Search Results (58)

In the realm of click-type reactions and their application to bioorthogonal chemistry in living organisms, metal-free [3+2] cycloadditions involving mesoionic rings and strained cycloalkynes have gained increasing attention and potentiality in recent years. While there has been a significant accretion of experimental data, biological assays, and assessments of reaction mechanisms, some pieces of the tale are still missing. For instance, which structural and/or stereoelectronic effects are actually interlocked and which remain unplugged. With the advent of data-driven methods, including machine learning simulations, quantitative estimations of relevant observables and their correlations will explore better the chemical space of these transformations. Here we unveil a series of linear relationships, such as Hammett-type correlations, as well as deviations of linearity, using the case study of phenylsydnone (and its 4-aryl-substituted derivatives) with a highly reactive bicyclo[6.1.0]nonyne carbinol. Through accurate estimation of activation barriers and prediction of rate constants, our findings further increase the significance of integrating strain release and electronic effects in organic reactivity. Moreover, such results could pave the way to use mesoionics cycloadditions as probes for measuring the extent of delocalization-assisted strain release, which can be applied to related reactions involving dipoles and strained rings. Full article

Micromachines, small-scale engineered devices prepared to carry out exact tasks at the micro level, have garnered great interest across different fields such as drug delivery, chemical synthesis, and biomedical applications. In emerging applications, micromachines have indicated great potential in advancing click chemistry, a highly selective and efficient chemical technique widely applied in materials science, bioconjugation, and pharmaceutical development. Click chemistry, distinguished by its rapid reaction rates, high efficiency, and bioorthogonality, serves as a robust method for molecular assembly and functionalization. Incorporating micromachines into click chemistry processes paves the way for precise, automated, and scalable chemical synthesis. These tiny devices can effectively transport reactants, boost reaction efficiency through localized mixing, and enable highly exact site-specific modifications. Moreover, micromachines driven by external forces such as magnetic fields, ultrasound, or chemical fuels provide exceptional control over reaction conditions, significantly enhancing the selectivity and efficiency of click reactions. In this review, we explore the interaction between micromachines and click chemistry, showcasing recent advancements, potential uses, and future prospects in this cross-disciplinary domain. By leveraging micromachine-supported click chemistry, scientists can surpass conventional reaction constraints, opening doors to groundbreaking innovations in materials science, drug discovery, and beyond. Full article

Enzyme immobilization plays a critical role in enhancing the efficiency and sustainability of biocatalysis, addressing key challenges such as limited enzyme stability, short shelf life, and difficulties in recovery and recycling, which are pivotal for green chemistry and industrial applications. Classical approaches, including adsorption, entrapment, encapsulation, and covalent bonding, as well as advanced site-specific methods that integrate enzyme engineering and bio-orthogonal chemistry, were discussed. These techniques enable precise control over enzyme orientation and interaction with carriers, optimizing catalytic activity and reusability. Key findings highlight the impact of immobilization on improving enzyme performance under various operational conditions and its role in reducing process costs through enhanced stability and recyclability. The review presents numerous practical applications of immobilized enzymes, including their use in the pharmaceutical industry for drug synthesis, in the food sector for dairy processing, and in environmental biotechnology for wastewater treatment and dye degradation. Despite the significant advantages, challenges such as activity loss due to conformational changes and mass transfer limitations remain, necessitating tailored immobilization protocols for specific applications. The integration of immobilization with modern biotechnological advancements, such as site-directed mutagenesis and recombinant DNA technology, offers a promising pathway for developing robust, efficient, and sustainable biocatalytic systems. This comprehensive guide aims to support researchers and industries in selecting and optimizing immobilization techniques for diverse applications in pharmaceuticals, food processing, and fine chemicals. Full article

Homogeneous antibody–drug conjugates (ADCs) exhibit significantly improved pharmacological properties compared to their heterogeneous counterparts. Site-specific conjugation of the payload to the IgG required for homogeneity can be achieved using enzymes. One example is microbial transglutaminase (MTGase), which can selectively perform transamidation on the Q295 residue of human Fc when N297 glycans are removed. As a result, two modifications can be introduced per IgG molecule; however, achieving higher drug-to-antibody ratios (DARs) requires the use of branched linkers. While several such linkers have been reported, little information is available on the relationship between linker structure and ADC properties. To address this gap, we synthesized two branched amino triazide linkers, differing by a PEG₄ fragment inserted after the branching point, which were used to prepare two homogeneous trastuzumab-based DAR 6 ADCs (a “short” and a “long” one). This was achieved by a two-step process consisting of enzymatic linker conjugation followed by bioorthogonal coupling with a cleavable linker bearing monomethyl auristatin E (MMAE). Two other trastuzumab–MMAE conjugates were used as controls: a heterogeneous DAR 6 ADC, made using conventional thiol–maleimide chemistry, and a homogeneous DAR 2 ADC. We found that, while the four conjugates had identical affinity for HER2, their cytotoxicity differed significantly: the “long” homogeneous DAR 6 ADC was just as active as its heterogeneous counterpart, but the “short” DAR 6 ADC was an order of magnitude less potent, inferior even to the DAR 2 conjugate. Our findings indicate that the length of the branched linker critically affects the cytotoxic activity of ADCs, possibly due to steric hindrance influencing the rate of linker cleavage by lysosomal enzymes. Full article

Several studies have reported the successful use of bio-orthogonal catalyst nanoparticles (NPs) for cancer therapy. However, the delivery of the catalysts to the target tissues in vivo remains an unsolved challenge. The combination of catalytic NPs with extracellular vesicles (EVs) has been proposed as a promising approach to improve the delivery of therapeutic nanomaterials to the desired organs. In this study, we have developed a nanoscale bio-hybrid vector using a CO-mediated reduction at low temperature to generate ultrathin catalytic Pd nanosheets (PdNSs) as catalysts directly inside cancer-derived EVs. We have also compared their biodistribution with that of PEGylated PdNSs delivered by the EPR effect. Our results indicate that the accumulation of PdNSs in the tumour tissue was significantly higher when they were administered within the EVs compared to the PEGylated PdNSs. Conversely, the amount of Pd found in non-target organs (i.e., liver) was lowered. Once the Pd-based catalytic EVs were accumulated in the tumours, they enabled the activation of a paclitaxel prodrug demonstrating their ability to carry out bio-orthogonal uncaging chemistries in vivo for cancer therapy. Full article

STED nanoscopy allows for the direct observation of dynamic processes in living cells and tissues with diffraction-unlimited resolution. Although fluorescent proteins can be used for STED imaging, these labels are often outperformed in photostability by organic fluorescent dyes. This feature is especially crucial for time-lapse imaging. Unlike fluorescent proteins, organic fluorophores cannot be genetically fused to a target protein but require different labeling strategies. To achieve simultaneous imaging of more than one protein in the interior of the cell with organic fluorophores, bioorthogonal labeling techniques and cell-permeable dyes are needed. In addition, the fluorophores should preferentially emit in the red spectral range to reduce the potential phototoxic effects that can be induced by the STED light, which further restricts the choice of suitable markers. In this work, we selected five different cell-permeable organic dyes that fulfill all of the above requirements and applied them for SPIEDAC click labeling inside living cells. By combining click-chemistry-based protein labeling with other orthogonal and highly specific labeling methods, we demonstrate two-color STED imaging of different target structures in living specimens using different dye pairs. The excellent photostability of the dyes enables STED imaging for up to 60 frames, allowing the observation of dynamic processes in living cells over extended time periods at super-resolution. Full article

Over the past few decades, photodynamic therapy (PDT) has evolved as a minimally invasive treatment modality offering precise control over cancer and various other diseases. To address inherent challenges associated with PDT, researchers have been exploring two promising avenues: the development of intelligent photosensitizers activated through light-induced energy transfers, charges, or electron transfers, and the disruption of photosensitive bonds. Moreover, there is a growing emphasis on the bioorthogonal delivery or activation of photosensitizers within tumors, enabling targeted deployment and activation of these intelligent photosensitive systems in specific tissues, thus achieving highly precise PDT. This concise review highlights advancements made over the last decade in the realm of light-activated or bioorthogonal photosensitizers, comparing their efficacy and shaping future directions in the advancement of photodynamic therapy. Full article

Vaccines typically work by eliciting an immune response against larger antigens like polysaccharides or proteins. Small molecules like nicotine, on their own, usually cannot elicit a strong immune response. To overcome this, anti-nicotine vaccines often conjugate nicotine molecules to a carrier protein by carbodiimide crosslinking chemistry to make them polymeric and more immunogenic. The reaction is sensitive to conditions such as pH, temperature, and the concentration of reactants. Scaling up the reaction from laboratory to industrial scales while maintaining consistency and yield can be challenging. Despite various approaches, no licensed anti-nicotine vaccine has been approved so far due to the susboptimal antibody titers. Here, we report a novel approach to conjugate maleimide-modified nicotine hapten with a disulfide bond-reduced carrier protein in an organic solvent. It has two advantages compared with other approaches: (1) The protein was unfolded to make the peptide conformation more flexible and expose more conjugation sites; (2) thiol–maleimide “click” chemistry was utilized to conjugate the disulfide bond-reduced protein and maleimide-modified nicotine due to its availability, fast kinetics, and bio-orthogonality. Various nicotine conjugate vaccines were prepared via this strategy, and their immunology effects were investigated by using MPL and QS-21 as adjuvants. The in vivo study in mice showed that the nicotine–BSA conjugate vaccines induced high anti-nicotine IgG antibody titers, compared with vaccines prepared by using traditional condensation methods, indicating the success of the current strategy for further anti-nicotine or other small-molecule vaccine studies. The enhancement was more significant by using MPL and QS-21 than that of traditional aluminum adjuvants. Full article

Bio-orthogonal chemistry provides a powerful tool for drug delivery systems due to its ability to generate therapeutic agents in situ, minimizing off-target effects. Bio-orthogonal transition metal catalysts (TMCs) with stimuli-responsive properties offer possibilities for controllable catalysis due to their spatial-, temporal-, and dosage-controllable properties. In this paper, we fabricated a stimuli-responsive bio-orthogonal catalysis system based on an enhanced green fluorescent protein (EGFP)–nanozyme (NZ) complex (EGFP-NZ). Regulation of the catalytic properties of the EGFP-NZ complex was directly achieved by modulating the ionic strength of the solution. The dielectric screening introduced by salt ions allows the dissociation of the EGFP-NZ complex, increasing the access of substrate to the active site of the NZs and concomitantly increasing nanozyme activity. The change in catalytic rate of the NZ/EGFP = 1:1 complex was positively correlated with salt concentration from 0 mM to 150 mM. Full article

“Click” cycloadditions offer effective pathways for the modifications of supramolecular structures, polymers, and nanomaterials. These reactions include bioorthogonal mechanisms that do not interfere with the biological processes, providing a type of chemistry to operate directly in living environments, such as cells and animals. As a result, the “click” cycloadditions represent highly and selective tools for tailoring the properties of nanomedicine scaffolds, expanding the efficacy of multiple therapeutic strategies. We focused this minireview on the bioorthogonal cycloadditions, presenting an insight into the strategies to modify nanostructured biomedical scaffolds inside living systems. We organized the contributions according to the three main mechanisms of “click” cycloadditions: strain-promoted sydnone-alkyne, tetrazine ligation, and strain-promoted [3+2] azido-alkyne. Full article

The administration of therapeutics to peripheral nerve tissue is challenging due to the complexities of peripheral neuroanatomy and the limitations imposed by the blood–nerve barrier (BNB). Therefore, there is a pressing need to enhance delivery effectiveness and implement targeted delivery methods. Recently, erythrocyte-derived exosomes (Exos) have gained widespread attention as biocompatible vehicles for therapeutics in clinical applications. However, engineering targeted Exos for the peripheral nervous system (PNS) is still challenging. This study aims to develop a targeted Exo delivery system specifically designed for presynaptic terminals of peripheral nerve tissue. The clostridium neurotoxin, tetanus toxin-C fragment (TTC), was tethered to the surface of red blood cell (RBC)-derived Exos via a facile and efficient bio-orthogonal click chemistry method without a catalyst. Additionally, Cyanine5 (Cy5), a reactive fluorescent tag, was also conjugated to track Exo movement in both in vitro and in vivo models. Subsequently, Neuro-2a, a mouse neuronal cell line, was treated with dye-labeled Exos with/without TTC in vitro, and the results indicated that TTC-Exos exhibited more efficient accumulation along the soma and axonal circumference, compared to their unmodified counterparts. Further investigation, using a mouse model, revealed that within 72 h of intramuscular administration, engineered TTC-Exos were successfully transported into the neuromuscular junction and sciatic nerve tissues. These results indicated that TTC played a crucial role in the Exo delivery system, improving the affinity to peripheral nerves. These promising results underscore the potential of using targeted Exo carriers to deliver therapeutics for treating peripheral neuropathies. Full article

Chimeric antigen receptor T cell (CAR-T) therapy has been applied in the treatment of B-cell lymphoma; however, CAR-T manufacturing requires virus- or non-virus-based genetic modification, which causes high manufacturing costs and potential safety concerns. Antibody–cell conjugation (ACC) technology, which originated from bio-orthogonal click chemistry, provides an efficient approach for arming immune cells with cancer-targeting antibodies without genetic modification. Here, we applied ACC technology in Vγ9Vδ2 T (γδ2 T) cells to generate a novel off-the-shelf CD20-targeting cell therapy ACE1831 (rituximab-conjugated γδ2 T cells) against relapsed/refractory B-cell lymphoma. ACE1831 exhibited superior cytotoxicity against B-cell lymphoma cells and rituximab-resistant cells compared to γδ2 T cells without rituximab conjugation. The in vivo xenograft study demonstrated that ACE1831 treatment strongly suppressed the aggressive proliferation of B-cell lymphoma and prolonged the survival of tumor-bearing mice with no observed toxicity. Mass spectrometry analysis indicated that cell activation receptors including the TCR complex, integrins and cytokine receptors were conjugated with rituximab. Intriguingly, the antigen recognition of the ACC-linked antibody/receptor complex stimulated NFAT activation and contributed to ACE1831-mediated cytotoxicity against CD20-expressing cancer cells. This study elucidates the role of the ACC-linked antibody/receptor complex in cytotoxicity and supports the potential of ACE1831 as an off-the-shelf γδ2 cell therapy against relapsed/refractory B-cell lymphoma. Full article

All known organisms encode 20 canonical amino acids by base triplets in the genetic code. The cellular translational machinery produces proteins consisting mainly of these amino acids. Several hundred natural amino acids serve important functions in metabolism, as scaffold molecules, and in signal transduction. New side chains are generated mainly by post-translational modifications, while others have altered backbones, such as the β- or γ-amino acids, or they undergo stereochemical inversion, e.g., in the case of D-amino acids. In addition, the number of non-canonical amino acids has further increased by chemical syntheses. Since many of these non-canonical amino acids confer resistance to proteolytic degradation, they are potential protease inhibitors and tools for specificity profiling studies in substrate optimization and enzyme inhibition. Other applications include in vitro and in vivo studies of enzyme kinetics, molecular interactions and bioimaging, to name a few. Amino acids with bio-orthogonal labels are particularly attractive, enabling various cross-link and click reactions for structure-functional studies. Here, we cover the latest developments in protease research with non-canonical amino acids, which opens up a great potential, e.g., for novel prodrugs activated by proteases or for other pharmaceutical compounds, some of which have already reached the clinical trial stage. Full article

The recently developed compound, tetramethylthiocycloheptyne sulfoximine (TMTHSI), has shown to be a promising strained alkyne for strain-promoted azide–alkyne cycloaddition (SPAAC), metal-free click chemistry. This research explores the properties of TMTHSI-based compounds via three aspects: (1) large-scale production, (2) unique stability in acidic conditions and its subsequent use in peptide synthesis, and (3) the functionalization of antibodies. Here, it is shown that (1) scale-up is achieved on a scale of up to 100 g. (2) TMTHSI is remarkably stable against TFA allowing for the site-specific functionalization of peptides on resin. Finally, (3) the functionalization of an antibody with a model payload is very efficient, with antibody conjugation demonstrating more beneficial features such as a high yield and limited hydrophobicity as compared to other alkyne reagent conjugates. These results illustrate the high potential of TMTHSI for diverse bioconjugation applications, with production already being GMP-compatible and a highly efficient conversion resulting in attractive costs of goods. Full article

Hydrogels are widely used in stem cell therapy due to their extensive tunability and resemblance to the extracellular matrix (ECM), which has a three-dimensional (3D) structure. These features enable various applications that enhance stem cell maintenance and function. However, fast and simple hydrogel fabrication methods are desirable for stem cells for efficient encapsulation and to reduce adverse effects on the cells. In this study, we present a one-pot double-crosslinked hydrogel consisting of polyethylene glycol (PEG) and collagen, which can be prepared without the multi-step sequential synthesis of each network, by using bio-orthogonal chemistry. To enhance the adipogenic differentiation efficiency of adipose-derived stem cells (ADSCs), we added degradable components within the hydrogel to regulate matrix stiffness through cell-mediated degradation. Bio-orthogonal reactions used for hydrogel gelation allow rapid gel formation for efficient cell encapsulation without toxic by-products. Furthermore, the hybrid network of synthetic (PEG) and natural (collagen) components demonstrated adequate mechanical strength and higher cell adhesiveness. Therefore, ADSCs grown within this hybrid hydrogel proliferated and functioned better than those grown in the single-crosslinked hydrogel. The degradable elements further improved adipogenesis in ADSCs with dynamic changes in modulus during culture and enabled the retrieval of differentiated cells for potential future applications. Full article