title: Expeditious chemoenzymatic synthesis of homogeneous N-glycoproteins carrying defined oligosaccharide ligands.
abstract: An efficient chemoenzymatic method for the construction of homogeneous N-glycoproteins was described that explores the transglycosylation activity of the endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) with synthetic sugar oxazolines as the donor substrates. First, an array of large oligosaccharide oxazolines were synthesized and evaluated as substrates for the Endo-A-catalyzed transglycosylation by use of ribonuclease B as a model system. The experimental results showed that Endo-A could tolerate modifications at the outer mannose residues of the Man3GlcNAc-oxazoline core, thus allowing introduction of large oligosaccharide ligands into a protein and meanwhile preserving the natural, core N-pentasaccharide (Man3GlcNAc2) structure in the resulting glycoprotein upon transglycosylation. In addition to ligands for galectins and mannose-binding lectins, azido functionality could be readily introduced at the N-pentasaccharide (Man3GlcNAc2) core by use of azido-containing Man3GlcNAc oxazoline as the donor substrate. The introduction of azido functionality permits further site-specific modifications of the resulting glycoproteins, as demonstrated by the successful attachment of two copies of alphaGal epitopes to ribonuclease B. This study reveals a broad substrate specificity of Endo-A for transglycosylation, and the chemoenzymatic method described here points to a new avenue for quick access to various homogeneous N-glycoproteins for structure-activity relationship studies and for biomedical applications.
authors.affiliation: Institute of Human Virology, Department of Biochemistry & Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Ochiai
authors.forename: Wei
authors.initials: W
authors.lastname: Huang
authors.forename: Lai-Xi
authors.initials: LX
authors.lastname: Wang
mesh_terms: D001173:Arthrobacter / Q000201:enzymology; D002384:Catalysis; D003500:Cyclization; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism*; D037102:Lectins / Q000737:chemistry; D008024:Ligands; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism*; D008958:Models, Molecular; D015394:Molecular Structure; D009844:Oligosaccharides / Q000737:chemistry*; D010080:Oxazoles / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D006023:Glycoproteins; D037102:Lectins; D008024:Ligands; D009844:Oligosaccharides; D010080:Oxazoles; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase