Quantum Chemical Calculations and Experimental Validation of the Photoclick Reaction for Fluorescent Labeling of the 5’ cap of Eukaryotic mRNAs

Bioorthogonal click reactions are powerful tools to specifically label biomolecules in living cells. Considerable progress has been made in site-specific labeling of proteins and glycans in complex biological systems, but equivalent methods for mRNAs are rare. We present a chemo-enzymatic approach to label the 5’ cap of eukaryotic mRNAs using a bioorthogonal photoclick reaction. Herein, the N7-methylated guanosine of the 5’ cap is enzymatically equipped with an allyl group using a variant of the trimethylguanosine synthase 2 from Giardia lamblia (GlaTgs2). To elucidate whether the resulting N²-modified 5’ cap is a suitable dipolarophile for photoclick reactions, we used Kohn–Sham density functional theory (KS-DFT) and calculated the HOMO and LUMO energies of this molecule and nitrile imines. Our in silico studies suggested that combining enzymatic allylation of the cap with subsequent labeling in a photoclick reaction was feasible. This could be experimentally validated. Our approach generates a turn-on fluorophore site-specifically at the 5’ cap and therefore presents an important step towards labeling of eukaryotic mRNAs in a bioorthogonal manner.     

Cell–cell interactions via non-covalent click chemistry

Metabolic glycoengineering with unnatural sugars became a valuable tool for introducing recognition markers on the cell membranes via bioorthogonal chemistry. By using this strategy, we functionalized the surface of tumor and T cells using complementary artificial markers based on both β-cyclodextrins (β-CDs) and adamantyl trimers, respectively. Once tied on cell surfaces, the artificial markers induced cell–cell adhesion through non-covalent click chemistry. These unnatural interactions between A459 lung tumor cells and Jurkat T cells triggered the activation of natural killer (NK) cells thanks to the increased production of interleukin-2 (IL-2) in the vicinity of cancer cells, leading ultimately to their cytolysis. The ready-to-use surface markers designed in this study can be easily inserted on the membrane of a wide range of cells previously submitted to metabolic glycoengineering, thereby offering a simple way to investigate and manipulate intercellular interactions.

We designed complementary artificial markers that were introduced on the surface of cells previously modified by metabolic glycoengineering. These recognition markers enable unnatural cell–cell adhesion through non-covalent click chemistry.     

Second-generation difluorinated cyclooctynes for copper-free click chemistry

The 1,3-dipolar cycloaddition of azides and activated alkynes has been used for site-selective labeling of biomolecules in vitro and in vivo. While copper catalysis has been widely employed to activate terminal alkynes for [3 + 2] cycloaddition, this method, often termed "click chemistry", is currently incompatible with living systems because of the toxicity of the metal. We recently reported a difluorinated cyclooctyne (DIFO) reagent that rapidly reacts with azides in living cells without the need for copper catalysis. Here we report a novel class of DIFO reagents for copper-free click chemistry that are considerably more synthetically tractable. The new analogues maintained the same elevated rates of [3 + 2] cycloaddition as the parent compound and were used for imaging glycans on live cells. These second-generation DIFO reagents should expand the use of copper-free click chemistry in the hands of biologists.     

Synthesis,characterization, and antimicrobial activity of new bis-1,2,3-triazol-H-yl-substituted 2-arylbenzimidazoles

New bis-1,2,3-triazol-H-yl-substituted 2-aryl benzimidazoles VIa-VIp were synthesized from O- and N-bis-propargyl substituted 2-arylbenzimidazoles using “click chemistry.” The newly synthesized compounds were characterized by IR, NMR, and mass spectra. These compounds were screened for their activity against bacterial and fungal organisms.     

Synthesis of novel carbohydrate-based valine-derived formamide organocatalysts by CuAAC click chemistry and their application in asymmetric reduction of imines with trichlorosilane

Novel organocatalysts combining carbohydrate and N-formyl-l-valine derivatives were prepared by Cu^II-catalyzed diazo transfer and Cu^I-catalyzed azide–alkyne 1,3-dipolar cycloaddition CuAAC click chemistry. It was found that the carbohydrate-based valine-derived formamide organocatalyst had high catalytic activity for the asymmetric reduction of imines with trichlorosilane. The reduction can proceed at room temperature in toluene in high yield (up to 98%) and with excellent enantioselectivity (up to 94%). ‘CuAAC’ click chemistry is a bridge to link N-formyl-l-valine derived organocatalysts with carbohydrates.     

Surface functionalization with redox active molecule-based imidazolium via click chemistry

In this study, the redox active molecule N-ferrocenylmethyl-N-propargylimidazolium bromide was immobilized onto the surface of an electrode. The surface modification was performed by coupling the electrochemical reduction of the 4-azidophenyldiazonium generated in situ with a copper(I) catalyzed click chemistry reaction. Surface and electrochemical investigations suggest the attachment of a monolayer of redox active molecules containing an ionic liquid framework onto the electrode surface. Furthermore, scanning electrochemical microscopy studies revealed the conductive behavior of the attached ferrocenyl moieties on the ITO surface.     

Targeted Ultrasound‐Assisted Cancer‐Selective Chemical Labeling and Subsequent Cancer Imaging using Click Chemistry

Metabolic sugar labeling followed by the use of reagent‐free click chemistry is an established technique for in vitro cell targeting. However, selective metabolic labeling of the target tissues in vivo remains a challenge to overcome, which has prohibited the use of this technique for targeted in vivo applications. Herein, we report the use of targeted ultrasound pulses to induce the release of tetraacetyl N‐azidoacetylmannosamine (Ac₄ManAz) from microbubbles (MBs) and its metabolic expression in the cancer area. Ac₄ManAz‐loaded MBs showed great stability under physiological conditions, but rapidly collapsed in the presence of tumor‐localized ultrasound pulses. The released Ac₄ManAz from MBs was able to label 4T1 tumor cells with azido groups and significantly improved the tumor accumulation of dibenzocyclooctyne (DBCO)‐Cy5 by subsequent click chemistry. We demonstrated for the first time that Ac₄ManAz‐loaded MBs coupled with the use of targeted ultrasound could be a simple but powerful tool for in vivo cancer‐selective labeling and targeted cancer therapies.     

Cathepsin B‐Specific Metabolic Precursor for In Vivo Tumor‐Specific Fluorescence Imaging

Recently, metabolic glycoengineering with bioorthogonal click reactions has focused on improving the tumor targeting efficiency of nanoparticles as delivery vehicles for anticancer drugs or imaging agents. It is the key technique for developing tumor‐specific metabolic precursors that can generate unnatural glycans on the tumor‐cell surface. A cathepsin B‐specific cleavable substrate (KGRR) conjugated with triacetylated N‐azidoacetyl‐d ‐mannosamine (RR‐S‐Ac₃ManNAz) was developed to enable tumor cells to generate unnatural glycans that contain azide groups. The generation of azide groups on the tumor cell surface was exogenously and specifically controlled by the amount of RR‐S‐Ac₃ManNAz that was fed to target tumor cells. Moreover, unnatural glycans on the tumor cell surface were conjugated with near infrared fluorescence (NIRF) dye‐labeled molecules by a bioorthogonal click reaction in cell cultures and in tumor‐bearing mice. Therefore, our RR‐S‐Ac₃ManNAz is promising for research in tumor‐specific imaging or drug delivery.     

A pH-responsive soluble polymer-based homogeneous system for fast and highly efficient N-glycoprotein/glycopeptide enrichment and identification by mass spectrometry

Liquid phase homogeneous reactions using soluble polymer supports have found numerous applications in homogeneous catalysis and organic synthesis because of their advantages of no interface mass transfer limitation and a high conversion rate. However, their application in analytical separation is limited by the inefficient/inconvenient recovery of the target molecules from the extremely complex biological samples. Here, we report a stimuli-responsive polymer system for facile and efficient enrichment of trace amounts of biomolecules from complex biological samples. The soluble polymer supports provide a homogeneous reaction system with fast mass transfer and facilitate interactions between the supports and the target molecules. More importantly, the stimuli-responsive polymers exhibit reversible self-assembly and phase separation under pH variations, which leads to facial sample recovery with a high yield of the target biomolecules. The stimuli-responsive polymer is successfully applied to the enrichment of low abundant N-glycoproteins/glycopeptides, which play crucial roles in various key biological processes in mammals and are closely correlated with the occurrence, progression and metastasis of cancer. N-Glycoprotein is coupled to the stimuli-responsive polymer using the reported hydrazide chemistry with pre-oxidation of the oligosaccharide structure. Highly efficient enrichment of N-glycoproteins/N-glycopeptides with >95% conversion rate is achieved within 1 h, which is eight times faster than using solid/insoluble hydrazide enrichment materials. Mass spectrometry analysis achieves low femtomolar identification sensitivity and obtained 1317 N-glycopeptides corresponding to 458 N-glycoproteins in mouse brain, which is more than twice the amount obtained after enrichment using commercial solid/insoluble materials. These results demonstrate the capability of this “smart” polymer system to combine stimuli-responsive and target-enrichment moieties to achieve improved identification of key biological and disease related biomolecules.     

Stronger together for in-cell translation: natural and unnatural base modified mRNA

The preparation of highly modified mRNAs and visualization of their cellular distribution are challenging. We report in-cell application of in vitro transcribed mRNA containing natural base modifications and site-specifically introduced artificial nucleotides. Click chemistry on mRNA allows visualization in cells with excellent signal intensities. While non-specific introduction of reporter groups often leads to loss in mRNA functionality, we combined the benefits from site-specificity in the 3′-UTR incorporated unnatural nucleotides with the improved translation efficiency of the natural base modifications Ψ and 5mC. A series of experiments is described to observe, quantify and verify mRNA functionality. This approach represents a new way to visualize mRNA delivery into cells and monitor its spread on a cellular level and translation efficiency. We observed increased protein expression from this twofold chemically modified, artificial mRNA counterbalancing a reduced transfection rate. This synergetic effect can be exploited as a powerful tool for future research on mRNA therapeutics.

Introducing unnatural base modifications site-specifically into the 3′-UTR of an mRNA bearing natural base modifications allows efficient visualization in cells by click chemistry. An enhanced protein expression in cells is observed from this twofold modified mRNA.     

Impacts of gold nanoparticle charge and ligand type on surface binding and toxicity to Gram-negative and Gram-positive bacteria

Although nanomaterials facilitate significant technological advancement in our society, their potential impacts on the environment are yet to be fully understood. In this study, two environmentally relevant bacteria, Shewanella oneidensis and Bacillus subtilis, have been used as model organisms to elucidate the molecular interactions between these bacterial classes and Au nanoparticles (AuNPs) with well-controlled and well-characterized surface chemistries: anionic 3-mercaptopropionic acid (MPA), cationic 3-mercaptopropylamine (MPNH₂), and the cationic polyelectrolyte poly(allylamine hydrochloride) (PAH). The data demonstrate that cationic, especially polyelectrolyte-wrapped AuNPs, were more toxic to both the Gram-negative and Gram-positive bacteria. The levels of toxicity observed were closely related to the percentage of cells with AuNPs associated with the cell surface as measured in situ using flow cytometry. The NP concentration-dependent binding profiles were drastically different for the two bacteria strains, suggesting the critical role of bacterial cell surface chemistry in determining nanoparticle association, and thereby, biological impact.     

Glycoengineering artificial receptors for microglia to phagocytose Aβ aggregates

Oligomeric and fibrillar amyloid-β (Aβ) are principally internalized via receptor-mediated endocytosis (RME) by microglia, the main scavenger of Aβ in the brain. Nevertheless, the inflammatory cascade will be evoked after vast Aβ aggregate binding to pattern recognition receptors on the cell membrane, which then significantly decreases the expression of these receptors and further deteriorate Aβ deposition. This vicious circle will weaken the ability of microglia for Aβ elimination. Herein, a combination of metabolic glycoengineering and self-triggered click chemistry is utilized to engineer microglial membranes with ThS as artificial Aβ receptors to promote microglia to phagocytose Aβ aggregates. Additionally, to circumvent the undesirable immune response during the process of the bioorthogonal chemistry reaction and Aβ-microglial interaction, Mn-porphyrin metal–organic frameworks (Mn-MOFs) with superoxide dismutase (SOD) and catalase (CAT) mimic activity are employed to carry N-azidoacetylmannosamine (AcManNAz) and eradicate over-expressed reactive oxygen species (ROSs). The artificial Aβ receptors independent of a signal pathway involved in immunomodulation as well as Mn-MOFs with antioxidant properties can synergistically promote the phagocytosis and clearance of Aβ with significantly enhanced activity and negligible adverse effects. The present study will not only provide valuable insight into the rational design of the microglial surface engineering strategy via bioorthogonal chemistry, but also hold great potential for other disease intervention associated with receptor starvation.

A combination of metabolic glycoengineering and self-triggered click chemistry is utilized to engineer a microglial membrane with ThS as artificial Aβ receptors to promote microglia to phagocytose Aβ aggregates.     

Convergent chemo-enzymatic synthesis of mannosylated glycopeptides; targeting of putative vaccine candidates to antigen presenting cells

The combination of solid phase peptide synthesis and endo-β-N-acetylglucosaminidase (ENGase) catalysed glycosylation is a powerful convergent synthetic method allowing access to glycopeptides bearing full-length N-glycan structures. Mannose-terminated N-glycan oligosaccharides, produced by either total or semi-synthesis, were converted into oxazoline donor substrates. A peptide from the human cytomegalovirus (CMV) tegument protein pp65 that incorporates a well-characterised T cell epitope, containing N-acetylglucosamine at specific Asn residues, was accessed by solid phase peptide synthesis, and used as an acceptor substrate. High-yielding enzymatic glycosylation afforded glycopeptides bearing defined homogeneous high-mannose N-glycan structures. These high-mannose containing glycopeptides were tested for enhanced targeting to human antigen presenting cells (APCs), putatively mediated via the mannose receptor, and for processing by the APCs for presentation to human CD8+ T cells specific for a 9-mer epitope within the peptide. Binding assays showed increased binding of glycopeptides to APCs compared to the non-glycosylated control. Glycopeptides bearing high-mannose N-glycan structures at a single site outside the T cell epitope were processed and presented by the APCs to allow activation of a T cell clone. However, the addition of a second glycan within the T cell epitope resulted in ablation of T cell activation. We conclude that chemo-enzymatic synthesis of mannosylated glycopeptides enhances uptake by human APCs while preserving the immunogenicity of peptide epitopes within the glycopeptides, provided those epitopes are not themselves glycosylated.     

Facile synthesis of dendrimer-like star-branched poly(isopropylacrylamide) via combination of click chemistry and atom transfer radical polymerization

We report a facile synthesis method of dendrimer-like star-branched poly(N-isopropylacrylamide) (PNIPAM) via the combination of click chemistry and atom transfer radical polymerization (ATRP) by employing the arm-first approach. First, the α-azido-ω-chloro-heterodifunctionalized building block, N ₃-PNIPAM-Cl (G0-Cl), was synthesized via ATRP by 3-azidopropyl 2-chloropropionate as the initiator. Taking advantage of click chemistry, the first generation (G1) of dendrimer-like star-branched PNIPAM, G1-(Cl)₃, was facilely prepared via the click coupling reaction between G0-Cl and tripropargylamine. For the construction of second generation (G2) dendrimer-like star-branched PNIPAM, G2-(Cl)₆, terminal chloride moieties of G1-(Cl)₃ were first converted to azide, and then reacted with excess tripropargylamine to give G1-(alkynyl)₆; G2-(Cl)₆ was subsequently prepared via click reaction between G1-(alkynyl)₆ and G0-Cl. Gel permeation chromatography (GPC) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry were employed to confirm the successful construction of dendrimer-like star-branched polymers. The unique thermal phase transition behavior of this dendrimer-like star-branched polymer in aqueous solutions was further investigated by turbidimetry and micro-differential scanning calorimetry (Micro-DSC).     

Synthesis of “Clickable” Nano Metal‐organic Framework by Generic Postsynthetic Modification Route

Organic azides have been somewhat popularized due to their pivotal role in the emerging field of “click chemistry”. A simple approach has been used for the synthesis of uniform nano Fe‐MIL‐88B‐NH₂, and a generic postsynthetic modification route has been developed for the synthesis of azide‐modified nano Fe‐MIL‐88B‐N₃. The approach also has been used to synthesize the azide‐modified IRMOF‐3(‐N₃). These new azide‐modified Fe‐MIL‐88B‐N₃ nanocrystals hold promising potential for the applications in the fields of “click chemistry”, nanotechnology devices and nano composite membranes.     

The first chemical synthesis of boronic acid-modified DNA through a copper-free click reaction

The first chemical incorporation of the boronic acid group into DNA using a copper-free click reagent was reported. Compared with the PCR-based method, this approach allows for site-specific incorporation and synthesis on a larger scale.     

1H‐1,2,3‐Triazole: From Structure to Function and Catalysis

The heterocyclic family of azoles have recently become one of the most widely used members of the N‐heterocycles; the most prominent one being 1H‐1,2,3‐triazole and its derivatives. The sudden growth of interest in this structural motif was sparked by the advent of click chemistry, first described in the early 2000s. From the early days of click chemistry, when the accessibility of triazoles made them into one of the most versatile linkers, interest has slowly turned to the use of triazoles as functional building blocks. The presence of multiple N‐coordination sites and a highly polarized carbon atom allows for metal coordination and the complexation of anions by both hydrogen and halogen bonding. Exploitation of these multiple binding sites makes it possible for triazoles to be used in various functional materials, such as metallic and anionic sensors. More recently, triazoles have also shown their potential in catalytic systems, thus increasing their impact far beyond the initial purpose of click chemistry. This report gives an overview of the structure, functionalities, and use of triazoles with a focus on their use in catalytic systems.     

Diels-Alder cycloaddition for fluorophore targeting to specific proteins inside living cells

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.     

Impregnated copper ferrite on mesoporous graphitic carbon nitride: An efficient and reusable catalyst for promoting ligand‐free click synthesis of diverse 1,2,3‐triazoles and tetrazoles

Magnetic CuFe₂O₄/g‐C₃N₄ hybrids were synthesized through a facile method and their catalytic performances were evaluated in click chemistry for the first time. The structural and morphological characterization of prepared materials was carried out by different techniques such as X‐ray diffraction, high‐resolution transmission electron microscopy, field emission scanning electron microscopy, Fourier infrared spectroscopy, vibrating sample magnetometry, thermogravimetric analysis, and N₂ adsorption–desorption analysis (Brunauer–Emmett–Teller surface area). The utilization of magnetic CuFe₂O₄/g‐C₃N₄ enabled superior performance in the one‐pot azide–alkyne cycloaddition reaction in water using alkyl halides and epoxides as azide precursors without the need of any additional agents. The present system is broad in scope and especially practical for the synthesis of macrocyclic triazoles and also tetrazoles. In addition, the catalytic system highly fulfills the demands of “green click chemistry” with its convenient conditions, especially easy access to a variety of significant products in low catalyst loading and simple work‐up and isolation procedure.