The broad goal of this proposal is to use fast in vivo click chemistries to enable biomedical imaging with affinity ligands such as monoclonal antibodies. The candidate, Neal Devaraj, has received extensive training in the field of chemistry and now wishes to transition to biomedical research. His immediate goal is to further develop skill sets through immersion in translational research and to make significant contributions to the field of in vivo imaging. Long term, the candidate wishes to make a full transition from chemist to independent biomedical research scientist. The candidate proposes to carry out the proposal at the Harvard/MGH Center for Systems Biology under the mentorship of Dr. Ralph Weissleder MD PhD. In addition to the research proposal, his career development plan includes supplemental coursework, mentored readings, journal clubs, attendance and presentation at several seminars and conferences, practical exposure to the imaging sciences, and close formal interaction with both his mentor and an advisory committee consisting of Prof. Robert Langer, Prof. Moungi Bawendi, and Prof. Marcelo Di Carli. The research plan aims to develop platform chemistries to allow site specific in vivo targeting of affinity ligands once they have homed to their target (i.e. decoupled administration of affinity ligand and imaging reporter). Though a large number of targeting ligands have been developed to date, target-reporter constructs have inherent pharmacokinetics which are undesirable for in vivo imaging. To achieve site specific coupling, the candidate proposes to work with the recently introduced tetrazine/strained dienophile cycloaddition. This extremely rapid reaction is highly selective, irreversible, and can be performed in physiological conditions. The proposal aims to build on this cutting-edge technology and to develop generic in vivo click reactions for molecular imaging with positron emission tomography (PET). The specific aims are to 1) develop and characterize a panel of fast click reactions for radioisotope labeling and 2) pursue in vivo click labeling for biomedical imaging. To test this method, the candidate will create dienophile affinity ligands against A33 glycoprotein, a surface persistent marker that is highly expressed in human colon cancers. Mice bearing primary and metastatic colon cancer will be injected with dienophile affinity ligands. After several days of clearance, a small, readily cleared tetrazine imaging probe will be administered. Imaging with PET will be used to quantify probe localization and the method will be optimized and compared to other methods such as direct labeling of affinity ligands and the metabolic reporter 18fluorodeoxglucose. If successful, the development of in vivo click chemistry for biomedical imaging will provide a nearly universal methodology for in vivo imaging of virtually any targeting affinity ligand.