Multi-Tag: A modular platform of bioorthogonal probes for multi-modal (glyco)protein analysis

ABSTRACT Bioorthogonal chemistry is a well-established concept for tagging and analyzing targets of interest even in living cells, tissue or organisms. In particular glycans, which are, as a posttranslational modification, not amenable to genetic engineering, became analytically accessible through the establishment of metabolic oligosaccharide engineering and subsequent bioorthogonal tagging of chemical probes. Since many essential cellular processes involve glycoproteins, it is not surprising that especially aberrant glycosylation has been associated with the pathology of many diseases. Investigation of aberrant glycosylation in a disease background is complicated by the heterogeneity of glycans and dynamic changes in their composition. In order to create a meaningful information depth, it can be beneficial to analyze the same sample with different analytical methods. This becomes even more relevant for samples with limited access. Most of the currently existing bioorthogonal probes are designed for use in only one type of experiment. These design restrictions are mainly based on the limited synthetic accessibility of more complex bioorthogonal probes. Multi-step syntheses are often time consuming and cost-inefficient. Here, we introduce a fast and easily manageable strategy for the synthesis of complex bioorthogonal probes that allow an application in multiple coordinated experiments. Using established principles and conditions of solid-phase peptide synthesis, we combine different functional building blocks to generate multi-functional bioorthogonal probes (named Multi-Tag s). We show the easy synthesis of several multi-modal probes and demonstrate their applicability and versatility in exemplary assays.