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 non-equivalent (X ≠ Y). We report here a general solution to this long-standing problem with four examples of individual 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 achiral parent bisphosphonic acids to P,C-dimorpholinamide derivatives (7) of their (R)-mandelic acid monoesters (6), 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 the “ portal ” diastereomers 10, which were readily coupled to morpholine-activated dGMP. Removal of the chiral auxiliary by H2 (Pd/C) afforded the four individual diastereomeric nucleotides (12), which were characterized by 31P, 1H and 19F NMR, and by MS. After treatment with Chelex®-100 to remove traces of paramagnetic ions, at pH ~10 the diastereomer pairs 12a and 12b exhibit discrete Pα and Pβ
31P resonances. The more upfield Pα and more downfield Pβ resonances (and also the more upfield 19F NMR resonance in 12b) are assigned to the (R) configuration at the Pβ-CHX-Pγ carbons, based on the absolute configurations of the individual diastereomers as determined by X-ray crystallographic structures of their ternary complexes with DNA-pol β.
Recently, we synthesized the first individual β,γ-CHX dGTP diastereomers ((R)- or (S)-CHX; X = F or Cl), and determined their structures in ternary complexes with pol β. We now report stereospecificity by pol β on the mixed β,γ-CHX diastereomer pairs using NMR and on the separate diastereomers using transient kinetics. For both the F and Cl diastereomers, the R isomer is favored over the S isomer for G•C correct incorporation, with stereospecificities [(kpol/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. Stereopreference for the (R)-CHF dGTP diastereomer was abolished for kpol but not Kd 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, design of CXY deoxyribonucleotide analogues to enhance interaction is a possible strategy to inhibit BER selectively in cancer cells.
Eight novel single amino acid (6–11) and dipeptide (12, 13) tyrosine P-O esters of cyclic cidofovir ((S)-cHPMPCa, 4) and its cyclic adenine analog ((S)-cHPMPA, 3) were synthesized and evaluated as prodrugs. In vitro IC50 values for the prodrugs vs vaccinia, cowpox, human cytomegalo- and herpes simplex type 1 viruses were similar to those for the parent drugs ((S)-HPMPC, 2, (S)-HPMPA, 1; IC50 0.3 – 30 µM); there were no cytoxicity with KB or HFF cells at ≤ 100 µM. The prodrugs exhibited a wide range of half-lives in rat intestinal homogenate at pH 6.5 (<30 – 1732 min) with differences of 3–10× between phostonate diastereomers. The tyrosine-alkylamide derivatives of 3 and 4 were the most stable. (L)-Tyr-NHiBu cHPMPA (11) was converted in rat or mouse plasma solely to two active metabolites and had significantly enhanced oral bioavailability vs parent drug 1 in a mouse model (39 % vs <5 %).
The configuration at phosphorus in cyclic (S)-HPMPC (1, Cidofovir) and (S)-HPMPA (2) phenyl ester (5 and 6, respectively) diastereomers ((Rp)-5, (Rp)-6, (Sp)-6) were determined by X-ray crystallography and correlated to their 1H and 31P NMR spectra in solution. (Rp)-5 and (Rp)-6 have chair conformations with the nucleobase substituent equatorial and the P-OPh axial. Perhaps surprisingly, (Sp)-6 is (a, a) in the crystal and exists largely as an equilibrium of (a, a)/ (e, e) conformers in chloroform or acetonitrile.
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