To View Your Biomolecule, Click inside the Cell

Abstract

The surging development of bioorthogonal chemistry has profoundly transformed chemical biology over the last two decades.I nvolving chemical partners that specifically react together in highly complex biological fluids,t his branch of chemistry nowa llows researchers to probe biomolecules in their natural habitat through metabolic labelling technologies.C hemical reporter strategies include metabolic glycan labelling,s ite-specific incorporation of unnatural amino acids in proteins,and post-synthetic labelling of nucleic acids.W hile amajority of literature reports mark cell-surface exposed targets, implementing bioorthogonal ligations in the interior of cells constitutes amore challenging task. Owing to limiting factors such as membrane permeability of reagents,fluorescence background due to hydrophobic interactions and off-target covalent binding, and suboptimal balance between reactivity and stability of the designed molecular reporters and probes,these strategies need mindful planning to achieve success. In this review,w ediscuss the hurdles encountered when targeting biomolecules localized in cell organelles and give an easily accessible summary of the strategies at hand for imaging intracellular targets.

Switching azide and alkyne tags on bioorthogonal reporters in metabolic labeling of sialylated glycoconjugates: a comparative study

“…Here we show that ManNAz is a good alternative for the labeling of cell surface sialylated GCs, especially for the colon cell lines whose proliferation is sensitive to high concentration of Ac 4 ManNAz (≥ 100 µM). It may come from the perturbation of intracellular pH due to acetic acid release into cells, and the cross-reactivity with cysteine residues of cytosolic proteins 23,28 . In addition, Ac 4 ManNAz metabolic incorporation results in a diffuse fluorescence in the cytoplasm and nucleus of HT29 and HCT116 cells.…”

“…1). Cytosolic UDP-GlcNAz could be further used for O-GlcNAc modification proteins catalyzed by the nuclear and cytosolic enzyme, O-GlcNAc transferase (OGT) 23 . Interestingly, OGT is overexpressed in colon cancer cells 24 ; this could explain in part the higher cytosolic and nuclear signal observed in the two cancer cell lines cultured with Ac 4 ManNAz, compared to the non-cancerous CCD841 cell line.…”

“…CuAAC is the only bioorthogonal reaction in which alkynes and azides can be introduced indifferently either on the metabolic analog or on the probe for the ligation, making this reaction very flexible and adaptable to various labeling strategies. In contrast, only azides are adapted for a bioorthogonal ligation via SPAAC reaction, which is easier to apply in living systems than CuAAC due to the absence of copper associated cytotoxicity, but generally suffers from poorer kinetics and specificity especially in the intracellular environment 23 . Albeit numerous works using MOE to label sialylated glycans have been reported, the comparison of alkyne-or azidefunctionalized ManNAc and Sia free monosaccharides has not been done so far.…”