Two Scaffolds from Two Flips: (α,β)/(β,γ) CH₂/NH “Met-Im” Analogues of dTTP

Novel α,β-CH₂ and β,γ-NH (<b>1a</b>) or α,β-NH and β,γ-CH₂ (<b>1b</b>) “Met-Im” dTTPs were synthesized via monodemethylation of triethyl-dimethyl phosphorimido-bisphosphonate synthons (<b>4a</b>, <b>4b</b>), formed via a base-induced [1,3]-rearrangement of precursors (<b>3a</b>, <b>3b</b>) in a reaction with dimethyl or diethyl phosphochloridate. Anomerization during final bromotrimethylsilane (BTMS) deprotection after Mitsunobu conjugation with dT was avoided by microwave conditions. <b>1a</b> was 9-fold more potent in inhibiting DNA polymerase β, attributed to an NH-group interaction with R183 in the active site

Mapping Functional Substrate–Enzyme Interactions in the pol β Active Site through Chemical Biology: Structural Responses to Acidity Modification of Incoming dNTPs

We report high-resolution crystal structures of DNA polymerase (pol) β in ternary complex with a panel of incoming dNTPs carrying acidity-modified 5′-triphosphate groups. These novel dNTP analogues have a variety of halomethylene substitutions replacing the bridging oxygen between Pβ and Pγ of the incoming dNTP, whereas other analogues have alkaline substitutions at the bridging oxygen. Use of these analogues allows the first systematic comparison of effects of 5′-triphosphate acidity modification on active site structures and the rate constant of DNA synthesis. These ternary complex structures with incoming dATP, dTTP, and dCTP analogues reveal the enzyme’s active site is not grossly altered by the acidity modifications of the triphosphate group, yet with analogues of all three incoming dNTP bases, subtle structural differences are apparent in interactions around the nascent base pair and at the guanidinium groups of active site arginine residues. These results are important for understanding how acidity modification of the incoming dNTP’s 5′-triphosphate can influence DNA polymerase activity and the significance of interactions at arginines 183 and 149 in the active site

Transition State in DNA Polymerase β Catalysis: Rate-Limiting Chemistry Altered by Base-Pair Configuration

Kinetics studies of dNTP analogues having pyrophosphate-mimicking β,γ-pCXYp leaving groups with variable X and Y substitution reveal striking differences in the chemical transition-state energy for DNA polymerase β that depend on all aspects of base-pairing configurations, including whether the incoming dNTP is a purine or pyrimidine and if base-pairings are right (T•A and G•C) or wrong (T•G and G•T). Brønsted plots of the catalytic rate constant (log­(<i>k</i>_pol)) versus p<i>K</i>_a4 for the leaving group exhibit linear free energy relationships (LFERs) with negative slopes ranging from −0.6 to −2.0, consistent with chemical rate-determining transition-states in which the active-site adjusts to charge-stabilization demand during chemistry depending on base-pair configuration. The Brønsted slopes as well as the intercepts differ dramatically and provide the first direct evidence that dNTP base recognition by the enzyme–primer–template complex triggers a conformational change in the catalytic region of the active-site that significantly modifies the rate-determining chemical step

Effect of β,γ-CHF- and β,γ-CHCl-dGTP Halogen Atom Stereochemistry on the Transition State of DNA Polymerase β

Recently, we synthesized the first individual β,γ-CHX-dGTP diastereomers [(<i>R</i>)- or (<i>S</i>)-CHX, where X is F or Cl] and determined their structures in ternary complexes with DNA polymerase β (pol β). We now report stereospecificity by pol β on the mixed β,γ-CHX diastereomer pairs using nuclear magnetic resonance and on the separate diastereomers using transient kinetics. For both the F and Cl diastereomers, the <i>R</i> isomer is favored over the <i>S</i> isomer for G·C correct incorporation, with stereospecificities [(<i>k</i>_pol/<i>K</i>_d)_<i>R</i>/(<i>k</i>_pol/<i>K</i>_d)_<i>S</i>] of 3.8 and 6.3, respectively, and also for G·T misincorporation, with stereospecificities of 11 and 7.8, respectively. Stereopreference for the (<i>R</i>)-CHF-dGTP diastereomer was abolished for <i>k</i>_pol but not <i>K</i>_d with mutant pol β (R183A). These compounds constitute a new class of stereochemical probes for active site interactions involving halogen atoms. As Arg183 is unique in family X pols, the design of CXY deoxyribonucleotide analogues to enhance interaction is a possible strategy for inhibiting BER selectively in cancer cells

Probing DNA Base-Dependent Leaving Group Kinetic Effects on the DNA Polymerase Transition State

We examine the DNA polymerase β (pol β) transition state (TS) from a leaving group pre-steady-state kinetics perspective by measuring the rate of incorporation of dNTPs and corresponding novel β,γ-CXY-dNTP analogues, including individual β,γ-CHF and -CHCl diastereomers with defined stereochemistry at the bridging carbon, during the formation of right (R) and wrong (W) base pairs. Brønsted plots of log <i>k</i>_pol versus p<i>K</i>_a4 of the leaving group bisphosphonic acids are used to interrogate the effects of the base identity, the dNTP analogue leaving group basicity, and the precise configuration of the C-X atom in <i>R</i> and <i>S</i> stereoisomers on the rate-determining step (<i>k</i>_pol). The dNTP analogues provide a range of leaving group basicity and steric properties by virtue of monohalogen, dihalogen, or methyl substitution at the carbon atom bridging the β,γ-bisphosphonate that mimics the natural pyrophosphate leaving group in dNTPs. Brønsted plot relationships with negative slopes are revealed by the data, as was found for the dGTP and dTTP analogues, consistent with a bond-breaking component to the TS energy. However, greater multiplicity was shown in the linear free energy relationship, revealing an unexpected dependence on the nucleotide base for both A and C. Strong base-dependent perturbations that modulate TS relative to ground-state energies are likely to arise from electrostatic effects on catalysis in the pol active site. Deviations from a uniform linear Brønsted plot relationship are discussed in terms of insights gained from structural features of the prechemistry DNA polymerase active site