Efficient Chemoenzymatic Synthesis of N-Glycans with a β1,4-Galactosylated Bisecting GlcNAc Motif

Abstract

In human serum immunoglobulin G (IgG), a rare modification of biantennary complex N-glycans lead to a β1,4-galactosylated bisecting GlcNAc branch. We found that the bisecting GlcNAc on a biantennary core-fucosylated N-glycan was enzymatically galactosylated under stringent reaction conditions. Further optimizations led to an efficient enzymatic approach to this particular modification for biantennary substrates. Notably, tri- and tetra-antennary complex N-glycans were not converted by bovine galactosyltransferase. An N-glycan with a galactosylated bisecting GlcNAc was linked to a lanthanide binding tag. The pseudo-contact shifts (PCS) obtained from the corresponding Dy-complex were used to calculate the conformational preferences of the rare N-glycan. Besides two extended conformations only a single folded conformation was found.

Keywords: N-glycans; glycobiology; glycosylation; immunoglobulin; paramagnetic NMR spectroscopy.

Scheme 1

Biantennary N‐glycan with unmodified bisecting GlcNAc (A); complex N‐glycans found on human serum IgG bearing a β1,4‐galactosylated bisecting GlcNAc motif (B, C); hybrid N‐glycans from 19 A glycoprotein with a galactosylated bisecting GlcNAc motif (D, E).

Scheme 2

Synthesis of N‐glycan 3, deprotection to hemiacetal 4 and enzymatic galactosylation to 5 and 6. The numbering of the residues for NMR assignments is shown for 6.

Scheme 3

The panel of bisected N‐glycan azides (7–11) was enzymatically galactosylated under dilute (1.) and subsequently under stringent (2.) conditions. Transfer of an additional galactose to the bisecting GlcNAc was observed only in the case of the biantennary N‐glycans 17 and 18. (n. d.=not detected).

Scheme 4

Synthesis of the trigalactosylated bisected N‐glycan azide 17, conjugation with lanthanide binding tag 18, deprotection and formation of the diamagnetic complex 20  a and the paramagnetic complex 20  b.

Scheme 5

A), B) HSQC sections of the anomeric region of the diamagnetic (20  a) and paramagnetic complex (20  b); the three galactose signals are within the dotted lines. C) PCS of paramagnetic complex 20  b. D) Relative percentages of the main conformations calculated from PCS.