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: (S)- and (R)-β,γ-CHCl-dGTP (12a-1/12a-2) and (S)- and (R)-β,γ-CHF-dGTP (12b-1/12b-2). Central to their preparation was conversion of the prochiral parent bisphosphonic acids to the P,C-dimorpholinamide derivatives 7 of their (R)-mandelic acid monoesters, which provided access to the individual diastereomers 7a-1, 7a-2, 7b-1, and 7b-2 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 12, 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 12a,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 12b) are assigned to the R 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.