Asparagine-linked glycans are involved in complex regulation and signaling, and play a critical role in disease. This is because small differences in this post translational modification dramatically change the function of any biomolecule. To fully unravel and leverage the role of glycosylation in human health, a wide array of glycan standards is required to facilitate this research. There is a critical need to customize N-glycan standards, but the chemical structure of glycans makes this process challenging. This is because glycans are defined by the variation in the type of monomeric saccharide unit, the position of the linkage between adjacent saccharide monomers, and the chain branching. Even simple glycan standards are costly, and this cost increases dramatically with increasing structural complexity. The objective of this project is to bridge this technology gap by providing a tool to customize enzyme- based N-glycan remodeling through an automated nanoscale and microscale approach. A capillary electrophoresis method is adapted to trim and rebuild glycan structures within minutes using a single- capillary or 8-capillary instrument. The heart of this system is a thermally reversible nanogel that sustains and integrates enzyme conversion of a heterogeneous substrate into a standardized glycoform product. Once synthesized, these glycans are easily conjugated to any biomolecule resulting in a custom glycoform. The proposed technology is ideal for small scale processing because the small volume of the 25-100 µm inner diameter fused silica electrophoresis capillary is compatible with the 0.5 to 100 µg quantities of each glycoform needed to create an experimental test set, or even a molecular library. A major advantage to in-line enzyme reactions is that it reduces the volume of a bench top process to the nanoliter regime. By scaling down the volume of the enzyme conversion, diffusion limited processes are less significant. An attractive feature of nanogels is that an enzyme can be pseudo- immobilized within the highly viscous gel without using covalent chemistry to anchor the enzyme. This new strategy for processing of N-linked glycan structures is facilitated through two independent approaches that differ in the quantity of product that is made. Aim 1 creates a discrete stepwise modification through the successive use of trimming (exoglycosidase) and building (transferase) enzymes. This enables automated production of microgram quantities of research grade N-glycans. Aim 2 transfers and scales up the enzymatic processing using continuous feed and parallel reaction capillaries. The proposed activities are significant because the speed and automation of the electrophoresis-based foundry yield previously unattainable flexibility in chemical processing. This new tool provides the standards needed for individual researchers to obtain direct information about the relationship between complex variations in glycosylation and physiological effect.

Project Narrative This proposal addresses the compelling need for new strategies to customize the glycosylation of biological standards. Enabling technology that overcomes current limitations in the field will allow scientists to create biomolecules in support of research on human health.