Chemoselective Ligation Reactions Research Articles

Protocol Considerations for In Vitro Metabolic Glycoengineering of Non-Natural Glycans.

Metabolic glycoengineering (MGE) refers to a technique where non-natural monosaccharide analogs are introduced into living biological systems. Once inside a cell, these compounds intercept a targeted biosynthetic glycosylation pathway and in turn are metabolically incorporated into cell-surface-displayed oligosaccharides, where they can modulate a host of biological activities or be exploited as tags for bioorthogonal and chemoselective ligation reactions. Over the past decade, azido-modified monosaccharides have become the go-to analogs for MGE; at the same time, analogs with novel chemical functionalities continue to be developed. Therefore, one emphasis of this article is to describe a general approach for analog selection and then provide protocols to ensure safe and efficacious analog usage by cells. Once cell-surface glycans have been successfully remodeled by MGE methodology, the stage is set for probing changes to the myriad cellular responses modulated by these versatile molecules. This manuscript concludes by detailing how one of these detection methods-flow cytometry-can be successfully utilized to quantify MGE analog incorporation and set the stage for numerous follow-up applications. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Incubation of cells with sugar analogs Support Protocol: Routine growth and maintenance of Jurkat cells Basic Protocol 2: Cell viability assays Basic Protocol 3: Periodate-resorcinol assay to measure analog uptake and incorporation into metabolic pathways Basic Protocol 4: Quantitation of cell-surface glycoconjugates.

Oxime and thiazolidine chemoselective ligation reactions: a green method for cotton functionalization

During the last years, the need to create textile materials provided with peculiar properties has grown significantly. In particular, new textiles are studied to be a first protection in the prevention of living organisms from pathogens. In this regard, modifying a textile material with biologically active compounds, such as antibacterial or antiviral peptides would be useful for many applications. Our work shows a study on the possibility of modifying cotton fabrics with peptides using thiazolidine and oxime chemoselective ligations. For this purpose, an enzymatic oxidation of cellulose in a heterogeneous phase and the possibility to reuse the oxidation solution for multiple times was successfully applied. Model peptides have been designed and synthesized in order to set up the conditions for conjugating peptides to cotton via either thiazolidine or oxime bond. A systematic study of the time, pH, and quantities needed for the best reaction conditions has been conducted. The efficiency and stability of the two chemoselective ligation bonds have been studied and compared.Graphical abstract

Wittig reagents for chemoselective sulfenic acid ligation enables global site stoichiometry analysis and redox-controlled mitochondrial targeting.

Triphenylphosphonium ylides, known as Wittig reagents, are one of the most commonly used tools in synthetic chemistry. Despite their considerable versatility, Wittig reagents have not yet been explored for their utility in biological applications. Here we introduce a chemoselective ligation reaction that harnesses the reactivity of Wittig reagents and the unique chemical properties of sulfenic acid, a pivotal post-translational cysteine modification in redox biology. The reaction, which generates a covalent bond between the ylide nucleophilic α-carbon and electrophilic γ-sulfur, is highly selective, rapid and affords robust labelling under a range of biocompatible reaction conditions, which includes in living cells. We highlight the broad utility of this conjugation method to enable site-specific proteome-wide stoichiometry analysis of S-sulfenylation and to visualize redox-dependent changes in mitochondrial cysteine oxidation and redox-triggered triphenylphosphonium generation for the controlled delivery of small molecules to mitochondria.

β-Turn Mimics by Chemical Ligation.

We report a simple reductive amination protocol to ligate two peptides, while simultaneously installing a β-turn mimic at the ligation junction. This strategy uses commercially available materials, mild chemical conditions, and a chemoselective ligation reaction of unprotected peptide substrates accessed through standard solid phase methods. This system was implemented in a designed β-hairpin system, and biophysical analysis demonstrates effective mimicry of the β-turn.

Electron Microscopic Detection of Single Membrane Proteins by a Specific Chemical Labeling.

SummaryElectron microscopy (EM) is a technology that enables visualization of single proteins at a nanometer resolution. However, current protein analysis by EM mainly relies on immunolabeling with gold-particle-conjugated antibodies, which is compromised by large size of antibody, precluding precise detection of protein location in biological samples. Here, we develop a specific chemical labeling method for EM detection of proteins at single-molecular level. Rational design of α-helical peptide tag and probe structure provided a complementary reaction pair that enabled specific cysteine conjugation of the tag. The developed chemical labeling with gold-nanoparticle-conjugated probe showed significantly higher labeling efficiency and detectability of high-density clusters of tag-fused G protein-coupled receptors in freeze-fracture replicas compared with immunogold labeling. Furthermore, in ultrathin sections, the spatial resolution of the chemical labeling was significantly higher than that of antibody-mediated labeling. These results demonstrate substantial advantages of the chemical labeling approach for single protein visualization by EM.

Site-Specific Conjugation for Fully Controlled Glycoconjugate Vaccine Preparation.

Glycoconjugate vaccines are formed by covalently link a carbohydrate antigen to a carrier protein whose role is to achieve a long lasting immune response directed against the carbohydrate antigen. The nature of the sugar antigen, its length, its ratio per carrier protein and the conjugation chemistry impact on both structure and the immune response of a glycoconjugate vaccine. In addition it has long been assumed that the sites at which the carbohydrate antigen is attached can also have an impact. These important issue can now be addressed owing to the development of novel chemoselective ligation reactions as well as techniques such as site-selective mutagenesis, glycoengineering, or extension of the genetic code. The preparation and characterization of homogeneous bivalent pneumococcal vaccines is reported. The preparation and characterization of homogeneous bivalent pneumococcal vaccines is reported. A synthetic tetrasaccharide representative of the serotype 14 capsular polysaccharide of Streptococcus pneumoniae has been linked using the thiol/maleimide coupling chemistry to four different Pneumococcal surface adhesin A (PsaA) mutants, each harboring a single cysteine mutation at a defined position. Humoral response of these 1 to 1 carbohydrate antigen/PsaA conjugates have been assessed in mice. Our results showed that the carbohydrate antigen-PsaA connectivity impacts the anti-carrier response and raise questions about the design of glycoconjugate vaccine whereby the protein plays the dual role of immunogen and carrier.

Modern Ligation Methods to Access Natural and Modified Proteins.

Proteins and peptides are gaining increasing interest as tools and targets in fundamental research and drug discovery. Growing research applications have prompted the need for methodologies that produce homogenous peptide and protein material. The development of efficient, chemoselective ligation reactions using unprotected peptide fragments presents a key solution for this challenging task. This review outlines modern ligation methods that enable the synthesis of both native, and also labelled or post-translationally modified peptides and proteins. The ligation methods herein discussed focus on the formation of the backbone amide bond.

Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method.

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for systemic energy homeostasis. Primary hepatocytes isolated from the murine liver constitute a valuable biological tool to understand the functional properties or alterations occurring in the liver. Herein we describe a method for the isolation and culture of primary mouse hepatocytes by performing a two-step collagenase perfusion technique and discuss their utilization for investigating protein metabolism. The liver of an adult mouse is sequentially perfused with ethylene glycol-bis tetraacetic acid (EGTA) and collagenase, followed by the isolation of hepatocytes with the density gradient buffer. These isolated hepatocytes are viable on culture plates and maintain the majority of endowed characteristics of hepatocytes. These hepatocytes can be used for assessments of protein metabolism including nascent protein synthesis with non-radioactive reagents. We show that the isolated hepatocytes are readily controlled and comprise a higher quality and volume stability of protein synthesis linked to energy metabolism by utilizing the chemo-selective ligation reaction with a Tetramethylrhodamine (TAMRA) protein detection method and western blotting analyses. Therefore, this method is valuable for investigating hepatic nascent protein synthesis linked to energy homeostasis. The following protocol outlines the materials and methods for the isolation of high-quality primary mouse hepatocytes and detection of nascent protein synthesis.

Covalent Surface Modification of Lipid Nanoparticles by Rapid Potassium Acyltrifluoroborate Amide Ligation.

Because of the recent increasing demand for the synthetic biomimetic nanoparticles as in vivo carriers of drugs and imaging probes, it is very important to develop reliable, stable, and orthogonal methods for surface functionalization of the particles. To address these issues, in this study, a recently reported chemoselective amide-forming ligation reaction [potassium acyltrifluoroborate (KAT) ligation] was employed for the first time, as a mean to provide the surface functionalization of particles for creating covalent attachments of bioactive molecules. A KAT derivative of oleic acid (OA-KAT, 1) was added to a mixture of three lipid components (triolein, phosphatidyl choline, and cholesteryl oleate), which have been commonly used as substrates for lipid nanoparticles. After sonication and extrusion in a buffer, successfully obtained lipid nanoparticles containing OA-KAT (NP-KAT) resulted to be well-dispersed with mean diameters of about 40-70 nm by dynamic light scattering. After preliminary confirmation of the fast and efficient KAT ligation in a solution phase using the identical reaction substrates, the "on-surface (on-particle)" KAT ligation on the NP-KAT was tested with an N-hydroxylamine derivative of fluorescein 2. The ligation was carried out in a phosphate buffer (10 mM, pH 5.2) at room temperature with reactant concentration ranges of 250 μM. Reaction efficiency was evaluated based on the amount of boron (determined by inductively coupled plasma mass spectrometry) and fluorescein (determined by fluorescence emission) in the particles before and after the reaction. As a result, the reaction proceeded in a significantly efficient way with ca. 40-50% conversion of the OA-KAT incorporated in the particles. Taken together with the fact that KAT ligation does not require any additional coupling reagents, these results indicated that the "on-surface" chemical functionalization of nanoparticles by KAT ligation is a useful method and represents a powerful and potentially versatile tool for the production of nanoparticles with a variety of covalently functionalized biomolecules and probes.

Site-Specific Antibody Functionalization Using Tetrazine-Styrene Cycloaddition.

Biologics, such as antibody-drug conjugates, are becoming mainstream therapeutics. Consequently, methods to functionalize biologics without disrupting their native properties are essential for identifying, characterizing, and translating candidate biologics from the bench to clinical practice. Here, we present a method for site-specific, carboxy-terminal modification of single-chain antibody fragments (scFvs). ScFvs displayed on the surface of yeast were isolated and functionalized by combining intein-mediated expressed protein ligation (EPL) with inverse electron-demand Diels-Alder (IEDDA) cycloaddition using a styrene-tetrazine pair. The high thiol concentration required to trigger EPL can hinder the subsequent chemoselective ligation reactions; therefore, the EPL reaction was used to append styrene to the scFv, limiting tetrazine exposure to damaging thiols. Subsequently, the styrene-functionalized scFv was reacted with tetrazine-conjugated compounds in an IEDDA cycloaddition to generate functionalized scFvs that retain their native binding activity. Rapid functionalization of yeast surface-derived scFv in a site-directed manner could find utility in many downstream laboratory and preclinical applications.

Chemoselective and Bioorthogonal Ligation Reactions. Concepts and Applications. 2 Volumes Edited by W. Russ Algar, Philip Dawson, and Igor L. Medintz.

Chemoselective and Bioorthogonal Ligation Reactions. Concepts and Applications. 2 Volumes Edited by W. Russ Algar, Philip Dawson, and Igor L. Medintz.

One-Pot Self-Assembly of Peptide-Based Cage-Type Nanostructures Using Orthogonal Ligations.

The designed arrangement of biomolecular entities within monodisperse nanostructures is an important challenge toward functional biomaterials. We report herein a method for the formation of water-soluble peptide-based cages using orthogonal ligation reactions-acylhydrazone condensation and thiol-maleimide addition. The results show that using preorganized cyclic peptides and heterobifunctional spacers as building blocks and a set of orthogonal and chemoselective ligation reactions enable cage formation in one pot from six components and through eight reactions. Molecular modelling simulations reveal the structural dynamics of these structures. Finally, we exploited the reactional dynamics of the acylhydrazone by demonstrating the controlled dissociation of the cage through directed component exchange.

Chemoselective ligation reaction of N-acetylglucosamine (NAG) with hydrazide functional probes to determine galactosyltransferase activity by MALDI mass spectrometry.

Quantification of β-1,4-galactosyltransferase (β-1,4-GT) activity is of considerable significance in the diagnosis of various cancers including lung and ovarian cancer. We report here the use of synthetic β-N-acetylglucosamine (NAG) ligands that contain hydrazide functional groups to determine galactosyltransferase activity by mass spectrometry. With hydrazide-linked β-d-NAG as the acceptor, the activity of β-1,4-GT is quantified by matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF) mass spectrometry (MS) with high efficiency. Using the disulfide moiety in a 3,3'-dithiodipropionic acid dihydrazide (DTP)-linked β-d-NAG probe, Au nanoparticles (AuNPs) are employed for enriching DTP-linked β-d-NAG after enzymatic reaction, and the ligand-bound AuNPs are subsequently deposited on a MALDI plate for analysis. In addition, we have demonstrated that a perfluorocarbon (PF) labeled β-NAG-ligand can be useful for surface-based enzymatic reaction with a perfluorooctadecanethiol (PFDT)-covered gold surface. Using the ratiometric method, the conversion rate of β-1,4-GT is determined to be 0.83 ± 0.03, which shows a high level of activity. This is the first work that uses hydrazide-linked β-d-NAG for activity analysis, providing a new surface-based MS approach to determine enzyme activity in a potentially high-throughput manner.

Critical evaluation and rate constants of chemoselective ligation reactions for stoichiometric conjugations in water.

Chemoselective ligation reactions have contributed immensely to the development of organic synthesis and chemical biology. However, the ligation of stoichiometric amounts of large molecules for applications such as protein-protein conjugates is still challenging. Conjugation reactions need to be fast enough to proceed under dilute conditions and chemoselective in the presence of unprotected functional groups; the starting materials and products must be stable under the reaction conditions. To compare known ligation reactions for their suitability under these conditions, we determined the second-order rate constants of ligation reactions using peptide substrates with unprotected functional groups. The reaction conditions, the chemoselectivity of the reactions, and the stability of the starting materials and products were carefully evaluated. In some cases, the stability could be improved by modifying the substrate structure. These data obtained under the ligation conditions provide a useful guide to choose an appropriate ligation reaction for synthesis of large molecules by covalent ligation reactions of unprotected substrates in water.

Stabilizing Impact of N-Glycosylation on the WW Domain Depends Strongly on the Asn-GlcNAc Linkage

N-glycans play important roles in many cellular processes and can increase protein conformational stability in specific structural contexts. Glycosylation (with a single GlcNAc) of the reverse turn sequence Phe-Yyy-Asn-Xxx-Thr at Asn stabilizes the Pin 1 WW domain by -0.85 ± 0.12 kcal mol(-1). Alternative methods exist for attaching carbohydrates to proteins; some occur naturally (e.g., the O-linkage), whereas others use chemoselective ligation reactions to mimic the natural N- or O-linkages. Here, we assess the energetic consequences of replacing the Asn linkage in the glycosylated WW domain with a Gln linkage, with two natural O-linkages, with two unnatural triazole linkages, and with an unnatural α-mercaptoacetamide linkage. Of these alternatives, only glycosylation of the triazole linkages stabilizes WW, and by a smaller amount than N-glycosylation, highlighting the need for caution when using triazole- or α-mercaptoacetamide-linked carbohydrates to mimic native N-glycans, especially where the impact of glycosylation on protein conformational stability is important.

Preparation of Carbohydrate Arrays by Using Diels–Alder Reactions with Inverse Electron Demand

Carbohydrate microarrays are an emerging tool for the high-throughput screening of carbohydrate-protein interactions that represent the basis of many biologically and medicinally relevant processes. The crucial step in the preparation of carbohydrate arrays is the attachment of carbohydrate probes to the surface. We examined the Diels-Alder reaction with inverse-electron-demand (DARinv) as an irreversible, chemoselective ligation reaction for that purpose. After having shown the efficiency of the DARinv in solution, we prepared a series of carbohydrate-dienophile conjugates that were printed onto tetrazine-modified glass slides. Binding experiments with fluorescently labeled lectins proved successful and homogeneous immobilization was achieved by the DARinv. For immobilization of nonfunctionalized reducing oligosaccharides we developed a bifunctional chemoselective linker that enabled the attachment of a dienophile tag to the oligosaccharides through oxime ligation. The conjugates obtained were successfully immobilized on glass slides. The presented strategies for the immobilization of both synthetic carbohydrate derivatives and unprotected reducing oligosaccharides facilitate the preparation of high-quality carbohydrate microarrays by means of the chemoselective DARinv. This concept can be readily adapted for the preparation of other biomolecule arrays.

ChemInform Abstract: Organic Synthesis Without Stoichiometric Reagents: A Guiding Principle for Reaction Development

AbstractReview: 66 refs.

Organic Synthesis without Stoichiometric Reagents: A Guiding Principle for Reaction Development

A common theme of our research program is the development of new organic transformations that operate under catalytic conditions or as ligation reactions that do not require the addition of any reagents or other additives. Our catalysis program features the transient generation of reactive species from alpha-functionalized aldehydes via intramolecular redox reactions using N-heterocyclic carbenes as multifunctional catalysts. This approach makes possible the catalytic generation of enolates, homoenolates, and activated carboxylates and their application to diastereo- and enantioselective transformation. Intermolecular redox couplings are key to a general, highly chemoselective amide-forming ligation reaction and its use for oligopeptide synthesis. The concepts behind these transformation and examples of their use as well as current and future directions of our research program are presented.

Experimental Design Considerations for In Vitro Non‐Natural Glycan Display via Metabolic Oligosaccharide Engineering

Metabolic oligosaccharide engineering (MOE) refers to a technique where non-natural monosaccharide analogs are introduced into living biological systems. Once inside a cell, these compounds intercept a targeted biosynthetic glycosylation pathway and in turn are metabolically incorporated into cell-surface-displayed oligosaccharides where they can modulate a host of biological activities or be exploited as "tags" for bio-orthogonal and chemoselective ligation reactions. Undertaking a MOE experiment can be a daunting task based on the growing repertoire of analogs now available and the ever increasing number of metabolic pathways that can be targeted; therefore, a major emphasis of this article is to describe a general approach for analog design and selection and then provide protocols to ensure safe and efficacious analog usage by cells. Once cell-surface glycans have been successfully remodeled by MOE methodology, the stage is set for probing changes to the myriad cellular responses modulated by these versatile molecules. Curr. Protoc. Chem. Biol. 2:171-194 © 2010 by John Wiley & Sons, Inc.

Metabolic glycoengineering: Sialic acid and beyond

This report provides a perspective on metabolic glycoengineering methodology developed over the past two decades that allows natural sialic acids to be replaced with chemical variants in living cells and animals. Examples are given demonstrating how this technology provides the glycoscientist with chemical tools that are beginning to reproduce Mother Nature's control over complex biological systems - such as the human brain - through subtle modifications in sialic acid chemistry. Several metabolic substrates (e.g., ManNAc, Neu5Ac, and CMP-Neu5Ac analogs) can be used to feed flux into the sialic acid biosynthetic pathway resulting in numerous - and sometime quite unexpected - biological repercussions upon nonnatural sialoside display in cellular glycans. Once on the cell surface, ketone-, azide-, thiol-, or alkyne-modified glycans can be transformed with numerous ligands via bioorthogonal chemoselective ligation reactions, greatly increasing the versatility and potential application of this technology. Recently, sialic acid glycoengineering methodology has been extended to other pathways with analog incorporation now possible in surface-displayed GalNAc and fucose residues as well as nucleocytoplasmic O-GlcNAc-modified proteins. Finally, recent efforts to increase the "druggability" of sugar analogs used in metabolic glycoengineering, which have resulted in unanticipated "scaffold-dependent" activities, are summarized.