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ChemInform Abstract: Bioorthogonal Chemistry: Strategies and Recent Developments
Abstract
The use of covalent chemistry to track biomolecules in their native environment-a focus of bioorthogonal chemistry-has received considerable interest recently among chemical biologists and organic chemists alike. To facilitate wider adoption of bioorthogonal chemistry in biomedical research, a central effort in the last few years has been focused on the optimization of a few known bioorthogonal reactions, particularly with respect to reaction kinetics improvement, novel genetic encoding systems, and fluorogenic reactions for bioimaging. During these optimizations, three strategies have emerged, including the use of ring strain for substrate activation in the cycloaddition reactions, the discovery of new ligands and privileged substrates for accelerated metal-catalysed reactions, and the design of substrates with pre-fluorophore structures for rapid "turn-on" fluorescence after selective bioorthogonal reactions. In addition, new bioorthogonal reactions based on either modified or completely unprecedented reactant pairs have been reported. Finally, increasing attention has been directed toward the development of mutually exclusive bioorthogonal reactions and their applications in multiple labeling of a biomolecule in cell culture. In this feature article, we wish to present the recent progress in bioorthogonal reactions through the selected examples that highlight the above-mentioned strategies. Considering increasing sophistication in bioorthogonal chemistry development, we strive to project several exciting opportunities where bioorthogonal chemistry can make a unique contribution to biology in the near future.
... [1][2][3][4][5][6][7][8][9] To be effective in vivo, and hence maximise the clinical impact of the approach, it is essential that the reactions proceed at relatively fast reaction rates under physiological conditions. [10,11] Moreover, the bioorthogonal moieties must be nontoxic and stable in vivo. [12,13] As a consequence of these challenges, fewer in vivo applications of bioorthogonal reactions have been reported compared with in vitro applications. ...
... Many excellent reviews detail the types of bioorthogonal reactions developed so far, [11,[14][15][16] and their in vitro applications, with a focus on diagnosis [17] and therapy. [18] Recent bioorthogonal chemistry strategies to image and detect biomolecules in intracellular compartments have also been reviewed. ...
... [29][30][31] Over the past 20 years, bioorthogonal chemistry has continued to facilitate many significant advances in chemical biology for example by allowing biomolecules to be studied in their native environment. [10,11,[14][15][16]32] Herein we discuss and critically appraise the bioorthogonal reactions that are reported to be successfully used in vivo, and provide the context for the development by highlighting the key milestones in the development of each reaction. ...
Bioorthogonal chemistry involves selective biocompatible reactions between functional groups that are not normally present in biology. It has been used to probe biomolecules in living systems, and has advanced biomedical strategies such as diagnostics and therapeutics. In this review, the challenges and opportunities encountered when translating in vitro bioorthogonal approaches to in vivo settings are presented, with a focus on methods to deliver the bioorthogonal reaction components. These methods include metabolic bioengineering, active targeting, passive targeting, and simultaneously used strategies. The suitability of bioorthogonal ligation reactions and bond cleavage reactions for in vivo applications is critically appraised, and practical considerations such as the optimum scheduling regimen in pretargeting approaches are discussed. Finally, we present our own perspectives for this area and identify what, in our view, are the key challenges that must be overcome to maximise the impact of these approaches.
... Biological processes in the living systems are extremely complicated but are also largely needed for the unveiling in natural and biomedical exploration conditioning. To study the molecular details of natural processes active natural examinations toward these processes ate needed, for which colourful bioconjugation strategies are largely-demanded and constructed to develop these examinations [1][2][3][4][5]. Monoclonal antibodies and inheritable Fluorescent protein mixtures represent typical natural strategies for bioconjugation and explication on the places of specific proteins in dynamic cellular mechanisms. ...
Bioorthogonal chemistry represents a class of high-yielding chemical reactions that proceed rapidly and selectively in biological environments without side reactions towards endogenous functional groups.Bioorthogonal chemistry represents a class of high-yielding chemical reactions that proceed rapidly and selectively in biological environments without side reactions towards endogenous functional groups. Bioorthogonal chemistry allows organic synthesis ordinarily performed in a laboratory to be performed in living organisms and cells. Thus it helps in increasing the sustainability of the environment. Bioorthogonal chemistry is a set of methods using the chemistry of non-native functional groups to explore and understand biology in living organisms.
... The driving force behind the development of bioorthogonal chemistry lies in the need for chemical tools that can seamlessly with the complexity of living systems. Traditional chemical reactions often suffer from limitations such as low selectivity, poor biocompatibility, and interference with cellular processes [18]. The journey of bioorthogonal chemistry began with the introduction of Staudinger ligation, a reaction that laid the groundwork for subsequent advances in the field [19]. ...
Bioorthogonal chemistry has emerged as a pivotal field in molecular science, offering transformative tools for applications in drug discovery, imaging, and molecular biology. This review provides a comprehensive analysis of recent advancements in bioorthogonal chemistry, emphasizing key innovations, practical applications, and future research directions. We explore state-of-the-art bioorthogonal reactions, including Staudinger ligation, strain-promoted azide-alkyne cycloaddition (SPAAC), and tetrazine ligation, detailing their mechanisms, advantages, and limitations. The review highlights significant innovations such as novel fluorogenic probes, improved catalysts, and enhanced reaction conditions that have expanded the utility and efficiency of these reactions. Practical applications are examined, showing how these advances have revolutionized fields like live-cell imaging, targeted drug delivery, and molecular labeling. Looking to the future, we discuss emerging trends and potential research avenues, including the integration of bioorthogonal chemistry with other advanced technologies and the development of new reaction methodologies. This review provides a detailed overview of the current state of bioorthogonal chemistry and outlines its future potential, serving as a valuable resource for researchers and practitioners in the field.
... Furthermore, from this perspective, the formed probe-protein covalent conjugate shares many features with naturally occurring protein post-translational modifications (PTMs) offering productive exchange of the techniques and experimental setups used in both fields ( Figure 2B) [33,34]. The second prerequisite required for a probe used in a chemical proteomic study is an embedded bioorthogonal handle, for example a terminal alkyne or azide, which is able to react chemoselectively with a tag facilitating unambiguous identification by a selected analytical technique, for example LC-MS/MS ( Figure 1) [35][36][37]. A covalent bond between a probe and a protein is needed because of denaturing conditions used during the analysis. ...
Identification of interactions between proteins and natural products or similar active small molecules is crucial for understanding of their mechanism of action on a molecular level. To search elusive, often labile, and low-abundant conjugates between proteins and active compounds, chemical proteomics introduces a feasible strategy that allows to enrich and detect these conjugates. Recent advances in mass spectrometry techniques and search algorithms provide unprecedented depth of proteome coverage and the possibility to detect desired modified peptides with high sensitivity. The chemical ‘linker’ connecting an active compound–protein conjugate with a detection tag is the critical component of all chemical proteomic workflows. In this review, we discuss the properties and applications of different chemical proteomics linkers with special focus on their fragmentation releasing diagnostic ions and how these may improve the confidence in identified active compound–peptide conjugates. The application of advanced search options improves the identification rates and may help to identify otherwise difficult to find interactions between active compounds and proteins, which may result from unperturbed conditions, and thus are of high physiological relevance.
... While affinity-based protein-RNA interactions have been employed extensively, covalent linkage between proteins and RNA offers distinct advantages, such as irreversible binding and enhanced stability through direct linkage of an exposed end, protecting the molecule from exonuclease degradation. However, covalent linkage also presents unique challenges, as it often requires synthetic methods that rely on the chemical modification of surface exposed residues in order to facilitate the formation of a stable protein-RNA bond, which could inhibit the labeled protein's biological function [5][6][7][8] . ...
Replication-initiating HUH-endonucleases (Reps) are enzymes that form covalent bonds with single-stranded DNA (ssDNA) in a sequence specific manner to initiate rolling circle replication. These nucleases have been co-opted for use in biotechnology as sequence specific protein-ssDNA bioconjugation fusion partners dubbed ‘HUH-tags’. Here, we describe the engineering and in vitro characterization of a series of laboratory evolved HUH-tags capable of forming robust sequence-directed covalent bonds with unmodified RNA substrates. We show that promiscuous Rep-RNA interaction can be enhanced through directed evolution from nearly undetectable levels in wildtype enzymes to robust reactivity in final engineered iterations. Taken together, these engineered HUH-tags represent a promising platform for enabling site-specific protein-RNA covalent bioconjugation in vitro, potentially mediating a host of new applications and offering a valuable addition to the HUH-tag repertoire.
... Cysteine residues forming intramolecular disulfide bridges and thioether alkylation in thiol groups can be utilized for selective modification, particularly single-site modification, owing to the low proportion of cysteine in proteins [7,8]. By contrast, lysine residues, which show a high frequency in nature, are more popular than cysteine in multiple modification methods because of the amine group (-NH 2 ) [3]. The end products could be amide, urea, thiourea, sulfonamide, or amine following reductive amination from a Schiff base [9]. ...
During the initial phases of extracellular vesicle (EV) research, researchers faced many obstacles, including a lack of standard methods for the separation and characterization of EVs and efficient tools to study EV functions in cell-cell communication. With rapid and intensive development in the EV research field, the demand for novel methods to investigate EVs has increased. Under certain circumstances, the features of natural EVs cannot meet research or application requirements. Therefore, natural EVs must be functionalized using biological, chemical, physical, or engineering methods. In this chapter, we focus on the principle of applying chemical biology methodologies and tools to regulate or engineer EVs in order to meet future development demands. In particular, the bioorthogonal reaction in click chemistry, small-molecule modulators, polypeptide probes, and posttranslational and epigenetic modifications have potential applications in the modulation of EVs. Chemical biology is a subdiscipline that utilizes chemical techniques to solve biological problems. In general, chemical biology in EV research is suitable for precise sensing, labeling, and regulation of EVs. In this section, we discuss the principles, methods, and technologies of different EV fields and provide examples of applications of chemical biology in EV research.
... [29,30] These reactions have been successfully employed as a bioorthogonal click reaction that efficiently and selectively occurs in molecularly crowded biological environments. [31][32][33][34][35] Consequently, tetrazine-based fluorogenic dyes have been extensively developed, with significant efforts aiming at enhancing their emission enhancement ratio (I AC /I BC , where I AC and I BC are the photoluminescence intensities after and before click reactions) of the post-click product. To achieve this, amplifying the emission-quenching efficiency of the tetrazine unit is of utmost importance. ...
Fluorescence imaging, a key technique in biological research, frequently utilizes fluorogenic probes for precise imaging in living systems. Tetrazine is an effective emission quencher in fluorogenic probe designs, which can be selectively damaged upon bioorthogonal click reactions, leading to considerable emission enhancement. Despite significant efforts to increase the emission enhancement ratio ( I AC / I BC ) of tetrazine‐functionalized fluorogenic probes, the influence of molecular aggregation on the emission properties has been largely overlooked in these probe designs. In this study, we reveal that an ultrahigh I AC / I BC can be realized in the aggregate system when tetrazine is paired with aggregation‐induced emission (AIE) luminogens. Tetrazine amplifies its quenching efficiency upon aggregation and drastically reduce background emissions. Subsequent click reactions damage tetrazine and trigger significant AIE, leading to considerably enhanced I AC / I BC . We further showcase the capability of these ultra‐fluorogenic systems in selective imaging of multiple organelles in living cells. We term this unique fluorogenicity of AIE luminogen‐quencher complexes with amplified dark‐bright states as “Matthew effect” in aggregate emission, potentially providing a universal approach to attain ultrahigh I AC / I BC in diverse fluorogenic systems.
... The iEDDA reaction has been successfully employed as a bioorthogonal reaction that can selectively occur in the molecularly crowded environment inside biological systems. 23 Consequently, tetrazine-based fluorogenic dyes have been extensively developed, with significant efforts aiming at enhancing their emission enhancement ratio (IAC/IBC, where IAC and IBC are the photoluminescence intensities after and before click reactions) by increasing tetrazine quenching efficiency. For example, initial studies started in the last decade focused on Förster Resonance Energy Transfer (FRET) 24,25 or through-bond energy transfer (TBET) 26,27 to direct n-π* emission quenching and ultrahigh IAC/IBC (>1000-fold) was achieved in blue to green probes via connecting fluorophore and tetrazine in proximity distance. ...
Fluorescent imaging, a key technique in life science research, frequently utilizes fluorogenic probes for precise imaging in living systems. Tetrazine is an effective emission quencher in the design of fluorogenic probes, which can be selectively damaged upon bioorthogonal click reactions, leading to considerable emission enhancement. Despite significant efforts to increase the emission enhancement ratio (IAC/IBC) of tetrazine-functionalized fluorogenic probes, the influence of molecular aggregation on the emission properties has been largely overlooked in the design of these probes. In this study, we reveal that an ultrahigh IAC/IBC can be realized in the aggregate system when tetrazine is paired with aggregation-induced emission luminogens (AIEgens). Tetrazine can increase its quenching efficiency upon aggregation and drastically reduce background emissions. Subsequent click reactions damage tetrazine and trigger significant AIE, leading to considerably enhanced IAC/IBC. We further showcase the capability of these ultra-fluorogenic systems in selective imaging of multiple organelles in living cells. We propose the term "Matthew Effect in Aggregate Emission" to describe the unique fluorogenicity of these probes, potentially providing a universal approach to attain ultrahigh emission enhancements in diverse fluorogenic aggregate systems.
... Bioorthogonal reporters have occupied an immense space parallel to bioconjugation in chemical biology, drug discovery, and cell biology. [1] Notably, bioorthogonal reactions have facilitated research on understanding cellular biochemical [2,3] and metabolic pathways, [4,5] drug discovery, [6] disease diagnosis and treatment, [7,8] and biomaterial applications. [9,10] A bioorthogonal reaction minimally interferes with the biological process and follows stringent criteria. ...
Finding an ideal bioorthogonal reaction that responds to a wide range of biological queries and applications is of great interest in biomedical applications. Rapid diazaborine (DAB) formation in water by the reactions of ortho‐carbonyl phenylboronic acid with α‐nucleophiles is an attractive conjugation module. Nevertheless, these conjugation reactions demand to satisfy stringent criteria for bioorthogonal applications. Here we show that widely used sulfonyl hydrazide (SHz) offers a stable DAB conjugate by combining with ortho‐carbonyl phenylboronic acid at physiological pH, competent for an optimal biorthogonal reaction. Remarkably, the reaction conversion is quantitative and rapid (k2>10³ M⁻¹ s⁻¹) at low micromolar concentrations, and it preserves comparable efficacy in a complex biological milieu. DFT calculations support that SHz facilitates DAB formation via the most stable hydrazone intermediate and the lowest energy transition state compared to other biocompatible α‐nucleophiles. This conjugation is extremely efficient on living cell surfaces, enabling compelling pretargeted imaging and peptide delivery. We anticipate this work will permit addressing a wide range of cell biology queries and drug discovery platforms exploiting commercially available sulfonyl hydrazide fluorophores and derivatives.
... into X-yne click polymerizations, such as strain-promoted alkyne-nitrone cycloaddition, [132] strain-promoted alkynenitrile oxide cycloadditions, [133] inverse electron demand Diels-Alder reaction, [134][135][136][137] Diels-Alder reaction, [138] and sydnone-alkyne cycloaddition reaction. [139][140][141] Current Xyne click polymerizations are primarily proceeded based on symmetric monomers that contain two or more identical functional groups. ...
Alkyne‐based click polymerizations have been nurtured into a powerful synthetic technique for the preparation of new polymers with advanced structures and versatile properties. Among them, the emerging thiol‐yne, hydroxyl‐yne, and amino‐yne click polymerizations have made remarkable progress from reactions to applications. These polymerizations avoid the usage of inherently dangerous monomers and are safer to operate than the classical azide‐alkyne click polymerization (AACP), making them more prospective for diverse applications. To greatly promote the new alkyne‐based click polymerizations beyond AACP, a new concept of “X‐yne click polymerization” is proposed to unify them, where “X” denotes the monomers that can react with alkynes under mild reaction conditions, including thiols, alcohols, amines, and other promising ones. In this review, we mainly present a brief account of the progress of X‐yne click polymerization and discuss in detail the challenges and opportunities in this field.
... To optimise the cyclooctyne structure, electron-withdrawing groups were used to ensure lower energy of the alkyne's lowest unoccupied molecular orbital [69,74]. Here, sp 2 -hybridised atoms on the cyclooctyne ring increased the reaction rate between azides and cyclooctyne [69,75]. Notably, research from Gold et al., introduced the importance of electronic activation between the azide and cyclooctyne, affecting the stabilisation and reaction rate [69,76]. ...
Abstract
Vaccination is the most effective intervention for infectious diseases. Immunization with pathogen-derived antigens stimulates the adaptive immune response, protecting against infections or cancers. Subunit vaccines, consisting of single or multiple microbial components (e.g. peptide, protein), are safer than traditional vaccines, reducing the risk of autoimmune and allergic reactions through their refined formulations. Synthetic chemical methods combined with recombinant engineering are involved in the bulk production of antigens economically. However, poor immunogenicity of subunit vaccines requires conjugation with potent adjuvants and drug delivery agents for the induction of strong humoral and cellular immunity. Bioconjugation, a technique which includes at least one antigenic biomolecule (e.g. carbohydrate, proteins, peptides, or nucleic acid) is conjugated with an adjuvant leading to a stimulation of a robust and antigen-specific immune response. Discussed here are some of the common bioconjugation constituents used to enhance the immunogenicity of subunit vaccines.
Keywords
Click reactionsImmune responseLipidsLipopeptidesPeptidesPolymeric materialsSaponin-based adjuvantsToll-like receptors
... To this effect, bioorthogonal chemistry has long been utilized to covalently attach probes in a way that does not exhibit cross reactivity with the natural system. [19][20][21][22] Metabolic incorporation of functionalized sugar analogs, so called metabolic oligosaccharide engineering (MOE), 23,24 followed by tagging them with analytical tools has previously been established to analyze glycoproteins. 25,26 To this end, bioorthogonal probes equipped with fluorophores or affinity tags have been developed to analyze glycans and glycoproteins in applications, such as live cell imaging 27 or glycoproteomics. ...
Bioorthogonal chemistry is a well-established concept for tagging and analyzing targets of interest even in living cells, tissue or organisms. In particular glycans, which are, as a posttranslational modification, not amenable to genetic engineering, became analytically accessible through the establishment of metabolic oligosaccharide engineering and subsequent bioorthogonal tagging of chemical probes. Since many essential cellular processes involve glycoproteins, it is not surprising that especially aberrant glycosylation has been associated with the pathology of many diseases. Investigation of aberrant glycosylation in a disease background is complicated by the heterogeneity of glycans and dynamic changes in their composition. In order to create a meaningful information depth, it can be beneficial to analyze the same sample with different analytical methods. This becomes even more relevant for samples with limited access. Most of the currently existing bioorthogonal probes are designed for use in only one type of experiment. These design restrictions are mainly based on the limited synthetic accessibility of more complex bioorthogonal probes. Multi-step syntheses are often time consuming and cost-inefficient. Here, we introduce a fast and easily manageable strategy for the synthesis of complex bioorthogonal probes that allow an application in multiple coordinated experiments. Using established principles and conditions of solid-phase peptide synthesis, we combine different functional building blocks to generate multi-functional bioorthogonal probes (named Multi-Tags). We show the easy synthesis of several multi-modal probes and demonstrate their applicability and versatility in exemplary assays.
... One approach that has been gaining traction in the study of carbohydrate and lipid studies has been the use of so-called bioorthogonal reagents in combination with cell-compatible click chemistry [18][19][20] . ...
System level analysis of single cell data is rapidly transforming the field of immunometabolism. However, metabolic profiling of single cells and small populations by flow and mass cytometry is extremely limited by the availability of specific reagents such as antibodies and fluorescently nutrient analogues. Given the competitive demand for nutrients in pathogenic microenvironments including sites of infection, tumours and autoinflammation, there is a need to understand how and when immune cells access these nutrients. Fluorescent-tagging of nutrients is one approach to study nutrient transport but is extremely limited in its usefulness as tagging usually changes the transport characteristics and transporter specificity of the nutrient. Herein, we developed a completely new approach for single cell analysis of nutrient uptake where a fluorophore is attached to a functionalized amino acid after it has been transported across the plasma membrane and is within the cell. This in-cell biorthogonal labelling ensures that bona fide transport has been measured. System ASC transporter SLC1A5/ASCT2 transports multiple amino acids, most notably the crucial fuel glutamine, and has essential roles in supporting immune metabolism, signalling and function. This flow cytometry assay allows for rapid, sensitive, and quantitative measurement of SLC1A5-mediated uptake, which we used to interrogate the transport capacity of the complex immune subpopulations within the thymus, at a single cell resolution previously 'unreachable'. Taken together, our findings provide an easy procedure to assess which cells support their function via SLC1A5 mediated uptake of amino acids in a sensitive single cell assay. This assay is a significant addition to the single-cell metabolic toolbox required to decode the metabolic landscape of complex immune microenvironments
... making it more efficient when binding with TCO sites onto the surface of nanoparticles (Chang et al., 2010). Different from Cu (I)-catalyzed 1, 3dipolar cycloaddition of azides and alkynes reaction using dibenzoazacyclooctyne (Azide/DBCO), TCO/Tz reaction does not require the catalysis process with metal ions (Ramil and Lin, 2013). The previous study has demonstrated that TCO/Tz reaction was more efficient than Azide/DBCO for labeling when detecting Staphylococcus aureus (Jewett and Bertozzi, 2010). ...
Phages are uniquely suited for bacterial detection due to their low cost and ability to recognize live bacteria. Herein, our work establishes the proof-of-concept detection of Salmonella in orange juice based on a phage-mediated portable magnetic relaxation switching (MRS) biosensor. The limit of quantification (LOQ) could reach 5 CFU/mL (95% confidence interval [CI]: 4-7, N=4) with a linear range of 10²-10⁸ CFU/mL, which has improved 10-fold than that without bioorthogonal signal amplification. The recovery rate of the phage-based MRS biosensor was 95.0% (95% confidence interval [CI]: 89.0%-100.9%, N=6). The specificity of the phage-based MRS biosensor was 100% without false-positive results. In addition, this sensor was able to detect <10 CFU per 25 mL of Salmonella in orange juice with 4-h pre-enrichment. The result from the phage-based MRS biosensor is consistent with that from the standard plate count method. This sensor provides a reliable and ultrasensitive detection platform for pathogens.
... 93 The second-order reaction rates for DBCO groups with azide groups are around 1 M-1 s -1 , which are nearly one thousand times higher than a simple cyclooctyne. 94 Lanthanide luminescence shows its great potential for biomedical imaging and analyses. 68 Emission from lanthanides mostly feature much longer lifetimes (0.5-10 millisecond) than those from common organic luminophores (1-100 nanosecond) so the time-resolved detection is available which improves the signal-to-noise ratio largely. ...
This thesis presents my works in designing, preparing, and characterizing several types of novel lanthanide complexes for application in the fields of photodynamic theranostics. All the reported products are working as integrated systems on which various functions can be achieved simultaneously. Although the terms “lanthanide complexes” and “photodynamic theranostics” are relatively broad research fields. The specific interests are focused on utilizing lanthanides and their ligands in possible clinical imaging and photodynamic therapies (PDT). Lanthanides to be discussed here mainly are Eu(III), Gd(III), and Yb(III) based on their unique spectroscopic properties. The diagnostic methods to be used are molecular imaging by visible/near-infrared light and magnetic resonance imaging. The possible medical effects are justified by the generation of singlet oxygen, an active species in PDT.The first chapter provides descriptions of several topics related to the fundamental concepts or techniques applied in my projects. Although some sections do not cover the evolution of all theories or the operation of every instrument, all key elements associated with the ensuing discussion are elucidated. To be specific, the origin of special properties of lanthanides and their ligands (porphyrins) are introduced; the development of preparing/characterizing lanthanide complexes are reported; and the principles of modern photodynamic theranostics are explained.The second chapter is my first project in which a series of lanthanide-porphyrin double-decker complexes were prepared and characterized with advanced techniques (for example, scanning tunneling microscopy, transient absorption spectroscopy, and low-temperature NIR emission spectroscopy). With the strategic molecular design, the reported complexes showed advantageous theranostic performance over the previously reported analogs and some commercially available compounds.In chapter III, one ever-present goal is to explore multimodal theranostic agents with the best combination of imaging techniques and medical treatment. My effort is to introduce several porphyrin-cyclen bimetallic complexes. The ligand featuring PDT ability is capable of chelating one or two metals and reveals synergistic effects. For the gallium-gadolinium complexes, a possible PET/MRI coupled imaging ability could be achieved with singlet oxygen generation simultaneously.The last chapter is to fill the gap of bioorthogonal chemistry, a rising field investigating chemistry reactions that undergo on mild conditions and with high specificity. However, there are only a few reports concerning the instant monitor of bioorthogonal reactions by luminescent emission. Therefore, a Eu(III)-based complex was prepared which can process a typical bioorthogonal reaction, and the resulting expansion in the ligands guaranteed efficient sensitization from the chromophore to the lanthanide center, and then strong visible emission. In summary, all my three projects were proposed and finished for the purpose of investigating the potential of lanthanide complexes as photodynamic theranostic agents. Different molecular strategies have been applied to explore the optimal ligands improving both the spectroscopic performance of lanthanides and the PDT effects of porphyrins.
... Numerous chemical tagging approaches in combination with bioorthogonal chemistry enable incorporation of chemical probes into proteins in cells and organisms. 3,[15][16][17][18] Although these approaches are versatile and robust, most of these approaches rely on exogenous expression of protein of interest (POI) with genetic tags or non-canonical amino acids. 2,6,8,[19][20][21][22] The advance in selective labeling of endogenous proteins facilitates affinitybased protein proling, 23 proteomic analysis of cellular organelles and protein complexes, 24 target identication, 25 and diagnosis. ...
Chemical modification of proteins is enormously useful for characterizing protein function in complex biological systems and for drug development. Selective labeling of native or endogenous proteins is challenging owing to the existence of distinct functional groups in proteins and in living systems. Chemistry for rapid and selective labeling of proteins remains in high demand. Here we have developed novel affinity labeling probes using benzotriazole (BTA) chemistry. We showed that affinity-based BTA probes selectively and covalently label a lysine residue in the vicinity of the ligand binding site of a target protein with a reaction half-time of 28 s. The reaction rate constant is comparable to the fastest biorthogonal chemistry. This approach was used to selectively label different cytosolic and membrane proteins in vitro and in live cells. BTA chemistry could be widely useful for labeling of native/endogenous proteins, target identification and development of covalent inhibitors.
... The synthesis and methods for making these reagents and biomaterials are described in detail. Because bioorthogonal chemical reactions have emerged as excellent tools for biolabeling, [68][69][70][71][72][73][74][75] we discussed the combination between bioorthogonal reactions and diazonium salt reagents. We also outline the future directions for crosslinking of plant viruses via multi-diazonium reagents. ...
Tobacco mosaic virus (TMV) is a rod-shaped hollow plant viral nanoparticle (300 nm × 18 nm) and exhibits abundant amino acid residues on the surface of capsid proteins for facile chemical labeling. The use of TMV as a nano-template to produce materials with multiple functions has received particular attention in the past decade. In addition, TMV can be largely produced in gram-scale quantities and is also considered much safe for mammals. Hence, using TMV as building blocks to assemble biomaterials (e.g., hydrogels) has emerged as an attractive field for biomedical applications. This minireview details up-to-date research on the development of bench-stable diazonium reagents and their applications for TMV labeling and crosslinking. The strategy for the preparation of virus-based hydrogels is highlighted. We hope that this review will inspire the development of a large number of plant virus-based biomaterials for various applications in the near future.
... Click chemistry is widely used in the combination of materials science and biology, such as polymer synthesis (Voigt et al. 2019), bioconjugation (Colombo et al. 2012), and biological imaging . Because of no affection on the structure of nanoparticles, click chemistry has been developed as an excellent strategy for surface modification (Ramil and Lin 2013); among them, Cu(I)-catalyzed 1,3-dipolar cycloaddition of azides and alkynes (CuAAC) is the most popular reaction (Alonso et al. 2015). Coordination-based recognition involves amino acid-mediated, sulfhydryl compound-mediated, and ligand exchange reaction. ...
Rheumatoid arthritis is a chronic and systemic autoimmune disease with aggressive and symmetrical polyarthritis as the main clinical manifestation, which has a considerably high incidence and mortality. In clinics, traditional drug therapy often produces toxicity to gastrointestinal and immunosuppression-mediated infection, which has greatly promoted the research and application of drugs based on nanomaterials in the diagnosis and treatment of rheumatoid arthritis. In recent years, nanotechnology has shown great advantages in developing novel therapeutic and diagnostic systems in the field of inflammation-related diseases. Among them, metal nanoparticles can be applied as therapeutic agents and drug delivery vectors for the treatment of rheumatoid arthritis (RA), as well as achieve quantitative analysis of RA markers rapidly attributed to the extra optical and magnetic effects of metal nanoparticles. The purpose of this review is to introduce the application of metal nanoparticles in rheumatoid arthritis, focusing on its latest progress, challenges, and future development prospects.
... Finally, progress in bio-orthogonal chemistry and the combination with nanotechnology open exciting new possibilities for the tracking and targeted elimination of cancer cells [377][378][379][380][381]. The potential of bioorthogonal chemistry/NP combinations to overcome in vivo barriers of cancer nanomedicine have been discussed recently [379][380][381]. ...
Nanomaterials are at the forefront of health research and development. Among different nanomaterials, nanoparticles are especially promising for cancer theranostics. However, despite great potential, the clinical translation of nano-based applications continues to face obstacles. A major hurdle to the localized eradication of tumors is the efficient targeting of nanomaterials to the desired tissues and cells. In particular, nanoparticle properties and the route of administration impact the efficacy of precision nanomedicine. This review focuses on nanoparticles that have been produced for the detection and treatment of cancer. Common and tissue-specific barriers that limit the accumulation of nanoparticles in malignant tumors are discussed. The in-depth discussion focuses on the physicochemical properties of nanoparticles and the surface modifications that achieve efficient accumulation at tumor sites. Furthermore, limitations of current strategies and open questions are presented. The review concludes with an outlook on future directions and the trajectories that will drive the field forward to advance nano-oncology in the clinic.
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Azide-alkyne cycloaddition of cyclooct-2-yn-1-ol and 2-(azidophenyl)boronic acid proceeded rapidly at room temperature with complete regioselectivity to afford a triazole having a boronate ester group. The secondary covalent interaction of the...
We report quantitative details of addition reactions of a strained trans ‐cyclooctene with a series of oxadiazinones, analogues of the bioorthogonally fruitful tetrazines. Both experimental and computational studies of these ligations reveal their sensitivity to differing electronic character of oxadiazinone substituents. Achieving rate constants up to 9.5 L mol ⁻¹ s ⁻¹ , oxadiazinones demonstrate untapped potential for bioorthogonal applications.
Methods rooted in chemical biology have contributed significantly to studies of integral membrane proteins. One recent key approach has been the application of genetic code expansion (GCE), which enables the site-specific incorporation of noncanonical amino acids (ncAAs) with defined chemical properties into proteins. Efficient GCE is challenging, especially for membrane proteins, which have specialized biogenesis and cell trafficking machinery and tend to be expressed at low levels in cell membranes. Many eukaryotic membrane proteins cannot be expressed functionally in E. coli and are most effectively studied in mammalian cell culture systems. Recent advances have facilitated broader applications of GCE for studies of membrane proteins. First, AARS/tRNA pairs have been engineered to function efficiently in mammalian cells. Second, bioorthogonal chemical reactions, including cell-friendly copper-free “click” chemistry, have enabled linkage of small-molecule probes such as fluorophores to membrane proteins in live cells. Finally, in concert with advances in GCE methodology, the variety of available ncAAs has increased dramatically, thus enabling the investigation of protein structure and dynamics by multidisciplinary biochemical and biophysical approaches. These developments are reviewed in the historical framework of the development of GCE technology with a focus on applications to studies of membrane proteins.
Bioorthogonal chemistry has provided an elaborate arsenal to manipulate native biological processes in living systems. As the great advancement of nanotechnology in recent years, bioorthogonal nanozymes are innovated to tackle the challenges that emerged in practical biomedical applications. Bioorthogonal nanozymes are uniquely positioned owing to their advantages of high customizability and tunability, as well as good adaptability to biological systems, which bring exciting opportunities for biomedical applications. More intriguingly, the great advancement in nanotechnology offers an exciting opportunity for innovating bioorthogonal catalytic materials. In this comprehensive review, the significant progresses of bioorthogonal nanozymes are discussed with both spatiotemporal controllability and high performance in living systems, and highlight their design principles and recent rapid applications. The remaining challenges and future perspectives are then outlined along this thriving field. It is expected that this review will inspire and promote the design of novel bioorthogonal nanozymes, and facilitate their clinical translation.
Fluorescence‐assisted tools based on organic molecules have been extensively applied to interrogate complex biological processes in a non‐invasive manner with good sensitivity, high resolution, and rich contrast. However, the signal‐to‐noise ratio is an essential factor to be reckoned with during collecting images for high fidelity. In view of this, the wash‐free strategy is proven as a promising and important approach to improve the signal‐to‐noise ratio, thus a thorough introduction is presented in the current review about wash‐free fluorescent tools based on organic molecules. Firstly, generalization and summarization of the principles for designing wash‐free molecular fluorescent tools (WFTs) are made. Subsequently, to make the thought of molecule design more legible, a wash‐free strategy is highlighted in recent studies from four diverse but tightly binding aspects: (1) special chemical structures, (2) molecular interactions, (3) bio‐orthogonal reactions, (4) abiotic reactions. Meanwhile, biomedical applications including bioimaging, biodetection, and therapy, are ready to be accompanied by. Finally, the prospects for WFTs are elaborated and discussed. This review is a timely conclusion about wash‐free strategy in the fluorescence‐guided biomedical applications, which may bring WFTs to the forefront and accelerate their extensive applications in biology and medicine.
The bioorthogonal retro‐Cope elimination reaction of linear alkynes R3C−C≡C−X (R3 = combinations of H, MeO, F; X = H, F, Cl, Br, I) with N,N‐dimethylhydroxylamine was quantum chemically investigated using relativistic density functional theory at ZORA‐BP86/TZ2P. This novel reaction can be tuned through judicious substitution of the alkyne at both the terminal and propargylic position to render second‐order kinetics that rival and out‐compete strain‐promoted variants. Activation strain and quantitative molecular orbital analyses reveal that, both upon terminal or propargylic substitution of propyne, the main effect of substituting H for X is a lowering of the propyne LUMO which stabilizes the HOMO–LUMO interactions and thus the transition state. In the case of terminal substitution with larger halogens (X = Cl, Br, I), a secondary effect interferes: steric repulsion with these larger halogens is absorbed into a longer forming C⋯N bond leading to a more asynchronous reaction accompanied by less (not more) steric Pauli repulsion.
Although tumor models have revolutionized perspectives on cancer aetiology and treatment, current cell culture methods remain challenges in constructing organotypic tumor with in vivo‐like complexity, especially native characteristics, leading to unpredictable results for in vivo responses. Herein, we developed the bioorthogonal nanoengineering strategy (BONE) for building photothermal dynamic tumor spheroids. In this process, biosynthetic machinery incorporated bioorthogonal azide reporters into cell surface glycoconjugates, followed by reacting with multivalent click ligand (ClickRod) that was composed of hyaluronic acid‐functionalized gold nanorod carrying dibenzocyclooctyne moieties, resulting in rapid construction of tumor spheroids. We identified BONE could effectively assemble different cancer cells and immune cells together to construct heterogenous tumor spheroids. Particularly, ClickRod exhibited favourable photothermal activity, which precisely promoted cell activity and shaped physiological microenvironment, leading to formation of dynamic features of original tumor, such as heterogeneous cell population and pluripotency, different maturation levels, and physiological gradients. Importantly, BONE not only offered a promising platform for investigating tumorigenesis and therapeutic response, but improved establishment of subcutaneous xenograft model under mild photo‐stimulation, thereby significantly advancing cancer research. Therefore, we present the first bioorthogonal nanoengineering strategy for developing dynamic tumor models, which have the potential for bridging gaps between in vitro and in vivo research.
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Bioorthogonal reactions are a class of chemical reactions that can be carried out in living organisms without interfering with other reactions, possessing high yield, high selectivity, and high efficiency. Since the first proposal of the conception by Professor Carolyn Bertozzi in 2003, bioorthogonal chemistry has attracted great attention and has been quickly developed. As an important chemical biology tool, bioorthogonal reactions have been applied broadly in biomedicine, including bio-labeling, nucleic acid functionalization, drug discovery, drug activation, synthesis of antibody–drug conjugates, and proteolysis-targeting chimeras. Given this, we summarized the basic knowledge, development history, research status, and prospects of bioorthogonal reactions and their biomedical applications. The main purpose of this paper is to furnish an overview of the intriguing bioorthogonal reactions in a variety of biomedical applications and to provide guidance for the design of novel reactions to enrich bioorthogonal chemistry toolkits.
System-level analysis of single-cell data is rapidly transforming the field of immunometabolism. Given the competitive demand for nutrients in immune microenvironments, there is a need to understand how and when immune cells access these nutrients. Here, we describe a new approach for single-cell analysis of nutrient uptake where we use in-cell biorthogonal labeling of a functionalized amino acid after transport into the cell. In this manner, the bona fide active uptake of glutamine via SLC1A5/ASCT2 could be quantified. We used this assay to interrogate the transport capacity of complex immune subpopulations, both in vitro and in vivo. Taken together, our findings provide an easy sensitive single-cell assay to assess which cells support their function via SLC1A5-mediated uptake. This is a significant addition to the single-cell metabolic toolbox required to decode the metabolic landscape of complex immune microenvironments.
Self-assembled systems, like polymeric micelles, have become great facilitators for conducting organic reactions in aqueous media due to their broad potential applications in green chemistry, as well as biomedical applications. Massive strides have been taken to improve the reaction scope of such systems to the point of performing multistep synthesis of active pharmaceutical ingredients using micelles. Considering these important advancements, we sought to study the relationships between the architecture of the amphiphiles and the reactivity of their PdII loaded micellar nanoreactors in conducting depropargylation. Towards this goal, we designed and synthesized a series of isomeric polyethylene glycol (PEG)-dendron amphiphiles with different dendritic architectures but with identical degree of hydrophobicity and hydrophilic to lipophilic balance (HLB). We observed that the dendritic architecture, which serves as the main binding site for the PdII ions, has greater influence on the reactivity than the hydrophobicity of the dendron. These trends remained constant for two different propargyl containing substrates, highlighting the robustness of the obtained results. Density functional theory (DFT) calculations of simplified models of the dendritic blocks revealed the different binding modes of the various dendritic architectures to PdII ions, which could explain the difference in the reactivity of the nanoreactors in this study. Our results show how tuning the internal architecture of amphiphiles and the resulting orientation of the chelating moieties can play a major role in controlling the reactivity of the nanoreactors.
Click reactions have gained enormous popularity among chemists for their ambient reaction parameters and wide application in various frontier research fields. Photo-initiated click reactions add another dimension of spatiotemporal control which ensures fruitful bioorthogonal reaction. Several cycloaddition reactions, enlisted in the category of ‘photo click’ reactions due to the fast kinetics and ambient reaction conditions, are widely utilized by scientists for bioorthogonal conjugation. In this review, various types of 1,3-dipolar cycloaddition reactions and their applications in the field of protein bioconjugation are discussed.
Bioorthogonal chemistry is a rising field investigating chemical reactions in physiological environments with high specificity. However, only very few examples concern the real‐time monitoring of bioorthogonal reactions by luminescence or magnetic relaxivity. To fill this gap, herein, the Eu(III)‐based complex is reported as a small‐molecule optical imaging agent which shows off–on luminescence and provides quantitative analysis for the progress of the bioorthogonal reaction. The characteristic signal is achieved through efficient energy harvesting and transferring to the Eu(III) from the expansion of the conjugated system of the antenna. Moreover, the gadolinium(III) counterpart significantly enhances relaxivity in magnetic resonance imaging (MRI) after the bioorthogonal reaction since the rotational correlation time is shortened with increased molecular sizes and weights.
Extracellular vesicles have received a great interest as safe biocarriers in biomedical engineering. There is a need to develop more efficient delivery strategies to improve localized therapeutic efficacy and minimize off-target adverse effects. Here, exosome mimetics (EMs) are reported for bone targeting involving the introduction of hydroxyapatite-binding moieties through bioorthogonal functionalization. Bone-binding ability of the engineered EMs is verified with hydroxyapatite-coated scaffolds and an ex vivo bone-binding assay. The EM-bound construct provided a biocompatible substrate for cell adhesion, proliferation, and osteogenic differentiation. Particularly, the incorporation of Smoothened agonist (SAG) into EMs greatly increased the osteogenic capacity through the activation of hedgehog signaling. Furthermore, the scaffold integrated with EM/SAG significantly improved in vivo reossification. Lastly, biodistribution studies confirmed the accumulation of systemically administered EMs in bone tissue. This facile engineering strategy could be a versatile tool to promote bone regeneration, offering a promising nanomedicine approach to the sophisticated treatment of bone diseases.
In a combinatorial approach, a family of ruthenium(II) azido complexes [Ru(N3)(N∧N)(terpy)]PF6 with terpy = 2,2':6',2″-terpyridine and N∧N as a bidentate chelator derived from 2,2'-biypridine and its 4,4'-disubstituted derivatives, 2,2'-bipyrimidine, and 1,10-phenanthroline were reacted with different internal and terminal alkynes to give access to a total of 7 × 7 = 49 triazolato complexes in a room-temperature catalyst-free iClick reaction. The reactants were mixed in a repurposed high-performance liquid chromatography (HPLC) autosampler, and the reaction progress was monitored by direct injection into an electrospray mass spectrometer. The ratio of the peak intensities of [Ru(N3)(N∧N)(terpy)]+ and [Ru(triazolato)(N∧N)(terpy)]+ was converted to a colored heat map for facile visual inspection of the conversion ratio. By automated multiple injections of the reaction mixture in fixed time intervals and plotting peak intensities over reaction time, pseudo-first-order rate constants were easily determined. Finally, nonoverlapping isotope patterns of the azido starting materials and triazolato products enabled multiplexed parallel determination of rate constants for four different ruthenium(II) azido complexes from a single sample vial, thereby reducing experiment time by 75%.
Chlorpyrifos (CPF) is the most frequently found organophosphate pesticide residue in solid food samples and can cause increasing public concerns about potential risks to human health. Traditional detection signals of such small molecules are mostly generated by target-mediated indirect conversion, which tends to be detrimental to sensitivity and accuracy. Herein, a novel magnetic relaxation switching detection platform was developed for target-mediated direct and sensitive detection of CPF with a controllable aggregation strategy based on a bioorthogonal ligation reaction between tetrazine (Tz) and trans-cyclooctene (TCO) ligands. Under optimal conditions, this sensor can achieve a detection limit of 37 pg/mL with a broad linear range of 0.1-500 ng/mL in 45 min, which is approximately 51-fold lower than that of the gas chromatography analysis and 13-fold lower than that of the enzyme-linked immunosorbent assay. The proposed click chemistry-mediated controllable aggregation strategy is direct, rapid, and sensitive, indicating great potential for residue screening in food matrices.
Monoclonal antibodies (mAbs) are valuable therapeutic tools for targeted therapies to attack tumor cells but preserve healthy tissues, therefore resulting in fewer side effects. With the rise of genetic engineering, recombinant human or humanized mAbs are available in the market for the treatment of cancer, either in monotherapy, in association or conjugated with other drugs. Thus, monovalent or bispecific mAbs find applicability for their antitumor cytotoxic activity, as well as for being able to be used in cancer immunotherapy. Although some antibody-drug conjugates are commercially available, immunoconjugates with nanoparticles are less developed. In this context, nanoparticles play an important role for improving drug delivery, allowing for controlled release and site-specific delivery, both passively, through the enhanced permeation and retention effect, and actively, through the functionalization of nanoparticles with antibodies or antibody fragments with high affinity for receptors overexpressed on tumor cells. The bioconjugation can be performed mainly by adsorption or covalent binding or through the use of adapter molecules. Immobilization of antibodies on the surface of nanoparticles must ensure the desired amount of antibodies per nanoparticle and proper antibody orientation and generate a stable binding in order to preserve its biological activity. In this chapter, the main strategies for conjugating antibodies to nanoparticles through covalent bonds, such as the chemistry of carbodiimide, maleimide, and click, and non-covalent bonds such as adsorption and the biotin-avidin system will be discussed. Herein, we will address the development of monoclonal antibodies, the functionalization strategies, and antibody-receptor-targeted nanoparticles of different compositions, such as lipid, polymeric, and inorganic, focusing on their preparation techniques, physicochemical characterization, and in vitro and in vivo biological activity. Overall, the main bioconjugation technique is provided by the maleimide chemistry, particularly employed in the functionalized of lipid nanoparticles, such as liposomes, the most advanced nanosystem. Cell culture studies have revealed that the functionalized nanoparticle undergoes specific and efficient uptake via receptor-mediated endocytosis. Nanoparticle immunoconjugates have also shown promising for cancer treatment following the success of preclinical studies with cancer xenografts; however, clinical trials have yet to show efficacy and safety.KeywordsAntibodiesCancerFunctionalizationNanoparticlesTargeting
Imaging biomolecules, such as glycans, in living systems remains a formidable chemical challenge. However, significant progress has been made over the past ten years, with the ground-breaking development of the bioorthogonal chemical reporter strategy. In this context, complex biomolecules are fitted with a non-native chemical functionality (reporter) that can react selectively with a complementary bioorthogonal probe for detection. In order to image these biomolecules in real time, fluorogenic probes, non fluorescent reagents that produce highly fluorescent products, have recently been developed. This last decade, the metal-free 1,3-dipolar cycloaddition between cyclooctynes and azides have been elegantly employed for biomolecules imaging in living systems. However, azides suffer from limitations such as their reduction by endogenous cellular thiols. Consequently, we have investigated the use of more stable 1,3-dipoles as new chemical reporters for fluorescent biomolecule imaging. In addition, we also developed novel bioorthogonal fluorogenic probes with improved fluorescence properties such as high quantum yields and red-shifted fluorescence emission for potential applications in living animals.
Finding an ideal biorthogonal reaction that responds to a wide range of biological queries and applications is of great interest in biomedical applications. Rapid diazaborine (DAB) formation in water by the reactions of ortho-carbonyl phenylboronic acid with α-nucleophiles is an attractive conjugation module. Yet such reactions demand stringent criteria to satisfy biorthogonal applications. Here we show that widely used sulfonyl hydrazide (SHz) offers a stable DAB conjugate by combining with ortho-carbonyl phenylboronic acid at physiological pH, competent for an optimal biorthogonal reaction. The reaction conversion is quantitative and rapid (k2 >103 M-1s-1) at low micromolar concentrations, and it preserves comparable efficacy in a complex biological milieu. Further, DFT calculations support that SHz facilitated DAB formation runs via the lowest energy transition state and provides the most stable conjugate product while considering other biocompatible α-nucleophiles. This conjugation is extremely efficient on living cell surfaces, enabling compelling pretargeted imaging and peptide delivery. We anticipate this work will permit addressing a wide range of cell biology queries and drug discovery platforms exploiting commercially available sulfonyl hydrazide fluorophores and derivatives.
Novel approaches to targeted cancer therapy that combine improved efficacy of current chemotherapies while minimising side effects are highly sought after. The development of single-chain polymeric nanoparticles (SCPNs) as bio-orthogonal...
Chemical modification of proteins is enormously useful for characterizing protein function in complex biological systems and for drug development. Selective labeling of native or endogenous proteins is challenging owing to the existence of distinct functional groups in proteins and in living systems. Chemistry for rapid and selective labeling of proteins remains in high demand. Here we have developed novel affinity labeling probes using benzotriazole (BTA) chemistry. We showed that affinity-based BTA probes selectively and covalently label a lysine residue in the vicinity of the ligand binding site of a target protein with a reaction half-time of 28 s. The reaction rate constant is comparable to the fastest biorthogonal chemistry. This approach was used to selectively label different cytosolic and membrane proteins in vitro and in live cells. BTA chemistry could be widely useful for labeling of native/endogenous proteins, target identification and development of covalent inhibitors.
Lipid-based formulations provide a nanotechnology platform that is widely used in a variety of biomedical applications because it has several advantageous properties including biocompatibility, reduced toxicity, relative ease of surface modifications, and the possibility for efficient loading of drugs, biologics, and nanoparticles. A combination of lipid-based formulations with magnetic nanoparticles such as iron oxide was shown to be highly advantageous in a growing number of applications including magnet-mediated drug delivery and image-guided therapy. Currently, lipid-based formulations are prepared by multistep protocols. Simplification of the current multistep procedures can lead to a number of important technological advantages including significantly decreased processing time, higher reaction yield, better product reproducibility, and improved quality. Here, we introduce a one-pot, single-step synthesis of drug-loaded magnetic multimicelle aggregates (MaMAs), which is based on controlled flow infusion of an iron oxide nanoparticle/lipid mixture into an aqueous drug solution under ultrasonication. Furthermore, we prepared molecular-targeted MaMAs by directional antibody conjugation through an Fc moiety using Cu-free click chemistry. Fluorescence imaging and quantification confirmed that antibody-conjugated MaMAs showed high cell-specific targeting that was enhanced by magnetic delivery.
Deuterium labeling has been widely used for SRS metabolic imaging. It is the minimum label that causes little perturbation to the biochemical property of target molecules. In this chapter, we introduce the applications of deuterium-probed SRS metabolic imaging in living organisms. We first review recent developments of two SRS imaging techniques that enable simultaneous visualization of the metabolic dynamics of a variety of biomolecules (lipids, protein, and DNA) in living organisms. One novel technique uses D2O as the deuterium source and combines it with SRS (DO-SRS) microscopy for metabolic imaging. The other uses deuterated glucose for visualizing various newly synthesized biomolecules by spectral tracing of deuterium (STRIDE) in the living organism. Next, we overview a volumetric tissue clearing-enhanced SRS imaging technique that increases imaging depth by 10-fold. At last, we review the applications of SRS for imaging protein metabolic dynamics in mouse tissues and organs, by in vivo intracarotid injection of the deuterated amino acid (D-AA).
Stimulated Raman scattering (SRS) with vibrational imaging is gaining increasing popularity in mapping biomolecular species in living systems. Toward higher detection sensitivity, molecular specificity, and ability to track dynamic processes, SRS microscopy can be coupled with small and Raman-active vibrational probes to enable bioorthogonal chemical imaging. Many vibrational probes bearing triple bonds, conjugation systems, and stable isotopes have been developed and demonstrated. Due to the desirable features of tiny size and minimal labeling, vibrational probes are regarded as reliable Raman reporters and employed to disentangle a multitude of biological processes. This chapter reviews a series of vibrational probes reported to date for SRS imaging and their labeling strategies. Meanwhile, various studies using bioorthogonal SRS imaging in areas ranging from cell and tissue metabolism, pharmacokinetics, chemical sensing, to cell screening and phenotyping are discussed. The development of vibrational probes paved the way for a deeper understanding of complex biological systems.
Quick and clean: A method for Pd-catalyzed Suzuki-Miyaura cross-coupling to iododeoxyuridine (IdU) in DNA is described. Key to the reactivity is the choice of the ligand and the buffer. A covalent [Pd]-DNA intermediate was isolated and characterized. Photocrosslinking probes were generated to trap proteins that bind to epigenetic DNA modifications. © 2013 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Cross-metathesis (CM) has recently emerged as a viable strategy for protein modification. Here, efficient protein CM has been demonstrated through biomimetic chemical access to Se-allyl selenocysteine (Seac), a metathesis reactive amino acid substrate, via dehydroalanine. On-protein reaction kinetics reveal a rapid reaction with rate constants of Seac-mediated-CM comparable or superior to off-protein rates of many current bioconjugations. This use of Se-relayed Seac CM on proteins has now enabled reactions with substrates (allyl GlcNAc, N-allyl acetamide) that were previously not possible for the corresponding sulfur-analogue. This CM strategy was applied to histone proteins to install a mimic of acetylated lysine (KAc, an epigenetic marker). The resulting synthetic H3 was successfully recognized by antibody that binds natural H3-K9Ac. Moreover, Cope-type selenoxide elimination allowed this putative marker (and function) to be chemically expunged regenerating an H3 that can be rewritten to complete a chemically-enabled 'writer(CM)-eraser(ox)-rewrite(CM)' cycle.
Seeing the sugar coating: N-Acetyl-glucosamine and mannosamine derivatives tagged with an isonitrile group are metabolically incorporated into cell-surface glycans and can be detected with a fluorescent tetrazine. This bioorthogonal isonitrile-tetrazine ligation is also orthogonal to the commonly used azide-cyclooctyne ligation, and so will allow simultaneous detection of the incorporation of two different sugars.
Synthetic procedures for the construction of fluorogenic azido-labels were developed. Photophysical properties were elaborated by experimental and theoretical investigations. Of the newly synthesized fluorogenic and bioorthogonally applicable dyes two were selected on the basis of their fluorogenic performance and further subjected to in vitro and in vivo studies. Both tags exhibited excellent fluorogenic properties as in aqueous medium, the azide form of the selected dyes is virtually non-fluorescent, while their "clicked" triazole congeners showed intense fluorescence. One of these labels showed a very large Stokes shift. To the best of our knowledge this is the first reported case of mega-Stokes type of fluorogenic labels. These studies have justified that these two fluorogenic tags are remarkably suitable for bioorthogonal tagging schemes. The developed synthetic approach together with the theoretical screen of possible fluorogenic tags will enable the generation of libraries of such tags in the future.
Strain-promoted alkyne-nitrone cycloadditon (SPANC) was optimized as a versatile strategy for dual functionalization of peptides and proteins. The usefulness of the dual labeling protocol is first exemplified by the simultaneous introduction of a chloroquine and a stearyl moiety, two endosomal escape-improving functional groups, into the cell-penetrating peptide hLF (human lactoferrin). Additionally, we demonstrate that dual labeling of proteins is feasible by combining metal-free and copper-catalyzed click chemistry. First, SPANC is applied to enhanced green fluorescent protein to introduce both biotin and a terminal alkyne. The terminal acetylene then serves as a convenient anchor point for the CuAAC reaction with azido-containing fluorescein, thereby demonstrating the potential of combined SPANC and CuAAC for the straightforward, dual functionalization of proteins.
Building with PEGs: PEG-boronic acids, in the presence of simple Pd sources, are capable of acting as direct and effective Suzuki reagents in 70-98 % yield. When combined with non-natural amino acids, they allow efficient and direct, site-selective PEGylation of proteins at predetermined positions under biologically compatible conditions without the need for exogenous ligands.
Suzuki-Miyaura cross-coupling has been used to couple novel carbohydrate-based boronic acids, site-selectively, to the surface of E. coli at an unnatural amino acid. In this way, benign metal-catalyzed cellular switching allowed modulation of interactions with biomolecular partners via prokaryotic O-glycosylation mimics.
A systematic study of the ring-closing metathesis (RCM) of unprotected oxytocin and crotalphine peptide analogues in water is reported. The replacement of cysteine with S-allyl cysteine enables RCM to proceed readily in water containing excess MgCl(2) with 30% t-BuOH as a co-solvent. The presence of the sulfur atom is vital for efficient aqueous RCM to occur, with non-sulfur containing analogues undergoing RCM in low yields.
The heteroatom helps! The introduction of an endocyclic sulfur atom enables fine-tuning of the reactivity and stability of thiacycloalkynes for copper-free click chemistry. The stabilizing effect of the endocyclic sulfur atom allows the use of highly activated seven-membered rings as reagents for bioorthogonal copper-free click chemistry.
By simply stirring in water, organic azides and terminal alkynes are readily and cleanly converted into 1,4-disubstituted 1,2,3-triazoles through a highly efficient and regioselective copper(I)-catalyzed process (see scheme for an example).
Selective chemical reactions that are orthogonal to the diverse functionality of biological systems have become important tools in the field of chemical biology. Two notable examples are the Staudinger ligation of azides and phosphines and the Cu(I)-catalyzed [3 + 2] cycloaddition of azides and alkynes ("click chemistry"). The Staudinger ligation has sufficient biocompatibility for performance in living animals but suffers from phosphine oxidation and synthetic challenges. Click chemistry obviates the requirement of phosphines, but the Cu(I) catalyst is toxic to cells, thereby precluding in vivo applications. Here we present a strain-promoted [3 + 2] cycloaddition between cyclooctynes and azides that proceeds under physiological conditions without the need for a catalyst. The utility of the reaction was demonstrated by selective modification of biomolecules in vitro and on living cells, with no apparent toxicity.
Full details on the reactivity of the title compound as 4π component in inverse-type Diels–Alder reactions, including kinetic data, are reported. Donor-substituted alkynes, alkenes, donor-substituted and unsubstituted cycloalkenes, ketene acetals and aminals, as well as several cyclic enol ethers were used as dienophiles in these investigations. A number of 4-mono- and 4,5-disubstituted pyridazines can easily be obtained by this method.
The reactivity of more than 40 open chain and cyclic dienophiles was studied with and as electron poor dienes in inverse type DIELS-ALDER reactions.
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The cycloaddition rate of cyclooctyne to a number of N-containing heterocyclic compounds can be correlated with the reduction potentials of these dienes provided steric substituent effects are approximately equal.
A 405 nm light-activatable terthiophene-based tetrazole was designed that reacts with a fumarate dipolarophile with the second-order rate constant k2 exceeding 10(3) M(-1) s(-1). The utility of this laser-activatable tetrazole in imaging microtubules in a spatiotemporally controlled manner in live cells was demonstrated.
The poly(ethylene glycol) ester of bromo-, iodo-, and triflate-para-substituted benzoates are smoothly cross-coupled with aryl boronic acids (Suzuki reaction) under "ligandless" palladium acetate catalysis in water. The reaction proceeds without organic cosolvent under conventional thermal conditions (70 degrees C, 2 h) and under microwave irradiation (75 W, 2-4 min). The polymeric support remains stable under both reaction conditions. Whereas conventional thermal conditions induced ester cleavage (up to 45%), this side reaction is suppressed when microwave conditions are employed. Aryl nonaflates give fair yields under these conditions. Non-polymer-bound aryl halides form biaryls in good to excellent yields in water/poly(ethylene glycol) mixtures under microwave irradiation (4 min, 75 W).
Suzuki-coupling, a representative cross-coupling reaction between small molecules, can proceed on a protein surface in aqueous solution under mild conditions. The modified protein produced by the coupling reaction maintained its native like structure and function. In addition, fluorescent dye appended-protein acts as a fluorescent biosensor.
Bei der Umsetzung von Triphenyl-phosphin-methylen und seinen in der Methylengruppe substituierten Derivaten mit Aldehyden und Ketonen wird der doppelt gebundene Sauerstoff gegen die Methylenreste ausgetauscht, wobei Triphenyl-phosphinoxyd und die entsprechenden ungesättigten Verbindungen entstehen. Der mit dieser neuen Methode sich abzeichnende präparative Fortschritt liegt darin, daβ die C=C–Bindung ohne Verschiebung am Ort der ursprünglichen C=O–Bindung ausgebildet wird.
We report the facile preparation of palladacycles as storable arylpalladium(ii) reagents from acetanilides via cyclopalladation. The palladacycles exhibit good stability in PBS buffer and are capable of functionalizing a metabolically encoded HPG-containing protein, thus providing a new type of biocompatible organometallic reagent for selectively functionalizing the alkyne-encoded proteins.
Being a cyctoalkyne and a bicyclo[n.1.0]‐derivative at the same time is a unique feature of the compounds 1a and 1b. Both were generated from the corresponding tricyclic selenadiazoles. 1 is isolable, 1b can be trapped. (Figure Presented.)
Spatial and temporal control over chemical and biological processes, both in terms of "tuning" products and providing site-specific control, is one of the most exciting and rapidly developing areas of modern science. For synthetic chemists, the challenge is to discover and develop selective and efficient reactions capable of generating useful molecules in a variety of matrices. In recent studies, light has been recognized as a valuable method for determining where, when, and to what extent a process is started or stopped. Accordingly, this Minireview will present the fundamental aspects of light-induced click reactions, highlight the applications of these reactions to diverse fields of study, and discuss the potential for this methodology to be applied to the study of biomolecular systems.
Palladium, a key transition metal in advancing modern organic synthesis, mediates diverse chemical conversions including many carbon-carbon bond formation reactions between organic compounds. However, expanding palladium chemistry for conjugation of biomolecules such as proteins, particularly within their native cellular context, is still in its infancy. Here we report the site-specific protein labeling inside pathogenic Gram-negative bacterial cells via a ligand-free palladium-mediated cross-coupling reaction. Two rationally designed pyrrolysine analogues bearing an aliphatic alkyne or an iodophenyl handle were first encoded in different enteric bacteria, which offered two facial handles for palladium-mediated Sonogashira coupling reaction on proteins within these pathogens. A GFP-based bioorthogonal reaction screening system was then developed, allowing evaluation of both the efficiency and the biocompatibilty of various palladium reagents in promoting protein-small molecule conjugation. The identified simple compound-Pd(NO3)2 exhibited high efficiency and biocompatibility for site-specific labeling of proteins in vitro and inside living E. coli cells. This Pd-mediated protein coupling method was further utilized to label and visualize a Type-III Secretion (T3S) toxin-OspF in Shigella cells. Our strategy may be generally applicable for imaging and tracking various virulence proteins within Gram-negative bacterial pathogens.
Copper(I)-catalyzed azide-alkyne cycloaddition has become a commonly employed method for the synthesis of complex molecular
architectures under challenging conditions. Despite the widespread use of copper-catalyzed cycloaddition reactions, the mechanism
of these processes has remained difficult to establish due to the involvement of multiple equilibria between several reactive
intermediates. Real-time monitoring of a representative cycloaddition process via heat-flow reaction calorimetry revealed
that monomeric copper acetylide complexes are not reactive toward organic azides unless an exogenous copper catalyst is added.
Furthermore, crossover experiments with an isotopically enriched exogenous copper source illustrated the stepwise nature of
the carbon–nitrogen bond-forming events and the equivalence of the two copper atoms within the cycloaddition steps.
There is an increasing interest in the use of bioorthogonal ligation to advance biomedical research through selective labeling of biomolecules in living systems. Accordingly, discovering new reactions to expand the toolbox of bioorthogonal chemistry is of particular interest to chemical biologists. Herein we report a new bioorthogonal ligation enabled by click hetero-Diels-Alder (HDA) cycloaddition of in situ-generated o-quinolinone quinone methides and vinyl thioethers. This reaction is highly selective and proceeds smoothly under aqueous conditions. The functionalized vinyl thioethers are small and chemically stable in vivo, making them suitable for use as bioorthogonal chemical reporters that can be effectively coupled to various biomolecules. We utilized this bioorthogonal ligation for site-specific labeling of proteins as well as imaging of bioactive small molecules inside live cells.
One is not enough: Terminal alkenes are used as chemical reporters and ligation partners for 1,2,4,5-tetrazines in a Diels-Alder reaction with inverse electron demand (DARinv). Combination with strain-promoted azide-alkyne cycloaddition (SPAAC) allows the visualization of two different glycan structures in one experiment.
Aldehyde- and ketone-functionalized proteins are appealing substrates for the development of chemically modified biotherapeutics and protein-based materials. Their reactive carbonyl groups are typically conjugated with α-effect nucleophiles, such as substituted hydrazines and alkoxyamines, to generate hydrazones and oximes, respectively. However, the resulting C=N linkages are susceptible to hydrolysis under physiologically relevant conditions, which limits the utility of such conjugates in biological systems. Here we introduce a Pictet-Spengler ligation that is based on the classic Pictet-Spengler reaction of aldehydes and tryptamine nucleophiles. The ligation exploits the bioorthogonal reaction of aldehydes and alkoxyamines to form an intermediate oxyiminium ion; this intermediate undergoes intramolecular C-C bond formation with an indole nucleophile to form an oxacarboline product that is hydrolytically stable. We used the reaction for site-specific chemical modification of glyoxyl- and formylglycine-functionalized proteins, including an aldehyde-tagged variant of the therapeutic monoclonal antibody Herceptin. In conjunction with techniques for site-specific introduction of aldehydes into proteins, the Pictet-Spengler ligation offers a means to generate stable bioconjugates for medical and materials applications.
Chemical reporters are unique functional groups that can be used to label biomolecules in living systems. Only a handful of broadly applicable reporters have been identified to date, owing to the rigorous demands placed on these functional groups in biological settings. We describe here a new chemical reporter-cyclopropene-that can be used to target biomolecules in vitro and in live cells. A variety of substituted cyclopropene scaffolds were synthesized and found to be stable in aqueous solution and in the presence of biological nucleophiles. Furthermore, some of the cyclopropene units were metabolically introduced into cell surface glycans and subsequently detected with covalent probes. The small size and selective reactivity of cyclopropenes will facilitate efforts to tag diverse collections of biomolecules in vivo.
An efficient aqueous Wittig reaction was enabled on protein bioconjugation for the first time. By investigating the reaction on small molecules, peptides, and proteins, a site-specific reaction targeting "aldehyde tag" was presented. A variety of functional groups could be introduced into the protein of interest.
Fluorogenic probes activated by bioorthogonal chemical reactions can enable biomolecule imaging in situations where it is not possible to wash away unbound probe. One challenge for the development of such probes is the a priori identification of structures that will undergo a dramatic fluorescence enhancement by virtue of the chemical transformation. With the aid of density functional theory calculations reported previously by Nagano and co-workers, we identified azidofluorescein derivatives that were predicted to undergo an increase in fluorescence quantum yield upon Cu-catalyzed or Cu-free cycloaddition with linear or cyclic alkynes, respectively. Four derivatives were experimentally verified in model reactions, and one, a 4-azidonaphthylfluorescein analogue, was further shown to label alkyne-functionalized proteins in vitro and glycoproteins on cells with excellent selectivity. The azidofluorescein derivative also enabled cell imaging under no-wash conditions with good signal above background. This work establishes a platform for the rational design of fluorogenic azide probes with spectral properties tailored for biological imaging.
Tosylhydrazones of a number of α,β-unsaturated aldehydes and ketones have been prepared. On reaction with sodium methoxide in aprotic media at 160-220° alkyl-substituted cyclopropenes are formed. The yields vary from excellent to poor depending mainly on the degree of β-substitution of the tosylhydrazone. The sequence: tosylhydrazone → diazoalkene → alkenylcarbene → cyclopropane is proposed as the most suitable description of the multistep reaction.
We just click: Genetic incorporation of a cyclopropene amino acid CpK site-specifically into proteins in E. coli and mammalian cells was achieved using an orthogonal aminoacyl-tRNA synthetase/tRNA(CUA) pair (CpKRS/MbtRNA(CUA) ). Cyclopropene exhibited fast reaction kinetics in the photoclick reaction and allowed rapid (ca. 2 min) labeling of proteins.
Visualizing biomolecules by fluorescent tagging is a powerful method for studying their behaviour and function inside cells. We prepared and genetically encoded an unnatural amino acid (UAA) that features a bicyclononyne moiety. This UAA offered exceptional reactivity in strain-promoted azide-alkyne cycloadditions. Kinetic measurements revealed that the UAA reacted also remarkably fast in the inverse-electron-demand Diels-Alder cycloaddition with tetrazine-conjugated dyes. Genetic encoding of the new UAA inside mammalian cells and its subsequent selective labeling at low dye concentrations demonstrate the usefulness of the new amino acid for future imaging studies.
Genetic code expansion for unnatural amino acid mutagenesis has, until recently, been limited to cell culture. We demonstrate the site-specific incorporation of unnatural amino acids into proteins in Drosophila melanogaster at different developmental stages, in specific tissues and in a subset of cells within a tissue. This approach provides a foundation for probing and controlling processes in this established metazoan model organism with a new level of molecular precision.
Rasche Folgereaktionen vereitelten bislang die Isolierung substituierter Nitrilimine. Benz-phenylhydrazid-chlorid (3) tauscht nur in Anwesenheit von Triäthylamin mit Triäthylammoniumchlorid-36Cl das Chlor aus. Identische Orientierungsverhältnisse bei Cycloadditionen an Zimtsäure-methylester bzw. Crotonsäure-methylester sowie übereinstimmende Konkurrenzkonstanten bei der Umsetzung mit Dipolarophilen-Paaren beweisen das Auftreten ein und derselben Zwischenstufe bei folgenden Reaktionen: Thermolyse und Photolyse des 2.5-Diphenyl-tetrazols (1), Umsetzungen von Benz-phenylhydrazid-chlorid (3) mit tert. Amin sowie von [α-Nitro-benzyliden]-phenylhydrazin (8) mit Triäthylamin und Natriumjodid. Nur das freie Diphenylnitrilimin genügt den strukturellen Anforderungen.
In contrast to the very large number of special methods applicable to syntheses in the heterocyclic series, relatively few general methods are available. The 1,3-dipolar addition offers a remarkably wide range of utility in the synthesis of five-membered heterocycles. Here the “1,3-dipole”, which can only be represented by zwitterionic octet resonance structures, combines in a cycloaddition with a multiple bond system – the “dipolarophile” – to form an uncharged five-membered ring. Although numerous individual examples of this reaction were known, some even back in the nineteenth century, fruitful development of this synthetic principle has been achieved only in recent years.
ortho-Quinone methides (o-QMs) are emerging as highly useful intermediates, the inherent reactivity of which can be used in linchpin reactions for the construction of complex natural products. This review encompasses the major contributions in this field, exemplifying the major strategies and reactivity modes which can be applied. Orchestrating reactions: ortho-Quinone methides (o-QMs) are highly labile, often transient species, the inherent reactivity of which can be used for orchestrating linchpin reactions in total synthesis. A review of their use in natural product syntheses is presented.
Rapid, site-specific labeling of proteins with diverse probes remains an outstanding challenge for chemical biologists. Enzyme-mediated labeling approaches may be rapid but use protein or peptide fusions that introduce perturbations into the protein under study and may limit the sites that can be labeled, while many "bioorthogonal" reactions for which a component can be genetically encoded are too slow to effect quantitative site-specific labeling of proteins on a time scale that is useful for studying many biological processes. We report a fluorogenic reaction between bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) and tetrazines that is 3-7 orders of magnitude faster than many bioorthogonal reactions. Unlike the reactions of strained alkenes, including trans-cyclooctenes and norbornenes, with tetrazines, the BCN-tetrazine reaction gives a single product of defined stereochemistry. We have discovered aminoacyl-tRNA synthetase/tRNA pairs for the efficient site-specific incorporation of a BCN-containing amino acid, 1, and a trans-cyclooctene-containing amino acid 2 (which also reacts extremely rapidly with tetrazines) into proteins expressed in Escherichia coli and mammalian cells. We demonstrate the rapid fluorogenic labeling of proteins containing 1 and 2 in vitro, in E. coli , and in live mammalian cells. These approaches may be extended to site-specific protein labeling in animals, and we anticipate that they will have a broad impact on labeling and imaging studies.
The genetic incorporation of one azide-containing and one keto-containing noncanonical amino acid into a protein at amber and ochre mutation sites respectively, followed by their orthogonal reactions with hydroxylamine-containing and cyclooctyne-containing dyes allows highly efficient one-pot site-specific dual labeling of the protein in a catalyst-free fashion.
Bring your own copper: Copper-chelating azides undergo much faster click reactions (CuAAC) than nonchelating azides under a variety of biocompatible conditions. This kinetic enhancement allows site-specific protein labeling to be performed on the surface of living cells with only 10-40 μM Cu(I) /Cu(II) . Detection sensitivity was also increased for CuAAC detection of alkyne-modified proteins and RNA.
Under tension: A set of genetically encoded unnatural amino acids can be used for biocompatible site-specific labeling of proteins with fluorogenic dyes. The new compounds have norbornene and trans-cyclooctene units that react with tetrazine derivatives in an inverse-electron-demand Diels-Alder cycloaddition (left in picture). The technique offers fast labeling that is orthogonal to labeling through azide-cyclooctyne click reaction (right).
Three at the same time: A ligation strategy combining tetrazine-norbornene cycloaddition, Staudinger-Bertozzi ligation, and copper(I)-catalyzed click reaction was used to label the three catalytic activities of the proteasome simultaneously in a single experiment. The orthogonality of the three ligation reactions enables selective monitoring of multiple targets at the same time in complex biological samples.
The site-specific incorporation of bioorthogonal groups via genetic code expansion provides a powerful general strategy for site-specifically labelling proteins with any probe. However, the slow reactivity of the bioorthogonal functional groups that can be encoded genetically limits the utility of this strategy. We demonstrate the genetic encoding of a norbornene amino acid using the pyrrolysyl tRNA synthetase/tRNA(CUA) pair in Escherichia coli and mammalian cells. We developed a series of tetrazine-based probes that exhibit 'turn-on' fluorescence on their rapid reaction with norbornenes. We demonstrate that the labelling of an encoded norbornene is specific with respect to the entire soluble E. coli proteome and thousands of times faster than established encodable bioorthogonal reactions. We show explicitly the advantages of this approach over state-of-the-art bioorthogonal reactions for protein labelling in vitro and on mammalian cells, and demonstrate the rapid bioorthogonal site-specific labelling of a protein on the mammalian cell surface.
Bioorthogonal ligation methods with improved reaction rates and less obtrusive components are needed for site-specifically labeling proteins without catalysts. Currently no general method exists for in vivo site-specific labeling of proteins that combines fast reaction rate with stable, nontoxic, and chemoselective reagents. To overcome these limitations, we have developed a tetrazine-containing amino acid, 1, that is stable inside living cells. We have site-specifically genetically encoded this unique amino acid in response to an amber codon allowing a single 1 to be placed at any location in a protein. We have demonstrated that protein containing 1 can be ligated to a conformationally strained trans-cyclooctene in vitro and in vivo with reaction rates significantly faster than most commonly used labeling methods.
Strain-promoted cycloadditions of cyclic nitrones with biaryl-aza-cyclooctynone (BARAC) proceed with rate constants up to 47.3 M(-1) s(-1), this corresponds to a 47-fold rate enhancement relative to reaction of BARAC with benzyl azide and a 14-fold enhancement over previously reported strain promoted alkyne-nitrone cycloadditions (SPANC). Studies of the SPANC reaction using the linear free energy relationship defined by the Hammett equation demonstrated that the cycloaddition reaction has a rho value of 0.25 ± 0.04, indicating that reaction is not sensitive to substituents and thus should have broad applicability.
The inverse-electron-demand Diels-Alder cycloaddition between trans-cyclooctenes and tetrazines is biocompatible and exceptionally fast. We utilized this chemistry for site-specific fluorescence labeling of proteins on the cell surface and inside living mammalian cells by a two-step protocol. Escherichia coli lipoic acid ligase site-specifically ligates a trans-cyclooctene derivative onto a protein of interest in the first step, followed by chemoselective derivatization with a tetrazine-fluorophore conjugate in the second step. On the cell surface, this labeling was fluorogenic and highly sensitive. Inside the cell, we achieved specific labeling of cytoskeletal proteins with green and red fluorophores. By incorporating the Diels-Alder cycloaddition, we have broadened the panel of fluorophores that can be targeted by lipoic acid ligase.
Benign C-C bond formation at various sites in cell-surface channels has been achieved through Suzuki-Miyaura coupling of genetically positioned unnatural amino acids containing aryl halide side chains. This enabled site-selective cell surface manipulation of Escherichia coli ; the phosphine-free catalyst caused no cell death at required Pd loadings, suggesting future in vivo application of catalytic metal-mediated bond formation in more complex organisms.