Direct synthesis and characterisation of multi‐dendritic peptides for use as immunogens

Abstract

The direct synthesis and subsequent rapid characterisation of multiple antigen peptides (MAPs) for use as immunogens has presented difficulties, partly because of the formation of incomplete or truncated peptide sequences during the synthetic procedure. Therefore, many researchers have resorted to ligation procedures for the synthesis of MAP constructs. This article describes a method to improve the yield of MAP constructs by direct synthesis methods, as well as a general procedure that enables easier characterisation of the synthetic products. In particular, during the synthesis of MAP constructs, a capping procedure was introduced after each amino acid coupling step, thus improving significantly the yield of the desired multi-dendritic peptidic immunogens. Through the use of this capping procedure, problems arising from the incomplete amino acid residue coupling at the point of synthesis were minimised, and any deletion peptides which formed could be eliminated more readily during the subsequent purification procedures. In addition, previous difficulties in purification and characterisation of MAP construct by, e.g. electrospray mass spectroscopy (ES-MS), often led to the multi-dendritic peptidic immunogens being used without full characterisation after dialysis and recovery of the product(s). This article describes an enzymatic (tryptic) digestion method with the MAP construct, followed by characterisation of the enzymatic digest by reversed phase high-performance liquid chromatography-ES-MS. With this method, fragments of the MAP construct cleaved at specific amino acid residue sites (e.g. lysine or arginine) within the sequence of the parent peptide can be readily determined and the kinetics of the digestion easily followed. This enzymatic digestion procedure thus provides a facile approach to confirm that all of the multi-dendritic arms of the purified MAP construct have been equivalently elongated during the peptide synthesis and that consequently the purified construct structure contains the correct peptide sequence.