Synthesis of Fractalkine (CDF) and its structual analysis by new 15N labeled NMR method
For elucidating the effect of oligosaccharide on glycoprotein, the Hien and coworkers synthesized fractalkine (CDF) having complex type and high mannose type oligosaccharide (Fig9: Total chemical synthesis of CDF and structural analysis by using specific and gradient of 15N-isotope concentration).[28] Furthermore, they conducted structural analyses of the glycoproteins using stable isotope labeling with position specific and gradient of 15N-isotope concentration (SGI). CDF consists of 76 amino acids and has N-linked glycan at Asn9. According to her synthetic scheme she synthesized glycopeptide thioesters 81 having complex type nonasaccharide and high-mannose type saccharide by either standard Fmoc or Boc protocol respectively. Then four segments 80-83 were sequential coupled by NCL and then deprotection were performed. Finally, deprotection of Acm protecting groups afforded the full-length glycopeptide. After oxidative folding process afforded the folded two types of glycosyl CDF 85 with either highmannose or complex type glycan, in which thirteen 15N-labeled amino acids were inserted at specific positions. They also synthesized non-glycosyl CDF in the same manner. CD spectra and enzymatic disulfide bond mapping confirmed that both glycosylated CDF 85 and non-glycosylated CDF were correctly folded. The SGI labeling protocol enabled us to assign specific amino acids to the 1H-15N HSQC spectra and investigated the dynamics of local conformational properties of three synthetic CDFs. According to the HSQC combined with TOCSY and NOESY methods, glycosylation did not affect conformational changes of CDF at room temperature. However, T1 values indicated that the glycan motif influenced dynamics at the local conformation. As the result, temperature varied circular dichroism CD spectra and T1 values indicated that oligosaccharides might inhibit protein fluctuation and stabilize protein structure.
