Expanding the Strain-Promoted 1,3-Dipolar Cycloaddition Arsenal for a More Selective Bioorthogonal Labeling in Living Cells

The advent of bioorthogonal chemistry, more importantly the strain-promoted 1,3-dipolar cycloaddition of cyclooctynes with azides to give stable triazoles, has opened new avenues for the study of biomolecules in their native environment. While much effort has been focused on improving the kinetics of these reactions, very little attention has been given to their bioselectivity. In this review, we will take you on our journey that led us to develop new cyclooctyne probes with enhanced physical attributes, including water solubility, cell-surface labeling selectivity and fluorogenic capabilities. We then went on to expand the bioorthogonal chemistry toolbox by focusing on the development of new chemical reporters. Here you will read about our work investigating the use of other 1,3-dipoles, including nitrile oxides and diazo reagents, for the labeling of biomolecules, and more recently highly biostable sydnones that can be exploited to selectively influence glycan-processing enzymes.     

Bioorthogonal Phosphines: Then and Now

Bioorthogonal chemistry traces its roots to a seminal report by Saxon and Bertozzi, who described a modified Staudinger reaction between organic azides and triaryl phosphines. This finding not only inspired several biological pursuits, but also launched an entire field of reaction discovery. Over the years, much effort has been directed at identifying alternative bioorthogonal transformations with organic azides; less work has focused on leveraging triaryl phosphines for new reaction development. The landscape has changed in recent years, with the generation of faster-reacting Staudinger probes and novel classes of bioorthogonal reagents. This perspective covers newly developed phosphine-based chemistries and their application in biological settings. We focus, in particular, on reactions with cyclopropenones and related analogs. These transformations feature unique mechanisms that are broadening the scope of bioorthogonal reactivity.     

Bioorthogonal chemical reporters for analyzing protein lipidation and lipid trafficking

Protein lipidation and lipid trafficking control many key biological functions in all kingdoms of life. The discovery of diverse lipid species and their covalent attachment to many proteins has revealed a complex and regulated network of membranes and lipidated proteins that are central to fundamental aspects of physiology and human disease. Given the complexity of lipid trafficking and the protein targeting mechanisms involved with membrane lipids, precise and sensitive methods are needed to monitor and identify these hydrophobic molecules in bacteria, yeast, and higher eukaryotes. Although many analytical methods have been developed for characterizing membrane lipids and covalently modified proteins, traditional reagents and approaches have limited sensitivity, do not faithfully report on the lipids of interest, or are not readily accessible. The invention of bioorthogonal ligation reactions, such as the Staudinger ligation and azide-alkyne cycloadditions, has provided new tools to address these limitations, and their use has begun to yield fresh insight into the biology of protein lipidation and lipid trafficking. In this Account, we discuss how these new bioorthogonal ligation reactions and lipid chemical reporters afford new opportunities for exploring the biology of lipid-modified proteins and lipid trafficking. Lipid chemical reporters from our laboratory and several other research groups have enabled improved detection and large-scale proteomic analysis of fatty-acylated and prenylated proteins. For example, fatty acid and isoprenoid chemical reporters in conjunction with bioorthogonal ligation methods have circumvented the limited sensitivity and hazards of radioactive analogues, allowing rapid and robust fluorescent detection of lipidated proteins in all organisms tested. These chemical tools have revealed alterations in protein lipidation in different cellular states and are beginning to provide unique insights in mechanisms of regulation. Notably, the purification of proteins labeled with lipid chemical reporters has allowed both the large-scale analysis of lipidated proteins as well as the discovery of new lipidated proteins involved in metabolism, gene expression, and innate immunity. Specific lipid reporters have also been developed to monitor the trafficking of soluble lipids; these species are enabling bioorthogonal imaging of membranes in cells and tissues. Future advances in bioorthogonal chemistry, specific lipid reporters, and spectroscopy should provide important new insight into the functional roles of lipidated proteins and membranes in biology.     

Staudinger Ligation and Reactions – From Bioorthogonal Labeling to Next-Generation Biopharmaceuticals

In this review, we highlight groundbreaking discoveries and applications of Staudinger reactions in the molecular life sciences, starting from the engineering of the Staudinger ligation as a bioorthogonal reaction until most recent applications in modern bioconjugation methods to generate next-generation biopharmaceuticals. Bioorthogonal reactions refer to a set of chemoselective transformations in biological environments able to take place in presence of naturally occurring functional groups. The Staudinger ligation set a new paradigm of such transformations, resulting in the development of various labeling and bioconjugation strategies for the modification of (bio-)molecules of interest.     

Phosphine-Activated Lysine Analogues for Fast Chemical Control of Protein Subcellular Localization and Protein SUMOylation

The Staudinger reduction and its variants have exceptional compatibility with live cells but can be limited by slow kinetics. Herein we report new small-molecule triggers that turn on proteins through a Staudinger reduction/self-immolation cascade with substantially improved kinetics and yields. We achieved this through site-specific incorporation of a new set of azidobenzyloxycarbonyl lysine derivatives in mammalian cells. This approach allowed us to activate proteins by adding a nontoxic, bioorthogonal phosphine trigger. We applied this methodology to control a post-translational modification (SUMOylation) in live cells, using native modification machinery. This work significantly improves the rate, yield, and tunability of the Staudinger reduction-based activation, paving the way for its application in other proteins and organisms.     

Cycloadditions of Trans-Cyclooctenes and Nitrones as Tools for Bioorthogonal Labelling

Trans-cyclooctenes (TCOs) represent interesting and highly reactive dipolarophiles for organic transformations including bioorthogonal chemistry. Herein we show that TCOs react rapidly with nitrones and that these reactions are bioorthogonal. Kinetic analysis of acyclic and cyclic nitrones with strained-trans-cyclooctene (s-TCO) shows fast reactivity and demonstrates the utility of this cycloaddition reaction for bioorthogonal labelling. Labelling of the bacterial peptidoglycan layer with unnatural d -amino acids tagged with nitrones and s-TCO-Alexa488 is demonstrated. These new findings expand the bioorthogonal toolbox, and allow TCO reagents to be used in bioorthogonal applications beyond tetrazine ligations for the first time and open up new avenues for bioorthogonal ligations with diverse nitrone reactants.     

Bioorthogonal chemistry for site-specific labeling and surface immobilization of proteins

Understanding protein structure and function is essential for uncovering the secrets of biology, but it remains extremely challenging because of the high complexity of protein networks and their wiring. The daunting task of elucidating these interconnections requires the concerted application of methods emerging from different disciplines. Chemical biology integrates chemistry, biology, and pharmacology and has provided novel techniques and approaches to the investigation of biological processes. Among these, site-specific protein labeling with functional groups such as fluorophors, spin probes, and affinity tags has greatly facilitated both in vitro and in vivo studies of protein structure and function. Bioorthogonal chemical reactions, which enable chemo- and regioselective attachment of small-molecule probes to proteins, are particularly attractive and relevant for site-specific protein labeling. The introduction of powerful labeling techniques also has inspired the development of novel strategies for surface immobilization of proteins to create protein biochips for in vitro characterization of biochemical activities or interactions between proteins. Because this process requires the efficient immobilization of proteins on surfaces while maintaining structure and activity, tailored methods for protein immobilization based on bioorthogonal chemical reactions are in high demand. In this Account, we summarize recent developments and applications of site-specific protein labeling and surface immobilization of proteins, with a special focus on our contributions to these fields. We begin with the Staudinger ligation, which involves the formation of a stable amide bond after the reaction of a preinstalled azide with a triaryl phosphine reagent. We then examine the Diels-Alder reaction, which requires the protein of interest to be functionalized with a diene, enabling conjugation to a variety of dienophiles under physiological conditions. In the oxime ligation, an oxyamine is condensed with either an aldehyde or a ketone to form an oxime; we successfully pursued the inverse of the standard technique by attaching the oxyamine, rather than the aldehyde, to the protein. The click sulfonamide reaction, which involves the Cu(I)-catalyzed reaction of sulfonylazides with terminal alkynes, is then discussed. Finally, we consider in detail the photochemical thiol-ene reaction, in which a thiol adds to an ene group after free radical initiation. Each of these methods has been successfully developed as a bioorthogonal transformation for oriented protein immobilization on chips and for site-specific protein labeling under physiological conditions. Despite the tremendous progress in developing such transformations over the past decade, however, the demand for new bioorthogonal methods with improved kinetics and selectivities remains high.     

Well-defined dibenzocyclooctyne end functionalized polymers from atom transfer radical polymerization

The dibenzocyclooctyne end functionalized agent 1 was designed as atom transfer radical polymerization (ATRP) initiator. The ATRP was then explored on three types of monomers widely used in free radical polymerization: methyl methacrylate, styrene, and acrylates (n-butyl acrylate and tert-butyl acrylate). The living polymerization behaviors were obtained for the methyl methacrylate and styrene monomers. The SPAAC click reactivity of dibenzocyclooctyne end group were demonstrated by successfully reacting with azide functionalized small chemical agents and polymers. Various topological polymers such as block and brush polymers were produced from strain-promoted azide-alkyne cycloaddition reaction (SPAAC) using the resultant dibenzocyclooctyne end functionalized poly(methyl methacrylate)/polystyrene as building blocks. For the acrylates, however, the polymerization did not hold the living characteristics with the dibenzocyclooctyne end functionalized ATRP initiator 1.     

Azide: A Unique Dipole for Metal‐Free Bioorthogonal Ligations

Covalently bound azide on a (small) organic molecule or a (large) biomolecular structure has proven an important handle for bioconjugation. Azides are readily introduced, small, and stable, yet undergo smooth ligation with a range of reactive probes under mild conditions. In particular, the potential of azides to undergo metal‐free reactions with strained unsaturated systems has inspired the development of an increasing number of reactive probes, which are comprehensively summarized here. For each individual probe, the synthetic preparation is described, together with reaction kinetics and the full range of applications, from materials science to glycoprofiling. Finally, a qualitative and quantitative comparison of azido‐reactive probes is provided.     

Step-growth polymerization and ‘click’ chemistry: The oldest polymers rejuvenated

Since its discovery in 2001, copper catalyzed azide-alkyne ‘click’ chemistry has been extensively used in polymer chemistry to modify polymeric materials and create advanced polymer structures by efficient coupling reactions. Surprisingly, the contribution of this Huisgen cycloaddition reaction to industrially important commodity polymers, prepared by step-growth polymerization, was not existing until recently. Nevertheless, since many decades academic and industrial research was focused on finding attractive synthetic pathways to introduce large contents of different reactive functional groups in several polymer classes such as polyesters and polyurethanes. Because of the high tolerance of azide-alkyne coupling reactions to a wide variety of functional groups and to extreme reaction conditions often used in step-growth polymerizations, the straightforward synthesis of alkyne-containing building blocks created an ideal platform to modify and broaden the physico-chemical properties of step-growth polymers by choosing readily available low and high molecular weight azide components. This feature article provides a comprehensive review covering the strategies toward ‘click’-functionalization of several classes of industrially important step-growth polymers.     

Exploiting the substrate tolerance of farnesyltransferase for site-selective protein derivatization

The site-selective modification of proteins with a functional group is an important biochemical technique, but covalent attachment of a desired group to a chosen site is complicated by the reactivity of other amino acid side chains, often resulting in undesired side reactions. One potential solution to this problem involves exploiting the activity of protein-modifying enzymes that recognize a defined protein sequence. Protein farnesyltransferase (FTase) covalently attaches an isoprenoid moiety to a cysteine unit in the context of a short C-terminal sequence that can be easily grafted onto recombinant proteins. Here we describe the synthesis of four phosphoisoprenoids functionalized with biotin, azide, or diene groups. These phosphoisoprenoids bound to FTase with affinities comparable to that of the native substrate. With the exception of the biotin-functionalized analogue, all the phosphoisoprenoids generated could be transferred to peptide and protein substrates by FTase. Unlike proteins modified with farnesyl moieties, Ypt7 prenylated with (2E,6E)-8-(azidoacetamido)-3,7-dimethylocta-2,6-dienyl groups did not oligomerize and showed no detectable increase in hydrophobicity. To assess the suitability of the functionalized isoprenoids for protein modifications they were further derivatized, both by Diels-Alder cycloaddition with 6-maleimidohexanoic acid and by Staudinger ligation with a phosphine. We demonstrate that the Staudinger ligation proceeds more rapidly and is more efficient than the Diels-Alder cycloaddition. Our data validate the use of FTase as a protein-modification tool for biochemical and biotechnological applications.     

An Azide‐Modified Nucleoside for Metabolic Labeling of DNA

Metabolic incorporation of azido nucleoside analogues into living cells can enable sensitive detection of DNA replication through copper(I)‐catalyzed azide–alkyne cycloaddition (CuAAC) and strain‐promoted azide–alkyne cycloaddition (SPAAC) “click” reactions. One major limitation to this approach is the poor chemical stability of nucleoside derivatives containing an aryl azide group. For example, 5‐azido‐2′‐deoxyuridine (AdU) exhibits a 4 h half‐life in water, and it gives little or no detectable labeling of cellular DNA. In contrast, the benzylic azide 5‐(azidomethyl)‐2′‐deoxyuridine (AmdU) is stable in solution at 37 °C, and it gives robust labeling of cellular DNA upon addition of fluorescent alkyne derivatives. In addition to providing the first examples of metabolic incorporation into and imaging of azide groups in cellular DNA, these results highlight the general importance of assessing azide group stability in bioorthogonal chemical reporter strategies.     

New poly(acrylic acid) containing segmented copolymer structures by combination of “click” chemistry and atom transfer radical polymerization

In this paper, the combination of atom transfer radical polymerization (ATRP) of 1-ethoxyethyl acrylate (EEA) and the copper(I) catalyzed “click” 1,3-dipolar cycloaddition reaction of azides and terminal alkynes was evaluated as a method to synthesize diverse amphiphilic copolymer structures. Using the 1-ethoxyethyl protecting group strategy, the application field was broadened with the synthesis of complex polymer structures containing poly(acrylic acid) (PAA) segments. A modular approach has been used: polymers with alkyne functionalities as well as azide functionalities have been synthesized. These polymers were subsequently “clicked” together to yield block copolymers. Furthermore, graft copolymers were synthesized by grafting alkyne-containing polymers onto a polymer backbone with multiple azide functions using the combination of ATRP and “click” reactions.     

pH-responsive biodegradable amphiphilic networks

Copper-mediated azide-alkyne Huisgen's 1,3-dipolar cycloaddition is a “click” reaction that was successfully used to prepare pH-responsive, amphiphilic and biodegradable networks. Indeed, this reaction proved to be very efficient in the “one pot” grafting of amino alkyne onto azide containing poly(?-caprolactone) and the cross-linking of these chains by α,ω-dialkynyl poly(ethylene oxide). The pH-controlled release of guests hosted during the cross-linking step was illustrated with an entrapped model dye.     

Tetrazine-Triggered Release of Carboxylic-Acid-Containing Molecules for Activation of an Anti-inflammatory Drug

In addition to its use for the study of biomolecules in living systems, bioorthogonal chemistry has emerged as a promising strategy to enable protein or drug activation in a spatially and temporally controlled manner. This study demonstrates the application of a bioorthogonal inverse electron-demand Diels–Alder (iEDDA) reaction to cleave trans-cyclooctene (TCO) and vinyl protecting groups from carboxylic acid-containing molecules. The tetrazine-mediated decaging reaction proceeded under biocompatible conditions with fast reaction kinetics (<2 min). The anti-inflammatory activity of ketoprofen was successfully reinstated after decaging of the nontoxic TCOprodrug in live macrophages. Overall, this work expands the scope of functional groups and the application of decaging reactions to a new class of drugs.     

KO~tBu促进的有机叠氮和末端炔烃的环加成反应改进研究

文章系统研究了叔丁醇钾促进下芳基叠氮和取代苯乙炔的环加成反应。在等摩尔数的叔丁醇钾作用下,当溶剂为DMF时,反应效果最佳,产率高达92%。其他的芳基叠氮和苯乙炔的反应研究结果表明,芳基叠氮和苯乙炔在此环加成反应中具有良好的反应适应性。     

Contribution of “click chemistry” to the synthesis of antimicrobial aliphatic copolyester

A straightforward strategy is proposed to impart antimicrobial properties to biodegradable poly(oxepan-2-one) (poly(?-caprolactone) or PCL), which is based on the grafting of pendant ammonium salts by “click” chemistry. First, statistical copolymerization of 3-chlorooxepan-2-one (α-chloro-?-caprolactone or αCl?CL) with oxepan-2-one (?-caprolactone or ?CL) was initiated by 2,2-dibutyl-2-stanna-1,3-dioxepane (DSDOP). In a second step, pendant chlorides were converted into azides by reaction with sodium azide (NaN₃). Finally, quaternary ammonium containing alkynes were quantitatively added to the pendant azide groups of PCL by the copper-catalyzed Huisgen's 1,3-dipolar cycloaddition, which is a typical “click” reaction. An alternative two-step strategy based on the cycloaddition of the amine containing alkyne onto the pendant azides, followed by quaternization turned out to be less efficient. The antimicrobial activity was analyzed by the “shaking flask method” in the presence of Escherichia coli (E. coli).     

Quick Click: The DNA‐Templated Ligation of 3′‐O‐Propargyl‐ and 5′‐Azide‐Modified Strands Is as Rapid as and More Selective than Ligase

The copper(I)‐mediated azide–alkyne cycloaddition (CuAAC) of 3′‐propargyl ether and 5′‐azide oligonucleotides is a particularly promising ligation system because it results in triazole linkages that effectively mimic the phosphate–sugar backbone of DNA, leading to unprecedented tolerance of the ligated strands by polymerases. However, for a chemical ligation strategy to be a viable alternative to enzymatic systems, it must be equally as rapid, as discriminating, and as easy to use. We found that the DNA‐templated reaction with these modifications was rapid under aerobic conditions, with nearly quantitative conversion in 5 min, resulting in a k_obs value of 1.1 min^?1, comparable with that measured in an enzymatic ligation system by using the highest commercially available concentration of T4 DNA ligase. Moreover, the CuAAC reaction also exhibited greater selectivity in discriminating C:A or C:T mismatches from the C:G match than that of T4 DNA ligase at 29 °C; a temperature slightly below the perfect nicked duplex dissociation temperature, but above that of the mismatched duplexes. These results suggest that the CuAAC reaction of 3′‐propargyl ether and 5′‐azide‐terminated oligonucleotides represents a complementary alternative to T4 DNA ligase, with similar reaction rates, ease of setup and even enhanced selectivity for certain mismatches.     

A need for speed: genetic encoding of rapid cycloaddition chemistries for protein labelling in living cells

Rapid reactions: Several reactants for strain-promoted cycloaddition reactions have been genetically encoded as the side chains of noncanonical amino acids. This results in decisive improvements for the fluorescent labelling of intracellular proteins such as quantitative turnover, completion of labelling reactions within minutes, fluorogenic effects and even partial orthogonality for multicolour labelling.     

Correlation of Structure and Sensitivity in Inorganic Azides III. A Mechanistic Interpretation of Impact Sensitivity Dependency on Non‐bonded Nitrogen to Nitrogen Distance

Previously established correlations between impact sensitivity and minimum, non‐bonded, nitrogen to nitrogen distances in inorganic azides, imply that there must be a mechanism in operation, which can predict the non reaction of alkali metal azides and the violent decomposition of copper, silver, and lead azides. This paper examines the molecular orbitals used for bonding in the azides. The orbital energy level diagram indicates that the highest occupied molecular orbital, HOMO, are two, π type, non‐bonded orbitals, each occupied by an electron pair. The electron density lobes for these π type orbitals protrude into the space beyond both ends of the azide ion. These orbitals can overlap with ‘p’ and ‘d’ type orbitals on the metal cation, facilitating the transfer of the electron back to the metal cation; an integral part of the decomposition reaction. If an exciton is generated on the azide ion, the electron can migrate, via the extended three center MO, to the metal cation, leaving the positive hole on the terminal nitrogen atom. A similar hole on an adjacent azide, would allow the non‐bonding orbitals on each azide to interact. As the distance between neighboring azide ions decreases, it is postulated that these, non‐bonding, π type orbitals start to overlap and become bonding orbitals between adjacent azide ions. This process forms an unstable N₆ moiety, which leads to the formation of three nitrogen molecules from two original azide ions. Thus, a feasible mechanism for the reaction can explain the observation that azides with non‐bonded nitrogen to nitrogen distances of >300 nm do not show impact sensitivity but, as this distance decreases below 300 nm, the sensitivity increases. The non impact sensitive azides could respond to thermal stimulus, which increases the thermal motion, thus reducing the critical nitrogen to nitrogen non‐bonded distance and reducing the energy for exciton production. Further work is required on the energy changes for such a reaction.