Bioorthogonal Chemistry Research Articles

A bispecific, crosslinking lectibody activates cytotoxic T cells and induces cancer cell death.

Aberrant glycosylation patterns play a crucial role in the development of cancer cells as they promote tumor growth and aggressiveness. Lectins recognize carbohydrate antigens attached to proteins and lipids on cell surfaces and represent potential tools for application in cancer diagnostics and therapy. Among the emerging cancer therapies, immunotherapy has become a promising treatment modality for various hematological and solid malignancies. Here we present an approach to redirect the immune system into fighting cancer by targeting altered glycans at the surface of malignant cells. We developed a so-called "lectibody", a bispecific construct composed of a lectin linked to an antibody fragment. This lectibody is inspired by bispecific T cell engager (BiTEs) antibodies that recruit cytotoxic T lymphocytes (CTLs) while simultaneously binding to tumor-associated antigens (TAAs) on cancer cells. The tumor-related glycosphingolipid globotriaosylceramide (Gb3) represents the target of this proof-of-concept study. It is recognized with high selectivity by the B-subunit of the pathogen-derived Shiga toxin, presenting opportunities for clinical development. The lectibody was realized by conjugating an anti-CD3 single-chain antibody fragment to the B-subunit of Shiga toxin to target Gb3+ cancer cells. The reactive non-canonical amino acid azidolysine (AzK) was inserted at predefined single positions in both proteins. The azido groups were functionalized by bioorthogonal conjugation with individual linkers that facilitated selective coupling via an alternative bioorthogonal click chemistry reaction. In vitro cell-based assays were conducted to evaluate the antitumoral activity of the lectibody. CTLs, Burkitt´s lymphoma-derived cells and colorectal adenocarcinoma cell lines were screened in flow cytometry and cytotoxicity assays for activation and lysis, respectively. This proof-of-concept study demonstrates that the lectibody activates T cells for their cytotoxic signaling, redirecting CTLs´ cytotoxicity in a highly selective manner and resulting in nearly complete tumor cell lysis-up to 93%-of Gb3+ tumor cells in vitro. This research highlights the potential of lectins in targeting certain tumors, with an opportunity for new cancer treatments. When considering a combinatorial strategy, lectin-based platforms of this type offer the possibility to target glycan epitopes on tumor cells and boost the efficacy of current therapies, providing an additional strategy for tumor eradication and improving patient outcomes.

Making functional connections: A summary of the Nobel Prize in Chemistry 2022

<p indent="0mm">The Nobel Prize in Chemistry 2022 was awarded to three scientists for their work in the development of click chemistry and bioorthogonal chemistry: Carolyn R. Bertozzi from Stanford University, Morten Meldal from the University of Copenhagen, and K. Barry Sharpless from Scripps Research. Notably, this is the second time Sharpless was awarded this prize. In 2001, he received his first Nobel Prize for the development of catalytic asymmetric oxidation reactions. This year’s prize, however, is in a completely different area, as will be summarized below. For both click chemistry and bioothogonal chemistry, their focus is on making easy connections between molecules and snapping them together. Although modern chemistry has experienced substantial development since its birth in the eighteenth century, dealing with complex molecules and systems remains a challenging and laborious process that requires skill and knowledge. Synthesis has become the bottleneck for the discovery of molecular functions. In 2001, Sharpless and co-workers coined the concept of click chemistry, by defining a set of stringent criteria so that few good reactions could be sorted out for making easy and modular connections: Molecules could be linked together simply like buckling up through a “click”. Later in 2002, Sharpless group published the first click-type reaction, namely the copper-catalyzed azide-alkyne cycloaddition (CuAAC). The reaction is silent without catalyst due to the high kinetic barrier, but become as fast as a “click” to generate triazoles in the presence of copper(I) with a strong thermodynamic driving force. Independently, Meldal made a similar discovery during the peptide synthesis. Over the years, CuAAC has proved itself to be a reliable tool for connecting chemical units of various forms together, nearly under any circumstances with high fidelity. In the late 1990s, in Bertozzi’s search for chemical tools for sugar labeling on the surface of living cell, she realized that the key for successful utilization of a chemical handle was the non-interfering nature of the involved reactions with the surrounding biological environments. She termed this kind of non-interfering reactivity as bioothogonal chemistry. By metabolically incorporating unnatural sialic acids to the cell surface, including the azide derivatives, Bertozzi successfully used Staudinger reaction to attach imaging probes on the cell with amide connections. The iconic breakthrough took place in 2004 when click chemistry was merged with bioothogonal chemistry for the first time. Bertozzi developed a stain-promoted azide-alkyne cycloaddition (SPAAC), which gave improved connection capability for biorthogonal cell imaging without the need of a catalyst, nor any compromise on bioorthogonality. The impact of click chemistry and bioorthogonal chemistry is tremendous and ever-growing. They have inspired the innovation of many connection tools, such as the inverse electron-demand Diels-Alder reaction, photoclick chemistry, and the sulfur(VI)-fluoride exchange (SuFEx) reaction. Apart from serving as tools for tracking and manipulating biomolecules and related events <italic>in vivo</italic>, they have also been used for material sciences and medicinal chemistry. For instance, sacituzumab govitecan is a Trop-2-directed antibody and topoisomerase inhibitor drug conjugate approved by the FDA for the treatment of metastatic triple-negative breast cancer and metastatic urothelial cancer. The key linkage is a CuAAC-derived 1,2,3-triazole. In short, there remain a lot of challenges in chemistry and the related fields to be addressed through diverse strategies and chemical approaches nowadays. The mission of chemistry is to bring the greatest benefit to mankind through continuous innovation and integration.

LGBTQ+ chemists react to Carolyn Bertozzi’s Nobel win

When I first heard Carolyn Bertozzi speak at the American Chemical Society national meeting in the spring of 2017, I was a closeted college sophomore, still in the early stages of my journeys with chemistry and queerness. Bertozzi represented to me what she has for so many others: a shining example of what it means to be a successful, captivating, and proudly out chemist . In the years that followed, I would come to learn just how much of a powerhouse Bertozzi is, developing the field of bioorthogonal chemistry while championing diversity in science, technology, engineering, and mathematics as a gay woman. When I sat down to interview Bertozzi for my podcast , My Fave Queer Chemist , in the summer of 2021, a lot had changed since that first time I heard her speak. I was almost halfway through my PhD at the University of Michigan, my journey with

Dithiol-Activated Bioorthogonal Chemistry for Endoplasmic Reticulum-Targeted Synergistic Chemophototherapy.

The controlled intracellular release of nitrite is still an unmet challenge due to the lack of bio-friendly donors, and the antitumor effect of nitrite is limited by its physiologically inert activity. Herein, we designed benzothiadiazole-based organic nitrite donors that are stable against bio-relevant species but selectively respond to dithiol species through SN Ar/intramolecular cyclization tandem reactions in the aqueous media. The bioorthogonal system was established to target the endoplasmic reticulum (ER) of liver cancer HepG2 cells. The nitrite and nonivamide were coupled to induce elevation of intracellular levels of calcium ions as well as reactive oxygen/nitrogen species, which resulted in ER stress and mitochondrial dysfunction. We demonstrated that a combination of photoactivation and "click to release" strategy could enhance antitumor effect in cellular level and show good potential for cancer precision therapy.

Dithiol‐Activated Bioorthogonal Chemistry for Endoplasmic Reticulum‐Targeted Synergistic Chemophototherapy

AbstractThe controlled intracellular release of nitrite is still an unmet challenge due to the lack of bio‐friendly donors, and the antitumor effect of nitrite is limited by its physiologically inert activity. Herein, we designed benzothiadiazole‐based organic nitrite donors that are stable against bio‐relevant species but selectively respond to dithiol species through SNAr/intramolecular cyclization tandem reactions in the aqueous media. The bioorthogonal system was established to target the endoplasmic reticulum (ER) of liver cancer HepG2 cells. The nitrite and nonivamide were coupled to induce elevation of intracellular levels of calcium ions as well as reactive oxygen/nitrogen species, which resulted in ER stress and mitochondrial dysfunction. We demonstrated that a combination of photoactivation and “click to release” strategy could enhance antitumor effect in cellular level and show good potential for cancer precision therapy.

A Cell Selective Fluoride-Activated MOF Biomimetic Platform for Prodrug Synthesis and Enhanced Synergistic Cancer Therapy.

As a burgeoning bioorthogonal reaction, the fluoride-mediated desilylation is capable of prodrug activation. However, due to the reactions lack of cell selectivity and unitary therapy modality, this strongly impedes their biomedical applications. Herein, we construct a cancer cell-selective biomimetic metal-organic framework (MOF)-F platform for prodrug activation and enhanced synergistic chemodynamic therapy (CDT). With cancer cell membranes camouflage, the designed biomimetic nanocatalyst displays preferential accumulation to homotypic cancer cells. Then, pH-responsive nanocatalyst releases fluoride ions and ferric ions. For activation of our designed prodrug tert-butyldimethyl silyl (TBS)-hydroxycamptothecin (TBSO-CPT), fluoride ions can desilylate TBS and cleave the designed silyl ether linker to synthesize the OH-CPT (10-hydroxycamptothecin) drug molecule, which effectively kills cancer cells. Intriguingly, the bioorthogonal-synthesized OH-CPT drug upregulates intracellular H2O2 by activating nicotinamide adenine dinucleotide phosphate oxidase (NOX), amplifying the released iron induced Fenton reaction for synergistic CDT. Both in vitro and in vivo studies demonstrate our strategy presents a versatile fluoride-activated bioorthogonal catalyst for cancer cell-selective drug synthesis. Our work may accelerate the biomedical applications of fluoride-activated bioorthogonal chemistry.

Tetrazine bioorthogonal chemistry derived in vivo imaging.

Bioorthogonal chemistry represents plenty of highly efficient and biocompatible reactions that proceed selectively and rapidly in biological situations without unexpected side reactions towards miscellaneous endogenous functional groups. Arise from the strict demands of physiological reactions, bioorthogonal chemical reactions are natively selective transformations that are rarely found in biological environments. Bioorthogonal chemistry has long been applied to tracking and real-time imaging of biomolecules in their physiological environments. Thereinto, tetrazine bioorthogonal reactions are particularly important and have increasing applications in these fields owing to their unique properties of easily controlled fluorescence or radiation off-on mechanism, which greatly facilitate the tracking of real signals without been disturbed by background. In this mini review, tetrazine bioorthogonal chemistry for in vivo imaging applications will be attentively appraised to raise some guidelines for prior tetrazine bioorthogonal chemical studies.

Bio-Orthogonal Chemistry Conjugation Strategy Facilitates Investigation of N-methyladenosine and Thiouridine Guide RNA Modifications on CRISPR Activity.

The CRISPR-Cas9 system is an important genome editing tool that holds enormous potential toward the treatment of human genetic diseases. Clinical success of CRISPR technology is dependent on the incorporation of modifications into the single-guide RNA (sgRNA). However, chemical synthesis of modified sgRNAs, which are over 100 nucleotides in length, is difficult and low-yielding. We developed a conjugation strategy that utilized bio-orthogonal chemistry to efficiently assemble functional sgRNAs containing nucleobase modifications. The described approach entails the chemical synthesis of two shorter RNA oligonucleotides: a 31-mer containing tetrazine (Tz) group and a 70-mer modified with a trans-cyclooctene (TCO) moiety. The two oligonucleotides were conjugated to form functional sgRNAs. The two-component conjugation methodology was utilized to synthesize a library of sgRNAs containing nucleobase modifications such as N1-methyladenosine (m1A), N6-methyladenosine (m6A), 2-thiouridine (s2U), and 4-thiouridine (s4U). The impact of these RNA modifications on overall CRISPR activity were investigated in vitro and in Cas9-expressing HEK293T cells.

A dark intermediate in the fluorogenic reaction between tetrazine fluorophores and trans-cyclooctene

Fluorogenic labeling via bioorthogonal tetrazine chemistry has proven to be highly successful in fluorescence microscopy of living cells. To date, trans-cyclooctene (TCO) and bicyclonyne have been found to be the most useful substrates for live-cell labeling owing to their fast labeling kinetics, high biocompatibility, and bioorthogonality. Recent kinetic studies of fluorogenic click reactions with TCO derivatives showed a transient fluorogenic effect but could not explain the reaction sequence and the contributions of different intermediates. More recently, fluorescence quenching by potential intermediates has been investigated, suggesting their occurrence in the reaction sequence. However, in situ studies of the click reaction that directly relate these observations to the known reaction sequence are still missing. In this study, we developed a single-molecule fluorescence detection framework to investigate fluorogenic click reactions. In combination with data from ultra-performance liquid chromatography-tandem mass spectrometry, this explains the transient intensity increase by relating fluorescent intermediates to the known reaction sequence of TCO with fluorogenic tetrazine dyes. More specifically, we confirm that the reaction of TCO with tetrazine rapidly forms a fluorescent 4,5-dihydropyridazine species that slowly tautomerizes to a weakly fluorescent 1,4-dihydropyridazine, explaining the observed drop in fluorescence intensity. On a much slower timescale of hours/days, the fluorescence intensity may be recovered by oxidation of the intermediate to a pyridazine. Our findings are of importance for quantitative applications in fluorescence microscopy and spectroscopy as the achieved peak intensity with TCO depends on the specific experimental settings. They clearly indicate the requirement for more robust benchmarking of click reactions with tetrazine dyes and the need for alternative dienophiles with fast reaction kinetics and stable fluorescence emission to further applications in advanced fluorescence microscopy.

External stimuli-responsive gasotransmitter prodrugs: Chemistry and spatiotemporal release.

Gasotransmitters like nitric oxide, carbon monoxide, and hydrogen sulfide with unique pleiotropic pharmacological effects in mammals are an emerging therapeutic modality for different human diseases including cancer, infection, ischemia-reperfusion injuries, and inflammation; however, their clinical translation is hampered by the lack of a reliable delivery form, which delivers such gasotransmitters to the action site with precisely controlled dosage. The external stimuli-responsive prodrug strategy has shown tremendous potential in developing gasotransmitter prodrugs, which affords precise temporospatial control and better dose control compared with endogenous stimuli-sensitive prodrugs. The promising external stimuli employed for gasotransmitter activation range from photo, ultrasound, and bioorthogonal click chemistry to exogenous enzymes. Herein, we highlight the recent development of external stimuli-mediated decaging chemistry for the temporospatial delivery of gasotransmitters including nitric oxide, carbon monoxide, hydrogen sulfide and sulfur dioxide, and discuss the pros and cons of different designs.

P-38 - Optimized synthesis of tri(hydroxypyridinone)-tetrazine and 68Ga-radiolabeling for bioorthogonal chemistry

P-38 - Optimized synthesis of tri(hydroxypyridinone)-tetrazine and 68Ga-radiolabeling for bioorthogonal chemistry

Discovering glycoRNA: Traditional and Non‐Canonical Approaches to Studying RNA Modifications

AbstractChemical biology opens the door to understanding biological processes in many areas, but nowhere has it had a greater impact on defining a field than in glycobiology. The Wolf and Nobel Prize‐winning work by Carolyn Bertozzi and coworkers to conceptualize bioorthogonal chemistry, enable it on living cells, deploy it with metabolic chemical reporters (MCRs) such as N‐azidoacetylmannosamine‐tetraacylated (Ac4ManNAz), has allowed researchers to elucidate the structure, functions, and regulation of complex glycans across various domains of life. In this review, we highlight how our work using Ac4ManNAz to discover glycosylated RNA (glycoRNA) shed the traditional paradigms of RNA post‐transcriptional modification discovery. While our understanding of RNA modifications has historically been expanded with unbiased chemical tools, the creative use of Ac4ManNAz for the targeted study of RNA's intersection with glycobiology has advanced Bertozzi's impact into the world of RNA.

The Nobel Prize in Chemistry 2022: Fulfilling Demanding Applications with Simple Reactions.

The Nobel Prize in Chemistry 2022 was awarded jointly to Carolyn R. Bertozzi, Morten Meldal, and K. Barry Sharpless "for the development of click chemistry and bioorthogonal chemistry". Such rapid, selective, and nontoxic covalent reactions that link two components together under benign conditions have led to a paradigm shift in basic research and practical applications.

Imaging and Editing the Phospholipidome.

Membranes are multifunctional supramolecular assemblies that encapsulate our cells and the organelles within them. Glycerophospholipids are the most abundant component of membranes. They make up the majority of the lipid bilayer and play both structural and functional roles. Each organelle has a different phospholipid composition critical for its function that results from dynamic interplay and regulation of numerous lipid-metabolizing enzymes and lipid transporters. Because lipid structures and localizations are not directly genetically encoded, chemistry has much to offer to the world of lipid biology in the form of precision tools for visualizing lipid localization and abundance, manipulating lipid composition, and in general decoding the functions of lipids in cells.In this Account, we provide an overview of our recent efforts in this space focused on two overarching and complementary goals: imaging and editing the phospholipidome. On the imaging front, we have harnessed the power of bioorthogonal chemistry to develop fluorescent reporters of specific lipid pathways. Substantial efforts have centered on phospholipase D (PLD) signaling, which generates the humble lipid phosphatidic acid (PA) that acts variably as a biosynthetic intermediate and signaling agent. Though PLD is a hydrolase that generates PA from abundant phosphatidylcholine (PC) lipids, we have exploited its transphosphatidylation activity with exogenous clickable alcohols followed by bioorthogonal tagging to generate fluorescent lipid reporters of PLD signaling in a set of methods termed IMPACT.IMPACT and its variants have facilitated many biological discoveries. Using the rapid and fluorogenic tetrazine ligation, it has revealed the spatiotemporal dynamics of disease-relevant G protein-coupled receptor signaling and interorganelle lipid transport. IMPACT using diazirine photo-cross-linkers has enabled identification of lipid-protein interactions relevant to alcohol-related diseases. Varying the alcohol reporter can allow for organelle-selective labeling, and varying the bioorthogonal detection reagent can afford super-resolution lipid imaging via expansion microscopy. Combination of IMPACT with genome-wide CRISPR screening has revealed genes that regulate physiological PLD signaling.PLD enzymes themselves can also act as tools for precision editing of the phospholipid content of membranes. An optogenetic PLD for conditional blue-light-stimulated synthesis of PA on defined organelle compartments led to the discovery of the role of organelle-specific pools of PA in regulating oncogenic Hippo signaling. Directed enzyme evolution of PLD, enabled by IMPACT, has yielded highly active superPLDs with broad substrate tolerance and an ability to edit membrane phospholipid content and synthesize designer phospholipids in vitro. Finally, azobenzene-containing PA analogues represent an alternative, all-chemical strategy for light-mediated control of PA signaling.Collectively, the strategies described here summarize our progress to date in tackling the challenge of assigning precise functions to defined pools of phospholipids in cells. They also point to new challenges and directions for future study, including extension of imaging and membrane editing tools to other classes of lipids. We envision that continued application of bioorthogonal chemistry, optogenetics, and directed evolution will yield new tools and discoveries to interrogate the phospholipidome and reveal new mechanisms regulating phospholipid homeostasis and roles for phospholipids in cell signaling.

Enzymatically Triggered Deprotection and Cross-Linking of Thiolated Alginate-Based Bioinks

Thiolated polymers are widely used in hydrogels for drug delivery, tissue engineering, and biofabrication. The oxidation of thiols is spontaneous, resulting in the formation of disulfide bridges and cross-linking of polymers. The cross-linking process is, however, difficult to control and is initiated directly when the thiolated components are exposed to ambient conditions, which significantly complicates handling of the materials. Here, we show a fully bioorthogonal enzyme-mediated thiol-based chemistry for dynamic covalent cross-linking of carbohydrate-based hydrogels that circumvents the problems with uncontrolled thiol oxidation. Alginate was modified with cysteine residues, protected by an enzyme-labile thiol-protecting group (Phacm). Releasing the Phacm group by penicillin G acylase generates free thiols that oxidize under physiological conditions, resulting in a reversible cross-linking and formation of hydrogels with tunable stiffness. Prior to deprotection, the components can be exposed to ambient conditions. The enzyme-triggered deprotection and subsequent gelation allows for encapsulation of cells and 3D bioprinting of cell-laden hydrogel structures. Remaining deprotected thiols enabled postprinting modifications and hydrogel self-healing. The proposed hydrogel synthesis strategy significantly increases the versatility of thiol-based cross-linking chemistries and provides new possibilities to generate dynamic covalent hydrogels for a broad range of biomedical applications.

Synthetic Organelles for Multiple mRNA Selective Genetic Code Expansions in Eukaryotes.

Engineering new functionalities into living eukaryotic systems is one of the main goals of synthetic biology. To this end, often enzyme evolution or de novo protein design is employed, whicheach have their own advantages and disadvantages. As complimentary tools, we recently developed orthogonally translating and film-like synthetic organelles that allow to create new enzyme functionalities based on spatial separation. We applied this technology to genetic code expansion (GCE) and showed that it is possible to equip eukaryotic cells with multiple orthogonal genetic codes that enable the specific reprogramming of distinct translational machineries, each with single-residue precision.In this protocol, we describe how synthetic organelles can be used to perform mRNA selective GCE and how they can be further developed to allow the simultaneous incorporation of distinct noncanonical amino acids (ncAAs) into selected proteins and how this can be used to label proteins selectively with fluorescent dyes via bioorthogonal chemistry.

A Motor-Based Carbonaceous Nanocalabash Catalyst for Deep-Layered Bioorthogonal Chemistry.

Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction has been widely regarded as a promising avenue in bioorthogonal chemistry. The emerging heterogeneous copper catalysts have been developed with a series of exciting applications such as in situ activation of prodrugs because of their excellent stability and biocompatibility. However, due largely to the complex biophysical barriers in living organisms, most synthetic bioorthogonal drugs cannot penetrate deep pathological tissues. Especially in biofilm-associated infections, the biofilms severely block the penetration of traditional antimicrobial agents and increase the antibiotic resistance, which makes it extremely difficult to eliminate the biofilms. Inspired by self-propelled biological motors, such as enzymes, herein, we develop a NIR light-controllable carbonaceous nanocalabash (CNC) motor catalyst with good biocompatibility for active targeted synthesis of drugs inside the biofilms as a robust general-purpose bioorthogonal platform. Under the NIR laser, the CNC motor catalysts display a rapid autonomous motion and generate active molecules in the deep biofilm layers, removing the biofilms and eradicating the shielded bacteria. Our work will shed light on developing a robust bioorthogonal platform for active targeted synthesis of drugs in deep-layered living systems.

Signal Amplification Pretargeted PET/Fluorescence Imaging Based on Human Serum Albumin-Encapsulated GdF3 Nanoparticles for Diagnosis of Ovarian Cancer.

Different modal imaging techniques could be complementary in tumor diagnosis. Human serum albumin (HSA)-encapsulated GdF3 nanoparticles were developed as T1 magnetic resonance imaging (MRI) contrast agents. However, no significant T1 enhancement in the tumor site of the SKOV3 human ovarian cancer xenograft tumor model was observed within 3 h after injection of tetrazine-modified GdF3@HSA NPs through small-animal MRI. After intravenous injection of 18F (or Cy7)-labeled Reppe anhydride, pretargeted positron emission tomography (PET) (near-infrared (NIR) fluorescence) imaging was used to reveal the pharmacokinetics of GdF3@HSA NPs in the SKOV3 xenograft mouse model to locate the tumor. The probe based on Reppe anhydride achieved rapid ligation with tetrazine-modified GdF3@HSA nanoparticles (NPs), which accumulated in tumor through Reppe anhydride/tetrazine bioorthogonal chemistry. This pretargeting strategy enabled excellent tumor visualization and quantification at an early period after nanoparticle injection (3 h p.i.), while the MRI images with significant T1 enhancement could be obtained until 24 h after injection of Gd-based contrast agents only. In vivo pretargeted multimodal imaging based on the tetrazine/Reppe anhydride system using HSA-encapsulated GdF3 nanoparticles would be beneficial for amplification of the imaging signal in the disease site and enhancing diagnostic efficiency.

Affinity Bioorthogonal Chemistry (ABC) Tags for Site‐Selective Conjugation, On‐Resin Protein‐Protein Coupling, and Purification of Protein Conjugates

AbstractThe site‐selective functionalization of proteins has broad application in chemical biology, but can be limited when mixtures result from incomplete conversion or the formation of protein containing side products. It is shown here that when proteins are covalently tagged with pyridyl‐tetrazines, the nickel‐iminodiacetate (Ni‐IDA) resins commonly used for His‐tags can be directly used for protein affinity purification. These Affinity Bioorthogonal Chemistry (ABC) tags serve a dual role by enabling affinity‐based protein purification while maintaining rapid kinetics in bioorthogonal reactions. ABC‐tagging works with a range of site‐selective bioconjugation methods with proteins tagged at the C‐terminus, N‐terminus or at internal positions. ABC‐tagged proteins can also be purified from complex mixtures including cell lysate. The combination of site‐selective conjugation and clean‐up with ABC‐tagged proteins also allows for facile on‐resin reactions to provide protein‐protein conjugates.

Affinity Bioorthogonal Chemistry (ABC) Tags for Site-Selective Conjugation, On-Resin Protein-Protein Coupling, and Purification of Protein Conjugates.

The site-selective functionalization of proteins has broad application in chemical biology, but can be limited when mixtures result from incomplete conversion or the formation of protein containing side products. It is shown here that when proteins are covalently tagged with pyridyl-tetrazines, the nickel-iminodiacetate (Ni-IDA) resins commonly used for His-tags can be directly used for protein affinity purification. These Affinity Bioorthogonal Chemistry (ABC) tags serve a dual role by enabling affinity-based protein purification while maintaining rapid kinetics in bioorthogonal reactions. ABC-tagging works with a range of site-selective bioconjugation methods with proteins tagged at the C-terminus, N-terminus or at internal positions. ABC-tagged proteins can also be purified from complex mixtures including cell lysate. The combination of site-selective conjugation and clean-up with ABC-tagged proteins also allows for facile on-resin reactions to provide protein-protein conjugates.