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A New Strategy for the Synthesis of Dinucleotides Loaded with Glycosylated Amino Acids—Investigations on in vitro Non‐natural Amino Acid Mutagenesis for Glycoprotein Synthesis
Author(s) -
Röhrig Christoph H.,
Retz Oliver A.,
Hareng Lars,
Hartung Thomas,
Schmidt Richard R.
Publication year - 2005
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200500079
Subject(s) - amino acid , biochemistry , transfer rna , mutagenesis , protein biosynthesis , aminoacylation , in vitro , chemistry , translation (biology) , biology , rna , mutation , messenger rna , gene
Abstract The in vitro non‐natural amino acid mutagenesis method provides the opportunity to introduce non‐natural amino acids site‐specifically into proteins. To this end, a chemically synthesised aminoacylated dinucleotide is enzymatically ligated to a truncated suppressor transfer RNA. The loaded suppressor tRNA is then used in translation reactions to read an internal stop codon. Here we report an advanced and general strategy for the synthesis of the aminoacyl dinucleotide. The protecting group pattern developed for the dinucleotide facilitates highly efficient aminoacylation, followed by one‐step global deprotection. The strategy was applied to the synthesis of dinucleotides loaded with 2‐acetamido‐2‐deoxy‐glycosylated amino acids, including N ‐ and O ‐β‐glycosides and O ‐ and C ‐α‐glycosides of amino acids, thus enabling the extension of in vitro non‐natural amino acid mutagenesis towards the synthesis of natural glycoproteins of high biological interest. We demonstrate the incorporation of the glycosylamino acids—although with low suppression efficiency—into the human interleukin granulocyte‐colony stimulating factor (hG‐CSF), as verified by the ELISA technique.