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While bioorthogonal reactions are routinely employed in living cells and organisms, their application within individual organelles remains limited. In this review, we highlight diverse examples of bioorthogonal reactions used to investigate the roles of biomolecules and biological processes as well as advanced imaging techniques within cellular organelles. These innovations hold great promise for therapeutic interventions in personalized medicine and precision therapies. We also address existing challenges related to the selectivity and trafficking of subcellular dynamics. Organelle-targeted bioorthogonal reactions have the potential to significantly advance our understanding of cellular organization and function, provide new pathways for basic research and clinical applications, and shape the direction of cell biology and medical research.

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Inspired by the principle of in situ self-assembly, the development of enzyme-activated molecular nanoprobes can have a profound impact on targeted tumor detection. However, despite their intrinsic promise, obtaining an optical readout of enzyme activity with high specificity in native milieu has proven to be challenging. Here, a fundamentally new class of Raman-active self-assembling bioorthogonal enzyme recognition (nanoSABER) probes for targeted tumor imaging is reported. This class of Raman probes presents narrow spectral bands reflecting their vibrational fingerprints and offers an attractive solution for optical imaging at different bio-organization levels. The optical beacon harnesses an enzyme-responsive peptide sequence, unique tumor-penetrating properties, and vibrational tags with stretching frequencies in the cell-silent Raman window. The design of nanoSABER is tailored and engineered to transform into a supramolecular structure exhibiting distinct vibrational signatures in presence of target enzyme, creating a direct causality between enzyme activity and Raman signal. Through the integration of substrate-specific for tumor-associated enzyme legumain, unique capabilities of nanoSABER for imaging enzyme activity at molecular, cellular, and tissue levels in combination with machine learning models are shown. These results demonstrate that the nanoSABER probe may serve as a versatile platform for Raman-based recognition of tumor aggressiveness, drug accumulation, and therapeutic response.

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Bioorthogonal chemistry is a promising toolbox for dissecting biological processes in the native environment. Recently, bioorthogonal reactions have attracted considerable attention in the medical field for treating diseases, since this approach may lead to improved drug efficacy and reduced side effects via in situ drug synthesis. For precise biomedical applications, it is a prerequisite that the reactions should occur in the right locations and on the appropriate therapeutic targets. In this minireview, we highlight the design and development of targeted bioorthogonal reactions for precise medical treatment. First, we compile recent strategies for achieving target-specific bioorthogonal reactions. Further, we emphasize their application for the precise treatment of different therapeutic targets. Finally, a perspective is provided on the challenges and future directions of this emerging field for safe, efficient, and translatable disease treatment.

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Targeted killing multidrug-resistant bacteria with high efficiency is urgently needed for the treatment of infection with minimal collateral damage. Herein, a new near-infrared (NIR) fluorescence nanoprobe is designed and synthesized with aggregation-induced emission (AIE) features, which also is excellent reactive oxygen species (ROS) generator. The as-prepared AIE nanoparticles (NPs) present outstanding sterilizing rate on methicillin-resistant Staphylococcus aureus (MRSA) and kanamycin-resistant Escherichia coli (KREC). Meanwhile, considering the differences in the surface structure of animal cells and bacteria, a non-invasive image-guided strategy for precise treatment of bacterial infection has been successfully implemented based on bioorthogonal reaction which can perform and control unnatural chemical reactions inside living organisms. The AIE NPs are thus specifically trapped on the bacterial surface while not on the normal cells, realizing real-time tracking of the infected site distribution in vivo and guiding photodynamic therapy (PDT) for eliminating bacteria in inflammation region. That significantly improves the accuracy and sterilization rate of bacterial-infected wounds with negligible side effects. The investigation developed a potential antibacterial agent and also provides an instructive way for targeting treatment based on bioorthogonal reaction.

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We explored a bioorthogonal approach to release drugs from stimuli-responsive micelles inside tumor cells. The concept relies on sydnonimine-based micelles that undergo quantitative cleavage in presence of cyclooctynes, hence releasing their content within living cells. Four cleavable micelles were developed to allow massive burst release of Entinostat, a potent histone deacetylase inhibitor, following their internalization inside cancer cells. A comparative study on the influence of the bioorthogonal-mediated versus passive drug release from micelles was carried out. The results indicated that a fast release of the drug triggered a stronger antiproliferative activity on tumor cells compared to the passive diffusion of the drug from the micelles core. These finding may be of great interest for the development of new nanomedicines.

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Phage display facilitates the evolution of peptides and proteins for affinity selection against targets, but it is mostly limited to the chemical diversity provided by the naturally encoded amino acids. The combination of phage display with genetic code expansion allows the incorporation of noncanonical amino acids (ncAAs) into proteins expressed on the phage. In this method, we describe incorporation of one or two ncAAs in a single-chain fragment variable (scFv) antibody in response to amber or quadruplet codon. We take advantage of the pyrrolysyl-tRNA synthetase/tRNA pair to incorporate a lysine derivative and an orthogonal tyrosyl-tRNA synthetase/tRNA pair to incorporate a phenylalanine derivative. The encoding of novel chemical functionalities and building blocks in proteins displayed on phage provides the foundation for further phage display applications in fields such as imaging, protein targeting, and the production of new materials.

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The late-stage functionalization of peptides and proteins holds significant promise for drug discovery and facilitates bioorthogonal chemistry. This selective functionalization leads to innovative advances in in vitro and in vivo biological research. However, it is a challenging endeavor to selectively target a certain amino acid or position in the presence of other residues containing reactive groups. Biocatalysis has emerged as a powerful tool for selective, efficient, and economical modifications of molecules. Enzymes that have the ability to modify multiple complex substrates or selectively install nonnative handles have wide applications. Herein, we highlight enzymes with broad substrate tolerance that have been demonstrated to modify a specific amino acid residue in simple or complex peptides and/or proteins at late-stage. The different substrates accepted by these enzymes are mentioned together with the reported downstream bioorthogonal reactions that have benefited from the enzymatic selective modifications.

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Cell membrane camouflaged nanoparticles have been widely used in the field of drug leads discovery attribute to their unique biointerface targeting function. However, random orientation of cell membrane coating does not guarantee effective and appropriate binding of drugs to specific sites, especially when applied to intracellular regions of transmembrane proteins. Bioorthogonal reactions have been rapidly developed as a specific and reliable method for cell membrane functionalization without disturbing living biosystem. Herein, inside-out cell membrane camouflaged magnetic nanoparticles (IOCMMNPs) were accurately constructed via bioorthogonal reactions to screen small molecule inhibitors targeting intracellular tyrosine kinase domain of vascular endothelial growth factor recptor-2. Azide functionalized cell membrane acted as a platform for specific covalently coupling with alkynyl functionalized magnetic Fe3O4 nanoparticles to prepare IOCMMNPs. The inside-out orientation of cell membrane was successfully verified by immunogold staining and sialic acid quantification assay. Ultimately, two compounds, senkyunolide A and ligustilidel, were successfully captured, and their potential antiproliferative activities were further testified by pharmacological experiments. It is anticipated that the proposed inside-out cell membrane coating strategy endows tremendous versatility for engineering cell membrane camouflaged nanoparticles and promotes the development of drug leads discovery platforms.

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Cell membrane camouflaged nanoparticles have been widely used in the field of drug leads discovery attribute to their unique biointerface targeting function. However, random orientation of cell membrane coating does not guarantee effective and appropriate binding of drugs to specific sites, especially when applied to intracellular regions of transmembrane proteins. Bioorthogonal reactions have been rapidly developed as a specific and reliable method for cell membrane functionalization without disturbing living biosystem. Herein, inside-out cell membrane camouflaged magnetic nanoparticles (IOCMMNPs) were accurately constructed via bioorthogonal reactions to screen small molecule inhibitors targeting intracellular tyrosine kinase domain of vascular endothelial growth factor recptor-2. Azide functionalized cell membrane acted as a platform for specific covalently coupling with alkynyl functionalized magnetic Fe3O4 nanoparticles to prepare IOCMMNPs. The inside-out orientation of cell membrane was successfully verified by immunogold staining and sialic acid quantification assay. Ultimately, two compounds, senkyunolide A and ligustilidel, were successfully captured, and their potential antiproliferative activities were further testified by pharmacological experiments. It is anticipated that the proposed inside-out cell membrane coating strategy endows tremendous versatility for engineering cell membrane camouflaged nanoparticles and promotes the development of drug leads discovery platforms.

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Although various methods for selective protein tagging have been established, their ap plications are limited by the low fluorescent tagging efficiency of specific terminal regions of the native proteins of interest (NPIs). In this study, the highly sensitive fluorescence imaging of single NPIs was demonstrated using a eukaryotic translation mechanism involving a free carboxyl group of a cell-permeable fluorescent dye. In living cells, the carboxyl group of cell-permeable fluorescent dyes reacted with the lysine residues of acceptor peptides (AP or AVI-Tag). Genetically encoded recognition demonstrated that the efficiency of fluorescence labeling was nearly 100%. Nickel-nitrilotriacetic acid (Ni-NTA) beads bound efficiently to a single NPI for detection in a cell without purification. Our labeling approach satisfied the necessary conditions for measuring fluorescently labeled NPI using universal carboxyl fluorescent dyes. This approach is expected to be useful for resolving complex biological/ecological issues and robust single-molecule analyses of dynamic processes, in addition to applications in ultra-sensitive NPIs detection using nanotechnology.

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Methods that site-selectively attach multivalent carbohydrate moieties to proteins can be used to generate homogeneous glycodendriproteins as synthetic functional mimics of glycoproteins. Here, we study aspects of the scope and limitations of some common bioconjugation techniques that can give access to well-defined glycodendriproteins. A diverse reactive platform was designed via use of thiol-Michael-type additions, thiol-ene reactions, and Cu(I)-mediated azide-alkyne cycloadditions from recombinant proteins containing the non-canonical amino acids dehydroalanine, homoallylglycine, homopropargylglycine, and azidohomoalanine.

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The development of new bioorthogonal reactions with mutual orthogonality to classic bioorthogonal reactions such as the strain-promoted azide-alkyne click reaction and the inverse-electron-demand Diels-Alder reaction is of great importance in providing chemical tools for multiplex labelling of live cells. Here we report the first anionic cycloaddend-promoted bioorthogonal cycloaddition reaction between phenanthrene-9,10-dione and furan-2(3H)-one derivatives, where the high polarity of water is exploited to stabilize the highly electron-rich anionic cycloaddend. The reaction is metal- and strain-free, which proceeds rapidly in aqueous solution and on live cells with a second-order rate constant up to 119 M-1 s-1 . The combined utilization of this reaction together with the two other widely used bioorthogonal reactions allows for mutually orthogonal labelling of three types of proteins or three groups of living cells in one batch without cross-talking. Such results highlight the great potential for multiplex labelling of different biomolecules in live cells.

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Recent advances in materials science and engineering highlight the importance of designing sophisticated biomaterials with well-defined architectures and tunable properties for emerging biomedical applications. Click chemistry, a powerful method allowing specific and controllable bioorthogonal reactions, has revolutionized our ability to make complex molecular structures with a high level of specificity, selectivity, and yield under mild conditions. These features combined with minimal byproduct formation have enabled the design of a wide range of macromolecular architectures from quick and versatile click reactions. Furthermore, copper-free click chemistry has resulted in a change of paradigm, allowing researchers to perform highly selective chemical reactions in biological environments to further understand the structure and function of cells. In living systems, introducing clickable groups into biomolecules such as polysaccharides (PSA) has been explored as a general approach to conduct medicinal chemistry and potentially help solve healthcare needs. De novo biosynthetic pathways for chemical synthesis have also been exploited and optimized to perform PSA-based bioconjugation inside living cells without interfering with their native processes or functions. This strategy obviates the need for laborious and costly chemical reactions which normally require extensive and time-consuming purification steps. Using these approaches, various PSA-based macromolecules have been manufactured as building blocks for the design of novel biomaterials. Clickable PSA provides a powerful and versatile toolbox for biomaterials scientists and will increasingly play a crucial role in the biomedical field. Specifically, bioclick reactions with PSA have been leveraged for the design of advanced drug delivery systems and minimally invasive injectable hydrogels. In this review article, we have outlined the key aspects and breadth of PSA-derived bioclick reactions as a powerful and versatile toolbox to design advanced polymeric biomaterials for biomedical applications such as molecular imaging, drug delivery, and tissue engineering. Additionally, we have also discussed the past achievements, present developments, and recent trends of clickable PSA-based biomaterials such as 3D printing, as well as their challenges, clinical translatability, and future perspectives.

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Supramolecular assemblies are an emerging class of nanomaterials for drug delivery systems (DDS), while their unintended retention in the biological milieu remains largely unsolved. To realize the prompt clearance of supramolecular assemblies, the bioorthogonal reaction to disassemble and clear the supramolecular assemblies within living cells is investigated here. A series of tetrazine-capped assembly precursors which can self-assemble into nanofibers and hydrogels upon enzymatic dephosphorylation are designed. Such an enzyme-instructed supramolecular assembly process can perform intracellularly. The time-dependent accumulation of assemblies elicits oxidative stress and induces cellular toxicity. Tetrazine-bearing assemblies react with trans-cyclooctene derivatives, which lead to the disruption of π-π stacking and induce disassembly. In this way, the intracellular self-assemblies disassemble and are deprived of potency. This bioorthogonal disassembly strategy leverages the biosafety aspect in developing nanomaterials for DDSs.

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Despite more than 20 years of work since the lipid raft concept was proposed, the existence of these nanostructures remains highly controversial due to the lack of noninvasive methods to investigate their native nanorganization in living unperturbed cells. There is an unmet need for probes for direct imaging of nanoscale membrane dynamics with high spatial and temporal resolution in living cells. In this paper, a bioorthogonal-based cholesterol probe (chol-N3 ) is developed that, combined with nanoscopy, becomes a new powerful method for direct visualization and characterization of lipid raft at unprecedented resolution in living cells. The chol-N3 probe mimics cholesterol in synthetic and cellular membranes without perturbation. When combined with live-cell super-resolution microscopy, chol-N3 demonstrates the existence of cholesterol-rich nanodomains of <50 nm at the plasma membrane of resting living cells. Using this tool, the lipid membrane structure of such subdiffraction limit domains is identified, and the nanoscale spatiotemporal organization of cholesterol in the plasma membrane of living cells reveals multiple cholesterol diffusion modes at different spatial localizations. Finally, imaging across thick organ samples outlines the potential of this new method to address essential biological questions that were previously beyond reach.

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Despite the great advances in solid-phase peptide synthesis (SPPS), the incorporation of certain functional groups into peptide sequences is restricted by the compatibility of the building blocks with conditions used during SPPS. In particular, the introduction of highly reactive groups used in modern bioorthogonal reactions into peptides remains elusive. Here, we present an optimized synthetic protocol enabling installation of two strained dienophiles, trans-cyclooctene (TCO) and bicyclononyne (BCN), into different peptide sequences. The two groups enable fast and modular post-synthetic functionalization of peptides, as we demonstrate in preparation of peptide-peptide and peptide-drug conjugates. Due to the excellent biocompatibility, the click-functionalization of the peptides can be performed directly in live cells. We further show that the introduction of both clickable groups into peptides enables construction of smart, multifunctional probes that can streamline complex chemical biology experiments such as visualization and pull-down of metabolically labeled glycoconjugates. The presented strategy will find utility in construction of peptides for diverse applications, where high reactivity, efficiency and biocompatibility of the modification step is critical.

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Tailored ruthenium sandwich complexes bearing photoresponsive arene ligands can efficiently promote azide-thioalkyne cycloaddition (RuAtAC) when irradiated with UV light. The reactions can be performed in a bioorthogonal manner in aqueous mixtures containing biological components. The strategy can also be applied for the selective modification of biopolymers, such as DNA or peptides. Importantly, this ruthenium-based technology and the standard copper-catalyzed azide-alkyne cycloaddition (CuAAC) proved to be compatible and mutually orthogonal.

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Pretargeted imaging has emerged as a promising approach to advance nuclear imaging of malignant tumors. Herein, we combine the enzyme-mediated fluorogenic reaction and in situ self-assembly with the inverse electron demand Diels-Alder (IEDDA) reaction to develop an activatable pretargeted strategy for multimodality imaging. The trans-cyclooctene (TCO) bearing small-molecule probe, P-FFGd-TCO, can be activated by alkaline phosphatase and in situ self-assembles into nanoaggregates (FMNPs-TCO) retained on the membranes, permitting to (1) amplify near-infrared (NIR) fluorescence (FL) and magnetic resonance imaging (MRI) signals, and (2) enrich TCOs to promote IEDDA ligation. The Gallium-68 (68 Ga) labeled tetrazine can readily conjugate the tumor-retained FMNPs-TCO to enhance radioactivity uptake in tumors. Strong NIR FL, MRI, and positron emission tomography (PET) signals are concomitantly achieved, allowing for pretargeted multimodality imaging of ALP activity in HeLa tumor-bearing mice.

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Twenty-five years ago, GFP revolutionized the field of cell biology by enabling scientists to visualize, for the first time, proteins in living cells. However, when it comes to current, state-of-the-art imaging technologies, fluorescent proteins (such as GFP) have several limitations that result from their size and photophysics. Over the past decade, an elegant, alternative approach, which is based on the direct labeling of proteins with fluorescent dyes and is compatible with live-cell and super-resolution imaging applications, has been introduced. In this approach, an unnatural amino acid that can covalently bind a fluorescent dye is incorporated into the coding sequence of a protein. The protein of interest is thereby site-specifically fluorescently labeled inside the cell, eliminating the need for protein- or peptide-labeling tags. Whether this labeling approach will change cell biology research is currently unclear, but it clearly has the potential to do so. In this short review, a general overview of this approach is provided, focusing on the imaging of site-specifically labeled proteins in mammalian tissue culture cells, and highlighting its advantages and limitations for cellular imaging.

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Antibody-protein conjugates have been useful tools for studying biological systems and also played important roles in developing therapeutics and diagnostics. In particular, because of the increased interest in therapeutics of complexity higher than monoclonal antibodies, various methods have been reported for generating bispecific antibodies, immunotoxins, and antibody-enzyme conjugates in which antibodies are site-specifically conjugated with other proteins. Compared with conjugating antibodies with synthetic molecules, controlling the modification sites is difficult in the antibodies conjugated with protein cargos due to the presence of several reactive groups in both molecules. Enzymatic reactions are often used to generate antibody-protein conjugates owing to their high specificity for both reactants and products. Chemical modifications involving genetic introduction of natural or unnatural amino acid residues have also been used for site-specific conjugation of antibodies. Recent studies have developed methods to modify native antibodies using peptides having affinity for antibodies, and these methods do not need antibody engineering for conjugation reactions. In this review, we have summarized enzymatic and chemical approaches to generate site-specific antibody-protein conjugates.

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