Click Chemistry-Based Force Spectroscopy Revealed Enhanced Binding Dynamics of Phosphorylated HMGB1 to Cisplatin-DNA.

Cisplatin, a cornerstone in cancer chemotherapy, is known for its DNA-binding capacity and forms lesions that lead to cancer cell death. However, the repair of these lesions compromises cisplatin's effectiveness. This study investigates how phosphorylation of HMGB1, a nuclear protein, modifies its binding to cisplatin-modified DNA (CP-DNA) and thus protects it from repair. Despite numerous methods for detecting protein-DNA interactions, quantitative approaches for understanding their molecular mechanism remain limited. Here, we applied click chemistry-based single-molecule force spectroscopy, achieving high-precision quantification of the interaction between phosphorylated HMGB1 and CP-DNA. This method utilizes a synergy of click chemistry and enzymatic ligation for precise DNA-protein immobilization and interaction in the system. Our results revealed that HMGB1 binds to CP-DNA with a significantly high rupture force of ∼130 pN, stronger than most natural DNA-protein interactions and varying across different DNA sequences. Moreover, Ser14 is identified as the key phosphorylation site, enhancing the interaction's kinetic stability by 35-fold. This increase in stability is attributed to additional hydrogen bonding suggested by molecular dynamics (MD) simulations. Our findings not only reveal the important role of phosphorylated HMGB1 in potentially improving cisplatin's therapeutic efficacy but also provide a precise method for quantifying protein-DNA interactions.

Customizable Click Biochemistry Strategy for the Design and Preparation of Glucagon-like Peptide-1 Conjugates and Coagonists.

The development of oligomeric glucagon-like peptide-1 (GLP-1) and GLP-1-containing coagonists holds promise for enhancing the therapeutic potential of the GLP-1-based drugs for treating type 2 diabetes mellitus (T2DM). Here, we report a facile, efficient, and customizable strategy based on genetically encoded SpyCatcher-SpyTag chemistry and an inducible, cleavable self-aggregating tag (icSAT) scheme. icSAT-tagged SpyTag-fused GLP-1 and the dimeric or trimeric SpyCatcher scaffold were designed for dimeric or trimeric GLP-1, while icSAT-tagged SpyCatcher-fused GLP-1 and the icSAT-tagged SpyTag-fused GIP were designed for dual GLP-1/GIP (glucose-dependent insulinotropic polypeptide) receptor agonist. These SpyCatcher- and SpyTag-fused protein pairs were spontaneously ligated directly from the cell lysates. The subsequent icSAT scheme, coupled with a two-step standard column purification, resulted in target proteins with authentic N-termini, with yields ranging from 35 to 65 mg/L and purities exceeding 99%. In vitro assays revealed 3.0- to 4.1-fold increased activities for dimeric and trimeric GLP-1 compared to mono-GLP-1. The dual GLP-1/GIP receptor agonist exhibited balanced activity toward the GLP-1 receptor or the GIP receptor. All the proteins exhibited 1.8- to 3.0-fold prolonged half-lives in human serum compared to mono-GLP-1 or GIP. This study provides a generally applicable click biochemistry strategy for developing oligomeric or dual peptide/protein-based drug candidates.

In situ crosslinkable multi-functional and cell-responsive alginate 3D matrix via thiol-maleimide click chemistry.

In vivo, cells interact with the extracellular matrix (ECM), which provides a multitude of biophysical and biochemical signals that modulate cellular behavior. Inspired by this, we explored a new methodology to develop a more physiomimetic polysaccharide-based matrix for 3D cell culture. Maleimide-modified alginate (AlgM) derivatives were successfully synthesized using DMTMM to activate carboxylic groups. Thiol-terminated cell-adhesion peptides were tethered to the hydrogel network to promote integrin binding. Rapid and efficient in situ hydrogel formation was promoted by thiol-Michael addition "click" chemistry via maleimide reaction with thiol-flanked protease-sensitive peptides. Alginate derivatives were further ionically crosslinked by divalent ions present in the medium, which led to greater stability and allowed longer cell culture periods. By tailoring alginate's biofunctionality we improved cell-cell and cell-matrix interactions, providing an ECM-like 3D microenvironment. We were able to systematically and independently vary biochemical and biophysical parameters to elicit specific cell responses, creating custom-made 3D matrices. DMTMM-mediated maleimide incorporation is a promising approach to synthesizing AlgM derivatives that can be leveraged to produce ECM-like matrices for a broad range of applications, from in vitro tissue modeling to tissue regeneration.

Development of Novel Immobilized Copper-Ligand Complex for Click Chemistry of Biomolecules.

Copper-catalyzed azide-alkyne cycloaddition click (CuAAC) reaction is widely used to synthesize drug candidates and other biomolecule classes. Homogeneous catalysts, which consist of copper coordinated to a ligand framework, have been optimized for high yield and specificity of the CuAAC reaction, but CuAAC reaction with these catalysts requires the addition of a reducing agent and basic conditions, which can complicate some of the desired syntheses. Additionally, removing copper from the synthesized CuAAC-containing biomolecule is necessary for biological applications but inconvenient and requires additional purification steps. We describe here the design and synthesis of a PNN-type pincer ligand complex with copper (I) that stabilizes the copper (I) and, therefore, can act as a CuAAC catalyst without a reducing agent and base under physiologically relevant conditions. This complex was immobilized on two types of resin, and one of the immobilized catalyst forms worked well under aqueous physiological conditions. Minimal copper leaching was observed from the immobilized catalyst, which allowed its use in multiple reaction cycles without the addition of any reducing agent or base and without recharging with copper ion. The mechanism of the catalytic cycle was rationalized by density functional theory (DFT). This catalyst's utility was demonstrated by synthesizing coumarin derivatives of small molecules such as ferrocene and sugar.

Site-Specific Conjugation of Bottlebrush Polymers to Therapeutic Protein via Bioorthogonal Chemistry.

Achieving efficient and site-specific conjugation of therapeutic protein to polymer is crucial to augment their applicability in the realms of biomedicine by improving their stability and enzymatic activity. In this study, we exploited tetrazine bioorthogonal chemistry to achieve the site-specific conjugation of bottlebrush polymers to urate oxidase (UOX), a therapeutic protein for gout treatment. An azido-functionalized zwitterionic bottlebrush polymer (N3-ZBP) using a "grafting-from" strategy involving RAFT and ATRP methods was synthesized, and a trans-cyclooctene (TCO) moiety was introduced at the polymer end through the strain-promoted azide-alkyne click (SPAAC) reaction. The subsequent coupling between TCO-incorporated bottlebrush polymer and tetrazine-labeled UOX using a fast and safe bioorthogonal reaction, inverse electron demand Diels-Alder (IEDDA), led to the formation of UOX-ZBP conjugates with a 52% yield. Importantly, the enzymatic activity of UOX remained unaffected following polymer conjugation, suggesting a minimal change in the folded structure of UOX. Moreover, UOX-ZBP conjugates exhibited enhanced proteolytic resistance and reduced antibody binding, compared to UOX-wild type. Overall, the present findings reveal an efficient and straightforward route for synthesizing protein-bottlebrush polymer conjugates without compromising the enzymatic activity while substantially reducing proteolytic degradation and antibody binding.

Modular Synthesis of PEG-Dendritic Block Copolymers by Thermal Azide-Alkyne Cycloaddition with Internal Alkynes and Evaluation of their Self-Assembly for Drug Delivery Applications.

Linear-dendritic block copolymers assemble in solution due to differences in the solubility or charge properties of the blocks. The monodispersity and multivalency of the dendritic block provide unparalleled control for the design of drug delivery systems when incorporating poly(ethylene glycol) (PEG) as a linear block. An accelerated synthesis of PEG-dendritic block copolymers based on the click and green chemistry pillars is described. The tandem composed of the thermal azide-alkyne cycloaddition with internal alkynes and azide substitution is revealed as a flexible, reliable, atom-economical, and user-friendly strategy for the synthesis and functionalization of biodegradable (polyester) PEG-dendritic block copolymers. The high orthogonality of the sequence has been exploited for the preparation of heterolayered copolymers with terminal alkenes and alkynes, which are amenable for subsequent functionalization by thiol-ene and thiol-yne click reactions. Copolymers with tunable solubility and charge were so obtained for the preparation of various types of nanoassemblies with promising applications in drug delivery.

Click Chemistry with Cell-Permeable Fluorophores Expands the Choice of Bioorthogonal Markers for Two-Color Live-Cell STED Nanoscopy.

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.

Synthesis of eco-friendly multifunctional dextran microbeads for multiplexed assays.

There has been an increasing demand for simultaneous detection of multiple analytes in one sample. Microbead-based platforms have been developed for multiplexed assays. However, most of the microbeads are made of non-biodegradable synthetic polymers, leading to environmental and human health concerns. In this study, we developed an environmentally friendly dextran microbeads as a new type of multi-analyte assay platform. Biodegradable dextran was utilized as the primary material. Highly uniform magnetic dextran microspheres were successfully synthesized using the Shirasu porous glass (SPG) membrane emulsification technique. To enhance the amount of surface functional groups for ligand conjugation, we coated the dextran microbeads with a layer of dendrimers via a simple electrostatic adsorption process. Subsequently, a unique and efficient click chemistry coupling technique was developed for the fluorescence encoding of the microspheres, enabling multiplexed detection. The dextran microbeads were tested for 3-plex cytokine analysis, and exhibited excellent biocompatibility, stable coding signals, low background noise and high sensitivity.

Efficient one-pot assembly of higher-order DNA nanostructures by chemically conjugated branched DNA.

Chemically conjugated branched DNA was successfully synthesized by a copper-free click reaction to construct sophisticated and higher-order polyhedral DNA nanostructures with pre-defined units in one pot, which can be used as an efficient nanoplatform to precisely organize multiple gold nanoparticles in predesigned patterns.

Acidic Derivatization of Thiols Using Diethyl 2-Methylenemalonate: Thiol-Michael Addition Click Reaction for Simultaneous Analysis of Cysteine and Cystine in Culture Media Using LC-MS/MS.

Cysteine (Cys) and its oxidized form, cystine (Cys2), play crucial roles in biological systems and have considerable applications in cell culture. However, Cys in cell culture media is easily oxidized to Cys2, leading to solubility issues. Traditional analytical methods struggle to maintain the oxidation states of Cys and Cys2 during analysis, posing a significant challenge to accurately measuring and controlling these compounds. To effectively control the Cys and Cys2 levels, a rapid and accurate analytical method is required. Here, we screened derivatizing reagents that can react with Cys even under acidic conditions to realize a novel analytical method for simultaneously determining Cys and Cys2 levels. Diethyl 2-methylenemalonate (EMM) was found to possess the desired traits. EMM, characterized by its dual electron-withdrawing attributes, allowed for a rapid reaction with Cys under acidic conditions, preserving intact information for understanding the functions of target compounds. Combined with LC-MS/MS and an internal standard, this method provided high analytical accuracy in a short analytical time of 9 min. Using the developed method, the rapid oxidation of Cys in cell culture media was observed with the headspace of the storage container considerably influencing Cys oxidation and Cys2 precipitation rates. The developed method enabled the direct and simplified analysis of Cys behavior in practical media samples and could be used in formulating new media compositions, ensuring quality assurance, and real-time analysis of Cys and Cys2 in cell culture supernatants. This novel approach holds the potential to further enhance the media performance by enabling the timely optimal addition of Cys.

Identification of Cellular Protein Targets of a Half-Sandwich Iridium(III) Complex Reveals Its Dual Mechanism of Action via Both Electrophilic and Oxidative Stresses.

Identification of intracellular targets of anticancer drug candidates provides key information on their mechanism of action. Exploiting the ability of the anticancer (C∧N)-chelated half-sandwich iridium(III) complexes to covalently bind proteins, click chemistry with a bioorthogonal azido probe was used to localize a phenyloxazoline-chelated iridium complex within cells and profile its interactome at the proteome-wide scale. Proteins involved in protein folding and actin cytoskeleton regulation were identified as high-affinity targets. Upon iridium complex treatment, the folding activity of Heat Shock Protein HSP90 was inhibited in vitro and major cytoskeleton disorganization was observed. A wide array of imaging and biochemical methods validated selected targets and provided a multiscale overview of the effects of this complex on live human cells. We demonstrate that it behaves as a dual agent, inducing both electrophilic and oxidative stresses in cells that account for its cytotoxicity. The proposed methodological workflow can open innovative avenues in metallodrug discovery.

Protein click chemistry and its potential for medical applications.

A revolution in chemical biology occurred with the introduction of click chemistry. Click chemistry plays an important role in protein chemistry modifications, providing specific, sensitive, rapid, and easy-to-handle methods. Under physiological conditions, click chemistry often overlaps with bioorthogonal chemistry, defined as reactions that occur rapidly and selectively without interfering with biological processes. Click chemistry is used for the posttranslational modification of proteins based on covalent bond formations. With the contribution of click reactions, selective modification of proteins would be developed, representing an alternative to other technologies in preparing new proteins or enzymes for studying specific protein functions in different biological processes. Click-modified proteins have potential in diverse applications such as imaging, labeling, sensing, drug design, and enzyme technology. Due to the promising role of proteins in disease diagnosis and therapy, this review aims to highlight the growing applications of click strategies in protein chemistry over the last two decades, with a special emphasis on medicinal applications.

Design, synthesis, and docking study of saccharin N-triazolyl glycoconjugates.

To achieve better-repurposed motifs, saccharin has been merged with biocompatible sugar molecules via a 1,2,3-triazole linker, and ten novel 1,2,3-triazole-appended saccharin glycoconjugates were developed in good yield by utilizing modular CuAAC click as regioselective triazole forming tool. The docking study indicated that the resulting hybrid molecules have an overall substantial interaction with the CAXII macromolecule. Moreover, the galactose triazolyl saccharin analogue 3h has a binding energy of -8.5 kcal/mol with 5 H-bonds, and xylosyl 1,2,3-triazolyl saccharin analogue 3d has a binding energy of -8.2 kcal/mol with 6 H-bond interactions and have exhibited the highest binding interaction with the macromolecule system.

Smart Biointerfaces via Click Chemistry-Enabled Nanopatterning of Multiple Bioligands and DNA Force Sensors.

Nanoscale biomolecular placement is crucial for advancing cellular signaling, sensor technology, and molecular interaction studies. Despite this, current methods fall short in enabling large-area nanopatterning of multiple biomolecules while minimizing nonspecific interactions. Using bioorthogonal tags at a submicron scale, we introduce a novel hole-mask colloidal lithography method for arranging up to three distinct proteins, DNA, or peptides on large, fully passivated surfaces. The surfaces are compatible with single-molecule fluorescence microscopy and microplate formats, facilitating versatile applications in cellular and single-molecule assays. We utilize fully passivated and transparent substrates devoid of metals and nanotopographical features to ensure accurate patterning and minimize nonspecific interactions. Surface patterning is achieved using bioorthogonal TCO-tetrazine (inverse electron-demand Diels-Alder, IEDDA) ligation, DBCO-azide (strain-promoted azide-alkyne cycloaddition, SPAAC) click chemistry, and biotin-avidin interactions. These are arranged on surfaces passivated with dense poly(ethylene glycol) PEG brushes crafted through the selective and stepwise removal of sacrificial metallic and polymeric layers, enabling the directed attachment of biospecific tags with nanometric precision. In a proof-of-concept experiment, DNA tension gauge tether (TGT) force sensors, conjugated to cRGD (arginylglycylaspartic acid) in nanoclusters, measured fibroblast integrin tension. This novel application enables the quantification of forces in the piconewton range, which is restricted within the nanopatterned clusters. A second demonstration of the platform to study integrin and epidermal growth factor (EGF) proximal signaling reveals clear mechanotransduction and changes in the cellular morphology. The findings illustrate the platform's potential as a powerful tool for probing complex biochemical pathways involving several molecules arranged with nanometer precision and cellular interactions at the nanoscale.

Thermally Triggered Triazolinedione-Tyrosine Bioconjugation with Improved Chemo- and Site-Selectivity.

A bioconjugation strategy is reported that allows the derivatization of tyrosine side chains through triazolinedione-based "Y-clicking". Blocked triazolinedione reagents were developed that, in contrast to classical triazolinedione reagents, can be purified before use, can be stored for a long time, and allow functionalization with a wider range of cargoes and labels. These reagents are bench-stable at room temperature but steadily release highly reactive triazolinediones upon heating to 40 °C in buffered media at physiological pH, showing a sharp temperature response over the 0 to 40 °C range. This conceptually interesting strategy, which is complementary to existing photo- or electrochemical bioorthogonal bond-forming methods, not only avoids the classical synthesis and handling difficulties of these highly reactive click-like reagents but also markedly improves the selectivity profile of the tyrosine conjugation reaction itself. It avoids oxidative damage and "off-target" tryptophan labeling, and it even improves site-selectivity in discriminating between different tyrosine side chains on the same protein or different polypeptide chains. In this research article, we describe the stepwise development of these reagents, from their short and modular synthesis to small-molecule model bioconjugation studies and proof-of-principle bioorthogonal chemistry on peptides and proteins.

The Effect of Lipid Composition on the Liposomal Delivery of Camptothecin Developed by Active Click Loading.

In the present study, we investigated the role of lipid composition of camptothecin (CPT)-loaded liposomes (CPT-Lips) to adjust their residence time, drug distribution, and therefore the toxicities and antitumor activity. The CPT was loaded into liposomes using a click drug loading method, which utilized liposomes preloaded with GSH and then exposed to CPT-maleimide. The method produced CPT-Lips with a high encapsulation efficiency (>95%) and sustained drug release. It is shown that the residence times of CPT-Lips in the body were highly dependent on lipid compositions with an order of non-PEGylated liposomes of unsaturated lipids < non-PEGylated liposomes of saturated lipids < PEGylated liposomes of saturated lipids. Interestingly, the fast clearance of CPT-Lips resulted in significantly decreased toxicities but did not cause a significant decrease in their in vivo antitumor activity. These results suggested that the lipid composition could effectively adjust the residence time of CPT-Lips in the body and further optimize their therapeutic index, which would guide the development of a liposomal formulation of CPT.

Chemical Proteomics Strategies for Analyzing Protein Lipidation Reveal the Bacterial O-Mycoloylome.

Protein lipidation dynamically controls protein localization and function within cellular membranes. A unique form of protein O-fatty acylation in Corynebacterium, termed protein O-mycoloylation, involves the attachment of mycolic acids─unusually large and hydrophobic fatty acids─to serine residues of proteins in these organisms' outer mycomembrane. However, as with other forms of protein lipidation, the scope and functional consequences of protein O-mycoloylation are challenging to investigate due to the inherent difficulties of enriching and analyzing lipidated peptides. To facilitate the analysis of protein lipidation and enable the comprehensive profiling and site mapping of protein O-mycoloylation, we developed a chemical proteomics strategy integrating metabolic labeling, click chemistry, cleavable linkers, and a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method employing LC separation and complementary fragmentation methods tailored to the analysis of lipophilic, MS-labile O-acylated peptides. Using these tools in the model organism Corynebacterium glutamicum, we identified approximately 30 candidate O-mycoloylated proteins, including porins, mycoloyltransferases, secreted hydrolases, and other proteins with cell envelope-related functions─consistent with a role for O-mycoloylation in targeting proteins to the mycomembrane. Site mapping revealed that many of the proteins contained multiple spatially proximal modification sites, which occurred predominantly at serine residues surrounded by conformationally flexible peptide motifs. Overall, this study (i) discloses the putative protein O-mycoloylome for the first time, (ii) yields new insights into the undercharacterized proteome of the mycomembrane, which is a hallmark of important pathogens (e.g., Corynebacterium diphtheriae, Mycobacterium tuberculosis), and (iii) provides generally applicable chemical strategies for the proteomic analysis of protein lipidation.

Ultrasensitive and Multiplexed Protein Imaging with Clickable and Cleavable Fluorophores.

Single-cell spatial proteomic analysis holds great promise to advance our understanding of the composition, organization, interaction, and function of the various cell types in complex biological systems. However, the current multiplexed protein imaging technologies suffer from low detection sensitivity, limited multiplexing capacity, or are technically demanding. To tackle these issues, here, we report the development of a highly sensitive and multiplexed in situ protein profiling method using off-the-shelf antibodies. In this approach, the protein targets are stained with horseradish peroxidase (HRP) conjugated antibodies and cleavable fluorophores via click chemistry. Through repeated cycles of target staining, fluorescence imaging, and fluorophore cleavage, many proteins can be profiled in single cells in situ. Applying this approach, we successfully quantified 28 different proteins in human formalin-fixed paraffin-embedded (FFPE) tonsil tissue, which represents the highest multiplexing capacity among the tyramide signal amplification (TSA) methods. Based on their unique protein expression patterns and their microenvironment, ∼820,000 cells in the tissue are classified into distinct cell clusters. We also explored the cell-cell interactions between these varied cell clusters and observed that different subregions of the tissue are composed of cells from specific clusters.

Visual Detection of LPS at the Femtomolar Level Based on Click Chemistry-Induced Gold Nanoparticles Electrokinetic Accumulation.

Lipopolysaccharide (LPS) presents a significant threat to human health. Herein, a novel method for detecting LPS was developed by coupling hybridization chain reaction (HCR), gold nanoparticles (AuNPs) agglutination (AA) triggered by a Cu(I)-catalyzed azide-alkyne cycloaddition click chemistry (CuAAC), and electrokinetic accumulation (EA) in a microfluidic chip, termed the HCR-AA-EA method. Thereinto, the LPS-binding aptamer (LBA) was coupled with the AuNP-coated Fe3O4 nanoparticle, which was connected with the polymer of H1 capped on CuO (H1-CuO) and H2-CuO. Upon LPS recognition by LBA, the polymers of H1- and H2-CuO were released into the solution, creating a "one LPS-multiple CuO" effect. Under ascorbic acid reduction, CuAAC was initiated between the alkyne and azide groups on the AuNPs' surface; then, the product was observed visually in the microchannel by EA. Finally, LPS was quantified by the integrated density of AuNP aggregates. The limit of detections were 29.9 and 127.2 fM for water samples and serum samples, respectively. The levels of LPS in the injections and serum samples by our method had a good correlation with those from the limulus amebocyte lysate test (r = 0.99), indicating high accuracy. Remarkably, to popularize our method, a low-cost, wall-power-free portable device was developed, enabling point-of-care testing.

A highly sensitive and selective fluorescent biosensor for breast cancer derived exosomes using click reaction of azide-CD63 aptamer and alkyne-polymer dots.

Exosomes have gained recognition as valuable reservoirs of biomarkers, holding immense potential for early cancer detection. Consequently, there is a pressing need for the development of an economical and highly sensitive exosome detection methodology. In this work, we present a fluorescence method for breast cancer-derived exosome detection based on Cu-triggered click reaction of azide-modified CD63 aptamer and alkyne functionalized Pdots. The detection threshold for the exosomes obtained from the breast cancer serum was determined to be 6.09 × 107 particles per µL, while the measurable range spanned from 6.50 × 107 to 1.30 × 109 particles per µL. The employed methodology achieved notable success in accurately distinguishing breast cancer patients from healthy individuals through serum analysis. The application of this method showcases the significant potential for early exosome analysis in the clinical diagnosis of breast cancer patients.