Biological imaging has broad applications in basic research and in medicine, with new technologies capable of broad human and economic impact. The development of methods that can probe specific processes in complex solutions, in cells, or in organisms can substantially increase our understanding of complexity in biology and can be ultimately used to develop new approaches to identify changes that are associated with disease. In this work, new approaches to detect the functions of proteins and the structure of proteins are developed, via the incorporation of multiple fluorine atoms into proteins. Fluorine is exceptionally useful for imaging because it is not normally present in biological environments and because it can be specifically detected by magnetic resonance approaches such as MRI. Thus, addition of fluorine to molecules that are in complex solutions, such as inside cells, allows the specific identification of that fluorinated molecule in a sea of other non-fluorinated molecules that are not seen by fluorine magnetic resonance, as well as changes to those molecules that occur during biological processes. This proposal involves the development of highly fluorinated amino acids, to allow sensitive detection, and their incorporation in proteins and application to identify specific biological processes, including the actions of enzymes and other proteins, that are important in changes in cells that define development and disease. This approach should be broadly applicable to the identification of changes and functions of proteins by magnetic resonance. The technologies developed herein will be distributed to other researchers for maximum impact and may be commercialized, contributing to economic development. Undergraduate researchers will contribute significantly to all aspects of this project, training students who go on to careers in diverse areas and applications of science, including research, teaching, medicine, policy, business, and law. Undergraduate and graduate students trained in this project will have broad cross-disciplinary education, with preparation for diverse applications in their careers.
With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Neal Zondlo of the University of Delaware to develop approaches to examine specific enzymatic activities and protein structure using F-19 NMR with fluorinated amino acids. Probing biological processes in real time in situ is a substantial challenge whose solution has broad potential applications in imaging and in understanding basic biology. The development of new and general approaches to imaging that allow the identification of specific intracellular or extracellular events will allow new explorations and interventions in basic and applied biological research. General methods to the application of fluorinated amino acids for the interrogation of biological processes in situ with high sensitivity will be developed. F-19 imaging has enormous potential because of its specificity (high signal to noise due to the absence of fluorine in vivo; application to detect specific molecular events), its high magnetic sensitivity comparable to proton, and its application using commercial proton NMR and MRI instruments. The potential of F-19 magnetic imaging is currently substantially limited by a need to achieve increased sensitivity for diverse applications and for the development of specific probes of defined biological processes. An ideal approach to enhance specificity and sensitivity of F-19 magnetic resonance spectroscopy would involve the incorporation of an intense fluorine signal in native ligands in a manner that is minimally disruptive of structure. Methods to incorporate perfluoro-tert-butyl groups into peptides as novel amino acids will be developed. Perfluoro-tert-butyl groups have 9 equivalent fluorines, and thus have a 9-fold increase in signal-to-noise over single fluorines. At least as importantly, perfluoro-tert-butyl groups are sharp singlets by NMR, further increasing signal-to-noise and operational simplicity, meaning that most existing proton-based instrumentation can readily be adjusted to detect peptides containing perfluoro-tert-butyl groups. This approach will be used to detect kinase activity in real time by F-19 NMR, with potential applications to imaging diverse biological processes. In addition, approaches to probe cis-trans isomerism by NMR in complex solutions will be developed, using 4,4-difluoroproline, an amino acid, which is a sensitive F-19 NMR probe of the isomerization state of proline amide bonds. The approaches developed may be broadly applicable to probing processes in solution, in cells, and potentially in vivo. While this work is initially focused on applications in kinases and cell signaling, it is also applicable to other enzymatic modifications of proteins, including proteases and cell surface ligands, as well as to protein-protein interactions. This work has the potential to open new and sensitive approaches to imaging.
