David Keen

Abstract:

Defect engineering is a powerful tool that can be used to tailor the properties of metal–organic frameworks (MOFs). Here, we incorporate defects through ball milling to systematically vary the porosity of the giant pore MOF, MIL-100 (Fe). We show that milling leads to the breaking of metal–linker bonds, generating additional coordinatively unsaturated metal sites, and ultimately causes amorphisation. Pair distribution function analysis shows the hierarchical local structure is partially retained, even in the amorphised material. We find that solvents can be used to stabilise the MIL-100 (Fe) framework against collapse, which leads to a substantial retention of porosity over the non-stabilised material

Abstract:

This three-year Ph.D. research project is the result of the collaboration among the research group of Prof. Guido Ennas from University of Cagliari (Italy), expert in innovative and green synthesis approaches of micro- and nanomaterials, including mechanochemistry and sonochemistry, and the research group of Dr. Sarah Hudson from University of Limerick (Ireland), expert in biocatalysts and drug-delivery systems. The research focuses on the development of innovative synthesis routes for the preparation of proteins/enzymes – Metal Organic Frameworks (MOFs) hybrid composite materials for biomedical applications. In particular, alternative sonochemical and mechanochemical methods have been explored for the one-pot synthesis of glucose oxidase – iron(III) trimesate composites under eco- and bio-compatible conditions. Conventional harsh synthesis conditions have been overcome in order to minimise enzyme denaturation and activity loss, while retaining structural and textural features of the MOF. Beside their biocompatibility and low cost, one of the advantages of using iron(III) trimesate materials to immobilise biomolecules is their peroxidase-mimic behaviour. Indeed, such MOFs do not act as passive supports for the immobilisation of glucose oxidase, but also perform as enzyme-mimics, avoiding the immobilisation of additional peroxidase enzymes. Hybrid glucose oxidase – iron(III) trimesate composites prepared via mechanochemical and sonochemical approaches under mild conditions were applied as sensitive biosensors for glucose colorimetric detection

Abstract:

Acetylcholinesterase (AChE) catalyzes hydrolysis of acetylcholine thereby terminating cholinergic nerve impulses for efficient neurotransmission. Human AChE (hAChE) is a target of nerve agent and pesticide organophosphorus compounds that covalently attach to the catalytic Ser203 residue. Reactivation of inhibited hAChE can be achieved with nucleophilic antidotes, such as oximes. Understanding structural and electrostatic (i.e. protonation states) determinants of the catalytic and reactivation processes is crucial to improve design of oxime reactivators. Here we report X-ray structures of hAChE conjugated with a reversible covalent inhibitor 4K-TMA (4K-TMA:hAChE) at 2.8 ​Å resolution and of 4K-TMA:hAChE conjugate with oxime reactivator methoxime, MMB4 (4K-TMA:hAChE:MMB4) at 2.6 ​Å resolution, both at physiologically relevant room temperature, as well as cryo-crystallographic structure of 4K-TMA:hAChE at 2.4 ​Å resolution. 4K-TMA acts as a substrate analogue reacting with the hydroxyl of Ser203 and generating a reversible tetrahedral hemiketal intermediate that closely resembles the first tetrahedral intermediate state during hAChE-catalyzed acetylcholine hydrolysis. Structural comparisons of room temperature with cryo-crystallographic structures of 4K-TMA:hAChE and published mAChE complexes with 4K-TMA, as well as the effect of MMB4 binding to the peripheral anionic site (PAS) of the 4K-TMA:hAChE complex, revealed only discrete, minor differences. The active center geometry of AChE, already highly evolved for the efficient catalysis, was thus indicative of only minor conformational adjustments to accommodate the tetrahedral intermediate in the hydrolysis of the neurotransmitter acetylcholine (ACh). To map protonation states in the hAChE active site gorge we collected 3.5 ​Å neutron diffraction data paving the way for obtaining higher resolution datasets that will be needed to determine locations of individual hydrogen atoms.