Almost all eukaryotic cell surfaces and secreted proteins are modified by complex carbohydrates, known as glycans, which regulate processes ranging from protein folding and cell–cell communication to immune responses and fertilization. Aberrant glycosylation is linked to inflammation, autoimmunity, allergy, and cancer. Diverse, well-defined glycan collections are essential for studying glycan-binding proteins, serving as reference standards for glycan structure determination, and acting as probes for glycoconjugate biosynthesis. Large, well-defined glycan libraries are increasingly important for Glycomics, Glycan Profiling, and Glycoproteomics.

This article highlights a study by Chakraborty, Minder, Prudden, and Boons published in the Journal of the American Chemical Society (2026) that introduces the first automation platform capable of preparing sulfated polylactosamines and asymmetric multiantennary complex N-glycans by integrating enzymatic glycosylation with chemical manipulation in sequential cycles.

The Challenge of Automated Glycan Synthesis

Chemical oligosaccharide synthesis faces a lack of general glycosylation protocols; in particular, the installation of 1,2-cis linkages often produces anomeric mixtures that are difficult to resolve after multiple cycles. Enzymatic synthesis using glycosyltransferases offers regio- and stereoselectivity and broad substrate scope, but automated implementations have been limited to relatively simple oligosaccharides. Immobilized-enzyme microfluidic reactors, one-pot multienzyme cascades, and a prior sulfonate-based catch-and-release tag each encounter limitations: enzymes are fragile, buffer systems cannot be flexibly switched, and the basic elution conditions required for product release are incompatible with base-sensitive functional groups needed for stop-and-go strategies. As a result, asymmetrically branched glycans and highly charged structures such as sulfated glycans could not previously be prepared automatically.

A Nap-Tag Catch-and-Release Strategy

The new platform replaces the sulfonate tag with an anomeric 2-naphthylmethyl (Nap) tag. The Nap group provides sufficient hydrophobicity to capture oligosaccharides of widely varying complexity, including charged derivatives, on a reverse-phase C18 resin. Washing with water removes buffer salts, excess sugar nucleotide, and all reaction components, while elution with 40% aqueous acetonitrile releases the product into a small volume. The neutral catch-and-release conditions are compatible with the base-labile trifluoroacetamido (TFA) groups central to stop-and-go synthesis. A custom 3D-printed evaporation zone removes acetonitrile under airflow, leaving the product in water ready for the next cycle.

Three standardized purification modules are used depending on the step. SPE1 combines Ni-NTA capture of the His6-tagged enzyme with C18 catch-and-release for enzymatic reactions. SPE2 uses only C18 catch-and-release for aqueous chemical transformations. SPE3 adds an evaporation step before C18 capture to remove organic solvent and prevent premature product elution. A single set of catch-and-release conditions thus serves glycans ranging from a dimer to a septendecimer.

Fig. 1 (a) Sulfonate Catch and Release Tag for Automated Enzymatic Synthesis of Complex Glycan; (b) Versatile 2-Naphthylmethyl Tag for the Chemoenzymatic Synthesis of Complex Glycans; (c) All SPE Modules. (Chakraborty, et al. 2026)

Automated Workflow on a Glycosynthesizer

The workflow runs on a Chemspeed ISYNTH AI Swing workstation controlled through Autosuite software. A robotic arm transfers liquid between a cooled reagent rack, a temperature-controlled reactor block, and an SPE cartridge rack without manual intervention. A cooled rack stores sensitive enzymes and sugar nucleotides, and the integrated evaporation zone provides continuous airflow with heating to remove volatile organic solvents. The platform processes up to 30 mg of material and can perform parallel reactions, offering a route to larger-scale synthesis.

Fig. 2 Schematic representation of the glycosynthesizer based on a Chemspeed AI Swing Isynth platform. (Chakraborty, et al. 2026)

Chemical Transformations for Stop-and-Go Synthesis

To build asymmetric architectures, the platform introduces the unnatural donor UDP-GlcNHTFA. Enzymatic incorporation yields a GlcNHTFA residue whose TFA group is removed by mild base to give GlcNH2, which resists further enzymatic modification and acts as a stopping point. The free amine can be converted into an azide (GlcN3) by imidazole-1-sulfonyl azide, protected as a Boc derivative, or reacetylated to the natural GlcNAc at the appropriate stage. Azide reduction with trimethylphosphine and Boc deprotection with trifluoroacetic acid are also performed inline. These orthogonal chemical handles let a single branching point be masked, elaborated elsewhere, and then unmasked for selective extension.

Automated Synthesis of Sulfated Glycans

The prior sulfonate tag could not handle highly charged glycans because of excessive binding to the ion-exchange resin. The Nap tag overcomes this. Starting from a common intermediate, the team automated the synthesis of sulfated polyLacNAc structures, exploiting the mutual exclusivity of α1,3-fucosides and galactose 6-sulfates. Sequential use of glycosyltransferases and sulfotransferases, with CHST2 and CHST1 providing regioselective 6-O-sulfation, delivered disulfated glycans in high per-step yield. Structural integrity was confirmed by two-dimensional NMR, which resolved the characteristic downfield shifts of 6-O-sulfated residues and α2,3-linked sialic acids.

Fig. 3 Automated Synthesis of Sulfated polyLacNAc Derivatives. (Chakraborty, et al. 2026)

Automated Synthesis of Asymmetric N-Glycans

The platform was extended to asymmetrically branched N-Glycans. A bi-antennary glycan with distinct α1,3- and α1,6-antennae was assembled in 11 automated cycles with an average yield of 93% per step, using GlcNH2 as a stopping point to direct selective extension. A second bi-antennary target, selectively fucosylated on a tri-LacNAc chain, required 17 cycles across SPE1, SPE2, and SPE3, with GlcNH2 and GlcN3 serving as orthogonal stopping points, and reached an average yield of about 86% per step. The most demanding demonstration was a tri-antennary N-glycan carrying a unique appendage at each antenna, built from an intermediate bearing GlcNAc, GlcNH2, and GlcN3 as complementary noncanonical residues; the 15-cycle sequence gave an average yield of 89% per step. In total, only 11 recombinant human glycosyl- and sulfo-transferases were required to access these complex structures.

Fig. 4 Automated Chemoenzymatic Synthesis of an Asymmetrical Bi-Antennary Glycan Using a Stop-and-Go Strategy and SPE1 and SPE2 Modules. (Chakraborty, et al. 2026)

Outlook

This work establishes the first automation platform that couples enzymatic and chemical steps for glycan assembly, enabling sulfated polyLacNAcs and asymmetric multiantennary N-glycans to be prepared without manual intervention. The modular architecture of glycans means a small set of transferases can generate large structural diversity, and double couplings and optional offline LC-MS checks make multistep syntheses reliable. The authors note that future integration of automated LC-MS feedback control and machine-learning-based Synthesis planning could further streamline the platform. Expansion of the available noncanonical sugar nucleotides and engineered glycosyltransferases will broaden the accessible chemical space. Removal of the Nap tag by hydrogenation yields oligosaccharides ready for microarray printing or bioconjugation.

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• N-Glycan Profiling Service
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• Glycan Separation and Purification Service
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• Glycomic Characterization Service
• Custom Carbohydrate Synthesis
• Glycan Microarray Assay
• Oligosaccharide Analysis Service
• Glycoprotein Quantification

Reference

1. Chakraborty, S., et al. (2026). An Automation Platform for the Chemoenzymatic Synthesis of Complex Sulfated and Branched Glycans. Journal of the American Chemical Society. DOI: 10.1021/jacs.5c22181.