Gamma-fluoromethotrexate (
FMTX) is a poorly glutamylated mimic of the anti-
cancer drug methotrexate (MTX) which is useful in studies of the roles of MTX poly-gamma-
glutamates. A second chiral center occurs at C-4 of the 4-fluoroglutamate used to synthesize
FMTX and, as a consequence,
FMTX occurs as both D,L-erythro and D,L-threo diastereomers. The interaction of both diastereomers with intracellular
dihydrofolate reductase has been examined in the human
leukemia cell line CCRF-CEM, using a centrifugal column technique. Measurements of the rate at which radiolabel was displaced from [3H]MTX-saturated
dihydrofolate reductase following
suspension of the cells in unlabeled
drug indicated that MTX and the erythro isomer of
FMTX gave essentially the same rate of displacement; the rate of displacement by the threo isomer of
FMTX was slower, but the interpretation of these data was ambiguous since the rate of transport of threo-
FMTX may have been limiting. In reciprocal experiments in which
dihydrofolate reductase was saturated with [3H]erythro-
FMTX, the erythro isomer and MTX again behaved equivalently in terms of displacement. When
dihydrofolate reductase was saturated with [3H]threo-
FMTX, the radiolabel was clearly displaced at a much faster rate than either other radiolabel regardless of whether the displacing agent was MTX or the isomer. These results indicate a distinct stereospecificity for interaction of inhibitor with
dihydrofolate reductase in which the threo isomer has a faster off-rate. Of the two
FMTX diastereomers, the erythro isomer thus most closely mimics the properties of MTX.