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Schiff base, an important family of reaction in click chemistry, has received significant attention in the formation of self-healing hydrogels in recent years. Schiff base reversibly reacts even in mild conditions, which allows hydrogels with self-healing ability to recover their structures and functions after damages. Moreover, pH-sensitivity of the Schiff base offers the hydrogels response to biologically relevant stimuli. Different types of Schiff base can provide the hydrogels with tunable mechanical properties and chemical stabilities. In this review, we summarized the design and preparation of hydrogels based on various types of Schiff base linkages, as well as the biomedical applications of hydrogels in drug delivery, tissue regeneration, wound healing, tissue adhesives, bioprinting, and biosensors.

Defining protein–protein interactions (PPIs) in their native environment is crucial to understanding protein structure and function. Cross-linking–mass spectrometry (XL-MS) has proven effective in capturing PPIs in living cells; however, the proteome coverage remains limited. Here, we have developed a robust in vivo XL-MS platformto facilitate in-depth PPI mapping by integrating a multifunctional MS-cleavable cross-linker with sample preparation strategies and high-resolution MS. The advancement of click chemistry–based enrichment significantly enhanced the detection of cross-linked peptides for proteome-wide analyses. This platform enabled the identification of 13,904 unique lysine–lysine linkages from in vivo cross-linked HEK 293 cells, permitting construction of the largest in vivo PPI network to date, comprising 6,439 interactions among 2,484 proteins. These results allowed us to generate a highly detailed yet panoramic portrait of human interactomes associated with diverse cellular pathways. The strategy presented here signifies a technological advancement for in vivo PPI mapping at the systems level and can be generalized for charting protein interaction landscapes in any organisms.

The copper(I)-catalyzed azide−alkyne cycloaddition (CuAAC) reaction is considered to be the most representative ligation process within the context of the “click chemistry” concept. This CuAAC reaction, which yields compounds containing a 1,2,3-triazole core, has become relevant in the construction of biologically complex systems, bioconjugation strategies, and supramolecular and material sciences. Although many CuAAC reactions are performed under homogenous conditions, heterogenous copper-based catalytic systems are gaining exponential interest, relying on the easy removal, recovery, and reusability of catalytically copper species. The present review covers the most recently developed copper-containing heterogenous solid catalytic systems that use solid inorganic/organic hybrid supports, and which have been used in promoting CuAAC reactions. Due to the demand for 1,2,3-triazoles as non-classical bioisosteres and as framework-based drugs, the CuAAC reaction promoted by solid heterogenous catalysts has greatly improved the recovery and removal of copper species, usually by simple filtration. In so doing, the solving of the toxicity issue regarding copper particles in compounds of biological interest has been achieved. This protocol is also expected to produce a practical chemical process for accessing such compounds on an industrial scale.

We report a covalent chemistry-based hepatocellular carcinoma (HCC)-specific extracellular vesicle (EV) purification system for early detection of HCC by performing digital scoring on the purified EVs. Earlier detection of HCC creates more opportunities for curative therapeutic interventions. EVs are present in circulation at relatively early stages of disease, providing potential opportunities for HCC early detection. We develop an HCC EV purification system (i.e., EV Click Chips) by synergistically integrating covalent chemistry-mediated EV capture/release, multimarker antibody cocktails, nanostructured substrates, and microfluidic chaotic mixers. We then explore the translational potential of EV Click Chips using 158 plasma samples of HCC patients and control cohorts. The purified HCC EVs are subjected to reverse-transcription droplet digital PCR for quantification of 10 HCC-specific mRNA markers and computation of digital scoring. The HCC EV-derived molecular signatures exhibit great potential for noninvasive early detection of HCC from at-risk cirrhotic patients with an area under receiver operator characteristic curve of 0.93 (95% CI, 0.86 to 1.00; sensitivity = 94.4%, specificity = 88.5%). Extracellular vesicles (EVs) are present in circulation at relatively early stages of disease, providing potential opportunities for early cancer diagnosis. Here, the authors report a covalent chemistry-based hepatocellular carcinoma (HCC)-specific EV purification system for early detection of HCC by performing digital scoring on the purified EVs.

As a result of the biological activities of natural flavonoids, several synthetic strategies aiming to obtain analogues with improved potency and/or pharmacokinetic profile have been developed. Since the triazole ring has been associated with several biological activities and metabolic stability, hybridization with a 1,2,3-triazole ring has been increasingly reported over the last years. The feasible synthesis through copper (I) catalyzed azide-alkyne cycloaddition (CuAAC) has allowed the accomplishment of several hybrids. Since 2017, almost 700 flavonoid hybrids conjugated with 1,2,3-triazole, including chalcones, flavones, flavanones and flavonols, among others, with antitumor, antimicrobial, antidiabetic, neuroprotective, anti-inflammatory, antioxidant, and antifouling activity have been reported. This review compiles the biological activities recently described for these hybrids, highlighting the mechanism of action and structure–activity relationship (SAR) studies.

The explosively-growing applications of the Cu-catalyzed Huisgen 1,3-dipolar cycloaddition reaction between organic azides and alkynes (CuAAC) have stimulated an impressive number of reports, in the last years, focusing on recoverable variants of the homogeneous or quasi-homogeneous catalysts. Recent advances in the field are reviewed, with particular emphasis on systems immobilized onto polymeric organic or inorganic supports.

Site-specific labeling of proteins with small fluorophores is advantageous for imaging. Lemke et al . describe how to site-specifically label membrane proteins with organic fluorophores by incorporating non-canonical amino acids via Amber suppression technology. We describe a protocol for the rapid labeling of cell-surface proteins in living mammalian cells using click chemistry. The labeling method is based on strain-promoted alkyne-azide cycloaddition (SPAAC) and strain-promoted inverse-electron–demand Diels–Alder cycloaddition (SPIEDAC) reactions, in which noncanonical amino acids (ncAAs) bearing ring-strained alkynes or alkenes react, respectively, with dyes containing azide or tetrazine groups. To introduce ncAAs site specifically into a protein of interest (POI), we use genetic code expansion technology. The protocol can be described as comprising two steps. In the first step, an Amber stop codon is introduced—by site-directed mutagenesis—at the desired site on the gene encoding the POI. This plasmid is then transfected into mammalian cells, along with another plasmid that encodes an aminoacyl-tRNA synthetase/tRNA (RS/tRNA) pair that is orthogonal to the host's translational machinery. In the presence of the ncAA, the orthogonal RS/tRNA pair specifically suppresses the Amber codon by incorporating the ncAA into the polypeptide chain of the POI. In the second step, the expressed POI is labeled with a suitably reactive dye derivative that is directly supplied to the growth medium. We provide a detailed protocol for using commercially available ncAAs and dyes for labeling the insulin receptor, and we discuss the optimal surface-labeling conditions and the limitations of labeling living mammalian cells. The protocol involves an initial cloning step that can take 4–7 d, followed by the described transfections and labeling reaction steps, which can take 3–4 d.

Since its conceptualization, click chemistry in all its variants has proven to be a superior synthesis protocol, compared to conventional methods, for forming new covalent bonds under mild conditions, orthogonally, and with high yields. If a term like reactive resilience could be established, click reactions would be good examples, as they perform better under increasingly challenging conditions. Particularly, highly hindered couplings that perform poorly with conventional chemistry protocols—such as those used to conjugate biomacromolecules (e.g., proteins and aptamers) or multiple drugs onto macromolecular platforms—can be more easily achieved using click chemistry principles, while also promoting high stereoselectivity in the products. In this review, three molecular platforms relevant in the field of nanomedicine are considered: polymers/copolymers, cyclodextrins, and fullerenes, whose functionalization poses a challenge due to steric hindrance, either from the intrinsic bulk behavior (as in polymers) or from the proximity of confined reactive sites, as seen in cyclodextrins and fullerenes. Their functionalization with biologically active groups (drugs or biomolecules), primarily through copper-catalyzed azide–alkyne cycloaddition (CuAAC), strain-promoted azide–alkyne cycloaddition (SPAAC), inverse electron-demand Diels–Alder (IEDDA) and thiol–ene click reactions, has led to the development of increasingly sophisticated systems with enhanced specificity, multifunctionality, bioavailability, delayed clearance, multi-targeting, selective cytotoxicity, and tracking capabilities—all essential in the field of nanomedicine.

The 2022 Nobel Prize in Chemistry recognized the development of biorthogonal chemical ligation reactions known as click chemistry in biomedicine. This concept has catalyzed significant progress in sensing and diagnosis, chemical biology, materials chemistry, and drug discovery and delivery. In proteomics, the ability to incorporate a click tag into proteins has propelled development of powerful new methods for selective enrichment of protein complexes that inform understanding of protein networks. It also has had a strong influence on the ability to enrich for protein post-translational modifications. This feature article summarizes the impacts of biorthogonal click chemistry on proteomics. Graphical Abstract

Although a plethora of chemistries have been developed to selectively decorate protein molecules, novel strategies continue to be reported with the final aim of improving selectivity and mildness of the reaction conditions, preserve protein integrity, and fulfill all the increasing requirements of the modern applications of protein conjugates. The targeting of the protein N-terminal alpha-amine group appears a convenient solution to the issue, emerging as a useful and unique reactive site universally present in each protein molecule. Herein, we provide an updated overview of the methodologies developed until today to afford the selective modification of proteins through the targeting of the N-terminal alpha-amine. Chemical and enzymatic strategies enabling the selective labeling of the protein N-terminal alpha-amine group are described.