Uncovering a Latent Ligation Site for Glycopeptide Synthesis

Abstract

Glycosylation, one of the most important posttranslational modifications, plays an important role in a variety of biological events.[1] Oligosaccharides on glycoproteins exhibit structural heterogeneity, which makes it difficult to elucidate the relationship between the oligosaccharide structure and the function of the glycoprotein.Chemical synthesis is one of the powerful approaches for obtaining homogeneous glycoproteins. [2] We have already reported the synthesis of a glycoprotein with a homogeneous N-linked complex-type oligosaccharide.[3] This synthesis employed native chemical ligation (NCL) to perform peptide-segment coupling. NCL relies on the thiol-exchange reaction between a peptide with an a-thioester group at the C terminus and another peptide with a cysteine residue at the N terminus and on the subsequent intramolecular acyl transfer.[4] However, occasionally, the cysteine residue is not properly located or does not exist in the target protein. To take this potential difficulty into consideration, a long glycopeptide sequence that is 30-50 amino acids from one cysteine site to another cysteine site occasionally needs to be synthesized for glycoprotein synthesis by the NCL method. The synthesis of such a glycopeptide with an N-linked glycopeptide is not easy to perform [2,5] and requires an appropriate amount of N-linked complex-type oligosaccharides; therefore, there is greater difficulty in glycoprotein synthesis than in simple protein synthesis.To examine NCL without a cysteine residue in a long target peptide, reduction methods changing the sulfhydryl group of cysteine to a hydrogen atom after NCL and utilizing an auxiliary group have been developed. [6] In the latter method, the amino acid sequence at the ligation site is limited for performance. For the development of a widely usable method in glycopeptide synthesis, we have also explored suitable NCL approaches; this endeavor enabled us to find a new ligation position at the serine site in the consensus sequence NXS (X: any amino acid except for proline), by which an asparagine residue is generally incorporated in an Nlinked oligosaccharide. This sequence is found in glycoproteins along with the NXT sequence.[1] In order to use the serine site for a new NCL, we have examined the new concept and attempted the conversion of a cysteine residue into a serine residue after NCL. For such a technique, it was necessary to explore concise reaction sequences. As a result, we found the possibility of using a CNBr cleavage method at a methylcysteine site, which could be obtained by specific methylation of cysteine.[7] Herein, we report a new chemical ligation approach at serine sites, which relies on the conversion of a cysteine residue into a serine residue after NCL.The strategy is shown in Scheme 1. After NCL (product A), the conversion of cysteine into serine was performed by the following reactions: S methylation of cysteine with methyl 4-nitrobenzenesulfonate (product B) and intramolecular rearrangement by activation with CNBr in 80 % HCOOH solution...

Advances in chemical ligation strategies for the synthesis of glycopeptides and glycoproteins

“…99 Rather than using a ligation-desulfurisation sequence, the method relies on the conversion of a cysteine residue to a native serine side chain after a traditional NCL reaction has taken place. The strategy involves an S-selective methylation of the cysteine side chain with O -N acyl shift under slightly basic conditions to complete the conversion of cysteine to serine.…”

“…(B) Synthesis of a model glycopeptide 62 bearing a complex N-linked glycan via NCL followed by conversion of the ligation site cysteine to serine. 99 SMe 2 and TFA was performed after the cysteine to serine conversion to regenerate the native methionine side chain. 101 The cysteine to serine conversion was attempted on glycopeptide 60 bearing a complex-type N-linked asialooligosaccharide 102 which was initially prepared by a standard NCL reaction (Scheme 21B).…”

Ligations of O-acyl threonine units to give native peptides via 5-, 8-, 9- and 10-membered cyclic transition states

“…2,[17][18][19][20][21][22] In addition, the low abundance of cysteine (1.7% of the residues in protein sequences) is a major drawback of this methodology since NCL is restricted to Cysteine residues. To address this limitation, efficient synthetic approaches have been developed 20,[23][24][25][26][27][28][29][30][31] including our recent report on classic O-to N-acyl shifts via a 8 and 11-membered transition states in O-acyl serine 32 and 12-to 19-membered cyclic transition states in O-acyl tyrosinepeptides. 33 Thus, "traceless" chemical ligation involving O-to N-acyl shift (at Ser and Tyr site)…”

Novel proteoglycan glycotechnology: chemoenzymatic synthesis of chondroitin sulfate-containing molecules and its application

“…Several strategies for glycopeptides and glycoproteins have been reported [11][12][13][14][15][16][17][18][19], but these methods are difficult to use for proteoglycans. Because the molecular weight of ChS ranges from 6.5 to 3.5×10 4 , and furthermore its hydroxyl groups are highly and randomly sulfated [2,20], it is difficult to artificially reproduce the ChS in homogeneity that is found in the natural compounds.…”