Synthesis and Enzymatic Deprotection of Biodegradably Protected Dinucleoside‐2′,5′‐monophosphates: 3‐(Acetyloxy)‐2,2‐bis(ethoxycarbonyl)propyl Phosphoesters of 3′‐O‐(Acyloxymethyl)adenylyl‐2′,5′‐adenosines

Abstract

As a first step towards a viable prodrug strategy for short oligoribonucleotides, such as 2-5A and its congeners, adenylyl-2',5'-adenosines bearing a 3-(acetyloxy)-2,2-bis(ethoxycarbonyl)propyl group at the phosphate moiety, and an (acetyloxy)methyl- or a (pivaloyloxy)methyl-protected 3'-OH group of the 2'-linked nucleoside have been prepared. The enzyme-triggered removal of these protecting groups by hog liver carboxyesterase at pH 7.5 and 37° has been studied. The (acetyloxy)methyl group turned out to be too labile for the 3'-O-protection, being removed faster than the phosphate-protecting group, which results in 2',5'- to 3',5'-isomerization of the internucleosidic phosphoester linkage. In addition, the starting material was unexpectedly converted to the 5'-O-acetylated derivative. (Pivaloyloxy)methyl group appears more appropriate for the purpose. The fully deprotected 2',5'-ApA was accumulated as a main product, although, even in this case, the isomerization of the starting material takes place.

Synthesis and Deprotection of Biodegradably and Thermally Protected Dinucleoside‐2′,5′‐Monophosphate Prodrug Model of 2‐5A

“…The data clearly shows that the now reported protection group strategy is superior compared to the earlier reported one for the dimers 2a and b . During 1 day, approximately 80% of 1 was converted to the desired unprotected phosphodiester 9 the amount of which was finally about 90% ( Fig.…”

“…With 2a and b , 2′,5′ApA, in turn, accumulated only as a minor product during 1 day (20% and 5%, respectively), since the isomerization to 3′,5′‐isomer and cleavage of P–O5′ bond markedly competed with the deprotection ( Figs. and ) . The removal of 4‐(acetylthio)‐2‐(ethoxycarbonyl)‐3‐oxo‐2‐methylbutyl from 1 proceeds more than one order of magnitude faster ( t 1/2 = 6.3 min; k = 1.84 × 10 −3 s −1 ) than that of 3‐[(acetyloxy)methoxy]‐2,2‐bis(ethoxycarbonyl)propyl from 2a ( t 1/2 = 395 min; k = 2.9 × 10 −5 s −1 ).…”

“…The adenosine building block 6a was synthesized analogously as reported earlier . Accordingly, 5′‐OH and N 6 ‐amino functions of adenosine were first 4‐methoxytritylated to afford 5 ( Scheme ).…”

“… RP ‐ HPLC traces for the HLE ‐catalyzed hydrolysis of 2b at pH 7.5 and 37.0 °C ( I = 0.1 m with NaCl) …”

“…We have previously studied on removal of esterase labile protecting groups from dimeric adenylyl‐2′,5′‐adenosines (2′,5′‐ApA) ( 2a and b ) and from 2‐5A trimers (pA2′p5′A2′p5′A) ( 3a and b ) using 3‐acetyloxy‐2,2‐bis(ethoxycarbonyl)propyl protection for the phosphate moieties and pivaloyloxymethyl (PivOCH 2 ) or acetyloxymethyl (AcOCH 2 ) groups for the 3′‐OH ( Fig. ).…”