Nature uses disulfide bonds and some unusual inter-strand chemical crosslinks to rigidify protein structure and achieve specific function. Inspired by Nature, protein scientists have employed genetic code expansion technology to introduce latent electrophilic amino acids into protein structure for specific crosslinking with nearby nucleophilic residues via the proximity-driven reactions. Herein, we describe the experimental protocol for recombinant production of orthogonal crosslinked monobodies afforded by a genetically encoded β-lactam-lysine (BeLaK) and their characterization by gel electrophoresis and mass spectrometry. When introduced to the N-terminal β-strand of a series of supercharged monobodies, BeLaK enables efficient inter-strand crosslinking with a proximal lysine on a neighboring β-strand. Compared to its non-crosslinked counterpart, a BeLaK-crosslinked, +18-charged monobody showed higher thermal stability and greater cell permeability. The discovery of this BeLaK-crosslinked, rigidified immunoglobulin fold should facilitate the design of cell-permeable monobodies as potential protein-based therapies targeting the intracellular signaling proteins.

Bioorthogonal reactions offer a powerful tool for site-specific labeling of biomolecules in living systems. Among them, the recently reported hydrazonyl sultone (HS)-bicyclo[6.1.0]non-4-yne (BCN) ligation reaction stands out for its fast reaction kinetics and tunable aqueous stability. Herein, we describe the experimental protocols of using HS-BCN ligation for site-specific modification of a recombinant nanobody in vitro and a G protein-coupled receptor (GPCR) on a live mammalian cell surface. These protocols include the genetic encoding of BCN-lysine (BCNK) into the target protein, bioorthogonal modification of the BCNK-encoded proteins, and characterization of the reaction rate and selectivity. Together with the robust genetic encoding of the strained alkyne BCN in any protein structure, the HS-BCN ligation reaction promises to expand the capabilities of bioorthogonal chemistry to enable facile modifications of domain antibodies in vitro for diagnostic applications and selective fluorescent labeling of GPCRs for biophysical studies of receptor dynamics in live cells.

Dysregulated reactive oxygen species (ROS) are implicated in various diseases, positioning NADPH oxidase enzymes (NOXs) as attractive therapeutic targets. However, progress in tool compound discovery has been hindered by rational optimization strategies that can improve isoform selectivity. Starting from a nonselective but well-behaved NOX inhibitor (VAS2870), we have discovered a first-in-class NOX5 selective inhibitor through minor functionalization on a benzoxazolethiol moiety, which is released upon covalent modification to the target enzyme. These unexpected findings showcase a unique strategy for optimizing SNAr covalent inhibitors and offer new avenues for the development of isoform-selective NOX inhibitors.

Domain antibodies such as monobodies provide an attractive immunoglobin fold for evolving high-affinity protein binders targeting the intracellular proteins implicated in cell signalling. However, it remains a challenge to endow cell permeability to these small and versatile protein binders. Here, we report a streamlined approach combining orthogonal crosslinking afforded by a genetically encoded β-lactam-lysine (BeLaK) and genetic supercharging to generate cell-penetrating monobodies. When introduced to the N-terminal β-strand of a series of supercharged monobodies, BeLaK enabled efficient inter-strand crosslinking with the neighbouring lysine. Compared to its non-crosslinked counterpart, a BeLaK-crosslinked, +18-charged monobody exhibited enhanced thermostability and greater cellular uptake at 40 nM. Moreover, this structurally rigidified, supercharged monobody inhibited ERK1/2 phosphorylation in KYSE-520 esophageal cancer cell line at sub-micromolar concentration, indicating significant endosomal escape after endocytosis. Together, the discovery of this BeLaK-encoded, rigidified immunoglobin fold should facilitate the design of cell-penetrating monobodies targeting intracellular signalling proteins.

We report a phosphine-mediated cyclodehydration procedure for the facile synthesis of N-aryl/alkyl-substituted hydrazonyl sultones, a class of bioorthogonal reagents, from the readily prepared vicinal sulfo-acyl hydrazides in moderate to good yields. The aqueous stability and bioorthogonal reactivity of these hydrazonyl sultones toward bicyclo[6.1.0]non-4-yn-9-ylmethanol were investigated, revealing key structural requirements for hydrazonyl sultones to possess balanced stability and reactivity.

We report the synthesis of a series of hydrazonyl sultones (HS) containing an ortho-CF3 group, a five- or six-membered sultone ring, and a varying N-aryl substituent, and characterization of their aqueous stability and reactivity toward bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) in a 1,3-dipolar cycloaddition reaction. To avoid the purification of highly polar intermediates, we employed two protecting groups in our synthetic schemes. Most HS were obtained in moderate to good yields with optimized reaction conditions. The X-ray structure analysis of two HS revealed that the partially negative-charged fluorine atoms in CF3 electrostatically shield the electrophilic nitrile imine (NI) center from a nucleophilic attack, underpinning their extraordinary aqueous stability. In addition, the N-aryl substituents further modulate HS reactivity and stability, with the electron-rich six-membered HS displaying excellent aqueous stability and increased cycloaddition reactivity. The utility of these improved HS reagents was demonstrated through fast and selective modification of a BCNK-encoded nanobody with second-order rate constants as high as 1488 M-1 s-1 in phosphate-buffered saline--ethanol (9:1), representing the fastest HSB--CN ligation reported in the literature.

Compared to the disulfide bond, other naturally occurring intramolecular crosslinks have received little attention, presumably due to their rarity in the vast protein space. Here we presented examples of natural non-disulfide crosslinks, which we refer to as orthogonal crosslinks, emphasizing their effect on protein topology and function. We summarize recent efforts on expanding orthogonal crosslinks by using either the enzymes that catalyze protein circularization or the genetic code expansion strategy to add electrophilic amino acids site-specifically in proteins. The advantages and disadvantages of each method are discussed, along with their applications to generate novel protein topology and function. In particular, we highlight our recent work on spontaneous orthogonal crosslinking, in which a carbamate-based crosslink was generated in situ, and its applications in designing orthogonally crosslinked domain antibodies with their topology-mimicking bacterial adhesins.

Domain antibodies such as monobodies provide an attractive immunoglobin fold for evolving high-affinity binders targeting the intracellular proteins implicated in cell signaling. However, it remains challenging to endow cell permeability to these small and versatile protein binders. Here, we report a streamlined strategy combining orthogonal crosslinking mediated by a genetically encoded beta-lactam-lysine (BeLaK) and genetic supercharging to generate cell-penetrating monobodies. When BeLaK was introduced site-specifically to the N-terminal beta-strand of a panel of supercharged monobodies, it enabled efficient interstrand crosslinking with a nearby lysine, generating the rigidified analogs. Compared to the non-crosslinked counterparts, the BeLaK-crosslinked supercharged monobodies exhibited higher thermostability and enhanced cellular uptake at concentrations as low as 40 nM. Most significantly, a +11 charged, orthogonally crosslinked monobody showed significant endosomal escape after endocytosis. The discovery of this stabilized immunoglobin fold should facilitate the design of cell-permeable domain antibodies for targeting intracellular proteins.

Here we report the design and synthesis of a new class of bioorthogonal reagents called hydrazonyl sultones (HS) that serve as stable tautomers of highly reactive nitrile imines (NI). Compared to the photogenerated NI, HS display a broad range of aqueous stability and tunable reactivity in a 1,3-dipolar cycloaddition reaction, depending on substituents, sultone ring structure, and solvent conditions. DFT calculations have provided vital insights into the HS → NI tautomerism, including a base-mediated anionic tautomerization pathway and a small activation barrier. Comparative kinetic analysis of tetrazole vs HS-mediated cycloadditions reveals that a tiny fraction of the reactive NI (∼15 ppm) is present in the tautomeric mixture, underpinning the extraordinary stability of the six-membered HS. We further demonstrate the utilities of HS in selective modification of bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN)-lysine-containing nanobodies in phosphate buffered saline and fluorescent labeling of a BCN-lysine-encoded transmembrane glucagon receptor on live cells.

Light-induced bioorthogonal reactions offer spatiotemporal control over selective biomolecular labeling. This review covers the recent advances in the design of photo-activatable reagents for bioorthogonal conjugation reactions in living systems. These reagents are stable in the absence of light, but transformed into reactive species upon light illumination, which then undergo rapid ligation reactions. The light wavelength has been tuned from ultraviolet to near infrared to enable efficient photo-activation in reactions in deep tissues. The most prominent photo-activatable reagents are presented, including tetrazoles, tetrazines, 9,10-phenanthrenequinone, diarylsydnones, and others. A particular focus is on the strategies for improving reaction kinetics and biocompatibility accomplished through careful molecular engineering. The utilities of these photo-activatable reagents are illustrated through a broad range of biological applications, including in vivo protein labeling, positron emission tomography (PET) imaging, responsive hydrogels, and fluorescence microscopy. The further development and optimization of these biocompatible photo-activatable reagents should lead to new chemical biology strategies for studying biomolecular structure and function in living systems.

Recent clinical trials have revealed that the chimeric peptide hormones simultaneously activating glucagon-like peptide-1 receptor (GLP-1R) and glucose-dependent insulinotropic polypeptide receptor (GIPR) demonstrate superior efficacy in glycemic control and body weight reduction, better than those activating the GLP-1R alone. However, the linear peptide-based GLP-1R/GIPR dual agonists are susceptible to proteolytic cleavage by common digestive enzymes present in the gastrointestinal tract and thus not suitable for oral administration. Here, we report the design and synthesis of biaryl-stapled peptides, with and without fatty diacid attachment, that showed potent GLP-1R/GIPR dual agonist activities. Compared to a linear peptide dual agonist and semaglutide, the biaryl-stapled peptides displayed drastically improved proteolytic stability against the common digestive enzymes. Furthermore, two stapled peptides showed excellent efficacy in an oral glucose tolerance test in mice, owing to their potent receptor activity in vitro and good pharmacokinetics exposure upon subcutaneous injection. By exploring a more comprehensive set of biaryl staplers, we expect that this stapling method could facilitate the design of the stapled peptide-based dual agonists suitable for oral administration.

Here we report the design of N2 -carboxy-4-aryl-1,2,3-triazole-lysines (CATKs) and their site-specific incorporation into proteins via genetic code expansion. When introduced into the protein dimer interface, CATKs permitted spontaneous, proximity-driven, site-selective crosslinking to generate covalent protein dimers in living cells, with phenyl-bearing CATK-1 exhibiting high reactivity toward the proximal Lys and Tyr. Furthermore, when introduced into the N-terminal β-strand of either a single-chain VHH antibody or a supercharged monobody, CATK-1 enabled site-specific, inter-strand, orthogonal crosslinking with a proximal Tyr located on the opposing β-strand. Compared with a non-crosslinked monobody, the orthogonally crosslinked monobody displayed improved cellular uptake and enhanced proteolytic stability against an endosomal enzyme. The robust crosslinking reactivity of CATKs should facilitate the design of novel protein topologies with improved physicochemical properties.

Fast bioorthogonal reactions are sought after because of their superior performance in labeling low-abundance biomolecules in native cellular environments. An attractive strategy to increase reaction kinetics is to access the reactive intermediates through photochemical activation. To this end, significant progress was made in the last few years in harnessing two highly reactive intermediates-nitrile imine and tetrazine-generated through photoinduced ring rupture and catalytic photooxidation, respectively. The efficient capture of these reactive intermediates by their cognate reaction partners has enabled bioorthogonal fluorescent labeling of biomolecules in live cells.

To study GPCR conformational dynamics in live cells, here we report an integrated approach combining enzymatic SNAP-tagging with bioorthogonal chemistry for dual fluorescent labeling of GLP-1R. The resulting GLP-1R conformational biosensors permit a FRET-based analysis of the receptor subdomain movement in response to ligand stimulation in live cells.

Bioorthogonal chemistry represents a class of high-yielding chemical reactions that proceed rapidly and selectively in biological environments without side reactions towards endogenous functional groups. Rooted in the principles of physical organic chemistry, bioorthogonal reactions are intrinsically selective transformations not commonly found in biology. Key reactions include native chemical ligation and the Staudinger ligation, copper-catalysed azide-alkyne cycloaddition, strain-promoted [3+2] reactions, tetrazine ligation, metal-catalysed coupling reactions, oxime and hydrazone ligations as well as photoinducible bioorthogonal reactions. Bioorthogonal chemistry has significant overlap with the broader field of 'click chemistry' - high-yielding reactions that are wide in scope and simple to perform, as recently exemplified by sulfuryl fluoride exchange chemistry. The underlying mechanisms of these transformations and their optimal conditions are described in this Primer, followed by discussion of how bioorthogonal chemistry has become essential to the fields of biomedical imaging, medicinal chemistry, protein synthesis, polymer science, materials science and surface science. The applications of bioorthogonal chemistry are diverse and include genetic code expansion and metabolic engineering, drug target identification, antibody-drug conjugation and drug delivery. This Primer describes standards for reproducibility and data deposition, outlines how current limitations are driving new research directions and discusses new opportunities for applying bioorthogonal chemistry to emerging problems in biology and biomedicine.

The third intracellular loop (ICL3) in the cytosolic face of glucagon receptor (GCGR) experiences significant conformational transition during receptor activation. It thus offers an attractive site for the introduction of organic fluorophores in our efforts to construct fluorescence-based GPCR biosensors. Herein, we report our confocal microscopic study of intracellular fluorescent labeling of ICL3 using a bioorthogonal chemistry strategy. Our approach involves the site-specific introduction of a strained alkene amino acid into the ICL3 through genetic code expansion, followed by a highly specific inverse electron-demand Diels-Alder reaction with the fluorescent tetrazine probes. Among the three strained alkene amino acids examined, both SphK and 2'-aTCOK offered successful fluorescent labeling of GCGR ICL3 with the appropriate tetrazine probes. At the same time, 4'-TCOK gave high background fluorescence due to its intracellular retention. The fluorescent tetrazine probes were designed following a computational model for background-free intracellular fluorescent labeling; however, their performance varied significantly in live-cell imaging as the strong non-specific signals interfered with the specific ones. Among all GCGR ICL3 mutants bearing a strained alkene, the H339SphK/2'-aTCOK mutants provided the best reaction partners for the BODIPY-Tz1/4 reagents in the bioorthogonal labeling reactions. The results from this study highlight the challenges in identifying bioorthogonal reactant pairs suitable for intracellular labeling of low-abundance receptors in live-cell imaging studies.

Here we report the design of a superfast bioorthogonal ligation reactant pair comprising a sterically shielded, sulfonated tetrazole and bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN). The design involves placing a pair of water-soluble N-sulfonylpyrrole substituents at the C-phenyl ring of diphenyltetrazoles to favor the photoinduced cycloaddition reaction over the competing nucleophilic additions. First-principles computations provide vital insights into the origin of the tetrazole-BCN cycloaddition's superior kinetics compared to the tetrazole-spirohexene cycloaddition. The tetrazole-BCN cycloaddition also enabled rapid bioorthogonal labeling of glucagon receptors on live cells in as little as 15 s.

In this method paper, we describe the protocols for selective labeling of GCGR, a member of the class B GPCR family regulating glucose homeostasis, in live cells. A two-step procedure is presented in which a strained alkene chemical reporter is inserted into any desired location within the GPCR in the first step, followed by a robust bioorthogonal ligation reaction with a fluorophore-conjugated tetrazine or tetrazole reagent in the second step. The amber codon suppression strategy was adopted for site-specific incorporation of the strained alkene reporter, either spirohexene or trans-cyclooctene, in HEK293T cells. Subsequently, the inverse electron-demand Diels-Alder reaction with an AF647-conjugated 3,6-di (2-pyridyl)-S-tetrazine (DpTz) was performed with the alkene-encoded GCGR on live-cell surface. Alternatively, a photo-induced cycloaddition with a Cy5-conjugated, sterically shielded tetrazole was carried out, giving rise to faster fluorescent labeling along with excellent selectivity. Owing to their robust reaction kinetics and excellent chemoselectivity, the bioorthogonal labeling protocols described here could be readily adapted to labeling any accessible protein targets, e.g., membrane proteins, in live cells.

The merging of click chemistry with discrete photochemical processes has led to the creation of a new class of click reactions, collectively known as photoclick chemistry. These light-triggered click reactions allow the synthesis of diverse organic structures in a rapid and precise manner under mild conditions. Because light offers unparalleled spatiotemporal control over the generation of the reactive intermediates, photoclick chemistry has become an indispensable tool for a wide range of spatially addressable applications including surface functionalization, polymer conjugation and cross-linking, and biomolecular labeling in the native cellular environment. Over the past decade, a growing number of photoclick reactions have been developed, especially those based on the 1,3-dipolar cycloadditions and Diels-Alder reactions owing to their excellent reaction kinetics, selectivity, and biocompatibility. This review summarizes the recent advances in the development of photoclick reactions and their applications in chemical biology and materials science. A particular emphasis is placed on the historical contexts and mechanistic insights into each of the selected reactions. The in-depth discussion presented here should stimulate further development of the field, including the design of new photoactivation modalities, the continuous expansion of λ-orthogonal tandem photoclick chemistry, and the innovative use of these unique tools in bioconjugation and nanomaterial synthesis.