Conjugated molecularly imprinted polymers based on covalent organic frameworks: Fluorescent sensing platform for specific capture of urea and elimination of ethyl carbamate.

This study described the preparation of an azide covalent organic framework-embedded molecularly imprinted polymers (COFs(azide)@MIPs) platform for urea adsorption and indirect ethyl carbamate (EC) removal from Chinese yellow rice wine (Huangjiu). By modifying the pore surface of COFs using the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, COFs(azide) with a high fluorescence quantum yield and particular recognition ability were inventively produced. In order to selectively trap urea, the COFs(azide) were encased in an imprinted shell layer via imprinting technology. With a detection limit (LOD) of 0.016 µg L-1 (R2 = 0.9874), the COFs(azides)@MIPs demonstrated a good linear relationship with urea in the linear range of 0-5 µg L-1. Using real Huangjiu samples, the spiking recovery trials showed the viability of this sensing platform with recoveries ranging from 88.44 % to 109.26 % and an RSD of less than 3.40 %. The Huangjiu processing model system achieved 38.93 % EC reduction by COFs(azides)@MIPs. This research will open up new avenues for the treatment of health problems associated with fermented alcoholic beverages, particularly Huangjiu, while also capturing and removing hazards coming from food.

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.

Post-click labeling enables highly accurate single cell analyses of glucose uptake ex vivo and in vivo.

Cellular glucose uptake is a key feature reflecting metabolic demand of cells in physiopathological conditions. Fluorophore-conjugated sugar derivatives are widely used for monitoring glucose transporter (GLUT) activity at the single-cell level, but have limitations in in vivo applications. Here, we develop a click chemistry-based post-labeling method for flow cytometric measurement of glucose uptake with low background adsorption. This strategy relies on GLUT-mediated uptake of azide-tagged sugars, and subsequent intracellular labeling with a cell-permeable fluorescent reagent via a copper-free click reaction. Screening a library of azide-substituted monosaccharides, we discover 6-azido-6-deoxy-D-galactose (6AzGal) as a suitable substrate of GLUTs. 6AzGal displays glucose-like physicochemical properties and reproduces in vivo dynamics similar to 18F-FDG. Combining this method with multi-parametric immunophenotyping, we demonstrate the ability to precisely resolve metabolically-activated cells with various GLUT activities in ex vivo and in vivo models. Overall, this method provides opportunities to dissect the heterogenous metabolic landscape in complex tissue environments.

Access to Reverse Glycosyl Azides and Rare Sugar-Based Glycosyl Azides via Radical Decarboxylative Azidation: Divergent Synthesis of 4'-C-Azidonucleosides as Potential Antiviral Agents.

The radical decarboxylative azidation of structurally diverse uronic acids has been established as an efficient approach to reverse glycosyl azides and rare sugar-derived glycosyl azides under the action of Ag2CO3, 3-pyridinesulfonyl azide, and K2S2O8. The power of this method has been highlighted by the divergent synthesis of 4'-C-azidonucleosides using Vorbrüggen glycosylation of nucleobases with 4-C-azidofuranosyl acetates. The antiviral assessment of the resulting nucleosides revealed one compound as a potential inhibitor of covalently closed circular DNA.

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.

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.

Bioorthogonal Radiolabeling of Azide-Modified Bacteria Using [18F]FB-sulfo-DBCO.

Purpose: This study was motivated by the need for better positron emission tomography (PET)-compatible tools to image bacterial infection. Our previous efforts have targeted bacteria-specific metabolism via assimilation of carbon-11 labeled d-amino acids into the bacterial cell wall. Since the chemical determinants of this incorporation are not fully understood, we sought a high-throughput method to label d-amino acid derived structures with fluorine-18. Our strategy employed a chemical biology approach, whereby an azide (-N3) bearing d-amino acid is incorporated into peptidoglycan muropeptides, with subsequent "click" cycloaddition with an 18F-labeled strained cyclooctyne partner. Procedures: A water-soluble, 18F-labeled and dibenzocyclooctyne (DBCO)-derived radiotracer ([18F]FB-sulfo-DBCO) was synthesized. This tracer was incubated with pathogenic bacteria treated with azide-bearing d-amino acids, and incorporated 18F was determined via gamma counting. In vitro uptake in bacteria previously treated with azide-modified d-amino acids was compared to that in cultures treated with amino acid controls. The biodistribution of [18F]FB-sulfo-DBCO was studied in a cohort of healthy mice with implications for future in vivo imaging. Results: The new strain-promoted azide-alkyne cycloaddition (SPAAC) radiotracer [18F]FB-sulfo-DBCO was synthesized with high radiochemical yield and purity via N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB). Accumulation of [18F]FB-sulfo-DBCO was significantly higher in several bacteria treated with azide-modified d-amino acids than in controls; for example, we observed 7 times greater [18F]FB-sulfo-DBCO ligation in Staphylococcus aureus cultures incubated with 3-azido-d-alanine versus those incubated with d-alanine. Conclusions: The SPAAC radiotracer [18F]FB-sulfo-DBCO was validated in vitro via metabolic labeling of azide-bearing peptidoglycan muropeptides. d-Amino acid-derived PET radiotracers may be more efficiently screened via [18F]FB-sulfo-DBCO modification.

Strain-Promoted Cycloadditions in Lipid Bilayers Triggered by Liposome Fusion.

Due to the variety of roles served by the cell membrane, its composition and structure are complex, making it difficult to study. Bioorthogonal reactions, such as the strain promoted azide-alkyne cycloaddition (SPAAC), are powerful tools for exploring the function of biomolecules in their native environment but have been largely unexplored within the context of lipid bilayers. Here, we developed a new approach to study the SPAAC reaction in liposomal membranes using azide- and strained alkyne-functionalized Förster resonance energy transfer (FRET) dye pairs. This study represents the first characterization of the SPAAC reaction between diffusing molecules inside liposomal membranes. Potential applications of this work include in situ bioorthogonal labeling of membrane proteins, improved understanding of membrane dynamics and fluidity, and the generation of new probes for biosensing assays.

Reflections on a Copenhagen-Minneapolis Axis in Bioorganic Chemistry.

The international peptide community rejoiced when one of its most distinguished members, Morten Meldal of Denmark, shared the 2022 Nobel Prize in Chemistry. In fact, the regiospecific solid-phase "copper(I)-catalyzed 1,3-dipolar cycloaddition of terminal alkynes to azides" (CuACC) reaction-that formed the specific basis for Meldal's recognition-was reported first at the 17th American Peptide Symposium held in San Diego in June 2001. The present perspective outlines intertwining conceptual and experimental threads pursued concurrently in Copenhagen and Minneapolis, sometimes by the same individuals, that provided context for Meldal's breakthrough discovery. Major topics covered include orthogonality in chemistry; the dithiasuccinoyl (Dts) protecting group for amino groups in α-amino acids, carbohydrates, and monomers for peptide nucleic acids (PNA); and poly(ethylene glycol) (PEG)-based solid supports such as PEG-PS, PEGA, and CLEAR [and variations inspired by them] for solid-phase peptide synthesis (SPPS), solid-phase organic synthesis (SPOS), and combinatorial chemistry that can support biological assays in aqueous media.

3-Acyl-4-Pyranone as a Lysine Residue-Selective Bioconjugation Reagent for Peptide and Protein Modification.

Chemoselective protein modification plays extremely important roles in various biological, medical, and pharmaceutical investigations. Mimicking the mechanism of the chemoselective reaction between natural azaphilones and primary amines, this work successfully simplified the azaphilone scaffold into much simpler 3-acyl-4-pyranones. Examinations confirmed that these slim-size mimics perfectly kept the unique reactivity for selective conjugation with the primary amines including lysine residues of peptides and proteins. The newly developed pyranone tool presents remarkably increased aqueous solubility and compatible second-order rate constant by comparison with the original azaphilone. Additional advantages also include the ease of biorthogonal combinative use with a copper-catalyzed azide-alkyne Click reaction, which was conveniently applied to decorate lysozyme with neutral-, positive- and negative-charged functionalities in parallel. Moderate-degree modification of lysozyme with positively charged quaternary ammoniums was revealed to increase the enzymatic activities.

Real-time Visualization of Transcribed mRNA via Click Chemistry in a Liposomal Space.

We present a CuAAC (Copper-Catalyzed Azide-Alkyne Cycloaddition) reaction protocol designed for the visualization of mRNA. To achieve this, we synthesized stable mRNA molecules incorporating the modified nucleoside analog, EU, a crucial element for fluorophore attachment. Leveraging this modified mRNA, we successfully executed the CuAAC reaction, wherein the pro-fluorophore, coumarin, was conjugated to EU on the mRNA through our meticulously designed CuAAC process. This innovative approach resulted in the emission of fluorescence, enabling both precise quantification and visual observation of mRNA. Furthermore, we demonstrated the feasibility of concurrent mRNA synthesis and visualization by seamlessly integrating the CuAAC reaction mix into the mRNA transcription process. Additionally, our novel methodology opens avenues for prospective real-time monitoring of mRNA transcription within artificial cells. These advancements hold significant promise for expanding our comprehension of fundamental cellular processes and finding applications across diverse biological contexts in the future.

Semisynthesis and characterization of versatile azide intermediates using sodium alginate and its homopolymeric derivatives as starting material.

Alginate, a polyuronic biopolymer composed of mannuronic and guluronic acid units, contain hydroxyl and carboxyl groups as targeting modification sites to obtain structures with new and/or improved biological properties. The copper-catalyzed azide-alkyne cycloaddition (CuAAC) is a versatile click reaction for polymer functionalization, but it typically requires a "pre-click" modification to introduce azide or alkyne groups. Here, we described a straightforward chemical path to selectively modify alginate carboxyl groups producing versatile azido derivatives through N-acylation using 3-azydopropylamine. The resulting azide-functionalized polysaccharides underwent click chemistry to yield amino derivatives, confirmed by NMR and FTIR analyses. The 1H NMR spectrum reveals a characteristic triazole group signal at 8.15 ppm. The absence of the azide FTIR band for all amino derivatives, previously observed for the N-acylation products, indicated reaction success. Antibacterial and antioxidant assessments revealed that the initial polysaccharide lacks E. coli inhibition, while the click chemistry-derived amine products exhibit growth inhibition at 5.0 mg/mL. Lower molecular weight derivatives demonstrate superior DPPH scavenging ability, particularly amino-derivatives (24-33 % at 1.2 mg/mL). This innovative chemical pathway offers a promising strategy for developing polysaccharide structures with enhanced properties, demonstrating potential applications in various fields.

An array of femtoliter wells for sensitive detection of copper using click chemistry.

Sensitive detection of copper ion (Cu2+), which is of great importance for environmental pollution and human health, is crucial. In this study, we present a highly sensitive method for measuring Cu2+ in an array of femtoliter wells. In brief, magnetic beads (MBs) modified with alkyne groups were bound to the azide groups of biotin-PEG3-azide (bio-PEG-N3) via Cu+-catalyzed click chemistry. Cu+ in the click chemistry reaction was generated by reducing Cu2+ with sodium ascorbate. Following the ligation, the surface of the MBs was modified with biotin, which could be labeled with streptavidin-ß-galactosidase (SßG). The MBs complex was then suspended in ß-galactosidase substrate fluorescein-di-ß-d-galactopyranoside (FDG), and loaded into the array of femtoliter wells. The MBs sank into the wells due to gravity, and the resulting fluorescent product, generated from the reaction between SßG on the surface of the MBs and FDG, was confined within the wells. The number of fluorescent wells increased with higher Cu2+ concentrations. The bright-field and fluorescent images of the wells were acquired using an inverted fluorescent microscope. The detection limit of this assay for Cu2+ was 1 nM without signal amplification, which was 103 times lower than that of traditional fluorescence detection assays.

An oxidative photocyclization approach to the synthesis of Securiflustra securifrons alkaloids.

Securines and securamines are cytotoxic alkaloids that contain reactive alkene and heterocyclic residues embedded in skeletons comprising four to six oxidized rings. This structural complexity imparts a rich chemistry to the isolates but has impeded synthetic access to the structures in the nearly three decades since their isolation. We present a flexible route to eight isolates that exemplify the three skeletal classes of metabolites. The route proceeds by the modular assembly of the advanced azides 38 and 49 (13 steps, 6 to 10% yield), sequential oxidative photocyclizations, and late-stage functional group manipulations. With this approach, the targets were obtained in 17 to 19 steps, 12 to 13 purifications, and 0.5 to 3.5% overall yield. The structure of an advanced intermediate was elucidated by microcrystal electron diffraction (MicroED) analysis. The route will support structure-function and target identification studies of the securamines.

Bioinspired Selenium-Nitrogen Exchange (SeNEx) Click Chemistry Suitable for Nanomole-Scale Medicinal Chemistry and Bioconjugation.

Click chemistry is a powerful molecular assembly strategy for rapid functional discovery. The development of click reactions with new connecting linkage is of great importance for expanding the click chemistry toolbox. We report the first selenium-nitrogen exchange (SeNEx) click reaction between benzoselenazolones and terminal alkynes (Se-N to Se-C), which is inspired by the biochemical SeNEx between Ebselen and cysteine (Cys) residue (Se-N to Se-S). The formed selenoalkyne connection is readily elaborated, thus endowing this chemistry with multidimensional molecular diversity. Besides, this reaction is modular, predictable, and high-yielding, features fast kinetics (k2≥14.43 M-1 s-1), excellent functional group compatibility, and works well at miniaturization (nanomole-scale), opening up many interesting opportunities for organo-Se synthesis and bioconjugation, as exemplified by sequential click chemistry (coupled with ruthenium-catalyzed azide-alkyne cycloaddition (RuAAC) and sulfur-fluoride exchange (SuFEx)), selenomacrocycle synthesis, nanomole-scale synthesis of Se-containing natural product library and DNA-encoded library (DEL), late-stage peptide modification and ligation, and multiple functionalization of proteins. These results indicated that SeNEx is a useful strategy for new click chemistry developments, and the established SeNEx chemistry will serve as a transformative platform in multidisciplinary fields such as synthetic chemistry, material science, chemical biology, medical chemistry, and drug discovery.

Immobilization of Adenosine Derivatives onto Cellulose Nanocrystals via Click Chemistry for Biocatalysis Applications.

Adenosine triphosphate (ATP) is a central molecule of organisms and is involved in many biological processes. It is also widely used in biocatalytic processes, especially as a substrate and precursor of many cofactors─such as nicotinamide adenine dinucleotide phosphate (NADP(H)), coenzyme A (CoA), and S-adenosylmethionine (SAM). Despite its great scientific interest and pivotal role, its use in industrial processes is impeded by its prohibitory cost. To overcome this limitation, we developed a greener synthesis of adenosine derivatives and efficiently selectively grafted them onto organic nanoparticles. In this study, cellulose nanocrystals were used as a model combined with click chemistry via a copper-catalyzed azide/alkyne cycloaddition reaction (CuAAC). The grafted adenosine triphosphate derivative fully retains its biocatalytic capability, enabling heterobiocatalysis for modern biochemical processes.

1H-1,2,3-triazolyl-1,6-naphthyridin-7(6H)-ones as Potential Fluorescent Nucleoside Analogues: Synthesis and Optical Properties.

In this article, we present the synthesis and the optical properties of three original molecules as potential fluorescent ribonucleoside analogues incorporating a 1,6-naphthyridin-7(6H)-one scaffold as a fluorescent nucleobase and a 1,2,3-triazole as a linkage. The nucleosides were prepared via a Cu alkyne-azide cycloaddition (CuAAC) reaction between a ribofuranosyl azide and a 4-ethynylpyridine partner. Construction of substituted 1,6-naphthyridin-7(6H)-ones was achieved through two additional steps. Optical property studies were investigated on nucleoside analogues. Powerful fluorescence properties have been evidenced with a remarkable change of emissivity depending on the polarity of the solvent, making these molecules suitable as a new class of artificial fluorescent nucleosides for investigating enzyme binding sites as well as probing nucleic acids. In addition, we are convinced that such analogues could be of great interest in the search for new antiviral or antitumoral drugs based on nucleosides.