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  • A further generally applicable approach for

    2022-01-15

    A further generally applicable approach for the synthesis of N-linked glycopeptides involves the convergent Lansbury aspartylation of glycosylamines. Here, the detrimental formation of aspartimides during the activation of the side chain aspartate can be prevented through the use of Ser/Thr pseudoproline motifs on the adjacent residue within the Asn-X-Ser/Thr N-glycosylation consensus sequence. The coupling of the sugar works equally well in solution [29] and on the solid phase [30] (Figure 1a). The bottleneck in the synthesis of N-glycopeptides in many cases is the availability of full-length N-glycans or the corresponding oxazolines needed for enzymatic glycan remodeling. The use of a biantennary N-glycan from egg yolk as a readily available starting material has thus become popular for many research groups. Various improved methods exist for the isolation of the parent sialoglycopeptide [31,32]. Additional complex-type N-glycans can be obtained by semisynthetic modification of this BMS 299897 versatile oligosaccharide [33,]. Depending on the structure of the desired N-glycan a chemoenzymatic synthesis may be required [35, 36, 37, 38,]. The subsequent incorporation into glycopeptides can be carried out after anomeric functionalization of the free N-glycan as a glycosyl amine or an azide (Figure 3).
    Synthesis of N-glycoproteins Having gained access to glycopeptide fragments using suitable combinations of the methods presented above, a number of N-glycoproteins have been synthesized and successfully refolded to the native structures. Chemically synthesized erythropoietin (EPO) with three biantennary sialylated N-glycans in native positions (24, 38, 83) displayed efficient protein folding, whereas the EPO glycopeptides having only one or two N-glycans showed only reduced folding efficiency (Figure 4a) [40,]. These experiments indicated that glycans can affect protein solubility and aggregation during the folding process. The cytokine CCL1 having a biantennary N-glycan was synthesized together with its non-glycosylated mirror image D-protein. Only the quasi-racemic mixture of the L-glycoprotein and the non-glycosylated D-protein gave crystals, which were analyzed by X-ray crystallography [42]. The use of a synthetic glycopeptide hydrazide as the central segment was crucial for the efficient N-terminal to C-terminal ligation of interleukin-6 (IL-6) using a semisynthetic approach (Figure 4b). A robust multistep refolding and purification protocol for IL-6 glycoforms was established [43] and enabled the synthesis of a library of natural IL-6 glycoforms. IL-8 bearing a high-mannose N-glycan has also been synthesized both in native and misfolded forms by intentional scrambling of the two disulfide bonds. The homogeneous misfolded glycoprotein was used as a substrate for understanding the enzymes involved in the glycoprotein quality control system (Figure 4c) []. A small library of glycoforms of the lysosomal sphingolipid adapter protein saposin D (SapD) was obtained after solving the difficult synthesis of the two hydrophobic segments. Assays based on the interaction of SapD with membranes showed that the various bioactivities of the glycoprotein were dependent on the size and type of its carbohydrate moiety (Figure 4d) []. Whenever the parent N-glycoprotein has only a single N-glycosylation site the enzymatic preparation of homogenous glycoforms by ENGase mediated deglycosylation and reglycosylation using N-glycan oxazolines has become a more routine strategy (Figure 1b). For example, this methodology has recently been employed for the chemoenzymatic preparation of phosphorylated glycoforms of RNase [46,47]. The glycan remodeling of erythropoietin with three N-glycosylation sites led to selective reglycosylation of two sites using a mutant ENGase [48]. Using mutant ENGases the reglycosylation of GlcNAc-glycoproteins was successfully extended by the Wang group to multiantennary N-glycan oxazolines [49] and core fucosylated protein acceptors including recombinant therapeutic antibodies [50,51]. Recently, the efficient expression of a therapeutic antibody has been established in glycoengineered yeast strains followed by enzymatic remodeling with a complex-type N-glycan [] (Figure 1a). A rationally designed fucoligase utilizing fucosyl fluoride as a substrate is capable of efficiently installing α1,6-linked core fucose on N-glycopeptides and N-glycoproteins thus overcoming the restrictive acceptor specificity of the natural fucosyltransferase FUT8 []. In cases where the parent N-glycoprotein is not readily available the corresponding GlcNAc protein can also be assembled by ligation methods and refolded prior to elongation of the N-glycan by transglycosylation. Following this route N-glycosylated murine saposin C [54] and the Ig domain of Tim-3 [55] have recently been prepared.