Synthesis of asymmetrical multiantennary human milk oligosaccharides

Abstract

Despite mammalian glycans typically having highly complex asymmetrical multiantennary architectures, chemical and chemoenzymatic synthesis has almost exclusively focused on the preparation of simpler symmetrical structures. This deficiency hampers investigations into the biology of glycan-binding proteins, which in turn complicates the biomedical use of this class of biomolecules. Herein, we describe an enzymatic strategy, using a limited number of human glycosyltransferases, to access a collection of 60 asymmetric, multiantennary human milk oligosaccharides (HMOs), which were used to develop a glycan microarray. Probing the array with several glycan-binding proteins uncovered that not only terminal glycoepitopes but also complex architectures of glycans can influence binding selectivity in unanticipated manners. N- and O-linked glycans express structural elements of HMOs, and thus, the reported synthetic principles will find broad applicability.

Keywords: chemoenzymatic synthesis; glycosyltransferases; human milk oligosaccharides; protein–glycan interactions.

Fig. 1.

Structural diversity of HMOs. (A) Simple HMOs. (B) Structural diversity of multiantennary HMOs. (C) Enzymatic strategy for the synthesis of asymmetric multiantennary HMOs. (D) Example of a chemical structure of an asymmetric multiantennary HMO prepared by the enzymatic approach.

Fig. 2.

Lactose linker conjugations and enzymatic synthesis of HMOs. (A) Lactose-linker complex. (B) Introducing structural asymmetry. (C) Extended poly-LacNAc β6 branch. (D) Selective fucosylation. Reagents and conditions: a, 0.25 M CH₃CO₂Na, pH 4.2, 37 °C; b, B3GNT2, UDP-GlcNAc; c, GalT1, UDP-Gal; d, GCNT2, UDP-GlcNAc; e, ST6GAL1, CMP-Neu5Ac; f, FUT1, GDP-Fuc; g, B3GALT5, UDP-Gal; h, FUT3, GDP-Fuc; and i, FUT5, GDP-Fuc.

Fig. 3.

Monosaccharide directing groups for selective Le^x installation. Reagents and conditions: a, ST6GAL1, CMP-Neu5Ac; b, FUT3, GDP-Fuc; c, sialidase from Arthrobacter ureafaciens; d, FUT1, GDP-Fuc; and e, microbial α1–2 fucosidase.

Fig. 4.

Synthesis of an asymmetric, triantennary HMO. Reagents and conditions: a, GCNT2, UDP-GlcNAc; b, FUT1, GDP-Fuc; c, GalT1, UDP-Gal; and d, B3GNT2, UDP-GlcNAc.

Fig. 5.

HMO library members. The compounds are organized according to an increase in complexity. B, biantennary glycan; L, linear glycan; and T, triantennary glycan. See SI Appendix, Table S2, for correlation of library identifier with compound synthesis number.

Fig. 6.

Screening of the HMO library. Microarray results of the HMO library at 100 µM. (A) Galectin-9 (3 µg⋅mL⁻¹). (B) V. cholera toxin subunit B₅ (100 µg⋅mL⁻¹). (C) Porcine rotaviral strain CRW-8 VP8* (200 µg⋅mL⁻¹). RFU, relative fluorescence units.