Completing the β,γ-CXY-dNTP Stereochemical Probe Toolkit: Synthetic Access to the dCTP Diastereomers and ³¹P and ¹⁹F NMR Correlations with Absolute Configurations

Abstract

Nucleoside 5′-triphosphate (dNTP) analogues in which the β,γ-oxygen is mimicked by a CXY group (β,γ-CXY-dNTPs) have provided information about DNA polymerase catalysis and fidelity. Definition of CXY stereochemistry is important to elucidate precise binding modes. We previously reported the (R)- and (S)-β,γ-CHX-dGTP diastereomers (X = F, Cl), prepared via P,C-dimorpholinamide CHCl (6a, 6b) and CHF (7a, 7b) bisphosphonates (BPs) equipped with an (R)-mandelic acid as a chiral auxiliary, with final deprotection using H2/Pd. This method also affords the β,γ-CHCl-dTTP (11a, 11b), β,γ-CHF (12a, 12b), and β,γ-CHCl (13a, 13b) dATP diastereomers as documented here, but the reductive deprotection step is not compatible with dCTP or the bromo substituent in β,γ-CHBr-dNTP analogues. To complete assembly of the toolkit, we describe an alternative synthetic strategy featuring ethylbenzylamine or phenylglycine-derived chiral BP synthons incorporating a photolabile protecting group. After acid-catalyzed removal of the (R)-(+)-α-ethylbenzylamine auxiliary, coupling with activated dCMP and photochemical deprotection, the individual diastereomers of β,γ-CHBr- (33a, 33b), β,γ-CHCl- (34a, 34b), β,γ-CHF-dCTP (35a, 35b) were obtained. The β,γ-CH­(CH3)-dATPs (44a, 44b) were obtained using a methyl (R)-(−)-phenylglycinate auxiliary. 31P and 19F NMR Δδ values are correlated with CXY stereochemistry and pK a2–4 values for 13 CXY-bisphosphonic acids and imidodiphosphonic acid are tabulated.

Uridine Bisphosphonates Differentiate Phosphoglycosyl Transferase Superfamilies

“…This purification yielded individual, but not yet stereochemically-defined, epimers of S-25cα and R-25cα, primed for the final synthetic step. 25 Compound 9 was not converted to the corresponding α-GlcNAc-C(CH3)2 derivative due to the weak inhibition of the PGTs observed for this UBP precursor.…”

“…With these opportunities in mind, we describe here the modular syntheses of a series of uridine 5'bisphosphonate (CXY-UBP) and uridine 5'-bisphosphonate-N-acetylglucosamine (GlcNAc-CXY-UBP) analogues (Figure 2) in which the central diphosphate (P-O-P) oxygen is replaced by a substituted methylene (P-CXY-P), wherein X/Y = H/H, F/F, Cl/Cl, CH3/CH3, (S)-H/F and (R)-H/F. The α,β-CXY-and β,γ-bisphosphonate analogues of deoxynucleotides [25][26][27][28][29] have been previously used as probes of nucleic acid polymerase structure and function. [30][31][32][33] In addition to introduction of a non-hydrolyzable bisphosphonate mimicking the natural diphosphate, the CXY substitution allows variation in such properties as POH/POacidity, P-O/P-C bond lengths, P-O-P/P-C-P bond angles, 34 and the effect of steric perturbation adjacent to the CXY group.…”

Kinetic Effects of β,γ-Modified Deoxynucleoside 5′-Triphosphate Analogues on RNA-Catalyzed Polymerization of DNA

“…As probes for the mechanism of various protein DNA polymerases, a family of dNTP analogues in which the β,γ-bridging oxygen is replaced by a substituted methylene (CXY) or imido (NH) group has been synthesized − to study the effect of the leaving group structure on the kinetics of templated polymerization reactions. These nucleotide probes generally react in the same manner as standard dNTPs, resulting in the formation of a phosphodiester linkage between the 3′-hydroxyl of a template-bound primer and the α-phosphate of the incoming dNTP.…”

“…Taking advantage of a rich toolkit of β,γ-bridging O -substituted dGTP and dCTP analogues that span a broad range of acidity for the bisphosphonate leaving group, ,,− , the rate of deoxynucleotide incorporation was determined as described above. The dihalo-substituted series, which includes the CF 2 , CFCl, CCl 2 , and CBr 2 analogues, was especially informative, with the p K a4 of the leaving group spanning a range of 7.8 to 9.3.…”

“…The pK a4 values of the bisphosphonate or imidodiphosphate leaving groups, as previously determined experimentally, 20 are 7.8 for CF 2 , 8.4 for CFCl, 8.8 for CCl 2 , 8.9 for O (pyrophosphate), 9.0 for CHF, 9.3 for CBr 2 , 9.5 for CHCl, 9.7 for NH, 9.9 for CHBr, 10.5 for CH 2 , and 11.6 for CHCH3 . Where X has a stereogenic center,18,20 the dNTP analogue is a ∼1:1 mixture of diastereomers 20.…”