Native N-glycopeptide thioester synthesis through N→S acyl transfer

Graphical abstract We describe experiments that give us a sense of the impact of native chemical ligation, on N-glycopeptide thioester synthesis via N→S acyl transfer.


N-linked glycopeptide synthesis of H-AEN((OAc) 3 (GlcNAc)ITTGC-OH, 3
Fmoc-Cys(Trt)-NovaSyn®TGT resin (0.2 mmolg -1 loading) (0.5 g, 0.1 mmol) was deprotected with 20% The samples were centrifuged at 3000 rpm for 15 minutes and the supernatant was decanted off and discarded. The process was repeated again and the crude peptide was left to dry in a fume hood for an hour. Semi-preparative reverse phase HPLC was used to purify the peptide (gradient: 5 to 60% acetonitrile in water over 45 minutes

Synthesis of glycopeptide thioester of H-AEN((OAc) 3 (GlcNAc)ITTG-SCH 2 CH 2 SO 3 H
In 5  The reaction was repeated as described above using a peptide concentration of 0.2mg/mL (5 mg in 25 mL) in a round bottomed flask, heated on an oil bath. The reaction was also repeated using 0.2 mg/mL of peptide with 10% acetic acid instead of sodium phosphate buffer to give a pH ≈ 2.
1H NMR spectra of 3 (lower trace) and 4 (upper trace) and stored at 4 °C overnight. A white precipitate formed and the sample was centrifuged at 3000 rpm for 10 minutes. The process was repeated, resuspending the protein in cold distilled water (8 mL) and allowed to precipitate at 4 °C overnight. The crude protein was then collected by centrifugation and then treated with hydrazine hydrate (5% v/v) and DTT (50 mM) in 6 M guanidine hydrochloride (3 mL) at room temperature with gentle agitation. After an hour, LCMS indicated complete deacetylation of the starting material and the protein was isolated through dilution with water and precipitation as illustrated above.

Model Peptide Synthesis
The model peptide, was derived from the C-terminal sequence of green fluorescent protein (GFP) and was synthesised on Rink Amide resin to afford the peptide carboxamide.
Electrospray mass spectrometry (carried out on a Waters Aquity UPLC-SQD MS system with an applied voltage of 60 V) confirmed that these new components contained the epimeric peptide 7 (tGFP-HC D -OH) after 6 hours in the reactions with more than 0.75 eq D-cysteine hydrochloride. This was observed with 1 mass unit difference from the starting material due to the C-terminal comprising a carboxylic acid in place of the carboxamide of the starting peptide, calculated mass,