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Antibody-drug conjugates (ADCs) are an emerging class of anticancer agents which combine the cell-targeting properties of a monoclonal antibody and the cytotoxicity of a small molecule drug. The antibody, cytotoxin and the covalent linker which connects the two units must be optimised for the ADC to have favourable pharmacokinetic, toxicology, and overall pharmacological profiles. The first project describes the design, synthesis, and evaluation of vinylheteroarene linkers for the post-translational modification of proteinogenic cysteine residues. It was hypothesised that the variable heteroarene ring electronics would greatly affect the conjugate addition reaction rate and resulting bioconjugate stability. Thus, a panel of vinylheteroarenes with differing heterocyclic groups were synthesised and evaluated for their suitability. Using small molecule model systems, vinylheteroarene linkers were assessed for cysteine reactivity and chemoselectivity, and conjugate stability to identify the optimum linker. The lead linker was utilised to modify a number of protein substrates with various functionality, including the synthesis of a homogeneous, stable and biologically active ADC. The reagent was also efficient in labelling proteome-wide cysteines in cell lysates. The efficiency and selectivity of these reagents as well as the stability of the products makes them suitable for the generation of biotherapeutics or studies in chemical biology. The second project describes studies towards using (+)-discodermolide as a cytotoxic payload in ADCs. Discodermolide is a polyketide natural product which displays potent activity as an antimitotic agent and is known to be effective against a range of malignancy types. It was desired was to attach discodermolide to an antibody using a cleavable linker which would enable traceless release of the cytotoxin. The alcohol groups in discodermolide presented an opportunity to achieve this in a chemically tractable way. Thus, a valine-alanine-para-aminobenzylcarbonyl-hemiaminal linker was designed for the selective cleavage and payload release by lysosomal cathepsin enzymes. Importantly, this linker strategy allows the synthesis of linker-payloads in one synthetic step, making this a promising approach for the rapid assembly of ADCs using unexplored alcohol cytotoxins. Using this linker, a discodermolide ADC was successfully synthesised which can be used for biological evaluation.

Chemical mutagenesis is defined as the post-expression interconversion of amino-acid (AA) sidechains. This approach enabled the first ever point-mutations in proteins and has seen a resurgence of interest over the last two decades. This thesis reports on two disparate applications of chemical mutagenesis within the context of Alzheimer's disease (AD). First, exploring the capabilities of utilizing chemical mutagenesis to enable a structure-activity relationship study on a single-domain antibody (SdAb) beyond the 20 canonical AAs. The starting SdAb was designed to target the Amyloid-b peptide (Ab) and has a nascent ability to inhibit its aggregation, a key pathological process in AD. Utilizing the synthetically versatile non-canonical AA dehydroalanine (Dha), we explore diverse sidechains at key residues along the binding loop of the SdAb and identify a non-canonical sidechain that potently enhances the inhibition of Ab's primary nucleation. The second application focuses on the other major protein implicated in the pathology of AD: Tau. In AD, tau aggregates into ordered amyloid fibrils and contains a plethora of post-translational modifications (PTMs), many of which are speculated to have an impact on the pathogenesis of AD as well as other so-called tauopathies. In order to facilitate the accurate study of tau PTMs, an easily accessible method for their site- selective installation is needed. To this end, we again explore the utilisation of Dha to install accurate PTM mimetics for phosphorylation, lysine acetylation, and lysine dimethylation at diverse sites along tau. This approach produced homogenous products that accurately recapitulated known behaviours and allowed for the initial characterisation of poorly investigated PTMs. The characterisation of S199 phosphorylation, a known AD associated modification, through this approach provides the first evidence for its potential role in regulating tau's primary function of microtubule polymerisation and stabilisation.

Chemistry plays an essential role in biological processes within all living organisms and better understanding of how specific chemical bonds can be either formed or broken gives chemical biologists opportunities to control and regulate these processes with high precision. Therefore, with growing importance of the field of chemical biology, expansion of the toolbox of available strategies for the formation or cleavage of specific chemical bonds under biocompatible conditions is needed for future advances in diverse topics in life sciences. The presented work aims to contribute to this growing field with a versatile method for the preparation of site-specific and plasma stable DNA-protein conjugates which are suitable for cellular applications. Using the method developed, various types of ONs can be easily conjugated to a range of proteins via solvent accessible cysteine residues, including therapeutic full-length IgG antibodies. Moreover, in order to optimise and simplify the characterisation of prepared DNA-protein conjugates, an improved and straightforward LC-MS method for the analysis of bioconjugation reactions between proteins and ONs was developed. Prepared and characterised DNA-antibody conjugates were then employed as probes in cellular imaging using a super- resolution DNA-PAINT method or fluorescence microscopy. Furthermore, this thesis reports new types of metal-mediated bond-cleavage reactions triggered by an active catalytic palladium species which can be either prepared as a complex or directly generated in living cells under biocompatible conditions. The cleavage reactions described are then used for on-demand extra- or intracellular release of highly cytotoxic small molecule drugs bearing phenolic hydroxyl or amine functional groups. Essentially, the strategy presented not only allows the control of the biological activity of small molecules, but also the reactivity of palladium species involved in palladium-assisted bioorthogonal reactions. In summary, the methods and findings presented in this thesis contribute to the portfolio of chemical transformation strategies available in the interdisciplinary field of chemical biology and provide new opportunities for future therapeutic applications.

Bioorthogonal reactions, due to its excellent selectivity and time-efficiency, have emerged as a popular tool for protein and cell probing. Among all the bioorthogonal reactions, the inverse electron-demand Diels-Alder reaction (IEDDA) reaction has its advantage of bearing the fastest kinetics. Although the IEDDA reaction drew considerable attention in chemical biology in the last decade, challenges lie in finding the suitable dienophiles. Strained dienophiles, for example, trans-cyclooctene derivatives, can undergo ultrafast IEDDA reactions and therefore have been extensively developed. Unstrained alkenes and alkynes, however, have not been well investigated as IEDDA handles. In general, unstrained dienophiles are more straightforward to synthesise compared with strained dienophiles, therefore they are more accessible to researchers. In addition, the absence of a highly reactive bond makes unstrained dienophiles inert to biological nucleophiles, which allows effectively cellular labelling. In this dissertation, I described three different unstrained dienophiles for different biological purposes. Allyl handle is thiol-stable and non-toxic, which was utilised to label apoptotic cells in a pre-targeting manner. Enol ethers can react with tetrazines to decage protected amino acids and prodrugs. Potassium arylethynyltrifluoroborate, as a novel dienophile, was shown to react fast with pyridyl tetrazines controllably and this new IEDDA was applied to label proteins site-selectively and to fluorescently label two proteins orthogonally. In addition to IEDDA reactions, other bioorthogonal reactions were also developed using these versatile unstrained handles. Allyl-bearing amino acids and proteins can undergo an acetophenone-mediated hetero-[2+2] photocycloaddition with maleimide derivatives, expanding the toolbox of photo-triggered chemistry for protein modification. The potassium arylethynyltrifluoroborate handle was also found reactive in copper(I)-catalyzed alkyne-azide cycloaddition reaction (CuAAC) and showcased the huge potential for protein labelling and multicolour cellular labelling.

Acriflavine (ACF) is a Topoisomerase 2 (Top2) poison, a class of drugs which stall Top2 during its reaction cycle causing the formation of persistent DNA breaks to which Top2 remains covalently bound. Deletion of ULS1 causes sensitivity to ACF, with cells showing activation of the Rad53 DNA damage checkpoint. Uls1 is a chromatin remodelling enzyme also implicated in the regulation of levels of SUMO conjugated proteins. We show that Uls1 has both a genetic and physical interaction with Top2, with uls1Δ sensitivity to ACF being linked to Top2 activity. Analysis of Uls1 and Top2 localisation genome wide via ChIP-seq reveals areas where the two proteins co-localise, with Top2 enrichment on chromatin being altered upon deletion of ULS1. At these areas, the presence of Uls1 prevents accumulation of Top2 upon addition of ACF. Our data suggests that Uls1 is required for regulation of stalled Top2. Top2 poisons are used therapeutically as anti-cancer drugs, however these drugs have been implicated in the formation of secondary cancers due to chromosomal translocations arising during the repair of Top2 generated double strand breaks (DSB). The use of dual targeted therapies where a Top2 poison is paired with an inhibitor of another pathway that increases sensitivity to the Top2 poison allows a lower dose to be used, therefore reducing harmful side effects. Our work looked to identify Top2 poison sensitive pathways in S. cerevisiae, where non-essential and essential gene mutants were assayed for sensitivity to ACF. This allowed a comprehensive analysis of 83% of the genes in S. cerevisiae, identifying novel genes within the areas of DNA repair, DNA replication, transcription, chromatin structure, protein modification/degradation, cell division/cell cycle and cellular organisation/cytoskeleton as being important in the response to this bulky adduct.

In this thesis will be described research towards the development of bioorthogonal bond-cleavage reactions, and their applications in targeted drug delivery (Figure 1). The first project relates to the development of a palladium mediated bond-cleavage or "decaging" reaction which can cause a propargyl carbamate to decompose and release an amine. This was further developed by the incorporation of a protein modification handle which allowed an amine-bearing drug to be covalently ligated to a protein by a palladium-cleavable linker. This chemistry was demonstrated by the conjugation of the anticancer drug doxorubicin to a tumour targeted anti-HER2 nanobody. The drug could then be delivered to cancer cells upon addition of a palladium complex. The second project relates to the development of a platinum mediated bond-cleavage reaction. This was developed with the aim of using platinum-containing anticancer drugs - such as cisplatin - as a catalyst to cause drug release reactions in tumours. In this reaction an alkyne-containing amide can decompose to release an amine upon addition of platinum complexes, and was applied to the release of prodrugs of the cytotoxins monomethylauristatin E and 5-fluorouracil in cancer cells. A cisplatin-cleavable antibody-drug conjugate was designed and synthesised, and progress towards its biological evaluation will be discussed.

Post-translational modifications (PTMs), which biochemically modify proteins to generate diversity and control functionality, are important in the field of chemical biology research. However, typical bioconjugation methods involving nucleophilic addition of cysteine and lysine residues are limited by their lack of site-specificity. In this thesis, we have demonstrated the site-selective chemical modification of a variety of proteins through protein halogenation and subsequent pallado-biology strategies. The "Tag-and-Modify" approach, developed previously in our group and involving the creation of a protein tag for further manipulation, is a useful tool for site-selective chemical modification. Here, we envisioned extending its utility to a novel concept of [18F]-radiolabelling of proteins involving direct electrophilic fluorination, thus forming a C-F bond at a specific modification site and leading to the generation of protein species applicable for in vivo [18F]-PET imaging and diagnosis. We also explored several alternative methods for the installation of C-Br/C-I bonds onto a protein of interest. Our first attempts involved the biosynthetic incorporation of a synthetic bromotryptophan into the protein, however these processes were unsuccessful. As a workaround, we found IPy2BF4, an iodinating agent, to be very effective as a direct method for the site-specific installation of C-I bonds on tyrosine/histidine residues of proteins. We subsequently demonstrated novel and efficient palladium-catalysed cross-couplings of the resultant iodinated tyrosine/histidine moieties, leading to the creation of new C-C bonds. This approach was compatible with a wide range of functionally diverse boronic acids, using a water-soluble and phosphine-ligand-free Pd-complex catalyst under fully aqueous conditions. Our novel, successful method for C-C bond formation onto proteins is potentially applicable to the initial investigation of challenging in vivo Pd cross-coupling of thyroglobulin, based on our achievement of protein labelling under ex vivo conditions. In summary, we are able to create new chemical tools for the site-selective/site-specific chemical modification of proteins, enabling their use as biological probes.

Medical imaging is an important area of scientific research with the principle aim of accurately diagnosing diseases to guide more effective treatment. Magnetic resonance imaging (MRI) provides advantageous high spatial resolution and deep tissue visualization. However, 1H MRI can suffer from low signal-to-noise ratios due to strong background signals from abundant, endogenous molecules such as water and fat. 19F MRI overcomes this drawback by directly detecting administered 19F-labelled agents without any background signal. This thesis reports the design and development of a new fluorinated agent for medical imaging. The bacteriophage Qβ, which comprises 180 copies of its coat protein, was used as the scaffold for the imaging agent. The fluorinated amino acid trifluoromethionine (TFM) was incorporated into the Qβ coat protein by the sense codon reassignment method using auxotrophic E. coli. This marks the first example of introducing fluorine into a virus by genetic modification. Reports of previous attempts to incorporate TFM into proteins have shown low to good incorporation levels (the maximum being 82%). Here, extensive optimisation of the protein production conditions resulted in unprecedentedly high incorporation levels (96%). Analysis of the modified Qβ-F by agarose gel electrophoresis, dynamic light scattering and transmission electron microscopy showed the coat protein self-assembles into virus-like particles (VLPs), essentially indistinguishable from the wild-type Qβ VLPs. The intact Qβ-F VLP gave no 19F NMR signal. This was rationalised in terms of the VLP's high molecular weight and resulting fast transverse relaxation. Stimulating disassembly of the VLP by addition of dithiothreitol (DTT), sodium dodecyl sulphate (SDS), or a combination of the two resulted in the development of 19F NMR peaks. Multimers and monomers of the disassembled Qβ-F were distinguished by 19F NMR and 19F DOSY. This result was verified by measuring the 19F NMR of a prepared sample of alkylated Qβ-F monomer and comparing its chemical shift value with that of peaks from the 19F NMR of the disassembled Qβ-F VLP. Finally, initial in vivo experiments were carried out in order to investigate the biodistribution of the Qβ-F particle. After injection of Qβ-F VLPs into rats, organs were dissected and homogenized. The Qβ-F particle was found to accumulate predominantly in the liver. 19F NMR of the liver after sucrose density gradient ultracentrifugation only showed a signal on incubation with DTT, demonstrating that the Qβ-F particle remained intact in vivo.

Site-selective protein modification has become an important tool to study protein functions in chemical biology. In the preliminary work, allyl sulfides were found to be reactive substrates in aqueous cross-metathesis (CM) enabling the first examples of protein modification via this approach. In order to access the enhanced CM reactivity of allyl sulfide on proteins, facile chemical methods to install S-allyl cysteine on protein surface were developed. In particular, a cysteine-specific allylating reagent – allyl selenocyanate was used on protein substrate for the first time. The substrate scope of allyl sulfide-tagged proteins and factors that affect the outcome of CM was also investigated. A range of metathesis substrates containing different olefin tether of various lengths were screened; allyl ethers were found to be most suitable as CM partners. By reducing the steric hindrance around the allyl sulfide on protein surface through a chemical spacer, the rate and conversion of metathesis reaction on proteins was greatly enhanced. Moreover, allyl selenides were found to be more reactive than allyl sulfides in CM and enabled reactions with substrates that were previously impossible for the corresponding sulfur-analogue. Through this work, substrate selection guidelines for successful metathesis reaction on proteins were established. Rapid Se-relayed CM was further investigated through biomimetic chemical access to Se-allyl selenocysteine (Seac) via dehydroalanine. On-protein reaction kinetics revealed rate constants of Seac-mediated CM to be comparable or superior to off-protein rates of many current bioconjugations. This CM strategy was applied to histone proteins to install a mimic of acetylated lysine (K9Ac, an epigenetic marker). The resulting synthetic H3 was successfully recognized by antibody that binds natural H3-K9Ac. A Cope-type selenoxide elimination subsequently allowed the removal of such modification to regenerate dehydroalanine. Finally, preliminary research efforts towards metabolic incorporation of allyl sulfide-containing amino acid into proteins, and CM on cell surfaces were discussed.

Chemical modification of proteins is critical for many areas of biochemistry and medicine. Several methods for site-selective protein modification are reported in this Thesis that are useful in accessing both natural and artificial protein architectures. Multiple, complementary methods for the conversion of cysteine to dehydroalanine are described. Dehydroalanine is used as a general precursor to several post-translational modifications and glycosylation, polyprenylation, phosphorylation, and lysine methylation and acetylation are all accessible. These modifications and their mimics were explored on multiple proteins, including histone proteins. Unnatural modifications were also explored. The first examples of olefin metathesis and Suzuki-Miyaura cross-coupling on protein substrates are reported. Allyl sulfides were discovered to be remarkably reactive substrates in olefin metathesis, allowing use of this reaction in water and on proteins. For Suzuki-Miyaura cross-coupling, a new catalyst is described that is fully compatible with proteins. Both olefin metathesis and cross-coupling allow the formation of carbon-carbon bonds on proteins. The prospects of these transformations in chemical biology are discussed. Finally, a novel strategy is reported for the installation of natural, unnatural, and post-translationally modified amino acid residues on proteins. This technology relies on addition of carbon radicals to dehydroalanine. This method of "chemical mutagenesis" is anticipated to complement standard genetic manipulation of protein structure.

The ability to construct site-specific protein bioconjugates has a range of applications in basic and applied science. Many biorthogonal chemistries have been developed to modify proteins at well-defined sites, but these often require the introduction of artificial functionality into a protein sequence, a laborious process that comes at the cost of protein expression. An alternative approach targets native functionality present in a protein sequence, such as the N- or C-terminus, for which most proteins have one. Herein I developed a suite of enzymatic methods for the modification of protein termini all using the enzyme tyrosinase. In an initial demonstration I showed that tyrosinase can oxidize small molecule phenols, generating a reactive o-quinone intermediate, which covalently modifies protein N-termini. This strategy has a number of key advantages, including the use of catalytic amounts of commercially available enzyme and easily accessible reagents. This work laid the foundation for a new research area in the lab focused on the development of tyrosinase-mediated bioconjugations. I then worked in a collaborative team effort to extend this approach to protein C-termini, where tyrosinase could be used to generate site-specific o-quinones at introduced tyrosine-tags. These could couple to free thiols on a different protein, allowing for the rapid and facile synthesis of protein-protein conjugates. I ultimately applied this method towards the modification of the cell surface, where I used tyrosinase to synthesize nanobody-natural killer conjugates that could elicit targeted cell killing. Together, these methods have enabled the synthesis of complex bioconjugates that were previously difficult to access.

Proteins and enzymes perform important functions of life. Further expanding and regulating their function, proteins are dynamically decorated with posttranslational modifications (PTMs). Because of the large number of modifications and pathways involved, it is challenging to isolate a protein with a specific modification from natural sources. Besides native PTMs, artificial modifications can augment the properties and efficacy of proteins for their use in pharmaceutical applications and research. To determine the characteristics of natively existing modifications and to create modified protein products, efficient strategies for their preparation are required. Protein modification in a controlled manner is a difficult task because of the richness of functional groups and chemical environments that the protein structure harbors. While chemical modification of proteins generally offers chemoselectivity for a specific functional group, enzymatic approaches frequently proceed with high site specificity and operate under mild reaction conditions. Such chemoenzymatic methods rely on pathways that operate on proteins, and substrate analogs that can be utilized by these enzymes. Many existing protocols, however, are restricted to modification at the protein termini, rely on non-peptidic metabolites, or require large recognition domains. This dissertation describes the development and application of lysine acylation using conjugating enzymes (LACE), a chemoenzymatic strategy to site-specifically modify folded proteins at internal lysine residues. LACE relies on a minimal genetically encoded tag (four residues) recognized by the E2 small ubiquitin-like modifier-conjugating enzyme Ubc9 (Ube2I), and peptide or protein thioesters. Together, this approach obviates the need for E1 and E3 enzymes, enabling isopeptide formation with just Ubc9 in a programmable manner. Our studies demonstrate that LACE accepts a series of functionalized thioester probes, ranging from synthetic molecules to entire protein domains. The short tag size allowed the modification of diverse substrates, ranging from monomeric to multimeric proteins, and combination of the method with existing strategies for one-pot dual modifications. Importantly, LACE enabled the site-specific installation of native ubiquitin and ISG15 in a programmable manner from entirely recombinant sources in one step. In a complementary approach, this dissertation describes the development of a workflow towards the identification of sequence-specific redox reactivity of cysteine residues. To this end, a solid-supported combinatorial peptide library was prepared, and a high-throughput screen combined with semi-automated peptide sequencing was established for the identification and characterization of library hits.

In the study of this Ph.D. dissertation, two research topics related to zwitterionic materials have been investigated. Even though the applications have different objectives, the unique properties that existed in zwitterionic materials have, including charge property and antifouling property, have been used in both of research. The first topic is the development of zwitterionic-peptides gene delivery system. The gene delivery system, with high efficacy, low toxicity, long blood circulation time and targeting the specific cancer cell, is investigated. The second topic is the functionalization of protein therapeutics with zwitterionic polymers. The protein therapeutics with better solubility, stability, and activity is developed.The non-viral gene delivery system is under research due to their low toxicity, low immunogenic and large DNA loading size in gene therapy. Peptides gene delivery system is reported with the cationic charge and buffering effect which overcomes the barrier and delivery DNA into the nucleus. In our group, the economic dextran-peptide hybrid gene delivery system was developed with high transfection efficiency and low toxicity. The first topic of my research was expanded as a continuous work under the same research interest. The effect of the design of peptides length, zwitterionic group and targeting group was studied for the optimization objects on achieving low toxicity, transfection efficiency and blood circulation time, which was summarized into three research projects under this topic. The system was adjusted by the peptides length for toxicity and economic purpose. The system was functionalized with the zwitterionic group for improved stability, enhanced endosomal escape and longer blood circulation time. The system was also conjugating with targeting ligand for targeting gene delivery. It was found that the shorter length of peptides will not provide enough charge to form stable micelle with report DNA. The zwitterionic functionalized material have lower toxicity and lower transfection efficiency compared to non-zwitterionic functionalized dextran-peptides. Moreover, the transfection is considered as high compared to PEI. The micelle stability during pH titration was improved. The folate functionalized zwitterionic gene delivery system did not show any promoted transfection efficiency in this time.Currently, the protein therapeutics are under investigation with their unique treatment effect. There are still many challenges from the development to the application for protein therapeutics. One of the challenges is that the protein desaturates during production and application which will increase the product price and reduce the shelf life and decrease the bioactivity. To solve this problem, the formulation and functionalization are studied. In our group, the functionalization of the protein with zwitterionic chains for better solubility, stability and uninfluenced bioactivity is under study. The second research topic of my work is to functionalize the protein with the zwitterionic group by precise designed peptides chain. With the DNA recombinant technique, the functionalized protein is produced in one pot expression. The protein stability during varies conditions is under investigation. It includes thermostability, pH stability, salt, and urea resistance.

In this research, peptides and protein containing serine on the N-terminus underwent site-selective modification following organocatalyzed bioconjugation that offered an additional functional group. It was shown that transforming the N-terminus serine to an aldehyde allowed site-specific bioconjugation to occur by utilizing the well-known Henry reaction. This method also grants a safer pathway for bioconjugation utilizing “green-chemistry” and biocompatible conditions. Amino acids and amino acid derived organocatalysts were utilized in the Henry reaction resulting in yields of up to 86 % conversion. Promising preliminary results were achieved in this research using peptides and myoglobin as the bioconjugation targets. Further investigation to be performed includes the analysis of the final product, as well as, applying this methodology to a number of other proteins.

Supramolecular chemistry is a diverse field that encompasses host-guest interactions, mechanical bond formation, metal coordination, and other noncovalent chemistries. The design strategies of supramolecular chemistry have been implemented in a diverse set of applications, but has seen limited use in the field of chemical biology. By combining supramolecular chemistry with chemical biology, new discoveries and advances could be made that would not be possible without a supramolecular approach. This work therefore sought to combine supramolecular strategies and chemical biology for applications in protein modification and nanomedicine. By using cucurbituril host-guest chemistry, a supramolecular-promoted copper-free click chemistry reaction was developed for protein modification. Cucurbituril host-guest chemistry was additionally explored as a selective protecting group for either the N terminus in the case of cucurbit[7]uril (CB7), or as a lysine protecting group in the case of cucurbit[6]uril (CB6). Supramolecular interactions were further utilized in the design of new turn-on diagnostic probes for 129Xe NMR/MRI detection of cancer, via the rotaxane-mediated suppression, and subsequent activation of Xe-CB6 host-guest interactions. Lastly, self-assembled protein-based nanomaterials were explored for applications in chemotherapeutic drug delivery to glioblastomas, with a special focus on how the morphology of protein-based nanomaterials can affect their biodistributions and drug delivery efficacies. Throughout this work, supramolecular interactions are interwoven with applications and techniques in chemical biology to achieve new advances that would not be possible otherwise.

The imbalance between the production of reactive oxygen/nitrogen species (ROS/RNS) and their consumption by antioxidants leads to excess free radicals and peroxides, which can attack various components of cell or the entire organism. Polyunsaturated fatty acids (PUFAs) are especially vulnerable to free radical mediated oxidation. The resulting oxidative degradation of PUFAs gives reactive bifunctional aldehyde such as 4-hydroxy-2-nonenal (4-HNE) as well as polyoxygenated products with the full carbon chain of the fatty acid such as epoxyketooctadecenoic acids (EKODEs). Many of lipid peroxidation (LPO) products are highly electrophilic and can subsequently react with biological nucleophiles including protein side chains and DNA bases, consequently leading to enzyme inactivation and gene mutation. A number of metabolic fates of LPO products have been discovered and are critical for counteracting the oxidative damage. In addition, parallel catabolic pathways of 4-HNE were identified while the mechanism was not fully elucidated yet.The first goal of my work was to generate a better understanding of the transformations involved in the catabolic pathways of 4-HNE. An LC-MS/MS based assay was developed to probe the presence of 4-hydroxy-acyl-CoA kinase activity, which generates 4-phosphoacyl-CoA (4-P-acyl-CoA) as a key intermediate during the isomerization of 4-hydroxy-acyl-CoA to 3-hydroxy-acyl-CoA. This assay was used to guide the purification of the kinase from liver tissues. A number of purification techniques including ammonium sulfate precipitation, fast protein liquid chromatography (FPLC) and SDS-PAGE were utilized. Protein sequencing of the resulting protein fractions gave five kinase candidates. Sedoheptulose kinase, the most relevant candidate with oxidative stress was tested for 4-hydroxy-acyl-CoA kinase activity.In addition to the catabolic fates of LPO products, we also investigated the chemical nature and biological consequences of the protein modification by these molecules. A series of EKODE model compounds were synthesized and applied in the reaction with model nucleophiles to identify the adducts. Michael adducts of cysteine and histidine were discovered as well as an unexpected ring-opening products in the reaction of EKODE II model compound with cysteine analogues. The mechanism was found to be an intra-molecular rearrangement of the Michael adduct, which was revealed by the reactions of EKODE II derivatives with butanethiol and confirmed by our analytical tools including LC-MS and UV spectrometer. The reactions of EKODEs with HSA were also explored using LC-MS based assays, where Cys-34, His-67, His-146, and His-440 were found to be the most reactive residues. Those findings led us to further investigate the relevance of EKODE-Cys adducts in aging and disease state. Polyclonal antibodies were produced and characterized, and subsequently immunoassays were developed to detect and quantify EKODE-Cys adducts in biological samples. An H2O2 concentration dependent increase of EKODE-Cys adducts was identified in neuroblastoma cells. The accumulation was also identified in human brain tissues during aging process as well as ischemic rat heart tissues.

The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are ligand-gated glutamatergic ion channels that mediate most excitatory neurotransmission in the brain. Alterations in AMPAR synaptic accumulation mediate synaptic plasticity, including long-term potentiation, long-term depression and homeostatic synaptic plasticity. AMPAR abundance in neurons is determined by balanced processes of protein translation and degradation. Changes in AMPAR function and trafficking have direct impacts on synaptic transmission and cognitive functions. However, the molecular mechanisms regulating AMPAR expression and dynamics in neurons remain largely unknown. In this thesis, two molecular mechanisms that regulate AMPAR translation and protein stability through two different signaling pathways, 5' adenosine monophosphate-activated protein kinase (AMPK) and sirtuin 2 (SIRT2), are described. It is shown that SIRT2, a NAD+-dependent protein deacetylase, directly controls AMPAR stability by regulating AMPAR acetylation. For the first time, we discovered that AMPARs are subject to lysine acetylation, a novel form of post-translational modification for glutamate receptors. Under basal conditions, AMPARs are highly acetylated at their intracellular C termini, which protects against ubiquitination to antagonize AMPAR endocytosis and degradation, leading to prolonged receptor half-life. SIRT2 is also identified as the enzyme responsible for AMPAR deacetylation. Knockdown of SIRT2 led to elevated AMPAR acetylation and reduced ubiquitination, and consequently, increased AMPAR levels and synaptic transmission. SIRT2 knockout mice displayed weakened synaptic plasticity and impaired learning and memory. Resveratrol is a phytoalexin that has been shown to increase AMPAR expression and synaptic accumulation in neurons. The resveratrol effect on AMPAR expression is independent of sirtuin 1, the conventional target of resveratrol, but rather is mediated by AMPK and its downstream phosphoinositide 3-kinase (PI3K)/Akt pathway. Application of the AMPK activator, 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR), to neurons mimics the effects of resveratrol on both signaling and AMPAR expression. The resveratrol-induced increase in AMPAR expression results from elevated protein synthesis through the AMPK-PI3K pathway activation. These studies describe novel regulatory mechanisms responsible for the control of AMPAR protein amount and subcellular distribution in neurons, providing insights into our understanding of synaptic plasticity, brain function and neurological disorders.

With highly evolved structures and function, proteins have an extraordinarily diverse range of capabilities. In order to take advantage of their unparalleled specificity in the field of chemical biology, bioconjugation methods can be used to produce synthetically modified proteins. As applications for protein-based materials are becoming ever-increasingly complex, there is a constant need for methodologies that can covalently modify protein substrates. More specifically, there is a requirement for reliable and chemoselective reactions that result in well-defined bioconjugates that are modified in a single location. We have developed a protein transamination strategy that uses N-methylpyridinium-4-carboxaldehyde benzenesulfonate salt (Rapoport's salt) to oxidize the N-terminal amine to a ketone or aldehyde functionality. We have identified high-yielding conditions for this reaction, such as N-terminal sequence and pH, and shown its applicability in the modification of several protein systems. In addition, we have used N-terminal protein modification methodologies in the synthesis of protein-DNA conjugates, towards the goal of developing a generalizable DNA-directed protein immobilization platform. Overall, the work presented herein expands the toolkit of methodologies available for building protein-based materials.

As one of the core building blocks of life, proteins play a central role in our efforts to understand, control, and engineer the natural world. Chemical modification of proteins facilitates these efforts by allowing manipulation of protein properties and creation of new materials. A variety of methods for the modification of proteins exist, and collectively these methods have enabled the synthesis of drugs, the study of biological systems, and the creation of new materials. Unfortunately the exquisite control provided by many of these methods does not extend to oligomeric proteins, which are found ubiquitously in nature. These proteins often have symmetries and structures that make them particularly appealing for use in materials science and pharmaceutical applications, yet their multivalency makes their controlled modification difficult. This thesis describes the development of a method for protein modification in which control over the number of modification sites is exerted through noncovalent interactions. This method is able to control the level of modification of both monomeric and small oligomeric proteins. Further development revealed that this method also discriminates between different modification sites, such that it could be used to site-specifically modify proteins or monitor bioconjugation reactions. Finally, the principles used in the construction of these bioconjugation strategies were applied to the recovery of enzymes from industrial reactions.

Protein microarrays are becoming increasingly popular as platforms for applications ranging from bioanalyte detection (biosensors and diagnostics) to enzyme activity profiling and even the biosynthesis of natural products and their analogs in vitro. Selective and efficient protein immobilization methods are essential for such applications, and herein we describe the first example of such an immobilization that exploits the self-catalysis and binding properties of the commonly-used glutathione transferase (GST) purification tag.A library of 25 analogs of the known GST substrate 1-chloro-2,4-dinitrobenzene (CDNB) was synthesized, and 20 of these compounds bearing bioorthogonal linker substituents were screened for glutathione (GSH) conjugation activity with the GST from the helminth worm Schistosoma japonicum. The Michaelis-Menten kinetics for several enzymatic reactions were studied, and it was observed that improved aqueous solubility and strongly electron-withdrawing ring substituents accelerate the conjugation reaction, providing hints for the design of a second generation of GST substrates. The regiochemistry of the SNAr reaction was also studied; it was found that conjugation of GSH is favored at the 4-position of the aryl ring, a result that was rationalized by noting the increased electrophilicity of that position due to resonance effects.An alkyne-bearing CDNB analog was chosen to serve as a linker for the immobilization of SjGST on an azide-functionalized glass microscope slide. A “click” reaction was performed to modify the surface with the CDNB analog, and subsequent treatment with SjGST and GSH led to an array of immobilized protein that was characterized via contact-angle microscopy, fluorescence immunolabeling, and atomic force microscopy. Arrays of SjGST fabricated in this way showed an 18-fold higher fluorescence detection signal compared to non-oriented proteins, and a 6-fold increase in signal versus conventionally-immobilized GSH. As a first step toward the creation of a biosynthetic microarray, a GST-tagged isonitrile synthase, IsnA, was cloned, expressed, and immobilized using this technique. Future experiments will be directed towards the creation of a functional biosynthetic chip that can produce several cyanobacterial natural products of interest to our lab.

Cells are highly complex bio-nanoreactors comprised of a complex medium where multiple multistep reactions occur simultaneously across the cell. To prevent interference and degradation of these catalytic pathways, cells compartmentalise. Compartmentalisation achieves control of synthetic pathways at specific sites in the cell (organelles). In nature, compartmentalisation is demonstrated in plants by the organelle, chloroplast, which is responsible for photosynthesis. Photosynthesis is one of the most important biological reactions, responsible for sustaining life. In this thesis, the development of light harvesting bioconjugates as artificial chloroplasts is reported. Donor-acceptor light activated bioconjugates based on ruthenium(II)-bisterpyridine complexes (Ru(II)) is covalently linked to a redox metalloprotein cytochrome c (cyt c), which was prepared using maleimide-cysteine chemistry. These bioconjugates are capable of undergoing electron transfer upon photoactivation using ~480 nm light. In order to compartmentalise the Ru(II)-cyt c bioconjugates to construct artificial organelles, polymersomes (polymer vesicles) were formed based on a novel method developed in this project based on the polyelectrolyte, polystyrene-b-poly(acrylic acid) (PS-b-PAA). Polymersomes could be induced in the presence of positively charged biomolecules including cyt c and green fluorescent protein. In the presence of simple salts or negatively charged biomolecules such as calmodulin, micelles form. A primitive synthetic chloroplast capable of converting light energy into chemical energy by generating a proton gradient upon photo-excitation was developed. An artificial photosynthetic-respiratory hybrid was achieved by replacing chlorophyll (photosensitiser) with Ru(II)-cyt c. This light-harvesting bioconjugate as well as its natural electron acceptor, cytochrome c oxidase, is encapsulated in the synthetic polymersome membrane of the diblock copolymer PS-b-PAA, reminiscent of a photosynthetic membrane construct. The successful self-assembly of this complex light-harvesting enzyme cascade within membranes of polymeric vesicles were characterised using confocal laser-scanning microscopy (CLSM), TEM and cryo-TEM. Upon light-activation, cytochrome c oxidase (reduced) is capable of pumping protons across the membrane to generate an electrochemical gradient which is detected using a fluorescent pH dye encapsulated within the aqueous core of the polymersome.

As applications of protein-based materials become increasingly complex, there is a growing need for an expanded set of bioconjugation reactions that can attach synthetic components to defined locations on protein substrates. Although the introduction of artificial amino acids is a useful method for achieving site-selective protein modification, there are advantages to targeting native amino acids that can be introduced without specialized expression systems. This work describes the discovery, development, and application of new oxidation-based bioconjugation methods that target the native electron-rich aromatic amino acids, tyrosine and tryptophan. These methods include a peptide modification strategy that uses synthetic reagents to site-selectively introduce and modify oxidized tryptophan residues, as well as series of protein modification strategies that use cerium(IV) ammonium nitrate (CAN) to oxidatively couple tyrosine and tryptophan residues to electron rich aniline derivatives. One of these reactions, a CAN-mediated oxidative coupling between anisidine derivatives and tyrosine residues, has been applied towards the synthesis of a novel protein-based material and the site-selectivity of this reaction has been explored using combinatorial peptides libraries. Overall, these unprecedented modification strategies target these under-utilized amino acids with excellent chemoselectivity, greatly augmenting the important and growing list of useful bioconjugation techniques.

Abstract: Triticale (×Triticosecale Wittmackk) is being actively explored as a feedstock for bioethanol production in Canada. As the main co-product of bioethanol processing, triticale distillers grain contains 20-43% protein (dry basis), and mainly used as animal feed. The purpose of this study was to find new uses of triticale protein as adhesive. Proteins from triticale distillers wet grains (DWG) and distillers dried grains with solubles (DDGS) were extracted by five methods: pH shifting method, 60% ethanol, alkaline ethanol, glacial acetic acid, and enzyme-aided extraction. Extraction with alkaline ethanol and glacial acetic acid gave higher protein contents (~61–65%) than those obtained from other extraction methods (~23–24%); however, enzyme-aided extraction using Protex 6L yielded 75–82% protein at a content of 43–57%. To prepare adhesives, proteins were modified by NaOH, urea and glutaraldehyde; effects of modifications on protein structure were analyzed by FTIR, and their adhesion properties were measured by automated bonding evaluation system (ABES II). The highest (p

Metal catalyzed oxidation of protein involves a reaction between hydrogen peroxide and protein-bound metal ions. Production of the highly-reactive hydroxyl radical leads to oxidative damage in the immediate vicinity of the bound-metal ion. To examine the relationship between protein structure and oxidative damage a series of site-directed mutants of hen egg white lysozyme (HEWL) were created. This project focuses on generation of the site directed mutant where residue His15 will be altered to a serinyl residue (H15S), also work on the preliminary purification and characterization of the putative mutant would be touched on.

Diabetes is a strong risk factor for several chronic diseases and for premature mortality. Among other complications, the diabetic patient develops cardiomyopathy due to several metabolic, biochemical and ultrastructural changes involving the cardiac tissue. This myocardial dysfunction not only is considered one of the most serious and life threatening factors, but also worsens the prognosis of the diabetic patient in the event of other cardiac insults such as ischemia and myocardial infarction. Numerous studies have shown that diabetic cardiomyopathy involves several functional alterations within the cardiac myocyte. In this respect, mitochondria have received a great deal of attention as they represent the main cellular source of energy. The decline in the respiratory function of mitochondria from diabetic tissues has been mainly attributed to defects in several enzymes of the mitochondrial electron transport chain. However, the presence and the extent of these defects are controversial due to several factors, including the different diabetic animal models used, the parameters considered in classifying animals as diabetic or not, and tissues utilized to isolate mitochondria. In addition, the extent of the reported defects in electron transport in diabetic mitochondria is not sufficient to explain per se the differences in respiration observed between diabetic and non diabetic mitochondria. A unifying factor between diabetes and mitochondrial dysfunction is the existence of cellular oxidative damage. Several studies have shown that diabetes is in fact a state of oxidative stress. The mechanisms by which oxidative stress occurs and the generation of reactive oxygen species increases under diabetic conditions are not fully elucidated. Because mitochondria are one of the main cellular sources of ROS, it is believed that mitochondrial dysfunction in diabetic tissues largely contributes to the increased oxidative damage present in diabetic tissues. One of the main consequences of oxidative damage is the production of lipid peroxidation products, including aldehydes, from phospholipids of the biological membranes as a result of reactions involving free radicals. The generated lipid peroxidation products are highly toxic to the cells and can cause structural and functional modifications to cellular proteins. Based on these considerations we hypothesize that the existent mitochondrial defects in diabetic hearts, specifically in the electron transport chain, is a major source of ROS. In turn, ROS can react with and modify unsaturated lipids from the mitochondrial membrane, thus generating the lipid peroxidation product HNE, which can further react with key mitochondrial proteins or enzymes. This sequence of events can represent an important mechanism by which mitochondrial respiratory function is altered in diabetic hearts and contribute to the development of diabetic cardiomyopathy. To test this hypothesis, we used a streptozotocin diabetic rat model to address the following specific aims: 1) To examine the mitochondrial oxygen consumption as an index of mitochondrial respiratory function over a time course of 12 weeks of diabetes; 2) To determine the development of oxidative stress in diabetic mitochondria by detecting HNE modified mitochondrial proteins; 3) To identify specific HNE-modified mitochondrial proteins, and determine the functional consequence of their modification; and 4) To ascertain whether this process can be prevented or reversed by insulin administration. The elucidation of these main points can help to understand the nature of the defect(s) in mitochondria function in diabetic hearts, and the consequence of oxidative damage on key mitochondrial proteins.