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  • An alternative to the chemoenzymatic


    An alternative to the chemoenzymatic preparation of glycoproteins is the production through total chemical synthesis or semi-synthesis [2,3,]. Many successful glycoprotein syntheses are based on native chemical ligation (NCL) [11], a reaction where unprotected peptides and glycopeptides of any length can be coupled chemoselectively. The reaction requires only a C-terminal thioester and an N-terminal cysteine functionality on the peptides and is compatible with aqueous and organic solvent systems. The appropriately functionalized peptides can be obtained chemically or recombinantly [12]. Longer protein sequences often require multiple ligations [13], for which several variations have been developed. Since the solid phase synthesis of longer peptides is generally demanding, the incorporation of thioesters or glycans can leads to additional challenges that need to be investigated and optimized separately. Cysteine is of relatively low overall abundance in proteins (1–2%) and is frequently not placed properly for native chemical ligation. In these cases, β-mercapto-amino nefiracetam australia analogues are employed at the N-terminus followed by a desulfurization step after ligation. [14, 15, 16,,]. Alternatively, chemically attached auxiliaries with thiol groups can serve as a surrogate for an N-terminal cysteine. The notoriously low reactivity when employing auxiliaries at the N-terminus was recently overcome by a novel type of auxiliary, which is cleaved via radical desulfurization and is efficient also for ligation sites other than glycine [19]. Since peptide thioesters are labile and susceptible to epimerization the use of peptide hydrazides as stable precursors for thioesters has been widely adopted [20] and is fully compatible with glycopeptides.
    Ligation methods for N-glycopeptides Some examples of different ligation approaches to N-linked glycopeptides and glycoproteins are outlined in Figure 2. A benzylated GlcNAc-Asn building block containing a β-thiol auxiliary was employed as a surrogate for N-terminal cysteine in the multi-segment synthesis of a GM2 activator protein bearing a GlcNAc residue [21] (Figure 2a). Peptides with C-terminal salicylesters selectively react with peptides bearing an N-terminal Ser or Thr moiety giving a ligation intermediate with a pseudoproline moiety. This Ser/Thr ligation was shown to be compatible with glycosylation and native chemical ligation [] (Figure 2b). A conceptually novel approach especially designed for N-glycopeptides is based on a thiolated auxiliary, which can be installed on the reducing end of GlcNAc or maltotriose. The labile N-glycosides were ligated with peptides bearing an N-terminal aspartic acid with a side chain thioester. The auxiliary of the resulting N-glycopeptide permits a second ligation with peptide thioesters [23] and can be removed with a final TFA treatment (Figure 2c). By introducing a photolabile auxiliary with solubility-enhancing PEG-chains on the N-terminus of mucin-type glycopeptides their enzymatic elongation using glycosyltransferases and subsequent isolation was successfully achieved. After ligation the auxiliary was removed by irradiation [24]. The pH-dependent solubility enhancing properties of picolylesters were crucial for the synthesis and purification of highly insoluble segments in the course of the synthesis of O-glycosylated interleukin-2 following the thioester method [25].
    Synthesis of N-glycopeptides The solid phase synthesis of O-glycopeptides needs to be carried out with preformed glycosyl amino acids since convergent attachment of the sugar with control of the absolute stereochemistry at the anomeric center is very challenging. Special care is also required with the preformed glycosylamino acid blocks due to the tendency of O-glycosyl serine derivatives to undergo epimerization after activation [26]. The synthesis of N-glycopeptides faces different challenges. For example, the synthesis of N-glycopeptide thioesters on the solid phase using glycosyl amino acids can be problematic due to side reactions on the free OH-groups of the sugar or through unfavorable interactions of the sugar with the resin. The limitation in the length of a given glycopeptide target that can be generated through linear SPPS can be circumvented by racemization-free segment couplings on the solid phase [27]. Sialylated N-glycans linked to asparagine, that can be prepared readily from natural sources, can be efficiently incorporated into glycopeptide thioesters following the in situ neutralization Boc solid phase synthesis [28] (Figure 1a).