A highly pure MUC1-derived glycopeptide dendrimer of 22 kDa was prepared by a sequential segment coupling, achieved by an N-alkylcysteine (NAC)-assisted thioesterification. The glycopeptide having C-terminal NAC was prepared by the Fmoc method and converted to the thioester by 3-mercaptopropionic acid treatment. The thioester was condensed with a lysine trimer carrying NAC to afford tetramer, which was then converted to the thioester. Two tetramers were condensed with ethylenediamine to give the octameric glycopeptide dendrimer.
The native chemical ligation (NCL) method is generally used for the synthesis of (glyco)proteins.[1] It efficiently condenses peptide segments without the protection of functional groups, except for the terminal carboxyl group of the carboxyl component by the thioester group. Thus, the segments can be easily prepared by the solid-phase method or recombinant DNA technology. Recently, the preparation of peptide thioesters by the recombinant DNA technique has also been realized by intein technology.[2] The major problem in NCL is that a cysteine residue is required at the ligation site to achieve chemoselective coupling. Since the natural abundance of the cysteine residue in proteins is low (1.5 %), the ligation site is severely limited by this problem. Thus, much effort has been made to develop thiol auxiliary groups to overcome this limitation. [3] In initial studies, [3a-d,f-i] the a-amino group was used to anchor thiol auxiliary groups, which were cleaved after the ligation. However, this strategy might lead to incomplete ligation as a result of steric hindrance, and thus has been mainly used where the ligation site contains at least one glycine residue. The other strategies used thiol groups at the side-chain functional groups, which were subsequently converted to proteinogenic amino acid forms. In these strategies the ligation seems to proceed efficiently, as the acyl group in the thioester intermediate is transferred to the primary amino group through five-, six-, or even larger-membered rings using the thiol group on carbohydrate moieties. [3i,m] These strategies also require additional reaction for the removal of auxiliary groups, which is mainly carried out by metal-based reduction. However, the reaction might not be compatible when applied to the synthesis of peptides having various functional groups, especially sulfur-containing groups. Recently, Wan and Danishefsky developed the free-radical reduction of thiol groups, which will be useful for (glyco)peptide synthesis by the NCL method.[3o]Herein, we focused on the use of a mercaptomethyl group on the side-chain hydroxy groups of serine and threonine, which is a thiohemiacetal that is generally regarded as too labile to be used as an auxiliary group. In return, an additional deprotection step for this auxiliary group would not be required, as it is spontaneously hydrolyzed after the ligation. The use of the hydroxy groups of serine and threonine residues provides far more candidates for the ligation site, since these amino acids appear in proteins more frequently (ca. 12 % in total) than cysteine. A general route for the new method is shown in Scheme 1. The mercaptomethyl group is generated in situ by the reduction of a disulfide-protected parent peptide. Then the ligation with N-terminal thioester is performed, followed by the S-to N-acyl shift via a sevenmembered ring. After ligation, the mercaptomethyl group is hydrolyzed without any extra deprotection steps. If this concept functions well, all reactions can be performed in one pot, thus making t...
The native chemical ligation (NCL) method is generally used for the synthesis of (glyco)proteins.[1] It efficiently condenses peptide segments without the protection of functional groups, except for the terminal carboxyl group of the carboxyl component by the thioester group. Thus, the segments can be easily prepared by the solid-phase method or recombinant DNA technology. Recently, the preparation of peptide thioesters by the recombinant DNA technique has also been realized by intein technology.[2] The major problem in NCL is that a cysteine residue is required at the ligation site to achieve chemoselective coupling. Since the natural abundance of the cysteine residue in proteins is low (1.5 %), the ligation site is severely limited by this problem. Thus, much effort has been made to develop thiol auxiliary groups to overcome this limitation. [3] In initial studies, [3a-d,f-i] the a-amino group was used to anchor thiol auxiliary groups, which were cleaved after the ligation. However, this strategy might lead to incomplete ligation as a result of steric hindrance, and thus has been mainly used where the ligation site contains at least one glycine residue. The other strategies used thiol groups at the side-chain functional groups, which were subsequently converted to proteinogenic amino acid forms. In these strategies the ligation seems to proceed efficiently, as the acyl group in the thioester intermediate is transferred to the primary amino group through five-, six-, or even larger-membered rings using the thiol group on carbohydrate moieties. [3i,m] These strategies also require additional reaction for the removal of auxiliary groups, which is mainly carried out by metal-based reduction. However, the reaction might not be compatible when applied to the synthesis of peptides having various functional groups, especially sulfur-containing groups. Recently, Wan and Danishefsky developed the free-radical reduction of thiol groups, which will be useful for (glyco)peptide synthesis by the NCL method.[3o]Herein, we focused on the use of a mercaptomethyl group on the side-chain hydroxy groups of serine and threonine, which is a thiohemiacetal that is generally regarded as too labile to be used as an auxiliary group. In return, an additional deprotection step for this auxiliary group would not be required, as it is spontaneously hydrolyzed after the ligation. The use of the hydroxy groups of serine and threonine residues provides far more candidates for the ligation site, since these amino acids appear in proteins more frequently (ca. 12 % in total) than cysteine. A general route for the new method is shown in Scheme 1. The mercaptomethyl group is generated in situ by the reduction of a disulfide-protected parent peptide. Then the ligation with N-terminal thioester is performed, followed by the S-to N-acyl shift via a sevenmembered ring. After ligation, the mercaptomethyl group is hydrolyzed without any extra deprotection steps. If this concept functions well, all reactions can be performed in one pot, thus making t...
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