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

Department of Chemistry, University of Southern California, Los Angeles, California 90089, USA.
Journal of the American Chemical Society (Impact Factor: 12.11). 03/2012; 134(21):8734-7. DOI: 10.1021/ja300218x
Source: PubMed


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 H(2) (Pd/C) gave the four individual diastereomeric nucleotides 12, which were characterized by (31)P, (1)H, and (19)F 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)(31)P resonances. The more upfield P(α) and more downfield P(β) resonances (and also the more upfield (19)F 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.

Download full-text


Available from: Samuel Wilson, Feb 07, 2014
  • [Show abstract] [Hide abstract]
    ABSTRACT: We describe a general method for the elongation of nucleoside oligophosphate chains by means of cyanoethyl (CE) phosphorimidazolides. Though the method requires a phosphorylation and subsequent deprotection reaction, both steps could be achieved in one pot without isolation/purification of the initial phosphorylation product. We have also found that pyrophosphate bond formation by this method is significantly accelerated by microwave irradiation.
    No preview · Article · Sep 2012 · Organic Letters
  • [Show abstract] [Hide abstract]
    ABSTRACT: Early studies on chemical synthesis of biological molecules can be seen to progress to preparation and biological evaluation of phosphonates as analogues of biological phosphates, with emphasis on their isosteric and isopolar character. Work with such mimics progressed into structural studies with a range of nucleotide-utilising enzymes. The arrival of metal fluorides as analogues of the phosphoryl group, PO 3−, for transition state (TS) analysis of enzyme reactions stimulated the symbiotic deployment of 19F NMR and protein crystallography. Characteristics of enzyme transition state analogues are reviewed for a range of reactions. From the available MFx species, trifluoroberyllate gives tetrahedral mimics of ground states (GS) in which phosphate is linked to carboxylate and phosphate oxyanions. Tetrafluoroaluminate is widely employed as a TS mimic, but it necessarily imposes octahedral geometry on the assembled complexes, whereas phosphoryl transfer involves trigonal bipyramidal (tbp) geometry. Trifluoromagnesate (MgF 3−) provides the near-ideal solution, delivering tbp geometry and correct anionic charge. Some of the forty reported tbp structures assigned as having AlF 30 cores have been redefined as trifluoromagnesate complexes. Transition state analogues for a range of kinases, mutases, and phosphatases provide a detailed description of mechanism for phosphoryl group transfer, supporting the concept of charge balance in their TS and of concerted-associative pathways for biocatalysis. Above all, superposition of GS and TS structures reveals that in associative phosphoryl transfer, the phosphorus atom migrates through a triangle of three, near-stationary, equatorial oxygens. The extension of these studies to near attack conformers further illuminates enzyme catalysis of phosphoryl transfer.
    No preview · Article · Oct 2012 · Biochemistry (Moscow)
  • [Show abstract] [Hide abstract]
    ABSTRACT: Recently, we synthesized the first individual β,γ-CHX-dGTP diastereomers [(R)- or (S)-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 R isomer is favored over the S isomer for G·C correct incorporation, with stereospecificities [(k(pol)/K(d))(R)/(k(pol)/K(d))(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 k(pol) but not K(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.
    No preview · Article · Oct 2012 · Biochemistry
Show more