. Unfortunately, harsh reaction conditions are required during the MBH reaction and unpredictable product stereospecificity have hampered the widespread application of this reaction. Catalysis of the MBH reaction by enzymes has the potential to allow the reaction to occur at ambient conditions, while offering scope for improving the stereospecificity. This thesis focussed on the enzyme design of a MBH enzyme using thermostable fructose-1,6- bisphosphate (FBP) aldolases as scaffolds. These enzymes were chosen because there are common features between the aldol and MBH reactions, both making use of an enol intermediate to attack the aldehyde. In addition, aldolases typically accept a wide variety of substrates. Thermostable aldolases were selected for increased temperature tolerance creating a more desirable catalyst for industrial purposes. Thermoproteus tenax FBP aldolase (TtFBPA; WT and W144L, W144Y, K177A variants) and Staphylococcus carnosus FBP aldolase (ScFruA) were expressed and purified from E. coli. While the retro-aldol reaction catalysed by these enzymes could be easily monitored, the reverse reaction (aldol synthesis) is more difficult to quantify. Multiple methodologies for high throughput spectrophotometric detection of aldol activity were developed as a method of monitoring constructs made during directed evolution of the FBP aldolases. However, none of these proved successful in robustly determining aldol activity. The dihydroxyacetone phosphate (DHAP) mimic 1-hydroxy-3-buten-2-one phosphate (HBOP) was used to assay for MBH catalysis. While crystallographic studies with TtFBPA suggest that HBOP is bound to W144L TtFBPA in a manner compatible with the MBH reaction. NMR studies could not detect any corresponding activity. This suggests further protein engineering will be required to evolve this FBP aldolase to an MBH catalyst. In addition, our crystallographic and NMR studies with TtFBPA reveals this enzyme is capable of catalysing the formation of both FBP and tagatose-1,6-bisphosphate (TBP).Additionally, we determined the first structure of ScFruA. Interestingly, NMR experiments suggested ScFruA lacks significant control of the stereospecificity of the aldol condensation reaction and appears to catalyse the formation of FBP, TBP, xyluose-1,6- bisphosphate and psicose-1,6-bisphosphate. We conclude that while FPB aldolases could indeed provide useful scaffolds for the development of an MBH catalyst, the enzymes lack any inherent activity, necessitating the need for future creation of variants. The success of this approach will depend on the ability to screen mutant libraries for MBH product formation.

Understanding the internal organization of cells from the molecular up to organelle level is a current challenge for biology if we are to better comprehend the mechanisms by which cellular processes occur. A prevailing view of the cell interior is that biochemical reactions and molecular movements are dominated by random diffusion. However, in addition to being compartmented into organelles, cells may well be organized at a finer level. Proteins may localise to specific areas within sub-cellular compartments, by associating with each other, the cytoskeleton and organelle membranes. Thus giving rise to distinct microcompartments. Considerable in vitro evidence exists for such interactions between enzymes and larger cellular components. This strengthens the idea that cells may be microcompartmented. However, little in vivo evidence supports this hypothesis, especially in plants. As a test-case for the concept of microcompartmentation this project investigated the sub-cellular distribution of the glycolytic enzyme fructose-bisphosphate aldolase in Arabidopsis thaliana; the ultimate aim being to establish whether it is microcompartmented in vivo and, further, to test the potential function(s) of such microcompartmentation.

The fructose 1,6-bisphosphate type I aldolase from the thermophilic organism Thermoproteus tenax has been expressed and purified by heat treatment. The aldolase aldehyde substrate scope was investigated using electrospray mass spectrometry to detect the formation of any aldol products. Parameters affecting aldolase activity, including temperature, buffer pH and solvent additive were investigated. The synthesis of an aldehyde with an attached fluorescent reporter group was performed for potential use in the screening of mutant aldolases for aldol or Baylis-Hillman activity. The synthesis of 1-hydroxy-3-buten-2-one phosphate, an analogue of dihydroxyacetone phosphate, capable of participating in Baylis-Hillman reactions, was achieved in 5 steps from 3-buten-1-ol. This analogue was used in the investigation of the wild type aldolase and several mutant aldolases for Baylis-Hillman activity. X-ray crystallographic data was obtained for the wild type enzyme and the Trp144Leu mutant aldolase with 1-hydroxy-3-buten-2-one phosphate bound at their active sites. In the wild type aldolase, the substrate was found to bind in a similar manner to dihydroxyacetone phosphate, with the formation of a Schiff base with the Lys177 amino acid residue at the enzyme active site. In the Trp144Leu mutant aldolase, Lys177 has added in Michael fashion to the enone functionality of the bound substrate forming an enolate instead of forming a Schiff base. Both forms of the bound substrate are potentially capable of participating in Baylis-Hillman reactions. The enzymes have yet to be fully investigated for Baylis-Hillman activity.

Fluorine is a critically important element for the design of safe and effective drugs in the modern pharmaceutical industry. As the field of synthetic methodology for fluorination of organic compounds has flourished, biocatalysis has also been rapidly adopted as a means to achieve high reaction selectivity and process sustainability. However, the areas of biocatalysis and fluorine chemistry have rarely intersected directly, owing to the rarity of fluorine-processing enzymes in Nature. We envision that this methodology gap can be bridged by the action of carbon-carbon bond forming enzymes upon non-native fluorinated substrates, thus transforming simple organofluorine building blocks into more complex organofluorines. In this dissertation, we describe the application of enzymatic aldol addition to fluoro-aldol reactions that furnish value-added organofluorine products bearing fluorine stereocenters. Our development of a novel platform for biocatalytic organofluorine synthesis began with the discovery that Type II pyruvate aldolases of the HpcH family use fluoropyruvate as an alternative nucleophilic substrate. After studying the stereoselectivity, kinetics, and mechanism of the fluoropyruvate aldol reaction, the afforded products were converted to diversely functionalized organofluorines through downstream reactions. Next, the HpcH system was expanded beyond fluoropyruvate to generalized β-fluoro-α-ketoacids as nucleophiles. The investigation of these challenging substrates, aided by rational active site engineering, resulted in the first synthesis of tertiary fluorides via biocatalytic C-C bond formation. Usefulness of the HpcH platform was demonstrated with preparative syntheses of products relevant to drug fragments and bioactive natural products. Finally, we studied Type I aldolases with the unique ability to use simple aldehydes and ketones as nucleophiles. Their activity on fluorinated ketones may enable the efficient synthesis of fluorinated sugars, which are applicable to pharmaceuticals and chemical biology. Taken together, this dissertation delineates a selective and sustainable biocatalytic approach to access the unique properties conferred by fluorination, which may have broad implications for improved process design towards important organofluorines.

Fructose 1,6-bisphosphate aldolase, or simply aldolase, is a key enzyme involved in the glucose and fructose metabolic pathways. Vertebrate aldolases exists in nature as extremely stable tetramers. While essential for energy metabolism, aldolase is involved in numerous “moonlighting functions,” many of which involve the ability to bind F-actin. Evidence suggests that the quaternary structure of aldolase may have evolved for such moonlighting activities in the cell. Though aldolase was one of the first actin-binding proteins discovered, the exact structure and biological functions of this interaction have remained elusive. Electron microscopy studies demonstrate that a minimum of a dimer is required to bind and decorate actin filaments, but only tetrameric aldolase is capable of binding and crosslinking F-actin into bundles. Experiments are described that will lead to a high-definition structure of the aldolase-F-actin complex and resolution of the binding interface used by aldolase. These include efforts to create chimeric aldolase tetramers using in vitro protein hybridization and modular cloning approaches. Understanding this critical interaction is necessary for mechanistic models explaining aldolase moonlighting functions. The significance of aldolase’s association with F-actin in vivo has only more recently been explored. Studies with two other aldolase binding partners—Wiskott-Aldrich Syndrome protein (WASP) and Sorting Nexin 9 (SNX9), suggest that aldolase may be moonlighting as a scaffold protein during late stage endocytic events in the cell. The hypothesis that aldolase has a regulatory role in endocytosis is tested. Precise coordination and efficiency of recruited proteins are hallmarks of clathrin-mediated endocytosis, and a scaffolding role for aldolase could serve at least two functions: 1) to sequester the active domains of WASP and SNX9 prior to vesicle scission, and 2), enable sufficient concentration of WASP and SNX9 at sites of endocytosis via association with F-actin. This type of regulation would sequester each protein until the exact function is needed, allowing release at the appropriate time points as well as in the proper sequence. Uptake assays performed using two different markers and cell lines show upregulation of endocytosis after knockdown of aldolase. A model is proposed for participation by aldolase in the dynamics of this process.

Human fructose-bisphosphate aldolase A (ALDOA) is a key enzyme in glycolysis that catalyzes the conversion of fructose 1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. ALDOA has also been shown to play key moonlighting roles associated with cellular structure, motility and proliferation. This aldolase isoenzyme is significantly overexpressed in a number of human tumors and has been shown to have oncogenic potential. We have demonstrated that ALDOA overexpression has further inference through cycling of hypoxia inducible transcription factor-1 (HIF-1) regulation. This feed-forward loop mediated through AMPK is a new target to consider in the irregular hypoxic environment of solid tumors. The body of work that follows presents five different methods of targeting and inhibiting ALDOA function. Using small molecule probes, the function and regulation of these iterations is characterized, towards better understanding of the role of ALDOA in cells and in cancer

Carbohydrate represents a class of biomacromolecules which is quite different from polynucleotide and polypeptide, given its branching character and non-template based biosynthesis. Carbohydrates exist in cells or on cell surfaces mainly as glycoconjugates: conjugation of a sugar chain (glycan) with a protein (glycoprotein), a lipid (glycolipid) or both lipid and protein. Cell surface carbohydrates are of paramount significance in biochemical recognition processes and carbohydrate complexes have been widely used in pharmaceutical areas such as prevention of infection, neutralization of toxins and cancer immunotherapy.It is well known that nucleic acids are biosynthesized using dNTP (or NTP) and proteins using activated amino acids. Our focus on this research (Chapters 1-4) is to find a facile way to produce the key building blocks for enzymatic carbohydrate synthesis---sugar nucleotide donors. We discovered a pathway in which N-acetylglucosamine/N-acetylgalactosamine (GlcNAc/GalNAc) and their analogues were phosphorylated by a 1-kinase called NahK to generate sugar-1-phosphates in good yields. These sugar-1-phosphates were subsequently pyrophosphorylated by GlmU from bacteria or AGX1 from mammals to give the corresponding sugar nucleotide donor molecules. We thus obtained a library of UDP-GlcNAc/GalNAc analogues on a preparative scale since these involved enzymes proved to be rather promiscuous. With the modified sugar nucleotide donors in hand, we are primed to incorporate the structurally modified GlcNAc/GalNAc analogues into different carbohydrate-containing biomolecules to either investigate carbohydrate metabolic pathways or construct a glycorandomized library of new glycoforms.In addition, the high stereoselectivity of aldolases in C-C bond construction confers upon them tremendous applications as synthetic biocatalysts. Among the aldolases, dihydroxyacetone phosphate (DHAP)-dependent aldolases are particularly attractive as a set of four possible diastereomers of vicinal diols can be synthesized conferring upon them the potential to be used in the synthesis of rare sugars and other hydroxylated natural products. Unfortunately, the strict requirement for the donor substrate DHAP, a rather expensive and unstable compound, limits aldolase use in large-scale preparation. Therefore, the capability to generate DHAP from inexpensive sources could ultimately broaden the scope of aldolase reactions making it an attractive challenge. In Chapter 5, we employed DHAP-dependent aldolases in the synthesis of several rare sugars via a one-pot four enzyme reaction system in which DHAP is generated from the oxidation of cheap DL-glycerol 3-phosphate. Subsequently, the DHAP generated in situ is coupled with glyceraldehydes by aldolases to give different rare sugars. More significantly, we used engineered bacteria as ‘chemists’ to carry out the aldolase reactions in vivo. The bacteria were fed with green starting materials (e.g. glycerol or glucose) and DHAP is generated in vivo via bacterial glycolysis. Desired rare sugars could thus be produced by simple fermentation and isolated from the culture media.

The investigations carried out in my doctoral studies focus on (1) applications of pyridinium salt photochemical and tandem ene-yne-ene metathesis reactions to the synthesis of the natural products (+)-lactacystin and lepadiformine C, and (2) the preparation of a novel inhibitor for the class II Giardia lamblia fructose-1,6-bisphosphate aldolase. New developments in the area of pyridinium salt photochemistry were explored in the context of approaches to the natural product (+)-lactacystin. The results of this effort show that irradiation of a substituted 1,2-cyclopenta-fused pyridinium salt in aqueous solution followed by treatment with sodium bicarbonate leads to selective production of an unusual, structural and functional complex, and stereodefined tetracyclic carbamate. This substance contains structural features and a functionality array that would make it applicable as a late stage intermediate in a synthesis of the proteosome inhibitor, lactacystin. A novel strategy was developed for the synthesis of lepadiformine C, a structurally interesting, biologically active marine alkaloid derived from the marine organism Claveline moluccensis. that has a tricyclic structure. The approach, which begins with l-proline, relies on implementation of a ruthenium-alkylidene catalyzed, tandem ene-yne-ene metathesis process to construct the tricyclic structure. Class I and class II fructose-1,6-bisphosphate aldolases (FBPA), enzymes that exhibit no amino acid sequence homology and utilize different catalytic mechanisms, promote the retro-aldol conversion of fructose-1,6-bisphosphate (FBP) to dihydroxyacetone phosphate and D-glyceraldehyde-3-phosphate as part of energy producing glycolysis pathways in bacteria, protists and humans. The mammalian class I FBPA employs a Schiff base mechanism, involving an active site lysine amine group, whereas the parasitic protozoan Giardia lamblia relies on a class II FBPA that utilizes an active site Zn+2 to stabilize the forming enolate of dihdroxyacetone phosphate. One subgoal of my studies was to develop a novel strategy for the design of inhibitors of the class II Giardia lamblia FBP (glFBPA). As part of an overall effort in this area, potential inhibitors that possess Zn+2 binding 3-hydroxy-2-pyridone moieties were designed and prepared. The inhibitory properties of these substances against glFBPA were determined. The results show that the structure-based inhibitor design is effective in identifying new 3-hydroxy-2-pyridone based glFBPA inhibitors that have modestly tight (low micromolar) binding affinities.

E. coli 2-keto-4-hydroxyglutarate (KHG) aldolase is a Class I lysine-type aldolase that catalyzes the reversible cleavage of 2-keto-hydroxyglutarate with formation of pyruvate plus glyoxylate. The mechanism of action involves Schiff-base formation between pyruvate or KHG and the (epsilon)-amino group of an active-site lysine residue as well as transfer of a proton from the C-4 hydroxyl of KHG to the C-3 of pyruvate by an amphoteric group. A positively charged entity could also be in the active-site to facilitate interaction with the anionic substrates. The data presented in this thesis delineate the complete amino acid sequence of E. coli KHG aldolase and also identify Lys-133, Glu-45, and Arg-49 as aminoacyl residues required for catalytic activity. These residues most likely participate in the active-site of the aldolase in the roles indicated above. Incubation of E. coli KHG aldolase with ('14)C pyruvate in the presence of NaCNBH(,3) results in the incorporation of one mol of ('14)C per mol of enzyme subunit. Digestion of this enzyme-adduct with trypsin, followed by purification of the peptides, allowed for the isolation of a unique radioactive peptide. Its amino acid sequence showed that the pyruvate-binding (i.e., Schiff-base forming) lysine residue is located at position 133 in the intact enzyme. E. coli KHG aldolase activity is lost when the enzyme is reacted with bromopyruvate; saturation kinetics are observed. The substrates, pyruvate and KHG, protect the enzyme from inactivation. Both facts suggest that the reagent is active-site specific. Incubation of the aldolase with 3-('14)C bromopyruvate is associated with a concomitant loss of enzymatic activity and esterification of Glu-45; if the enzyme is denatured in the presence of excess bromopyruvate, Cys-159 and Cys-180 are also alkylated. Blocking the active-site lysine residue with pyruvate prevents Glu-45 from being esterified but does not eliminate alkylation of these two cysteine residues. Woodward's Reagent K was also found to inactivate the aldolase under conditions that are usually specific for carboxyl group modification. This aldolase is also inactivated by 1,2-cyclohexanedione. Loss of enzymatic activity occurs concomitantly with modification of one arginine residue per enzyme subunit. Treatment of the aldolase with the arginine-specific reagent, 4-(oxyacetyl)phenoxyacetic acid, followed by digestion with trypsin allowed for the isolation of a unique peptide and the identification of Arg-49 as the specific residue involved.

Procedures for the isolation and characterization of Ascaris glyceraldehyde-3-phosphate dehydrogenase and actin are described. The properties of these proteins, including molecular weights, isoelectric points, kinetics, peptide maps, and amino acid compositions, are strikingly similar to the respective proteins from rabbit muscle.

The muscle aldolase from Ambystoma tigrinum has been purified 73-fold to a final specific activity of 13.2 units per mg. The purified enzyme appeared to be homogenous by ultracentrifugation and electrophoretic criteria. A molecular weight of 159,000 + 1000 was determined by gel filtration on Sephadex G-200 and high speed sedimentation equilibrium ultracentrifugation. The enzyme migrated identically with rabbit muscle aldolase when subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and is apparently a tetramer of nearly identical subunits of approximately 40,000 MW. The catalytic constants of the salamander enzyme were similar to those reported for other muscle aldolases with the exception of the unusually low Fru-P2/FlP ratio.

A procedure is described for the purification of human erythrocyte aldolase (EC 4.1.2.13). The process involves a specific substrate elution of the enzyme from phosphocellulose followed by a reverse ammonium sulfate fractionation.

Aldolase from human heart has been purified 128-fold to a final specific activity of 11.52 units per mg. The purification procedure employed column chromatography on phosphocellulose.