Current Approaches for RNA Labeling in Vitro and in Cells Based on Click Reactions

Over recent years, click reactions have become recognized as valuable and flexible tools to label biomacromolecules such as proteins, nucleic acids, and glycans. Some of the developed strategies can be performed not only in aqueous solution but also in the presence of cellular components, as well as on (or even in) living cells. These labeling strategies require the initial, specific modification of the target molecule with a small, reactive moiety. In the second step, a click reaction is used to covalently couple a reporter molecule to the biomolecule. Depending on the type of reporter, labeling by the click reaction can be used in many different applications, ranging from isolation to functional studies of biomacromolecules. In this minireview, we focus on labeling strategies for RNA that rely on the click reaction. We first highlight click reactions that have been used successfully to label modified RNA, and then describe different strategies to introduce the required reactive groups into target RNA. The benefits and potential limitations of the strategies are critically discussed with regard to possible future developments.     

Protein Iodination by Click Chemistry

Two steps to click iodine : We have developed a two‐step reaction for protein iodination using click chemistry. With this method, which is summarized in the scheme, covalent attachment of a stable iodine‐containing aromatic azide moiety to an alkyne‐containing protein was achieved.

     

Synthesis of Ferrocene-containing Polyacetylenes by Click Chemistry

Ferrocene-containing polyacetylenes are synthesized in good yields by Cu-catalyzed click reactions of azido moieties of poly[(6-chloro-1-phenyl-1-hexyne)-co-(6-azido-1-phenyl-1-hexyne)] and poly{1-[(4-azidohexyloxy)phenyl]-2-phenylacetylene} with ethynylferrocene. All the organometallic polymers are completely soluble in common organic solvents such as chloroform, THF and toluene. Spectroscopic analyses reveal that all the azido functional groups have cyclized with ethynylferrocene. With the incorporation of ferrocene rings into the polyacetylene structure, the resulting polymers show enhanced thermal stability and redox activity. This communication is dedicated to Professor Ian Manners and his scientific accomplishments     

Ultramild Protein‐Mediated Click Chemistry Creates Efficient Oligonucleotide Probes for Targeting and Detecting Nucleic Acids

Functionalized synthetic oligonucleotides are finding growing applications in research, clinical studies, and therapy. However, it is not easy to prepare them in a biocompatible and highly efficient manner. We report a new strategy to synthesize oligonucleotides with promising nucleic acid targeting and detection properties. We focus in particular on the pH sensitivity of these new probes and their high target specificity. For the first time, human copper(I)‐binding chaperon Cox17 was applied to effectively catalyze click labeling of oligonucleotides. This was performed under ultramild conditions with fluorophore, peptide, and carbohydrate azide derivatives. In thermal denaturation studies, the modified probes showed specific binding to complementary DNA and RNA targets. Finally, we demonstrated the pH sensitivity of the new rhodamine‐based fluorescent probes in vitro and rationalize our results by electronic structure calculations.     

Capturing the Direct Binding of CFTR Correctors to CFTR by Using Click Chemistry

Cystic fibrosis (CF) is a lethal genetic disease caused by the loss or dysfunction of the CF transmembrane conductance regulator (CFTR) channel. F508del is the most prevalent mutation of the CFTR gene and encodes a protein defective in folding and processing. VX‐809 has been reported to facilitate the folding and trafficking of F508del‐CFTR and augment its channel function. The mechanism of action of VX‐809 has been poorly understood. In this study, we sought to answer a fundamental question underlying the mechanism of VX‐809: does it bind CFTR directly in order to exert its action? We synthesized two VX‐809 derivatives, ALK‐809 and SUL‐809, that possess an alkyne group and retain the rescue capacity of VX‐809. By using Cu^I‐catalyzed click chemistry, we provide evidence that the VX‐809 derivatives bind CFTR directly in vitro and in cells. Our findings will contribute to the elucidation of the mechanism of action of CFTR correctors and the design of more potent therapeutics to combat CF.     

Assessing the Delivery of Molecules to the Mitochondrial Matrix Using Click Chemistry

Mitochondria are central to health and disease, hence there is considerable interest in developing mitochondria‐targeted therapies that require the delivery of peptides or nucleic acid oligomers. However, progress has been impeded by the lack of a measure of mitochondrial import of these molecules. Here, we address this need by quantitatively detecting molecules within the mitochondrial matrix. We used a mitochondria‐ targeted cyclooctyne (MitoOct) that accumulates several‐ hundredfold in the matrix, driven by the membrane potential. There, MitoOct reacts through click chemistry with an azide on the target molecule to form a diagnostic product that can be quantified by mass spectrometry. Because the membrane potential‐dependent MitoOct concentration in the matrix is essential for conjugation, we can now determine definitively whether a putative mitochondrion‐targeted molecule reaches the matrix. This “ClickIn” approach will facilitate development of mitochondria‐targeted therapies.     

点击化学在硅及玻璃表面改性中的应用

综述了点击化学方法对几种硅材料表面的化学改性,包括硅片、硅纳米粒子、硅烷玻璃、微通道和玻璃等的表面修饰作用。通过叠氮-炔环加成作用将功能分子固定到基材表面,赋予表面生物活性、防污性以及良好的吸附性能等,有望将硅基材的应用范围扩展至生物传感器、防污表面和吸附材料等领域。     

An Efficient Bioorthogonal Strategy Using CuAAC Click Chemistry for Radiofluorinations of SNEW Peptides and the Role of Copper Depletion

The EphB2 receptor is known to be overexpressed in various types of cancer and is therefore a promising target for tumor cell imaging by positron emission tomography (PET). In this regard, imaging could facilitate the early detection of EphB2‐overexpressing tumors, monitoring responses to therapy directed toward EphB2, and thus improvement in patient outcomes. We report the synthesis and evaluation of several fluorine‐18‐labeled peptides containing the SNEW amino acid motif, with high affinity for the EphB2 receptor, for their potential as radiotracers in the non‐invasive imaging of cancer using PET. For the purposes of radiofluorination, EphB2‐antagonistic SNEW peptides were varied at the C terminus by the introduction of L ‐cysteine, and further by alkyne‐ or azide‐modified amino acids. In addition, two novel bifunctional and bioorthogonal labeling building blocks [¹⁸F]AFP and [¹⁸F]BFP were applied, and their capacity to introduce fluorine‐18 was compared with that of the established building block [¹⁸F]FBAM. Copper‐assisted Huisgen 1,3‐dipolar cycloaddition, which belongs to the set of bioorthogonal click chemistry reactions, was used to introduce both novel building blocks into azide‐ or alkyne‐modified SNEW peptides under mild conditions. Finally, the depletion of copper immediately after radiolabeling is a highly important step of this novel methodology.     

Modulation of Mitochondriotropic Properties of Cyanine Dyes by in Organello Copper‐Free Click Reaction

Cyanine (Cy) dyes show a general propensity to localize in polarized mitochondria. This mitochondriotropism was used to perform a copper‐free click reaction in the mitochondria of living cells. The in organello reaction of dyes Cy3 and Cy5 led to a product that was easily traceable by Förster resonance energy transfer (FRET). As determined by confocal laser scanning microscopy, the Cy3–Cy5 conjugate showed enhanced retention in mitochondria, relative to that of the starting compounds. This enhancement of a favorable property can be achieved by synthesis in organello, but not outside mitochondria.     

Vinyl Sulfonates: A Click Function for Coupling‐and‐Decoupling Chemistry and their Applications

The term coupling‐and‐decoupling (CAD) chemistry refers to applications in which efficient bond formation and subsequent cleavage between two moieties is required. Within this context, the scope of the vinyl sulfonate (VSO) group as an efficient tool for CAD chemistry is reported. The coupling step relies on the click features of the Michael‐type addition of diverse nucleophiles to vinyl sulfonates as a valuable methodology. The feasibility of this strategy has been proved by the high yields obtained in mild conditions with model VSO derivatives. Cleavage of the resulting sulfonate adducts either through nucleophilic substitution with different nucleophiles (for alkyl VSO groups) or through hydrolysis (for both alkyl and aryl VSO) are successful strategies for the decoupling step, the former being the most promising, as the reaction proceeds under milder conditions with thiol nucleophiles. Moreover, the click VSO coupling chemistry proves to be orthogonal with the click CuAAC reaction, which enables the VSO‐CAD methodology for the preparation of hetero‐bifunctional clickable and cleavable linkers for double click modular strategies. The potential of the VSO‐CAD chemistry is demonstrated in two biologically relevant examples: the decoupling of sulfonates with glutathione (GSH) under conditions compatible with those of living systems; and the synthesis of homo‐ and heterogeneous multivalent glycosylated systems from 1‐thio and 1‐azido or 1‐azidoethyl sugar derivatives and bis‐vinyl sulfonates (homo systems) or alkynyl‐VSO bifunctional clickable‐cleavable linkers (hetero systems). As proof of concept, the cleavable character of these multivalent systems was demonstrated by using one of them as a reversible linker for the non‐covalent assembling and chemical decoupling of two model lectins.

     

Diaminoterephthalate turn-on fluorescence probes for thiols--tagging of recoverin and tracking of its conformational change

Diaminoterephthalates with a maleimide moiety were synthesized and used as fluorescence dyes for sensing thiols. Whereas these "NiWa Blue" dyes showed no emission, the conjugate addition of a thiol to the maleimide group turned on a fluorescence at about 400 nm when irradiating the dye at 338 nm. The neuronal-calcium sensor protein recoverin possesses a single cysteine residue at position 39, which reacts with NiWa Blue, and is therefore labeled by a fluorophore with an emission at about 440 nm. In the absence of Ca(2+), irradiation at 280 nm of a tryptophan residue in close proximity to Cys-bound NiWa Blue lead to strong FRET, which was detected by emission of the dye at 440 nm. In the presence of Ca(2+), the protein holds a conformation with distal Trp and Cys residues, thus FRET of irradiated Trp to Cys-bound NiWa Blue was significantly weakened.     

PNA Molecular Beacons Assembled by Post‐Synthetic Click Chemistry Functionalization

To avoid the tedious synthesis of functionalized peptide nucleic acid (PNA) monomers for probe development, we proposed a simple approach to modify PNA oligomers by post‐synthetic on‐resin click chemistry. PNA molecular beacons (MBs) were prepared by incorporation of azide‐containing monomers into the oligomer by automatic solid‐phase peptide synthesis and subsequent derivatization with pyrene moieties by copper‐catalyzed azide–alkyne cycloaddition. Two pyrene‐based quencher‐free PNA molecular beacons, a stemless MB and one possessing a stem–loop structure, targeting a portion of the cystic fibrosis gene, were successfully synthesized by using this method. Fluorescence studies showed that the stem–loop MB exhibited better discrimination of changes in excimer/monomer ratios as compared to the stemless MB construct.     

Characterization of the Secretome of a Specific Cell Expressing Mutant Methionyl-tRNA Synthetase in Co-Culture Using Click Chemistry

Co-culture system, in which two or more distinct cell types are cultured together, is advantageous in that it can mimic the environment of the in vivo niche of the cells. In this study, we presented a strategy to analyze the secretome of a specific cell type under the co-culture condition in serum-supplemented media. For the cell-specific secretome analysis, we expressed the mouse mutant methionyl-tRNA synthetase for the incorporation of the non-canonical amino acid, azidonorleucine into the newly synthesized proteins in cells of which the secretome is targeted. The azidonorleucine-tagged secretome could be enriched, based on click chemistry, and distinguished from any other contaminating proteins, either from the cell culture media or the other cells co-cultured with the cells of interest. In order to have more reliable true-positive identifications of cell-specific secretory bodies, we established criteria to exclude any identified human peptide matched to bovine proteins. As a result, we identified a maximum of 719 secreted proteins in the secretome analysis under this co-culture condition. Last, we applied this platform to profile the secretome of mesenchymal stem cells and predicted its therapeutic potential on osteoarthritis based on secretome analysis.     

Multimerization of cRGD Peptides by Click Chemistry: Synthetic Strategies,Chemical Limitations,and Influence on Biological Properties

Integrin α_νβ₃ is overexpressed on endothelial cells of growing vessels as well as on several tumor types, and so integrin‐binding radiolabeled cyclic RGD pentapeptides have attracted increasing interest for in vivo imaging of α_νβ₃ integrin expression by positron emission tomography (PET). Of the cRGD derivatives available for imaging applications, systems comprising multiple cRGD moieties have recently been shown to exhibit highly favorable properties in relation to monomers. To assess the synthetic limits of the cRGD‐multimerization approach and thus the maximum multimer size achievable by using different efficient conjugation reactions, we prepared a variety of multimers that were further investigated in vitro with regard to their avidities to integrin α_νβ_3. The synthesized peptide multimers containing increasing numbers of cRGD moieties on PAMAM dendrimer scaffolds were prepared by different click chemistry coupling strategies. A cRGD hexadecimer was the largest construct that could be synthesized under optimized reaction conditions, thus identifying the current synthetic limitations for cRGD multimerization. The obtained multimeric systems were conjugated to a new DOTA‐based chelator developed for the derivatization of sterically demanding structures and successfully labeled with ⁶⁸Ga for a potential in vivo application. The evaluated multimers showed very high avidities—increasing with the number of cRGD moieties—in in vitro studies on immobilized α_νβ₃ integrin and U87MG cells, of up to 131‐ and 124‐fold, respectively, relative to the underivatized monomer.     

A Compact and Synthetically Accessible Fluorine‐18 Labelled Cyclooctyne Prosthetic Group for Labelling of Biomolecules by Copper‐Free Click Chemistry

A new fluorine‐containing azadibenzocyclooctyne (ADIBO‐F) was designed using a synthetically accessible pathway. The fluorine‐18 prosthetic group was prepared from its toluenesulfonate precursor and isolated in 21–35 % radiochemical yield in 30 minutes of synthetic time. ADIBO‐F has been incorporated into azide‐functionalized, cancer‐targeting peptides through a strain‐promoted alkyne–azide cycloaddition with high radiochemical yields and purities. The final products are novel peptide‐based positron emission tomography (PET) imaging agents that possess high affinities for their targets, growth hormone secretagogue receptor 1a (GHSR‐1a) and gastrin‐releasing peptide receptor (GRPR), with IC₅₀ values of 9.7 and 0.50 nm , respectively. This is a new and rapid labelling option for the incorporation of fluorine‐18 into biomolecules for PET imaging.