Folate-mediated drug delivery: Effect of alternative conjugation chemistry

When utilized as a macromolecular drug targeting ligand, folic acid (Pte-Gl u) has traditionally been coupled to peptides, proteins' and lipids via one of its two carboxylate groups fortuitously located within a distal glutamy l moiety, It has been assumed in the literature that the gamma-glutamyl car boxylate of Pte-Glu is the preferred conjugation site for macromolecules en during endocytosis via the folate-binding protein receptor. However, it is also possible that the steric placement of the attached macromolecule aroun d the vitamin's pteridine moiety may be the more influential parameter cont rolling this delivery mechanism. Using solid-phase chemistries, we have syn thesized dipeptide derivatives of pteroic acid for the purpose of identifyi ng the preferred site onto which a macromolecule can be chemically attached without compromising its endocytosis potential. Thus, using fluorescent an d radiolabeled conjugates, we have determined that macromolecules attached to Pte-Glu by tither an alpha- or gamma-glutamyl linkage could associate wi th receptor-bearing cells at virtually identical levels. We further discove red that removal of the remaining un-conjugated glutamyl carboxylate had no inhibitory effect on cell uptake; and, the cytotoxicity of related momordi n toxin conjugates were comparable among the various pteroate derivatives t ested. From these observations we suggest that the preparation of endocytos is-competent pteroate-macromolecule conjugates is strongly influenced by th e steric environment around the ligand's para-aminobenzoic acid moiety, and that no selective isomeric (i.e. alpha Glu versus gamma Glu) conjugation r equirement necessarily exists.