Chemoenzymatic Methods for the Synthesis of Glycoproteins

Abstract

Glycosylation is one of the most prevalent posttranslational modifications that profoundly affects the structure and functions of proteins in a wide variety of biological recognition events. However, the structural complexity and heterogeneity of glycoproteins, usually resulting from the variations of glycan components and/or the sites of glycosylation, often complicates detailed structure-function relationship studies and hampers the therapeutic applications of glycoproteins. To address these challenges, various chemical and biological strategies have been developed for producing glycan-defined homogeneous glycoproteins. This review highlights recent advances in the development of chemoenzymatic methods for synthesizing homogeneous glycoproteins, including the generation of various glycosynthases for synthetic purposes, endoglycosidase-catalyzed glycoprotein synthesis and glycan remodeling, and direct enzymatic glycosylation of polypeptides and proteins. The scope, limitation, and future directions of each method are discussed.

Figure 1.

Structures and symbols of common monosaccharide units found in eukaryotic systems.

Figure 2.

Chemical structures and symbol representations of representative glycan-amino acid linkages found in eukaryotic glycoproteins: (a) complex type N-glycan; (b) core 2 O-glycan; (c) GlcNAc-glycosylation; (d) proteoglycan linkage; (e) linkage strcuture of GPI anchored proteins. In many cases, the monossaccharide units in the above structures can be subjected to further carbohydrate or various noncarbohydrate modifications.

Figure 3.

Biosynthesis of eukaryotic N-glycoproteins.

Figure 4.

Biosynthesis of O-glycoproteins with typical O-glycan core structures.

Figure 5.

Major approaches for synthesizing homogeneous glycoproteins.

Figure 6.

Catalytic mechanisms of different types of glycosidases. R¹ and R² could be sugar or other moieties. In the case of the substrate-assisted mechanism, X is an essential residue to facilitate the formation or stability of reaction intermediate and could be an asparagine or aspartic acid residue.

Figure 7.

Catalytic mechanisms of glycosynthases. R, R¹, and R² could be sugar or other moieties.

Figure 8.

Catalytic mechanism of S-glycoligase and O-glycoligase.

Figure 9.

Catalytic mechanism of glycosylation via ENGase mutants derivated from distinct GH families. R could be sugar or other moieties. X could be alanine, glutamine, methionine, or histidine residue.

Figure 10.

Structures of the synthetic glycosylated CMV-peptides derived from a human cytomegalovirus (CMV).

Figure 11.

Enzymatic synthesis of glycoproteins carrying eukaryotic N-glycans by coupling engineered C. jejuni PglB N-glycosylation pathway in E. coli with in vitro enzymatic glycan-remodeling.

Figure 12.

Enzymatic protein glycosylation by engineeering eukaryotic N-glycan assembly coupled with PglB-catalyzed N-glycan transfer in E. coli.

Figure 13.

C. jejuni PglB mediated enzymatic synthesis of glycoconjugate vaccines carrying bacterial O-antigens.

Scheme 1.

Glycosynthase-Catalyzed Modification of Glycopeptide and Glycoprotein

Scheme 2.

Synthesis of Thio-neoglycoprotein by Chemical Ligation and Subsequent Transglycosylation via a Thioglycoligase

Scheme 3.

Glycoligase-Catalyzed Direct Core Fucosylation of Glycopeptide and Glycoprotein

Scheme 4.

Sugar Oxazolines as Donor Substrates for ENGase-Catalyzed Synthesis of N-Glycopeptides

Scheme 5.

Chemoenzymatic Synthesis of Homogeneous Glycoproteins by ENGase-Catalyzed Glcosylation Remodeling with Sugar Oxazolines

Scheme 6.

Chemoenzymatic Synthesis of Homogenous Glycoforms Ribonuclease B by ENGase-Derived Glycosynthases

Scheme 7.

Chemoenzymatic Synthesis of CD52 Glycopeptide Antigens Containing Both N- and O-Glycans

Scheme 8.

Synthesis of CD52 Glycopeptide Carrying Fucosylated Bi- And Triantennary N-Glycan Using Endo-F3 D165A

Scheme 9.

Chemoenzymatic Synthesis of Glycopramlintides Using ENGase Glycosynthase Mutants

Scheme 10.

Chemoenzymatic Synthesis of HIV-1 V1 V2 Glycopeptides Carrying Defined N-Glycans

Scheme 11.

Convergent Chemoenzymatic Installation of Two Distinct N-Glycans in the V1 V2 Cyclic Peptide

Scheme 12.

Chemoenzymatic Synthesis of V3 Glycopeptides Carrying Two N-Glycans

Scheme 13.

Chemoenzymatic Synthesis of a Three-Component Glycopeptide Immunogen

Scheme 14.

Chemoenzymatic Synthesis of Hydrophobic Glycoprotein Saposin C

Scheme 15.

Enzymatic Synthesis of Phosphorylated RNase B Containing M6P Moieties

Scheme 16.

Chemical Synthesis of M6P-Containing High-Mannose N-Glycan Oxazolines as Enzyme Substrates

Scheme 17.

Chemoenzymatic Glycosyiation Remodeling of Ribonuclease B with M6P N-Glycans

Scheme 18.

Glycan Remodeling of Human Erythropoietin (EPO)

Scheme 19.

Chemeonzymatic Synthesis of Various IgG-Fc Glycoforms

Scheme 20.

Chemoenzymatic Glyco-Remodeling of Therapeutic Antibodies Using Endoglycosidases and Glycosynthase Mutants

Scheme 21.

Chemical Synthesis of Oligosaccharide Oxazolines

Scheme 22.

Direct Conversion of Unprotected Oligosaccharide Containing a Reducing End GlcNAc Moiety to Oligosaccharide Oxazoline in Water

Scheme 23.

Synthesis of Glucose-Containing High-Mannose N-Glycan Oxazoline

Scheme 24.

Semisynthesis of Sialyl Biantennary Complex Type N-Glycan Oxazoline (SCT-ox)

Scheme 25.

Semisynthesis of High Mannose Type N-Glycan (Man9-ox)

Scheme 26.

Semisynthesis of Triantennary Complex Type N-Glycan Oxazoline (TCT-ox)

Scheme 27.

Chemoenzymatic Synthesis of Und-PP-Trisacchandes Carrying Site-Specific Azido Groups

Scheme 28.

In Vitro Enzymatic Synthesis of N-Glycopeptides Carrying Site-Specific Azido Groups Using C. jejuni PglB

Scheme 29.

In Vitro Chemoenzymatic Synthesis of N-Glycopeptides Using PglB as the Enzyme and Synthetic Lipid-Linked GlcNAc as the Donor Substrates

Scheme 30.

Enzymatic Synthesis of Glycopeptidesbearing Complex N-Glycans by Coupling ApNGT and Endo-glycosidases

Scheme 31.

Enzymatic Synthesis of Glycopeptides Carrying Natural Eukaryotic N-Glycans Scheme

Scheme 32.

Site-Specific Enzymatic Synthesis of Glyco-Peptide/Protein Carrying O-Linked Eukaryotic N-Glycans

Scheme 33.

Chemoenzymatic Synthesis of MUC1 Glycoproteins Carrying Multiple Tn, T, and STn O-Glycans