In the first part of the thesis, a series of six modified 2'-deoxyadenosine triphosphates, bearing small functional groups (chloro, amino, methyl, vinyl, ethynyl and phenyl) at position 2 of adenine, was designed and synthesised. They were then tested as substrates for DNA polymerases in enzymatic synthesis of minor-groove modified DNA. The 2-phenyl modified dATP was the only triphosphate unable to be incorporated, meaning that the phenyl group is already too big for minor-groove incorporations. All of the other tested nucleotides were good substrates for tested DNA polymerases [KOD XL, Vent(exo-) and Bst LF] affording minor- groove modified DNA bearing one or four modifications. The vinyl- and ethynyl-modified DNAs were then used for post-synthetic modification of DNA minor groove with fluorescent labels utilising click reactions. Ethynyl group reacted in copper-catalysed alkyne-azide cycloaddition (CuAAC), whereas the vinyl group participated in thiol-ene reaction. This procedure allowed for the attachment of big functional groups otherwise unable to be installed into the DNA minor groove using direct enzymatic incorporation. The second part of the thesis was devoted to the study of 2-alkylamino-2'- deoxyadenosine triphosphates and their use in enzymatic synthesis of base-modified ONs and DNA....

Peptide nucleic acids (PNAs) were used as encoding tags to enable the analysis of peptide libraries by PNA/DNA hybridisation onto DNA microarrays. This allowed entire peptide libraries to be organised and sorted in a two dimensional format whereby all library members could be interrogated and analysed on a one-byone basis. In this thesis, PNA-encoded peptide libraries, generated by split-and-mix library synthesis, were screened for a variety of functions. Peptide sequences identified from the screening of a PNA-encoded library were analysed in detail as the first specific substrates for chymopapain. A new PNAencoded library consisting of D-amino acids was synthesised and screened with a number of proteases in attempts to identify novel/unusual substrates. PNA-encoded libraries were also used in the screening of peptide libraries for other activities. Thus substrates for catalyst-free Hüisgen cycloaddition were identified following the reaction between an alkyne modified peptide library and azidofluorescein, while cell-penetrating peptides were identified by hybridization of an internalized encoded library onto a DNA microarray.

The objective of this project was to prepare alkyne-functionalized polymer latexes using surfactant-free emulsion polymerization, and then functionalize these polymer latexes with three quaternary ammonium azides via Cu(I)-catalyzed azide/alkyne cycloaddition (CuAAC) in order to produce antimicrobial polymer latexes. Three quaternary ammonium azides with different linear alkyl chain lengths (C4, C8 and C12) were successfully synthesised in high yield ( > 70%) using established procedures, and their purity determined by elemental analysis, Fourier transform infrared (FTIR) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy. Alkyne-functionalized polymer latexes were prepared via surfactant-free emulsion polymerization using 2,2'-azobis(2-methylpropionamidine)dihydrochloride (AIBA) as initiator, [2-(methaccryloyloxy)ethyl]trimethylammonium chloride (MATMAC) as cationic comonomer, propargyl methacrylate (PMA) to provide the alkyne groups, and for some latexes, ethylene glycol dimethacrylate (EGDMA) as crosslinking comonomer. The effects of temperature and the concentrations of AIBA, MATMAC, PMA and EGDMA on monomer conversion, the rate of polymerization, particle diameter and colloidal stability have been investigated. The studies showed that the very high rates of polymerization were due to high values of the number of radicals per particle (in the range 3-2300). The observations also determined that the reaction conditions required to produce small particles (diameter of 150-350 nm) of narrow size distribution were: 75 oC reaction temperature, AIBA at 0.2 wt% to the total mass of monomer, MATMAC level of smaller or equal to 12 mol% to total monomer (including MATMAC), and EGDMA level of < 2.0 mol% to total monomer (excluding EGDMA). Three series of alkyne-functionalized polymer latexes have been synthesised using these conditions: non-crosslinked (NCL), crosslinked (CL) and core-shell (CS). All the latex particles were functionalized with the three quaternary ammonium azides by CuAAC. Zeta potential analysis, FTIR and Raman spectroscopy analysis confirmed the success of the click reactions. The quantitative analysis of FTIR and Raman spectra showed similar values of conversion of click reaction for both NCL and CL particles, indicating NCL and CL particles have similar swellability. The data also showed that significantly higher click reaction conversions were achieved for CS particles (around 60%) than for NCL/CL particles (less than 40%), which indicates that the click reaction only occurred at the surface of particles and that a higher proportion of alkyne groups are located on the surface of CS particles than on NCL/CL particles. The antimicrobial properties of all QAAs, MATMAC, NCL, CL and CS polymer latexes against E. coli bacteria (ATCC 25922) have been investigated using a modified liquid microdilution method in M9 medium, which was shown not to affect latex colloidal stability. It was found that all the polymer latexes showed much higher antimicrobial activities (MIC 6.5-75 µg ml-1) than many antimicrobial polymers reported recently in the literature (MIC 100-2000 µg ml-1); (Ganewatta, M.S. and C.B. Tang, Controlling macromolecular structures towards effective antimicrobial polymers. Polymer, 2015. 63: p. A1-A29). Polymer latexes with clicked-on QAAs showed significantly higher antimicrobial activities than the original latexes. The magnification of the increase in antimicrobial properties of CS particles after click reaction (~3.5 times) was greater than for NCL/CL particles (~2.5 times), showing that a larger amount of QAAs have been clicked onto the surface of CS particles than NCL/CL particles and that the clicked-on QAAs enhance the antimicrobial activity significantly.

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Protein adsorption on solid surfaces is a common yet complicated phenomenon that is not fully understood. Self-assembled monolayers have been utilized in many studies, as well-defined model systems for studying protein-surface interactions in the atomic level. Various strategies, including the use of single component SAMs[1, 2], combinations of long and short alkanethiolates with methyl- and hydroxyl- terminal groups[3, 4], and using mixtures of alkanethiolates with similar chain length and varying terminal functional group [5] have been used to effectively control the surface wettability and determine the effect of surface composition and wettability on protein adsorption. In this dissertation we report key new findings on the effect of surface density of functional groups on protein adsorption phenomenon. In The first phase of this research, we developed a novel approach for preparation of low-density self-assembled monolayers(LD-SAMs) on gold surfaces, based on radical-initiated thiol-yne click chemistry. This approach provides exceptional adsorbate stability and conformational freedom of interfacial functional groups, and is readily adapted for low-density monolayers of varied functionality. The resulting monolayers have two distinct phases: a highly crystalline head phase adjacent to the gold substrate, and a reduced density tail phase, which is in contact with the environment. First, we investigated the feasibility of the proposed chemistry in solution-phase. In this approach, we synthesized “Y” shaped carboxylate-terminated thiol adsorbates via radical-initiated thiol-yne reaction. The LD-SAMs were then prepared through immersion of gold substrates into the solution of synthesized adsorbate molecules in hexane. The chemical structuring and electrochemical properties of resultant LD-SAMs were analyzed and compared with those of analogous traditional well-packed monolayers, using techniques such as Fourier transform infrared spectroscopy, ellipsometry, electrochemical impedance spectroscopy, reductive desorption, and contact angle goniometry. Characterization results indicated that resulting LD-SAMs have a lower average crystallinity, and higher electrochemical stability compared to well-packed monolayers. In addition, using a three-electrode system, we were able to show a reversible change in LD-SAM surface wettability, in response to an applied voltage. This remodeling capacity confirms the low density of the surface region of LD-SAM coatings. The second area of work was focused on using the developed chemistry in solid-phase. The solid-phase approach minimized the required synthesis steps in solution-phase method, and used the photo-initiated thiol-yne click-reaction for grafting of acid-terminated alkynes to thiol-terminated monolayers on a gold substrate to create similar LD-SAMs as what were prepared through solution-phase process. We characterized the resulting monolayers and compared them to analogous well-packed SAMs and the also low-density monolayers prepared through the solution phase approach. The results confirmed the proposed two-phase structure, with a well-packed phase head phase and a loosely-packed tail phase. In addition, the electrochemical studies, indicated that the resultant monolayers were less stable than the monolayers prepared via solution-phase, but they are yet significantly more stable than typical well-packed monolayers. The less stability of these monolayers were attributed to the partial desorption of adsorbates from the gold substrate due to UV irradiation during the grafting process. Building on the established chemistry, we studied the effect of lateral packing density of functional groups in a monolayer on the adsorption of Bovine serum albumin protein. we used surface plasmon resonance spectroscopy (SPR) and spectroscopic ellipsometry, to evaluate BSA adsorption on carboxylate‑, hydroxyl-, or alkyl- terminated LD-SAMs. It was found that for the LD-SAMs, the magnitude of protein adsorption is consistently higher than that of a pure component, well-packed SAM for all functionalities studied. In addition, it was seen that the magnitude of BSA adsorption the LD-SAMs, was consistently higher than that of a pure component, well-packed SAM for all functionalities studied. The difference of protein adsorption on LD-SAMs and SAMs can not be associated to difference in lateral packing density, unless we eliminate the impact of other contributing factors in protein adsorption such as surface energy. In order to better understand the impact of packing density on protein-surface interactions, we prepared the mixed SAMs of (carboxylate/alkyl) and (hydroxyl/alkyl) with matching surface energy as the carboxylate and hydroxyl terminated LD-SAMs. It was found that the energy-matched mixed SAMs of carboxylate and hydroxyl functionality adsorbed more protein than the LD-SAMs. However, an opposite trend was seen for the alkyl surfaces, where surface energies are comparable for LD-SAMs and pure component SAMs, indicating that BSA proteins have higher affinity for methyl- terminated LD-SAMs than well-packed SAMs.

Poly(N-isopropylacrylamide-co-acrylic acid) (pNIPAm-AAc) copolymeric multi-responsive microgels demonstrate responsivity to temperature, pH, and ionic strength. A temperature-programmed polymerization protocol is proposed for the synthesis of large pNIPAm-AAc microgel particles with a hydrodynamic diameter of 2~5 μm. Immediately after preparation of concentrated pNIPAm-AAc dispersions in closed system, the average hydrodynamic diameter is smaller than the unperturbed diameter probably due to osmotic de-swelling effect. During the aging process, pNIPAm-AAc microgel particles start to swell while their dynamics slow down. The snapshots of phase behavior of pNIPAm-AAc microgel dispersions at different pH values are illustrated. The formation of crystalline phase should follow a nonergodic path in which microgel particles swell to the extent that they build up weak attractive interaction to allow them to associate while maintaining the opportunity of rearrangement to minimize local Gibbs free energy. The age-dependent thermostability of pNIPAm-AAc microgel dispersions suggests strong attractive interactions evolve between particles during aging-convoluted crystallization. Finally, to introduce multiple biological "handle"s on the microgel particles for biomedical applications, the Cu(I)-catalyzed azide-terminal alkyne 1,3-dipolar cycloaddition, also called Sharpless-Meldal "click" reaction, is used to functionalize pNIPAm-AAc microgel particles.

Polyhomologation is a powerful method to prepare polyethylene-based materials with controlled molecular weight, topology and composition. This dissertation focuses on the discovery of new synthetic routes to prepare polyethylene-based macromolecular architectures by combining polyhomologation with highly orthogonal and efficient linking reactions such as Diels Alder, copper-catalyzed azide-alkyne cycloaddition (CuAAC), and Glaser. Taking advantage of functionalized polyhomologation initiators, as well as of the efficient coupling chemistry, we were able to synthesize various types of polymethylene (polyethylene)-based materials with complex architectures including linear co/terpolymers, graft terpolymers, and tadpole copolymers. In the first project, a facile synthetic route towards well-defined polymethylene-based co/terpolymers, by combining the anthracene/maleimide Diels–Alder reaction with polyhomologation, is presented. For the synthesis of diblock copolymers the following approach was applied: (a) synthesis of α-anthracene-ω-hydroxy-polymethylene by polyhomologation using tri (9 anthracene-methyl propyl ether) borane as the initiator, (b) synthesis of furan-protected-maleimide-terminated poly(ε-caprolactone) or polyethylene glycol and (c) Diels–Alder reaction between anthracene and maleimide-terminated polymers. In the case of triblock terpolymers, the α-anthracene-ω-hydroxy polymethylene was used as a macroinitiator for the ring-opening polymerization of D, L-lactide to afford an anthracene-terminated PM-b-PLA copolymer, followed by the Diels–Alder reaction with furan-protected maleimide-terminated poly (ε-caprolactone) or polyethylene glycol to give the triblock terpolymers. The synthetic methodology is general and potentially applicable to a range of polymers. The coupling reaction applied in the second project of this dissertation was copper-catalyzed “click” cycloaddition of azides and alkynes (CuAAC). Novel well-defined polyethylene-based graft terpolymers were synthesized via the “grafting onto” strategy by combining nitroxide-mediated radical polymerization (NMP), polyhomologation and copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC). Three steps were involved in this approach: (a) synthesis of alkyne-terminated polyethylene-b-poly(ε-caprolactone) (PE-b-PCL-alkyne) block copolymers (branches) by esterification of PE-b-PCL-OH with 4-pentynoic acid; the PE-b-PCL-OH was obtained by polyhomologation of dimethylsulfoxonium methylide to afford PE-OH, followed by ring opening polymerization of ε-caprolactone using PE-OH as a macroinitiator (b) synthesis of random copolymers of styrene (St) and 4-chloromethylstyrene (4-CMS) with various CMS contents, by nitroxide-mediated radical copolymerization (NMP), and conversion of chloride to azide groups by reaction with sodium azide (NaN3) (backbone) and (c) “click” linking reaction to afford the PE-based graft terpolymers. This method opens up new routes for the creation of polyethylene-based graft terpolymers by a combination of polyhomologation, NMP and CuAAC. The third project deals with the synthesis of polyethylene-based tadpole copolymer (c-PE)-b-PSt. Cyclic polymers represent a class of understudied polymer architecture mainly due to the synthetic challenges. Within this dissertation, a new method was reported for the synthesis of cyclic polymers in exceptionally high purity and yield. The main approaches to synthesize macrocycles are based on the end-to-end ring-closure (coupling) of homo difunctional linear precursors under high dilution. Our process relies on the preparation of well-defined linear α, ω-dihydroxy polyethylene and a bromide group at the middle of the chain through polyhomologation of ylide using functionalized initiator, followed by ATRP of styrene monomer. The two hydroxyl groups were transformed into alkyne groups, via esterification reaction, followed by Glaser reaction between terminal alkynes to afford the tadpole-shaped copolymers with PE ring and PSt tail. In Our PhD research, we also studied the self-assembly properties of the amphiphilic copolymers PM-b-PEG in aqueous solution by DLS, Cryo-TEM, and AFM. Furthermore, the critical micelle concentration (CMC) was estimated from the intensity of the pyrene emissions by the fluorescence technique. All the findings presented in this dissertation are emphasizing the utility of polyhomologation for the synthesis of well-defined polyethylene-based complex macromolecular architectures, almost impossible through other kind of polymerization including the catalytic polymerization of ethylene.

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