Protein immobilization confers the advantages of biological systems to a more chemical setting and has applications in catalysis, sensors, and materials development. While numerous immobilization techniques exist, it is optimal to develop a well-defined and chemically stable methodology to allow for full protein function. This paper describes the utilization of unnatural amino acid technologies to introduce bioorthogonal handles in a site-specific fashion for protein immobilization. To develop this approach a range of solid-supports, organic linkers, and protein immobilization sites have been investigated using a GFP reporter system. Overall, a sepharose resin derivatized with propargyl alcohol has afforded the highest yields of immobilized protein. Moreover, an unnatural amino acid residue protein context has been demonstrated, signifying a necessity to consider the protein site of immobilization. Finally, a resin-conferred stabilization was demonstrated in several organic solvents.

Fluorophores based on the BODIPY scaffold are prized for their tunable excitation and emission profiles, mild syntheses, and biological compatibility. Improving the water-solubility of BODIPY dyes remains an outstanding challenge. The development of water-soluble BODIPY dyes usually involves direct modification of the BODIPY fluorophore core with ionizable groups or substitution at the boron center. While these strategies are effective for the generation of water-soluble fluorophores, they are challenging to implement when developing BODIPY-based indicators: direct modification of BODIPY core can disrupt the electronics of the dye, complicating the design of functional indicators; and substitution at the boron center often renders the resultant BODIPY incompatible with the chemical transformations required to generate fluorescent sensors. In this study, we show that BODIPYs bearing a sulfonated aromatic group at the meso position provide a general solution for water-soluble BODIPYs. We outline the route to a suite of 5 new sulfonated BODIPYs with 2,6-disubstitution patterns spanning a range of electron-donating and -withdrawing propensities. To highlight the utility of these new, sulfonated BODIPYs, we further functionalize them to access 13 new, BODIPY-based, voltage-sensitive fluorophores (VF). The most sensitive of these BODIPY VF dyes displays a 48% ΔF/F per 100 mV in mammalian cells. Two additional BODIPY VFs show good voltage sensitivity (≥24% ΔF/F) and excellent brightness in cells. These compounds can report on action potential dynamics in both mammalian neurons and human stem cell-derived cardiomyocytes. Accessing a range of substituents in the context of a water-soluble BODIPY fluorophore provides opportunities to tune the electronic properties of water-soluble BODIPY dyes for functional indicators.

The utilization of unnatural amino acids (UAAs) in bioconjugations is ideal due to their ability to confer a degree of bioorthogonality and specificity. In order to elucidate optimal conditions for the preparation of bioconjugates with UAAs, we synthesized 9 UAAs with variable methylene tethers (2–4) and either an azide, alkyne, or halide functional group. All 9 UAAs were then incorporated into green fluorescent protein (GFP) using a promiscuous aminoacyl-tRNA synthetase. The different bioconjugations were then analyzed for optimal tether length via reaction with either a fluorophore or a derivatized resin. Interestingly, the optimal tether length was found to be dependent on the type of reaction. Overall, these findings provide a better understanding of various parameters that can be optimized for the efficient preparation of bioconjugates.