α,β-Difluoromethylene Deoxynucleoside 5′-Triphosphates: A Convenient Synthesis of Useful Probes for DNA Polymerase β Structure and Function

Abstract

Abstractα,β-Difluoromethylene deoxynucleoside 5'-triphosphates (dNTPs, N = A or C) are advantageously obtained via phosphorylation of corresponding dNDP analogues using catalytic ATP, PEP, nucleoside diphosphate kinase (NDPK) and pyruvate kinase (PK). DNA pol β K d values for the α,β-CF 2 and unmodified dNTPs, α,β-NH dUTP, and the a,β-CH 2 analogues of dATP and dGTP are discussed in relation to the conformations of α,β-CF 2 dTTP v. α,β-NH dUTP bound into the enzyme active site.In an ongoing multidisciplinary study of structure and function of DNA polymerase β, a eukaryotic enzyme primarily involved in filling short DNA gaps 1 , we required a series of α,β-methylene-substituted analogues of deoxynucleoside 5'-triphosphates (dNTPs). When the *Email Address: mckenna@usc.edu. Supporting Information Available General methods used; detailed synthetic procedures and characterization data for 1 -9; analytical and preparative HPLC methods; HPLC studies of conversions of 1 to 2, 2 to 3, 7 to 4, 4 to 5, 5 to 6; 1 H NMR spectra of 1-7 and 9; 31 P and 19 F NMR spectra of 2, 3, 5, 6, 9; 13 C NMR spectrum of 9; analytical HPLC traces for 3, 6 and 9; HRMS spectra for 3 and 6, and LRMS spectrum for 9; DNA synthesis gel analysis of 9 before and after HPLC purification; protocol and inhibition plot for inhibition of pol β dCTP incorporation by 6; crystallographic methods and statistics for the ternary pol β complex with 9; and supplementary literature references are available online at http://pubs.acs.org. P α -O-P β bridging oxygen in a natural mononucleotide substrate is replaced by an imido (NH) 2-4 or methylene (CXY) 4,5 group, the P-N or P-C bond should resist cleavage in the nucleotidyl transfer reaction catalyzed by the enzyme. As a result, these analogues will remain intact in stable ternary DNA complexes with the polymerase and therefore should be useful to probe pre-chemistry enzyme-complex function and structure, as recently shown with in an X-ray crystallographic study of α,β-NH dUTP with DNA pol β. 6 Information about such complexes provides a reference point for theoretical analysis of the chemical mechanism 7 for the complete transfer of a monophosphate nucleoside donor to the sugar acceptor in the active site. As probes for the mechanism of polymerase catalysis and its relationship to polymerase fidelity, α,β-methylene dNTP analogues permit exploration of stereoelectronic effects on active site interactions, by making appropriate substitutions X,Y on the adjacent P α CXY bridging carbon. The largest obtainable electron-withdrawing effect with minimal steric perturbation can be achieved using X,Y = F, resulting in analogues in which the bisphosphonate group is expected to be less basic than the pyrophosphate moiety in the natural dNTPs. 8,9 In this article, we describe the first synthesis of α,β-CF 2 dCTP 6, using a modified chemicalenzymological approach that also can be applied to synthesis of α,β-CF 2 dATP 3, affording these compounds in sufficient purity to virtually eliminate detectable contaminating substrate ...

Synthesis and biological evaluation of fluorinated deoxynucleotide analogs based on bis-(difluoromethylene)triphosphoric acid

“…Because Watson-Crick hydrogen bonding will not occur with the analog in this situation (i.e., would create an A-A mismatch), this result suggests that only the triphosphate portion of the analog and natural nucleotide are competing. This is also consistent with the weak binding affinity observed when the bridging O α;β of the incoming nucleotide is substituted with CF 2 (35). Thus, although the crystal structure indicates that the bisCF 2 -substituted analog is well tolerated within the polymerase active site, the primary conformation in solution does not permit Watson-Crick type hydrogen bonding.…”

“…The binding constant for the (α,β),(β,γ)-bisCF 2 dTTP analog was estimated through a steady-state inhibition analysis and found to be similar to (α,β)-CF 2 -dTTP analog (K i ¼ 1.4 mM) (35). Likewise, the (α,β),(β,γ)-bisCF 2 dATP bound weakly (K i ¼ 0.7 mM).…”

“…Only a few methods for attaching low nucleophilicity phosphates to the 5′-carbon of a nucleoside are known (14,15). These methods include the Mitsunobu reaction (8,(16)(17)(18), couplings promoted by DCC and other dehydrating agents (19,20), electrophilic phosphorylation developed by Yoshikawa, Ludwig, and Eckstein employing 2-chloro-4H-1,3, 2-benzodioxaphosphorin-4-one (21)(22)(23)(24), nucleophilic cleavage of phosphoryl anhydride (25), reactions involving phosphoramidates (19,(26)(27)(28)(29)(30)), Blackburn's method employing nucleophilic substitution at 5′ position (31)(32)(33), and enzymatic phosphorylation (20,34,35). All of these methods lack versatility and their success depends on many factors, such as nucleophilicity/electrophilicity of the phosphate source, steric bulkiness, stability of the product under acidic or basic conditions, etc.…”

“…However, the protected deoxyguanosine 5′-tosylate reacted with 11 very slowly, and the maximum conversion that could be achieved was 10% (Scheme 2). Interestingly, tributylammonium salt of BMF 4 TPA 13 showed absolutely no reactivity in Blackburn's method, although similar nucleophilic substitution with tributylammonium salt of difluoromethylene bisphosphonic acid proceeds with high yields (31)(32)(33)(34)(35). This could be due to intramolecular hydrogen bonding in BMF 4 TPA tributylammonium salt, which may significantly lower nucleophilicity of the anionic species.…”

“…Earlier structures of DNA pol β with incoming nucleoside triphosphate analogs with CF 2 substituted for O α;β or O β;γ indicated that this substitution is well tolerated (35,36). Recently, even O β;γ has been substituted by CXY group (X;Y ¼ H, F, Cl, Br, and /or CH 3 ) (37, 38).…”