Interactions of 2'-fluoro-substituted dUMP analogues with thymidylate synthase.
A series of 2'-fluoro-substituted dUMP/FdUMP analogues were synthesized, their interaction with human recombinant thymidylate synthase investigated, and structural (1)H and (19)F NMR study of the corresponding nucleosides performed. While 2'-F-dUMP (fluorine in the "down" configuration), in striking contrast to 2'-F-ara-UMP (fluorine in the "up" configuration) and 2',2''-diF-dUMP, showed substrate activity, 2'-F-ara-UMP and 2',2''-diF-dUMP were classic inhibitors, and 2',5-diF-ara-UMP 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 C(6) 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.
Ziemkowski
Przemysław
P
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 5A Pawińskiego Street, 02-106 Warszawa, Poland.
Felczak
Krzysztof
K
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 5A Pawińskiego Street, 02-106 Warszawa, Poland.
Poznański
Jarosław
J
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 5A Pawińskiego Street, 02-106 Warszawa, Poland.
Kulikowski
Tadeusz
T
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 5A Pawińskiego Street, 02-106 Warszawa, Poland.
Zieliński
Zbigniew
Z
Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur Street, 02-093 Warszawa, Poland.
Cieśla
Joanna
J
Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur Street, 02-093 Warszawa, Poland.
Rode
Wojciech
W
Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur Street, 02-093 Warszawa, Poland. Electronic address: rode@nencki.gov.pl.
D002241:Carbohydrates / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D003856:Deoxyuracil Nucleotides / Q000737:chemistry; D005461:Fluorine / Q000737:chemistry*; D005468:Fluorodeoxyuridylate / Q000737:chemistry*; D005663:Furans / Q000737:chemistry; D006027:Glycosides / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008956:Models, Chemical; D008968:Molecular Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013940:Thymidylate Synthase / Q000737:chemistry*
D002241:Carbohydrates; D003856:Deoxyuracil Nucleotides; D005663:Furans; D006027:Glycosides; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D005468:Fluorodeoxyuridylate; D005461:Fluorine; C007267:2'-deoxyuridylic acid; D013940:Thymidylate Synthase; D003545:Cysteine