UNPROTECTED PEPTIDES AS BUILDING-BLOCKS FOR BRANCHED PEPTIDES AND PEPTIDE DENDRIMERS

We describe two new site-specific ligation methods for preparing branc hed peptide dendrimers such as multiple antigen peptide (MAP), Both me thods are based on the general approach of exploiting the specific rea ction between a weak base and an aldehyde under acidic conditions so t hat unprotected peptides can be used as building blocks. A weak base s uch as benzoyl hydrazine or 1,2-amino thiol of cysteine was attached t o the N-terminal of an unprotected peptide as nucleophile to react wit h the alkyl aldehyde on the core matrix of MAP to form a stable hydraz one linkage or a five-membered thiazolidine ring, respectively. Two sy nthetic peptides rich in basic amino acids such as lysine and arginine were used as models in the ligation reactions in solution to give pep tide dendrimers containing four or eight copies of peptide immunogens. The resulting macromolecules with the MW ranging from 5 to 16 kDa wer e unambiguously characterized by laser-desorption mass spectrometry. F urthermore, we also optimized the conditions of these ligation reactio ns using elevated temperature and a water-miscible organic co-solvent to give a combination of rate enhancement about 10 fold. These optimiz ations allowed the ligation reactions to be completed in <4 h instead of 2-3 days. Our ligation approach also has the advantages of flexibil ity so that peptides can be attached through the amino or carboxyl ter minus to the core matrix, The phenyl hydrazone linkage and the five-me mbered ring were found to be stable at physiological pH suitable for i mmunization. Thus our results provide two practical and useful methods for the synthesis of macromolecular peptide dendrimers for vaccines, artificial proteins and enzymes. (C) Munksgaard 1995.