Total synthesis of glycosylated proteins

Abstract

Glycoproteins are an important class of naturally occurring biomolecules which play a pivotal role in many biological processes. They are biosynthesized as complex mixtures of glycoforms through post-translational protein glycosylation. This fact, together with the challenges associated with producing them in homogeneous form, has hampered detailed structure-function studies of glycoproteins as well as their full exploitation as potential therapeutic agents. By contrast, chemical synthesis offers the unique opportunity to gain access to homogeneous glycoprotein samples for rigorous biological evaluation. Herein, we review recent methods for the assembly of complex glycopeptides and glycoproteins and present several examples from our laboratory towards the total chemical synthesis of clinically relevant glycosylated proteins that have enabled synthetic access to full-length homogeneous glycoproteins.

Fig. 1

Common (β-GlcNAc)-N-linked asparagine and (α-GalNAc)/(β-GlcNAc)-O-linked serine/ threonine in glycosylated proteins

Scheme 1

Linear “cassette”-based approach for the synthesis of O-linked glycopeptides

Scheme 2

Lansbury aspartylation reaction facilitated by a pseudoproline motif for the convergent synthesis of N-linked glycopeptides

Scheme 3

Native chemical ligation (NCL)

Scheme 4

Total synthesis of antimicrobial O-glycoprotein diptericin by NCL

Scheme 5

NCL-based assembly of a complex model N-glycopeptide using an o-disulfide phenolic ester as latent thioester

Scheme 6

Synthesis of a complex model N-glycopeptide using an auxiliary-based, cysteine-free ligation strategy

Scheme 7

Non-cysteine ligation followed by desulfurization

Scheme 8

Synthesis of the α-subunit of human glycoprotein hormones, α-hGPH[–92]

Scheme 9

Synthesis of the β-subunit of human follicle stimulating hormone, β-hFSH[–111]

Scheme 10

Synthesis of β-subunit of human chorionic gonadotropin hormone, β-hCG[1–145]

Scheme 11

Synthesis of the bis-glycosylated GM-CSF glycoform

Scheme 12

Proposed synthetic strategy for convergent synthesis of erythropoietin, EPO[1–166] and ribbon diagram of the fully glycosylated protein

Scheme 13

Synthesis of EPO[125–166]

Scheme 14

Initial convergent synthesis of EPO

Scheme 15

Linear synthesis of erythropoietin, EPO[1–166]

Scheme 16

Synthesis of fully glycosylated EPO[1–166] bearing wild-type glycans at each of the four glycosylation sites