title: Alpha,beta-methylene-2'-deoxynucleoside 5'-triphosphates as noncleavable substrates for DNA polymerases: isolation, characterization, and stability studies of novel 2'-deoxycyclonucleosides, 3,5'-cyclo-dG, and 2,5'-cyclo-dT.
abstract: We report synthesis and characterization of a complete set of alpha,beta-methylene-2'-dNTPs (alpha,beta-m-dNTP; N = A, C, T, G, 12-15) in which the alpha,beta-oxygen linkage of natural dNTP was replaced by a methylene group. These nucleotides were designed to be noncleavable substrates for DNA polymerases. Synthesis entails preparation of 2'-deoxynucleoside 5'-diphosphate precursors, followed by an enzymatic gamma-phosphorylation. All four synthesized alpha,beta-m-dNTPs were found to be potent inhibitors of polymerase beta, with K i values ranging 1-5 microM. During preparation of the dG and dT derivatives of alpha,beta-methylene diphosphate, we also isolated significant amounts of 3,5'-cyclo-dG (16) and 2,5'-cyclo-dT (17), respectively. These novel 2'-deoxycyclonucleosides were formed via a base-catalyzed intramolecular cyclization (N3 --> C5' and O2 --> C5', respectively). In acidic solution, both 16 and 17 underwent glycolysis, followed by complete depurination. When exposed to alkaline conditions, 16 underwent an oxidative deamination to produce 3,5'- cyclo-2'-deoxyxanthosine (19), whereas 17 was hydrolyzed exclusively to dT.
authors.affiliation: Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, 41 Lower College Road, Kingston, Rhode Island 02881, USA.
authors.forename: Fengting
authors.initials: F
authors.lastname: Liang
authors.forename: Nidhi
authors.initials: N
authors.lastname: Jain
authors.forename: Troy
authors.initials: T
authors.lastname: Hutchens
authors.forename: David D
authors.initials: DD
authors.lastname: Shock
authors.forename: William A
authors.initials: WA
authors.lastname: Beard
authors.forename: Samuel H
authors.initials: SH
authors.lastname: Wilson
authors.forename: M Paul
authors.initials: MP
authors.lastname: Chiarelli
authors.forename: Bongsup P
authors.initials: BP
authors.lastname: Cho
mesh_terms: D002851:Chromatography, High Pressure Liquid; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003845:Deoxycytosine Nucleotides / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D015394:Molecular Structure; D013379:Substrate Specificity
chemical_list: D003845:Deoxycytosine Nucleotides; D003854:Deoxyribonucleotides; D004259:DNA-Directed DNA Polymerase