Besides nucleic acids, proteins and lipids, glycoconjugates are among the core building blocks of all living matter. They play a pivotal role in numerous physiological processes ranging from cell development, intracellular interactions, signaling to pathogenesis and infection. The complexity of sugars is reflected in the enormous diversity of glycoconjugates found in nature. It is this complexity, which makes their studies extremely challenging. The development of new tools helping us to unravel their structure and understand their function is therefore of high demand. The main goal of this work was to design and synthesize modified analogs of various sugars which, after metabolic incorporation, would allow for the visualization of the newly synthesized glycoconjugates using selective click labeling reaction. In this regard, we have succeeded in the synthesis of per-acetylated N-galacto, N-gluco and N-mannosamine derivatives containing 1,2,4-triazine reacting group. Optimization of the synthesis and purification steps was necessary to obtain the desired derivatives in sufficient purity. Our kinetic studies revealed that the 1,2,4-triazine moiety can react efficiently with trans-cyclooctene dienophiles. The successful incorporation of the triazine-containing sugars was verified upon reaction with trans-cyclooctene modified biotin probe and subsequent visualization of the click-conjugate using fluorescently labeled streptavidin. These experiments revealed that using our newly synthesized sugar derivatives it is possible to visualize intracellular glycoconjugates but not cell surface-associated ones. The major advantages of our method are fast reaction rates and no need for additional catalysis. This work is thus an important addition to already existing chemical biology tools enabling study of glycoconjugates and also a good starting point for future studies related to the use of 1,2,4-triazines as useful reagents in bioorthogonal reactions.
The main goal of this work was to design and synthesize modified analogs of various sugars which, after metabolic incorporation, would allow for the visualization of the newly synthesized glycoconjugates using selective click labeling reaction.