In this research proposal multinuclear (H1, C13, N15, F19, P31) magnetic resonance spectroscopy is used as a means of studying (a) the conformation of folates and folate antagonists in solution and (b) the specific modes by which folates, folate antagonists, and certain pyrimidine nucleotides interact with enzymatic systems: in particular, dihydrofolate reductase and thymidylate synthetase. Fourier Transform assisted multinuclear magnetic resonance techniques are employed to obtain chemical shifts, relaxation times, and isotropic spin coupling constants which will be used to identify the loci (atoms or molecular groups) of the folates, folate antagonists, and pyrimidine nucleotides which are intimately involved in the binding of these compounds to dihydrofolate reductase or thymidylate synthetase. To aid in these investigations, synthetic procedures are described for the regiospecific enrichment with C13 or N15 of tryptophan, the pyrimidine moiety of pyrimidine nucleotides, and the substituted pteridine ring, p-aminobenzoic acid, and L-glutamic acid portions of folic acid and related folate antagonists. Nmr procedures will also be used to characterize the behavior of thymidylate synthetase and dihydrofolate reductase which have been chemically modified at specific sites with agents bearing an nmr sensitive nucleus or which have been obtained from bacteria cultured in the presence of selectively enriched amino acids. BIBLIOGRAPHIC REFERENCE: J. A. Lyon, R. B. and P. D. Ellis, "Assignment of the Carbon-13 Nuclear Magnetic Resonance Spectra of Selected Folates," J. Magn. Resonance, 18, 293 (1975).