Differential reactivity of maleimide and bromoacetyl functions with thiols: Application to the preparation of liposomal diepitope constructs

The comparative reactivity of maleimide and bromoacetyl groups with thiols (2-mercaptoethanol, free cysteine; and cysteine residues present at the N-t erminus of peptides) was investigated in aqueous media. These studies were performed (i) with water-soluble functionalized model molecules, i.e., poly oxyethylene-based spacer arms that could also be coupled to lipophilic anch ors destined to be incorporated into liposomes, and (ii) with small unilame llar liposomes carrying at their surface these thiol-reactive functions. Ou r results indicate that an important kinetic discrimination (2-3 orders -of magnitude in terms of rate constants) can be achieved between the maleimid e and bromoacetyl functions when the reactions with thiols are performed at pH 6.5. The bromoacetyl function which reacts at higher pH values (e.g., p H 9.0) retained a high chemoselectivity; i.e., under conditions where it re acted appreciably with the thiols of, e.g., HS-peptides, it did react with other nucleophilic functions such as alpha- and epsilon-amino groups or imi dazole, which could also be present in peptides. This differential reactivi ty was applied to design chemically defined and highly immunogenic liposoma l diepitope constructs as synthetic vaccines, i;e., vesicles carrying at th eir surface two different peptides conjugated each to a specific amphiphili c anchor. This was realized by coupling sequentially at pH 6.5 and 9.0 two HS-peptides to preformed vesicles containing lipophilic anchors functionali zed with maleimide and bromoacetyl groups.