It is now clear that monoglutamyl folates, upon entering mammalian cells, are rapidly transformed to poly-gamma-glutamyl derivatives, that these compounds function as substrates for the folate dependent cellular enzymes and that mutational loss of the enzyme(s) that synthesize poly-gamma-glutamyl folates is a lethal deletion. The overall goal of this study is to evaluate poly-gamma-glutamate synthetase as a target enzyme for antifolate chemotherapy. The proposed work would (1) further delineate the role of the folate poly-gamma-glutamate synthetase enzyme system in celular metabolism, (2) study its distribution in normal mammalian tissues and various rodent and human tumor types, (3) attempt purification to homogeneity of the enzyme from human osteogenic sarcoma using newly synthesized inhibitors and normal substrates as ligands for affinity chromatography, (4) test potential transition state analogs of this enzymic reaction as inhibitors of the purified enzyme and of tumor cell growth in vitro, (5) test these compounds against relevant tumor models in vivo when inhibition of tumor cell growth in vitro warrants such testing and (6) characterize the alterations in folate metabolism induced by such inhibitors using mouse and human tumor cells in culture. This project offers a novel and rationally conceived class of antifol antimetabolites which differ from the classical antifols typified by methotrexate in the site of induced inhibition. As such, these agents hold promise in their own right, but also are of interest for use in combination chemotherapy with dihydrofolate reductase inhibitors or for use against tumors resistant to methotrexate.