The activity of inhibitors of dihydrofolate reductase (such as methotrexate) as cancer chemotherapeutic agents has led to the search for alternate target enzyme within the folate pathways that are essential for proliferating cells. Folylpolyglutamate synthetase has these characteristics and is being examined as a new target for folate antimetabolites. Previous studies have indicated that interaction of the glutamic acid side chain of the folate molecule is essential for binding of folate analogs bind poorly to folylpolyglutamate synthetase, a fact that indicates rigid structural requirements in the active site of this enzyme. One class of compounds, however, that are modified in the Omega-position have been found to be the first specific inhibitors of this enzyme. The overall goals of this work are to exploit this lead for the procurement of a potent inhibitor of folylpolyglutamate synthetase in tumor cells. Several new classes of folate analogs will be synthesized. The side chains of these compounds will differ from glutamic acid by modifications that will allow the testing of a number of hypotheses related to interactions between substrates and enzyme. We would modify the folate structure so as to : 1) alter the pKa of the Alpha- or Gamma-carboxyls; 2) allow the alkylation of any basic amino acid interacting with the Alpha-carboxyl of the folate substrate; 3) increase the binding of homocysteic acid analogs to enzymes; 4) replace the Alpha- or Gamma-carbosyls with other charged moieties; and 5) eliminate the Alpha- or Gamma-carboxyl in any analog that bound tightly to enzyme. These compounds will be studied as inhibitors of polyglutamation using partially purified mammalian folylpolyglutamate synthetase and also in intact mammalian tumor cells in culture. A compound that inhibits this process will be used to select for resistant cell lines. The cytotoxicity of new analogs will be tested against tumor cells in culture and in vivo.