Understanding immobilised enzymes by NMR spectroscopy

Enzyme immobilisation is the conversion of a soluble enzyme molecule into a solid particle form. This allows the recovery of the enzyme catalyst for its re-use and avoids protein contamination of the product streams. A better understanding of immobilised enzymes is necessary for their rational development. A more rational design can help enormously in the applicability of these systems in different areas, from biosensors to chemical industry. Immobilised enzymes are challenging systems to study and very little information is given by conventional biochemical analysis such as catalytic activity and amount of protein. Here, solid-state NMR has been applied as the main technique to study these systems and evaluate them more precisely. Various approaches are presented for a better understanding of immobilised enzymes, which is the aim of this thesis. Firstly, the requirements of a model system of study will be discussed. The selected systems will be comprehensibly characterised by a variety of techniques but mainly by solid-state NMR. The chosen system will essentially be the enzyme α-chymotrypsin covalently immobilised on two functionalised inorganic supports – epoxide silica and epoxide alumina – and an organic support – Eupergit®. The study of interactions of immobilised enzymes with other species is vital for understanding the macromolecular function and for predicting and engineering protein behaviour. The study of water, ions and inhibitors interacting with various immobilised enzyme systems is covered here. The interactions of water and sodium ions were studied by 17O and 23Na multiple-quantum techniques, respectively. Various pore sizes of the supports were studied for the immobilised enzyme in the presence of labelled water and sodium cations. Finally, interactions between two fluorinated inhibitors and the active site of the enzyme will be explored using 19F NMR, offering a unique approach to evaluate catalytic behaviour. These interactions will be explored by solution-state NMR firstly, then by solid-state NMR. NMR has the potential to give information about the state of the protein in the solid support, but the precise molecular interpretation is a difficult task.

The dehydrogenation reactions between LiH and organic amines for solid state hydrogen storage systems

Hydrogen is considered as an appealing alternative to fossil fuels in the pursuit of sustainable, secure and prosperous growth in the UK and abroad. However there exists a persisting bottleneck in the effective storage of hydrogen for mobile applications in order to facilitate a wide implementation of hydrogen fuel cells in the fossil fuel dependent transportation industry. To address this issue, new means of solid state chemical hydrogen storage are proposed in this thesis. This involves the coupling of LiH with three different organic amines: melamine, urea and dicyandiamide. In principle, thermodynamically favourable hydrogen release from these systems proceeds via the deprotonation of the protic N-H moieties by the hydridic metal hydride. Simultaneously hydrogen kinetics is expected to be enhanced over heavier hydrides by incorporating lithium ions in the proposed binary hydrogen storage systems. Whilst the concept has been successfully demonstrated by the results obtained in this work, it was observed that optimising the ball milling conditions is central in promoting hydrogen desorption in the proposed systems. The theoretical amount of 6.97 wt% by dry mass of hydrogen was released when heating a ball milled mixture of LiH and melamine (6:1 stoichiometry) to 320 °C. It was observed that ball milling introduces a disruption in the intermolecular hydrogen bonding network that exists in pristine melamine. This effect extends to a molecular level electron redistribution observed as a function of shifting IR bands. It was postulated that stable phases form during the first stages of dehydrogenation which contain the triazine skeleton. Dehydrogenation of this system yields a solid product Li2NCN, which has been rehydrogenated back to melamine via hydrolysis under weak acidic conditions. On the other hand, the LiH and urea system (4:1 stoichiometry) desorbed approximately 5.8 wt% of hydrogen, from the theoretical capacity of 8.78 wt% (dry mass), by 270 °C accompanied by undesirable ammonia and trace amount of water release. The thermal dehydrogenation proceeds via the formation of Li(HN(CO)NH2) at 104.5 °C; which then decomposes to LiOCN and unidentified phases containing C-N moieties by 230 °C. The final products are Li2NCN and Li2O (270 °C) with LiCN and Li2CO3 also detected under certain conditions. It was observed that ball milling can effectively supress ammonia formation. Furthermore results obtained from energetic ball milling experiments have indicated that the barrier to full dehydrogenation between LiH and urea is principally kinetic. Finally the dehydrogenation reaction between LiH and dicyandiamide system (4:1 stoichiometry) occurs through two distinct pathways dependent on the ball milling conditions. When ball milled at 450 RPM for 1 h, dehydrogenation proceeds alongside dicyandiamide condensation by 400 °C whilst at a slower milling speed of 400 RPM for 6h, decomposition occurs via a rapid gas desorption (H2 and NH3) at 85 °C accompanied by sample foaming. The reactant dicyandiamide can be generated by hydrolysis using the product Li2NCN.

Exploring coordination-driven self-assembly with autonomous chemical robots

The work presented herein focused on the automation of coordination-driven self assembly, exploring methods that allow syntheses to be followed more closely while forming new ligands, as part of the fundamental study of the digitization of chemical synthesis and discovery. Whilst the control and understanding of the principle of pre-organization and self-sorting under non-equilibrium conditions remains a key goal, a clear gap has been identified in the absence of approaches that can permit fast screening and real-time observation of the reaction process under different conditions. A firm emphasis was thus placed on the realization of an autonomous chemical robot, which can not only monitor and manipulate coordination chemistry in real-time, but can also allow the exploration of a large chemical parameter space defined by the ligand building blocks and the metal to coordinate. The self-assembly of imine ligands with copper and nickel cations has been studied in a multi-step approach using a self-built flow system capable of automatically controlling the liquid-handling and collecting data in real-time using a benchtop MS and NMR spectrometer. This study led to the identification of a transient Cu(I) species in situ which allows for the formation of dimeric and trimeric carbonato bridged Cu(II) assemblies. Furthermore, new Ni(II) complexes and more remarkably also a new binuclear Cu(I) complex, which usually requires long and laborious inert conditions, could be isolated. The study was then expanded to the autonomous optimization of the ligand synthesis by enabling feedback control on the chemical system via benchtop NMR. The synthesis of new polydentate ligands has emerged as a result of the study aiming to enhance the complexity of the chemical system to accelerate the discovery of new complexes. This type of ligand consists of 1-pyridinyl-4-imino-1,2,3-triazole units, which can coordinate with different metal salts. The studies to test for the CuAAC synthesis via microwave lead to the discovery of four new Cu complexes, one of them being a coordination polymer obtained from a solvent dependent crystallization technique. With the goal of easier integration into an automated system, copper tubing has been exploited as the chemical reactor for the synthesis of this ligand, as it efficiently enhances the rate of the triazole formation and consequently promotes the formation of the full ligand in high yields within two hours. Lastly, the digitization of coordination-driven self-assembly has been realized for the first time using an in-house autonomous chemical robot, herein named the ‘Finder’. The chemical parameter space to explore was defined by the selection of six variables, which consist of the ligand precursors necessary to form complex ligands (aldehydes, alkineamines and azides), of the metal salt solutions and of other reaction parameters – duration, temperature and reagent volumes. The platform was assembled using rounded bottom flasks, flow syringe pumps, copper tubing, as an active reactor, and in-line analytics – a pH meter probe, a UV-vis flow cell and a benchtop MS. The control over the system was then obtained with an algorithm capable of autonomously focusing the experiments on the most reactive region (by avoiding areas of low interest) of the chemical parameter space to explore. This study led to interesting observations, such as metal exchange phenomena, and also to the autonomous discovery of self assembled structures in solution and solid state – such as 1-pyridinyl-4-imino-1,2,3-triazole based Fe complexes and two helicates based on the same ligand coordination motif.