# Expanding the Glycomics toolbox: Chemo-enzymatic synthesis of well-defined N-glycan standards

> **NIH NIH R43** · VIAMUNE, INC. · 2021 · $274,486

## Abstract

Project Summary / Abstract:
Significance: N-linked protein glycosylation represents an important class of biomolecules receiving significant
attention for their application in disease diagnosis and therapeutic development. A major hurdle with rapid and
facile N-glycan structural identification stems from the lack of well-defined reagents that can be used as
bioanalytical reference material. The inability to robustly interpret protein N-glycosylation prevents development
of new technologies for uncovering biomarkers and hinders the advancement of recombinant glycoprotein
engineering, such as antibodies. Current commercially available reference material either provides a limited
number of N-glycans which are not representative of biological composition or lack details regarding exact
structural assembly. Our library will comprise the largest commercially available collection of well-defined N-
glycans and will represent a significant advancement to current available standards. Strategy: Taking advantage
of the strict substrate requirements of glycosyltransferases and hydrolases, an enzymatic approach will be
utilized to produce a collection of high purity structures. Specific Aim: This project describes a robust semi-
synthetic, enzymatic methodology for producing the 20 most abundant N-glycans found in human serum all
having an abundance greater than 1%, and together, comprise greater than 50% of the total serum N-glycome.
Each compound will have a purity of >99% and will contain a free-reducing anomeric terminus providing flexiblity
for use in a multitude of analytical techniques. Task 1: Extraction of a sialylated glycopeptide from commercially
available egg yolk powder and the preparation of an advanced intermediate which is common to all desired
targets. Task 2: Synthesis of bi-antennary targets with symmetric and asymmetric architectures. Task 3:
Synthesize a collection of tri-antennary, sialylated regio-isomers. Task 4: Development of a second product line
where each N-glycan contains one uniformly labeled 13C6 GlcNAc which can be used as an internal reference
for quantification. Preliminary Data: A scalable extraction strategy has been devised for accessing gram-
quantities of the desired sialylated glycopeptide starting material from egg yolk powder using robust extraction
technology. The synthetic methodologies to be employed in tasks 1 – 4 have been validated removing the need
for further optimization. Deliverables: When complete, the described library will represent the most
comprehensive collection of structurally defined N-glycan standards and will deliver an urgently needed tool to
the scientific community.

## Key facts

- **NIH application ID:** 10133101
- **Project number:** 5R43GM137724-02
- **Recipient organization:** VIAMUNE, INC.
- **Principal Investigator:** Anthony Prudden
- **Activity code:** R43 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $274,486
- **Award type:** 5
- **Project period:** 2020-04-01 → 2024-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10133101

## Citation

> US National Institutes of Health, RePORTER application 10133101, Expanding the Glycomics toolbox: Chemo-enzymatic synthesis of well-defined N-glycan standards (5R43GM137724-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10133101. Licensed CC0.

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