The Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA Pyl are extensively used to add non-canonical amino acids (ncAAs) to the genetic code of bacterial and eukaryotic cells. However, new ncAAs often require a cumbersome de novo engineering process to generate an appropriate PylRS/tRNA Pyl pair. We here report a strategy to predict a PylRS variant with novel properties. The designed polyspecific PylRS variant HpRS catalyzes the aminoacylation of 31 structurally diverse ncAAs bearing clickable, fluorinated, fluorescent, and for the first time biotinylated entities. Moreover, we demonstrated a site-specific and copper-free conjugation strategy of a nanobody by the incorporation of biotin. The design of polyspecific PylRS variants offers an attractive alternative to existing screening approaches and provides insights into the complex PylRS-substrate interactions.

Artificial metalloenzymes (ArMs) have high potential in biotechnological applications as they combine the versatility of transition-metal catalysis with the substrate selectivity of enzymes. An ideal host protein should allow high-yield recombinant expression, display thermal and solvent stability to withstand harsh reaction conditions, lack nonspecific metal-binding residues, and contain a suitable cavity to accommodate the artificial metal site. Moreover, to allow its rational functionalization, the host should provide an intrinsic reporter for metal binding and structural changes, which should be readily amendable to high-resolution structural characterization. Herein, we present the design, characterization, and de novo functionalization of a fluorescent ArM scaffold, named mTFP*, that achieves these characteristics. Fluorescence measurements allowed direct assessment of the scaffold’s structural integrity. Protein X-ray structures and transition metal Förster resonance energy transfer (tmFRET) studies validated the engineered metal coordination sites and provided insights into metal binding dynamics at the atomic level. The implemented active metal centers resulted in ArMs with efficient Diels–Alderase and Friedel–Crafts alkylase activities.

A mild aqueous protocol for palladium catalyzed Miyaura borylation of aryl iodides, aryl bromides and aryl chlorides with tetrahydroxydiboron (BBA) as a borylating agent is developed. The developed methodology requires low catalyst loading of Bedford-type palladacycle catalyst (0.05 mol %) and works best under mild reaction conditions at 40 °C in short time of 6 h in water. In addition, our studies show that for Miyaura borylation using BBA in aqueous condition, maintaining a neutral reaction pH is very important for reproducibility and higher yields of corresponding borylated products. Moreover, our protocol is applicable for a broad range of aryl halides, corresponding borylated products are obtained in excellent yields up to 93% with 29 examples demonstrating its broad utility and functional group tolerance.

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