A series of 2'-fluoro -substituted dUMP/FdUMP analogues were synthesized, their interaction with human recombinant thymidylate synthase investigated, and structural H-1 and F-19 NMR study of the corresponding nucleosides performed. While 2'-F-dUMP (fluorine in the "down" configuration), in striking contrast to 2'-2''-ara-UMP (fluorine in the "up" configuration) and 2',2"-diF-dUMP, showed substrate activity, 2'-2''-ara-UMP and 2',2"-diF-dUMP were classic inhibitors, and 2',5-diF-ara-UNIP behaved as a strong slow-binding inhibitor, suggesting the 2'-F substituent in the "up" position to interfere with the active center cysteine thiol addition to the pyrimidine Q6) and the pyrimidine C(5)-F to prevent this interference. In support, the direct through space heteronuclear coupling J(HF) was observed for the fluorine "up" derivatives, 2'-F-ara-U and 2',5-diF-ara-U, causing the splitting of the H(6) resonance lines. The absence of such splitting in 2',2"-diF-dUrd, indicating an unusual orientation of the base in relation to the furanose, was associated with an exceptionally weak interaction with the enzyme. (c) 2007 Elsevier Inc. All rights reserved.