Synthesis and Deprotection of Biodegradably and Thermally Protected Dinucleoside-2',5'-Monophosphate Prodrug Model of 2-5A.

Protected dinucleoside-2',5'-monophosphate has been prepared to develop a prodrug strategy for 2-5A. The removal of enzymatically and thermally labile 4-(acetylthio)-2-(ethoxycarbonyl)-3-oxo-2-methylbutyl phosphate protecting group and enzymatically labile 3'-O-pivaloyloxymethyl group was followed at pH 7.5 and 37 °C by HPLC from the fully protected dimeric adenosine-2',5'-monophosphate 1 used as a model compound for 2-5A. The desired unprotected 2',3'-O-isopropylideneadenosine-2',5'-monophosphate (9) was observed to accumulate as a major product. Neither the competitive isomerization of 2',5'- to a 3',5'-linkage nor the P-O5' bond cleavage was detected. The phosphate protecting group was removed faster than the 3'-O-protection and, hence, the attack of the neighbouring 3'-OH on phosphotriester moiety did not take place.

2,2-Disubstituted 4-acylthio-3-oxobutyl groups as esterase- and thermolabile protecting groups of phosphodiesters.

Five different 2,2-disubstituted 4-acylthio-3-oxobutyl groups have been introduced as esterase-labile phosphodiester protecting groups that additionally are thermolabile. The phosphotriesters 1-3 were prepared to determine the rate of the enzymatic and nonenzymatic removal of such groups at 37 °C and pH 7.5 by HPLC-ESI-MS. Additionally, (1)H NMR spectroscopic monitoring was used for structural characterization of the intermediates and products. When treated with hog liver esterase, these groups were removed by enzymatic deacylation followed by rapid chemical cyclization to 4,4-disubstituted dihydrothiophen-3(2H)-one. The rate of the enzymatic deprotection could be tuned by the nature of the 4-acylthio substituent, the benzoyl group and acetyl groups being removed 50 and 5 times as fast as the pivaloyl group. No alkylation of glutathione could be observed upon the enzymatic deprotection. The half-life for the nonenzymatic deprotection varied from 0.57 to 35 h depending on the electronegativity of the 2-substituents and the size of the acylthio group. The acyl group evidently migrates from the sulfur atom to C3-gem-diol obtained by hydration of the keto group and the exposed mercapto group attacks on C1 resulting in departure of the protecting group as 4,4-disubstituted 3-acyloxy-4,5-dihydrothiophene with concomitant release of the desired phosphodiester.

Synthesis and enzymatic deprotection of fully protected 2'-5' oligoadenylates (2-5A): towards a prodrug strategy for short 2-5A.

Fully protected pA2'p5'A2'p5'A trimers 1a and 1b have been prepared as prodrug candidates for a short 2'-5' oligoadenylate, 2-5A, and its 3'-O-Me analog, respectively. The kinetics of hog liver carboxyesterase (HLE)-triggered deprotection in HEPES buffer (pH 7.5) at 37° has been studied. The deprotection of 1a turned out to be very slow, and 2-5A never appeared in a fully deprotected form. By contrast, a considerable proportion of 1b was converted to the desired 2-5A trimer, although partial removal of the 3'-O-[(acetyloxy)methyl] group prior to exposure of the adjacent phosphodiester linkage resulted in 2',5'→3',5' phosphate migration and release of adenosine as side reactions.

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.

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.