Forging the Bioconjugate Toolbox: Development of Novel Probes for Orthogonal and Bioorthogonal Protein Functionalization

The field of bioconjugate chemistry is expansive and rapidly evolving, perennially introducing new strategies for labeling biological entities with high specificity. Advancement of bioconjugate protocols provides researchers with new tools to study complex biological systems, which ultimately expedites the rate of scientific discoveries, and even provides new insights important to development of novel therapeutics. One of the most popular sub-fields of bioconjugate chemistry pertains to modification and functionalization of proteins via small molecular probes for various applications including protein purification, fluorescent imaging, and therapeutic delivery. As such, our group has been dedicated to the development of molecular probes capable of versatile, orthogonal modification of proteins. Furthermore, we aimed to develop systems with reactivity that is triggered under specific chemical environments, which provides additional temporal and proximal selectivity. Herein, we provide an overview of bioconjugate and bioorthogonal protein labeling strategies and their associated applications. Furthermore, we highlight the development of several novel bioconjugate systems capable of site-selective protein modification of either tyrosine, or lysine. More specifically, we note the utility of aryl diazonium ions within bioconjugation, and the evolution and applications of protected aryl diazonium systems including triazabutadienes and triazenes. To that end, we first report the ability to fluorescently label proteins via a one-step protocol using our novel coumarin triazabutadienes in the presence of either mild acid, or UV irradiation. We next introduce a cyclooctyne triazabutadienes capable of rapid modification of proteins with a bioorthogonal handle for bifurcated azide functionalization. Inspired by our work with triazabutadiene, we show the repurposing of phenyldiazenyl triazenes to label proteins in an analogous fashion. Importantly, we reported for the first time, the ability to release aryl diazonium ions from phenyldiazenyl triazenes using mild UV irradiation. In addition to our work with aryl diazonium ions, we expanded our bioconjugate toolbox to include a novel amine reactive scaffold known as MaMa. While its discovery was serendipitous, we took advantage of its unique reactivity within alkaline conditions to label proteins with highly stable modifications in a pH specific manner. Taken together, the strategies and probes reported herein provide bioconjugate chemists with a numerous new option for orthogonal protein functionalization