Mikko Ora

Towards Nucleotide Prodrugs Derived from 2,2-Bis(hydroxymethyl)malonate and Its Congeners: Hydrolytic Cleavage of 2-Cyano-2-(hydroxymethyl)-3-methoxy-3-oxopropyl and 3-(Alkylamino)-2-cyano-2-(hydroxymethyl)-3-oxopropyl Protections from the Internucleosidic Phosphodiester and Phosphorothioate Linkages

Thymidylyl-(3′5′)-thymidine (TpT) and its stereoisomeric phosphoromonothioate analogs [P(R)]- and [P(S)]-Tp(s)T having the phosphate or thiophosphate linkage protected with a 2-cyano-2-{[(4,4′-dimethoxytrityl)oxy]methyl}-3-methoxy-3-oxopropyl group (see 5a,b), as well as [P(R)]-Tp(s)T bearing a S-(2-cyano-2-{[(4,4′-dimethoxytrityl)oxy]methyl}-3-oxo-3-[(2-phenylethyl)amino]propyl) protection (see 5c), were prepared. The kinetics of the cleavage of the protecting group from the corresponding detritylated compounds 6a–c was studied over a pH range from 2 to 7. All compounds undergo a hydroxide-ion-catalyzed reaction that releases the unprotected TpT (7a) or Tp(s)T (7b), in all likelihood by departure of the hydroxymethyl group as formaldehyde and concomitant elimination of the phosphodiester or phosphorothioate from the resulting carbanion. The half-life for the deprotection of 6a and 6b is ca. 6 s at pH 7 and 25°, and that of 6cca. 600 s. The reasonably fast release of Tp(s)T from 6c offers a novel method for temporary intrachain attachment of peptides to oligonucleotides to enhance the cellular uptake.

Derivatization of phosphopeptides with mercapto- and amino-functionalized conjugate groups by phosphate elimination and subsequent Michael addition

Kinetics of the beta-elimination of the phosphate group from H-Tyr-Ser(PO3H2)-Phe-OH and H-Tyr-Thr(PO3H2)-Phe-OH and subsequent addition of thiols and amines to the dehydroalaninyl and beta-methyldehydroalaninyl residues formed, were followed by RP HPLC under alkaline conditions in the absence and presence of Ba2+ ions. By this reaction sequence, the phosphoserinyl peptide was conjugated with mono-N-(2-mercaptoethyl)amide of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (4), a mercapto-functionalized pentapeptide, H-His-Gly-Gly-His-Gly-NH(CH2)4SH, and an amino-functionalized fluorescent dye, 5-dimethylaminonaphthalene-1-[N-(5-aminopentyl)]sulfonamide (dansyl cadaverine). The beta-methyldehydroalanine residue was, in turn, observed to be a poor Michael acceptor.

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 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.

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.

Hydrolytic reactions of 3′-N-phosphoramidate and 3′-N-thiophosphoramidate analogs of thymidylyl-3′,5′-thymidine

The diastereomeric thiophosphoramidate analogs [(R(P))- and (S(P))-3[prime or minute],5[prime or minute]-Tnp(s)T] and the phosphoramidate analog [3[prime or minute],5[prime or minute]-TnpT] of thymidylyl-3[prime or minute],5[prime or minute]-thymidine were prepared and their hydrolytic reactions over the pH-range 1-8 at 363.2 K were followed by RP HPLC. At pH < 6, an acid-catalyzed P-N3[prime or minute] bond cleavage (first-order in [H(+)]) takes place with both 3[prime or minute],5[prime or minute]-Tnp(s)T and 3[prime or minute],5[prime or minute]-TnpT, the former being about 12 fold more stable than the latter. At pH > 4, Tnp(s)T undergoes two competing pH-independent reactions, desulfurization (yielding TnpT) and depyrimidination (cleavage of the N-glycosidic bond) the rates of which are of the same order of magnitude. Also with 3[prime or minute],5[prime or minute]-TnpT the pH-independent depyrimidination competes with P-N3[prime or minute] cleavage at pH > 5.

Hydrolytic dethiophosphorylation and desulfurization of the monothioate analogues of uridine monophosphates under acidic conditions

The hydrolytic reactions of uridine 2′-, 3′- and 5′-phosphoromonothioates (2″-, 3′- and 5′-UMPS) under acidic and neutral conditions have been followed by HPLC. Under slightly acidic conditions (pH 2–5), only pH-independent dethiophosphorylation to uridine takes place. This reaction is 200- to 300-fold as fast as dephosphorylation of the corresponding uridine monophosphates (UMP), presumably due to higher stability of the thiometaphosphate monoanion compared to metaphosphate anion. At pH > 5, i.e. at pH > pKa2 of the thiophosphate moiety, the dethiophosphorylation is retarded with increasing basicity of the solution. At pH < 1, acid-catalysed desulfurization of 2′- and 3′-UMPS to an isomeric mixture of 2′/3′-UMP competes with their dethiophosphorylation. This reaction is suggested to proceed by a nucleophilic attack of the neighbouring hydroxy group on phosphorus. No such reaction occurs with 5′-UMPS. In contrast to 2′- and 3′-UMP, no sign of interconversion of 2′- and 3′-UMPS is detected.

3-Acetyloxy-2-cyano-2-(alkylaminocarbamoyl)propyl Groups as Biodegradable Protecting Groups of Nucleoside 5´-mono-Phosphates

Thymidine 5´-bis[3-acetyloxy-2-cyano-2-(2-phenylethylcarbamoyl)propyl]phosphate (1) has been prepared and the removal of phosphate protecting groups by hog liver carboxyesterase (HLE) at pH 7.5 and 37 °C has been followed by HPLC. The first detectable intermediates are the (RP)- and (SP)-diastereomers of the monodeacetylated triester 14, which subsequently undergo concurrent retro-aldol condensation to diester 4 and enzyme-catalyzed hydrolysis to the fully deacetylated triester 15. The former pathway predominates, representing 90% of the overall breakdown of 14. The diester 4 undergoes the enzymatic deacetylation 700 times less readily than the triester, but gives finally thymidine 5´-monophosphate as the desired main product. To elucidate the potential toxicity of the electrophilic 2-cyano-N-(2-phenylethyl)acrylamideby-product 17 released upon the deprotection, the hydrolysis of 1 has also been studied in the presence of glutathione (GSH).

Thio Effects as a Tool for Mechanistic Studies of the Cleavage of RNA Phosphodiester Bonds: The Chemical Basis

Replacement of one of the phosphorus-bound oxygen atoms with sulfur has extensively been used for elucidation of mechanistic details of the cleavage of RNA phosphodiester bonds by ribozymes. Since sulfur atom is larger, less electronegative, and more readily polarizable than oxygen, this substitution affects in many ways metal ion binding and the ease of formation and breakdown of the phosphorane intermediate/transition state obtained by the attack of the entering hydroxyl group on the phosphorus atom. The factors that may be altered by thio substitution include the geometry of the phosphorane intermediate, relative apicophilicities of the ligands, the leaving group property, hydrogen bonding, solvation, and the affinity to metal ions. Experimental studies and theoretical calculations on various model systems have been undertaken to obtain a solid chemical basis for the mechanistic interpretations based on thio effects in ribozyme catalysis. The results of such studies are surveyed in this chapter.

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.