Supporting 19F-centered NMR investigations across a range of biological applications

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The fluorine atom is almost never found in the natural molecules of life e.g. peptides, proteins, nucleic acids and enzyme cofactors. Therefore, carefully installing fluorine atoms into these biomolecules can provide a beacon with which to study their biochemical transformations, interactions with other biomolecules and changes in their shape using 19F NMR. NMR is a technique that provides information about the chemical environment in which a molecule or part of a molecule exists and 19F NMR is a particularly useful tool for biology because each different fluorine-containing molecule provides a distinct and measurable signal in different parts of the spectrum, meaning that we can perform real-time experiments simultaneously measuring multiple species in complex mixtures, unusual solvents e.g. biological fluids and in cells. Moreover, the absence of natural fluorine means that we can selectively observe only the events involving fluorinated biomolecules, providing a clear window into an otherwise very complex and crowded world at the molecular scale. One of the challenges in using NMR for biology has been the relatively low sensitivity of traditional methods. However, this can be overcome by i) reducing interfering background signals, ii) using NMR cryoprobes that improve signal-to-noise ratios, and iii) using novel methods to amplify the signal, finally allowing us to study dilute samples in biological environments. In this project, we aim to use the purchase of a dedicated cryo probe and new SHARPER methods developed at Edinburgh to significantly enhance the sensitivity for detection of fluorinated biomolecules by more than hundred-fold relative to the room temperature probes as a transformative central pillar for studies on biological systems. One aspect of this research that will exploit the exceptional sensitivity of the new cryoprobe and SHARPER methods is the development of new tools to attach fluorinated 'tags' to DNA and proteins, including patient-derived samples. Attaching a fluorinated reporter to a biomolecule for 19F NMR when there are no other 19F signals, allows us to clearly observe and measure the interactions between different DNA species at low micromolar-to-nanomolar concentrations due to distinctive changes in the signals. This will be used also to measure the different transient forms of proteins that exist, but cannot be observed, using fluorescence methods during protein folding and aggregation. We will also use the distinct and quantitative signals for fluorinated cages molecules to understand how tightly they bind to blood proteins. These outcomes will allow us to better understand the rules of life. Given the above benefits of 19F NMR, this is also an outstanding method to study biochemical reactions and transformations in real-time. This will be used to conveniently measure the biological reactivity and stability of new fluorinated Raman imaging tags and enzyme probes in whole cells and in cellular fluids. We will also use 19F NMR to understand how to harness biotechnology for the benefit of sustainable access to synthetic feedstocks by observing the transformation of fluorinated enzyme substrates into new products - importantly providing structural information on short-lived intermediate species that cannot be observed easily using other methods. The preparation of fluorinated molecules can be challenging and often requires the use of dangerous fluorine gas and complex apparatus. We will expand the biosynthetic toolbox to develop green artificial enzymes that can install fluorine atoms into new chemical building blocks under mild and safe conditions, which will revolutionise the preparation of fluorinated molecules. Overall, access to a dedicated cryoprobe, coupled with new analysis methods and synthetic fluorinated tools will release the untapped potential of 19F NMR as a tool with which to study dynamic biological processes.