Chemoenzymatic synthesis of glycopeptides and glycoproteins through endoglycosidase-catalyzed transglycosylation

Abstract

Homogeneous glycopeptides and glycoproteins are indispensable for detailed structural and functional studies of glycoproteins. It is also fundamentally important to correct glycosylation patterns for developing effective glycoprotein-based therapeutics. This review discusses a useful chemoenzymatic method that takes advantage of the endoglycosidase-catalyzed transglycosylation to attach an intact oligosaccharide to a polypeptide in a single step, without the need for any protecting groups. The exploration of sugar oxazolines (enzymatic reaction intermediates) as donor substrates has not only expanded substrate availability, but also has significantly enhanced the enzymatic transglycosylation efficiency. Moreover, the discovery of a novel mutant with glycosynthase-like activity has made it possible to synthesize homogeneous glycoproteins with full-size natural N-glycans. Recent advances in this highly convergent chemoenzymatic approach and its application for glycopeptide and glycoprotein synthesis are highlighted.

Figure 1

ENGase-catalyzed hydrolysis and transglycosylation.

Figure 2

Substrate-assisted mechanism for ENGase-catalyzed reactions.

Figure 3

Time-course of the transglycosylation reactions using Manp_sGlcpNAc-oxazoline as the donor substrate. The reactions were carried out in a phosphate buffer (pH 6.6) at 30 °C, using the GlcNpAc-pentapeptide as an acceptor (molar ratio of donor to acceptor, 2:1).

Figure 4

Preparation of GlcpNAc-functionalized proteins.

Scheme 1

ENGase-catalyzed synthesis of a gp120 fragment carrying the core trisaccharide.

Scheme 2

ENGase-catalyzed transglycosylation with the di- and tetrasacharide oxazolines.

Scheme 3

Chemoenzymatic synthesis of a cyclic V3 glycopeptide carrying two N-glycans.

Scheme 4

Endo-A-catalyzed transglycosylation with modified sugar oxazolines.

Scheme 5

Endo-M catalyzed transglycosylation with modified sugar oxazolines.

Scheme 6

Semi-synthesis of Manp₉GlcpNAc oxazoline.

Scheme 7

Transglycosylation with Endo-M and its mutants using Manp₉GlcpNAc-oxazoline.

Scheme 8

Enzymatic synthesis of a HIV-1 gp41 glycopeptide using mutant N175A.

Scheme 9

Synthesis of an unnatural hexasaccharide oxazoline.

Scheme 10

Glycosylation remodeling of bovine ribonuclease B.