De Capitani, Mario M.; Ramírez, Ana S.; Rossi, Lorenzo; Alexander, J. Andrew N.; De Lorenzo, Sabrina; Locher, Kaspar P.; Reymond, Jean-Louis (2023). Synthesis and characterisation of fluorescent substrates for eukaryotic protein N-glycosylation. Tetrahedron, 136, p. 133361. Elsevier Science 10.1016/j.tet.2023.133361
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Herein we report the synthesis of two fluorescently labelled analogues of C25 dolichol (Dol25) in which the terminal isoprene unit has been replaced by a dansyl or 7-amino-4-trifluoromethylcoumarin fluorophore, a transformation enabled by the regioselective epoxidation of the terminal olefin via its bromohydrin using the van Tamelen procedure. The lipid alcohols were phosphorylated and glycosylated to obtain lipid-linked chitobiose-α-diphosphates and a lipid-linked mannosyl-β-phosphate. Biochemical assays showed that these labelled substrates are accepted by eukaryotic protein N-glycosylation enzymes with rates comparable to the unlabelled substrates, reconstituting a major part of the pathway up to the lipid-linked dodeca-saccharide Glc1Man9GlcNAc2-PP-Dol25 and its transfer to an acceptor peptide catalysed by eukaryotic oligosaccharyltransferases (OSTs), namely the single-subunit OST STT3A from Trypanosoma brucei and the octameric OST complex from Saccharomyces cerevisiae. The fluorescent labels facilitate handling and purification of the lipid-linked glycosyl donors and acceptors and should facilitate further biochemical studies of protein glycosylation enzymes.
Item Type: |
Journal Article (Original Article) |
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Division/Institute: |
08 Faculty of Science > Department of Chemistry, Biochemistry and Pharmaceutical Sciences (DCBP) |
UniBE Contributor: |
De Capitani, Mario Michele Oreste, De Lorenzo Cardinal, Sabrina, Reymond, Jean-Louis |
Subjects: |
500 Science > 570 Life sciences; biology 500 Science > 540 Chemistry |
ISSN: |
0040-4020 |
Publisher: |
Elsevier Science |
Language: |
English |
Submitter: |
Sandra Tanja Zbinden Di Biase |
Date Deposited: |
11 Jan 2024 10:07 |
Last Modified: |
14 Jan 2024 02:44 |
Publisher DOI: |
10.1016/j.tet.2023.133361 |
BORIS DOI: |
10.48350/191465 |
URI: |
https://boris.unibe.ch/id/eprint/191465 |
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