Biodegradable Protections for Nucleoside 5′-Monophosphates: Comparative Study on the Removal of O-Acetyl and O-Acetyloxymethyl Protected 3-Hydroxy-2,2-bis(ethoxycarbonyl)propyl Groups

The applicability of 3-acetyloxy-2,2-bis(ethoxycarbonyl)propyl and 3-acetyloxymethoxy-2,2-bis(ethoxycarbonyl)propyl groups as biodegradable phosphate protecting groups for nucleoside 5′-monophosphates has been studied in a HEPES buffer at pH 7.5. Enzymatic deacetylation with porcine carboxyesterase triggers the removal of the resulting 3-hydroxy-2,2-bis(ethoxycarbonyl)propyl and 3-hydroxymethoxy-2,2-bis(ethoxycarbonyl)propyl groups by retro-aldol condensation and consecutive half acetal hydrolysis and retro-aldol condensation, respectively. The kinetics of these multistep deprotection reactions have been followed by HPLC, using appropriately protected thymidine 5′-monophosphates as model compounds. The enzymatic deacetylation of the 3-acetyloxymethoxy-2,2-bis(ethoxycarbonyl)propyl 5′-triester (2) is 25-fold faster than the deacetylation of its 3-acetyloxy-2,2-bis(ethoxycarbonyl)propyl-protected counterpart 1, and the difference in the deacetylation rates of the resulting diesters, 12b and 12a, is even greater. With 2, conversion to thymidine 5′-monophosphate (5′-TMP) is quantitative, while conversion of 1 to 5′-TMP is accompanied by formation of thymidine. Consistent with the preceding observations, quantitative release of 5′-TMP from 2 has been shown to take place in a whole cell extract of human prostate cancer cells.