Deoxynucleotide analog probes and model compounds for studying DNA polymerase structure and mechanism: synthesis and evaluation of alkyl-, azido-, and halomethylene bisphosphonate-substituted triphosphates

2012-05-31A variety of triphosphate analogs that replace a natural phosphate anhydride P-O-P linkage with a phosphonate P-CXY-P moiety have been synthesized for the characterization of DNA polymerase β (pol β) mechanism and structure. A suite of β,γ-CXY-dTTP compounds (X,Y = H, F, Cl, Br) was synthesized to complement a previous study conducted with series of β,γ-CXY-dGTP analogs. The high purity of the β,γ-CXY-dTTP following dual-pass HPLC purification is demonstrated by NMR, MS, and HPLC data and illustrates the merit of the DCC-mediated morpholidate coupling approach to Pᵦ-CXY-Pᵧ triphosphate mimics. ❧ Novel α-azido bisphosphonates [(RO)₂P(O)]₂CXN₃ (R = i-Pr, X = Me; R = i-Pr, X = H; R = H, X = Me; and R = H, X = H) were developed to examine the impact of the unique azido steric profile on the synthesis and bioactivity of α,β-CXN₃ and β,γ-CXN₃ dNTP analogs. For one example, α,β-CMeN₃-dATP, RP HPLC separated dADP precursors that were used to generate the first examples of nucleoside triphosphate analogs with isolated stereochemistry at an asymmetrically substituted bridging carbon. The acidity constants of the α-azido bisphosphonic acids were determined by potentiometric titration and selected reactions including reduction and UV photolysis are introduced. ❧ The α,β-C(Me)₂-dATP and (R/S)-α,β-CHF-dATP analogs were synthesized and, together with the α,β-CXN₃ dATP analogs, the binding affinities (Kd) with pol β were determined. (R/S)-α,β-CHF-dATP was crystallized with a DNA-pol β binary complex. X-ray structure analysis revealed only the (S)-α,β-CFH-dATP diastereomer was present in the enzyme active site. Analysis of this structure provides evidence for a non-covalent stabilizing interaction between an active site water bound to Asp276 and the fluorine in (S)-α,β-CFH-dATP. Molecular docking studies further demonstrate the importance of this structural water by suggesting a steric clash with the methyl group of α,β-C(Me)2-dATP. ❧ A series of novel β,γ-CH₂, -CHF, and -CF₂ bisphosphonophosphate alkyl monoesters were synthesized and used as model compounds intended to estimate the activation parameters for the non-enzymatic hydrolysis of the triphosphate Pα-O-Pᵦ moiety. ¹⁸O-labeled water experiments showed that the exclusive site of nucleophilic attack in these systems is at Pᵦ. Thus, the catalytic efficiency for a small class of diphosphokinases could be approximated and the upper limit for the rate of non-enzymatic hydrolysis at Pα established. Supplementary compounds that increase Pᵦ-O bond stability by substituting Pᵧ with a phenyl ring are introduced. Preliminary hydrolysis experiments of these compounds highlight the relative stability of the Pα-O anhydride bond in triphosphates