Genetically introducing novel chemical bonds into proteins provides innovative avenues for biochemical research, protein engineering, and biotherapeutic applications. Recently, latent bioreactive unnatural amino acids (Uaas) have been incorporated into proteins to covalently target natural residues through proximity-enabled reactivity. Aryl fluorosulfate is particularly attractive due to its exceptional biocompatibility and multitargeting capability via sulfur­(VI) fluoride exchange (SuFEx) reaction. Thus far, fluorosulfate-l-tyrosine (FSY) is the only aryl fluorosulfate-containing Uaa that has been genetically encoded. FSY has a relatively rigid and short side chain, which restricts the diversity of proteins targetable and the scope of applications. Here we designed and genetically encoded a new latent bioreactive Uaa, fluorosulfonyloxybenzoyl-l-lysine (FSK), in E. coli and mammalian cells. Due to its long and flexible aryl fluorosulfate-containing side chain, FSK was particularly useful in covalently linking protein sites that are unreachable with FSY, both intra- and intermolecularly, in vitro and in live cells. In addition, we created covalent nanobodies that irreversibly bound to epidermal growth factor receptors (EGFR) on cells, with FSK and FSY targeting distinct positions on EGFR to counter potential mutational resistance. Moreover, we established the use of FSK and FSY for genetically encoded chemical cross-linking to capture elusive enzyme–substrate interactions in live cells, allowing us to target residues aside from Cys and to cross-link at the binding periphery. FSK complements FSY to expand target diversity and versatility. Together, they provide a powerful, genetically encoded, latent bioreactive SuFEx system for creating covalent bonds in diverse proteins in vitro and in vivo, which will be widely useful for biological research and applications.

Genetically introducing covalent bonds into proteins in vivo with residue specificity is affording innovative ways for protein research and engineering, yet latent bioreactive unnatural amino acids (Uaas) genetically encoded to date react with one to few natural residues only, limiting the variety of proteins and the scope of applications amenable to this technology. Here we report the genetic encoding of (2R)-2-amino-3-fluoro-3-(4-((2-nitrobenzyl)oxy) phenyl) propanoic acid (FnbY) in E. coli and mammalian cells. Upon photoactivation, FnbY generated a reactive quinone methide (QM), which selectively reacted with nine natural amino acid residues placed in proximity in proteins directly in live cells. In addition to Cys, Lys, His and Tyr, photoactivated FnbY also reacted with Trp, Met, Arg, Asn and Gln, which are inaccessible with existing latent bioreactive Uaas. FnbY thus dramatically expanded the number of residues for covalent targeting in vivo. QM has longer half-life than the intermediates of conventional photo-crosslinking Uaas, and FnbY exhibited crosslinking efficiency higher than p-azido-phenylalanine. The photoactivatable and multi-targeting reactivity of FnbY with selectivity toward nucleophilic residues will be valuable for addressing diverse proteins and broadening the scope of applications through exploiting covalent bonding in vivo for chemical biology, biotherapeutics, and protein engineering.

Selenoproteins containing the 21 amino acid selenocysteine (Sec) exist in all three kingdoms of life and play essential roles in human health and development. The distinct low p K, high reactivity, and redox property of Sec also afford unique routes to protein modification and engineering. However, natural Sec incorporation requires idiosyncratic translational machineries that are dedicated to Sec and species-dependent, which makes it challenging to recombinantly prepare selenoproteins with high Sec specificity. As a consequence, the function of half of human selenoproteins remains unclear, and Sec-based protein manipulation has been greatly hampered. Here we report a new general method enabling the site-specific incorporation of Sec into proteins in E. coli. An orthogonal tRNA-ASecRS was evolved to specifically incorporate Se-allyl selenocysteine (ASec) in response to the amber codon, and the incorporated ASec was converted to Sec in high efficiency through palladium-mediated cleavage under mild conditions compatible with proteins and cells. This approach completely obviates the natural Sec-dedicated factors, thus allowing various selenoproteins, regardless of Sec position and species source, to be prepared with high Sec specificity and enzyme activity, as shown by the preparation of human thioredoxin and glutathione peroxidase 1. Sec-selective labeling in the presence of Cys was also demonstrated on the surface of live E. coli cells. The tRNA-ASecRS pair was further used in mammalian cells to incorporate ASec, which was converted into Sec by palladium catalyst in cellulo. This robust and versatile method should greatly facilitate the study of diverse natural selenoproteins and the engineering of proteins in general via site-specific introduction of Sec.

Small‐molecule crosslinkers are invaluable for probing biomolecular interactions and for crosslinking mass spectrometry. Existing chemical crosslinkers target only a small selection of amino acids, while conventional photo‐crosslinkers target almost all residues non‐specifically, complicating data analysis. Herein, we report photocaged quinone methide (PQM)‐based crosslinkers that target nine nucleophilic residues through Michael addition, including Gln, Arg, and Asn, which are inaccessible to existing chemical crosslinkers. PQM crosslinkers were used in vitro, in Escherichia coli, and in mammalian cells to crosslink dimeric proteins and endogenous membrane receptors. The heterobifunctional crosslinker NHQM could crosslink proteins to DNA, for which few crosslinkers exist. The photoactivatable reactivity of these crosslinkers and their ability to target multiple amino acids will enhance the use of chemical crosslinking for studies of protein–protein and protein–DNA networks and for structural biology.

Site-specific modification of proteins with functional molecules provides powerful tools for researching and engineering proteins. Here we report a new chemical conjugation method which photocages highly reactive but chemically selective moieties, enabling the use of protein-inert amines for selective protein modification. New amino acids FnbY and FmnbY, bearing photocaged quinone methides (QMs), were genetically incorporated into proteins. Upon light activation, they generated highly reactive QM, which rapidly reacted with amine derivatives. This method features a rare combination of desired properties including fast kinetics, small and stable linkage, compatibility with low temperature, photocontrollability, and widely available reagents. Moreover, labeling via FnbY occurs on the β-carbon, affording the shortest linkage to protein backbone which is essential for advanced studies involving orientation and distance. We installed various functionalities onto proteins and attached a spin label as close as possible to the protein backbone, achieving high resolution in double electron−electron paramagnetic resonance distance measurements.