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 pKa2-4 values for 13 CXY-bisphosphonic acids and imidodiphosphonic acid are tabulated.