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This article discusses a new method for efficiently modifying oligonucleotides with multiple sugars in a one-step reaction. The researchers used a bifunctional linker and Cu-catalyzed alkyne-azide click chemistry to achieve this modification. The method is fast, highly selective, and cost-effective, allowing for the synthesis of oligonucleotides with multiple sugar modifications. This research has implications for the development of nucleic acid therapeutics in the pharmaceutical industry. The study was supported by various funding sources and the data is available in the supporting information. [Extracted from the article]

This article explores the use of dual bioorthogonal and orthogonal metabolic labeling techniques to study interactions in biological environments. The researchers developed modified 2'-desoxynucleosides to overcome the challenge of cross-reactivity and successfully linked genomic DNA in cells with two different fluorogenic markers. The study demonstrates the potential for advanced metabolic labeling strategies for nucleic acids, particularly in living cells. The authors conducted experiments to investigate the reactivity and selectivity of certain nucleosides in two different reactions, IEDDA and photoclick, for potential use in DNA labeling. While successful DNA marking was achieved using the IEDDA reaction, attempts to mark DNA using the photoclick reaction were unsuccessful. The authors also tested the nucleosides for metabolic labeling of cellular DNA, but no successful labeling was observed. Overall, this study provides a foundation for future research in metabolic labeling strategies for nucleic acids, including experiments in living cells. [Extracted from the article]

Wir berichten über eine Dichtefunktionaltheorie (DFT)‐basierte Entdeckung von Ethinyl‐Triazolyl‐Phosphinaten (ETP) als eine neue Klasse elektrophiler Verbindungen für die selektive Biokonjugation von Cystein. Mit Hilfe der CuI‐katalysierten Azid‐Alkin‐Cycloaddition (CuAAC) in wässrigem Puffer konnten wir eine Vielzahl funktioneller elektrophiler Bausteine, darunter auch Proteine, aus Diethynylphosphinat herstellen. Wir verwendeten diese ETP‐Reagenzien, um fluoreszierende Peptid‐Konjugate für die Markierung von Rezeptoren auf lebenden Zellen sowie ein stabiles und biologisch aktives Antikörper‐Wirkstoff‐Konjugat zu erhalten. Darüber hinaus konnten wir ETP‐Elektrophile unter nativen Bedingungen in ein Azid‐haltiges Ubiquitin einbauen und ihr Potenzial für die Protein‐Protein‐Konjugation demonstrieren. Schließlich zeigen wir die exzellente Cystein‐Selektivität dieser neuen Klasse von Elektrophilen in Massenspektrometrie basierten, proteomweiten Reaktivitätsstudien und unterstreichen damit die generelle Anwendbarkeit in homogenen Biokonjugationsstrategien zur Verknüpfung zweier komplexer Biomoleküle. [ABSTRACT FROM AUTHOR]

Diethinylphosphinate wurden als bisfunktionelle Elektrophile für die ortsselektive Modifikation von Peptiden, Proteinen und Antikörpern entwickelt. Eine ihrer elektronenarmen Dreifachbindungen reagiert selektiv mit einem Thiol und positioniert eine elektrophile Einheit für eine nachfolgende intra‐ oder intermolekulare Reaktion mit einem anderen Thiol. Die erhaltenen Konjugate erwiesen sich im menschlichen Plasma und in Gegenwart kleiner Thiole als stabil. Wir zeigen weiter, dass diese Methode für die Generierung von funktionellen Proteinkonjugaten für den intrazellulären Transport geeignet ist. Schließlich wurde diese Reagenzklasse verwendet, um funktionale, homogen verbrückte Antikörper zu generieren, die spezifisch für ihr Ziel bleiben. Ihre modulare Synthese, Thiol‐Selektivität und die Stabilität der Konjugate machen Diethinylphosphinate zu idealen Kandidaten für die Proteinkonjugation für biologische und pharmazeutische Anwendungen. [ABSTRACT FROM AUTHOR]

Wir stellen die Biokonjugation von Polymeren vor, die durch RAFT‐Polymerisation mittels Hetero‐Diels‐Alder‐Cycloaddition durch ihren inhärenten terminalen Thiocarbonylthiorest mit einem dienmodifizierten Modellprotein synthetisiert wurden und keine spezifische funktionelle Endgruppe tragen. Die quantitative Konjugation erfolgt im Verlauf einiger Stunden bei Raumtemperatur und nahezu neutralem pH‐Wert und in Abwesenheit jeglichen Katalysators. Unsere Technologieplattform liefert thermoresponsive Biokonjugate, deren Aggregation allein durch die Polymerketten gesteuert wird. [ABSTRACT FROM AUTHOR]

Einwandige Kohlenstoffnanoröhren (SWCNTs) sind ein 1D‐Nanomaterial, das im nahen Infrarot (NIR, >800 nm) fluoresziert. In der Vergangenheit wurde kovalente Chemie zur Funktionalisierung von SWCNTs wenig genutzt, da sie die NIR‐Emission beeinträchtigt. Es sind jedoch bestimmte sp3‐Defekte (Quantendefekte) im Kohlenstoffgitter beobachtet worden, die die NIR‐Fluoreszenz erhalten und sogar zu einer neuen, rotverschobenen Emission führen. Hier berichten wir über Quantendefekte, die mittels lichtinduzierter Diazoniumchemie eingeführt wurden und als Ankerpunkte für Peptide und Proteine dienen. Wir zeigen, dass Maleimid‐Anker die Konjugation Cystein‐haltiger Proteine, z. B. eines GFP‐bindenden Nanobodys, ermöglichen. Darüber hinaus fungiert ein Fmoc‐geschützter Phenylalanin‐Defekt als Ausgangspunkt für die Konjugation von sichtbaren Fluorophoren zur Erzeugung mehrfarbiger SWCNTs und In‐situ‐Peptidsynthese direkt auf dem Nanoröhren. Daher stellen diese Quantendefekte eine vielseitige Plattform dar, um sowohl die photophysikalischen Eigenschaften der Nanoröhren als auch ihre Oberflächenchemie anzupassen. [ABSTRACT FROM AUTHOR]

An azanorbornadiene bromovinyl sulfone reagent for cysteine‐selective bioconjugation has been developed. Subsequent reaction with dipyridyl tetrazine leads to bond cleavage and formation of a pyrrole‐linked conjugate. The latter involves ligation of the tetrazine to the azanorbornadiene‐tagged protein through inverse electron demand Diels–Alder cycloaddition with subsequent double retro‐Diels–Alder reactions to form a stable pyrrole linkage. The sequence of site‐selective bioconjugation followed by bioorthogonal bond cleavage was efficiently employed for the labelling of three different proteins. This method benefits from easy preparation of these reagents, selectivity for cysteine, and stability after reaction with a commercial tetrazine, which has potential for the routine preparation of protein conjugates for chemical biology studies. [ABSTRACT FROM AUTHOR]

The conjugation of hydrophilic low‐fouling polymers to therapeutic molecules and particles is an effective approach to improving their aqueous stability, solubility, and pharmacokinetics. Recent concerns over the immunogenicity of poly(ethylene glycol) has highlighted the importance of identifying alternative low fouling polymers. Now, a new class of synthetic water‐soluble homo‐fluoropolymers are reported with a sulfoxide side‐chain structure. The incorporation of fluorine enables direct imaging of the homopolymer by 19F MRI, negating the need for additional synthetic steps to attach an imaging moiety. These self‐reporting fluoropolymers show outstanding imaging sensitivity and remarkable hydrophilicity, and as such are a new class of low‐fouling polymer for bioconjugation and in vivo tracking. [ABSTRACT FROM AUTHOR]

Photochemical ligation is important in biomaterials engineering for spatiotemporal control of biochemical processes. Such reactions however generally require activation by high energy UV or short wavelength blue light, which can limit their use as a consequence of the potential of these high energy light sources to damage living cells. Herein, we present an additive‐free, biocompatible, chemical ligation triggered by mild visible light. BODIPY dyes with a pendant thioether attached at the meso‐position undergo photolysis of the [C−S] bond under green light (λ=530 nm) excitation, producing an ion pair intermediate that can react specifically with a propiolate group. The utility of this photochemical ligation in materials science is demonstrated by the fabrication of hydrogels with specific architectures, photo‐immobilization of biomacromolecules, and live cell encapsulation within a hydrogel scaffold. [ABSTRACT FROM AUTHOR]

Nucleophile–nucleophile coupling is a challenging transformation in organic chemistry. Herein we present a novel umpolung strategy for α‐functionalization of aldehydes with nucleophiles. The strategy uses organocatalytic enamine activation and quinone‐promoted oxidation to access O‐bound quinol‐intermediates that undergo nucleophilic substitution reactions. These quinol‐intermediates react with different classes of nucleophiles. The focus is on an unprecedented organocatalytic oxidative α‐thiolation of aldehydes. The reaction scope is demonstrated for a broad range of thiols and extended to chemoselective bioconjugation, and applicable to a large variety of aldehydes. This strategy can also encompass organocatalytic enantioselective coupling of α‐branched aldehydes with thiols forming quaternary thioethers. Studies indicate a stereoselective formation of the intermediate followed by a stereospecific nucleophilic substitution reaction at a quaternary stereocenter, with inversion of configuration. [ABSTRACT FROM AUTHOR]

Squaramate‐linked 2′‐deoxycytidine 5′‐O‐triphosphate was synthesized and found to be good substrate for KOD XL DNA polymerase in primer extension or PCR synthesis of modified DNA. The resulting squaramate‐linked DNA reacts with primary amines to form a stable diamide linkage. This reaction was used for bioconjugations of DNA with Cy5 and Lys‐containing peptides. Squaramate‐linked DNA formed covalent cross‐links with histone proteins. This reactive nucleotide has potential for other bioconjugations of nucleic acids with amines, peptides or proteins without need of any external reagent. [ABSTRACT FROM AUTHOR]

Requirements for novel bioconjugation reactions for the synthesis of antibody–drug conjugates (ADCs) are exceptionally high, since conjugation selectivity as well as the stability and hydrophobicity of linkers and payloads drastically influence the performance and safety profile of the final product. We report Cys‐selective ethynylphosphonamidates as new reagents for the rapid generation of efficacious ADCs from native non‐engineered monoclonal antibodies through a simple one‐pot reduction and alkylation. Ethynylphosphonamidates can be easily substituted with hydrophilic residues, giving rise to electrophilic labeling reagents with tunable solubility properties. We demonstrate that ethynylphosphonamidate‐linked ADCs have excellent properties for next‐generation antibody therapeutics in terms of serum stability and in vivo antitumor activity. [ABSTRACT FROM AUTHOR]

The isocyano group is a structurally compact bioorthogonal functional group that reacts with tetrazines under physiological conditions. Now it is shown that bulky tetrazine substituents accelerate this cycloaddition. Computational studies suggest that dispersion forces between the isocyano group and the tetrazine substituents in the transition state contribute to the atypical structure–activity relationship. Stable asymmetric tetrazines that react with isonitriles at rate constants as high as 57 L mol−1 s−1 were accessible by combining bulky and electron‐withdrawing substituents. Sterically encumbered tetrazines react selectively with isonitriles in the presence of strained alkenes/alkynes, which allows for the orthogonal labeling of three proteins. The established principles will open new opportunities for developing tetrazine reactants with improved characteristics for diverse labeling and release applications with isonitriles. [ABSTRACT FROM AUTHOR]

The development of methods for conjugation of DNA to proteins is of high relevance for the integration of protein function and DNA structures. Here, we demonstrate that protein‐binding peptides can direct a DNA‐templated reaction, selectively furnishing DNA–protein conjugates with one DNA label. Quantitative conversion of oligonucleotides is achieved at low stoichiometries and the reaction can be performed in complex biological matrixes, such as cell lysates. Further, we have used a star‐like pentameric DNA nanostructure to assemble five DNA–Rituximab conjugates, made by our reported method, into a pseudo‐IgM antibody structure that was subsequently characterized by negative‐stain transmission electron microscopy (nsTEM) analysis. [ABSTRACT FROM AUTHOR]

A series of 2,2‐bis(hydroxymethyl)propionic acid dendrons of generation 2 through 8 having a strained cyclooctyne at the core and hydroxy groups at the periphery were prepared by a divergent method and used to functionalize azide‐decorated α‐chymotrypsin. The ability of the appended dendrons to selectively block enzyme activity (through a molecular sieving effect) was investigated using a small molecule substrate (benzoyl‐l‐tyrosine p‐nitroanilide), as well as two proteins of different size (casein and bovine serum albumin). Additionally, the ability of dendrons to block complexation with a chymotrypsin antagonist, α‐antichymotrypsin, was investigated, and it was found that the dendron coating effectively prevented inhibition by this antagonist. We found that a critical generation is required to achieve efficient sieving with bis‐MPA dendrons, which illustrates the importance of macromolecular architecture and size in the shielding of proteins. [ABSTRACT FROM AUTHOR]

We report here the development of a suite of biocompatible SuFEx transformations from the SOF4‐derived iminosulfur oxydifluoride hub in aqueous buffer conditions. These biocompatible SuFEx reactions of iminosulfur oxydifluorides (R‐N=SOF2) with primary amines give sulfamides (8 examples, up to 98 %), while the reaction with secondary amines furnish sulfuramidimidoyl fluoride products (8 examples, up to 97 %). Likewise, under mild buffered conditions, phenols react with the iminosulfur oxydifluorides (Ar‐N=SOF2) to produce sulfurofluoridoimidates (13 examples, up to 99 %), which can themselves be further modified by nucleophiles. These transformations open the potential for asymmetric and trisubstituted linkages projecting from the sulfur(VI) center, including versatile S−N and S−O connectivity (9 examples, up to 94 %). Finally, the SuFEx bioconjugation of iminosulfur oxydifluorides to amine‐tagged single‐stranded DNA and to BSA protein demonstrate the potential of SOF4‐derived SuFEx click chemistry in biological applications. [ABSTRACT FROM AUTHOR]

Quaternized vinyl‐ and alkynyl‐pyridine reagents were shown to react in an ultrafast and selective manner with several cysteine‐tagged proteins at near‐stoichiometric quantities. We have demonstrated that this method can effectively create a homogenous antibody–drug conjugate that features a precise drug‐to‐antibody ratio of 2, which was stable in human plasma and retained its specificity towards Her2+ cells. Finally, the developed warhead introduces a +1 charge to the overall net charge of the protein, which enabled us to show that the electrophoretic mobility of the protein may be tuned through the simple attachment of a quaternized vinyl pyridinium reagent at the cysteine residues. We anticipate the generalized use of quaternized vinyl‐ and alkynyl‐pyridine reagents not only for bioconjugation, but also as warheads for covalent inhibition and as tools to profile cysteine reactivity. [ABSTRACT FROM AUTHOR]

The need for atom‐precise biomolecule modification, and particularly the irreversible formation of covalent bonds to specific amino acids in proteins, has become an essential issue in the fields of pharmaceuticals and chemical biology. For example, antibody–drug conjugates (ADCs) are increasingly common entries into the clinical oncology pipeline. Herein, we report a new method of affinity peptide mediated regiodivergent functionalization (AJICAP™) that enables the synthesis of ADCs from native IgG antibodies. We succeeded in introducing thiol functional groups onto three lysine residues in IgGs using Fc affinity peptide reagents without antibody engineering. A cytotoxic molecule was then connected to the newly introduced thiol group, and both a surface plasmon resonance binding assay and in vivo xenograft mouse model results showed that the resulting ADC could selectively target and kill HER2‐positive cells. Our strategy provides a new approach for constructing complex antibody‐derived biomolecules. [ABSTRACT FROM AUTHOR]

Reported here is the synthesis of a class of semi‐oxamide vinylogous thioesters, designated STEFs, and the use of these agents as new electrophilic warheads. This work includes preparation of simple probes that contain this reactive motif as well as its installation on a more complex kinase inhibitor scaffold. A key aspect of STEFs is their reactivity towards both thiol and amine groups. Shown here is that amine conjugations in peptidic and proteinogenic samples can be facilitated by initial, fast conjugation to proximal thiol residues. Evidence that both the selectivity and the reactivity can be tuned by the structure of STEFs is provided, and given the unique ability of this functionality to conjugate by an addition‐elimination mechanism, STEFs are electrophilic warheads that could find broad use in chemical biology. [ABSTRACT FROM AUTHOR]

Although PEGylation reduces the immunogenicity of protein drugs to some extent, its limitations for highly immunogenic biotherapeutics have been demonstrated. Herein, a proactive strategy to alleviate the development of anti‐drug antibodies (ADAs) against protein drugs by immunomodulatory bioconjugation is reported. Rapamycin was conjugated to a PEGylated protein therapeutic via a cleavable disulfide linker. The conjugated rapamycin can be released from the bioconjugate and prevent immune responses once the bioconjugate is uptaken by antigen‐presenting cells. The immunomodulatory bioconjugate significantly reduced the titers of ADAs compared with a PEGylated protein. The inhibition of immune responses was specific to the conjugated antigen, avoiding systemic immune suppression and the risk of increased susceptibility to infections. The reported approach breaks the limitations of PEGylation by the proactive prevention of ADAs. [ABSTRACT FROM AUTHOR]

Abstract: In den vergangenen zwei Jahrzehnten hat sich die bioorthogonale Chemie zu einem beliebten Hilfsmittel entwickelt, um zentrenselektive Modifikationen an Proteinen zu erzielen. Dennoch sind nur einige wenige bioorthogonale Reaktionen bekannt, die zudem Limitierungen aufweisen können, wie etwa geringe Reaktionsgeschwindigkeiten, die Verwendung instabiler oder zytotoxischer Reagenzien und das Auftreten von Nebenreaktionen. Folglich besteht ein großes Interesse an der Erweiterung der Methodik in der bioorthogonalen Chemie. In diesem Zusammenhang wurden kürzlich Boronsäuren in den Bereich der bioorthogonalen Chemie eingeführt, welche in drei verschiedenen Strategien zum Einsatz kommen: 1) Bildung eines Boronsäureesters zwischen der Boronsäure und einem 1,2‐cis‐Diol; 2) Bildung eines Iminoboronates zwischen 2‐Acetyl‐/Formyl‐Arylboronsäure und Hydrazin‐/Hydroxylamin‐/Semicarbazid‐Derivaten; 3) Verwendung einer Boronsäure als Transfergruppe in einer Suzuki‐Miyaura‐Kreuzkupplung oder einer anderen Reaktion, in deren Verlauf die Boronylgruppe das Intermediat verlässt. In diesem Aufsatz fassen wir die Fortschritte der Anwendung von Boronsäuren in der bioorthogonalen Chemie zusammen, welche das zentrenspezifische Labeling von Proteinen ermöglicht, und vergleichen diese Methoden mit den gängigsten bioorthogonalen Reaktionen. [ABSTRACT FROM AUTHOR]

Abstract: Dual‐labeled biomolecules constitute a new generation of bioconjugates with promising applications in therapy and diagnosis. Unfortunately, the development of these new families of biologics is hampered by the technical difficulties associated with their construction. In particular, the site specificity of the conjugation is critical as the number and position of payloads can have a dramatic impact on the pharmacokinetics of the bioconjugate. Herein, we introduce dichlorotetrazine as a trivalent platform for the selective double modification of proteins on cysteine residues. This strategy is applied to the dual labeling of albumin with a macrocyclic chelator for nuclear imaging and a fluorescent probe for fluorescence imaging. [ABSTRACT FROM AUTHOR]

Abstract: Formylglycin‐generierende Enzyme haben in den letzten Jahren breite Anwendung in der bioorthogonalen Chemie gefunden. Sie katalysieren die ortsspezifische Oxidation eines Cysteinrestes zur Aldehyd‐haltigen Aminosäure Cα‐Formylglycin (FGly). FGly kann in jedem beliebigen Zielprotein generiert und anschließend für bioorthogonale Konjugationsreaktionen genutzt werden. Das prototypische Formylglycin‐generierende Enzym (FGE) und das Eisen‐Schwefel‐Protein AtsB zeigen leichte Variationen in ihren Erkennungssequenzen. Spezifische Motive in Peptiden und Proteinen wurden entworfen, die selektiv von diesen Enzymen adressiert werden. Die Kombination dieser Motive innerhalb eines Peptids oder rekombinanten Proteins ermöglichte die unabhängige und sukzessive Einführung von zwei FGly‐Resten und die Generierung hetero‐difunktionalisierter Proteinkonjugate. [ABSTRACT FROM AUTHOR]

Abstract: We report a site‐selective cysteine–cyclooctyne conjugation reaction between a seven‐residue peptide tag (DBCO‐tag, Leu‐Cys‐Tyr‐Pro‐Trp‐Val‐Tyr) at the N or C terminus of a peptide or protein and various aza‐dibenzocyclooctyne (DBCO) reagents. Compared to a cysteine peptide control, the DBCO‐tag increases the rate of the thiol–yne reaction 220‐fold, thereby enabling selective conjugation of DBCO‐tag to DBCO‐linked fluorescent probes, affinity tags, and cytotoxic drug molecules. Fusion of DBCO‐tag with the protein of interest enables regioselective cysteine modification on proteins that contain multiple endogenous cysteines; these examples include green fluorescent protein and the antibody trastuzumab. This study demonstrates that short peptide tags can aid in accelerating bond‐forming reactions that are often slow to non‐existent in water. [ABSTRACT FROM AUTHOR]

We report a fast Staudinger reaction between perfluoroaryl azides (PFAAs) and aryl phosphines, which occurs readily under ambient conditions. A rate constant as high as 18 m−1 s−1 was obtained between methyl 4-azido-2,3,5,6-tetrafluorobenzoate and methyl 2-(diphenylphosphanyl)benzoate in CD3CN/D2O. Furthermore, the iminophosphorane product was stable toward hydrolysis and aza-phosphonium ylide reactions. This PFAA Staudinger reaction proved to be an excellent bioothorgonal reaction. PFAA-derivatized mannosamine and galactosamine were successfully transformed into cell-surface glycans and efficiently labeled with phosphine-derivatized fluorophore-conjugated bovine serum albumin. [ABSTRACT FROM AUTHOR]

Copper-mediated coupling between alkynes to generate a structurally rigid, linear 1,3-diyne linkage has been known for over a century. However, the mechanistic requirement to simultaneously maintain CuI and an oxidant has limited its practical utility, especially for complex functional molecules in aqueous solution. We find that addition of a specific bpy-diol ligand protects unprotected peptides from CuII-mediated oxidative damage through the formation of an insoluble CuII gel which solves the critical challenge of applying Glaser coupling to substrates that are degraded by CuII. The generality of this method is illustrated through the conjugation of a series of polar and nonpolar labels onto a fully unprotected GLP-1R agonist through a linear 7 Å diynyl linker. [ABSTRACT FROM AUTHOR]

The isolation of specific nucleic acid sequences is a major bottleneck in molecular diagnostics. Magnetic beads/particles are typically used as solid supports for the capture of DNA targets to improve sample throughput but aggregate over time resulting in lower capture efficiency and obstruction of liquid handling devices. Herein, we describe a particle-free approach to sequence-specific DNA extraction using a magnetic liquid support and ion-tagged oligonucleotide (ITO) probes. ITO conjugates were synthesized with the highest yields ever achieved for the radical thiol-ene coupling of a substrate and oligonucleotide. In addition to distinguishing nucleotide mismatches, the ITO and magnetic liquid-based approach was more sensitive than a commercial magnetic bead-based method for the capture of target DNA from a pool of interfering genomic DNA. [ABSTRACT FROM AUTHOR]

Protein bioconjugation has been a crucial tool for studying biological processes and developing therapeutics. Sortase A (SrtA), a bacterial transpeptidase, has become widely used for its ability to site-specifically label proteins with diverse functional moieties, but a significant limitation is its poor reaction kinetics. In this work, we address this by developing proximity-based sortase-mediated ligation (PBSL), which improves the ligation efficiency to over 95 % by linking the target protein to SrtA using the SpyTag-SpyCatcher peptide-protein pair. By expressing the target protein with SpyTag C-terminal to the SrtA recognition motif, it can be covalently captured by an immobilized SpyCatcher-SrtA fusion protein during purification. Following the ligation reaction, SpyTag is cleaved off, rendering PBSL traceless, and only the labeled protein is released, simplifying target protein purification and labeling to a single step. [ABSTRACT FROM AUTHOR]

A mild method for the arylation of lysine in an unprotected peptide is presented. In the presence of a preformed biarylphosphine-supported palladium(II)-aryl complex and a weak base, lysine amino groups underwent C−N bond formation at room temperature. The process generally exhibited high selectivity for lysine over other amino acids containing nucleophilic side chains and was applicable to the conjugation of a variety of organic compounds, including complex drug molecules, with an array of peptides. Finally, this method was also successfully applied to the formation of cyclic peptides by macrocyclization. [ABSTRACT FROM AUTHOR]

Maleimide-mediated thiol-specific derivatization of biomolecules is one of the most efficacious bioconjugation approaches currently available. Alarmingly, however, recent work demonstrates that the resulting thiomaleimide conjugates are susceptible to breakdown via thiol exchange reactions. Herein, we report a new class of maleimides, namely o-CH2NH iPr phenyl maleimides, that undergo unprecedentedly rapid ring hydrolysis after thiol conjugation to form stable thiol exchange-resistant conjugates. Furthermore, we overcome the problem of low shelf lives of maleimide reagents owing to their propensity to undergo ring hydrolysis prior to bioconjugation by developing a photocaged version of this scaffold that resists ring hydrolysis. UV irradiation of thiol bioconjugates formed with this photocaged maleimide unleashes rapid thiomaleimide ring hydrolysis to yield the desired stable conjugates within 1 h under gentle, ice-cold conditions. [ABSTRACT FROM AUTHOR]

2′-Deoxyadenosine triphosphate (dATP) derivatives bearing diverse substituents (Cl, NH2, CH3, vinyl, ethynyl, and phenyl) at position 2 were prepared and tested as substrates for DNA polymerases. The 2-phenyl-dATP was not a substrate for DNA polymerases, but the dATPs bearing smaller substituents were good substrates in primer-extension experiments, producing DNA substituted in the minor groove. The vinyl-modified DNA was applied in thiol-ene addition and the ethynyl-modified DNA was applied in a CuAAC click reaction to form DNA labelled with fluorescent dyes in the minor groove [ABSTRACT FROM AUTHOR]

Metal-organic frameworks (MOFs) are promising high surface area coordination polymers with tunable pore structures and functionality; however, a lack of good size and morphological control over the as-prepared MOFs has persisted as an issue in their application. Herein, we show how a robust protein template, tobacco mosaic virus (TMV), can be used to regulate the size and shape of as-fabricated MOF materials. We were able to obtain discrete rod-shaped TMV@MOF core-shell hybrids with good uniformity, and their diameters could be tuned by adjusting the synthetic conditions, which can also significantly impact the stability of the core-shell composite. More interestingly, the virus particle underneath the MOF shell can be chemically modified using a standard bioconjugation reaction, showing mass transportation within the MOF shell. [ABSTRACT FROM AUTHOR]

This report describes the synthesis and characterization of novel N-heterocyclic carbene (NHC)-gold(I) complexes and their bioconjugation to the CCRF-CEM-leukemia-specific aptamer sgc8c. Successful bioconjugation was confirmed by the use of fluorescent tags on both the NHC-AuI complex and the aptamer. Cell-viability assays indicated that the NHC-AuI-aptamer conjugate was more cytotoxic than the NHC-gold complex alone. A combination of flow cytometry, confocal microscopy, and cell-viability assays provided clear evidence that the NHC-AuI-aptamer conjugate was selective for targeted CCRF-CEM leukemia cells. [ABSTRACT FROM AUTHOR]

Enzyme-mediated protein modification often requires large amounts of biocatalyst, adding significant costs to the process and limiting industrial applications. Herein, we demonstrate a scalable and straightforward strategy for the efficient capture and recycling of enzymes using a small-molecule affinity tag. A proline variant of an evolved sortase A (SrtA 7M) was N-terminally labeled with lithocholic acid (LA)-an inexpensive bile acid that exhibits strong binding to β-cyclodextrin (βCD). Capture and recycling of the LA-Pro-SrtA 7M conjugate was achieved using βCD-modified sepharose resin. The LA-Pro-SrtA 7M conjugate retained full enzymatic activity, even after multiple rounds of recycling. [ABSTRACT FROM AUTHOR]

DNA‐bindende Proteine erlauben die sequenzspezifische Modifikation von DNA‐basierten Nanostrukturen. Hier untersuchen wir die Eignung von Relaxaseproteinen – speziell TrwC, TraI und MobA – für die Nanofabrikation. Relaxasen sind Komponenten des bakteriellen Konjugationsmechanismus, bei dem Plasmide zwischen Bakterien ausgetauscht werden, wobei sie an DNA durch eine kovalente Phosphotyrosinbindung binden. Wir untersuchen die Anbindung der Relaxasen an zwei prototypische DNA‐Origamistrukturen – stabförmige Sechs‐Helix‐Bündel und flache rechteckige Origami‐Platten. Die Proteine zeigen orthogonale Anbindungseigenschaften mit Ausbeuten von 40–50 % pro Bindestelle, vergleichbar mit alternativen Funktionalisierungsmethoden. Die Ausbeuten variieren zwischen den Origamistrukturen und hängen auch von der Position der Bindestellen ab. Aufgrund ihrer Spezifität für einzelsträngige Zielsequenzen und ihrer orthogonalen Anbindeeigenschaften sind Relaxasen eine einzigartige Erweiterung des molekularen Werkzeugkastens für die Modifikation von DNA‐Nanostrukturen. Relaxaseproteine sind in der Natur am konjugativen Transfer von Plasmiden zwischen Bakterien beteiligt. Hier wird gezeigt, dass ihre Spezifität für einzelsträngige Zielsequenzen, ihre gegenseitige Orthogonalität und ihre Bindeeigenschaften dazu genutzt werden können, DNA‐Nanostrukturen sequenzspezifisch mit Proteinen zu funktionalisieren. [ABSTRACT FROM AUTHOR]

Michael addition reactions between biological thiols and endocyclic olefinic maleimides are extensively used for site-specific bioconjugation. The resulting thio-succinimidyl linkages, however, lack stability because of their susceptibility to thiol exchange. Reported herein is that in contrast to their endocyclic counterparts, exocyclic olefinic maleimides form highly stable thio-Michael adducts which resist thiol exchange at physiological conditions. A high-yielding approach for synthesizing a variety of exocyclic olefinic maleimides, by 4-nitrophenol-catalyzed solvent-free Wittig reactions, is reported. Mechanistic studies reveal that the catalyst facilitates the formation of the Wittig ylide intermediate through sequential proton donation and abstraction. Overall, this report details an improved thiol bioconjugation approach, a facile method for synthesizing exocyclic olefinic maleimides, and demonstrates that phenolic compounds can catalyze ylide formation. [ABSTRACT FROM AUTHOR]

Keywords: Antikörper-Wirkstoff-Konjugate (ADCs); Biokonjugation; Cysteine Profiling; Protein-Basierte Elektrophile; Protein-Protein Konjugation DE Antikörper-Wirkstoff-Konjugate (ADCs) Biokonjugation Cysteine Profiling Protein-Basierte Elektrophile Protein-Protein Konjugation 1 1 1 10/04/22 20221010 NES 221010 B The fast and the furious b : Ein DFT-Booster zur Beschleunigung der Cystein-Biokonjugation! In ihrem Forschungsartikel (e202205348) stellen Mu-Hyun Baik, Christian P. R. Hackenberger und Mitarbeiter Ethinyltriazolphosphinate (ETPs) als modulare Elektrophile für den unkomplizierten Einbau in komplexe Frachten und Proteine für modulare Protein- und Antikörpermodifizierungsstrategien vor (Titelbild: Barth van Rossum, FMP). [Extracted from the article]

Basierend auf der Kristallstruktur eines natürlichen Proteinsubstrats von mikrobieller Transglutaminase, einem Enzym, das die Vernetzung von Proteinen katalysiert, wurde eine Erkennungssequenz für ortsspezifische Konjugation rational entworfen. Dieses Strukturmimetikum einer nativen Konjugationsstelle wurde konformativ über eine intramolekulare Disulfidbrücke fixiert und ermöglichte dadurch die effiziente Konjugation eines Reportermoleküls an den therapeutischen monoklonalen Antikörper Cetuximab, ohne dessen Bindungseigenschaften zu beeinträchtigen. Ein neuer Marker für Proteinkonjugationen wurde auf Basis der Kristallstruktur eines natürlichen Substrats der mikrobiellen Transglutaminase (MTG) entworfen. Dieses konformativ eingeschränkte Strukturmimetikum einer natürlichen Konjugationsstelle ermöglicht ortspezifische, effiziente Proteinligation, wie für den Antikörper Cetuximab gezeigt. [ABSTRACT FROM AUTHOR]

Azido 18F-arenes are important and versatile building blocks for the radiolabeling of biomolecules via Huisgen cycloaddition ('click chemistry') for positron emission tomography (PET). However, routine access to such clickable agents is challenged by inefficient and/or poorly defined multistep radiochemical approaches. A high-yielding direct radiofluorination for azido 18F-arenes was achieved through the development of an ortho-oxygen-stabilized iodonium derivative (OID). This OID strategy addresses an unmet need for a reliable azido 18F-arene clickable agent for bioconjugation reactions. A ssDNA aptamer was radiolabeled with this agent and visualized in a xenograft mouse model of human colon cancer by PET, which demonstrates that this OID approach is a convenient and highly efficient way of labeling and tracking biomolecules. [ABSTRACT FROM AUTHOR]

We report the discovery of a peptide stapling and macrocyclization method using thiol-ene reactions between two cysteine residues and an α,ω-diene in high yields. This new approach enabled us to selectively modify cysteine residues in native, unprotected peptides with a variety of stapling modifications for helix stabilization or general macrocyclization. We synthesized stapled Axin mimetic analogues and demonstrated increased alpha helicity upon peptide stapling. We then synthesized stapled p53 mimetic analogues using pure hydrocarbon linkers and demonstrated their abilities to block the p53-MDM2 interaction and selectively kill p53 wild-type colorectal carcinoma HCT-116 cells but not p53 null cells. In summary, we demonstrated a robust and versatile peptide stapling method that could be potentially applied to both synthetic and expressed peptides. [ABSTRACT FROM AUTHOR]

The prevalence of bioconjugates in the biomedical sciences necessitates the development of novel mechanisms to facilitate their preparation. Towards this end, the translation of the Glaser-Hay coupling to an aqueous environment is examined, and its potential as a bioorthogonal conjugation reaction is demonstrated. This optimized, novel, and aqueous Glaser-Hay reaction is applied towards the development of bioconjugates utilizing protein expressed with an alkynyl unnatural amino acid. Unnatural amino acid technology provides a degree of bioorthognality and specificity not feasible with other methods. Moreover, the scope of the reaction is demonstrated through protein-small molecule couplings, small-molecule-solid-support couplings, and protein-solid-support immobilizations. [ABSTRACT FROM AUTHOR]

Macrocyclization is a broadly applied approach for overcoming the intrinsically disordered nature of linear peptides. Herein, it is shown that dichloroacetone (DCA) enhances helical secondary structures when introduced between peptide nucleophiles, such as thiols, to yield an acetone-linked bridge (ACE). Aside from stabilizing helical structures, the ketone moiety embedded in the linker can be modified with diverse molecular tags by oxime ligation. Insights into the structure of the tether were obtained through co-crystallization of a constrained S-peptide in complex with RNAse S. The scope of the acetone-linked peptides was further explored through the generation of N-terminus to side chain macrocycles and a new approach for generating fused macrocycles (bicycles). Together, these studies suggest that acetone linking is generally applicable to peptide macrocycles with a specific utility in the synthesis of stabilized helices that incorporate functional tags. [ABSTRACT FROM AUTHOR]

A unique two-step modular system for site-specific antibody modification and conjugation is reported. The first step of this approach uses enzymatic bioconjugation with the transpeptidase Sortase A for incorporation of strained cyclooctyne functional groups. The second step of this modular approach involves the azide-alkyne cycloaddition click reaction. The versatility of the two-step approach has been exemplified by the selective incorporation of fluorescent dyes and a positron-emitting copper-64 radiotracer for fluorescence and positron-emission tomography imaging of activated platelets, platelet aggregates, and thrombi, respectively. This flexible and versatile approach could be readily adapted to incorporate a large array of tailor-made functional groups using reliable click chemistry whilst preserving the activity of the antibody or other sensitive biological macromolecules. [ABSTRACT FROM AUTHOR]

We provide the first demonstration that isopeptide ligation, a noncanonical activity of the enzyme sortase A, can be used to modify recombinant proteins. This reaction was used in vitro to conjugate small molecules to a peptide, an engineered targeting protein, and a full-length monoclonal antibody with an exquisite level of control over the site of conjugation. Attachment to the protein substrate occurred exclusively through isopeptide bonds at a lysine ε-amino group within a specific amino acid sequence. This reaction allows more than one molecule to be site-specifically conjugated to a protein at internal sites, thereby overcoming significant limitations of the canonical native peptide ligation reaction catalyzed by sortase A. Our method provides a unique chemical ligation procedure that is orthogonal to existing methods, supplying a new method to site-specifically modify lysine residues that will be a valuable addition to the protein conjugation toolbox. [ABSTRACT FROM AUTHOR]