# An Automation Platform for the Chemoenzymatic Synthesis of Complex Sulfated and Branched Glycans

## TL;DR

This paper introduces an automated system for making complex glycans using a mix of enzymes and chemical reactions, enabling the creation of diverse sugar structures important for biological studies.

## Contribution

The novel contribution is an automation platform that enables the chemoenzymatic synthesis of complex sulfated and branched glycans using a modular approach.

## Key findings

- The platform successfully synthesizes sulfated polylactosamines and asymmetric multiantennary N-glycans.
- Only 11 recombinant human transferases were used to generate structurally diverse glycans.
- The system allows for chemical transformations to control enzymatic modification sites.

## Abstract

Diverse collections
of well-defined glycans are needed
to investigate
the molecular mechanisms by which these biomolecules mediate biological
and disease processes. Several automation approaches have been introduced
to accelerate the enzymatic synthesis of complex glycans. These methodologies
have, however, provided only relatively simple oligosaccharides due
to limitations of glycosyl transferase selectivity. Here, we describe
an automation platform that makes it possible, for the first time,
to prepare in an automated fashion sulfated polylactosamines and asymmetric
multiantennary complex N-glycans via sequential enzymatic
and chemical reaction cycles. It integrates glycosyltransferase catalyzed
glycosylations, the use of the unnatural sugar nucleotide donor 5′-diphosphate-2-deoxy-2-trifluoro-N-acetamido-glucose (UDP-GlcNHTFA), and chemical manipulations
including base-mediated trifluoroacetamido (TFA) removal, azido transfer
and azido reduction, tert-butyloxycarbonyl (Boc)
protection, acid mediated deprotection, and amine acylation. The latter
transformations are important for stop-and-go chemoenzymatic
synthetic strategies in which unnatural monosaccharides are introduced
to temporarily disable specific sites from enzymatic modification.
It is shown that, due to the modular architecture of glycans, a limited
number of glycosyl transferases can provide access to large numbers
of structurally diverse glycans. In this study, only 11 recombinant
human glycosyl- and sulfo transferases were employed to prepare highly
complex glycans. Removal of the Nap tag can be performed by hydrogenation
to give oligosaccharides that are ready for microarray printing or
bioconjugation.