Stereospecific Enzymatic Conversion of Boronic Acids to Amines.

Boronic acids and esters are highly regarded for their safety, unique reactivity, and versatility in synthesizing a wide range of small molecules, bioconjugates, and materials. They are not exploited in biocatalytic synthesis, however, because enzymes that can make, break, or modify carbon-boron bonds are rare. We wish to combine the advantages of boronic acids and esters for molecular assembly with biocatalysis, which offers the potential for unsurpassed selectivity and efficiency. Here, we introduce an engineered protoglobin nitrene transferase that catalyzes the new-to-nature amination of boronic acids using hydroxylamine. Initially targeting aryl boronic acids, we show that the engineered enzyme can produce a wide array of anilines with high yields and total turnover numbers (up to 99% yield and >4000 TTN), with water and boric acid as the only byproducts. We also demonstrate that the enzyme is effective with bench-stable boronic esters, which hydrolyze in situ to their corresponding boronic acids. Exploring the enzyme's capacity for enantioselective catalysis, we found that a racemic alkyl boronic ester affords an enantioenriched alkyl amine, a transformation not achieved with chemocatalysts. The formation of an exclusively unrearranged product during the amination of a boronic ester radical clock and the reaction's stereospecificity support a two-electron process akin to a 1,2-metallate shift mechanism. The developed transformation enables new biocatalytic routes for synthesizing chiral amines.

Ratiometric Oxygen Sensing with H-NOX Protein Conjugates.

Ratiometric sensors are self-referencing constructs that are functional in cells and tissues, and the read-out is independent of sensor concentration. One strategy for ratiometric sensing is to utilize two-color emission, where one component possesses analyte-dependent emission and the other is independent of analyte concentration, serving as an internal standard. In this way, the intensity ratio of the two components is a quantitative measure of the analyte. In this study, protein-based ratiometric oxygen sensors are prepared using the heme nitric oxide/oxygen-binding protein (H-NOX) from the thermophilic bacterium Caldanaerobacter subterraneus. The native heme cofactor is replaced with a Pd(II) or Pt(II) porphyrin as the oxygen-responsive phosphor. Mutagenesis is performed to incorporate a cysteine residue on the protein surface for thiol/maleimide coupling of the oxygen-insensitive dye, which serves as a Förster resonance energy transfer (FRET) donor for the porphyrin. While both Pd(II)- and Pt(II)-based sensors are responsive over biologically relevant ranges, the Pd sensor exhibits greater sensitivity at lower oxygen concentrations. Together, these sensors represent a new class of protein-based ratiometric oxygen sensors, and the modular platform allows the oxygen sensitivity to be tailored for a specific application. This proof-of-principle study has identified the key considerations and optimal methodologies to develop and subsequently refine protein-based ratiometric oxygen sensors.