Strain-promoted alkyne-azide cycloadditions (SPAAC) reveal new features of glycoconjugate biosynthesis

We have shown that 4-dibenzocyclooctynol (DIBO), which can easily be obtained by a streamlined synthesis approach, reacts exceptionally fast in the absence of a Cu(I) catalyst with azido-containing compounds to give stable triazoles. Chemical modifications of DIBO, such as oxidation of the alcohol to a ketone, increased the rate of strain promoted azide-alkyne cycloadditions (SPAAC). Installment of a ketone or oxime in the cyclooctyne ring resulted in fluorescent active compounds whereas this property was absent in the corresponding cycloaddition adducts; this provides the first example of a metal-free alkyne-azide fluoro-switch click reaction. The alcohol or ketone functions of the cyclooctynes offer a chemical handle to install a variety of different tags, and thereby facilitate biological studies. It was found that DIBO modified with biotin combined with metabolic labeling with an azido-containing monosaccharide can determine relative quantities of sialic acid of living cells that have defects in glycosylation (Lec CHO cells). A combined use of metabolic labeling/SPAAC and lectin staining of cells that have defects in the conserved oligomeric Golgi (COG) complex revealed that such defects have a greater impact on O-glycan sialylation than galactosylation, whereas sialylation and galactosylation of N-glycans was similarly impacted. These results highlight the fact that the fidelity of Golgi trafficking is a critical parameter for the types of oligosaccharides being biosynthesized by a cell. Furthermore, by modulating the quantity of biosynthesized sugar nucleotide, cells might have a means to selectively alter specific glycan structures of glycoproteins.     

An Extended Approach for the Development of Fluorogenic trans-Cyclooctene–Tetrazine Cycloadditions

Inverse-electron-demand Diels–Alder (iEDDA) cycloaddition between 1,2,4,5-tetrazines and strained dienophiles belongs among the most popular bioconjugation reactions. In addition to its fast kinetics, this cycloaddition can be tailored to produce fluorescent products from non-fluorescent starting materials. Here we show that even the reaction intermediates formed in iEDDA cycloaddition can lead to the formation of new types of fluorophores. The influence of various substituents on their photophysical properties and the generality of the approach with use of various trans-cyclooctene derivatives were studied. Model bioimaging experiments demonstrate the application potential of fluorogenic iEDDA cycloaddition.     

In Vivo Targeting through Click Chemistry

Targeting small molecules to diseased tissues as therapy or diagnosis is a significant challenge in drug delivery. Drug‐eluting devices implanted during invasive surgery allow the controlled presentation of drugs at the disease site, but cannot be modified once the surgery is complete. We demonstrate that bioorthogonal click chemistry can be used to target circulating small molecules to hydrogels resident intramuscularly in diseased tissues. We also demonstrate that small molecules can be repeatedly targeted to the diseased area over the course of at least one month. Finally, two bioorthogonal reactions were used to segregate two small molecules injected as a mixture to two separate locations in a mouse disease model. These results demonstrate that click chemistry can be used for pharmacological drug delivery, and this concept is expected to have applications in refilling drug depots in cancer therapy, wound healing, and drug‐eluting vascular grafts and stents.     

Nucleic Acid Bioconjugates in Cancer Detection and Therapy

Nucleoside‐ and nucleotide‐based chemotherapeutics have been used to treat cancer for more than 50 years. However, their inherent cytotoxicities and the emergent resistance of tumors against treatment has inspired a new wave of compounds in which the overall pharmacological profile of the bioactive nucleic acid component is improved by conjugation with delivery vectors, small‐molecule drugs, and/or imaging modalities. In this manner, nucleic acid bioconjugates have the potential for targeting and effecting multiple biological processes in tumors, leading to synergistic antitumor effects. Consequently, tumor resistance and recurrence is mitigated, leading to more effective forms of cancer therapy. Bioorthogonal chemistry has led to the development of new nucleoside bioconjugates, which have served to improve treatment efficacy en route towards FDA approval. Similarly, oligonucleotide bioconjugates have shown encouraging preclinical and clinical results. The modified oligonucleotides and their pharmaceutically active formulations have addressed many weaknesses of oligonucleotide‐based drugs. They have also paved the way for important advancements in cancer diagnosis and treatment. Cancer‐targeting ligands such as small‐molecules, peptides, and monoclonal antibody fragments have all been successfully applied in oligonucleotide bioconjugation and have shown promising anticancer effects in vitro and in vivo. Thus, the application of bioorthogonal chemistry will, in all likelihood, continue to supply a promising pipeline of nucleic acid bioconjugates for applications in cancer detection and therapy.     

Metabolic Remodeling of Cell‐Surface Sialic Acids: Principles,Applications, and Recent Advances

Cell‐surface sialic acids are essential in mediating a variety of physiological and pathological processes. Sialic acid chemistry and biology remain challenging to investigate, demanding new tools for probing sialylation in living systems. The metabolic glycan labeling (MGL) strategy has emerged as an invaluable chemical biology tool that enables metabolic installation of useful functionalities into cell‐surface sialoglycans by “hijacking” the sialic acid biosynthetic pathway. Here we review the principles of MGL and its applications in study and manipulation of sialic acid function, with an emphasis on recent advances.     

Site-Specific Small Molecule Labeling of an Internal Loop in JC Polyomavirus Pentamers Using the π-Clamp-Mediated Cysteine Conjugation

The major capsid protein VP1 of JC Polyomavirus assembles into pentamers that serve as a model for studying viral entry of this potentially severe human pathogen. Previously, labeling of viral proteins utilized large fusion proteins or non-specific amine- or cysteine-functionalization with fluorescent dyes. Imaging of these sterically hindered fusion proteins or heterogeneously labeled virions limits reproducibility and could prevent the detection of subtle trafficking phenomena. Here we advance the π-clamp-mediated cysteine conjugation for site-selective fluorescent labeling of VP1-pentamers. We demonstrate a one-step synthesis of a probe consisting of a bio-orthogonal click chemistry handle bridged to a perfluoro-biphenyl π-clamp reactive electrophile by a polyethylene glycol linker. We expand the scope of the π-clamp conjugation by demonstrating selective labeling of an internal, surface exposed loop in VP1. Thus, the π-clamp conjugation offers a general method to selectively bioconjugate tags-of-interest to viral proteins without impeding their ability to bind and enter cells.     

Modular Enzyme- and Light-Based Activation of Cyclopropene–Tetrazine Ligation

We describe a modular activation strategy for cyclopropene–tetrazine ligation. This activation strategy uses chemically diverse enzyme- or photolabile protecting groups as cyclopropene reactivity cages. The linkages between the caging groups and cyclopropene are through carbamates, thus permitting the application of diverse cages to allow bioorthogonal reactivity by administering enzymes or light.     

Phospha‐Michael Addition as a New Click Reaction for Protein Functionalization

A new type of click reaction between an alkyl phosphine and acrylamide was developed and applied for site‐specific protein labeling in vitro and in live cells. Acrylamide is a small electrophilic olefin that readily undergoes phospha‐Michael addition with an alkyl phosphine. Our kinetic study indicated a second‐order rate constant of 0.07 m ^?1 s^?1 for the reaction between tris(2‐carboxyethyl)phosphine and acrylamide at pH 7.4. To demonstrate its application in protein functionalization, we used a dansyl–phosphine conjugate to successfully label proteins that were site‐specifically installed with N^?‐acryloyl‐l ‐lysine and employed a biotin–phosphine conjugate to selectively probe human proteins that were metabolically labeled with N‐acryloyl‐galactosamine.