Palladium-Catalyzed Synthesis of 5‑Formyl-2'-Deoxycytidine, 5‑Carboxyl-2'-Deoxycytidine, and Nitrogen-15-Labeled Derivatives via Ex Situ-Generated Carbon Monoxide from Chloroform and Application in Isotope Dilution LC-MS/MS Analysis of DNA

Herein, we report a modular palladium-catalyzed carbonylative synthesis of unlabeled and ¹⁵N₃-labeled epigenetic DNA lesions 5-formyl-2'-deoxycytidine (5fdC) and 5-carboxyl-2'-deoxycytidine (5cadC) via ex situ CO generation from a KOH/CHCl₃ surrogate. For 5fdC synthesis, the protected 5-iodo-3',5'-(tert-butyl-dimethylsilyl)-2'-deoxycytidine was converted to the key intermediate, 5-formyl-3',5'-(tert-butyl-dimethylsilyl)-2'-deoxycytidine, through a vial-in-a-vial procedure (vial A: KOH, CHCl₃, and toluene; vial B: Pd₂(dba)₃, xantphos, Bu₃SnH [slow addition], toluene, and 65 °C) in a very good yield. An analogous approach was employed for the synthesis of the 5-methoxycarbonyl-2'-deoxycytidine precursor, with the carbonylative coupling conducted under modified conditions (vial A: KOH and CHCl₃ toluene; vial B: Pd₂(dba)₃, xantphos, Et₃N, and methanol, 55 °C). The KOH/CHCl₃ system provides significant safety advantages over gaseous CO allowing also synthetic efficiency at the microscale level. The ¹⁵N₃-labeled variants (4-amino-N1, N3-¹⁵N)-5fdC and (4-amino-, N1, N3-¹⁵N)-5cadC were synthesized with high isotopic purity and characterized by high-resolution mass (HRMS) and tandem mass (MS²) spectrometry. These labeled standards were subsequently employed in liquid chromatography-isotope dilution tandem mass spectrometry (LC-ID-MS/MS) that is used for accurate quantification of endogenous 5fdC and 5cadC in DNA. This methodology provides the synthetic stable isotope "heavy"-labeled compounds needed and a robust analytical platform for investigating the formation and biological roles of these oxidatively generated epigenetic DNA lesions in the dysregulation of gene expression.