Theses - Chemistry


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  • ItemOpen Access
    Exploration of novel linker scaffolds enabling the simultaneous rebridging of disulfide bonds for the synthesis of antibody-drug conjugates
    King, Thomas; King, Thomas [0000-0001-6992-8016]
    Antibody-drug conjugates (ADCs) are a class of targeted therapeutic, typically used for the treatment of cancers. By attaching drugs site-selectively to antibodies, off-target toxicity of the drug can be reduced, leading to significant benefits for the patient. In order to improve the homogeneity of ADCs, in particular their drug-to-antibody ratio (DAR) and the mixture of protein species present, a linker reagent known as TetraDVP, which simultaneously rebridges all interchain disulfide bonds in an IgG1 antibody, has been developed and was recently reported by the Spring Group. The work reported in this thesis explores the translation of this ‘all-in-one’ rebridging linker technique to the generation of azide-containing antibody-linker conjugates (ALCs). Synthesis of alternative linker scaffolds is reported, along with the corresponding assessment of their capabilities to rebridge the IgG1 antibody trastuzumab after interchain disulfide reduction. Application of post-conjugation click chemistry enables the synthesis of ADCs more efficiently than has been previously observed using TetraDVP linkers.
  • ItemEmbargo
    Understanding surface reactivity, local structure, and lithium-metal dendrite initiation in garnet solid electrolytes
    Vema, Sundeep; Vema, Sundeep [0000-0002-9894-5293]
    Solid electrolyte-based lithium-ion batteries can enable long lasting and safe energy storage devices with high energy densities. Out of the many solid electrolytes explored to date, doped Li₇La₃Zr₂O₁₂ (LLZO) garnets have high room temperature ionic conductivity and wide electrochemical stability making them promising candidates for commercial applications. Despite receiving much attention, the surface reactivity, regeneration, local structure of dopants in doped LLZO and the dendrite formation have not been systematically characterized and understood. This thesis elucidates the importance of the gas atmosphere and temperature for regeneration by tracking the surface of LLZO with near ambient pressure x-ray photoelectron spectroscopy and grazing incidence x-ray diffraction. From these results, a standardised protocol for handling LLZO is presented and a method to achieve a lithium metal - LLZO interface with low resistance is devised. The local structure of dopants in LLZO is clarified using ²⁷Al and ⁷¹Ga magic angle spinning magnetic resonance (MAS NMR) spectroscopy on LLZO and model compounds. The side-products on the surface of the grains and/or grain boundaries are demonstrated to give rise to multiple peaks in the MAS NMR spectra and it is shown that dopants occupy a single site in the LLZO lattice. The factors that give rise to these side-products is discussed and it is shown that the distribution of dopants in these side-products and LLZO significantly influences the ionic conductivity of LLZO. When LLZO is cycled with lithium metal in a symmetrical cell configuration (Li - LLZO - Li), continuous stripping and plating results in the formation of lithium metal filaments (dendrites) which initiate on the cathode, propagate through the solid electrolyte, and short-circuit the cell. By careful testing of symmetric cells under different cyclic current loading conditions, the critical current density (ICCD) at which the dendrites initiate, is shown to be protocol dependent. Unidirectional current experiments are shown to be a better way of estimating ICCD in solid electrolytes. Finally, the factors that determine the ICCD in solid electrolytes are identified by using an Onsager formalism to model the various non-equilibrium processes involved in lithium metal plating at the lithium metal electrode - electrolyte interface and possible solutions to increase ICCD in solid electrolytes are proposed.
  • ItemOpen Access
    Metal-organic frameworks and porous composites for photocatalytic and sensing applications
    Mtetwa, Sandile
    The sustainability and environmental issues associated with using fossil fuels threaten human health and the climate, making a move towards renewable energy critical. Metal-organic frameworks (MOFs) promise a solution. MOFs are a class of porous and crystalline materials comprised of inorganic building units, such as metals/metal oxide clusters, linked through organic ligands. As a way of producing green hydrogen, MOFs can be employed as photocatalysts which can absorb light energy, leading to the reduction of water to hydrogen with no carbon emissions. The current research used densified, monolithic MOFs (monoMOF) in contrast to conventional and widely reported powder MOFs. Such monoliths (prepared without binders or under high pressure) are mechanically robust and porous materials with the ability to host catalytically active nanoparticles (NPs). This project aimed to assess the performance of monoNH2-UiO-66 MOF as a photocatalytic agent for green hydrogen evolution from water, making it the first use of a densified, monolithic MOF as a photocatalyst for water-splitting. The MOF was sensitized with organic dyes to promote harvesting of the visible region of the solar spectrum and loaded with platinum (Pt) NP co-catalysts for proton reduction. Electron transfer and charge transport drive the photocatalytic process of evolving hydrogen via water-splitting. However, most reported MOFs utilized for this purpose are essentially insulating and their lack of conducting ability limits their performance in this application. Semiconductive and porous MOFs are an interesting class of material and, owing to their intrinsic or induced charge carrying properties, these MOFs are undoubtedly excellent candidates for photocatalytic applications. Interestingly, there is only limited evidence of this class of MOFs being used to drive the hydrogen evolution process and there is need to investigate this. Moreover, inherently conductive MOFs also offer suitability in the field of chemiresistive gas sensing. For this reason, the assessment of MOFs with charge carrying properties was conducted by collaborators who computationally screened published structures. Comprehensive high through-put screening yielded 1,692 MOFs which underwent DFT calculations on their electronic and conductive properties. Experimental validations of these DFT (low-level and high-level) calculated MOFs were attempted by synthesis of iv computationally shortlisted MOFs. The experimental validation processes assessed the conductivity, surface area and chemical stability of the short-listed MOFs and the correlation of their measured bandgaps with calculated values. Results obtained from experimental work suggested significant shortcomings in current standards of DFT prediction.
  • ItemEmbargo
    Functional Metal Oxide Coatings from Molecular Precursors for Energy Applications
    Riesgo Gonzalez, Victor; Riesgo Gonzalez, Victor [0000-0002-2433-8562]
    The ability to create and optimise new interfaces is essential to develop and optimise materials for use in sustainable energy storage and conversion technologies. In this thesis, the solution-deposition of coatings from molecular precursors is explored as a promising approach towards this end. First, a facile method for the deposition of electrocatalytically active zirconium-based films for photoelectrochemical water oxidation is developed. The films were derived from three novel alkoxy cage compounds containing Zr and a first-row transition metal (Co, Fe or Cu). The deposition of a Co-doped ZrO2 coating onto the BiVO4 photoanode lowers its onset potential by 0.12 V to 0.21 V vs. the reversible hydrogen electrode (RHE) and increases the maximum photocurrent density by ∼50% to 2.41 mA cm-2 compared to the uncoated BiVO4. In the next chapter, a new solution deposition method to coat the Li-ion battery cathode LiNi0.8Mn0.1Co0.1O2 (NMC811) with Al2O3 using aluminium isopropoxide (AIP) is developed. High-field solid-state nuclear magnetic resonance spectroscopy (SSNMR) probes the formation of γ-LiAlO2 at 600 °C and doping of aluminium into NMC811 starting at 500 – 600 °C. NMC811 coated with amorphous Al2O3 (200 – 400 °C) had a capacity retention comparable to pristine NMC811, while higher annealing temperatures led to more crystalline coatings and surface Al-doping which were found to increase the rate of degradation of NMC811 upon cycling. Finally, LiAlO2 coatings are deposited onto NMC811 using heterobimetallic alkoxides: LiAl[(OCH2Ph)4], LiAl[(OiPr)4] and LiAl[(OtBu)4]. The later showing the most promise as a coating precursor due to its high solubility in tetrahydrofuran (THF), low temperature decomposition (283 °C) and reaction with hydroxyl groups present on the surface of NMC811. This coating was tested on polycrystalline NMC811 (PC-NMC811) and Al2O3 coated single-crystal NMC811 (Al2O3/SC-NMC811). Significant improvements in capacity retention (17.2% more C/2 capacity retained after 107 cycles vs. Al2O3/SC-NMC811) were seen in the LiAlO2/Al2O3/SC-NMC811 system. Furthermore, coating PC-NMC811 that was previously degraded by soaking in water improved the capacity retention (50.1% more capacity retention at C/2 after 215 cycles vs. uncoated PC-NMC811 soaked in water and annealed at 400 °C) suggesting that the combination of a LiAlO2 coating and subsequent annealing step can recover NMC811 surfaces that have been previously degraded by soaking in water.
  • ItemEmbargo
    Photoelectrochemical and Chemoenzymatic Reforming for Sustainable Fuel Production
    Bhattacharjee, Subhajit; Bhattacharjee, Subhajit [0000-0003-0596-1073]
    The aggravating global problems of energy crisis, rising atmospheric greenhouse gas concentrations and accumulation of persistent waste have attracted the attention of scientists, policy-makers and global organisations to come up with effective and expeditious solutions to address these challenges. In this context, the development of sustainable technologies driven by renewable energy sources for the production of clean fuels and commodity chemicals from diverse waste feedstocks is an appealing approach towards creating a circular economy. Over the years, semiconductor photocatalysts based on TiO₂, CdS, carbon-nitrides (CNx) and carbon dots (CDs) have been widely used for the photocatalytic reforming (PC reforming) of pre-treated waste substrates to organic products, accompanied with clean hydrogen (H₂) generation. However, these conventional solar-driven processes suffer from major drawbacks such as low production rates, poor product selectivity, CO₂ release, challenging process and catalyst optimisation, and harsh waste pre-treatment conditions, which limit their commercial applicability. These challenges are tackled in this thesis with the introduction of new and efficient photoelectrochemical (PEC) and chemoenzymatic processes for reforming a diverse range of waste feedstocks to sustainable fuels. Solar-driven PEC reforming based on halide perovskite light-absorber is first developed as an attractive alternative to PC reforming. The PEC systems consist of a perovskite|Pt photocathode for clean H₂ production and a Cu-Pd alloy anode for reforming diverse waste streams, including pre-treated cellulosic biomass, polyethylene terephthalate (PET) plastics, and industrial by-product glycerol into industrially-relevant, value-added chemicals (gluconic acid, glycolic acid and glyceric acid) without any externally applied bias or voltage. Additionally, the single light-absorber PEC systems can also convert the airborne waste stream and greenhouse gas CO₂ to diverse products with the simultaneous reforming of PET plastics with no applied voltage. The perovskite-based photocathode enables the integration of different CO₂ reduction catalysts such as a molecular cobalt porphyrin, a Cu-In alloy and formate dehydrogenase enzyme, which produce CO, syngas and formate, respectively. The versatile PEC systems, which can be assembled in either a ‘two-compartment’ or standalone ‘artificial leaf’ configurations achieve 60‒90% oxidation product selectivity (with no over-oxidation) and >100 µmol cm‾² h‾¹ product formation rates, corresponding to 10²‒10⁴ times higher activity than conventional PC reforming systems. In addition to developing PEC platforms, this thesis also explores avenues for circumventing the harsh alkaline pre-treatment strategies (pH >13, 60‒80 ºC) adopted for photoreforming waste substrates. For this purpose, a chemoenzymatic pathway is introduced whereby PET and polycaprolactone plastics were deconstructed using functional enzymes under benign conditions (pH 6‒8, 37‒65 ºC), followed by PC reforming using Pt loaded TiO₂ (TiO₂|Pt) or Ni₂P loaded carbon-nitride (CNx|Ni₂P) photocatalysts. The chemoenzymatic reforming process demonstrates versatility in upcycling polyester films and nanoplastics for H₂ production at high yields reaching ∼10³‒10⁴ µmol gsub‾¹ and activities at >500 µmol gcat‾¹ h‾¹. The utilisation of enzyme pre-treated plastics also allowed the coupling of plastic reforming with photocatalytic CO₂-to-syngas conversion using a phosphonated cobalt bis(terpyridine) co-catalyst immobilised on TiO₂ (TiO₂|CotpyP). Finally, moving beyond solar-driven systems, a bio-electrocatalytic flow process is demonstrated for the conversion of microbe pre-treated food waste to ethylene (an important feedstock in the chemical industry) on graphitic carbon electrodes via succinic acid as the central intermediate. In conclusion, with its focus on improving efficiencies, achieving selective product formation, building versatile platforms, diversifying substrate and product scope, and reducing carbon footprint and economic strain, this thesis aims to bring sustainable waste-to-fuel technologies a step closer to commercial implementation.
  • ItemOpen Access
    Design and Synthesis of Thiamine Pyrophosphate (TPP) Analogues for Investigating the Structure-Activity Relationship (SAR) of Inhibitors of TPP-dependent Enzymes
    Chan, Hok Yan Alex
    Thiamine pyrophosphate (TPP), the bioactive form of vitamin B1, is an essential coenzyme needed for processes of cellular metabolism in all organisms. TPP-dependent enzymes all require the coenzyme TPP for catalytic activity, although individual members vary significantly in substrate preferences and biochemical reactions. To study these enzymes by chemical inhibition, the most popular way is to use thiamine/TPP analogues, which typically feature a neutral aromatic ring in place of TPP’s positive thiazolium ring. In response to the growing demand in understanding the roles of TPP-dependent enzymes and their pathways within biological systems, development of membrane-permeable thiamine analogues is pursued. In this PhD project, novel analogues of thiamine-based compounds have been designed, synthesised and evaluated on a variety of TPP-dependent enzymes. With the established structure-and-activity relationship of our analogues in hand, we developed membrane-permeable tools: some are selective to a given member while some possess broad inhibitory activities against the enzyme family.
  • ItemOpen Access
    Self-assembly and aggregation of glucagon-like peptide 1 and its analogues
    Přáda, Eva
    Aggregation and physical instability of peptide-based drugs poses a great challenge to the pharmaceutical industry. Glucagon-like peptide 1 (GLP-1) is a hormone that is used in the treatment of type-2 diabetes. However, GLP-1 has a short half-life *in vivo* and it is prone to aggregate which complicates its pharmaceutical usage. Strategies to overcome the short half-life *in vivo* include numerous chemical modifications of the native peptide. The focus of this Thesis is on the effect of two sets of chemical modification strategies, lipidation and C-terminal amidation, on the physical stability of the peptide. The first part of this work combines experimental and computational approaches to better understand the molecular basis of the aggregation of GLP-1 and its C-terminally amidated variant, GLP-1-Am. In particular, the off-pathway aggregation of GLP-1 and GLP-1-Am into disordered low-molecular weight oligomers is described. This process competes with the amyloid formation pathway and the addition of pre-formed off-pathway oligomers slightly slows down the fibrillation rate. Energy Landscape Theory was employed to investigate and rationalize the conformational behaviour and aggregation propensity of GLP-1 in different protonation states. Under all conditions studied, the GLP-1 energy landscape possesses a multi-funnel character with a variety of structurally different ensembles with low energy, which is a typical feature of intrinsically disordered proteins and aggregating systems. It is also shown that β-structure-containing conformations are more energetically favoured at acidic pH compared to neutral pH conditions, which agrees with a greater propensity of GLP-1 for aggregation at acidic pH which was observed experimentally. The second part of this Thesis focuses on the self-assembly and aggregation of lipidated analogues of GLP-1. Four lipidated GLP-1 analogues, which varied in the position of lipidation, and one additional analogue differing by the nature of the lipid moiety, were studied to establish the effect of the lipidation site and the lipid moiety on the physical stability of the peptide. The lipidation was shown to induce formation of large stable oligomers (i.e. > 7 monomeric units). The aggregation mechanism and kinetics were shown to be highly dependent on the lipidation position and the nature of the lipid moiety. Moreover, the aggregation kinetics of lipidated analogues were rarely observed to follow a classical nucleation-elongation mechanism but were rather likely to consist of more complex processes. Aggregates with a high content of β-sheet were formed by all analogues studied, however, they were distinct in their tertiary structure and aggregate morphology.
  • ItemOpen Access
    Analysis of Virtual Control Groups and Inter-species Concordance using Preclinical Toxicity Data
    Wright, Peter; Wright, Peter [0000-0003-1519-1404]
    Chapter 1 (Introduction). In line with the US EPA’s “Toxicity Testing in the 21st Century: A Vision and Strategy” and the Replacement, Refinement, and Reduction (3Rs) goals for animals in toxicity testing, the current two species preclinical testing paradigm for small molecules outlined in ICH M3 (R2) is under increasing pressure (Krewski et al., 2010). These pressures reflect not only the ethical concerns around animal testing (Prior et al., 2021) but also the lack of translatability of preclinical findings to humans. For example, despite animal testing forming the cornerstone of modern safety assessment (Greaves, 2012), undetected clinical safety risks (25.5%) were found to be the principal cause of drug attrition upon entering phase I clinical trials between 2000-2010 followed by efficacy (8.9%) (Waring et al., 2015). The high clinical failure rate is particularly pertinent due to the significant cost associated with phase I and II clinical trials (Bender and Cortés-Ciriano, 2020). This lack of translatability has given rise not only to questioning the external validity of increasingly standardised animal tests conducted with small sample sizes (Karp and Fry, 2021), but even whether use of animals for toxicity testing is scientific valid or is based on mere historical precedence (Monticello, 2015; Zbinden, 1993). Such concerns have driven changes in regulatory attitudes, such as the recent passing of the FDA Modernization Act 2021 which would remove the mandate for animal testing to evaluate the safety of drugs in the USA (Buchanan, 2021), and the European Parliament 2021 resolution to phase out animal testing for research, testing and education (Marshall et al., 2022). These developments follow in the wake of the 2006 REACH legislation in Europe and subsequent banning of animal testing for new cosmetic products (Yang et al., 2021), and aim to drive the development of New Approach Methodologies (NAMs) for the toxicity testing of drugs (Ball et al., 2022; Fischer et al., 2020; Parish et al., 2020). These issues have motivated two lines of research inquiry which aim to understand and increase the predictivity of clinical safety risks of which this thesis builds upon. Firstly, the potential use of Historical Control Data (HCD) in the form of Virtual Control Groups (VCGs) to replace or supplement Concurrent Control Groups (CCGs) in preclinical toxicity assessment. The scientific motivation of using HCD are its ability to increase the external validity of animal testing by providing context to findings through a better approximation of the biologically plausible range of outcomes relative to the small CGG resulting in the enhanced the assessment of treatment-related effects (Kluxen et al., 2021; Pinches et al., 2019; Steger-Hartmann et al., 2020). Moreover, from a 3Rs perspective the use of HCD preclinically has gained support as it has the potential to reduce animal usage by up to 25% (Steger-Hartmann et al., 2020). However, the idea has seen little uptake even though the use of HCD is well established in randomised human clinical trials (Berry et al., 2017) and that many studies are routinely performed under similar conditions. Despite several guidelines describing best practices for using HCD preclinically (Greim et al., 2003; Keenan et al., 2009; Kluxen et al., 2021), there is limited understanding of how variability and drift in HCD findings from CCGs could lead to differences in study outcomes across a large collection of studies (Steger-Hartmann et al., 2020). Therefore, we first aimed to conduct a large-scale retrospective analysis of the use of HCD in the form of VCGs and its potential impact on study outcomes (Chapter 3). Secondly, studies have been conducted to statistically quantify whether historical findings observed in preclinical studies are predictive for those later observed in humans, termed concordance analysis (Clark and Steger-Hartmann, 2018). However, publications have often come to contradictory conclusions with some authors claiming a lack of predictivity (Bailey et al., 2015, 2014, 2013; Van Norman, 2019), whilst others claiming their results support the current regulatory paradigm of animal testing (Monticello et al., 2017; Olson et al., 2000). However, studies have consistently demonstrated that observing the concordance of findings between preclinical animal models can lead to an increase in translatability of those findings to humans. However, these studies suffered from various limitations including small dataset size and a lack of control of experimental variables when comparing findings (Bailey et al., 2015). Therefore, we next aimed to quantify the inter-species concordance between preclinical findings whilst implementing methodological improvements and using a larger dataset (Chapter 4). Overall, both lines of inquiry were pursued through retrospective analyses of the eTOX preclinical toxicity dataset. Chapter 2 (Curation and Characterisation of the Histopathology and Pharmacokinetic Data in the eTOX Dataset) presents a methodology to curate the eTOX dataset, which is the largest preclinical toxicity dataset at the time of writing (Briggs et al., 2015; Cases et al., 2014; Sanz et al., 2017). This warranted its own research chapter as previous studies have highlighted the need to perform a multi-step curation of the dataset before any formal analysis is possible. The methodology included basic quality assurance regarding missing values and term standardisation as well as detailed steps required to handle the way in which the histopathology data were aggregated at the study, dose, time point, and severity grade level. We also discussed key characteristics of the dataset that have potential implications for the results presented in Chapter 3 and Chapter 4. Chapter 3 (Retrospective Analysis of the Potential use of Virtual Control Groups in Preclinical Toxicity Assessment using the eTOX Dataset) investigated the potential impact of replacing CCGs with VCGs based on HCD on preclinical toxicological study outcomes, namely histopathological finding treatment-relatedness designations. To this end, we developed a novel methodology whereby statistical predictions of treatment-relatedness using either CCGs or VCGs of varying covariate similarity to CCGs were compared to designations from original toxicologist reports; and changes in agreement were used to quantify changes in study outcomes. Generally, the best agreement was achieved when CCGs were replaced with VCGs with the highest level of covariate similarity, the same species, strain, sex, administration route, and vehicle. As HCD of increasing covariate dissimilarity were incorporated into VCGs we observed increasingly poor agreement and found this to be related to a concurrent increase in incidence rate divergence between HCD and CCGs. This result provided quantitative evidence that the CCG is the most relevant comparator for determining treatment-related findings, but more so systematically demonstrated that using increasingly heterogenous HCD leads to a divergence in study outcomes compared to when using the CCG. We therefore also presented the first identification of study covariates that impact study outcomes when using HCD to replace CCGs, which could help set future suitability criteria for the use of HCD in preclinical toxicity assessment. We next investigated a key choice when sampling HCD from a preclinical dataset, termed the Control Total Assumption, and found that assuming the lack of reporting of a finding to be equivalent to the absence of a finding systematically resulted in poorer agreement and a hyper-sensitivity to designate findings as treatment-related. Finally, although it is one of the largest and most comprehensive preclinical datasets, eTOX was found to lack sufficient documentation of study details previously highlighted as important when evaluating the suitability of HCD (Greim et al., 2003; Keenan et al., 2009; Kluxen et al., 2021). Therefore, we also highlight required features of future preclinical datasets to construct adequate VCGs which could potentially comply with future regulatory guidance. A more thorough analysis of study covariates gathered in Standard for Exchange of Nonclinical Data (SEND) format and their impact on study outcomes is currently being considered as part of an eTRANSAFE initiative (Steger-Hartmann et al., 2020). Overall, these results provide preliminary guidance for future industrial research into the VCG concept when sampling HCD from an internal or external database. Chapter 4 (Statistical Analysis of Preclinical Inter-species Concordance of Histopathological Findings in the eTOX Dataset) investigated the concordance of individual histopathological findings and target organ toxicities across four preclinical species in the eTOX dataset using likelihood ratios (LRs). Crucially, unlike previous studies this was done whilst using strict study comparability criteria; namely only comparing findings between studies with similar compound exposure (|ΔCmax| ≤ 1 log-unit), exposure duration, and animals of the same sex. We discovered 24 previously unreported significant inter-species associations between histopathological findings encoded by the HistoPATHology (HPATH) ontology. More associations with strong positive concordance (33% LR+ > 10) relative to strong negative concordance (12.5% LR- < 0.1) were identified. Of the top 10 most positively concordant associations, 60% were computed between different histopathological findings indicating potential differences in inter-species pathogenesis. We also observed low inter-species target organ toxicity concordance. For example, liver toxicity concordance in short-term studies between female rats and dogs observed an average LR+ of 1.84, and an average LR- of 0.73. This was corroborated by a similarly low concordance between rodents and non-rodents when analysing data for 75 candidate drugs from AstraZeneca. Overall, this work provides new statistically significant associations between preclinical species at comparable compound exposures which could be prioritised within ICH M3 (R2) due to their potential heightened predictivity for human risk (Monticello et al., 2017; Olson et al., 2000). However, we found that concordance is rare, particularly between the absence of findings. Therefore, given that findings observed in no one preclinical species can consistently translate to any other species, it is important to question how any species can be expected to consistently predict clinical safety risks in humans (Bailey et al., 2015). This is especially pertinent given the often-large exposure differentials resulting from the testing of animals up to the maximum tolerated dose which have previously been shown to be the predominant cause of false positive signals (Monticello et al., 2017). We expect that the growing body of research quantifying the discordance of toxicity between species will continue to drive the development of increasingly complex humanised NAMs. However, this is expected to be achieved stepwise through initially generating both NAM and animal data for specific endpoints to facilitate prospective validation and develop harmonised guidelines for their use in place of animal testing (Ball et al., 2022; Fischer et al., 2020; Parish et al., 2020).
  • ItemOpen Access
    The Initial Events of T-Cell Activation in Realistic Model Systems
    Koerbel, Markus; Koerbel, Markus [0000-0002-8344-8016]
    T cells are part of the cellular component of the adaptive immune system and specifically recognise foreign antigens in the body. They use their membrane-bound T-cell receptor (TCR) to bind small peptide antigens presented within the major histocompatibility complex (pMHC) on the surface of antigen presenting cells (APCs). Therefore, T-cell activation is crucially cell-cell contact dependent. The exact molecular mechanism that allows T cells to detect antigens with unprecedented selectivity and sensitivity remains controversial. This work investigates the T-cell decision-making in response to antigens. It focuses on how T cells can discriminate between foreign- and self-peptides in the initial cell-cell contacts formed with a target cell. This analysis relied on two crucial steps: the development of more realistic model systems to study those interactions, and advanced fluorescence microscopy to image and quantify the interactions in live T cells. Firstly, supported lipid bilayers (SLBs) as model surfaces for the APC membrane were improved by presenting a glycocalyx barrier and small adhesion protein CD58 alongside pMHC and ICAM-1. It was found that T cells use small membrane protrusions (microvilli) to penetrate the glycocalyx barrier, which were stabilised by CD58 and initiated signaling. A new image analysis was then developed that provided a framework to quantify T cell-APC interactions. The interaction could be classified into four stages and highlighted the presence of small contact zones throughout, supporting the kinetic segregation model of receptor triggering. Lastly, these concepts were transferred to real cell-cell contacts. Three-dimensional imaging showed T cells also responded to ImmTAC, a bispecific immunotherapeutic, presented on APCs via small contact zones during all stages of interaction. The results presented show the importance of small contact zones in the initial events of T-cell activation and provide a way to image and quantify them. This will help unravel the basis of TCR triggering and the development of novel immunotherapeutics.
  • ItemEmbargo
    Covalent Post-Assembly Modification of Metal-Organic Capsules
    Jahovic, Ilma
    Self-assembled molecular containers have been used for a range of applications, including molecular sensing, catalysis, and chemical separations. Post-assembly modification of these supramolecular structures has proven a useful strategy to impart the new functionality needed for these applications, circumventing the need to design and synthesize additional cage components. This thesis will detail the reactivity of anthracene-edged MII4L6 (MII = CoII, FeII, ZnII) tetrahedra with various reaction partners in hetero-Diels-Alder reactions and explore potential applications following these reactions. The first work demonstrates that iron(II) cages incorporate between one and five equivalents of singlet oxygen (1O2), whereas cobalt(II) capsules react fully and incorporate six equivalents of 1O2 . Structural changes following these reactions impacted the host-guest chemistry of the cobalt(II) cages, and binding studies using isothermal titration calorimetry are discussed. Specifically, the modified cages were found to bind the target guest molecules (pyrene, phenanthrene, β-endosulfan) more weakly than the parent cages. The cobalt(II) hosts could partially revert back to the precursor structures when subjected to high temperatures in the solid state, which suggested that such a mechanism might be used in the future to encapsulate and release molecular cargo on demand. The second work focuses on the reactivity of the anthracene and naphthalene moieties of the cages with a range of electron-deficient traizolinedione (TAD) derivatives in hetero-Diels-Alder reactions, and explores the potential for the reversibility of the transformation. Butyl-TAD was found to irreversibly react with the anthracene-edged cages, while the naphthalene- based cages remained untouched. This reduced reactivity of the naphthalene hosts was attributed to the increased degree of aromaticity of naphthalene compared to anthracene, allowing the anthracene cages to react with dienophiles. Unfortunately, the reaction was not found to be reversible under any of the tested conditions. Overall, this work presents the hetero-Diels-Alder reaction as a method for inducing covalent post-assembly modification on imine-based metal-organic cages. This is an important step toward developing transformations in which a single external stimulus (for instance singlet oxygen or an electrophile such as TAD) may be used to reversibly fine tune the binding of industrially-relevant molecules. This work could thus enable the creation of new materials that take up, and then release, specific molecules from mixtures.
  • ItemOpen Access
    Development of novel lysine targeting covalent inhibitors for Bruton’s tyrosine kinase and casein kinase 2 and synthesis of novel hybrid androgen receptor inhibitors
    Bizga Nicolescu, Radu Costin
    Cancer is the lead cause of death worldwide, accounting for 10 million deaths in 2020, or 1 in 6 deaths. While current therapies have significantly advanced in the last decade, they suffer from severe limitations such as resistance, lack of selectivity and life-threatening side effects. Herein we present three novel strategies of targeting prostate cancer, breast cancer and leukaemia. 1) The development of a novel class of hybrid compounds designed through covalently linking enzalutamide and EPI-001 through triazole-PEG linkers is reported. The compounds are accessed in 6 synthetic steps performed in parallel for the 5 final target compounds. The compounds display a 50-fold improvement in the cell killing potency compared to the gold standards of therapy, enzalutamide and EPI-001 (LC50 EPI = 85 μM, LC50 Enza = 65 μM, LC50 hybrid = 1.6 μM). The best in line compound was proven to exhibit its toxicity exclusively through AR mediated pathways, yielding the first-in-class hybrid AR inhibitor. 2) We report the design, computational validation and synthesis of a proposed lysine targeting allosteric CK2 inhibitor. The challenging synthetic steps towards the peptidomimetic electrophile are presented, along with the biological characterisation of the final target molecule. Protein mass spectrometry and tandem mass spectrometry studies indicate that the electrophile does not target Lys158, as intended, but that instead it forms a transient, water unstable, covalent bond with the protein. 3) We developed a novel class of lysine targeting covalent inhibitors, targeting the PH domain of BTK, a previously unreported targeting strategy. 3 families of analogues were rapidly constructed using an efficient 4 step synthetic strategy, yielding 23 analogues and 14 co-crystal structures with the BTK PH domain. The high percentage of crystal structures represents a success rate of 61%, which validates the fragment elaboration strategy, whereby all fragments covalently label Lys12. The binding selectivity was validated by protein mass spectrometry and differential scanning fluorimetry, whereby all analogues induce expected responses with the WT BTK, but fail to induce a response with the loss of function mutant R28C, as expected. A 6-fold improvement in binding affinity from the parent compound was achieved through a hit-to-lead optimisation campaign (160 μM to 30 μM).
  • ItemOpen Access
    Enzyme and Directed Evolution Technologies For Nerve Agent Neutralisation
    Briseño-Roa, Luis
    Owing to the magnitude of the utilisation of organophosphorus (OPs) insecticides and the possibility of using OPs nerve agents (NA) against civilian populations, the research and development of enzymes involved in the biotransformation and detoxification of OPs has attracted considerable attention in recent years. A number of enzymes have been identified that can catalyse the hydrolysis of OPs, including nerve agents. Two of the best characterised are Pseudomonas diminuta phosphotriesterase (PTE) and PON1, a mammalian member of the Serum paraoxonase (PONs) family. These enzymes have excellent catalytic properties towards some OPs, but relatively poor activities against others. It has been possible to alter PTE substrate specificity by rational site-mutagenesis, but with little improvements on the wild-type rates. A more successful approach has been the application of directed-evolution strategies. The aim of the present work has been to create variants of PTE with an increased catalytic efficiency towards OPs nerve agents. To this end, a directed evolution platform was developed to enable screening for organophosphatase activity. This methodology relies on the screening of Escherichia coli colonies transformed with PTE-variant libraries. Twelve fluorogenic NA analogues, with a 3-chloro-7-hydroxy-4-methylcoumarin leaving group, were tested for suitability as substrates for PTEs and PON1. Included in this series were analogues of the pesticides Paraoxon and Parathion, and the chemical warfare agents DFP, Dimefox, Tabun, Sarin, Cyclosarin, Soman, VX, and Russian-VX . These chemical surrogates have a similar structure but do not share the same physico-chemical properties as the nerve agents themselves. The directed evolution platform developed and used consisted of two parts. First, partially lysed Escherichia coli colonies were screened using the fluorogenic nerve agents analogues as probes. Second, the selected (positive) clones were grown in microplates filled with liquid medium, and their organophosphatase activity was measured in vivo. Several gene libraries were synthesised in each of which four codons of the residues forming PTE’s substrate binding site were selectively randomised. The PTE variant S5a was used as template for the libraries, as it expresses at 20-fold higher level than the wild type, in bacterial hosts, while retaining its kinetic properties for the wild-type substrate, Paraoxon. These libraries were screened using analogues of Russian-VX and Parathion as probes; approximately 106 clones were screened in total. The twenty most active variants, as determined in vivo, were expressed, purified, and their kinetic parameters for Paraoxon and the NA analogues were determined. PTE-S5a itself hydrolysed 8/VX, 9/Sarin and 10/Russian-VX analogues between 2.5 and 3.5 times more readily than PTE-wt. In contrast, towards 11/Soman and 12/Cyclosarin analogues its activity, was only 70% of that of the wild type enzyme. Three of the selected clones, PTE -A (I106T), C (I106L), and H (I106T/F132V/S308A/Y309W), exhibited a higher kcat than PTE-S5a towards Paraoxon. The latter exhibited a 5-fold increased in its turnover rate (31,016 s-1); this rate is higher than that of the in vitro evolved PTE-H5 (26,294 s-1). PTE variants A (I106T), C (I106L), D (I106A/F132G), E (I106V/F132L), and F (I106L/F132lG) exhibited between 2 and 4-fold increases in their kcat/KM towards the Paraoxon analogue relative to PTE-S5a. Variants Q (G60V/I106L/ S308G), S (G60V/I106M/L303E/S308E), and T(G60V/I106S/L303P/S308G) showed between 2 and 14-fold improvements in their activities towards Russian-VX, Soman and Cyclosarin analogues. The selectivity for this latter group towards phosphonate NA analogues increased up to 107-fold, relative to the wild type PTE. Each PTE monomer binds two divalent transition metal ions via a cluster of four histidines (His-55, His-57, His-201 and His-230) and one aspartate (Asp-301). In addition, the two metal ions are linked together by a carbamate functional group, formed by the carboxylation of the e-amino group of Lys-169 and a water (or hydroxide ion) from the solvent. A case study is presented in which using both site-directed mutagenesis and directed evolution strategies, the possibility of replacing the carboxylated lysine (Lys-169) by any other residue was assessed.
  • ItemOpen Access
    Heteroatom-Doped Graphitic Materials for Energy Storage
    Clark, Cassius
    The transition to a zero-carbon society will require a new generation of energy storage technology with high energy density. Central to this are novel electrode materials that possess high specific capacities (mAh g−1) and long cycling lifetimes. Modern lithium-ion batteries utilise graphite as an anode, being a cheap, safe and stable material. Sodium-ion batteries, a promising alternative to their lithium counterpart, utilise hard-carbon as an anode, a disordered matrix of graphene nano-sheets, due to the inability of sodium to bind effectively with pure graphite. However, the theoretical specific capacity of carbonaceous materials (372 mAh g−1 and 300 mAh g−1 for Li in graphite and Na in hard carbon, respectively) is low compared to other materials such as phosphorus (2596 mAh g−1 ) or silicon (3579 mAh g−1). Materials such as these come with their own caveats. High volume expansions, chemical instability, cost, or conductivity problems are a few potential issues encountered with Si or P anodes. Alternatively, chemical enhancement of graphite is considered an effective way of modifying the electrochemical ion-storage properties whilst retaining a high level of stability. Heteroatom substitution of carbon in a graphite lattice has been shown to produce high-capacity anode materials suitable for Li- and Na-ion batteries. This thesis develops an understanding of the structure and function of element-doped graphite within Li- and Na-ion batteries. Turbostratic doped graphitic materials were produced through pyrolysis of organic material. In Chapter 2, investigations into nitrogen-doping are made. Alterations to the synthetic procedure in combination with thorough structural and compositional analysis helps in understanding what factors make nitrogen-doped graphite effective as an anode. Notably, it was found that pyrolysis of organic precursors produced dense, solid spheres, previously thought to be hollow. Use of X-ray photoelectron spectroscopy, combined with ion-etching, revealed how the nitrogen dopant environment varied with increasing depth, and across annealing parameters. The effect of these environments on electrochemical performance could then be assessed. Chapter 3 focuses on boron as a dopant. The electron deficiency of boron compared to carbon is predicted to aid electron transfer, and subsequently facilitate intercalation of Li+ or Na+. Literature on boron-doped graphite largely considers systematic changes to the boron quantity present or focuses solely on applications. However, there is some debate about whether different precursors affect the final structure and performance of the graphite. In this chapter, a particular focus is on investigating the ability of different precursors to produce substitutionally-incorporated B-doped graphite. 11B solid-state nuclear magnetic resonance spectroscopy (SSNMR) combined with structural analysis are used to identify the phases boron is present in and how this is related to performance in Li- and Na-ion batteries. Relating the boron environments present to the voltage at which battery capacity is observed allows for the interpretation of how boron doping affects and facilitates the intercalation of Li+ and Na+ ions. In Chapter 4, an in-depth study of the function of phosphorus-doped graphite is presented. Ex-situ 31P SSNMR spectroscopic studies of doped-graphites, partially cycled to different voltages in Li- and Na-ion cells, led to proposed mechanisms of lithiation or sodiation. Furthermore, alternations to the synthetic procedure allowed reliable encapsulation of white phosphorus between graphene layers, enabling effective, reversible cycling of red phosphorus without degradation.
  • ItemOpen Access
    Molecular dynamics investigation of the long wavelength dielectric response of classical dipolar liquids using Bloch function methods
    Lloyd, Haydn
    Periodic Boundary Conditions (PBCs) are commonly utilised in many simulations of great importance in computational science, both as methods to accurately simulate perfect crystals, as well as to approximate macroscopic systems. When considering the latter case, the use of these boundary conditions restricts the wavevectors for which the Fourier components of quantities calculated from these systems can be non-zero. In particular, the minimum non-zero wavevector that can be examined, is inversely proportional to the size of the simulation cell. This means the examination of small, non-zero wavevectors can require the use of large systems, that may become prohibitively expensive to simulate. This can potentially inhibit the study of long-range contributions to the properties of the system. In this work, Bloch boundary conditions are introduced for systems that include charges. In these, the particle positions and momenta are periodic, as in PBCs, but the charge multipoles in image cells are changed in phase controlled, by a wavevector within the first Brillouin Zone, q, that is characteristic of the boundary conditions. In such cases, the accessible wavevectors are then all shifted by q, and so the minimum accessible wavevector is now q. A demonstration of how the Ewald summation is modified for energy and force calculations necessary for the simulation of these systems is given, explicitly for that of charges and dipoles, and a scheme to derive this for higher multipoles is given. This framework is then applied to the longitudinal and transverse dielectric constants of dipolar fluids subject to static electric fields, both Stockmayer and polarisable dipole fluids. This is used to verify the ratio of the transverse and longitudinal susceptibility being the dielectric constant, in the limit q goes to zero. A brief analysis of how this may be applied to systems with dynamic electric fields is also given.
  • ItemEmbargo
    Single-molecule techniques for mapping protein aggregates
    Lam, Yui
    Proteins in our body are usually folded to carry out their physiological functions. However, they can become misfolded and aggregated throughout the lifetime of a cell. These proteins can be degraded or cytotoxic, the latter of which leads to diseases such as neurodegeneration and cancers. Unfortunately, the compositions of native protein aggregates are still not fully known. Mapping them could help us understand the diseases they cause, and provide the basis for early diagnosis and monitoring therapy. Since protein aggregates are heterogeneous in sizes and structures, single-molecule techniques are required to unravel their compositions. Super-resolution microscopy is particularly useful, as these aggregates are smaller than the diffraction limit of light. Compared to conventional light microscopy, the fluorescence of the nearby molecules in super-resolution microscopy fluoresces at different times. With this, even though the fluorescence of the single fluorophore is still blurred by the diffraction limit (~200 nm), the fluorophore is inferred as in the centre of the blurred spot. Thus, the resolution in super-resolution microscopy can reach down to sub 20 nm. This allows us to super-resolve the protein aggregates and may therefore help us decipher the mysteries of disordered and aggregated proteins. However, applying super-resolution microscopy to these complexes is a challenge. Although there are already a range of developed methods, there are still important limitations. In this thesis, I present a series of advances in single-molecule techniques for mapping protein aggregates. Firstly, an advanced illumination module for super-resolution microscopy to facilitate accurate measurements was implemented and characterised. Functionalisation of antibodies, which are commonly used to map native protein aggregates in super-resolution microscopy, was then optimised. Next, single-molecule pull-down assays were investigated with the aim of capturing and imaging protein aggregates with higher selectivity, sensitivity, and speed on glass coverslips for super-resolution microscopy. Meanwhile, two classes of DNA-small molecule conjugates, with PET-ligand analogues and aggregate-targeting peptides, were synthesised and tested. These novel probes can potentially selectively target protein aggregates in super-resolution microscopy. Finally, a newly developed, state-of-the-art microscope for super-resolution microscopy, NanoPro, is presented. The theme that unites the presented work is improving single-molecule techniques for mapping protein aggregates.
  • ItemOpen Access
    Supramolecular Approaches to Viscoelastic Biomaterials and Their Applications
    Park, June
    Viscoelasticity encompasses the characteristics of both the shape-conserving elasticity and the shock-absorbing viscosity. The extracellular matrices (ECM), or the protein-polymer scaffolds surrounding the cells in our body, possess unique tissue-dependent viscoelasticity to protect and provide mechanical queues to the nearby cells. Viscoelasticity in polymeric hydrogels can be achieved in several ways, one of which is by incorporating the host-guest supramolecular components. The following works explore the application of cucurbit[8]uril (CB[8])-based host-guest chemistry to achieve tuneable viscoelasticity in hydrogels. First, an ECM-mimetic CB[8]-based biomaterial tuned to the viscoelasticity of the lung is developed for stem cell engraftment into the solid organs. The crosslinker designed with a MMP-cleavable sequence enabled the enzymatic and cell-mediated changes in the gel mechanical properties, supported the organoid culture, and facilitated the functional engraftment and differentiation of the lung stem cells in the mouse lungs, highlighting the importance of tuning the viscoelasticity and cell-responsiveness of stem cell-carrying biomaterials for engraftment into solid organs. The second work reports of biofabricating the flexible electronics through a parylene surfacemodification chemistry and a novel hybrid network consisting of CB[8] and 1-benzyl-3vinylimidazolium (BVI) host-guest crosslinkers and gelatin-methacrylate (GelMA). The gel was designed to match the stress relaxation profile of the brain, facilitating a more brain ECM-mimetic biomaterial that is resistant to the high strain of the flexible neural devices. Combined with the ability to photo-pattern and combine with the different types of cells, this work presented the potential ways of multiplexing different types of biomaterials and cells to augment the bioelectronics functionalities, opening a new era of regenerative bioelectronics. The final work describes a new coumarin-based monomer that can be incorporated into the various free-radical-polymerised and-crosslinked materials to achieve reversibly phototuneable viscoelasticity. Harnessing coumarin’s UV wavelength-dependent cyclo-additionvi and-reversion, an extraordinary range ( 0.01 seconds to 1 million seconds) of stress relaxation half time (τ1/2) and storage modulus (100-10,000 Pa) were achieved with UV irradiation alone without changing the molecular composition. Unlike the previous approaches of directly functionalising the coumarin onto the polymer backbones, generating a CB[8]-coumarin monomer provided a powerful platform to augment the existing materials to possess reversibly tuneable viscoelasticity.
  • ItemEmbargo
    The Exploration of a Strategy for Regioselective Arene Amination Utilising Non-Covalent Interactions
    Gillespie, James
    The formation of arene C-N bonds for the preparation of anilines is one of the most used reactions in industry and academia. Numerous methods which utilise reactive N-centred radicals for the synthesis of anilines from arenes have recently been developed. However, regiochemical control is a major challenge associated with these methods, with mixtures of regioisomers commonly obtained in most protocols. Non-covalent interactions have been used in a limited number of examples to control regioselectivity in radical reactions. We therefore decided to investigate whether non covalent interactions between an arene substrate bearing a suitable directing group and an incoming N-centred radical could be a viable strategy to selectively target the arene ortho position. Initial investigations focussed on using hydrogen bonding interactions between substrate and radical to direct radical addition to the arene ortho position. Whilst promising reactivity was seen in many cases, the regiochemical outcome was poor. Later studies focussed on the use of anionic substrates and investigating whether ion pairing interactions with a cationic N-centred radical could direct ortho-selective radical addition. It was found that reacting anionic phenylsulfamate substrates with reagents which generate aminium radical cations resulted in an ortho-selective radical amination, allowing access to ortho-phenylenediamine products. This work was subsequently expanded upon to include arenesulfonates and arenecarboxylates as substrates for ion pair-directed ortho-selective amination. In these cases, the reaction proceeds via rearrangement of readily accessible O- (arenesulfonyl)hydroxylamines and O-benzoylhydroxylamines, respectively, and constitutes a remarkably facile way to access the ortho-aminated products. This work provides a blueprint for further development of regioselective amination reactions and more generally showcases the potential for non-covalent interactions to control regioselectivity in radical chemistry.
  • ItemEmbargo
    Remodelling Surface Site Interaction Points
    Storer, Maria Chiara
    The Surface Site Interaction Point (SSIP) model describes the non-covalent interaction properties of molecules by abstracting a discrete number of points on the van der Waals surface of molecules. Each point is assigned a value based on empirical interaction scales and the calculated Molecular Electrostatic Potential (MEP). SSIPs have been used to provide predictions of partition coefficients, solvent effects on association constants for formation of intermolecular complexes, and the probability of cocrystal formation. In this thesis, secondary electrostatic interactions are shown to be highly overestimated at the van der Waals surface and to be more accurately described on electron density isosurfaces that lie closer to the nuclei. Interaction parameters calculated with these isosurfaces successfully account for the properties of arrays of multiple H-bond donor and acceptor groups in different configurations. Three MEP isosurfaces are required to describe soft H-bond acceptors, hard H-bond acceptors, and H-bond donors. The Atomic Surface Site Interaction Point (AIP) model has been developed to obtain interaction points on these three surfaces using empirical rules for different atom types. This new approach to obtain interaction sites ensures a correct description of secondary electrostatic interactions, an accurate placement of lone pairs, a consistent description of π-systems and a representation of short H-bond contacts with hard acceptors but longer contacts with soft acceptors. Partition data between n-hexadecane and water was used to fine-tune the AIP representation of non-polar functional groups, for which it is difficult to obtain accurate empirical interaction parameters. The phase transfer data is also used to analyse the effect of H-bond cooperativity on the AIPs of functional groups that can make more than one H-bond, like alcohols, ethers and carbonyls. Finally, two methods for fast calculation of AIPs are discussed. The first method is a fragment-based approach, which assigns AIPs of large compounds from the AIP representation of small molecules with matching substructures. The second approach relies on a neural network method developed by Astex to quickly calculate the MEP surface and obtain the AIPs. These methods extend the scope of the AIP model to describing large molecules or large libraries of compounds for applications such as virtual screening and modelling host-guest systems.
  • ItemOpen Access
    Dissipative Matsubara Dynamics
    Prada, Adam; Prada, Adam [0000-0003-3601-1265]
    The two most widely used path-integral chemical dynamics methods are Ring-polymer molecular dynamics (RPMD) and Centroid molecular dynamics (CMD). Both of these were originally proposed as heuristic approximations, which were justified by analytic limits and empirical evidence.In 2015, the Matsubara-dynamics theory was proposed as a controlled minimal approximation combining quantum statistics with classical dynamics, while conserving the quantum Boltzmann distribution. It was then shown that both RPMD and CMD are approximations to Matsubara dynamics, thus providing a firm theoretical ground for these methods. Unfortunately, a naive implementation of Matsubara dynamics is too computationally expensive to be a useful method because of a severe sign problem, which limited the number of Matsubara modes in a calculation to around 10 or less. Therefore, Matsubara dynamics has never been directly compared to exact quantum results with the exception of the harmonic oscillator and a linear correlation function cropped with a window function. In this work, we present results for up to 200 Matsubara modes and fully converged non-linear correlation functions. This was achieved by developing dissipative Matsubara dynamics — a way of implicitly including a bath of harmonic oscillators in a Matsubara dynamics simulation.We first show that the fact that the bath is harmonic allows its inclusion in the simulation without exacerbating the sign problem. Then we proceed to show that the presence of the bath even allows simulations of analytically continued Matsubara dynamics, which does not suffer from the sign problem but was previously impossible due to the presence of unstable trajectories. These simulations allow the inclusion of almost an order of magnitude more Matsubara modes than previously feasible. To further improve the stability, we introduce the “real-noise” approximation, which allows the simulation of up to ≈ 200 Matsubara modes. However, even this number is insufficient to converge non-linear operators. Therefore, we developed a harmonic correction for the tail of the Matsubara distribution, with which we were able to obtain converged non-linear correlation functions, which nearly perfectly match the exact quantum results. This is the first time such a comparison has been done and it provides a strong validation of the Matsubara-dynamics theory.
  • ItemOpen Access
    A Solid State NMR Investigation of Poly (ADP-ribose) and Its Involvement in Tissue Calcification
    Murgoci, Adrian
    The mechanisms of bone and vasculature biomineralisation are not fully understood. It is unclear how the calcium and phosphate ions are concentrated at the calcification site, and how the composition and structure of the mineral phase changes during maturation. This work aims to use solid state NMR spectroscopy to investigate the evolution of the mineral phase, and the interface between the apatite nanocrystals and the organic matrix at different time points on the mineralisation timescales in two in vitro models of biomineralisation, i.e. physiological bone mineralisation by MC3T3-E1 osteoblast-like cells (chapters 7 and 8) and pathological medial arterial calcification by bovine vascular smooth muscle cells (chapter 9). The advantage of solid state NMR to study biomineral in tissues is that it can be used on samples that are more or less in native state, without extensively dehydrating the tissue or removing the organic layers. However, in order to gain a holistic picture of the mineral at nanoscopic level, the calcified matrix samples studied by ssNMR have also been investigated by SEM/EDS and occasionally TEM, to explain the changes observed in the NMR spectra, and rationalise how the progression of mineralisation could occur in vivo. Another benefit of NMR spectroscopy is that no prior assumptions are needed about the composition of a sample to observe its components. The Duer group discovered that poly (ADP-ribose) (PAR), a biological polymer produced in response to DNA damage, is deposited in the calcified matrix of bone and pathologically mineralised arteries and could play an important role in mineralisation. PAR has affinity for calcium, forming “beads” that bind preferentially to the hole zones of collagen fibrils where mineralization is initiated. PAR mediates the biomimetic calcification of collagen fibrils in vitro in a periodic arrangement ofvi mineral density. The structure of PAR has been previously characterised by mass spectrometry following its isolation from different organs (but not calcified tissues like bones) via laborious and potentially damaging procedures. Chapter 6 aims to fully characterise the structure of poly (ADP-ribose) in 13C-enriched in vitro grown samples, without the requirement to extract it from its native environments, by determining the chemical shifts corresponding to the signals from the linear parts of the polymer, and potentially identify the chemical shifts of branching points and chain ends. Moreover, careful isotopic enrichment of cells and matrix with sugars or key amino acid residues help us define novel hypotheses about the interactions between collagen fibrils and PAR at a molecular level, and the involvement of the latter in the mineralisation of tissues (Chapter 8).