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

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-beta-glycosides and O- and C-alpha-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.

Efficient N‐Terminal Glycoconjugation of Proteins by the N‐End Rule

“…Aminooxy derivatives of various sugars were subsequently coupled to the keto group in the protein 15. A step further was the introduction of monoglycosylated amino acids into proteins in response to an amber stop codon,18, 19 to which additional sugar molecules were attached by the enzymatic action of glycosyltransferases 18. While this work provided a proof‐of‐principle for the use of an expanded genetic code in glycobiology, suppression‐based methodology is generally limited by low production yields and a technically expensive experimental setup.…”

Realizing the promise of chemical glycobiology

“…3b). In this regard, one of the 'killer apps' in the form of stop codon suppression by a glyco-amino acid has not yet been clearly demonstrated in a useable form, [53][54][55] but hope remains. The bacterial PglB oligosaccharyltransferase system has been nicely exploited in vivo to create valuable substrates using pathway recapitulation and repositioning of the appropriate DNXS/T consensus motif.…”

Site-Specific Incorporation of Glycosylated Serine and Tyrosine Derivatives into Proteins

“…12 We previously reported a general strategy for the synthesis of O-glycosylated seryl-pdCpAs and their ligation to truncated tRNAs, 13 and an alternative strategy has recently been described. 14 Further, the elaboration of modified glycoproteins in intact bacterial cells has also been described. 15 In the present report, we describe the extension of this methodology to the synthesis of Oglycosylated tyrosyl-pdCpAs and the corresponding misacylated tRNAs.…”