Streamlined gram-scale natural N-glycan production using reversible tagging after oxidative release of natural glycans

Abstract

The study of natural glycans is fundamental to modern glycomics; however, the functional analysis of these glycans is limited by their low natural abundance, structural heterogeneity, and the absence of efficient preparative-scale isolation methods. Here, we present a streamlined approach for the gram-scale production of complex natural N-glycans in their reducing form by combining oxidative release of natural glycans (ORNG) using household bleach, an innovative cleavable tag chemistry, and optimized multi-dimensional chromatography. Our ORNG process releases N-glycans from kilogram-scale of natural sources containing various glycoproteins, which can be efficiently attached with a 4-aminobenzoate tag, facilitating the selective separation of these glycans from other biomolecules. The tagged glycans can be purified via dry-load HILIC flash chromatography at gram scale followed by reverse-phase HPLC. Treatment with Oxone quantitatively cleaves the tag, regenerating the free reducing N-glycans in high yield. This method provides an accessible, gram-scale source of complex N-glycans, including complex asymmetric structures that are challenging to synthesize through conventional chemoenzymatic approaches. We believe this approach represents a versatile strategy for acquiring complex natural glycans, opening new avenues for the functional exploration of N-glycans in glycoscience.

Scheme 1

Regeneration of free reducing glycans from 2-aminobenzoic acid derivative-tagged glycans using NBS/Oxone. The reaction of 2-aminobenzoic acid derivative-tagged glycans with NBS/Oxone results in the release of free reducing glycans, accompanied by the formation of side products.

Fig. 1. Man₉GlcNAc₂-OH can be efficiently tagged with 4-aminobenzoic acid derivatives.

a Reaction scheme, b MS results, and (c) HPLC results of Man₉GlcNAc₂-OH tagged with para-aminobenzoic acid (PABA), ethyl 4-aminobenzoate (EPAB), and 2-(diethylamino)ethyl 4-aminobenzoate (DEAB) under standard reductive amination conditions. Green circles, Man; blue squares, GlcNAc.

Fig. 2. Regeneration of free reducing glycans from 4-aminobenzoic acid derivative-tagged glycans using Oxone.

Reaction scheme (a) and MS results (b) of free S2G2-M-OH regeneration from S2G2-M-PABA/EPAB/DEAB by Oxone. Green circles, Man; blue squares, GlcNAc; yellow circles, Gal; purple diamonds, Neu5Ac.

Fig. 3. Large-scale separation of tagged high-mannose glycans from lima beans.

a Overview of the procedure for large-scale separation of tagged high-mannose glycans from lima beans by ORNG method; b Lima beans N-glycan EPAB-conjugates flash chromatography profile on NH2 column; c MS of M5-M9 fractions from flash-HILIC purification; d Preparative HPLC separation profiles of M5-M9 fractions from flash-HILIC purification. 2 to 4 glycan structures were collected from each fractions.

Fig. 4. NMR spectrum of 16 purified high-mannose glycan-EPAB conjugates from lima beans.

We characterized high-mannose glycan-EPAB conjugates from lima beans using routine NMR, including the full assignment of anomeric protons. Green circles, Man; blue squares, GlcNAc; red triangles, Fuc; orange star, Xyl.

Fig. 5. NMR spectrum of selected distinct N-glycans separated from egg white powder.

7 selected N-glycans structures and their assignment of anomeric protons, H2 protons, and NAc protons. Green circles, Man; blue squares, GlcNAc; yellow circles, Gal.

Fig. 6. Preparation of complex-type N-glycans from spray-dried porcine plasma powder.

(a) Flash-DEAE separation profile of EPAB conjugated SDPP N-glycans; (b) C18-HPLC profile of E1-E5 from DEAE separation; (c) MS of purified free reducing glycan of S2G2F and S3G3F. Green circles, Man; blue squares, GlcNAc; yellow circles, Gal; red triangles, Fuc; pink diamonds, Neu5Ac.