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

β,γ-CHF- and β,γ-CHCl-dGTP Diastereomers: Synthesis, Discrete ³¹P NMR Signatures, and Absolute Configurations of New Stereochemical Probes for DNA Polymerases

Deoxynucleoside 5′-triphosphate analogues in which the β,γ-bridging oxygen has been replaced with a CXY group are useful chemical probes to investigate DNA polymerase catalytic and base-selection mechanisms. A limitation of such probes has been that conventional synthetic methods generate a mixture of diastereomers when the bridging carbon substitution is nonequivalent (X ≠ Y). We report here a general solution to this long-standing problem with four examples of β,γ-CXY dNTP diastereomers: (<i>S</i>)- and (<i>R</i>)-β,γ-CHCl-dGTP (<b>12a-1</b>/<b>12a-2</b>) and (<i>S</i>)- and (<i>R</i>)-β,γ-CHF-dGTP (<b>12b-1</b>/<b>12b-2</b>). Central to their preparation was conversion of the prochiral parent bisphosphonic acids to the P,C-dimorpholinamide derivatives <b>7</b> of their (<i>R</i>)-mandelic acid monoesters, which provided access to the individual diastereomers <b>7a-1</b>, <b>7a-2</b>, <b>7b-1</b>, and <b>7b-2</b> by preparative HPLC. Selective acidic hydrolysis of the P–N bond then afforded “portal” diastereomers, which were readily coupled to morpholine-activated dGMP. Removal of the chiral auxiliary by H₂ (Pd/C) gave the four individual diastereomeric nucleotides <b>12</b>, which were characterized by ³¹P, ¹H, and ¹⁹F NMR spectroscopy and by mass spectrometry. After treatment with Chelex-100 to remove traces of paramagnetic ions, at pH ∼10 the diastereomer pairs <b>12a</b>,<b>b</b> exhibit discrete P_α and P_β ³¹P resonances. The more upfield P_α and more downfield P_β resonances (and also the more upfield ¹⁹F NMR resonance in <b>12b</b>) are assigned to the <i>R</i> configuration at the P_β-CHX-P_γ carbons on the basis of the absolute configurations of the individual diastereomers as determined from the X-ray crystallographic structures of their ternary complexes with DNA and polymerase β

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