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

A 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 beta (pol beta) mechanism and structure. A suite of beta,gamma-CXY-dTTP compounds (X,Y = H, F, Cl, Br) was synthesized to complement a previous study conducted with series of beta,gamma-CXY-dGTP analogs. The high purity of the beta,gamma-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 Pbeta -CXY-Pgamma triphosphate mimics.;Novel alpha-azido bisphosphonates [(RO)2P(O)]2CXN 3 (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 alpha,beta-CXN3 and beta,gamma-CXN 3 dNTP analogs. For one example, alpha,beta-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 alpha-azido bisphosphonic acids were determined by potentiometric titration and selected reactions including reduction and UV photolysis are introduced.;The alpha,beta-C(Me)2-dATP and (R/S)-alpha,beta-CHF-dATP analogs were synthesized and, together with the alpha,beta-CXN3 dATP analogs, the binding affinities (Kd) with pol beta were determined. (R/S)-alpha,beta-CHF-dATP was crystallized with a DNA-pol beta binary complex. X-ray structure analysis revealed only the (S)-alpha,beta-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)-alpha,beta-CFH-dATP. Molecular docking studies further demonstrate the importance of this structural water by suggesting a steric clash with the methyl group of alpha,beta-C(Me) 2-dATP.;A series of novel beta,gamma-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 Palpha-O-Pbeta moiety. 18O-labeled water experiments showed that the exclusive site of nucleophilic attack in these systems is at Pbeta. 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 Palpha established. Supplementary compounds that increase Pbeta-O bond stability by substituting Pgamma; with a phenyl ring are introduced. Preliminary hydrolysis experiments of these compounds highlight the relative stability of the Palpha-O anhydride bond in triphosphates.