Abstract
13C and 1H NMR spectroscopy were applied on fungicide pharmacology in cellular level, and two-dimensional 1H NMR spectroscopy in molecular level. Mycelia of Pyricularia oryzae were incubated with [methyl-13C]methionine, and 13C NMR spectrum of the intact mycelia was measured. After 3hr incubation, N-methyl signal of choline appeared at 54.9ppm showing that methyltransfer from methionine into choline can be observed by the 13C NMR. Treatment with 350μM iprobenfos inhibited the choline biosynthesis. Isoprothiolane also inhibited the methyltransfer at the concentration of 140μM. The observed spin-spin relaxation time (T2) of intracellular water protons reflects the membrane water permeability. Effects of various types of fungicides on the membrane were, then, investigated by using the T2 of the water protons in the mycelial cells of Botrytis cinerea. Treatment of the cells with ionophores remarkably increased the membrane water permeability, which suggests that the water molecules exchange through the ion channels of the ionophores. Phosphatidylcholine biosynthesis inhibitors, as well as ergosterol biosynthesis inhibitors, slightly increased the membrane water permeability. This result indicates that phosphatidylcholine and ergosterol have no direct relation to the membrane water permeability though some secondary effects were observed through the change in the membrane structure. Some SH inhibitors increased the membrane water permeability probably by binding to the proteins at ion channels. The structure of the complex of a deoxyoctanucleotide, d(GCAATTGC)2, and berenil, a trypanosidal drug, was analyzed by two-dimensional 1H NMR spectroscopy. The nuclear Overhauser effects (NOE) between the two molecules and the ring current shifts revealed that berenil binds in the minor groove of d(GCAATTGC)2 retaining the overall B conformation of the octanucleotide duplex. It is likely that the complex has hydrogen bonds between the berenil amidine protons and the carbonyl oxygen of the external thymine or the purine nitrogen of the internal adenine.
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