Advancing bioconjugation through the design and synthesis of fluorogenic tools
Abstract
Bioconjugation is a foundational strategy across molecular biology, chemical biology, and diagnostic imaging, enabling the selective modification of biomolecules for visualization and tracking of labeling, molecular interactions, and reaction kinetics. Continued demand for improved bioconjugation methodologies has driven the development of innovative fluorescent tools. This dissertation aims to investigate the integration of fluorogenic strategies into a hetero- and a trifunctional crosslinker for bioconjugation. The first linker presented in this work can monitor the conjugation of two molecules of interest via a fluorescence “turn-on” resulting from hydrazone and oxime formation. The utility is demonstrated by tracking the conjugation of DOTA, a macrocyclic chelator used in radiopharmaceuticals, to a peptide of interest, however, it can be applied in alternative contexts such as heterobivalent applications. The second is a trifunctional linker that combines a near infrared (NIR) fluorophore with a DTPA derivative for bioconjugation to a monoclonal antibody for improved diagnostic imaging and accurate chelator-to-antibody ratio determination. By integrating fluorescence with nuclear imaging, these constructs merge the sensitivity of radiotracers and the resolution of fluorescence into single radiopharmaceuticals, while streamlining synthesis through a reduced number of conjugation steps.