Novel stereochemical probes for DNA polymerases: Nucleoside triphosphate beta,gamma-CXY analogues

2'-Deoxynucleoside 5'-triphosphate analogues with beta,gamma-bridging oxygen replaced by a CXY group are useful chemical probes to investigate DNA polymerase catalytic and base-selection mechanisms. A long-standing limitation of such probes has been that conventional synthetic methods generate the analogues as mixtures of two diastereomer components in equal amounts when the bridging carbon substitution is nonequivalent (X ≠ Y). X-ray structural studies of DNA polymerase beta (pol beta) carried out with ~1:1 mixture of beta,gamma-CXY dGTP diastereomers revealed stereospecific binding exclusively with the fluorine-containing diastereomer analogues. These findings provided strong impetus in investigating the effect of stereochemical interactions of the beta,gamma-diastereomers on pol beta kinetics, the stereochemical effect that could not be addressed in the previous linear free energy relationship (LFER) correlation plotting log(kpol) of the ~1:1 mixed diastereomers vs. the pKa4 of the prochiral bisphosphonate leaving group.;A general solution to the long-standing synthetic challenge was developed with eight examples of beta,gamma-CXY dNTP diastereomers: (S)- and (R)-CHCl, (S)- and (R)-CHF, and (S)- and (R)-CFCl dGTP diastereomers, as well as (S)- and (R)-CHCl dATP diastereomers. Central to their preparation was the synthesis of the HPLC separable diastereomeric bisphosphonate synthons, the P,C-dimorpholidate derivatives of the ( R)-mandelic acid monoesters of the parent prochiral bisphosphonic acids. Selective acidic hydrolysis of the P-N bond afforded "portal" diastereomers, which were readily coupled to morpholine-activated dGMP or dAMP. Removal of the chiral auxiliary by hydrogenolysis gave the individual diastereomeric nucleotides, of which the diastereomeric purities were confirmed by 31P and/or 19F NMR spectroscopy. After treatment with Chelex-100 to remove traces of paramagnetic ions, at pH ~10 the diastereomer pairs exhibit discrete P&agr; and Pbeta 31 P resonances, and discrete 19F resonances for fluorine-containing diastereomer pairs. The more upfield P&agr; and more downfield Pbeta resonances, as well as the more upfield 19F NMR resonances for the beta,gamma-CHF dGTP diastereomer, were assigned to the R configuration at the Pbeta-CHX-P gamma carbons, based on the absolute configurations of the individual diastereomers as determined from the X-ray crystallographic structures of their ternary complexes with DNA and polymerase beta.;An NMR method that was developed to identify the individual diastereomers in their mixture solutions was successfully applied to analyze kinetic assays in which the (R)- and (S)-beta,gamma-CHX dGTP (X = F, Cl, Br) diastereomers were competing for pol beta incorporation. Selective incorporation by pol beta was observed for all three diastereomer pairs, with stereospecificities of 3 (beta,gamma-CHF), 7 (beta,gamma-CHCl) and 2-3 (beta,gamma-CHBr). The (R)-stereoisomer was preferentially inserted for both the beta,gamma-CHF and beta,gamma-CHCl analogues. In the case of beta,gamma-CHBr analogue, although the absolute configuration of the preferred stereoisomer has not been identified yet, the preferred isomer also showed more upfield P&agr; and more downfield Pbeta resonances, similar to those of the (R)-CHF and (R)-CHCl stereoisomers.;Pre-steady-state kinetics of pol beta were performed with the fully characterized individual diastereomers of (R)- and (S)-beta,gamma-CHF dGTP and (R)- and (S)-beta,gamma-CHCl dGTP. For both diastereomer pairs, the (R)-stereoisomer was favored over the (S)-stereoisomer for G*C correct incorporation, with stereospecificities [(k pol/Kd)R/( kpol/Kd)S] of 3.8 and 6.3, respectively, and also for G*T misincorporation with stereospecificities of 11 and 7.8, respectively. The pre-steady state kinetics and direct competition assays showed similar stereospecificities with beta,gamma-CHCl and beta,gamma-CHF diastereomer pairs for G*C correct incorporation.