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A multi-nucleating azacrown conjugate ( 5a ) consisting of two 3,5-bis(1,5,9-triazacyclododecan-3-yloxymethyl)benzyl groups attached to 1 and 7 sites of cyclen was prepared and tested as an artificial ribonuclease. The conjugate in the presence of five equivalents of zinc nitrate expectedly showed uridine selectivity comparable to that 1,3,5-tris(1,5,9-triazacyclododecan-3-yl)benzene ( 2 ), a compound known to bind to two adjacent uridine residues and cleave the intervening phosphodiester bond. 5a was, however, unable to discriminate between two and three adjacent uridine residues, but cleaved oligonucleotides containing a UpU and UpUpU site at a comparable rate, even when present at sub-saturating concentrations.

Phosphoramidates 1 and 2 were synthesized by H-phosphonate methodology and subsequent oxidative amination with L-alanine methyl ester. The removal of the protecting groups at pH = 7.5 and 37 °C in the absence and presence of porcine liver esterase (PLE) or glutathione (GSH) was monitored by HPLC. The stability of phosphoramidate 1 was additionally studied at pH = 9 and 10. The reduction of the disulfide bond with glutathione from 2 triggers the removal of the protecting group by cyclization releasing quantitatively nucleoside 5′-{N-[(1S)-2-oxo-2-methoxy-1-methylethyl]phosphoramidate} (7) as the desired product. With 1, enzymatic deacetylation or acetyl migration from the sulfur atom to the adjacent hydrated oxo group followed by chemical cyclization produces 7. The S–S-bond-mediated dimerization (8) competes as a side reaction. Prolonged treatment, however, resulted in the conversion of the S–S dimer 8 into 7 that undergoes slow alanine methyl ester hydrolysis to form 10.

4'-(Hydroxymethyl)uridylyl-3',5'-thymidine, an RNA model bearing an extra hydroxymethyl group at the 4'-position of the 3'-linked nucleoside, has been prepared and its cleavage and isomerization reactions studied over a wide pH range (from 0 to 12). Overall, the pH-rate profiles of these reactions were very similar to those of uridylyl-3',5'-uridine (UpU) - only a very modest acceleration was observed under acidic and neutral conditions. Evidently, hydrogen bond assistance by the additional hydroxymethyl function does not play a significant role.

Formation of mixed-ligand Pd2+ complexes between canonical nucleoside 5'-monophosphates and five metal-ion-binding nucleoside analogs has been studied by 1H-NMR spectroscopy to test the ability of these nucleoside surrogates to discriminate between unmodified nucleobases by Pd2+-mediated base pairing. The nucleoside analogs studied included 2,6-bis(3,5-dimethylpyrazol-1-yl)-, 2,6-bis(1-methylhydrazinyl)- and 6-(3,5-dimethylpyrazol-1-yl)-substituted 9-(β-d-ribofuranosyl)purines 1–3, and 2,4-bis(3,5-dimethylpyrazol-1-yl)- and 2,4-bis(1-methylhydrazinyl)-substituted 5-(β-d-ribofuranosyl)-pyrimidines 4–5. Among these, the purine derivatives 1-3 bound Pd2+ much more tightly than the pyrimidine derivatives 4, 5 despite apparently similar structures of the potential coordination sites. Compounds 1 and 2 formed markedly stable mixed-ligand Pd2+ complexes with UMP and GMP, UMP binding favored by 1 and GMP by 2. With 3, formation of mixed-ligand complexes was retarded by binding of two molecules of 3 to Pd2+.

(4-Acetylthio-2,2-dimethyl-3-oxobutyl)-protected oligomeric phosphodiesters 1 and 2 were synthesized and removal of the protecting groups in the presence and absence of hog liver esterase was followed at pH 7.5 and 37 °C. Phosphotriesters 1 and 2 were successfully converted into the desired fully deprotected phosphodiesters 3 and 4, respectively. Some cleavage of internucleosidic P–O bonds took place, which reduced the yield of 3 and 4. Non-enzymatic removal of the protecting group was only modestly retarded by accumulation of negative charge on the molecule. With 1, the half-lives for the departure of the first and second protecting groups were 7.8 and 10.7 h, respectively, and with 2, 6.2 and 7.2 h, respectively. After 4 d, 70 % of both starting materials 1 and 2 were converted into the unprotected phosphodiester. The presence of hog liver esterase (2.6 units mL–1) resulted in fast removal of the first protecting group (τ1/2 2.7 min and 36 min with 1 and 2, respectively), but the appearance of fully deprotected 3 and 4 was accelerated only by a factor of 2, consistent with dramatic retardation of the enzymatic reaction upon accumulation of the negative charge.

68Ga labelled 2′-O-methyl oligoribonucleotides (anti-miR-15b) bearing one, three or seven d -galactopyranoside residues have been prepared and their distribution in healthy rats has been studied by positron emission tomography (PET). To obtain the heptavalent conjugate, an appropriately protected 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) precursor bearing a 4-[4-(4,4′-dimethoxytrityloxy)butoxy]phenyl side arm was first immobilized via a base labile linker to the support and the oligonucleotide was assembled on the detritylated hydroxyl function of this handle. A phosphoramidite building block bearing two phthaloyl protected aminooxy groups and one protected hydroxyl function was introduced into the 5′-terminus. One acetylated galactopyranoside was coupled as a phosphoramidite to the hydroxyl function, the phthaloyl protections were removed on-support and two trivalent galactopyranoside clusters were attached as aldehydes by on-support oximation. A two-step cleavage with aqueous alkali and ammonia released the conjugate in a fully deprotected form, allowing radiolabelling with 68Ga in solution. The mono- and tri-galactose conjugates were obtained in a closely related manner. In vivo imaging in rats with PET showed remarkable galactose-dependent liver targeting of the conjugates.

To learn more about the underlying principles of metal-ion-mediated recognition of nucleic acid bases, PdCl(+) complexes of six 2,6-disubstituted pyridines, viz. pyridine-2,6-dicarboxamide, its N(2),N(6)-dimethyl and N(2),N(6)-diisopropyl derivatives, 6-carbamoylpyridine-2-carboxylic acid, 6-aminomethylpyridine-2-carboxamide and its N(2)-methyl derivative, were prepared and their interaction with nucleoside 5'-monophosphate (NMP) was studied by (1)H NMR spectroscopy in D2O at pH7.2. The binding sites within the nucleobases were assigned on the basis of Pd(2+) induced changes in chemical shifts of the base moiety proton resonances. The mole fractions of NMPs engaged in mono- or dinuclear Pd(2+) complexes were determined at various concentrations by comparing the intensities of the aromatic and anomeric protons of the complexed and uncomplexed NMPs. Some of the pyridine complexes showed moderate discrimination between the NMPs.

Four metal-ion-binding nucleosides, viz. 2,6-bis(1-methylhydrazinyl)-9-(β-D-ribofuranosyl)-9H-purine (2a) and its N-acetylated derivative, 2b, 2,4-bis(3,5-dimethyl-1H-pyrazol-1-yl)-5-(β-D-ribofuranosyl)pyrimidine (3), and 2,4-bis(1-methylhydrazinyl)-5-(β-D-ribofuranosyl)pyrimidine (4) have been synthesized. The ability of these nucleosides and the previously prepared 2,6-bis(3,5-dimethyl-1H-pyrazol-1-yl)-9-(β-D-ribofuranosyl)-9H-purine to form Pd2+- and Hg2+-mediated complexes with uridine has been studied by 1H-NMR spectroscopy. To obtain additional support for the interpretation of the NMR data, comparative measurements on the ternary-complex formation between pyridine-2,6-dicarboxamide (5), pyrimidine nucleosides, and K2PdCl4 were carried out.

A dinucleoside-3',5'-phosphodiester model, 5'-amino-4'-aminomethyl-5'-deoxyuridylyl-3',5'-thymidine, incorporating two aminomethyl functions in the 4'-position of the 3'-linked nucleoside has been prepared and its hydrolytic reactions studied over a wide pH range. The amino functions were found to accelerate the cleavage and isomerization of the phosphodiester linkage in both protonated and neutral form. When present in protonated form, the cleavage of the 3',5'-phosphodiester linkage and its isomerization to a 2',5'-linkage are pH-independent and 50-80 times as fast as the corresponding reactions of uridylyl-3',5'-uridine (3',5'-UpU). The cleavage of the resulting 2',5'-isomer is also accelerated, albeit less than with the 3',5'-isomer, whereas isomerization back to the 3',5'-diester is not enhanced. When the amino groups are deprotonated, the cleavage reactions of both isomers are again pH-independent and up to 1000-fold faster than the pH-independent cleavage of UpU. Interestingly, the 2'- to 3'-isomerization is now much faster than its reverse reaction. The mechanisms of these reactions are discussed. The rate accelerations are largely accounted for by electrostatic and hydrogen-bonding interactions of the protonated amino groups with the phosphorane intermediate.

2-(3,5-Bis{[1,5,9-tris(trifluoroacetyl)-1,5,9-triazacyclododecan-3-yloxy]methyl}phenoxy)ethanol was synthesized and converted to a O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite building block, 12. 2′-O-Methyl oligoribonucleotides incorporating a 2-[(2S,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)ethyl 4-oxopentanoate or a 2-{2-[2-({[(2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]acetyl}amino)ethoxy]ethoxy}ethyl 4-oxopentanoate non-nucleosidic unit close to the 3′-terminus were assembled on a solid support, the 4-oxopentanoyl protecting groups were removed by treatment with hydrazinium acetate on-support, and 12 was coupled to the exposed OH function. The deprotected conjugates were purified by HPLC, and their ability to cleave a complementary RNA containing either uridine or some other nucleoside at the potential cleaving site was compared. Somewhat unexpectedly, conjugation to an oligonucleotide did not enhance the catalytic activity of the Zn2+bis(azacrown) complex and virtually abolished its selectivity towards the uridine sites.

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.

4'-C-[N,N-Di(4-pentyn-1-yl)aminomethyl]thymidine and 4'-C-[N-methyl-N-(4-pentyn-1-yl)aminomethyl]thymidine 3'-(2-cyanoethyl-N,N-diisopropyl)phosphoramidites (1, 2) were synthesized, and one or two such monomers were incorporated into a 15-mer oligodeoxyribonucleotide. After chain assembly, azido-functionalized ligands, including appropriate derivatives of 1,4-phenylenedimethaneamine, mannose, paromamine, and neomycin, were conjugated to the alkynyl groups by the click chemistry on a solid support. The influence of the 4'-modifications on the melting temperature with DNA and 2'-O-methyl RNA targets was studied. Oligonucleotides containing one to four mannose ligands in the central part of the chain (up to two 4'-C-[N,N-di(4-pentyn-1-yl)aminomethyl]thymidine units) form equally stable duplexes with complementary 2'-OMe RNA as the corresponding unmodified DNA sequence. At high salt content, the mannose conjugation is even stabilizing. On using a DNA target, a modest destabilization occurs. All the amino group bearing conjugates stabilized the duplexes, the DNA·DNA duplexes more than the DNA·2'-O-methyl RNA duplexes.

Clustered hyaluronan disaccharides were studied as mediators of cellular delivery of antisense oligonucleotides through receptor-mediated endocytosis. For this purpose, a synthetic route for preparation of an appropriately protected hyaluronic acid dimer bearing an aldehyde tether (1) was devised. Up to three non-nucleosidic phosphoramidite building blocks (2), each bearing two phthaloyl protected aminooxy groups, were then inserted into the 3'-terminus of the desired phosphorothioate oligodeoxyribonucleotide, and 6-FAM phosphoramidite was introduced into the 5'-terminus. After completion of the chain assembly, the aldehyde-tethered sugar ligands were attached to the deprotected aminooxy functions by on-support oximation. Three fluorescein-labeled phosphorothioate oligonucleotide glycoconjugates (28-30) containing two, four, or six hyaluronan disaccharides were prepared. The influence of the hyaluronan moieties on the cellular uptake of the thioated oligonucleotides was tested in a cell line expressing the hyaluronan receptor CD44. Specific uptake was not detected with this combination of multiple hyaluronan disaccharides.

The ability of the dinuclear Zn2+ complex of 1,4-bis[(1,5,9-triazacyclododecan-3-yloxy)methyl]benzene (L(1)) to promote the cleavage of the phosphodiester bond of dinucleoside-3',5'-monophosphates that contain a guanine base has been studied over a narrow pH range from pH 5.8 to 7.2 at 90 degrees C. Comparative measurements have been carried out by using the trinuclear Zn2+ complex of 1,3,5-tris[(1,5,9-triazacyclododecan-3-yloxy)methyl]benzene (L(2)) as a cleaving agent and guanylyl-3',5'-guanosine (5'-GpG-3') as a substrate. The strength of the interaction between the cleaving agent and the starting material has been elucidated by UV spectrophotometric titrations. The speciation and binding mode have been clarified by potentiometric titrations with hydrolytically stable 2'-O-methylguanylyl-3',5'-guanosine and 1H NMR spectroscopic measurements with guanylyl-3',5'-guanosine. The results show that the guanine base is able to serve as a site for anchoring for the Zn2+-azacrown moieties of the cleaving agents L(1) and L(2), analogously to uracil base. The interaction is, however, weaker than with the uracil base and, hence, only the 5'-GpG-3' site (in addition to 5'-GpU-3' and 5'-UpG-3' sites) is able to markedly modulate the phosphodiester cleavage by the Zn2+ complexes of di- and trinucleating azacrown ligands containing an ether oxygen as a potential H-bond-acceptor site.

A terpyridine or an imidazole unit has been tethered to an N -protected polyazacyclophane to give the appropriate N -monofunctionalized polyazacyclophane. After mild deprotection, four polyazacyclophanes incorporating a pendant group were obtained in satisfactory yields. Their preliminary cleavage ability of mRNA 5′- cap model was studied at pH 7.2.

The hydrolysis of a 5′ cap analogue, diadenosinyl-5′,5′-triphosphate (ApppA), and two dinucleoside monophosphates: adenylyl(3′,5′)adenosine (ApA) and uridylyl(3′,5′)uridine (UpU) promoted by an imidazolate-bridged heterobinuclear copper(II)–zinc(II) complex, Cu(II)-diethylenetriamino- μ -imidazolato–Zn(II)- tris(aminoethyl)amine trisperchlorate (denoted as Cu,Zn -complex in the followings) has been investigated. Kinetic measurements were performed in order to explore the effects of pH, the total concentration of the Cu,Zn-complex and temperature on the cleavage rate. The catalytic activity of the Cu,Zn-complex was quantified by pseudo-first-order rate constants obtained in the excess of the cleaving agent. The results show that the Cu,Zn-complex and its deprotonated forms have phosphoesterase activity and with ApppA the metal complex promoted cleavage takes place selectively within the triphosphate bridge.

Functionalized 2'-O-methyl oligoribonucleotides bearing two 3-(3-hydroxypropyl)-1,5,9-triazacyclododecane ligands attached via a phosphodiester linkage to a single non-nucleosidic building block have been prepared on a solid-support by conventional phosphoramidite chemistry. The branching units employed for the purpose include 2,2-bis(3-hydroxypropylaminocarbonyl)propane-1,3-diol, 2-hydroxyethyl 3'-O-(2-hydroxyethyl)-beta-D-ribofuranoside, and 2-hydroxyethyl 2'-O-(2-hydroxyethyl)-beta-D-ribofuranoside. Each of these has been introduced as a phosphoramidite reagent either into the penultimate 3'-terminal site or in the middle of the oligonucleotide chain. The dinuclear Zn2+ complexes of these conjugates have been shown to exhibit enhanced catalytic activity over their monofunctionalized counterpart, the 3'-terminal conjugate derived from 2-hydroxyethyl 3'-O-(2-hydroxyethyl)-beta-D-ribofuranoside being the most efficient cleaving agent. This conjugate cleaves an oligoribonucleotide target at a single phosphodiester bond and shows turnover and 1000-fold cleaving activity compared to the free monomeric Zn2+ chelate of 1,5,9-triazacyclododecane.

2,6-Bis(1,4,7,10-tetraazacyclododecan-1-ylmethyl)pyridine (11a) and 1,3-bis(1,4,7,10-tetraazacyclododecan-1-ylmethyl)benzene (11b) have been shown to accelerate at 50 mmol·L−1 concentration both the cleavage and mutual isomerization of uridylyl-3′,5′-uridine and uridylyl-2′,5′-uridine by up to two orders of magnitude. The catalytically active ionic forms are the tri- (in the case of 11b) tetra- and pentacations. The pyridine nitrogen is not critical for efficient catalysis, since the activity of 11b is even slightly higher than that of 11a. On the other hand, protonation of the pyridine nitrogen still makes 11a approximately four times more efficient as a catalyst, but only for the cleavage reaction. Interestingly, the respective reactions of adenylyl-3′,5′-adenosine were not accelerated, suggesting that the catalysis is base moiety selective.

Two novel peptide scaffolds, viz. cyclo[(N(alpha)-Alloc)Dpr-beta-Ala-(N(alpha)-Fmoc)Dpr-beta-Ala] (1) and cyclo[(N(alpha)-Alloc)Dpr-alpha-azido-beta-aminopropanoyl-(N(alpha)-Fmoc)Dpr-beta-Ala] (2), composed of orthogonally protected 2,3-diaminopropanoyl (Dpr) and beta-alanyl residues, have been described. Fmoc chemistry on a backbone amide linker derivatized resin has been used for the chain assembly. Selective removal of the 4-methyltrityl (Mtt) and 1-methyl-1-phenylethyl protections (PhiPr) exposes the beta-amino and carboxyl terminus, respectively, and on-resin cyclization then gives the desired orthogonally protected cyclo-beta-tetrapeptides (1 and 2). The alpha-amino groups, bearing the Fmoc and Alloc protections and the azide mask, allow stepwise orthogonal derivatization of these solid-supported cyclo-beta-tetrapeptide cores (1 and 2). This has been demonstrated by attachments of various sugar units [viz., acetyl- or toluoyl-protected carboxymethyl alpha-d-glycopyranosides (13-15) and methyl 6-O-(4-nitrophenoxycarbonyl)-alpha-d-glycopyranosides (22-24)] to obtain diverse di- and trivalent glycoclusters (33-42). Acidolytic release (TFA) from the support, followed by conventional NaOMe-catalyzed transesterification (33-40) or hydrazine-induced acyl substitution in DMF (41 and 42), gives the fully deprotected clusters (43-52) as final products.

Second-order rate constants for the hydroxide-ion-catalyzed imidazolium ring-opening of several mono- and dinucleosidic analogs of mRNA 5'-caphave been determined. Intramolecular stacking of the two nucleobases in the dinucleosidic analogs, m7GpppN (m7G = 7-methylguanosine, N = 5'-linked nucleoside), and electrostatic interaction between theN-alkylated imidazolium ring and phosphate moiety have been shown to shield the m7G moiety against the nucleophilic attack of hydroxide ion. In addition, the effect of methylation of the nucleobase amino groups and replacement of the 7-methyl group with other alkyl groups have been studied. The influence of all the structural modifications studied turned out to be modest, the cleavage rates of the most and least reactive analogs (with the exception of non-phosphorylated nucleosides) differing only by a factor of 5.

Mechanistic studies of the action of catalytic ribonucleic acids, ribozymes, are highly challenging, because even a slight structural change can dramatically affect the chain folding. This, in turn, alters the binding properties of the catalytic core, making identification of the real origin of the observed influence on rate difficult. Unambiguous structure-reactivity correlations based on studies with structurally simplified chemical models may help to distinguish between alternative mechanistic interpretations. The results of such model studies are reviewed. The topics include intramolecular cleavage of RNA phosphodiester bonds by solvent-derived species, general acids/bases and metal ions, effect of molecular environment on their hydrolytic stability and trinucleoside monophosphates as models for large ribozymes.

Four hexaazacyclophanes, 16a-d, incorporating two pyridine units and a (pyridin-2-yl)methyl or (quinolin-2-yl)methyl pendant group at one of the ring N-atoms have been prepared. The key step of the synthesis is an intermolecular cyclization of N,N-bis{[6-(tosyloxymethyl)pyridin-2-yl]methyl}-2-nitrobenzenesulfonamide (7) with either tert-butyl bis{2-[(2-nitrophenylsulfonyl)amino]ethyl}carbamate (2a) or tert-butyl bis{3-[(2-nitrophenylsulfonyl)amino]propyl}carbamate (2b) in the presence of anhydrous Cs(2)CO(3). Removal of the acid-labile tert-butoxycarbonyl protection then allows attachment of the pendant group by reductive alkylation to the exposed secondary amino group, and deprotection of the remaining aliphatic ring N-atoms completes the synthesis. The ability of the cyclophanes and their dinuclear Cu(2+) and Zn(2+) complexes to cleave the mRNA cap structure, m(7)G(5')pppG(5') (1), has been studied.

Straightforward synthesis for two pentaerythrityltetramine precursors, 2,2-bis(azidomethyl)propane-1,3-diamine (1) and 2-[N-(allyloxycarbonyl)aminomethyl]-2-azidomethylpropane-1,3-diamine (2), has been described. Both propane-1,3-diamines have been attached by reductive amination to a solid-supported backbone amide linker derived from 4-(4-formyl-3,5-dimethoxyphenoxy)butyric acid. The presence of the two methoxy substituents on the linker is essential to avoid cross-linking between two linkers. The remaining free primary amino group of the propane-1,3-diamine moiety may then be selectively acylated with an appropriately protected amino acid using conventional N,N-dicyclohexylcarbodiimide/1-hydroxybenzotriazole (DCC/HOBt) activation without any interference by the secondary amino function. The latter group may be subsequently acylated by an anhydride method. Sequential reduction of the azido group and removal of the allyloxycarbonyl protection from 2 allow further coupling of two different amino acids, and hence, this handle may be utilized in construction of branched structures containing four different amino acids or peptides. Solid-supported 1 may, in turn, be used for the synthesis of similar constructs containing two identical branches. It is worth noting that no acid-labile protecting groups are required in this approach, and hence, this dimension may be saved for the cleavage of the linker. The applicability of the scaffolds to library synthesis has been demonstrated by preparation of 11 pentaerythrityl-branched tetra- and octapeptides.

The formation of a disaccharide nucleoside (11) by O3'-glycosylation of 5'-O-protected 2'-deoxyadenosine or its N6-benzoylated derivative has been observed to be accompanied by anomerisation to the corresponding alpha-anomeric product (12). The latter reaction can be explained by instability of the N-glycosidic bond of purine 2'-deoxynucleosides in the presence of Lewis acids. An independent study on the anomerisation of partly blocked 2'-deoxyadenosine has been carried out. Additionally, transglycosylation has been utilized in the synthesis of 3'-O-beta-D-ribofuranosyl-2'deoxyadenosines and its alpha-anomer.

The effect of base sequence on the stability of the transition state (TS) of phosphodiester bond cleavage within linear single-stranded oligoribonucleotides has been studied in order to better understand why the reactivity of some phosphodiester bonds is enhanced compared to an unconstrained linkage. Molecular dynamics simulations of 3.0 ns were carried out for 14 oligonucleotides that contain in the place of the scissile phosphodiester bond a phosphorane structure mimicking the TS of the bond cleavage. The hydrolytic stability of the same oligonucleotides had previously been reported. Both the non-bridging oxyanions and the leaving 5[prime or minute]-oxygen of the pentacoordinated phosphorane moiety were observed to form hydrogen bonds with solvent water molecules in a similar way with all the compounds studied. In addition, water mediated hydrogen bonds between the phosphorane non-bridging oxyanions and the bases of the 3[prime or minute]-flanking sequence were detected with some of the compounds, but not with the most labile ones. Hence, it seems that the enhanced cleavage of some internucleosidic linkages does not result from the TS stabilisation by hydrogen bonding. With heterooligomers, the stacking of bases next to the cleavage site was observed to be enhanced on going from the initial state to the TS, whereas within uracil homooligomer, having initially negligible stacking, no change in the magnitude of stacking was seen. Accordingly, while strong stacking in the initial state is known to retard the phosphodiester bond cleavage, it may in the TS accelerate the reaction. Therefore, enhanced stacking on going from the initial state to transition state appears to be a factor that markedly contributes to the hydrolytic stability of phosphodiester bonds within oligonucleotides and may, at least partly, explain accelerated cleavage compared to fully unconstrained bonds, such as those in polyuridylic acid.

The ability of the dinuclear complexes of tdci [1,3,5-trideoxy-1,3,5-tris(dimethylamino)-cis-inositol] to promote the cleavage of the phosphodiester bonds of nucleoside 2',3'-cyclic monophosphates, dinucleoside monophosphates and polyribonucleotides has been studied. The homodinuclear copper(II) and zinc(II) complexes efficiently promote the hydrolysis of cyclic nucleotides. The second-order rate constant (k(2) approximately 0.44 M(-1) s(-1)) estimated for the cleavage of 2',3'-cAMP induced by dinuclear copper(II) complexes is about 107 times greater than that for the hydroxide-ion-catalysed reaction. The complex selectively cleaves the 2'O-P bond of 2',3'-cUMP and forms the 3'-product in 91 % yield. An equimolar mixture of copper(II), zinc(II) and tdci proved to be more efficient than either of the binary systems: a 7-20-fold rate enhancement was observed for the cleavage of 2',3'-cNMP substrates. The half-life for the hydrolysis of 2',3'-cAMP decreased from 300 days to five minutes at 25 degrees C when the concentration of each of the three components was 2.5 mM. In contrast to the copper(II) or zinc(II) complexes of tdci, the heterodinuclear species promoted the hydrolysis of several dinucleoside monophosphates. For two ApA isomers, cleavage of the 3',5'-bond was about 6.5 times faster than cleavage of the 2',5'-bond. On the basis of the kinetic data, a trifunctional mechanism is suggested for the heterodinuclear-complex-promoted cleavage of the phosphodiester bond. Double Lewis acid activation occurs when the metal ions bind to the phosphate oxygen atoms. In particular, a metal-bound hydroxide ion serves as a general base or a nucleophilic catalyst, and, presumably, a zinc(II)-bound aqua ligand behaves as a general acid and facilitates the departure of the leaving alkoxide group. The effect of the complexes on the hydrolysis of poly(U), poly(A) and type III native RNA was also investigated, and, for the first time, kinetic data on the cleavage of the phosphodiester bonds of polyribonucleotides by a dinuclear complex was obtained.

A straightforward method for the solid-supported synthesis of cryptand-like bicyclic peptides (1-5) on a backbone amide linker has been described. For the branching, two novel easily available building blocks, viz. N-(4-methoxytrityl)-N-(2-nitrobenzenesulfonyl)-protected N,N-bis(2-aminoethyl)-beta-alanine (6) and N-(9-fluorenylmethoxycarbonyl) protected iminodiacetic acid monoallyl ester (7), have been employed. The key steps of the synthesis are as follows: (i) stepwise coupling of one amino acid and 6 to the secondary amino group of the linker; (ii) removal of the 2-nitrobenzenesulfonyl group and SPPS by the Fmoc chemistry, using 7 as the penultimate and tert-butoxycarbonyl (Boc) protected glycine as the last amino acid; (iii) removal of the 4-methoxytrityl protection and subsequent SPPS by the Fmoc chemistry; (iv) removal of the allyl and Fmoc groups, followed by cyclization; and (v) removal of the Boc and tert-butyl groups, followed by cyclization. Final cleavage from the support and removal of benzyl-derived protecting groups gives the desired bicyclic products.

Four homodetic bicyclic peptides 9a−d, containing three parallel peptide chains, were synthesized on a hydroxymethyl-functionalized polystyrene support. α,α-Bis(aminomethyl)-β-alanine, bearing orthogonal protections (Alloc, Boc, Fmoc) on the three amino groups (1), was attached to the support through an H2N-Leu-Leu-Gly-OH spacer, and the peptide chains were assembled on the amino groups of 1 by either a stepwise coupling or coupling of a segment, keeping the amino protection within each chain unchanged. N-(4-Allyloxy-4-oxobutanoyl)iminodiacetic acid (2) was then coupled to the Fmoc-protected chain. The Boc protecting group was removed, and the exposed amino group was coupled with the remaining free carboxylic acid function of 2. Finally, the Alloc and allyl ester protections were removed from the carboxylic and amino functions, and a second cyclization was performed. Release from the support gave 9a−d as free carboxylic acids. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)

The effect of base sequence on the structure and flexibility of linear single-stranded RNA molecules and the influence of the base sequence on phosphodiester bond reactivity have been studied. Molecular dynamics simulations of 2.1 ns were carried out for nine chimeric oligonucleotides containing only one unsubstituted ribo unit, all the rest of sugars being 2'-O-methylated. The base sequence has recently been reported to make a big contribution to the reactivity of these compounds. A detailed examination of the interaction energies between the base moieties shows that base stacking is strongly context-dependent and cooperative. The strength of stacking at the site susceptible to chain cleavage by intramolecular transesterification was observed to be dependent on both the flanking bases of the cleavage site and those further apart in the molecule. The interaction energies between the bases in the vicinity of the scissile linkage were found to correlate well with the experimental phosphodiester bond cleavage rates: the stronger the bases close to the cleavage site are stacked, the slower the cleavage rate is.

The spirobicyclic peptides 2−6 were synthesized as stereoisomeric pairs using an orthogonally protected bis(aminomethyl)malonic acid building block 1 as the branching unit. Peptide 2 was synthesized by two methods. Either the chain assembly and first cyclization were carried out on a Wang resin, and the second cyclization in solution (Scheme 1), or the whole synthesis was performed on a solid-supported backbone amide linker derived from 4-alkoxybenzaldehyde (Scheme 3). The applicability of the latter method was further evaluated by synthesis of four additional spirobicyclic peptides 3−6.

The cleavage of short chimeric oligonucleotides containing only one reactive ribonucleoside unit, all other nucleosides being 2'-O-methylated, has been studied at pH 8.5 and 35 degrees C. Among the 20 different sequences that did not exhibit any tendency to form a defined secondary structure, the scissile 5'-UpA-3' and 5'-CpA-3' phosphodiester bonds experienced100- and up to 35-fold reactivity differences, respectively. Compared with dinucleoside monophosphates, both rate accelerations and retardations of more than one order of magnitude were observed. Even a change of a single base several nucleosides away from the scissile bond markedly affected the reaction rate. Duplex formation at the 3'- and/or 5'-side of the scissile bond was also studied and observed to be strongly rate retarding. The origin of the high sensitivity of phosphodiester bonds to the molecular environment is discussed.

Stereoisomeric uridylyl(3',5')uridines D,L-UpU and L,D-UpU were synthesised. Their cleavage was followed in the presence of acid, base and metal ion catalysts to study whether the stereochemistry affects the inherent reactivity of the internucleosidic phosphodiester bond, and whether the low molecular weight catalysts can distinguish between the substrates. The rate constants obtained were compared to those of D,D-UpU. The comparison shows that the stability of the phosphodiester bond does not depend on the stereochemistry of the sugar rings. In contrast slight reactivity differences are observed in the presence of metal ion catalysts, which suggests that selective cleavage of stereoisomeric substrates even by small molecular weight chemical catalysts may be possible.

Four different nucleoside phosphodiester protecting groups derived from bis(hydroxymethyl)-1,3-dicarbonyl compounds and their congeners have been prepared and introduced to 5′-O-pivaloylthymidine 3′-(2-methoxyethyl)phosphate and its monothioate analogs. Nucleoside phosphotriesters having either 2,2-bis(ethoxycarbonyl)-3-(4,4′-dimethoxytrityloxy)propyl (1a), 2-cyano-2-methoxycarbonyl-3-(4,4′-dimethoxytrityloxy)propyl (1b), 2,2-bis(cyano)-3-(4,4′-dimethoxytrityloxy)propyl (1c) or 2-acetyl-2-benzoyl-3-(4,4′-dimethoxytrityloxy)propyl (1d) protecting group have been prepared. Additionally were synthesized the O- and S-esterified phosphoromonothioate analogs of 1b. After removal of the dimethoxytrityl group under acidic conditions, each of the detritylated protecting groups is readily cleaved from the phosphate/thiophosphate moiety by a reaction suggested to involve a base-catalyzed retro-aldol condensation and following elimination of the phosphodiester from the formed carbanion intermediate. The kinetics of the hydroxide ion-catalyzed cleavage have been studied by HPLC over a pH range 2–7. The half-lives of the cleavage at pH 7 and 25 °C vary from 0.3 s (for 1c) to 5500 s (for 1a). The results confirm that these protecting groups have promising chemical properties for use in the development of antisense oligonucleotide pro-drug strategies.

This work studies the effect of RNA structure on the reactivity of RNA phosphodiester bonds. The aim of the study is to evaluate those structural parameters that affect reactivity to support the development of sequence-specific RNA cleaving agents. The cleavage of phosphodiester bonds within one- to five-nucleotide bulges has been studied in buffer solutions (pH 8.4), and in the presence of Zn2+ aqua ion (pH 6.4) or its 1,5,9-triazacyclododecane complex (pH 7.4). Molecular modelling has been applied to study the structure of the substrates. The basis of the reactivity and the catalytic potential of metal ion-based cleaving agents are discussed.

The hydrolysis of phosphodiester bonds of chimeric 2′-O-methyloligoribonucleotides was studied in buffer solutions. Pseudo-first-order rate constants for cleavage of phosphodiester bonds within hairpin loops were calculated and compared with those for cleavage of phosphodiester bonds within double-stranded stems and linear single-stranded oligonucleotides. No large differences in reactivity were observed: some of the hairpin structures studied were slightly less and others slightly more reactive than the linear reference. These results suggest that phosphodiester bonds within small hairpin loops are conformationally free to cleave by an in-line mechanism, but also that the secondary structure may influence the reactivity of phosphodiester bonds.

The course of hydrolysis of 3′-deoxy-3′-thioinosylyl-(3′ → 5′)-uridine (IspU) has been followed by HPLC over a wide pH-range. Two reactions of the internucleosidic thiophosphate linkage compete: (i) cleavage yielding thioinosine monophosphates and uridine, and (ii) isomerization to the 2′,5′-isomer of IspU. Under very acidic conditions, even acid-catalyzed depurination of the inosine moiety is observed. The stability of the thiophosphate linkage and the mechanisms of its rupture are discussed.

Novel non-nucleosidic phosphoramidite building block 2 was employed for multiple modification of oligonucleotides with hydrophobic octyl groups. Hydrophobie sites are attached via potentially biodegradable ester bonds that are demonstrated to withstand the conditions of DNA deprotection. The chimeric oligonucleotides are capable of forming triple helix complexes that are stabilixed by forming a hydrophobic

Six achiral N -phthaloyl protected α,α-disubstituted β-amino acids were synthesized, and used to construct a 96 compound library of 1,2,3,4-tetrahydroisoquinoline conjugates of β-peptides on a solid support. The library synthesis consisted of attachment of an appropriately 5- O -tethered 1,2,3,4-tetrahydroisoquinoline via the N -2 atom to a vinyl sulfonyl support, elongation of the deprotected 5- O -tether by repeated peptide coupling, quaternarization of N -2 with alkylamines and release of the β-peptide conjugate into solution with triethylamine.

A kinetic study on the cleavage of a number of mRNA 5′-cap analogs, m7GpppN and m7GppppG, with Cu2+ aquo ion has been performed. Time-dependent product distributions at various pH and metal ion concentrations have been determined by capillary zone electrophoresis, and these data have been used to calculate the rate constants for various parallel reactions of the breakdown of the cap analogs.

Several chimeric ribo/2'- O -methylribo oligonucleotides were synthesized and their hydrolytic cleavage studied in the presence of Mg2+, Zn2+, Pb2+and the 1,4,9-triaza-cyclododecane chelate of Zn2+(Zn2+[12]aneN3) to evaluate the importance of RNA secondary structure as a factor determining the reactivity of phosphodiester bonds. In all the cases studied, a phosphodiester bond within a 4-7 nt loop was hydrolytically more stable than a similar bond within a linear single strand, but markedly less stable than that in a double helix. With Zn2+and Zn2+[12]aneN3, the hydrolytic stability of a phosphodiester bond within a hairpin loop gradually decreased on increasing the distance from the stem. A similar but less systematic trend was observed with Pb2+. Zn2+- and Pb2+-promoted cleavage was observed to be considerably more sensitive to the secondary structure of the chain than that induced by Zn2+[12]aneN3. This difference in behaviour may be attributed to bidentate binding of uncomplexed aquo ions to two different phosphodiester bonds. Mg2+was observed to be catalytically virtually inactive compared with the other cleaving agents studied.

Concurrent isomerization of the ethyl, 2-ethoxyethyl, 2,2-dichloroethyl and 2,2,2-trichloroethyl esters of uridine 3′-phosphate to their 2′-counterparts and cleavage to uridine 2′,3′-cyclic phosphate have been studied over a wide pH-range (H0 = –0.2 to pH 9) at 363.2 K. The buffer-independent pH-rate profiles obtained show involvement of four distinct kinetic terms in the cleavage reaction, viz. dependence of rate on [H+][SH], [SH], [S–] and [S–][H+]–1 (S– denotes the diester monoanion). The βlg values (lg = leaving group) for the partial reactions are –0.04 ± 0.04, –0.19 ± 0.12, –0.59 ± 0.12 and –1.10 ± 0.05, respectively. The isomerization, in turn, shows dependence of rate on [H+][SH], [SH] and [S–], the βrg values (rg = remnant group) being –0.23 ± 0.04, –0.23 ± 0.11 and –0.03 ± 0.01. The mechanisms of various partial reactions are evaluated by comparing these values with those of specific and buffer catalyzed reactions of the corresponding 3′-phosphotriesters, regarded as mimetics of the neutral ionic form of diesters. Furthermore, the mechanistic significance of the dissimilar competition between the cleavage and isomerization with diesters and triesters is discussed.

RNA phosphodiester linkages undergo two transesterification reactions catalyzed by Bronsted acids or bases, viz., cyclization to a 2′,3′-cyclic phosphate with concomitant release of the 5′-linked nucleoside and isomerization to a 2′,5′-linkage. While these processes are approximately as fast under acidic conditions, the cyclization gradually takes over on going to alkaline solutions. At physiological pH, the isomerization still successfully competes with the chain cleavage. Nevertheless, protein enzymes, ribozymes, and metal ion complexes with some exceptions catalyze only the cleavage reaction. The mechanistic reasons for the dissimilar pH dependence and susceptibility to catalysis are discussed.

Several circular oligonucleotides were synthesized and characterized by electrospray ionization mass spectrometry. Experiments on termination of primer extension catalysed by DNA polymerases, Klenow fragment and Tth have demonstrated that a double helix forming circular 2'-deoxyribooligomer containing a 25mer sequence complementary to the target single-stranded DNA along with a 34mer random mismatching stretch appears to be a potent inhibitor of replication in vitro. Studies on inhibition of luciferase gene expression in a cell-free transcription-translation system have shown that a duplex forming circular 2'-deoxyribooligonucleotide containing a 25mer sequence complementary to the target mRNA and a 14mer random mismatching stretch can serve as an effective antisense compound as a standard linear complementary oligomer. Features of double helix forming circular oligonucleotides composed of 2'-deoxyribonucleosides seem to be useful for the design of new antigene and antisense agents.

A novel solid phase synthesis of 3′-phosphorylated oligonucleotides is described. The chain assembly is carried out by phosphoramidite strategy on solid support 2, which allows a mild and fast release of the oligonucleotide in solution. The applicability of the method is demonstrated by preparation of 3′-phosphorylated chimeric oligonucleotides containing methyl phosphotriester and methyl phosphonate internucleosidic linkages.

Hydrolytic reactions of the cis-phenyl ester of thymidine 3‘,5‘-cyclic monophosphate (1a) have been followed by HPLC over a wide pH range. Under acidic conditions (pH 80%) at pH 10, the hydrolysis is hydroxide-ion-catalyzed, yielding the three phosphodiester products in a 42:42:16 ratio ([2]:[3]:[4]). From pH 7 to 10, the pH−rate profile is nonlinear, possibly due to N3H de...

Pseudo first-order rate constants for the Zn2+ -promoted cleavage of 15 different dinucleoside monophosphates, 4 different ribo homopogmers and RNA III from baker's yeast have been determined. Furthermore, the distribution of various nucleosides at the 3′-and 5′-terminus of the oligomeric hdrolysls products of RNA has been quantified. On these bases, the effect of nearest neighbours on the metal-ion-promoted hydrolysis of the internucleosidic phosphodiester bonds of RNA is discussed.

1 H-NMR spectroscopy was applied to a study of the mode of interaction, in aqueous medium in the pH range 5.2–8.5 and at low and high temperatures, between several mono- and dinucleotide analogues of the mRNA cap m 7 GpppG and a selected tripeptide Trp-Leu-Glu, and a tetrapeptide Trp-Glu-Asp-Glu, the sequence of which corresponds to one of the suspected binding sites in the MRNA cap-binding protein (CBP). A program, GEOSHIFr, was developed, based on ring-current anisotropy theory, for analysis of experimentally observed changes in chemical shifts accompanying interactions between aromatic heterocyclic rings. This permitted quantitative evaluation of stacking interactions between the m 7 G cap and the tryptophan indole ring, and the relative orientations of the planes of the two rings, spaced about 3.2 A apart. The structures of the stacked complexes were determined. In particular, stacking between m 3 2,2,7 G (which has no free amino group for hydrogen bonding) and the indole ring is weaker and quite different from that between m 7 G and m 2 2,7 G and indole. With the dinucleotide cap-analogues, only the m 7 G component stacks with the indole ring, without disruption of intramolecular stacking. In contrast to numerous earlier reports, the calculated stacking interactions are quantitatively in accord with the values derived from fluorescence measurements. It also has been shown that the positively charged (cationic) form of m 7 G stacks much more efficiently with the indole ring than the zwitterionic form resulting from dissociation of the guanine ring NlH (p K a ≈ 7.3).

Hydrolyses of the two diastereomeric phosphoromonothioate analogs of uridine 2',3'-cyclic monophosphate [(R(P))- and (S(P))-2',3'-cUMPS] at 363.2 K have been followed by HPLC over pH-range 0-12. In aqueous alkali (pH9) only base-catalyzed endocyclic phosphoester hydrolysis to a nearly equimolar mixture of uridine 2'- and 3'-phosphoromonothioates (2'- and 3'-UMPS) takes place, analogously to the hydrolysis of uridine 2',3'-cyclic monophosphate (2',3'-cUMP). The (R(P))- and (S(P))-2',3'-cUMPS are hydrolyzed 50 and 30%, respectively, more slowly than 2',3'-cUMP. Under neutral and acidic conditions, desulfurization of the cyclic thiophosphates to 2',3'-cUMP competes with the phophoester hydrolysis, both reactions being acid-catalyzed at pH5. The desulfurization is most pronounced in strongly acidic solutions ([HCl]0.1 mol L(-)(1)), where more than 90% of the starting material is degraded via this route. At pH2, the thioates are considerably, i.e., more than 1 order of magnitude, more stable than 2',3'-cUMP. While the hydrolysis of 2',3'-cUMP is second-order in hydronium-ion concentration, that of 2',3'-cUMPS exhibits a first-order dependence. The reactivities of the two diastereomers are comparable with each other over the entire pH-range studied, the most significant difference being that the pH-independent desulfurization at pH5 is with the R(P)-isomer 5-fold faster than with the S(P)-isomer. In contrast to 2',3'-cUMP, depyrimidination of the starting material (i.e., release of the uracil base) competes with the hydrolysis of the thiophosphate moiety under neutral conditions (pH 6-8).