Synthesis and characterization of oligonucleotides containing 2'-fluorinated thymidine glycol as inhibitors of the endonuclease III reaction.
Endonuclease III (Endo III) is a base excision repair enzyme that recognizes oxidized pyrimidine bases including thymine glycol. This enzyme is a glycosylase/lyase and forms a Schiff base-type intermediate with the substrate after the damaged base is removed. To investigate the mechanism of its substrate recognition by X-ray crystallography, we have synthesized oligonucleotides containing 2'-fluorothymidine glycol, expecting that the electron-withdrawing fluorine atom at the 2' position would stabilize the covalent intermediate, as observed for T4 endonuclease V (Endo V) in our previous study. Oxidation of 5'- and 3'-protected 2'-fluorothymidine with OsO4 produced two isomers of thymine glycol. Their configurations were determined by NMR spectroscopy after protection of the hydroxyl functions. The ratio of (5R,6S) and (5S,6R) isomers was 3:1, whereas this ratio was 6:1 in the case of the unmodified sugar. Both of the thymidine glycol isomers were converted to the corresponding phosphoramidite building blocks and were incorporated into oligonucleotides. When the duplexes containing 2'-fluorinated 5R- or 5S-thymidine glycol were treated with Escherichia coli endo III, no stabilized covalent intermediate was observed regardless of the stereochemistry at C5. The 5S isomer was found to form an enzyme-DNA complex, but the incision was inhibited probably by the fluorine-induced stabilization of the glycosidic bond.
Division of Chemistry, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama, Toyonaka, Osaka 560-8531, Japan.
Yusuke
Y
Doi
Atsushi
A
Katafuchi
Yoshie
Y
Fujiwara
Kenichi
K
Hitomi
John A
JA
Tainer
Hiroshi
H
Ide
Shigenori
S
Iwai
D043224:Deoxyribonuclease (Pyrimidine Dimer) / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D007536:Isomerism; D009841:Oligonucleotides / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000138:chemical synthesis / Q000737:chemistry; D013379:Substrate Specificity; D013936:Thymidine / Q000031:analogs & derivatives* / Q000737:chemistry; D014764:Viral Proteins / Q000378:metabolism
C509335:1-(2-deoxy-2-fluororibofuranosyl)-5,6-dihydro-5,6-dihydroxythymine; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D009841:Oligonucleotides; D009943:Organophosphorus Compounds; D014764:Viral Proteins; C434331:phosphoramidite; C470698:endonuclease V, phage T4; D043224:Deoxyribonuclease (Pyrimidine Dimer); C106403:NTH protein, E coli; D013936:Thymidine