Bioconjugation chemistry generally refers to the covalent derivatisation of biomolecules. Derivatisation of cysteine’s thiol of peptides and proteins is a common method in bioconjugation chemistry as the thiolate is an excellent nucleophile in aqueous conditions. The propensity for thiols to oxidise in an aqueous environment necessitates the need for a disulfide reduction step prior to the addition of ligands derivatised with thiol alkylating linkers. Disulfide reducing agents such as tris(2-carboxyethyl)phosphine (TCEP) and tris(3-hydroxypropyl)phosphine (THPP) are disulfide reducing agents that are often marketed as being non-reactive with thiol alkylating reagents. The reaction of TCEP and THPP with thiol alkylation linkers was therefore investigated. Characterisation of reaction products and mechanistic studies revealed that TCEP and THPP both react with thiol alkylation reagents. A novel protocol was, therefore, developed utilising the Staudinger reaction to oxidise excess TCEP and THPP prior to the addition of thiol alkylating reagents. The protocol offers a simple “one-pot” method for effecting conjugate production via thiol alkylation, without the need for an intermediate purification step for the removal of excess disulfide reducing agents. 4-Vinyl pyridine (4-VP) derivatives were developed and explored as an alternative Michael acceptor class for thiol alkylation of peptides and proteins. The 4-VP derivatives exhibited high reactivity and specificity for thiol alkylation between pH = 7 and pH = 8. A selection of 4-VP linkers were subsequently functionalised with either carbohydrates or polyethylene glycol (PEG) and successfully utilised to produce peptide or protein conjugates via thiol alkylation reactions.

While the catalytically powerful [Cu,Zn] superoxide dismutase (SOD1) possesses great potential as a therapeutic, unfavorable properties in circulation limit its use in clinical medicine. The small, water soluble dimer is rapidly excreted by the kidney. Previous initiatives have been used to increase the mass of the enzyme (PEGylation, liposome encapsulation). This has resulted in highly heterogeneous mixtures of modified SOD1, which are difficult to characterize. Furthermore, these modified proteins have utilized foreign material that has shown to elicit an inflammatory response. We developed an improved strategy that creates a homogenous high molecular weight SOD1 based on combinations of the protein itself. This was accomplished through the addition of a site-specific, azide functionalized cross-linker to unmodified SOD1, followed by the conjugation of SOD dimers using CuAAC and a bis-alkyne linker to form a 64 kDa SOD tetramer. The final product, bis-SOD, presents the fully catalytic activity of the combined proteins.

While the catalytically powerful [Cu,Zn] superoxide dismutase (SOD1) possesses great potential as a therapeutic, unfavorable properties in circulation limit its use in clinical medicine. The small, water soluble dimer is rapidly excreted by the kidney. Previous initiatives have been used to increase the mass of the enzyme (PEGylation, liposome encapsulation). This has resulted in highly heterogeneous mixtures of modified SOD1, which are difficult to characterize. Furthermore, these modified proteins have utilized foreign material that has shown to elicit an inflammatory response. We developed an improved strategy that creates a homogenous high molecular weight SOD1 based on combinations of the protein itself. This was accomplished through the addition of a site-specific, azide functionalized cross-linker to unmodified SOD1, followed by the conjugation of SOD dimers using CuAAC and a bis-alkyne linker to form a 64 kDa SOD tetramer. The final product, bis-SOD, presents the fully catalytic activity of the combined proteins.

Dengue virus (DENV) is a mosquito-transmitted flavivirus that threatens approximately half of the world's population. In this dissertation, the use of bioorthogonal chemistry as a tool for researching emerging viral diseases, including DENV is explored. To this end, a bioorthogonally-modified amino acid was successfully installed within the proteome of DENV, which was used for the pull down of a known virus-protein interaction. This technology is intended to be broadly used for the determination of any virus-host interaction, through the installment of a non-perturbing modification that 1) does not hinder viral infectivity and 2) can be selectively discriminated by any complimentary probe. En route to using this technology, a new viral purification strategy was developed for DENV that reduces the overall purification time by 10 hours, and improves retention of virion infectivity. This method and a survey of other viral purification methods used with DENV is contained herein. Furthermore, a chemical scaffold that was repurposed for exploration of protein-protein crosslinking, namely for release of a reactive chemical warhead under acidic conditions, was used for the surface modification of DENV. This triazabutadiene probe was found to be activated by light. In this dissertation is reported the first time aryl diazonium ions for protein crosslinking have been generated on a protein or viral surface through UV-irradiation. The advantages and limitations of this chemistry are presented herein.

Semiconductor nanocrystals exhibit unique optical and physical properties that make them an attractive alternative to organic dyes for fluorescent bioapplications. Although significant advances have been made since their first reported use in biology a decade ago, it still remains a challenge to prepare high quality, biocompatible semiconductor nanocrystals. / In this thesis, studies are described with the aim to prepare robust, biocompatible semiconductor nanocrystals that exhibit each of the properties necessary for their implementation in biological applications. Two different approaches were investigated: ligand exchange and polymer encapsulation, and advances in each are presented. A heterobifunctional ligand suitable for bioconjugation, carboxyl terminated dihydrolipoic acid poly(ethylene glycol) (DHLA-PEG-COOH), was synthesised and characterised to prepare water-soluble, biocompatible semiconductor nanocrystals via ligand exchange. It was found that nanocrystals transferred into water using DHLA-PEG-COOH exhibit the same optical properties and colloidal stability as those prepared using DHLA-PEG. It was demonstrated that the surface charge of the nanocrystals may be controlled by altering the ratio of DHLA-PEG:DHLA-PEG- COOH ligands. In a different approach, colloidally stable, biocompatible nanocrystals were prepared via polymer encapsulation. It was found that by employing a low molecular weight polymer, biocompatible nanocrystals that exhibit a small hydrodynamic diameter could be realised. / Experimental results are presented on the conjugation of biocompatible nanocrystals to protein targets. It was found that while standard coupling chemistries yield protein-dye conjugates, these chemistries did not result in protein-nanocrystal conjugates. In order to overcome the drawbacks of standard coupling chemistries, which are susceptible to hydrolysis, a novel conjugation scheme utilising copper-free click chemistry is proposed. / Finally, the success of nanocrystals in bioapplications depends on the ability to characterise nanocrystal-protein conjugates. By means of analytical ultracentrifugation, data on the sedimentation properties of nanocrystals and nanocrystal-protein conjugates was obtained. Analysis of these data provided information on fundamental physical properties of biocompatible nanocrystals and nanocrystal-protein conjugates, in particular the core crystal size, hydrodynamic size, number of surface ligands and nanocrystal:protein stoichiometry. Such a precise, comprehensive characterisation of nanocrystals in general, and nanocrystal-protein conjugates in particular, will greatly facilitate their use in bioapplications.

The chemistry of fluorolkyl hypervalent iodine reagents has witnessed a great boast in recent years. These compounds are highly attractive as drug candidates, advanced materials and agrochemicals as described in detail in the Introduction. Despite this fact, applications of these reagents in biological studies are rather rare and under developed. The goal of this thesis is therefore the development of mild and metal-free methods in order to fill this gap. Two ways of application of fluoroalkyl hypervalent iodine reagents in labeling of biologically relevant compounds was explored. First, the applicability of previously reported parent Togni CF3 and their analogous tetrafluoroethyl reagents in radical fluoroalkylation of electron-rich substrates such as indole and pyrrole derivatives using sodium ascorbate as reductant was described. This afforded trifluoromethyl or 1,1,2,2-tetrafluoroethyl containing products in moderate to high yields. Next, same reagents were applied for labeling of several peptides and proteins bearing aromatic amino acids in their structure. This way, peptides and proteins containing electron-rich aromatics such as Trp, Phe, Tyr and His were reacted with fluoroalkyl groups with high selectivity toward Trp. In the second part of the work, a different approach of radical...

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Over the past decade, bioorthogonal chemistry has emerged as powerful tools used for tracking biomolecules within living systems. Despite the vast number of organic transformations in the literature, only select few reactions meet the stringent requirements of bioorthogonality. There is increasing demands to develop biocompatible reactions that display high specificity and exquisitely fast kinetics under physiological conditions. With the goal of increasing reaction rates as a means for reducing the concentrations of labelling reagents used for bioconjugation, we have developed metal-catalyzed and metal-free alkyne-nitrone cycloadditions as alternatives to azide-alkyne cycloadditions and demonstrate their applications for imaging cell surface proteins. The copper(I)-catalyzed alkyne-nitrone cycloaddition, also known as the Kinugasa reaction, is typically conducted with a Cu(I) catalyst in the absence of air. We have developed highly efficient micelle promoted multicomponent Kinugasa reactions in aqueous media to make the reaction faster and more efficient. Despite good product yields, the slow kinetics, limited substrate scope and competing side-reaction pathways precludes its practical applicability for biological labelling. We have designed and synthesized β-lactam alkyne probes obtained from these reactions for activity-based protein profiling of the activities of membrane proteins. Additionally, we report that alkyne tethered β-lactams serve as surface enhanced Raman spectroscopy (SERS) reporters bound to silver nanoparticles, and demonstrated that alkyne bound silver nanoparticles can be used for SERS imaging cell surface proteins. The strain-promoted alkyne-nitrone cycloaddition (SPANC) was also explored as a rapid alternative bioorthogonal reaction. We found that the reaction proceeded in high yield within aqueous media, and displayed rate enhancements that were 1-2 orders of magnitude faster than analogous reactions involving azides. The scope and kinetics of SPANC was evaluated in model reactions of various nitrones (acyclic and cyclic) with cyclooctynes, with the purpose of identifying stable nitrones that displayed intrinsically faster kinetics than azides in strain-promoted cycloadditions with cyclooctynes. Cyclic nitrones displayed good stability and exceptionally fast reactivity in these reactions. The SPANC reaction exhibited high selectivity in the presence of biological nucleophilic amino acid side chains and the presence of biological media did not adversely affect the reaction. We have utilized SPANC for highly specific labelling of proteins in vitro and for imaging ligand-receptor interactions on the surfaces of live cancer cells. The high selectivity, fast reaction rate, and aqueous compatibility of SPANC makes the reaction suitable for a variety of in vivo biological imaging applications.

Recent advancements in nanotechnology create a need for a better understanding of the underlying physical processes that lead to the different behavior of nanoscale structures in comparison to bulk materials. The influence of the surrounding environment on the physical and optical properties of nanoscale objects embedded inside them is of particular interest. This research is focused on the optical properties of semiconductor quantum dots which are zero-dimensional nanostructures. There are many investigation techniques for measuring the local parameters and structural characteristics of Quantum Dot structures. They include X-ray diffraction, Transmission Electron Microscopy, Wavelength Dispersive Spectroscopy, etc. However, none of these is suitable for the study of large areas of quantum dots matrices and substrates. The existence of spatial inhomogeneity in the quantum dots allows for a deeper and better understanding of underlying physical processes responsible in part icular for the observed changes in photoluminescence (PL) characteristics. Spectroscopic PL mapping can reveal areas of improved laser performance of InAs/InGaAs quantum dots structures. Establishing physical mechanisms responsible for two different types of spatial PL inhomogeneity in InAs/InGaAs quantum dots structures for laser applications was the first objective of this research. Most of the bio-applications of semiconductor quantum dots utilize their superior optical properties over organic fluorophores. Therefore, optimization of QD labeling performance with biomolecule attachment was another focus of this research. Semiconductor quantum dots suspended in liquids were investigated, especially the influence of surrounding molecules that may be attached or bio-conjugated to the quantum dots for specific use in biological reactions on the photoluminescence spectrum. Provision of underlying physical mechanisms of optical property instability of CdSe/ZnS quantum dots used for biologi cal applications was in the scope of this research. Bioconjugationand functionalization are the fundamental issues for bio-marker tagging application of semiconductor quantum dots. It was discovered that spatially resolved photoluminescence spectroscopy and PL photo-degradation kinetics can confirm the bioconjugation. Development of a methodology that will allow the spectroscopic confirmation of bio-conjugation of quantum dot fluorescent tags and optimization of their performance was the final goal for this research project.

Nature employs complex networks of protein-tailoring enzymes to effect the post-translational modification of proteins in vivo. By comparison, modern chemical methods rely upon either nonspecific labeling techniques or upon the genetic incorporation of bioorthogonal handles. To develop truly robust bioconjugates it is necessary to develop methods which possess the exquisite activity and specificity observed in biological catalysts. One attractive strategy to achieve this is the engineering of protein-tailoring enzymes possessing user-defined specificity and high catalytic efficiency. / Chemistry and Chemical Biology