Chemical Synthesis of GPI Glycan–Peptide Conjugates by Traceless Staudinger Ligation

Abstract

A new strategy has been developed for GPI glycan-peptide conjugate synthesis based upon a traceless Staudinger reaction between a peptide phosphinothioester and a GPI glycan azide. The strategy was first studied and optimized with simple peptides and GPI glycans, which offered excellent yields of the desired conjugates in both organic and aqueous solvents. It was then used to successfully synthesize an analog of the human CD52 antigen containing the whole CD52 peptide sequence and the conserved trimannose motif of all GPI anchors.

Chemical Synthesis and Semisynthesis of Lipidated Proteins

“…Although both sortase Aa nd NCL-based ligation strategies provide an efficient means to link synthetic GPI molecules to proteins,e ach requires the use of non-native protein-GPI linkages,t hrough either an additional peptidic recognition sequence (in the case of sortase ligation) or ar emnant Cys residue following the NCL step.W ith this in mind, and av iew to generating truly native protein-GPI constructs,Z hu and Guo developed am ethod for coupling GPI to peptides and proteins through the use of the traceless Staudinger ligation (Scheme 9C). [187] In an example of this approach, the CD52 peptide (85) was synthesized by Fmoc-SPPS on ah yper-acid-sensitive 2-CTC resin and, following cleavage from the resin, was converted into the respective phosphinothioester( 86); this could be further deprotected upon acid treatment to afford phosphinothioester 87.Ligation of either 86 or 87 with azidefunctionalized GPI (88)proceeded smoothly,thereby providing the human CD52 antigen bearing af ully native linkage between the protein and the GPI anchors (89). Thestrategic use of the traceless Staudinger ligation in this manner sets the scene for the generation of many more native GPI-anchored proteins in the future,i ncluding important proteins such as the CD48 antigen and carbonic anhydrase IV (both GPIanchored through aC -terminal Ser) or the Eph receptor ligand ephrin A5 (which is GPI-anchored through aCterminal Asn), all of which have not been studied in homogeneous form to date.K ey to such studies will be the implementation of recently developed predictive tools to identify new GPI-anchored proteins,such as PredGPI, [188] and the extension of phosphinothioester generation to larger and recombinant proteins,f or which intein-based methods could potentially prove an enabling technology.H owever, given that the traceless Staudinger ligation can suffer from slower reaction rates compared to other ligation methods,i ti s possible that such larger protein phosphinothioesters may not ligate as efficiently as the smaller peptide examples explored to date.…”

Glycoconjugations of Biomolecules by Chemical Methods

“…Whereas, the phosphate moiety tethered with azide moiety and further linked to a tri- and tetrasaccharide was chosen. Application of the traceless Staudinger ligation methodology led to the desired amide bond formation connecting the peptide fragment with the sugar fragment ( Scheme 9C ) (Zhu and Guo, 2017 ).…”

Semisynthesis of Functional Glycosylphosphatidylinositol‐Anchored Proteins

“…[8b] The Plasmodium GPIs are very heterogenous in the lipid part and contain an additional mannose (Man-IV) on the glycan core as the main modification ( Figure 1a). [11] Peptides and proteins have been attached mainly to GPI analogues by coupling fully protected peptides and glycans, [12] through Staudinger reaction and expressed-protein ligation, [13] and by transpeptidation with Sortase A. [14] However, access to GPI-APs containing an atural GPI limits the evaluation of the activity of these complex glycoconjugates.…”