Abstract
The O-linked β-N-acetylglucosamine (O-GlcNAc) modification is an abundant post-translational modification in eukaryotic cells, which plays a fundamental role in the activity of many cells and is associated with pathologies like type II diabetes, Alzheimer’s disease or some cancers. However, the precise connexion between O-GlcNAc-modified proteins and their function in cells is largely undefined for most cases. Confocal microscopy is a powerful and effective tool for in-cell elucidation of the function of biological molecules. Chemical labeling of non-ultraviolet or non-fluorescent carbohydrates with fluorescent tag is an essential step that makes intra-cellular microscopic inspection possible. Here we report a strategy based on the 1,3-dipolar cycloaddition, called click chemistry, between unnatural N-acetylglucosamine (GlcNAc) analogues Ac4GlcNAc (substituted with an azido group) and the corresponding fluorescent tag Ru(bpy)2(Phen-alkyne)Cl2 (4) to synthesize the fluorescent dye Ru(bpy)2(Phen-Ac4GlcNAc)Cl2 (5) under mild and neutral reaction conditions. Moreover, 5 showed good stability, desirable fluorescence characteristics, and exhibited rather low levels of cytotoxicity against sensitive MCF-7 cells. Additionally, we have achieved successful fluorescent imaging of 5 transported in living MCF-7 cells. Cell images displayed that proteins are potentially labelled with 5 in the cytoplasm.
Highlights
O-Linked β-N-acetylglucosamine (O-GlcNAc) glycosylation is an important regulation for the diverse cellular processes including transcription, cell division, and glucose homeostasis [1,2,3,4,5]
Compound 5 was synthesized by copper-catalyzed azide-alkyne cycloaddition (CuAAC)
Compound 5 was synthesized by copper-catalyzed azide-alkyne cycloaddition reaction of alkyne-dye 4 and azide-based O-GlcNAc glycosylation metabolite Ac4 GlcNAz (2)
Summary
O-Linked β-N-acetylglucosamine (O-GlcNAc) glycosylation is an important regulation for the diverse cellular processes including transcription, cell division, and glucose homeostasis [1,2,3,4,5].understanding the glycosylation of proteins is critical to elucidate the functional roles of proteins within the dynamic environment of cells in physiological and diseased states [6,7,8,9]. Due to the lack of specificity and selectivity of the antibody used, only a limited number of O-GlcNAc proteins can be detected [10] With this regard, great challenge remains to develop simple but efficient methods to monitor non-ultraviolet or non-fluorescent O-GlcNAc glycosylation in response to cellular stimuli [11]. Microscopic inspection is a powerful and widely used tool for high-resolution and non-invasive imaging of target molecules in their native environments [12]. With spectroscopic probes such as alkyne-functionalized rhodamine or 1,8-naphthalimide derivatives, imaging of O-GlcNAc proteins in Molecules 2018, 23, 1649; doi:10.3390/molecules23071649 www.mdpi.com/journal/molecules
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