Search

AMPA receptors (AMPARs) are the main drivers of excitatory glutamatergic transmission in the brain, central to synaptic plasticity, and are key drug targets. However, AMPARs are expressed in virtually every neuron in the central nervous system and are activated with complex temporal dynamics, making it difficult to determine their functional roles with sufficient precision. Here we describe a cell specific, light‐controllable competitive antagonist for the AMPA receptor called MP‐GluAblock that combines the temporal precision of a photo‐switchable ligand with the spatial and cellular specificity of a genetically‐encoded membrane‐anchor protein. This tool could pave the way for controlling endogenous AMPARs in neural circuits with cellular, spatial, and temporal specificity. [ABSTRACT FROM AUTHOR]

Antisense oligonucleotide (ASO) therapies hold significant promise in the realm of molecular medicine. By precisely targeting RNA molecules, ASOs offer an approach to modulate gene expression and protein production, making them valuable tools for treating a wide range of genetic and acquired diseases. As the precise intracellular targeting and delivery of ASOs is challenging, strategies for preparing ASO‐ligand conjugates are in exceedingly high demand. This work leverages the utility of native chemical ligation to conjugate ASOs with therapeutically relevant chemical modifications including locked nucleic acids and phosphorothioate backbone modifications to peptides and sugars via a stable amide linkage. A suite of post‐ligation functionalizations through modification of the cysteine ligation handle are highlighted, including chemoselective radical desulfurization, lipidation, and alkylation with a range of valuable handles (e.g. alkyne, biotin, and radionuclide chelating ligands), affording multifunctional constructs for further applications in biology and medicine. Application of the methodology to a clinically‐relevant triantennary‐GalNAc ASO conjugate and validation of its binding and functional activity underpins the applicability of the technique to oligonucleotide‐based therapeutics. [ABSTRACT FROM AUTHOR]

A new approach of dual visible light‐induced gold(III)‐catalyzed alkynylation and its application in selective modification of alkyne‐linked peptides has been developed. The bis‐cyclometalated gold(III) complex exhibited dual roles of (1) in situ generation of quinolizinium‐based photosensitizer (λem=500 – 594 nm) and (2) alkynylation of iminium ions. Under optimized conditions, alkynylated products were afforded in good yields up to 73 %. The application of this strategy in selective modification of alkyne‐linked peptides gave modified peptides in up to 67 % conversion. Our dual visible light/gold(III) catalysis exemplifies the potential of merging photocatalysis and transition metal catalysis to develop novel bioconjugation. [ABSTRACT FROM AUTHOR]

Nanostructured materials represent promising substrates for biocatalyst immobilization and activation. Cellulose nanocrystals (CNCs), accessible from waste and/or renewable sources, are sustainable and biodegradable, show high specific surface area for anchoring a high number of enzymatic units, and high thermal and mechanical stability. In this work, we present a holistic enzyme‐based approach to functional antibacterial materials by bioconjugation between the lysozyme from chicken egg white and enzymatic cellulose nanocrystals. The neutral CNCs were prepared by endoglucanase hydrolysis from Avicel. We explore the covalent immobilization of lysozyme on enzymatic CNCs and on their TEMPO oxidized derivatives (TO‐CNCs), comparing immobilization yields, material properties, and enzymatic activities. The materials were characterized by X‐ray diffractometry (XRD), attenuated total reflectance Fourier Transform infrared spectroscopy (ATR‐FTIR), bicinchoninic acid (BCA) assay, field‐emission scanning electron microscopy (FE‐SEM) and dynamic light scattering (DLS). We demonstrate the higher overall efficiency of the immobilization process carried out on TO‐CNCs, based on the success of covalent bonding and on the stability of the isolated bioconjugates. [ABSTRACT FROM AUTHOR]

Antibody–drug conjugates (ADCs) are a growing class of chemotherapeutic agents that have yielded striking clinical successes. However, the efficacy of ADCs often suffers from issues associated with tumor heterogeneity and resistance. To overcome these problems, a new generation of ADCs comprising a single monoclonal antibody with multiple different payloads attached, termed multi‐payload ADCs, have been developed. Here we deploy multiple orthogonal site‐specific protein modification strategies to generate highly homogeneous multi‐functionalised antibody conjugates comprising up to four different functionalities installed at four unique sites on the antibody. This work, which includes the use of a site‐specific cyclopropenone (CPO)‐based reagent, represents the first example of a homogeneous multi‐payload ADC with a payload count greater than two, and thereby facilitates the development of the next generation of ADCs. [ABSTRACT FROM AUTHOR]

Background: The incorporation of radionuclides into peptides and larger biomolecules requires efficient and sometimes biorthogonal reaction conditions, to which click chemistry provides a convenient approach. Methods: Traditionally, click-based radiolabeling techniques have focused on classical click chemistry, such as copper(I)-catalyzed alkyne-azide [3+2] cycloaddition (CuAAC), strain-promoted azide-alkyne [3+2] cycloaddition (SPAAC), traceless Staudinger ligation, and inverse electron demand Diels–Alder (IEDDA). Results: However, newly emerging click-based radiolabeling techniques, including tyrosine-click, sulfo-click, sulfur(VI) fluoride exchange (SuFEx), thiol-ene click, azo coupling, hydrazone formations, oxime formations, and RIKEN click offer valuable alternatives to classical click chemistry. Conclusions: This review will discuss the applications of these techniques in peptide radiochemistry. [ABSTRACT FROM AUTHOR]

Comprehensive Summary: Interfacing DNA oligonucleotides and DNA aptamers with gold nanoparticles has generated numerous functional hybrid materials for sensing, self‐assembly and drug delivery applications. Our lab has been working in this area for 15 years. In this article, the current understanding of the adsorption of DNA to gold nanoparticles is summarized, and related applications in bioconjugation of DNA to gold surface is described. In addition, problems of using gold nanoparticles to signaling aptamer binding are discussed. Finally, re‐selection of aptamers for previously reported targets using the library‐immobilization method is reviewed. What is the most favorite and original chemistry developed in your research group? My most favorite and original work is the study of biointerface chemistry between DNA oligonucleotides and gold nanoparticles enabling rapid DNA bioconjugation by lowering the pH and freezing. How do you get into this specific field? Could you please share some experiences with our readers? My PhD training was focused on catalytic DNA for the detection of metal ions, when I used gold nanoparticles to signal the reactions catalyzed by DNA. When I started my independent career in the University of Waterloo in 2009, I realized that there were many fundamental issues regarding gold nanoparticles and DNA to be studied. My first teaching assignment was a course named 'Surfaces and Interfaces'. By teaching this course, I learned a lot of surface science concepts that were later used in my research. That was the starting point for me to set up my own research program in this area. What is the most important personality for scientific research? Curiosity, careful observation, critical thinking, and keep trying. How do you keep balance between research and family? Research and personal life don't have to be always in conflict. New ideas may spark when I am in a relaxed family environment. What are your favorite journals? Journal of the American Chemical Society, Angewandte Chemie International Edition, Nucleic Acids Research, Analytical Chemistry, Langmuir. What are your hobbies? Running; traveling; watching movies. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae is the leading cause of neonatal sepsis and is increasingly difficult to treat owing to antibiotic resistance. Vaccination represents a tractable approach to combat this resistant bacterium; however, there is currently not a licensed vaccine. Surface polysaccharides, including O-antigens of lipopolysaccharide, have long been attractive candidates for vaccine inclusion. Herein we describe the generation of a bioconjugate vaccine targeting 7 predominant O-antigen subtypes in K. pneumoniae. Each bioconjugate was immunogenic in isolation, with limited cross-reactivity among subtypes. Vaccine-induced antibodies demonstrated varying degrees of binding to a wide variety of K. pneumoniae strains. Furthermore, serum from vaccinated mice induced complement-mediated killing of many of these strains. Finally, increased capsule interfered with the ability of O-antigen antibodies to bind and mediate killing of some K. pneumoniae strains. Taken together, these data indicate that this novel heptavalent O-antigen bioconjugate vaccine formulation exhibits limited efficacy against some, but not all, K. pneumoniae isolates. [ABSTRACT FROM AUTHOR]

The reaction between glycine-type aminonaphthol derivatives substituted with 2- or 1-naphthol and indole or 7-azaindole has been tested. Starting from 2-naphthol as a precursor, the reaction led to the formation of ring-closed products, while in the case of a 1-naphthol-type precursor, the desired biaryl ester was isolated. The synthesis of a bifunctional precursor starting from 5-chloro-8-hydroxyquinoline, morpholine, and ethyl glyoxylate via modified Mannich reaction is reported. The formed Mannich base 10 was subjected to give bioconjugates with indole and 7-azaindole. The effect of the aldehyde component and the amine part of the Mannich base on the synthetic pathway was also investigated. In favor of having a preliminary overview of the structure-activity relationships, the derivatives have been tested on cancer and normal cell lines. In the case of bioconjugate 16, as the most powerful scaffold in the series bearing indole and a 5-chloro-8-hydroxyquinoline skeleton, a potent toxic activity against the resistant Colo320 colon adenocarcinoma cell line was observed. Furthermore, this derivative was selective towards cancer cell lines showing no toxicity on non-tumor fibroblast cells. [ABSTRACT FROM AUTHOR]

A series of biotin-functionalized transition metal complexes was prepared by iClick reaction from the corresponding azido complexes with a novel alkyne-functionalized biotin derivative ([Au(triazolatoR,R′)(PPh3)], [Pt(dpb)(triazolatoR,R′)], [Pt(triazolatoR,R′)(terpy)]PF6, and [Ir(ppy)(triazolatoR,R′)(terpy)]PF6 with dpb = 1,3-di(2-pyridyl)benzene, ppy = 2-phenylpyridine, and terpy = 2,2′:6′,2′′-terpyridine and R = C6H5, R′ = biotin). The complexes were compared to reference compounds lacking the biotin moiety. The binding affinity toward avidin and streptavidin was evaluated with the HABA assay as well as isothermal titration calorimetry (ITC). All compounds exhibit the same binding stoichiometry of complex-to-avidin of 4:1, but the ITC results show that the octahedral Ir(III) compound exhibits a higher binding affinity than the square-planar Pt(II) complex. The antibacterial activity of the compounds was evaluated on a series of Gram-negative and Gram-positive bacterial strains. In particular, the neutral Au(I) and Pt(II) complexes showed significant antibacterial activity against Staphylococcus aureus and Enterococcus faecium at very low micromolar concentrations. The cytotoxicity against a range of eukaryotic cell lines was studied and revealed that the octahedral Ir(III) complex was non-toxic, while the square-planar Pt(II) and linear Au(I) complexes displayed non-selective micromolar activity. [ABSTRACT FROM AUTHOR]

As concern raised by the World Health Organization (WHO) of antibiotic‐resistant and bio‐defensive bacteria, a metal–organic framework (MOF) based optical biosensor came into consideration for precise, quick, and sensitive detection of Escherichia coli (E. coli) (ATCC 10799) using bacteriophage as a bio‐recognition element. In the present study, amine‐functionalized Fe‐based MOF, i.e., NH2‐MIL‐101(Fe), was synthesized by the solvothermal method (approx. 531–1106 nm in size and 20 mV zeta potentials by DLS) and further characterized by SEM, XRD, ATR‐FTIR, UV–VIS, and photoluminescent (PL) spectroscopy. The lytic bacteriophage was isolated from a sewage sample, purified, and concentrated using the ultra‐centrifugation method and achieved a high titer of 7.3 × 1012 PFU/ml. The concentration, stability, and accessible receptor binding domains (RBDs) of the biorecognition element for binding with their analytes play an important role in developing sensitive and specific biosensor systems. To fulfill the mentioned criteria, optimized glutaraldehyde concentration was estimated at 0.25%, at 30 °C for conjugating maximum bacteriophage titer of 8.6 × 105 PFU/ml for each 1 mg amine functionalized iron‐based MOF. The synthesized detection probe has shown excellent photoluminescence and antibacterial activity and achieved a detection limit of 652 CFU/ml over a bacterial detection concentration range from 5.78 × 101 to 5.78 × 106 CFU/ml for E. coli with 10–12 min of response time, high specificity, and long‐term stability even at room temperature. Therefore, it can be inferred that this MOF‐based strategy can be helpful in the specific and sensitive detection of various bacterial pathogens using bacteriophage as a bio‐recognition element. [ABSTRACT FROM AUTHOR]

Bioorthogonal click chemistry has played a transformative role in many research fields, including chemistry, biology, and medicine. Click reactions are crucial to produce increasingly complex bioconjugates, to visualize and manipulate biomolecules in living systems and for various applications in bioengineering and drug delivery. As biological (model) systems grow more complex, researchers have an increasing need for using multiple orthogonal click reactions simultaneously. In this review, we will introduce the most common bioorthogonal reactions and discuss their orthogonal use on the basis of their mechanism and electronic or steric tuning. We provide an overview of strategies to create reaction orthogonality and show recent examples of mutual orthogonal chemistry used for simultaneous biomolecule labeling. We end by discussing some considerations for the type of chemistry needed for labeling biomolecules in a system of choice. [ABSTRACT FROM AUTHOR]

The applications of fluorinated molecules in chemical biology are rapidly expanding driven by the unique properties of C−F bonds, leading to increased interest in methodologies for controlled introduction of this atom. In this study, we present the first method for late‐stage fluorination of tyrosine residues in proteins. Our results demonstrate that electrophilic fluorination using Selectfluor, a stable and non‐toxic reagent, offers a straightforward and cost‐effective method for labeling Cyanovirin‐N (CVN), a 101‐amino‐acid lectin with effective antiviral activity. Uni‐ and bidimensional 1H, 13C and 19F NMR analyses, along with mass spectrometry, revealed chemoselective fluorination of the three tyrosine residues in CVN without affecting its overall structure or mannose‐binding affinity. Additionally, we observed that other aromatic amino acids, such as tryptophan, phenylalanine, and histidine, are not fluorinated using this method. These findings advance our understanding of protein fluorination and its applications in studying structure, dynamics, and interactions, as well as other biological utilities. [ABSTRACT FROM AUTHOR]

Surface functionalization strategy is becoming a crucial bridge from magnetic nanoparticles (MNPs) to their broad bio-application. To realize the multiple functions of MNPs such as magnetic manipulation, target capture, and signal amplification in their use of electrochemical biosensing, co-crosslinking strategy was proposed here to construct dual-functionalized MNPs by combining ultra-sensitive redox moieties and specific biological probes. In this work, MNPs with a TEM size of 10 nm were synthesized by co-precipitation for amination and PEGylation to maintain colloid stability once dispersed in high-ionic-strength buffer (such as phosphate-buffered saline). Then, MNPs@IgG were prepared via the bis(sulfosuccinimidyl) suberate (BS3) cross-linker to conjugate these IgG onto the MNP surface, with a binding efficiency of 73%. To construct dual-functionalized MNPs, these redox probes of ferrocene-NHS (Fc) were co-crosslinked onto the MNP surface, together with IgG, by using BS3. The developed MNPs@Redox@IgG were characterized by SDS‒PAGE to identify IgG binding and by square wave voltammetry (SWV) to validate the redox signal. Additionally, the anti-CD63 antibodies were selected for the development of MNPs@anti-CD63 for use in the bio-testing of exosome sample capture. Therefore, co-crosslinking strategy paved a way to develop dual-functionalized MNPs that can be an aid of their potential utilization in diagnostic assay or electrochemical methods. [ABSTRACT FROM AUTHOR]

Spider silk is renowned for its exceptional mechanical properties, surpassing those of other natural and many synthetic fibers. Yet, replicating its remarkable properties through synthetic production remains a challenge. The variability in the mechanical properties of synthetic spider silks lacking protective coatings, exacerbated by factors such as spinning conditions and humidity levels, poses an additional challenge, impacting their application potential. Bioconjugation offers a versatile synthetic method to modify protein structures, enhancing their pharmacokinetics, solubility, stability, and immune response. In particular, polyethylene glycol (PEG)‐ylation has emerged as a successful strategy with numerous marketed PEG–protein conjugates. This study introduces synthetic spider silk—multiarm PEG bioconjugates, facilitating spidroin crosslinking, and chemical functionalization while retaining a biomimetic spinning approach. Two different examples demonstrate the potential of this approach to improve the fiber's tensile strength and extensibility, respectively, both leading to an increased toughness modulus. Furthermore, the approach could allow the tuning of fiber mechanical properties without developing a new mini‐spidroin construct and fiber coating with lipids attached to multiarm PEG, potentially mitigating the impact of environmental conditions on synthetic spider silk fibers. [ABSTRACT FROM AUTHOR]

This review delves into the innovative field of interfacial photoelectrochemical (iPEC) and photoelectrochemical (PEC) catalysis, the dynamic synthesis methodologies that seamlessly integrates electrochemical and photoredox catalysis for efficient and environmentally friendly reactions. Utilizing minute quantities of photocatalysts, visible light becomes a powerful tool, generating transient excited states to catalyze a spectrum of reactions through single‐electron oxidation or reduction events. The review categorizes recent advancements, highlighting applications in organic synthesis, late‐stage modifications, and the distinctive features of photoelectrochemical methodology. Despite being in its early stages, this synergistic approach holds great promise for propelling organic synthesis forward, with potential applications in large‐scale synthesis and diverse late‐stage functionalizations, including asymmetric catalysis and bioconjugation strategies for biomolecule modifications. [ABSTRACT FROM AUTHOR]

Hyaluronic acid (HA) is a natural, non‐sulfated glycosaminoglycan (GAG) present in ECM. It is involved in different biological functions with appealing properties in cosmetics and pharmaceutical preparations as well as in tissue engineering. Generally, HA has been electrospun in blends with natural or synthetic polymers to produce fibers having diameters in the order of nano and micro‐scale whose pores can host cells able to regenerate damaged tissues. In the last decade, a rich literature on electrospun HA‐based materials arose. Chemical modifications were generally introduced in HA scaffolds to favour crosslinking or conjugation with bioactive molecules. Considering the high solubility of HA in water, HA‐based electrospun scaffolds are cross‐linked to increase the stability in biological fluids. Crosslinking is necessary also to avoid the release of HA from the hybrid scaffold when implanted in‐vivo. Furthermore, to endow the HA based scaffolds with new chemical or biological properties, conjugation of bioactive molecules to HA was widely reported. Herein, we review the existing research classifying chemical modifications on HA and HA‐based electrospun fibers into three categories: i) in‐situ crosslinking of electrospun HA‐based scaffolds ii) off‐site crosslinking of electrospun HA‐based scaffolds; iii) conjugation of biofunctional molecules to HA with focus on peptides. [ABSTRACT FROM AUTHOR]

ABSTRACTTherapeutic bioconjugates are emerging as an essential tool to combat human disease. Site-specific conjugation technologies are widely recognized as the optimal approach for producing homogeneous drug products. Non-natural amino acid (nnAA) incorporation allows the introduction of bioconjugation handles at genetically defined locations. Escherichia coli (E. coli) is a facile host for therapeutic nnAA protein synthesis because it can stably replicate plasmids encoding genes for product and nnAA incorporation. Here, we demonstrate that by engineering E. coli to incorporate high levels of nnAAs, it is feasible to produce nnAA-containing antibody fragments and full-length immunoglobulin Gs (IgGs) in the cytoplasm of E. coli. Using high-density fermentation, it was possible to produce both of these types of molecules with site-specifically incorporated nnAAs at titers > 1 g/L. We anticipate this strategy will help simplify the production and manufacture of promising antibody therapeutics.

Photosynthetic light harvesting requires efficient energy transfer within dynamic networks of light-harvesting complexes embedded within phospholipid membranes. Artificial light-harvesting models are valuable tools for understanding the structural features underpinning energy absorption and transfer within chromophore arrays. Here, a method for attaching a protein-based light-harvesting model to a planar, fluid supported lipid bilayer (SLB) is developed.  The protein model consists of the tobacco mosaic viral capsid proteins that are gene-doubled to create a tandem dimer (dTMV). Assemblies of dTMV break the facial symmetry of the double disk to allow for differentiation between the disk faces. A single reactive lysine residue is incorporated into the dTMV assemblies for the site-selective attachment of chromophores for light absorption. On the opposing dTMV face, a cysteine residue is incorporated for the bioconjugation of a peptide containing a polyhistidine tag for association with SLBs. The dual-modified dTMV complexes show significant association with SLBs and exhibit mobility on the bilayer. The techniques used herein offer a new method for protein-surface attachment and provide a platform for evaluating excited state energy transfer events in a dynamic, fully synthetic artificial light-harvesting system.

A carboranylporphyrin of A3B-type bearing a single pentafluorophenyl ring was prepared through the regioselective nucleophilic aromatic substitution reaction of the p-fluorine atoms in 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin with 9-mercapto-m-carborane. The reaction of this porphyrin with sodium azide led to the selective substitution of the p-fluorine atom in the pentafluorophenyl substituent with an azide functionality which upon reduction with SnCl2 resulted in the formation of the corresponding porphyrin with an amino group. Pentafluorophenyl-substituted A3B-porphyrins were studied and transformed to thiol and amino-substituted compounds allowing for the preparation of porphyrins with different reactive groups such as hydroxy and amino derivatives capable for further functionalization and conjugation of these porphyrins to other substrates. In addition, conjugates containing maleimide or biotin entities in the structure of carborane A3B-porphyrin were also synthesized based on the amino-substituted A3B-porphyrin. The structures of the prepared carboranylporphyrins were determined by UV–vis, IR, 1H, 19F, 11B NMR spectroscopic data and MALDI mass spectrometry.

The article discusses the cyanopyridine-aminthiol click reaction, a bioconjugation method that allows for precise and selective chemical modifications in complex biological systems. The reaction is efficient, exhibits excellent chemoselectivity, and is stable under mild redox conditions. It is orthogonal to most endogenous reactants, except for cysteine itself and peptides or proteins with N-terminal cysteine residues. The reaction has been used for peptide macrocyclization, the synthesis of macrocyclic peptides, and the creation of DNA-encoded libraries. Future research is needed to explore its potential for broader bioconjugation purposes. [Extracted from the article]

Nucleic acids in the form of siRNA, antisense oligonucleotides or mRNA are currently explored as new promising modalities in the pharmaceutical industry. Particularly, the success of mRNA‐vaccines against SARS‐CoV‐2, along with the successful development of the first sugar‐modified siRNA therapeutics has inspired the field. The development of nucleic acid therapeutics requires efficient chemistry to link oligonucleotides to chemical structures that can improve stability, boost cellular uptake, or enable specific targeting. For the siRNA therapeutics currently in use, modification of the 3′‐end of the oligonucleotides with triple‐N‐acetylgalactosamine (GalNAc)3 was shown to be of significance. This modification is currently achieved through cumbersome multistep synthesis and subsequent loading onto the solid support material. Herein, we report the development of a bifunctional click‐reactive linker that allows the modification of oligonucleotides in a tandem click reaction with multiple sugars, regardless of the position within the oligonucleotide, with remarkable efficiency and in a one‐pot reaction. [ABSTRACT FROM AUTHOR]

Multiplexed bead assays for solution‐phase biosensing often encounter cross‐over reactions during signal amplification steps, leading to unwanted false positive and high background signals. Current solutions involve complex custom‐designed and costly equipment, limiting their application in simple laboratory setup. In this study, we introduce a straightforward protocol to adapt a multiplexed single‐bead assay to standard fluorescence imaging plates, enabling the simultaneous analysis of thousands of reactions per plate. This approach focuses on the design and synthesis of bright fluorescent and magnetic microspheres (MagSiGlow) with multiple fluorescent wavelengths serving as unique detection markers. The imaging‐based, single‐bead assay, combined with a scripted algorithm, allows the detection, segmentation, and co‐localization on average of 7500 microspheres per field of view across five imaging channels in less than one second. We demonstrate the effectiveness of this method with remarkable sensitivity at low protein detection limits (100 pg/mL). This technique showed over 85 % reduction in signal cross‐over to the solution‐based method after the concurrent detection of tumor‐associated protein biomarkers. This approach holds the promise of substantially enhancing high throughput biosensing for multiple targets, seamlessly integrating with rapid image analysis algorithms. [ABSTRACT FROM AUTHOR]

Dual bioorthogonal labeling enables the investigation and understanding of interactions in the biological environment that are not accessible by a single label. However, applying two bioorthogonal reactions in the same environment remains challenging due to cross‐reactivity. We developed a pair of differently modified 2′‐deoxynucleosides that solved this issue for dual and orthogonal labeling of DNA. Inverse‐electron demand Diels–Alder and photoclick reactions were combined to attach two different fluorogenic labels to genomic DNA in cells. Using a small synthetic library of 1‐ and 3‐methylcyclopropenyl‐modified 2′‐deoxynucleosides, two 2′‐deoxyuridines were identified to be the fastest‐reacting ones for each of the two bioorthogonal reactions. Their orthogonal reactivity could be evidenced in vitro. Primer extension experiments were performed with both 2′‐deoxyuridines investigating their replication properties as substitutes for thymidine and evaluating subsequent labeling reactions on the DNA level. Finally, dual, orthogonal and metabolic fluorescent labeling of genomic DNA was demonstrated in HeLa cells. An experimental procedure was developed combining intracellular transport and metabolic DNA incorporation of the two 2′‐deoxyuridines with the subsequent dual bioorthogonal labeling using a fluorogenic cyanine‐styryl tetrazine and a fluorogenic pyrene‐tetrazole. These results are fundamental for advanced metabolic labeling strategies for nucleic acids in the future, especially for live cell experiments. [ABSTRACT FROM AUTHOR]

With the advent of bioconjugation chemistry in the last two decades, highlighted by the Nobel Prize 2022, the quest for possible novel applications has been greatly intensified, broadening the prospects of these mostly simple, specific, and high‐yield reactions. The advancement of bioconjugation methods is anticipated to expand the scope of bioinstructive and bioadaptive materials science in the future. This perspective article will discuss the reactions developed in this research area over the last 10 years for coupling various biological entities such as polysaccharides, oligonucleotides, peptides, and proteins. Building on this, the impact of bioconjugation reactions in materials science and 3D printing, including their challenges and requirements is shown. Established procedures for modifying molecular structures such as Covalent and Metal Organic Frameworks (COF/MOF) or hybrid materials for biomedical applications and the scope for future research and optimization will be presented. [ABSTRACT FROM AUTHOR]

The compositional scope of polymer zwitterions has grown significantly in recent years and now offers designer synthetic materials that are broadly applicable across numerous areas, including supracolloidal structures, electronic materials interfaces, and macromolecular therapeutics. Among recent developments in polymer zwitterion syntheses are those that allow insertion of reactive functionality directly into the zwitterionic moiety, yielding new monomer and polymer structures that hold potential for maximizing the impact of zwitterions on the macromolecular materials chemistry field. This manuscript describes the preparation of zwitterionic choline phosphate (CP) methacrylates containing either aromatic or aliphatic thiols embedded directly into the zwitterionic moiety. The polymerization of these functional CP methacrylates by reversible addition‐fragmentation chain‐transfer methodology yields polymeric zwitterionic thiols containing protected thiol functionality in the zwitterionic units. After polymerization, the protected thiols are liberated to yield thiol‐rich polymer zwitterions which serve as precursors to subsequent reactions that produce polymer networks as well as polymer‐protein bioconjugates. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Paper-based biosensing platforms are the leading area of research today. In this work, a platform for biosensing applications with improved detection capability has been prepared using gold nanoparticles (AuNPs) incorporated in electrospun nanofibers. The computational study results demonstrated that the addition of AuNPs brings about better stability to the polymer complex, and the energy band gap was found to be lowered for the PVA-AuNPs (Eg = 3.57 eV) compared to PVA (Eg = 8.82 eV). Based on this data, AuNPs were incorporated into the polymer matrix by immersion and dispersion techniques. Different ratios of polyvinyl alcohol (PVA) to AuNPs have been prepared, and the optical, thermal, morphological, and structural properties of the substrates were evaluated to prepare a matrix with better biosensing capabilities. Improved photoluminescence emission intensity of the order of 2.5 times higher was observed for PVA-AuNPs (7:3) nanofibers compared to bare PVA nanofibers. The improved photoluminescence emission intensity of the polymer matrix can be used as a quantitative parameter for the diagnosis of several diseases. The field emission scanning electron microscopy (FESEM) and high-resolution transmission electron microscopy (HRTEM) analysis shows the successful encapsulation of AuNPs within the nanofibers with an average fiber diameter of 101 ± 21 nm and particle size of around 4.24 nm of Au NPs. The prepared PVA-AuNPs nanofibers showed stable luminescence properties (less than 10% variation) even after two months of storage at room temperature. The bioconjugation studies showed better photoluminescence emission intensity for the proposed substrate than the conventional nitrocellulose (NC) membrane. The functional performance of the modified NC membrane with electrospun nanofibers showed a three times higher response than the bare NC membrane. The present study may give new insight to use the gold-incorporated nanofibers as an additive element to the conventional NC membrane in order to bring out better bioconjugation competency with improved sensing properties. [ABSTRACT FROM AUTHOR]

While their broad utility in various chemistry fields were well recognized for decades, fluoroalcohols have recently emerged as a unique solvent system for bioconjugation development. This review describes examples and roles of fluoroalcohols such as trifluoroethanol (TFE) and hexafluoroisopropanol (HFIP) for chemical modification of biomolecules such as polypeptides, nucleic acids, and saccharides. Many chemical modification processes were facilitated by notable functions of those fluoroalcohols such as a proton shuttle, reversible adduct formation with reactive species, and compatibility with electrochemistry/photochemistry. The usefulness of the fluoroalcohol solvents can be even promoted by its combination with a different solvent system for reaction enhancement and protein stabilization. The collection of the various chemical transformations in this review is an indication of the rapid growth of the solvent-assisted bioconjugation field.

To address the current challenges in making bright, stable, and small DNA-functionalized quantum dots (QDs), we have developed a one-step ligand-exchange method to produce QD-DNA conjugates from commonly available hydrophobic QDs. We show that by systematically adjusting the reaction conditions such as ligand-to-nanoparticle molar ratio, pH, and solvent composition, stable and highly photoluminescent water-soluble QD-DNA conjugates with relatively high ligand loadings can be produced. Moreover, by site specifically binding these QD-DNA conjugates to a DNA origami template, we demonstrate that these bioconjugates have sufficient colloidal stability for DNA-directed self-assembly. Fluorescence quenching by an adjacent gold nanoparticle (AuNP) was demonstrated. Such QD-AuNP dimers may serve as biosensors with improved sensitivity and reproducibility. Moreover, our simple method can facilitate the assembly of QDs into more complex superlattices and discrete clusters that may enable novel photophysical properties.

The chemical bioconjugation of proteins has seen tremendous applications in the past decades, with the booming of antibody‐drug conjugates and their use in oncology. While genetic engineering has permitted to produce bespoke proteins featuring key (un−)natural amino acid residues poised for site‐selective modifications, the conjugation of native proteins is riddled with selectivity issues. Chemoselective strategies are plentiful and enable the precise modification of virtually any residue with a reactive side‐chain; site‐selective methods are less common and usually most effective on small and medium‐sized proteins. In this context, we studied the application of the Ugi multicomponent reaction for the site‐selective conjugation of amine and carboxylate groups on proteins, and antibodies in particular. Through an in‐depth mechanistic methodology work supported by peptide mapping studies, we managed to develop a set of conditions allowing the highly selective modification of antibodies bearing N‐terminal glutamate and aspartate residues. We demonstrated that this strategy did not alter their affinity toward their target antigen and produced an antibody‐drug conjugate with subnanomolar potency. Excitingly, we showed that the high site selectivity of our strategy was maintained on other protein formats, especially on anticalins, for which directed mutagenesis helped to highlight the key importance of a single lysine residue. [ABSTRACT FROM AUTHOR]

The term "in vivo ("in the living") chemistry" refers to chemical reactions that take place in a complex living system such as cells, tissue, body liquids, or even in an entire organism. In contrast, reactions that occur generally outside living organisms in an artificial environment (e.g., in a test tube) are referred to as in vitro. Over the past decades, significant contributions have been made in this rapidly growing field of in vivo chemistry, but it is still not fully understood, which transformations proceed efficiently without the formation of by‐products or how product formation in such complex environments can be characterized. Potential applications can be imagined that synthesize drug molecules directly within the cell or confer new cellular functions through controlled chemical transformations that will improve the understanding of living systems and develop new therapeutic strategies. The guiding principles of this contribution are twofold: 1) Which chemical reactions can be translated from the laboratory to the living system? 2) Which characterization methods are suitable for studying reactions and structure formation in complex living environments? [ABSTRACT FROM AUTHOR]

Iminoboronates and diazaborines are related classes of compounds that feature an imine ortho to an arylboronic acid (iminoboronate) or a hydrazone that cyclizes with an ortho arylboronic acid (diazaborine). Rather than acting as independent chemical motifs, the arylboronic acid impacts the rate of imine/hydrazone formation, hydrolysis, and exchange with competing nucleophiles. Increasing evidence has shown that the imine/hydrazone functionality also impacts arylboronic acid reactivity toward diols and reactive oxygen and nitrogen species (ROS/RNS). Untangling the communication between C=N linked functionalities and arylboronic acids has revealed a powerful and tunable motif for bioconjugation chemistries and other applications in chemical biology. Here, we survey the applications of iminoboronates and diazaborines in these fields with an eye toward understanding their utility as a function of neighboring group effects. [ABSTRACT FROM AUTHOR]

Curcumin, an anti-cancer compound found in the spice turmeric, is difficult to use because of its low water solubility. Poly(amidoamine) dendrimers are macromolecules with many nanoscale branches and extremely active amine groups on their surfaces, allowing them to attach to anticancer medicines such as curcumin. The goal of this study was to synthesize and evaluate the structural and anti-cancer properties of the poly(amidoamine)-curcumin dendrimer complex. Poly(amidoamine) dendrimer was synthesized up to the fourth generation (G4) and modified with glycidol to form the hydroxyl terminated dendrimer (G4–OH). Then, folic acid (FA) was utilized as a ligand to target the system specifically for cancer cell penetration. In addition to the physical loading of curcumin (Cu) on the FA-grafted dendrimer (G4–FA), curcumin was modified using ethyl bromoacetate, hydrolyzed with sodium hydroxide, and prepared to attach chemically to the hydroxyl terminated dendrimer (G4–Cu). FT-IR and 1H-NMR characterizations showed successful synthesis of the dendrimers as well as attachment of modified curcumin on the G4 surface. G4–FA revealed more effective performance in the release of loaded curcumin with release of 63.7% of loaded drug during 12 h. Using two cell lines of MG-63 and HT-29, the G4–Cu complex had the greatest growth inhibitory effect by increasing time among the derivatives, with the cell viability rate dropping by about 67%. These findings suggest that curcumin in combination with a fourth-generation dendrimer could be an effective cancer-fighting agent. The G4–Cu causes the complex to work more effectively, killing cancer cells to a greater extent, indicating the potential of the poly(amidoamine) dendrimer derivative for cancer treatments. [ABSTRACT FROM AUTHOR]

Fungal pathogens such as Candida albicans have significant impacts on women's health and the economy worldwide. Current detection methods often require access to laboratory facilities that are costly, inconvenient, and slow to access. This often leads to self-diagnosis, self-treatment and eventual antifungal resistance. We have created a rapid (within five minutes), cost-effective, and user-friendly method for the early detection of Candida albicans. Our platform utilises aptamer-tagged-gold-core-shell nanoparticles for Candida albicans detection based on the presence of 1,3-β-d glucan molecules. Nanoparticle aggregation occurs in the presence of Candida albicans fungal cells, causing a redshift in the UV-visible absorbance, turning from pink/purple to blue. This colour change is perceptible by the naked eye and provides a "yes"/"no" result. Our platform was also capable of detecting Candida albicans from individual yeast colonies without prior sample processing, dilution or purification. Candida albicans yeast cells were detected with our platform at concentrations as low as 5 × 105 cells within a 50 μL sample volume. We believe that this technology has the potential to revolutionise women's health, enabling women to test for Candida albicans accurately and reliably from home. This approach would be advantageous within remote or developing areas. [ABSTRACT FROM AUTHOR]

Surface modifications play a crucial role in enhancing the functionality of biomaterials. Different approaches can be followed in order to achieve the bioconjugation of drugs and biological compounds onto polymer surfaces. In this study, we focused on the immobilization of an amoxicillin antibiotic onto the surface of poly-L-lactic acid (PLLA) using a copper-free amino-yne click reaction. The utilization of this reaction allowed for a selective and efficient bioconjugation of the amoxicillin moiety onto the PLLA surface, avoiding copper-related concerns and ensuring biocompatibility. The process involved sequential steps that included surface activation via alkaline hydrolysis followed by an amidation reaction with ethylendiamine, functionalization with propiolic groups, and subsequent conjugation with amoxicillin via a click chemistry approach. Previous amoxicillin immobilization using tryptophan and fluorescent amino acid conjugation was carried out in order to determine the efficacy of the proposed methodology. Characterization techniques such as X-ray photoelectron spectroscopy (XPS), Attenuated Total Reflection (ATR)–Fourier Transform Infrared (FTIR) spectroscopy, surface imaging, water contact angle determination, and spectroscopic analysis confirmed the successful immobilization of both tryptophan and amoxicillin while maintaining the integrity of the PLLA surface. This tailored modification not only exhibited a novel method for surface functionalization but also opens avenues for developing antimicrobial biomaterials with improved drug-loading capacity. [ABSTRACT FROM AUTHOR]

Since the first report that 1,2,4,5‐tetrazines undergo a bioorthogonal reaction with dienophiles in the form of an inverse electron‐demand Diels‐Alder reaction, the demand for high‐yielding synthetic approaches towards them grew steadily. Despite this significant interest, tetrazines were predominantly accessed via the Pinner synthesis or other Pinner‐like reactions, significantly limiting available substrates. In particular, the synthesis of unsymmetrically substituted s‐tetrazines for the selective conjugation to another species presented a major challenge. To tackle these challenges, new and innovative high‐yielding transformations have been developed to widen the scope of accessible symmetric and unsymmetric tetrazines. For instance, Ni(II), Zn(II), and sulphur‐catalysed reactions between two nitriles and hydrazine were developed, which provide access to a wide range of (un‐)symmetric aryl s‐tetrazines. Also, amidines and orthoesters give tetrazines with alkyl substituents, whereas the usage of CH2Cl2 yields valuable H‐monosubstituted tetrazines. Methods using thiocarbohydrazides, terminal fluoroolefins, oxetane esters, or tosyl hydrazones are among the recent additions. Due to the high interest in tetrazine click‐chemistry approaches, some of which reach beyond inverse electron‐demand Diels‐Alder reactions, we expect this overview of synthetic routes to aid the exploration for further applications of tetrazines. Ultimately, we hope to provide a guide for chemists and chemical biologists for accessing functional s‐tetrazines. [ABSTRACT FROM AUTHOR]

There has been increasing interest in methods to generate synthetic lipid membranes as key constituents of artificial cells or to develop new tools for remodeling membranes in living cells. However, the biosynthesis of phospholipids involves elaborate enzymatic pathways that are challenging to reconstitute in vitro. An alternative approach is to use chemical reactions to non‐enzymatically generate natural or non‐canonical phospholipids de novo. Previous reports have shown that synthetic lipid membranes can be formed in situ using various ligation chemistries, but these methods lack biocompatibility and/or suffer from slow kinetics at physiological pH. Thus, it would be valuable to develop chemoselective strategies for synthesizing phospholipids from water‐soluble precursors that are compatible with synthetic or living cells Here, we demonstrate that amide‐forming ligations between lipid precursors bearing hydroxylamines and α‐ketoacids (KAs) or potassium acyltrifluoroborates (KATs) can be used to prepare non‐canonical phospholipids at physiological pH conditions. The generated amide‐linked phospholipids spontaneously self‐assemble into cell‐like micron‐sized vesicles similar to natural phospholipid membranes. We show that lipid synthesis using KAT ligation proceeds extremely rapidly, and the high selectivity and biocompatibility of the approach facilitates the in situ synthesis of phospholipids and associated membranes in living cells. [ABSTRACT FROM AUTHOR]

The photochemical thiol‐ene reaction is an efficient method for rapid and chemoselective formation of thioether linkages under mild conditions. It has found widespread use in small‐molecule synthesis as well as peptide and protein chemistry. While high‐throughput experimentation provides an invaluable tool for drug discovery, the considerable potential of the thiol‐ene reaction remains unexplored in this context. Herein, we report the development of nanomole‐scale photochemical thiol‐ene chemistry, performed using an automated approach in 1536‐well plates in a cost‐efficient custom reactor. Through careful reaction design and selection of reactants, this chemistry is applied to the lateral diversification of peptidic macrocycles to yield high purity crude mixtures. Selection of the photoinitiator 2,2‐dimethoxy‐2‐phenylacetophenone yields volatile breakdown products, which facilitates removal in vacuo. We demonstrate the use of this approach in late‐stage diversification of peptidic macrocycles with 96 examples averaging 95% conversion to the desired product. [ABSTRACT FROM AUTHOR]

AbstractVincamine, a natural chemical, was used as a reducing agent in the synthesis of IgG antibodies mediated biogenic gold nanoparticles (IgGAuNPs). Eventually, the synthesised IgGAuNPs were bioconjugated with the chemotherapeutic drug methotrexate (MTX-IgGAuNPs). The IgG isotype can target cancer cells through polymorphic Fc gamma receptors (FcγRs) and have therapeutic effects. They can restrict cell division by inhibiting different intracellular signal transduction pathways and activating NK cells and macrophages through antibody-dependent cellular cytotoxicity and macrophage-mediated antibody-dependent phagocytosis, respectively. Further, IgGAuNPs and MTX-IgGAuNPs were characterised by physical techniques. Moreover, 3D conformational changes in the structure of IgG were analysed by fluorescence spectroscopy during and after the synthesis of IgGAuNPs. Furthermore, the IgGAuNPs and MTX-IgGAuNPs were effective against lung cancer (A549 cells), while they were found to be non-toxic against normal cells (NRK cells). The effectiveness of IgGAuNPs and MTX-IgGAuNPs was examined by MTT cytotoxicity assay, DCFDA method for the production of ROS, and release of Cyt-c from the mitochondria for caspase-3-mediated apoptosis. Moreover, the confirmation of internalisation of particles into the nucleus was examined under the DAPI assay, and it was found that particles caused nuclear fragmentation, which was also an indication of apoptosis.

Proteome profiling by activated esters identified >9000 ligandable lysines but they are limited as covalent inhibitors due to poor hydrolytic stability. Here we report our efforts to design and discover a new series of tunable amine-reactive electrophiles (TAREs) for selective and robust labeling of lysine. The major challenges in developing selective probes for lysine are the high nucleophilicity of cysteines and poor hydrolytic stability. Our work circumvents these challenges by a unique design of the TAREs that form stable adducts with lysine and on reaction with cysteine generate another reactive electrophiles for lysine. We highlight that TAREs exhibit substantially high hydrolytic stability as compared to the activated esters and are non-cytotoxic thus have the potential to act as covalent ligands. We applied these alternative TAREs for the intracellular labeling of proteins in different cell lines, and for the selective identification of lysines in the human proteome on a global scale.

There are a limited number of stimuli-responsive biomaterials that are capable of delivering customizable dosages of a therapeutic at a specific location and time. This is especially true in tissue engineering and regenerative medicine applications, where it may be desirable for the stimuli-responsive biomaterial to also serve as a scaffolding material. Therefore, the purpose of this study was to engineer a traditionally non-stimuli responsive scaffold biomaterial to be thermally responsive so it could be used for on-demand drug delivery applications. Fibrin hydrogels are frequently used for tissue engineering and regenerative medicine applications, and they were functionalized with thermally labile oligonucleotide tethers using peptides from substrates for factor XIII (FXIII). The alpha 2-plasmin inhibitor peptide had the greatest incorporation efficiency out of the FXIII substrate peptides studied, and conjugates of the peptide and oligonucleotide tethers were successfully incorporated into fibrin hydrogels via enzymatic activity. Single-strand complement oligo with either a fluorophore model drug or platelet-derived growth factor-BB (PDGF-BB) could be released on demand via temperature increases. These results demonstrate a strategy that can be used to functionalize traditionally non-stimuli responsive biomaterials suitable for on-demand drug delivery systems (DDS).

Background: The incorporation of radionuclides into peptides and larger biomolecules requires efficient and sometimes biorthogonal reaction conditions, to which click chemistry provides a convenient approach. Methods: Traditionally, click-based radiolabeling techniques have focused on classical click chemistry, such as copper(I)-catalyzed alkyne-azide [3+2] cycloaddition (CuAAC), strain-promoted azide-alkyne [3+2] cycloaddition (SPAAC), traceless Staudinger ligation, and inverse electron demand Diels–Alder (IEDDA). Results: However, newly emerging click-based radiolabeling techniques, including tyrosine-click, sulfo-click, sulfur(VI) fluoride exchange (SuFEx), thiol-ene click, azo coupling, hydrazone formations, oxime formations, and RIKEN click offer valuable alternatives to classical click chemistry. Conclusions: This review will discuss the applications of these techniques in peptide radiochemistry.

The reaction between glycine-type aminonaphthol derivatives substituted with 2- or 1-naphthol and indole or 7-azaindole has been tested. Starting from 2-naphthol as a precursor, the reaction led to the formation of ring-closed products, while in the case of a 1-naphthol-type precursor, the desired biaryl ester was isolated. The synthesis of a bifunctional precursor starting from 5-chloro-8-hydroxyquinoline, morpholine, and ethyl glyoxylate via modified Mannich reaction is reported. The formed Mannich base 10 was subjected to give bioconjugates with indole and 7-azaindole. The effect of the aldehyde component and the amine part of the Mannich base on the synthetic pathway was also investigated. In favor of having a preliminary overview of the structure-activity relationships, the derivatives have been tested on cancer and normal cell lines. In the case of bioconjugate 16, as the most powerful scaffold in the series bearing indole and a 5-chloro-8-hydroxyquinoline skeleton, a potent toxic activity against the resistant Colo320 colon adenocarcinoma cell line was observed. Furthermore, this derivative was selective towards cancer cell lines showing no toxicity on non-tumor fibroblast cells.