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Comprehensive Summary: Protein‐protein interactions (PPIs) play a crucial role in drug discovery and disease treatment. However, the development of effective drugs targeting PPIs remains challenging due to limited methodologies for probing their spatiotemporal anisotropy. Here, we propose a single‐molecule approach using a unique force circuit to investigate PPI dynamics and anisotropy under mechanical forces. Unlike conventional techniques, this approach enables the manipulation and real‐time monitoring of individual proteins at specific amino acids with defined geometry, offering insights into molecular mechanisms at the single‐molecule level. The DNA force circuit was constructed using click chemistry conjugation methods and genetic code expansion techniques, facilitating orthogonal conjugation between proteins and nucleic acids. The SET domain of the MLL1 protein and the tail of histone H3 were used as a model system to demonstrate the application of the DNA force circuit. With the use of atomic force microscopy and magnetic tweezers, optimized assembly procedures were developed. The DNA force circuit provides an exceptional platform for studying the anisotropy of PPIs and holds promise for advancing drug discovery research targeted at PPIs. [ABSTRACT FROM AUTHOR]

Peptide and protein postmodification have gained significant attention due to their extensive impact on biomolecule engineering and drug discovery, of which cysteine‐specific modification strategies are prominent due to their inherent nucleophilicity and low abundance. Herein, the study introduces a novel approach utilizing multifunctional 5‐substituted 1,2,3‐triazine derivatives to achieve multifaceted bioconjugation targeting cysteine‐containing peptides and proteins. On the one hand, this represents an inaugural instance of employing 1,2,3‐triazine in biomolecular‐specific modification within a physiological solution. On the other hand, as a powerful combination of precision modification and biorthogonality, this strategy allows for the one‐pot dual‐orthogonal functionalization of biomolecules utilizing the aldehyde group generated simultaneously. 1,2,3‐Triazine derivatives with diverse functional groups allow conjugation to peptides or proteins, while bi‐triazines enable peptide cyclization and dimerization. The examination of the stability of bi‐triazines revealed their potential for reversible peptide modification. This work establishes a comprehensive platform for identifying cysteine‐selective modifications, providing new avenues for peptide‐based drug development, protein bioconjugation, and chemical biology research. [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]

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]

The combination of mass spectrometry and single crystal X‐ray diffraction of HPLC‐purified DNA‐stabilized silver nanoclusters (DNA‐AgNCs) is a powerful tool to determine the charge and structure of the encapsulated AgNC. Such information is not only relevant to design new DNA‐AgNCs with tailored properties, but it is also important for bio‐conjugation experiments and is essential for electronic structure calculations. Here, the efforts to determine the structure of a HPLC‐purified green emissive DNA‐AgNC are presented. Unfortunately, the original DNA‐AgNC, known to have four valence electrons, could not be crystallized. By modifying the stabilizing DNA sequence, while maintaining the original spectroscopic properties, several mutants could be successfully crystallized, and for one of them, single crystal X‐ray diffraction data provided insight into the silver positions. While the DNA conformation is not resolved, the described approach provides valuable insight into the class of green and dual emissive DNA‐AgNCs with four valence electrons. These results constitute a roadmap on how to improve crystallization and crystal quality for X‐ray diffraction measurements. [ABSTRACT FROM AUTHOR]

Protein functionalisation for the development of imaging agents and antibody drug conjugates still often relies on statistical amidation of the protein through accessible lysine and cysteine residues, requiring protein to protein conjugation optimisation and can potentially impact the overall function. To combat this, focus has turned to developing proteins that have noncanonical amino acids incorporated into their structure, allowing for site‐specific labelling and functionalisation. Herein we showcase the incorporation of non‐canonical amino acids bearing a tetrazine or azide orthogonal coupling modality into biologics targeted to the prostate‐specific membrane antigen and epidermal growth factor receptor respectively. The placement of these bioorthogonal residues into nanobody or single chain variable fragments (scFvs) is introduced by site‐directed mutagenesis of the protein‐coding DNA that allows for controlled insertion when these proteins are expressed. We show that bioorthogonal coupling of model compounds such as fluorophore or polymeric materials onto the protein does not significantly change the binding affinity, making these protein conjugation methods a powerful tool for development of simple customisable personalised targeted antibody‐drug conjugates where affinity is retained. [ABSTRACT FROM AUTHOR]

Rationale: The thiosuccinimide linker is widely used in the synthesis of bioconjugates. However, it is susceptible to hydrolysis and is transformed into its hydrolyzed and/or the isobaric thiazine forms, the latter of which is a fairly common product in a conjugate that contains a cysteinyl peptide. Matrix‐assisted laser desorption/ionization‐mass spectrometry (MALDI‐MS) and matrix‐assisted laser desorption/ionization‐tandem mass spectrometry (MALDI‐MS/MS) are useful for differentiating these isobaric species. Methods: Four cross‐linked peptides with thiosuccinimide linkers were synthesized. Analogs with linkers that were transformed into thiazine and/or the hydrolyzed thiosuccinimide linkers were then synthesized by incubating the samples at neutral or basic pH. All the cross‐linked peptides were purified using RP‐HPLC (reversed‐phase high‐performance liquid chromatography) and differentiated using MALDI‐MS, MALDI‐MS/MS, and ultraviolet photodissociation. Results: A cysteinyl peptide‐containing conjugate, the thiosuccinimide form, was largely transformed into the hydrolyzed or thiazine forms after incubation at neutral or basic pH. MALDI‐MS allowed the three forms to be differentiated: the thiosuccinimide and its hydrolysis product yielded two constituent peptides after reductive cleavage between the Cys and succinimide moieties; no fragment ions were produced from the thiazine form. In addition, MALDI‐MS/MS of the thiosuccinimide form yielded two pairs of complementary fragment ions via 1,4‐elimination: Cys‐SH and maleimide, and dehydro‐alanine and thiosuccinimide, which are different from those produced via reductive cleavage in MALDI‐MS. The thiazine form yielded fragment ions resulting from the cleavage of the newly formed amide bond in the linker that resulted from thiazine formation. Conclusions: The thiosuccinimide (but not thiazine) form of the cross‐linked peptide yielded individual constituent peptides using MALDI‐MS and MALDI‐MS/MS, showing specific 1,4‐elimination for the thiosuccinimide form and cleavage at the newly formed peptide bond via transcyclization for the thiazine form. [ABSTRACT FROM AUTHOR]

Metal‐containing peptide (bio−)conjugates have received continuous interest due to their enormous potential for bioinorganic and medicinal research. In many bioconjugates the chemical inertness of the metal‐containing units facilitates synthesis e. g. by copper‐catalyzed alkyne−azide cycloaddition or the formation of peptide bonds. However, when the metal complex contains labile ligands, which are often critical to their biological activity, the synthetic proceeding requires careful planning. Here, we report on the synthesis of a set of peptide bioconjugates with a platinum(II) core, coordinated through widely variable (O,S) chelating β‐hydroxydithiocinnamic ester and two monodentate ligands. We have evaluated the synthetic applicability of metal−peptide bioconjugation techniques between the model peptide Leu5−enkephalin and differently functionalized (O,S)Pt units. Within this, the type and position of anchor used at the β‐hydroxydithiocinnamic unit proved to be crucial for success, but equally important was the synthetic order of conjugation and complexation. In this work, synthetic approaches for the linkage of metal complexes that are coordinated by functionalized ligands towards peptides were explored. Two general methods to link the studied (O,S)Pt pharmacophore to the model peptide, Leu5−enkephalin, have been applied, namely the most prominent "click" reaction, CuAAC, and the linking via amide bonds. Overall, it could be shown that the structural motif of the (O,S)Pt compounds presented here offers multiple possibilities of derivatization, so that bioconjugation towards peptides can be made possible. Depending on the demands made on the resulting metal bioconjugate, both the dithioester and the aromatic unit of β‐hydroxydithiocinnamic esters can be used as anchor for peptides. However, from our results here, it can be concluded that anchoring at the aromatic subsite seems preferable from a synthetic point of view as the spatial distance of the reactive terminal functional group (alkyne or azide) to the metal center may avoid intramolecular reactions. Also, for using azide−alkyne click chemistry as a conjugation strategy, the (O,S) unit should contain the azide function and not the alkyne function, as triple bond hydration may occur. Classical amide‐bond conjugation also proved to be a suitable method in our hands when performed in solution. A coupling of the β‐hydroxydithiocinnamic unit to resin‐bound peptide would not be possible due to the typically harsh cleavage conditions. [ABSTRACT FROM AUTHOR]

Peptide and protein bioconjugation sees ever‐growing applications in the pharmaceutical sector. Novel strategies and reagents that can address the chemo‐ and regioselectivity issues inherent to these biomolecules, while delivering stable and functionalizable conjugates, are therefore needed. Herein, we introduce the crosslinking ethynylbenziodazolone (EBZ) reagent JW‐AM‐005 for the conjugation of peptides and proteins through the selective linkage of cysteine residues. This easily accessed compound gives access to peptide dimers or stapled peptides under mild and tuneable conditions. Applied to the antibody fragment of antigen binding (Fab) species, JW‐AM‐005 delivered rebridged proteins in a one‐pot three‐reaction process with high regioselectivity, outperforming the standard reagents commonly used for this transformation. [ABSTRACT FROM AUTHOR]

Mild strategies for the selective modification of peptides and proteins are in demand for applications in therapeutic peptide and protein discovery, and in the study of fundamental biomolecular processes. Herein, we describe the development of an electrochemical selenoetherification (e‐SE) platform for the efficient site‐selective functionalization of polypeptides. This methodology utilizes the unique reactivity of the 21st amino acid, selenocysteine, to effect formation of valuable bioconjugates through stable selenoether linkages under mild electrochemical conditions. The power of e‐SE is highlighted through late‐stage C‐terminal modification of the FDA‐approved cancer drug leuprolide and assembly of a library of anti‐HER2 affibody conjugates bearing complex cargoes. Following assembly by e‐SE, the utility of functionalized affibodies for in vitro imaging and targeting of HER2 positive breast and lung cancer cell lines is also demonstrated. [ABSTRACT FROM AUTHOR]

Mild strategies for the selective modification of peptides and proteins are in demand for applications in therapeutic peptide and protein discovery, and in the study of fundamental biomolecular processes. Herein, we describe the development of an electrochemical selenoetherification (e‐SE) platform for the efficient site‐selective functionalization of polypeptides. This methodology utilizes the unique reactivity of the 21st amino acid, selenocysteine, to effect formation of valuable bioconjugates through stable selenoether linkages under mild electrochemical conditions. The power of e‐SE is highlighted through late‐stage C‐terminal modification of the FDA‐approved cancer drug leuprolide and assembly of a library of anti‐HER2 affibody conjugates bearing complex cargoes. Following assembly by e‐SE, the utility of functionalized affibodies for in vitro imaging and targeting of HER2 positive breast and lung cancer cell lines is also demonstrated. [ABSTRACT FROM AUTHOR]

Background: Bispecific Abs (BsAbs) are useful class of Ab‐based drugs but with difficulty to fabricate them. Aims: Cross‐linking two Abs by click chemistry. Materials & Methods: Bis‐alkynylated divinylpyrimidine (DVP) was synthesized and incorporated into an Ab, which was then cross‐linked with azido‐modified Ab to produce BsAb. Results: The heterodimeric anti‐PD‐L1‐HER2 BsAbC was prepared by CuAAC and its formation was confirmed by SDS‐PAGE. BsAbC was purified by biotinylated HER2 antigen coated magnetic bead and was used to fabricate a microarray. In the microarray binding assay, the heterodimeric anti‐PD‐L1‐HER2 BsAbC showed binding activity with PD‐L1 and HER2 antigens. Discussion: This study represents the first proof‐of‐concept that bis‐alkynylated DVP crosslinker reagent can be used for conjugating half‐antibody fragments site‐selectively by CuAAC. A preliminary characterization revealed that this reagent predominantly incorporates a bioorthogonal functional handle into the desired half‐antibody fragments with minor amounts of a re‐bridged full‐length antibody. Conclusion: This study highlights the utility of DVP crosslinker reagents and validates the use of these molecules for the rapid assembly of switchable complex antibody bioconjugates that enables the control of the intended function of fabricated BsAbC. [ABSTRACT FROM AUTHOR]

Oxygen‐tolerant, aqueous copper‐mediated polymerization approaches are combined with click chemistry in either a sequential or a simultaneous manner, to enable the synthesis of multifunctional protein‐polymer conjugates. Propargyl acrylate (PgA) and propargyl methacrylate (PgMA) grafting from a bovine serum albumin (BSA) macroinitiator is thoroughly optimized to synthesize chemically addressable BSA‐poly(propargyl acrylate) and BSA‐poly(propargyl methacrylate) respectively. The produced multifunctional bioconjugates bear pendant terminal 1‐alkynes which can be readily post‐functionalized via both [3+2] Huisgen cycloaddition and thiol‐yne click chemistry under mild reaction conditions. Simultaneous oxygen‐tolerant, aqueous copper‐catalyzed polymerization, and click chemistry mediate the in situ multiple chemical tailoring of biomacromolecules in excellent yields. [ABSTRACT FROM AUTHOR]

We have developed a novel visible‐light‐catalyzed bioconjugation reaction, PhotoCLIC, that enables chemoselective attachment of diverse aromatic amine reagents onto a site‐specifically installed 5‐hydroxytryptophan residue (5HTP) on full‐length proteins of varied complexity. The reaction uses catalytic amounts of methylene blue and blue/red light‐emitting diodes (455/650 nm) for rapid site‐specific protein bioconjugation. Characterization of the PhotoCLIC product reveals a unique structure formed likely through a singlet oxygen‐dependent modification of 5HTP. PhotoCLIC has a wide substrate scope and its compatibility with strain‐promoted azide‐alkyne click reaction, enables site‐specific dual‐labeling of a target protein. [ABSTRACT FROM AUTHOR]

The great success of controlled radical polymerizations has encouraged researchers to develop more facile and robust approaches for surface‐initiated polymerizations (SIPs) to fabricate polymer brushes, even for non‐experts. In recent years, external‐stimuli‐mediated radical polymerization methods have come to the fore as SIPs because of their less rigorous synthetic procedures and high controllability, which expand the opportunities for synthesizing macromolecules with desired chemical compositions and structures, as well as tailor‐made polymers and bioconjugates that show broad applicability and physiological compatibility. This review discusses the latest developments in surface‐initiated polymerization methods, in particular, external‐stimuli mediated atom transfer radical polymerization (ATRP), photo‐induced polymerizations, and reversible addition‐fragmentation chain transfer (RAFT) polymerization, as well as other methods and their combination for the application in surface grafting. The implementation of these methods is of great interest due to their unique possibilities to temporally control a polymerization process, fast and straightforward polymerization, and environmentally benign features, which lead to established and emerging applications in biolubrication, antifouling, and biosensing. [ABSTRACT FROM AUTHOR]

We have developed a novel visible‐light‐catalyzed bioconjugation reaction, PhotoCLIC, that enables chemoselective attachment of diverse aromatic amine reagents onto a site‐specifically installed 5‐hydroxytryptophan residue (5HTP) on full‐length proteins of varied complexity. The reaction uses catalytic amounts of methylene blue and blue/red light‐emitting diodes (455/650 nm) for rapid site‐specific protein bioconjugation. Characterization of the PhotoCLIC product reveals a unique structure formed likely through a singlet oxygen‐dependent modification of 5HTP. PhotoCLIC has a wide substrate scope and its compatibility with strain‐promoted azide‐alkyne click reaction, enables site‐specific dual‐labeling of a target protein. [ABSTRACT FROM AUTHOR]

The great success of controlled radical polymerizations has encouraged researchers to develop more facile and robust approaches for surface‐initiated polymerizations (SIPs) to fabricate polymer brushes, even for non‐experts. In recent years, external‐stimuli‐mediated radical polymerization methods have come to the fore as SIPs because of their less rigorous synthetic procedures and high controllability, which expand the opportunities for synthesizing macromolecules with desired chemical compositions and structures, as well as tailor‐made polymers and bioconjugates that show broad applicability and physiological compatibility. This review discusses the latest developments in surface‐initiated polymerization methods, in particular, external‐stimuli mediated atom transfer radical polymerization (ATRP), photo‐induced polymerizations, and reversible addition‐fragmentation chain transfer (RAFT) polymerization, as well as other methods and their combination for the application in surface grafting. The implementation of these methods is of great interest due to their unique possibilities to temporally control a polymerization process, fast and straightforward polymerization, and environmentally benign features, which lead to established and emerging applications in biolubrication, antifouling, and biosensing. [ABSTRACT FROM AUTHOR]

Site‐specific functionalization of natural amino acid‐containing biological nanopores is pivotal in single molecule sensing. However, pore engineering methodologies are restricted to a limited choice and introduction of unnatural chemical components is extremely difficult. Herein we report the genetic code expansion (GCE) strategy to introduce unnatural amino acid (UAA) to an octameric Mycobacterium smegmatis porin A (MspA) nanopore. GCE allows for rapid and efficient introduction of bioorthogonal reactive site (i.e. azide) to the pore rim, and conjugation of single stranded DNA or lysozyme was demonstrated. The lysozyme‐conjugated pore was further used for the discrimination of different oligosaccharides, demonstrating a sensing capacity that a bare MspA nanopore does not possess. GCE with bioorthogonal handles, which has never been previously applied in the preparation of nanopores, is a versatile strategy for pore engineering and may further expand the application scenarios of nanopores. [ABSTRACT FROM AUTHOR]

Site‐specific functionalization of natural amino acid‐containing biological nanopores is pivotal in single molecule sensing. However, pore engineering methodologies are restricted to a limited choice and introduction of unnatural chemical components is extremely difficult. Herein we report the genetic code expansion (GCE) strategy to introduce unnatural amino acid (UAA) to an octameric Mycobacterium smegmatis porin A (MspA) nanopore. GCE allows for rapid and efficient introduction of bioorthogonal reactive site (i.e. azide) to the pore rim, and conjugation of single stranded DNA or lysozyme was demonstrated. The lysozyme‐conjugated pore was further used for the discrimination of different oligosaccharides, demonstrating a sensing capacity that a bare MspA nanopore does not possess. GCE with bioorthogonal handles, which has never been previously applied in the preparation of nanopores, is a versatile strategy for pore engineering and may further expand the application scenarios of nanopores. [ABSTRACT FROM AUTHOR]

The reaction of a series of electron‐deficient isoindolium‐based allenes with sulfhydryl compounds has been studied, leading to the formation of isoindolium‐based vinyl sulfides. The vinyl sulfides generated could be readily converted into the corresponding indanones and amines upon heating at 30–70 °C with good yields up to 61 %. The thermal cleavage reaction of vinyl sulfides was further studied for developing temperature‐sensitive systems. Notably, a novel FRET‐based fluorescent temperature sensor was designed and synthesized for temperature sensing at 50 °C, giving a 6.5‐fold blue fluorescence enhancement. Moreover, chemoselective bioconjugation of cysteine‐containing peptides with the isoindolium‐based allenes for the construction of multifunctional peptide bioconjugates was investigated. Thermal cleavage of isoindoliums on the modified peptides at 35–70 °C gave indanone bioconjugates with up to >99 % conversion. These results indicated the biocompatibility of this novel temperature‐sensitive reaction. [ABSTRACT FROM AUTHOR]

The reaction of a series of electron‐deficient isoindolium‐based allenes with sulfhydryl compounds has been studied, leading to the formation of isoindolium‐based vinyl sulfides. The vinyl sulfides generated could be readily converted into the corresponding indanones and amines upon heating at 30–70 °C with good yields up to 61 %. The thermal cleavage reaction of vinyl sulfides was further studied for developing temperature‐sensitive systems. Notably, a novel FRET‐based fluorescent temperature sensor was designed and synthesized for temperature sensing at 50 °C, giving a 6.5‐fold blue fluorescence enhancement. Moreover, chemoselective bioconjugation of cysteine‐containing peptides with the isoindolium‐based allenes for the construction of multifunctional peptide bioconjugates was investigated. Thermal cleavage of isoindoliums on the modified peptides at 35–70 °C gave indanone bioconjugates with up to >99 % conversion. These results indicated the biocompatibility of this novel temperature‐sensitive reaction. [ABSTRACT FROM AUTHOR]

Hyperbranched polymethacrylates were synthesized by green‐light‐induced atom transfer radical polymerization (ATRP) under biologically relevant conditions in the open air. Sodium 2‐bromoacrylate (SBA) was prepared in situ from commercially available 2‐bromoacrylic acid and used as a water‐soluble inibramer to induce branching during the copolymerization of methacrylate monomers. As a result, well‐defined branched polymethacrylates were obtained in less than 30 min with predetermined molecular weights (36 000

Hyperbranched polymethacrylates were synthesized by green‐light‐induced atom transfer radical polymerization (ATRP) under biologically relevant conditions in the open air. Sodium 2‐bromoacrylate (SBA) was prepared in situ from commercially available 2‐bromoacrylic acid and used as a water‐soluble inibramer to induce branching during the copolymerization of methacrylate monomers. As a result, well‐defined branched polymethacrylates were obtained in less than 30 min with predetermined molecular weights (36 000

Selective purification of biological materials for their detection in complex sample matrix is a general challenge for many researchers working in the field of diagnostics. Magnetic nanoparticles functionalized with biological molecules that impart biomolecular selectivity are therefore of major interest as capture probes thus allowing for magnetic separations. Nanoparticles can also be used for the enhanced detection of biomarkers. In this work, an ultrasensitive sandwich‐based impedimetric immunosensor was fabricated for the detection of C‐reactive protein antigen (CRPAg). Stable and oriented immobilization of anti‐CRP monoclonal antibody was achieved onto electrografted phenylethylamine derivatized with succinic anhydride and phenylboronic acid via carbodiimide chemistry. The detection of CRPAg was achieved using Electrochemical Impedance Spectroscopy (EIS). The enhancement of the impedance charge‐transfer resistance (RCT) signal was achieved using the sandwich approach. The anti‐CRP polyclonal antibody was immobilized in an oriented manner onto magnetic nanoparticles functionalized with phenylboronic acid. The increase in the change in charge‐transfer resistance (ΔRCT) values was linearly proportional to the concentration of CRPAg in the range 10 to 200 ng mL−1 covering the clinical range for CRPAg detection and with a detection limit of 0.34 ng mL−1. [ABSTRACT FROM AUTHOR]

N‐terminal Cys modification has been intensively studied to produce homogeneous bioconjugates essentially through two modes of reaction: reversible modification with the equilibrium shifted towards the formation of the desired conjugate or stable and irreversible conjugates. Herein, we report a new method of N‐terminal cysteine modification using O‐salicylaldehyde esters (OSAEs) through fast conjugation and irreversible deconjugation. These reagents can rapidly react with N‐terminal Cys at low‐micromolar concentration to form thiazolidines with subsequent hydrolysis of the ester moiety to the phenolic derivative. These phenolic thiazolidines can be hydrolyzed at acidic pH (≈4.5) to recover the intact N‐terminal Cys. Bioconjugation reactions using OSAEs offer controlled reversibility to as act as a protecting group for N‐terminal cysteines, allowing the modification of in‐chain residues without perturbing the N‐terminal Cys, which can then be deprotected and used as a conjugation site. [ABSTRACT FROM AUTHOR]

In recent years, clinical imaging with 89Zr-based radiopharmaceuticals has been gaining significant importance in nuclear medicine. This article provides the results of a comparative study of methods for obtaining 89Zr solutions using ZR, Chelex-100, and TBP resins in the form of [89Zr]Zr-oxalate, [89Zr]Zr-chloride, and [89Zr]Zr-citrate in terms of purification and labeling efficiency. All evaluated methods allowed us to obtain 89Zr with high yield (>90% in 1 ml). The chemical form of 89Zr has a significant influence on the radiolabeling efficiency of the deferoxamine (DFO) chelator and its derivatives. Compared with [89Zr]Zr-oxalate, the application of [89Zr]Zr-chloride and [89Zr]Zr-citrate solutions leads to a higher efficacy of [89Zr]Zr-DFO complex formation. It should be noted that the sequence of mixing of the reagents during the radiolabeling reaction and the residual concentration of oxalic acid appeared to be crucial in the case of [89Zr]Zr-chloride. According to the experimental data, radiolabeling of DFO and its derivatives is preferable to use more stable chemical forms of 89Zr, such as [89Zr]Zr-citrate. The concept will be applied in the further studies involving antibody-based bioconjugates. [ABSTRACT FROM AUTHOR]

Ortho‐phthalaldehyde has recently found wide potentials for protein bioconjugation and peptide cyclization. Herein, the second‐generation dialdehyde‐based peptide cyclization method is reported. The thiophene‐2,3‐dialdehyde (TDA) reacts specifically with the primary amine (from Lys side chain or peptide N‐terminus) and thiol (from Cys side chain) within unprotected peptides to generate a highly stable thieno[2,3‐c]pyrrole‐bridged cyclic structure, while it does not react with primary amine alone. This reaction is carried out in the aqueous buffer and features tolerance of diverse functionalities, rapid and clean transformation, and operational simplicity. The features allow TDA to be used for protein stapling and phage displayed peptide cyclization. [ABSTRACT FROM AUTHOR]

Recent advances in optical bioimaging have prompted the need for minimal chemical reporters that can retain the molecular recognition properties and activity profiles of biomolecules. As a result, several methodologies to reduce the size of fluorescent and Raman labels to a few atoms (e.g. single aryl fluorophores, Raman‐active triple bonds and isotopes) and embed them into building blocks (e.g. amino acids, nucleobases, sugars) to construct native‐like supramolecular structures have been described. The integration of small optical reporters into biomolecules has also led to smart molecular entities that were previously inaccessible in an expedite manner. In this article, we review recent chemical approaches to synthesize miniaturized optical tags as well as some of their multiple applications in biological imaging. [ABSTRACT FROM AUTHOR]

Recent advances in optical bioimaging have prompted the need for minimal chemical reporters that can retain the molecular recognition properties and activity profiles of biomolecules. As a result, several methodologies to reduce the size of fluorescent and Raman labels to a few atoms (e.g. single aryl fluorophores, Raman‐active triple bonds and isotopes) and embed them into building blocks (e.g. amino acids, nucleobases, sugars) to construct native‐like supramolecular structures have been described. The integration of small optical reporters into biomolecules has also led to smart molecular entities that were previously inaccessible in an expedite manner. In this article, we review recent chemical approaches to synthesize miniaturized optical tags as well as some of their multiple applications in biological imaging. [ABSTRACT FROM AUTHOR]

Several families of naturally occurring fatty acid conjugates are presented. Chemical and chemoenzymatic syntheses are exemplified for fatty acid conjugates with (poly)phenols, glycerol, hydroxyfatty acids, aminoacids and small peptides. [ABSTRACT FROM AUTHOR]

Protein‐polymer bioconjugates have shown great promise in biomedical and life science applications including drug delivery and diagnosis. The current bioconjugation strategies suffer from lack of efficiency and versatility. In this article, poly(styrene‐alt‐maleic anhydride) copolymers were first prepared by RAFT polymerization and characterized by different analytical techniques. Then, the poly(styrene‐alt‐maleic anhydride) precursors were functionalized with primary amine such as azidopropylamine and amino poly(ethylene glycol). The reaction of amino compounds with maleic anhydride was found to be a highly efficient, a versatile, and a facile chemical ligation reaction for the synthesis of macromolecules with quantitative yield under mild conditions. The main benefit is the incorporation of a wide range of functionality by easily changing the primary amine compound. For the amphiphilic graft copolymers based on poly(ethylene glycol), aggregation behavior in water was investigated. In a second part, azido‐functionalized polystyrene copolymers were used to prepare a new protein‐polymer bioconjugate by copper‐free click chemistry reaction. [ABSTRACT FROM AUTHOR]

Since the invention of the first biosensors 70 years ago, they have turned into valuable and versatile tools for various applications, ranging from disease diagnosis to environmental monitoring. Traditionally, antibodies have been employed as the capture probes in most biosensors, owing to their innate ability to bind their target with high affinity and specificity, and are still considered as the gold standard. Yet, the resulting immunosensors often suffer from considerable limitations, which are mainly ascribed to the antibody size, conjugation chemistry, stability, and costs. Over the past decade, aptamers have emerged as promising alternative capture probes presenting some advantages over existing constraints of immunosensors, as well as new biosensing concepts. Herein, we review the employment of antibodies and aptamers as capture probes in biosensing platforms, addressing the main aspects of biosensor design and mechanism. We also aim to compare both capture probe classes from theoretical and experimental perspectives. Yet, we highlight that such comparisons are not straightforward, and these two families of capture probes should not be necessarily perceived as competing but rather as complementary. We, thus, elaborate on their combined use in hybrid biosensing schemes benefiting from the advantages of each biorecognition element. [ABSTRACT FROM AUTHOR]

Peptides and proteins are becoming increasingly valuable as medicines, diagnostic agents and as tools for biomedical sciences. Much of this has been underpinned by the emergence of new methods for the manipulation and augmentation of native biomolecules. Perfluoroaromatic reagents are perhaps one of the most diverse and exciting tools with which to modify peptides and proteins, due principally to their nucleophilic substitution chemistry, high electron deficiency and the ability for their reactivity to be tuned towards specific nucleophiles. As discussed in this minireview, in recent years, perfluoroaromatic reagents have found applications as protecting groups or activating groups in peptide synthesis and as orthogonal handles for peptide modification. Furthermore, they have applications in chemoselective 'tagging', stapling and bioconjugation of peptides and proteins, as well as tuning of 'drug‐like' properties. This review will also explore possible future applications of these reagents in biological chemistry. [ABSTRACT FROM AUTHOR]

Residue‐selective bioconjugation methods for biomolecules are highly sought to expand the scope of their biological and medical applications. Inspired by the mechanism of the generation of natural vinylogous γ‐pyridones (vPDNs), we have developed a novel unique azaphilone‐based, activation‐free primary‐amine‐selective bioconjugation method for biomolecules. Our strategy allows facile functionalization of primary amine groups in peptides and proteins, including the clinically used therapeutic antibody trastuzumab, by generating a highly stable vPDN linkage. Excellent chemoselectivity toward primary amines also enables the azaphilone derivatives to specifically modify the lipid components of Gram‐positive bacteria while bypassing Gram‐negative bacteria and mammalian cells. The new method shows significant advantages including chemoselectivity, efficiency, flexibility and biocompatibility, and therefore provides a valuable addition to the current toolbox for biomolecule conjugation. [ABSTRACT FROM AUTHOR]

Residue‐selective bioconjugation methods for biomolecules are highly sought to expand the scope of their biological and medical applications. Inspired by the mechanism of the generation of natural vinylogous γ‐pyridones (vPDNs), we have developed a novel unique azaphilone‐based, activation‐free primary‐amine‐selective bioconjugation method for biomolecules. Our strategy allows facile functionalization of primary amine groups in peptides and proteins, including the clinically used therapeutic antibody trastuzumab, by generating a highly stable vPDN linkage. Excellent chemoselectivity toward primary amines also enables the azaphilone derivatives to specifically modify the lipid components of Gram‐positive bacteria while bypassing Gram‐negative bacteria and mammalian cells. The new method shows significant advantages including chemoselectivity, efficiency, flexibility and biocompatibility, and therefore provides a valuable addition to the current toolbox for biomolecule conjugation. [ABSTRACT FROM AUTHOR]

Cyanine dyes are exceptionally useful probes for a range of fluorescence‐based applications, but their photon output can be limited by trans‐to‐cis photoisomerization. We recently demonstrated that appending a ring system to the pentamethine cyanine ring system improves the quantum yield and extends the fluorescence lifetime. Here, we report an optimized synthesis of persulfonated variants that enable efficient labeling of nucleic acids and proteins. We demonstrate that a bifunctional sulfonated tertiary amide significantly improves the optical properties of the resulting bioconjugates. These new conformationally restricted cyanines are compared to the parent cyanine derivatives in a range of contexts. These include their use in the plasmonic hotspot of a DNA‐nanoantenna, in single‐molecule Förster‐resonance energy transfer (FRET) applications, far‐red fluorescence‐lifetime imaging microscopy (FLIM), and single‐molecule localization microscopy (SMLM). These efforts define contexts in which eliminating cyanine isomerization provides meaningful benefits to imaging performance. [ABSTRACT FROM AUTHOR]

Cyanine dyes are exceptionally useful probes for a range of fluorescence‐based applications, but their photon output can be limited by trans‐to‐cis photoisomerization. We recently demonstrated that appending a ring system to the pentamethine cyanine ring system improves the quantum yield and extends the fluorescence lifetime. Here, we report an optimized synthesis of persulfonated variants that enable efficient labeling of nucleic acids and proteins. We demonstrate that a bifunctional sulfonated tertiary amide significantly improves the optical properties of the resulting bioconjugates. These new conformationally restricted cyanines are compared to the parent cyanine derivatives in a range of contexts. These include their use in the plasmonic hotspot of a DNA‐nanoantenna, in single‐molecule Förster‐resonance energy transfer (FRET) applications, far‐red fluorescence‐lifetime imaging microscopy (FLIM), and single‐molecule localization microscopy (SMLM). These efforts define contexts in which eliminating cyanine isomerization provides meaningful benefits to imaging performance. [ABSTRACT FROM AUTHOR]

Design of reversible organelle‐like microcompartments formed by liquid–liquid phase separation in cell‐mimicking entities has significantly advanced the bottom‐up construction of artificial eukaryotic cells. However, organizing the formation of artificial organelle architectures in a spatiotemporal manner within complex primitive compartments remains scarcely explored. In this work, thermoresponsive hybrid polypeptide‐polymer conjugates are rationally engineered and synthesized, resulting from the conjugation of an intrinsically disordered synthetic protein (IDP), namely elastin‐like polypeptide, and synthetic polymers (poly(ethylene glycol) and dextran) that are widely used as macromolecular crowding agents. Cell‐like constructs are built using droplet‐based microfluidics that are filled with such bioconjugates and an artificial cytoplasm system that is composed of specific polymers conjugated to the IDP. The distinct spatial organizations of two polypeptide‐polymer conjugates and the dynamic assembly and disassembly of polypeptide‐polymer coacervate droplets in response to temperature are studied in the cytomimetic protocells. Furthermore, a monoblock IDP with longer length is concurrently included with bioconjugates individually inside cytomimetic compartments. Both bioconjugates exhibit an identical surfactant‐like property, compartmentalizing the monoblock IDP coacervates via temperature control. These findings lay the foundation for developing hierarchically structured synthetic cells with interior organelle‐like structures which could be designed to localize in desired phase‐separated subcompartments. [ABSTRACT FROM AUTHOR]

Many bioconjugation strategies for DNA oligonucleotides and antibodies suffer limitations, such as site‐specificity, stoichiometry and hydrolytic instability of the conjugates, which makes them unsuitable for biological applications. Here, we report a new platform for the preparation of DNA‐antibody bioconjugates with a simple benzoylacrylic acid pentafluorophenyl ester reagent. Benzoylacrylic‐labelled oligonucleotides prepared with this reagent can be site‐specifically conjugated to a range of proteins and antibodies through accessible cysteine residues. The homogeneity of the prepared DNA‐antibody bioconjugates was confirmed by a new LC‐MS protocol and the bioconjugate probes were used in fluorescence or super‐resolution microscopy cell imaging experiments. This work demonstrates the versatility and robustness of our bioconjugation protocol that gives site‐specific, well‐defined and plasma‐stable DNA‐antibody bioconjugates for biological applications. [ABSTRACT FROM AUTHOR]

Biomedical research has been empowered by tools that enable spatial and temporal control of biological systems. These have predominantly come from photocaged bioactive molecules (optochemical control; N. Ankenbruck, T. Courtney, Y. Naro, A. Deiters, Angew. Chem. Int. Ed. 2018, 57, 2768–2798) and light‐dependent proteins (optogenetic control; L. Fenno, O. Yizhar, K. Deisseroth, Annu. Rev. Neurosci. 2011, 34, 389–412). Recent advances in photocatalysis offer the opportunity to amplify these strategies by providing new dimension of biorthogonality. Photocatalysis also empowers bioconjugation with unprecedented reactivities enabling new crosslinking chemistry or biomolecule functionalization, while merging photocatalysis with biocatalysis extends the scope of both of these powerful classes of transformation. [ABSTRACT FROM AUTHOR]

Many bioconjugation strategies for DNA oligonucleotides and antibodies suffer limitations, such as site‐specificity, stoichiometry and hydrolytic instability of the conjugates, which makes them unsuitable for biological applications. Here, we report a new platform for the preparation of DNA‐antibody bioconjugates with a simple benzoylacrylic acid pentafluorophenyl ester reagent. Benzoylacrylic‐labelled oligonucleotides prepared with this reagent can be site‐specifically conjugated to a range of proteins and antibodies through accessible cysteine residues. The homogeneity of the prepared DNA‐antibody bioconjugates was confirmed by a new LC‐MS protocol and the bioconjugate probes were used in fluorescence or super‐resolution microscopy cell imaging experiments. This work demonstrates the versatility and robustness of our bioconjugation protocol that gives site‐specific, well‐defined and plasma‐stable DNA‐antibody bioconjugates for biological applications. [ABSTRACT FROM AUTHOR]

Mitochondria are key organelles that perform vital cellular functions such as those related to cell survival and death. The targeted delivery of different types of cargos to mitochondria is a well‐established strategy to study mitochondrial biology and diseases. Of the various existing mitochondrion‐transporting vehicles, most suffer from poor cytosolic entry, low delivery efficiency, limited cargo types, and cumbersome preparation protocols, and none was known to be universally applicable for mitochondrial delivery of different types of cargos (small molecules, proteins, and nanomaterials). Herein, two new cell‐penetrating, mitochondrion‐targeting ligands (named MitoLigand) that are capable of effectively "tagging" small‐molecule drugs, native proteins and nanomaterials are disclosed, as well as their corresponding chemoselective conjugation chemistry. Upon successful cellular delivery and rapid endosome escape, the released native cargos were found to be predominantly localized inside mitochondria. Finally, by successfully delivering doxorubicin, a well‐known anticancer drug, to the mitochondria of HeLa cells, we showed that the released drug possessed potent cell cytotoxicity, disrupted the mitochondrial membrane potential and finally led to apoptosis. Our strategy thus paves the way for future mitochondrion‐targeted therapy with a variety of biologically active agents. [ABSTRACT FROM AUTHOR]

Are chemical methods capable of precisely engineering the native proteins? Is it possible to develop platforms that can empower the regulation of chemoselectivity, site‐selectivity, modularity, protein‐specificity, and site‐specificity? This account delineates our research journey in the last ten years on the developments revolving around these questions. It will range from the realization of chemoselective and site‐selective labeling of reactivity hotspots to modular linchpin directed modification (LDM®) platform and site‐specific Gly‐tag® technology. Also, we outline a few biotechnology tools, including Maspecter®, that accelerated the detailed analysis of the bioconjugates and rendered a powerful toolbox for homogeneous antibody‐drug conjugates (ADCs). [ABSTRACT FROM AUTHOR]

Protein‐polymer bioconjugates present a way to make enzymes more efficient and robust for industrial and medicinal applications. While much work has focused on mono‐functional conjugates, that is, conjugates with one type of polymer attached such as poly(ethylene glycol) or poly(N‐isopropylacrylamide), there is a practical interest in gaining additional functionality by synthesizing well‐defined bifunctional conjugates in a hetero‐arm star copolymer architecture with protein as the core. Using ubiquitin as a model protein, a synthetic scheme is developed to attach two different polymers (oligo(ethylene oxide) methacrylate and N,N‐dimethylacrylamide) directly to the protein surface, using orthogonal conjugation chemistries and grafting‐from by photochemical living radical polymerization techniques. The additional complexity arising from attempts to selectively modify multiple sites led to decreased polymerization performance and indicates that initiators for continuous activator regeneration atom transfer radical polymerization and reversible addition‐fragmentation chain transfer polymerization are not well‐suited to bifunctional bioconjugates applications under the studied conditions. Nonetheless, the polymerization conditions preserve the native fold of the ubiquitin and enable production of a hetero‐arm star protein‐polymer bioconjugate. [ABSTRACT FROM AUTHOR]

The use of nanoparticle (NP) bioconjugates to control the activity of membrane ion channels has recently emerged as a new paradigm for the activation of electrically excitable cells. An NP‐based strategy is reported for the specific activation of channelrhodopsin C1V1 (ChR‐C1V1) expressed in the plasma membrane of HEK 293T/17 cells. Hydrophilic CdSe/ZnS core–shell semiconductor quantum dots (QDs) are self‐assembled to the exofacial face of recombinantly expressed ChR‐C1V1 by metal affinity‐driven interaction of the QD ZnS shell with an N‐terminal hexahistidine tag displayed on ChR‐C1V1. This configuration enables the Förster resonance energy transfer (FRET)‐based excitation and activation of the 11‐cis‐retinal moiety of ChR‐C1V1 using the QD as a light harvesting transducer/energy donor. It is shown that the specific laser‐induced opening of the ChR‐C1V1 channel wherein the photoexcited QD (405 nm excitation, 530 nm emission) iteratively activates ChR‐C1V1 channels as confirmed using the voltage‐sensitive dye (VSD) bis‐(1,3‐diethylthiobarbituric acid)trimethine oxonol (DiSBAC2(3)). In the absence of the QD transducer, excitation of ChR‐C1V1‐expressing cells at 405 nm results in no activation of ChR‐C1V1. The results demonstrate the ability to controllably interface QDs with living cells for the activation of ChR membrane proteins and detail a new NP‐bioconjugate hybrid system for the specific activation of ion channels. [ABSTRACT FROM AUTHOR]

Non‐steroidal anti‐inflammatory drugs (NSAIDs) are a powerful class of inhibitors targeting two isoforms of the family of cyclooxygenase enzymes (COX‐1 and COX‐2). While NSAIDs are widely used in the management of pain, in particular as a treatment for osteo‐ and rheumatoid arthritis, their long‐term use has been associated with numerous on‐ and off‐target effects. As the carboxylic acid moiety present in common NSAIDs is responsible for some of their adverse effects, but is not required for their anti‐inflammatory activity, we sought to mask this group through direct coupling to glucosamine, which is thought to prevent cartilage degradation. We report herein the conjugation of commonly prescribed NSAIDs to glucosamine hydrochloride and the use of molecular docking to show that addition of the carbohydrate moiety to the parent NSAID can enhance binding in the active site of COX‐2. In a preliminary, in vitro screening assay, the diclofenac‐glucosamine bioconjugate exhibited 10‐fold greater activity toward COX‐2, making it an ideal candidate for future in vivo studies. Furthermore, in an intriguing result, we observed that the mefenamic acid‐glucosamine bioconjugate displayed enhanced activity toward COX‐1 rather than COX‐2. [ABSTRACT FROM AUTHOR]

The catechol group of 3,4‐dihydroxyphenylalanine (L‐DOPA) derived from L‐tyrosine oxidation is a key post‐translational modification (PTM) in many protein biomaterials and has potential as a bioorthogonal handle for precision protein conjugation applications such as antibody–drug conjugates. Despite this potential, indiscriminate enzymatic modification of exposed tyrosine residues or complete replacement of tyrosine using auxotrophic hosts remains the preferred method of introducing the catechol moiety into proteins, which precludes many protein engineering applications. We have developed new orthogonal translation machinery to site‐specifically incorporate L‐DOPA into recombinant proteins and a new fluorescent biosensor to selectively monitor L‐DOPA incorporation in vivo. We show simultaneous biosynthesis and incorporation of L‐DOPA and apply this translation machinery to engineer a novel metalloprotein containing a DOPA‐Fe chromophore. [ABSTRACT FROM AUTHOR]

The catechol group of 3,4‐dihydroxyphenylalanine (L‐DOPA) derived from L‐tyrosine oxidation is a key post‐translational modification (PTM) in many protein biomaterials and has potential as a bioorthogonal handle for precision protein conjugation applications such as antibody–drug conjugates. Despite this potential, indiscriminate enzymatic modification of exposed tyrosine residues or complete replacement of tyrosine using auxotrophic hosts remains the preferred method of introducing the catechol moiety into proteins, which precludes many protein engineering applications. We have developed new orthogonal translation machinery to site‐specifically incorporate L‐DOPA into recombinant proteins and a new fluorescent biosensor to selectively monitor L‐DOPA incorporation in vivo. We show simultaneous biosynthesis and incorporation of L‐DOPA and apply this translation machinery to engineer a novel metalloprotein containing a DOPA‐Fe chromophore. [ABSTRACT FROM AUTHOR]

Site‐selective protein functionalization serves as an invaluable tool for investigating protein structures and functions in complicated cellular environments and accomplishing semi‐synthetic protein conjugates such as traceable therapeutics with improved features. Dual functionalization of proteins allows the incorporation of two different types of functionalities at distinct location(s), which greatly expands the features of native proteins. The attachment and crosstalk of a fluorescence donor and an acceptor dye provides fundamental insights into the folding and structural changes of proteins upon ligand binding in their native cellular environments. Moreover, the combination of drug molecules with different modes of action, imaging agents or stabilizing polymers provides new avenues to design precision protein therapeutics in a reproducible and well‐characterizable fashion. This review aims to give a timely overview of the recent advancements and a future perspective of this relatively new research area. First, the chemical toolbox for dual functionalization of proteins is discussed and compared. The strengths and limitations of each strategy are summarized in order to enable readers to select the most appropriate method for their envisaged applications. Thereafter, representative applications of these dual‐modified protein bioconjugates benefiting from the synergistic/additive properties of the two synthetic moieties are highlighted. [ABSTRACT FROM AUTHOR]

Widely used reagents in the peptide functionalization toolbox, Michael acceptors and N‐hydroxysuccinimide (NHS) activated esters, are combined in NHS‐activated acrylamides for efficient chemoselective amino‐sulfhydryl stapling on native peptides and proteins. NHS‐activated acrylamides allow for a fast functionalization of N‐terminal cysteines (k2=1.54±0.18×103 M−1 s−1) under dilute aqueous conditions, enabling selectivity over other nucleophilic amino acids. Additionally, the versatility of these new bioconjugation handles was demonstrated in the cross‐linking of in‐chain or C‐terminal cysteines with nearby lysine residues. NHS‐activated acrylamides are compatible with the use of other cysteine selective reagents, allowing for orthogonal dual‐modifications. This strategy was successfully applied to the late‐stage functionalization of peptides and proteins with a PEG unit, fluorescent probe, and cytotoxic agent. The level of molecular control offered by NHS‐activated acrylamides is expected to promote amino‐sulfhydryl stapling technology as a powerful strategy to design functional bioconjugates. [ABSTRACT FROM AUTHOR]