Stirring glycopeptides away from the constraints of solid-phase synthesis misconceptions

Glycosylation is one of the most prevalent post-translational modifications of proteins. Synthetic glycopeptides give access to protein fragments with well-defined glycosylation sites, providing a unique route to obtain relevant biochemical information. Since glycosylation can be extremely abundant and appear in different patterns, the assembly of glycopeptide libraries that manifest this variety is required. Several limitations in state-of-the-art solid-phase peptide synthesis make these processes less appropriate for the accelerated preparation of glycopeptides. Our lab has developed a highly-efficient method for glycopeptide synthesis, which employs high-shear mixing at a high temperature to obtain glycopeptides within minutes with minimal waste of building blocks. The development of this new process was not trivial. It encountered synthetic difficulties associated with the complexity of glycan chemistry, which were met by expanding the traditional technological boundaries and challenging common practices. In this perspective, we describe the thought process that has guided us through the development of this method. We illustrate the key role diffusion properties hold for the optimization of reactions and for streamlining and expediting the protocol. We then elaborate on how the ability to question some conceptual bottlenecks associated with SPPS conceptions was pivotal to the success of this project. We compare the presented study with other techniques that aim to accelerate the synthesis of glycopeptides. Finally, we describe the present and future possibilities of the strategy and how they may contribute to expanding the scope of glycopeptide research.