Direct Site Selective 19F- and 18F-labelling of Peptides and Proteins Towards "Zero Size - Zero Background" Bioimaging

Non-invasive imaging techniques now dominate disease diagnosis and can, in principle, provide vital knowledge of disease states at the molecular level. In this was accurate diagnosis will allow more appropriate and successful treatment plans. A myriad of imaging methods, perhaps more prominently magnetic resonance imaging, optical imaging, ultrasonography, positron emission tomography and X-ray computed tomography have become established over the past decades. In parallel, a range of small molecules, molecular complexes, micro- and nanoscale materials used as tracers, targeting or contrast agents has undergone intense diversification to support these technologies by enhancing signal. However, despite this remarkable progresses, there is a surprising lack in the use of molecular imaging agents - agents that not only give a better signal but also tell us about the molecular events associated with the disease. As a result, there has been slower progress towards the fundamental understanding of molecular processes in biology and diseases. We aim to bridge this 'disconnect' between the molecular and medicinal sciences through the development of a general method that will allow creation of innovative chemical tools to produce superior probes for 'molecular imaging'.

Access to precisely fluorine(F)-modified peptides and proteins is incredibly useful to allow the visualisation of biological phenomena in complex matrices including cellular lysates, whole cells, and even whole organisms. 19F Nuclear Magnetic Resonance (NMR) spectroscopy is a valuable tool in medicinal chemistry driving early lead discovery efforts, and 19F Magnetic Resonance Imaging (MRI) has been applied in proof-of-principle studies to cell tracking and for molecular imaging of biomarkers in preclinical models. In nuclear medicine, positron emission tomography (PET) is a powerful non-invasive imaging technique that can visualise whole organisms providing that one can access 18F-labelled mall molecules, peptides and proteins. 18F is a cyclotron-produced radioisotope that presents a range of advantageous physical properties including reasonable half life (just under two hours) and clean decay profile (97% positron emission). These remarkable applications have encouraged many scientists to develop methodologies to access fluorine-labelled biologics. From a reactivity viewpoint, fluorine is a "challenging"element of the periodic table, that has prevented the development of effective, precise and reproducible direct fluorine-peptide or fluorine-protein bond construction. To circumvent these difficulties, chemists have opted to modify firstly the peptide or protein to be labelled with a so-called prosthetic that is amenable to fluorine incorporation. However, such prosthetics can modify the structure of the native biologics to such a degree that function is affected in a detrimental way. As a result, direct, effective, precise and easy to implement methods for protein fluorination are in urgent demand.

Here, we propose to develop such a method to precisely fluorine-label peptide and proteins by pooling the expertise of the two applicants who together have extensive knowledge of fluorine chemistry and peptide/protein chemistry within the context of chemical biology and chemical medicine. The novelty of our approach is the development of a linker-prosthetic free technology that modifies peptide and proteins with the unnatural CH2SCF3 and CH2CF3 side-chains for application in bioimaging with the minimal symmetrical multi-fluorine group that is possible: CF3. Our proposed late stage 19F- and 18F-trifluoromethylation of peptide and proteins that relies on the availability of so-called 19F- and18F-Umemoto reagents is not only highly novel but could prove transformatory to imaging science as a tool.

Postranslational modification of proteins expands their structural and functional capabilities beyond those directly specified by the genetic code. In this context, access to precisely fluorine-modified proteins is incredibly useful for application in 19F NMR/MRI and in 18F PET bioimaging. Current methods towards fluorine-labelling of peptide and proteins employ often bulky prosthetics that could affect in a detrimental way the function of the biologics under investigation. Moreover, some fluorine markers suffer from stability problems, poor physicochemical properties, and long retention times of degradation products in the body, largely due to the instability issues of current fluorine labeling methods. The proposed research consists of developing novel F-labeling technologies that can serve both to label and to fine-tune the properties of 'biologics' both for superior bioimaging and function. We have designed a "zero-size zero background" method that will allow for direct precise and site-selective protein-CF3 bond construction. Validation studies will use readily available 19F prior to 18F radiochemistry enabling at each stage the generation of diversely applicable tools (NMR, MRI, PET). Our trifluoromethylation strategy targets cysteine or dehydroalanine residue, and will control both chemical reactivity and site-selectivity; these stringent conditions will be met with the availability of 18F-trifluoromethylation reagents of tunable reactivity. The novelty of our approach is the development of a linker-free technology that modifies peptide and proteins with the unnatural CH2SCF3 and CH2CF3 side-chains for application in bioimaging as the minimal symmetrical multi-fluorine group that is possible: CF3. The proposed late stage 19F- and 18F-trifluoromethylation of peptide and proteins with CF3-reagents, if successful, would represent a new departure in imaging science.

Economical/Societal Impact: Molecular bioimaging and medicine (MIM) contributes both to UK competitiveness and the quality of life. Accurate prognosis and the use of more efficient therapeutic solutions are essential components of modern medicine to enhance the patient's quality of life and reduce the societal costs of healthcare. A fundamental understanding of the dynamics, kinetics and dysregulation of biological and biochemical processes in vivo is critical for diagnosis, the treatment of a disease and predictions about the efficacy of a defined therapy. In this context, methods for "clean" labeling of peptides and proteins are urgently needed. This proposal offers a new method for 19F- and 18F-labeling of biologics and therefore contributes to the advance of MRI/PET imaging and its downstream benefits to society.

Beneficiaries (who and how): Upon completion of this research, a new trifluoromethylation method for the fluorine labeling of peptide and proteins via cystein and dehydroalanine residues will be made available for application in 19F NMR studies, 19F MRI and 18F PET bioimaging. Our zero size and zero background proposed methodology is distinctive in the sense that it has the potential to be highly site-selective, precise and reproducible, and does not require the use of perturbing linker and prosthetic that can impact detrimentally on biologics' function. Beneficiaries of this research include academics interested in proteins biology, proteins chemistry and in bioimaging, the biopharmaceutical industry that is big and growing rapidly, clinicians of course, and eventually patients. The applicants are well connected at Oxford (Mathematics, Physics and Life Science Division, Medical Science Division), in the UK and worldwide to ensure that potential beneficiaires within academia and industry will be aware of and can access the technology we propose to develop.

Pathway to dissemination: the applicants are committed to share broadly the results of this research with immediate publication and active participation at specialized workshop and broader (inter)national conferences. Whenever possible, presentation to non-chemical scientists regarding applications of our work will take place, with widespread publicity of the success of our science and collaborations (eg press and electronic media). Both applicants have a strong track record demonstrating proactive dissemination of their research.

People: The PDRA on this grant will gain experience in fluorine chemistry, 18F-radiochemistry, synthesis, peptide and proteins chemistry; he or she will be strongly encouraged to attend advanced courses, including business and entrepreneurship, scientific writing and presentation skills offered by the University of Oxford. This should ensure that the project produces a fully experienced PDRA, immediately employable in the scientific sector. Working at the frontiers of a highly interdisciplinary research program, he or she will be in great demand for industrial, teaching and/or academic vacancies.