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

Abstract

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.

Irreversible Inhibition of DNA Polymerase β by Small-Molecule Mimics of a DNA Lesion

“…B) Dependence on preincubation time of 1a (10 μM) with Pol β. No inhibitor, ●; Preincubation time 42 : 5, ; 20, ○; 40, ■; 60, .…”

Synthesis and Enzymatic Deprotection of Fully Protected 2′‐5′ Oligoadenylates (2‐5A): Towards a Prodrug Strategy for Short 2‐5A

“…Previous studies have shown that replacement of the 3-AcO group with a 3-[(acetyloxy)methoxy] group accelerated the first enzymatic deacetylation of bis-substituted thymidine 5'-monophosphate by a factor of 25, and the deacetylation of the resulting negatively charged diester even more [15]. As indicated above, 3'-O-[(pivaloyloxy)methyl] protection did not afford a viable prodrug strategy for 2-5A.…”

“…The present work is aimed at evaluating the feasibility of an enzymetriggered prodrug strategy for the trimeric 2-5A. Our previous studies [15] indicate that the removal of the second 3-(acetyloxy)-2,2-bis(ethoxycarbonyl)propyl group, i.e., conversion of phosphodiester to monoester, is exceedingly slow, but replacing the Ac group with an AcOCH 2 group markedly accelerates the reaction. The negatively charged phosphate group must be protected to enhance the internalization and the 3'-OH to prevent its attack on the neighboring protected phosphate linkage.…”

“…The crucial factor is the timing of the exposure of the 3'-OH and the adjacent phosphodiester linkage. [15], N 6 -(4methoxytrityl)-3'-O-[(pivaloyloxy)methyl]adenosin-2'-yl 2',3'-di-O-levulinoyl-N 6 -(4methoxytrityl)adenosin-5'-yl 3-(acetyloxy)-2,2-bis(ethoxycarbonyl)propyl phosphate (5a) [13], and 3'-O-[(acetyloxy)methyl]-N 6 -(4-methoxytrityl)adenosin-2'-yl 2',3'-di-Olevulinoyl-N 6 -(4-methoxytrityl)adenosin-5'-yl 3-(acetyloxy)-2,2-bis(ethoxycarbonyl)propyl phosphate (5b) [13] have been described earlier. The phosphate protecting group must be removed before the 3'-OH protection, since otherwise the extremely fast attack of the free 3'-OH group on the adjacent phosphotriester moiety would lead to isomerization of the 2',5' linkage to a 3',5' linkage and cleavage of the PÀO(5') bond [14].…”

Synthesis and Stability of Nucleoside 3′,5′‐Cyclic Phosphate Triesters Masked with Enzymatically and Thermally Labile Phosphate Protecting Groups

“…[12] The 4-acetylthio-2,2-dimethyl-3-oxobutyl group is also removed by esterases, but it is also thermolabile, and can thus be removed even if the enzymatic reaction is sluggish. [12] The 4-acetylthio-2,2-dimethyl-3-oxobutyl group is also removed by esterases, but it is also thermolabile, and can thus be removed even if the enzymatic reaction is sluggish.…”