DIRECT SYNTHESIS AND CHARACTERIZATION OF MULTI-DENDRITIC PEPTIDES FORUSE AS IMMUNOGENS

The direct synthesis and subsequent rapid characterisation of multiple antigen peptides (MAPs) for use as immunogens has presented difficult ies, partly because of the formation of incomplete or truncated peptid e sequences during the synthetic procedure. Therefore, many researcher s have resorted to ligation procedures for the synthesis of MAP constr ucts. This article describes a method to improve the yield of MAP cons tructs by direct synthesis methods, as well as a general procedure tha t enables easier characterisation of the synthetic products. In partic ular, during the synthesis of MAP constructs, a capping procedure was introduced after each amino acid coupling step, thus improving signifi cantly the yield of the desired multi-dendritic peptidic immunogens. T hrough the use of this capping procedure, problems arising from the in complete amino acid residue coupling at the point of synthesis were mi nimised, and any deletion peptides which formed could be eliminated mo re readily during the subsequent purification procedures. In addition, previous difficulties in purification and characterisation of MAP con struct by, e.g. electrospray mass spectroscopy (ES-MS), often led to t he multi-dendritic peptidic immunogens being used without full charact erisation after dialysis and recovery of the product(s). This article describes an enzymatic (tryptic) digestion method with the MAP constru ct, followed by characterisation of the enzymatic digest by reversed p hase high-performance liquid chromatography-ES-MS. With this method, f ragments of the MAP construct cleaved at specific amino acid residue s ites (e.g, lysine or arginine) within the sequence of the parent pepti de can be readily determined and the kinetics of the digestion easily followed. This enzymatic digestion procedure thus provides a facile ap proach to confirm that all of the multi-dendritic arms of the purified MAP construct have been equivalently elongated during the peptide syn thesis and that consequently the purified construct structure contains the correct peptide sequence. (C) Munksgaard 1997.