Synthesis and characterisation of fluorescent substrates for eukaryotic protein N-glycosylation

Capitani, Mario M. de; Ramírez, Ana S.; Rossi, Lorenzo; Alexander, J. Andrew N.; Lorenzo, Sabrina De; Locher, Kaspar P.; Reymond, Jean‐Louis

doi:10.1016/j.tet.2023.133361

Cited by 1 publication

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“…Our original synthesis of LLO 2b followed literature precedents for related synthetic LLOs, [3,[10][11][12][13] and comprised: 1) assembly of the C 25 -dolichol 3 in six steps from (S)-citronellol and its phosphorylation to lipid phosphate 4; 2) conversion of chitobiose to the peracetylated α-phosphate 7; and 3) coupling of the two phosphates, purification and deacetylation to LLO 2b. Strikingly, each of these three phases of the synthesis was low-yielding due to difficulties in a phosphate coupling step, namely the phosphorylation of the C 25 -dolichol 3 and of chitobiose acetate 5, as well as the pyrophosphate coupling to form 8 (Scheme 1).…”

Section: Identification Of Limiting Stepsmentioning

confidence: 99%

Optimizing Phosphate Couplings for Dolichyl Diphosphochitobiose to Enable Protein N‐Glycosylation Studies

Meirelles,

Reymond

2023

Helvetica Chimica Acta

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Herein we report an optimized synthesis for dolichyl diphosphochitobiose (GlcNAc2‐PP‐Dol25), a probe useful for biochemical and structural studies of protein N‐glycosylation in eukaryotic cells. We improved three phosphate coupling steps in terms of yields and reaction times, namely chitobiose phosphorylation, dolichol phosphorylation, and phosphate‐phosphate coupling, by adjusting reagents and conditions. We also developed an efficient preparative reverse‐phase HPLC purification protocol followed by ion exchange step to obtain the pure product as a stable sodium salt. These optimized procedures ensure a reliable supply of GlcNAc2‐PP‐Dol25 for enzymatic studies.

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