N-[N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithinyl]-L-phenylalanine (1), a carboxypeptidase A (CPA) cleavable prodrug was synthesized for use in an antibody directed strategy to improve the therapeutic selectivity of N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine (2), an extremely potent nonpoly-glutamatable DHFR inhibitor which is also highly cytotoxic. Compound 1 was shown by HPLC analysis to give a >99% yield of 2 upon incubation with bovine CPA (bCPA) for 20 min at 25 degrees C. In a spectrophotometric kinetic assay with 50 microM dihydrofolate as the competing substrate in the presence of 65 microM NADPH, 1+bCPA stoichiometrically inhibited recombinant human DHFR (rhDHFR) with a K(i) of 0.35 pM. In contrast, 1 without bCPA was a poor inhibitor of rhDHFR (K(i)>10 microM). In a 72 h growth inhibition assay against cultured CCRF-CEM human leukemic lymphoblasts, the growth inhibitory activities of 1+bCPA, 2+bCPA, and 2 alone were the same (IC(50) 1.3-1.4 nM), whereas 1 in the absence of bCPA was >100-fold less potent (IC(50) 155 nM).