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

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 H ₂ /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 , 3 4b ), β,γ-CHF-dCTP ( 35a , 35b ) were obtained. The β,γ-CH(CH ₃ )-dATPs ( 44a , 44b ) were obtained using a methyl ( R )-(-)-phenylglycinate auxiliary. ³¹ P and ¹⁹ F NMR Δδ values are correlated with CXY stereochemistry and p K _a2-4 values for 13 CXY-bisphosphonic acids and imidodiphosphonic acid are tabulated.