α,β-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

The 2’-deoxynucleoside 5’-triphosphate (dNTP) analogs with modifications in the triphosphate chain have been used as nucleotide probes in various biochemical and structural studies. Here we report synthesis and characterization of a complete set of α,β-methylene-2’-dNTPs (α,β-m-dNTP; N = A, C, T, G, 12-15), in which the α,β-oxygen linkage of natural dNTP was replaced isosterically by a methylene group. These nucleotides were designed to be non-cleavable DNA polymerase substrates. Our synthesis process entails preparation of 2’-deoxynucleoside 5’-diphosphate precursors by nucleophilic coupling of 5’-tosyl nucleosides and methylene-diphosphate, and a subsequent enzymatic γ-phosphorylation. All four synthesized α,β-m-dNTPs were found to be potent inhibitors of polymerase β with Ki values ranging from 1-5 μM. During preparation of the dG and dT derivatives of α,β-methylene diphosphate, we isolated significant amounts of 3,5’-anhydro-2’-deoxyguanosine (cyclo-dG, 16) and 2,5’-anhydro-2’-deoxythymidine (cyclo-dT, 17), respectively. These novel 2’-deoxycyclonucleosides were formed via a base-catalyzed deprotonation of the imino proton (N1-H and N3-H), followed by an intramolecular cyclization (N3 → C5’ and O2 → C5’, respectively). In acidic solution, the cyclonucleosides underwent glycolysis at N9, followed by complete depurination at N3. In the case of cyclo-dG, there existed an equilibrium between glycolysis and deglycolysis at the glycosidic linkage prior to complete depurination. When exposed to alkaline conditions, cyclo-dG underwent an oxidative deamination at C2 to produce 3,5’-anhydro-2’-deoxyxanthosine (cyclo-dX, 19), whereas cyclo-dT was hydrolyzed exclusively to dT via cleavage at the 2,5’-ether linkage.

Highly Potent and Selective Ectonucleotide Pyrophosphatase/Phosphodiesterase I Inhibitors Based on an Adenosine 5′-(α or γ)-Thio-(α,β- or β,γ)-methylenetriphosphate Scaffold

“…2′,3′ ′-(Methoxymethylidene)-5′-tosyladenosine, compound 8 , was phosphorylated with methylenediphosphonate bis(tributylammonium) salt to form 2′,3′-(methoxymethylidene)adenosine 5′- α , β -methylenediphosphonate, compound 9 . 28 Next the protecting group was removed with 10% HCl and then with 10% NH 4 OH. Activation of compound 10 with carbonyl diimidazole (CDI) for 4–5 h resulted in a phosphoroimidazolide intermediate, the yield of which was ca.…”

Insights into the Conformation of Aminofluorene-Deoxyguanine Adduct in a DNA Polymerase Active Site

“…In contrast, the binding of this analog to pol ␤ is tight (K i ϭ 0.9 M; dashed line) (57). The concentration of dCTP for these experiments/simulations was 20 M. B, shown is the interaction of a Bf active site lysine residue with the incoming nucleotide.…”

“…1B). These methylene analogs bound with high affinity to pol ␤ with no active site distortions permitting catalytic metal binding (56,57). dCMPcPP binds tightly to pol ␤ with K i of 0.9 M in unmodified DNA (57).…”

“…These methylene analogs bound with high affinity to pol ␤ with no active site distortions permitting catalytic metal binding (56,57). dCMPcPP binds tightly to pol ␤ with K i of 0.9 M in unmodified DNA (57). In the presence of dCMPcPP, the dG-FAF adduct exhibited two conformers B and B*, which indicates that the adducted nucleotide adopts an anti conformation, placing the aminofluorene moiety in the major groove.…”

Phosphonate analogues of nucleoside polyphosphates

“…30,51,116,117 In the example depicted in Scheme 58, synthesis of α,β-methylene-2´-deoxynucleoside 5´-triphosphates involves preparation of 2´-deoxynucleoside 5´-diphosphate precursors followed by an enzymatic -phosphorylation. 118 Enzymatic phosphorylation has been shown to be more efficient than the chemical approach for preparation of α,β-methylene-dNTPs. The α,β-methylene dNDP analogues examined are poor substrate for pyruvate kinase (PK).…”