Triple Orthogonal Labeling of Glycans by Applying Photoclick Chemistry

Abstract

Bioorthogonal labeling of multiple biomolecules is of current interest in chemical biology. Metabolic glycoengineering (MGE) has been shown to be an appropriate approach to visualizing carbohydrates. Here, we report that the nitrile imine–alkene cycloaddition (photoclick reaction) is a suitable ligation reaction in MGE. Using a mannosamine derivative with an acrylamide reporter group that is efficiently metabolized by cells and that quickly reacts in the photoclick reaction, we labeled sialic acids on the surface of living cells. Screening of several alkenes showed that a previously reported carbamate‐linked methylcyclopropene reporter that is well suited for the inverse‐electron‐demand Diels–Alder (DAinv) reaction has a surprisingly low reactivity in the photoclick reaction. Thus, for the first time, we were able to triply label glycans by a combination of DAinv, photoclick, and copper‐free click chemistry.

Advanced Bottom‐Up Engineering of Living Architectures

“…Cells with norbornene‐bearing surfaces have now been successfully generated, which could allow for light‐enabled precise spatial control of cell–cell interactions . However, although norbornene‐sugar incorporation efficiency is still limited in present attempts, other photocrosslinkable groups have shown promising results (i.e., acrylamide) . Cells exhibiting triple‐orthogonal surface engineering have also been reported, which could allow for more complex heterogenous configurations of cell–cell interactions .…”

“…However, although norbornene‐sugar incorporation efficiency is still limited in present attempts, other photocrosslinkable groups have shown promising results (i.e., acrylamide) . Cells exhibiting triple‐orthogonal surface engineering have also been reported, which could allow for more complex heterogenous configurations of cell–cell interactions . Alternatively, cell surface‐grafting strategies are starting to emerge, either as controlled radical polymerization or anchoring telechelic synthetic polymers, in order to re‐engineer cell surface functionality and interactions …”

Design and synthesis of metabolic chemical reporters for the visualization and identification of glycoproteins

“…HEK293T cells treated with all three molecules at once showed independent labeling of each, demonstrating that all three reactions can occur simultaneously with limited background. 102 Some salvage pathways are so tolerant of structural changes that the same sugar can be functionalized with two different bioorthogonal handles. Feng et al demonstrated this possibility with 9AzSiaNAlk and 9AzSiaNAz.…”

Chemical reporters to study mammalian O-glycosylation

“…Moving beyond the metabolic labelling of single types of monosaccharides, several groups have explored combinations of multiple bioorthogonal ligations for the simultaneous labelling of two glycan populations on the same cells [ 48 , 56 , 58 ]. Building on the latest advances in bioorthogonal chemistry, a triple labelling of cell surface glycans using ManNAc derivatives with three different tags has also been described [ 59 ]. The Vocadlo group used a dual bioorthogonal strategy to verify the existence of co-translational O-GlcNAcylation, a modification that had thus far been believed to occur only post-translationally [ 60 ].…”