Expanding and reprogramming the genetic code of cells and animals.

Genetic code expansion and reprogramming enable the site-specific incorporation of diverse designer amino acids into proteins produced in cells and animals. Recent advances are enhancing the efficiency of unnatural amino acid incorporation by creating and evolving orthogonal ribosomes and manipulating the genome. Increasing the number of distinct amino acids that can be site-specifically encoded has been facilitated by the evolution of orthogonal quadruplet decoding ribosomes and the discovery of mutually orthogonal synthetase/tRNA pairs. Rapid progress in moving genetic code expansion from bacteria to eukaryotic cells and animals (C. elegans and D. melanogaster) and the incorporation of useful unnatural amino acids has been aided by the development and application of the pyrrolysyl-transfer RNA (tRNA) synthetase/tRNA pair for unnatural amino acid incorporation. Combining chemoselective reactions with encoded amino acids has facilitated the installation of posttranslational modifications, as well as rapid derivatization with diverse fluorophores for imaging.

Bioorthogonal chemical labeling of endogenous neurotransmitter receptors in living mouse brains.

Neurotransmitter receptors are essential components of synapses for communication between neurons in the brain. Because the spatiotemporal expression profiles and dynamics of neurotransmitter receptors involved in many functions are delicately governed in the brain, in vivo research tools with high spatiotemporal resolution for receptors in intact brains are highly desirable. Covalent labeling by chemical reaction (chemical labeling) of proteins without genetic manipulation is now a powerful method for analyzing receptors in vitro. However, selective target receptor labeling in the brain has not yet been achieved. This study shows that ligand-directed alkoxyacylimidazole (LDAI) chemistry can be used to selectively tether synthetic probes to target endogenous receptors in living mouse brains. The reactive LDAI reagents with negative charges were found to diffuse well over the whole brain and could selectively label target endogenous receptors, including AMPAR, NMDAR, mGlu1, and GABAAR. This simple and robust labeling protocol was then used for various applications: three-dimensional spatial mapping of endogenous receptors in the brains of healthy and disease-model mice; multi-color receptor imaging; and pulse-chase analysis of the receptor dynamics in postnatal mouse brains. Here, results demonstrated that bioorthogonal receptor modification in living animal brains may provide innovative molecular tools that contribute to the in-depth understanding of complicated brain functions.

Manipulating complex chromatin folding via CRISPR-guided bioorthogonal chemistry.

Precise manipulation of chromatin folding is important for understanding the relationship between the three-dimensional genome and nuclear function. Existing tools can reversibly establish individual chromatin loops but fail to manipulate two or more chromatin loops. Here, we engineer a powerful CRISPR system which can manipulate multiple chromatin contacts using bioorthogonal reactions, termed the bioorthogonal reaction-mediated programmable chromatin loop (BPCL) system. The multiinput BPCL system employs engineered single-guide RNAs recognized by discrete bioorthogonal adaptors to independently and dynamically control different chromatin loops formation without cross-talk in the same cell or to establish hubs of multiway chromatin contacts. We use the BPCL system to successfully juxtapose the pluripotency gene promoters to enhancers and activate their endogenous expression. BPCL enables us to independently engineer multiway chromatin contacts without cross-talk, which provides a way to precisely dissect the high complexity and dynamic nature of chromatin folding.

Comprehensive insight on protein modification by V-type agent: A chemical proteomic approach employing bioorthogonal reaction.

Organophosphorus compounds (OPs) such as chemical agents and pesticides are posing critical threats to civilians due to their irreversible phosphonylation of diverse amino acids residues forming different protein adducts. However, traditional analytical approaches are quite limited in capturing the myriad of post-translational events that affect protein functions, especially in identifying the low-abundance OP adducts. Herein a systematic proteomic strategy based on a typical click-enrich-release-identify bioorthogonal operation was firstly developed by employing an alkynyl-tagged V-type agent probe (AVP) and a biotin-based azido-enrichment linker (BTP-N3 ). AVP targeting peptides from human serum albumin (HSA) or plasma were captured by BTP-N3 via CuAAC click reaction, enriched by streptavidin beads, released by selective alkaline hydrolysis of phenacyl ester bond, and subsequently sequenced by LC-MS/MS. This strategy has helped identifying 1115 unique OP adduction sites on 163 proteins in human plasma, and covers lots of OP adducts that cannot be achieved by traditional detection methods. The comprehensive coverage of novel OP substrates provided a general and sensitive approach to retrospective verification and/or dose assessment of toxic OPs.

Bioorthogonally Activatable Autophagy-Tethering Compounds for Aptamer-Guided Mitochondrial Degradation.

Although macroautophagy degradation targeting chimeras (MADTACs) have been demonstrated to be efficient in a broad spectrum from intracellular proteins to macromolecular complexes such as lipid droplets and the mitochondrion, MADTACs still face degradation of uncontrolled protein in normal cells and cause systemic toxicity, thus limiting their therapeutic applications. Herein, we employ bioorthogonal chemistry to develop a spatially controlled MADTACs strategy. Separated warheads display no activity in normal cells but can be activated by aptamer-based Cu nanocatalyst (Apt-Cu30) in tumors specifically. These in situ synthesized chimera molecules (bio-ATTECs) can degrade the mitochondrion in live tumor cells and subsequently induce autophagic cell death, which has been further demonstrated by lung metastasis melanoma murine models. To the best of our knowledge, this is the first bioorthogonal activated MADTAC in live cells for inducing autophagic tumor cell death, which may promote the development of cell-specific MADTACs for precision therapeutics by avoiding undesired side effects.

Cascade In Situ Self-Assembly and Bioorthogonal Reaction Enable the Enrichment of Photosensitizers and Carbonic Anhydrase Inhibitors for Pretargeted Cancer Theranostics.

We report here a tumor-pretargted theranostic approach for multimodality imaging-guided synergistic cancer PDT by cascade alkaline phosphatase (ALP)-mediated in situ self-assembly and bioorthogonal inverse electron demand Diels-Alder (IEDDA) reaction. Using the enzymatic catalysis of ALP that continuously catalyses the dephosphorylation and self-assembly of trans-cyclooctene (TCO)-bearing P-FFGd-TCO, a high density of fluorescent and magnetic TCO-containing nanoparticles (FMNPs-TCO) can be synthesized and retained on the membrane of tumor cells. They can act as 'artificial antigens' amenable to concurrently capture lately administrated tetrazine (Tz)-decorated PS (775NP-Tz) and carbonic anhydrase (CA) inhibitor (SA-Tz) via the fast IEDDA reaction. This two-step pretargeting process can further induce FMNPs-TCO regrowth into microparticles (FMNPs-775/SA) directly on tumor cell membranes, which is analyzed by bio-SEM and fluorescence imaging. Thus, efficient enrichment of both SA-Tz and 775NP-Tz in tumors can be achieved, allowing to alleviate hypoxia by continuously inhibiting CA activity and improving PDT of tumors. Findings show that subcutaneous HeLa tumors could be completely eradicated and no tumor recurred after irradiation with an 808 nm laser (0.33 W cm-2 , 10 min). This pretargeted approach may be applied to enrich other therapeutic agents in tumors to improve targeted therapy.

Systematic Structural Tuning Yields Hydrazonyl Sultones for Faster Bioorthogonal Protein Modification.

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 purification of highly polar intermediates, we employed two protecting groups in our synthetic schemes. Most HS were obtained in moderate to good yields under optimized reaction conditions. The X-ray crystal 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 1500 M-1 s-1 in phosphate-buffered saline-ethanol (9 : 1), representing the fastest HS-BCN ligation reported in the literature.

Bioorthogonal Probes for Analyzing Non-Enzymatic Post-Translational Modifications.

Non-enzymatic post-translational modifications (nPTMs) have been proposed as indicators of cellular stresses and diseases. Unfortunately, direct assessment of nPTMs in native environment is extremely challenging due to the heterogeneity of adducts and the lack of tagging tools. Given these challenges, bioorthogonal probes (BPs) have been developed for the analysis of nPTMs. The rationality is that BPs could selectively install azides or alkynes into nPTMs as a biorthogonal handle for the following enrichment or tracking. Herein, we review the state-of-art of BPs used for nPTMs studies, clarify their working principles, and highlight how they advance our understanding of the biological functions of nPTMs.

Sulfonyl Diazaborine 'Click' Chemistry Enables Rapid and Efficient Bioorthogonal Labeling.

Finding an ideal bioorthogonal reaction that responds to a wide range of biological queries and applications is of great interest in biomedical applications. Rapid diazaborine (DAB) formation in water by the reactions of ortho-carbonyl phenylboronic acid with α-nucleophiles is an attractive conjugation module. Nevertheless, these conjugation reactions demand to satisfy stringent criteria for bioorthogonal applications. Here we show that widely used sulfonyl hydrazide (SHz) offers a stable DAB conjugate by combining with ortho-carbonyl phenylboronic acid at physiological pH, competent for an optimal biorthogonal reaction. Remarkably, the reaction conversion is quantitative and rapid (k2 >103  M-1 s-1 ) at low micromolar concentrations, and it preserves comparable efficacy in a complex biological milieu. DFT calculations support that SHz facilitates DAB formation via the most stable hydrazone intermediate and the lowest energy transition state compared to other biocompatible α-nucleophiles. This conjugation is extremely efficient on living cell surfaces, enabling compelling pretargeted imaging and peptide delivery. We anticipate this work will permit addressing a wide range of cell biology queries and drug discovery platforms exploiting commercially available sulfonyl hydrazide fluorophores and derivatives.

Proximity-enhanced protein crosslinking through an alkene-tetrazine reaction.

The inverse electron demand Diels-Alder (iEDDA) reaction between a tetrazine and a strained alkene has been widely explored as useful bioorthogonal chemistry for selective labeling of biomolecules. In this work, we exploit the slow reaction between a non-conjugated terminal alkene and a tetrazine, and apply this reaction to achieving a proximity-enhanced protein crosslinking. In one protein subunit, a terminal alkene-containing amino acid was site-specifically incorporated in response to an amber nonsense codon. In another protein subunit, a tetrazine moiety was introduced through the attachment to a cysteine residue. Fast protein crosslinking was achieved due to a large increase in effective molarity of the two reactants that were brought to close proximity by the two interacting protein subunits. Such a proximity-enhanced protein crosslinking is useful for the study of protein-protein interactions.

Discovery of novel protein degraders based on bioorthogonal reaction-driven intracellular self-assembly strategy.

Proteolysis targeting chimera (PROTAC) is a promising therapeutic modality capable of degrading undruggable proteins and overcoming the shortcomings of traditional inhibitors. However, the molecular weight and pharmaceutical properties of PROTACs fall outside of a reasonable range. To overcome the inherent poor druggability of PROTACs, an intracellular self-assembly strategy based on bio-orthogonal reaction was proposed and applied in this study. Herein, two novel classes of intracellular precursors that can self-assemble into protein degraders through bio-orthogonal reactions were explored, including a novel class of E3 ubiquitin ligase ligands bearing tetrazine (E3L-Tz) and target protein ligands incorporated with norbornene (TPL-Nb). These two types of precursors could spontaneously undergo bio-orthogonal reactions in living cells, affording novel PROTACs. Among these precursors, the biological activities of PROTACs formed by target protein ligand with norbornene group (S4N-1) were more potent than others and degrade VEGFR-2, PDGFR-ß and EphB4. The results demonstrated that a highly specific bio-orthogonal reaction driven intracellular self-assembly strategy in living cells could be utilized to improve the degradation activity of PROTACs.

A Short Review of Research Progress on the Synthesis Approaches of Aza-Dibenzocyclooctyne Derivatives.

Cyclooctyne molecules have found wide applications in the strain-promoted azide-alkyne cycloaddition (SPAAC) reactions, which avoid the biotoxicity caused by the use of Cu(I) catalysts. Among the various cyclooctyne systems, dibenzocyclooctyne (DBCO) series have displayed the highest reaction activity. However, the synthesis processes of such structures are time-consuming, which to some extent limit their large-scale development and application. This review has summarized current synthesis routes of two DBCO molecules, aza-dibenzocyclooctyne (DIBAC) and biarylazacyclooctynone (BARAC).

Near Infrared Light-Triggered Photocatalytic Decaging for Remote-Controlled Spatiotemporal Activation in Living Mice.

Spatiotemporal manipulation of biological processes in living animals using noninvasive, remote-controlled stimuli is a captivating but challenging endeavor. Herein, we present the development of a biocompatible photocatalytic technology termed CAT-NIR, which uses external near infrared light (NIR, 740 nm) to trigger decaging reactions in living mice. The Os(II) terpyridine complex was identified as an efficient NIR photocatalyst for promoting deboronative hydroxylation reactions via superoxide generation in the presence of NIR light, resulting in the deprotection of phenol groups and the release of bioactive molecules under living conditions. The validation of the CAT-NIR system was demonstrated through the NIR-triggered rescue of fluorophores, prodrugs as well as biomolecules ranging from amino acids, peptides to proteins. Furthermore, by combining genetic code expansion and computer-aided screening, CAT-NIR could regulate affibody binding to the cell surface receptor HER2, providing a selective cell tagging technology through external NIR light. In particular, the tissue-penetrating ability of NIR light allowed for facile prodrug activation in living mice, enabling noninvasive, remote-controlled rescue of drug molecules. Given its broad adaptability, this CAT-NIR system may open new opportunities for manipulating the functions of bioactive molecules in living animals using external NIR light with spatiotemporal resolution.

A Protocol for Activated Bioorthogonal Fluorescence Labeling and Imaging of 4-Hydroxyphenylpyruvate Dioxygenase in Plants.

4-Hydroxyphenylpyruvate dioxygenase (HPPD) plays a crucial role in the synthesis of nutrients needed to maintain optimal plant growth. Its level is closely linked to the extent of abiotic stress experienced by plants. Moreover, it is also the target of commercial herbicides. Therefore, labeling of HPPD in plants not only enables visualization of its tissue distribution and cellular uptake, it also facilitates assessment of abiotic stress of plants and provides information needed for the development of effective environmentally friendly herbicides. In this study, we created a method for fluorescence labeling of HPPD that avoids interference with the normal growth of plants. In this strategy, a perylene-linked dibenzyl-cyclooctyne undergoes strain-promoted azide-alkyne cycloaddition with an azide-containing HPPD ligand. The activation-based labeling process results in a significant emission enhancement caused by the change in the fluorescent forms from an excimer to a monomer. Notably, this activated bioorthogonal strategy is applicable to visualizing HPPD in Arabidopsis thaliana, and assessing its response to multiple abiotic stresses. Also, it can be employed to monitor in vivo levels and locations of HPPD in crops. Consequently, the labeling strategy will be a significant tool in investigations of HPPD-related abiotic stress mechanisms, discovering novel herbicides, and uncovering unknown biological functions.

Visualization of Protein-Specific Glycation in Living Cells via Bioorthogonal Chemical Reporter.

Due to the lack of suitable chemical tools, probing the protein-specific glycation is highly challenging. Herein, we present a strategy based on glycation chemical reporter and proximity-induced FRET signal readout for visualizing protein-specific glycation in living cells. We first developed a bioorthogonal glucose analogue, 6-azido-6-deoxy-D-glucose (6AzGlc), as a novel glycation chemical reporter. Two types of DNA probes, glycation conversion probe and protein targeting probe, were designed to attach to glycation adducts and target proteins, respectively. After the protein was glycated by 6AzGlc, two DNA probes were sequentially applied to the target protein, triggering proximity-induced FRET signal readout. This strategy was successfully used to visualize glucose glycation of several proteins, including PD-L1 and integrin. More importantly, this strategy allowed us to analyze corresponding biological functions of glycated protein in the native environment.

Genetic Encoding of Cyanopyridylalanine for In-Cell Protein Macrocyclization by the Nitrile-Aminothiol Click Reaction.

Cyanopyridylalanines are non-canonical amino acids that react with aminothiol compounds under physiological conditions in a biocompatible manner without requiring added catalyst. Here we present newly developed aminoacyl-tRNA synthetases for genetic encoding of meta- and para-cyanopyridylalanine to enable the site-specific attachment of a wide range of different functionalities. The outstanding utility of the cyanopyridine moiety is demonstrated by examples of i) post-translational functionalization of proteins, ii) in-cell macrocyclization of peptides and proteins, and iii) protein stapling. The biocompatible nature of the protein ligation chemistry enabled by the cyanopyridylalanine amino acid opens a new path to specific in vivo protein modifications in complex biological environments.

Design and Development of a Bioorthogonal, Visualizable and Mitochondria-Targeted Hydrogen Sulfide (H2 S) Delivery System.

Hydrogen sulfide (H2 S) is an important endogenous gasotransmitter, but the targeted delivery and real-time feedback of exogenous H2 S are still challenging. With the aid of density functional theory (DFT) calculations, we designed a new 1,3-dithiolium-4-olate (DTO) compound, which can react with a strained alkyne via the 1,3-dipolar cycloaddition and the retro-Diels-Alder reaction to generate carbonyl sulfide (COS) as the precursor of H2 S, and a thiophene derivative with turn-on fluorescence. Moreover, the diphenylamino substituent in DTO greatly increases the mitochondrial targeting of this H2 S delivery system. Such a bioorthogonal click-and-release reaction has integrated three functions in one system for the first time: (1) in situ controllable H2 S release, (2) concomitant fluorescence response, and (3) mitochondria-targeted delivery. In addition, we investigated the mitochondrial membrane potential loss alleviation by using this system in H9c2 cells under oxidative stress.

In vivo organic synthesis by metal catalysts.

The metal-catalyzed reactions have given various chemical modifications that could not be achieved through basic organic chemistry reactions. In the past decade, many metal-mediated catalytic systems have carried out different transformations in cellulo, such as decaging of fluorophores, drug release, and protein conjugation. However, translating abiotic metal catalysts for organic synthesis in vivo, including bacteria, zebrafish, or mice, could encounter numerous challenges regarding their biocompatibility, stability, and reactivity in the complicated biological environment. In this review, we categorize and summarize the relevant advances in this research field by emphasizing the system's framework, the design of each transformation, and the mode of action. These studies disclose the massive potential of the emerging field and the significant applications in synthetic biology.

IEDDA: An Attractive Bioorthogonal Reaction for Biomedical Applications.

The pretargeting strategy has recently emerged in order to overcome the limitations of direct targeting, mainly in the field of radioimmunotherapy (RIT). This strategy is directly dependent on chemical reactions, namely bioorthogonal reactions, which have been developed for their ability to occur under physiological conditions. The Staudinger ligation, the copper catalyzed azide-alkyne cycloaddition (CuAAC) and the strain-promoted [3 + 2] azide-alkyne cycloaddition (SPAAC) were the first bioorthogonal reactions introduced in the literature. However, due to their incomplete biocompatibility and slow kinetics, the inverse-electron demand Diels-Alder (IEDDA) reaction was advanced in 2008 by Blackman et al. as an optimal bioorthogonal reaction. The IEDDA is the fastest bioorthogonal reaction known so far. Its biocompatibility and ideal kinetics are very appealing for pretargeting applications. The use of a trans-cyclooctene (TCO) and a tetrazine (Tz) in the reaction encouraged researchers to study them deeply. It was found that both reagents are sensitive to acidic or basic conditions. Furthermore, TCO is photosensitive and can be isomerized to its cis-conformation via a radical catalyzed reaction. Unfortunately, the cis-conformer is significantly less reactive toward tetrazine than the trans-conformation. Therefore, extensive research has been carried out to optimize both click reagents and to employ the IEDDA bioorthogonal reaction in biomedical applications.

Activation and Delivery of Tetrazine-Responsive Bioorthogonal Prodrugs.

Prodrugs, which remain inert until they are activated under appropriate conditions at the target site, have emerged as an attractive alternative to drugs that lack selectivity and show off-target effects. Prodrugs have traditionally been activated by enzymes, pH or other trigger factors associated with the disease. In recent years, bioorthogonal chemistry has allowed the creation of prodrugs that can be chemically activated with spatio-temporal precision. In particular, tetrazine-responsive bioorthogonal reactions can rapidly activate prodrugs with excellent biocompatibility. This review summarized the recent development of tetrazine bioorthogonal cleavage reaction and great promise for prodrug systems.