Synthetic and semi-synthetic approaches to unprotected N-glycan oxazolines

Abstract

N-Glycan oxazolines have found widespread use as activated donor substrates for endo-β-N-acetylglucosaminidase (ENGase) enzymes, an important application that has correspondingly stimulated interest in their production, both by total synthesis and by semi-synthesis using oligosaccharides isolated from natural sources. Amongst the many synthetic approaches reported, the majority rely on the fabrication (either by total synthesis, or semi-synthesis from locust bean gum) of a key Manβ(1-4)GlcNAc disaccharide, which can then be elaborated at the 3- and 6-positions of the mannose unit using standard glycosylation chemistry. Early approaches subsequently relied on the Lewis acid catalysed conversion of peracetylated N-glycan oligosaccharides produced in this manner into their corresponding oxazolines, followed by global deprotection. However, a key breakthrough in the field has been the development by Shoda of 2-chloro-1,3-dimethylimidazolinium chloride (DMC), and related reagents, which can direct convert an oligosaccharide with a 2-acetamido sugar at the reducing terminus directly into the corresponding oxazoline in water. Therefore, oxazoline formation can now be achieved in water as the final step of any synthetic sequence, obviating the need for any further protecting group manipulations, and simplifying synthetic strategies. As an alternative to total synthesis, significant quantities of several structurally complicated N-glycans can be isolated from natural sources, such as egg yolks and soy bean flour. Enzymatic transformations of these materials, in concert with DMC-mediated oxazoline formation as a final step, allow access to a selection of N-glycan oxazoline structures both in larger quantities and in a more expedient fashion than is achievable by total synthesis.

Keywords: DMC, ENGase; N-glycans; glycosyl oxazolines; oligosaccharides.

Scheme 1

The first ENGase-catalysed glycosylation of a GlcNAc acceptor using an N-glycan oxazoline as donor.

Scheme 2

Production of N-glycan oxazolines from peracetylated sugars using Lewis acids.

Scheme 3

Direct conversion of unprotected GlcNAc to a glycosyl oxazoline by treatment with DMC and Et₃N in water.

Scheme 4

Total synthesis of a truncated complex biantennary N-glycan oxazoline via an epimerisation approach and Lewis acid mediated oxazoline formation.

Scheme 5

Wangs’s total synthesis of an N-glycan oxazoline incorporating click handles, employing Crich direct β-mannosylation.

Scheme 6

Wangs’s total synthesis of an N-glycan dodecasaccharide oxazoline employing final step oxazoline formation with DMC.

Scheme 7

Production of a phosphorylated N-glycan oxazoline, employing final step oxazoline formation with DMC.

Scheme 8

Enzymatic degradation of locust bean gum, and chemical conversion into an N-glycan dodecasaccharide oxazoline.

Scheme 9

Production of a complex biantennary N-glycan oxazoline from hens’ eggs by semi-synthesis via isolation of SGP, enzymatic degradation, and final stage oxazoline formation.

Scheme 10

Production of a high mannose (Man-9) N-glycan oxazoline from soy bean flour.

Scheme 11

Production of a triantennary N-glycan oxazoline from bovine feruin by semi-synthesis.