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The cleavage of RNA phosphodiester bonds by RNase A and hammerhead ribozyme at neutral pH fundamentally differs from the spontaneous reactions of these bonds under the same conditions. While the predominant spontaneous reaction is isomerization of the 3',5'-phosphodiester linkages to their 2',5'-counterparts, this reaction has never been reported to compete with the enzymatic cleavage reaction, not even as a minor side reaction. Comparative kinetic measurements with structurally modified di-nucleoside monophosphates and oligomeric phosphodiesters have played an important role in clarification of mechanistic details of the buffer-independent and buffer-catalyzed reactions. More recently, heavy atom isotope effects and theoretical calculations have refined the picture. The primary aim of all these studies has been to form a solid basis for mechanistic analyses of the action of more complicated catalytic machineries. In other words, to contribute to conception of a plausible unified picture of RNA cleavage by biocatalysts, such as RNAse A, hammerhead ribozyme and DNAzymes. In addition, structurally modified trinucleoside monophosphates as transition state models for Group I and II introns have clarified some features of the action of large ribozymes.

The trafficking chaperone PDE6D (also referred to as PDEδ) has been nominated as a surrogate target for K-Ras4B (hereafter K-Ras). Arl2-assisted unloading of K-Ras from PDE6D in the perinuclear area is significant for correct K-Ras localization and therefore activity. However, the unloading mechanism also leads to the undesired ejection of PDE6D inhibitors. To counteract ejection, others have recently optimized inhibitors for picomolar affinities; however, cell penetration generally seems to remain an issue. To increase resilience against ejection, we engineered a “chemical spring” into prenyl-binding pocket inhibitors of PDE6D. Furthermore, cell penetration was improved by attaching a cell-penetration group, allowing us to arrive at micromolar in cellulo potencies in the first generation. Our model compounds, Deltaflexin-1 and -2, selectively disrupt K-Ras, but not H-Ras membrane organization. This selectivity profile is reflected in the antiproliferative activity on colorectal and breast cancer cells, as well as the ability to block stemness traits of lung and breast cancer cells. While our current model compounds still have a low in vitro potency, we expect that our modular and simple inhibitor redesign could significantly advance the development of pharmacologically more potent compounds against PDE6D and related targets, such as UNC119 in the future.

MicroRNAs (miRNAs) are endogenous, small, noncoding ribonucleic acids (RNAs) that bind to the 3′ untranslated regions of messenger RNAs (mRNAs) and induce translational repression or mRNA degradation. Although numerous studies have reported that miRNAs are of potential use for disease diagnostics and gene therapy, little is known about their fates in vivo. This study elucidated the whole-body distributions and kinetics of intravenously administered miRNA-targeting molecules in vivo by positron emission tomography (PET) imaging. A 22-mer sequence targeting miR-15b was conjugated with three different chelators and labeled with gallium-68 (68Ga). These tracers were compared with a scrambled 22-mer sequence; 22-mer with two single base substitutions; anti-miR-34 22-mer; hexathymidylate (T6), a 6-mer sequence; and an unconjugated chelator. miR-15b was chosen as a target because it is important for bone remodeling. All three 68Ga-labeled anti-miR-15b molecules had similar biodistributions and kinetics, and they all accumulated in the bones, kidneys, and liver. The bone accumulation of these tracers was the highest in the epiphyses of long tubular bones, maxilla, and mandible. By contrast, the scrambled 22-mer sequence, the 6-mer, and the unconjugated chelator did not accumulate in bones. PET imaging successfully elucidated the distributions and kinetics of 68Ga-labeled chelated miRNA-targeting molecules in vivo. This approach is potentially useful to evaluate new miRNA-based drugs.

Azidopropyl-modified precursors of chondroitin sulfate (CS) tetrasaccharides have been synthesized, which, after facile conversion to final CS structures, may be conjugated with alkyne-modified target compounds by a one-pot "click"-ligation. RP HPLC was used for the monitoring of the key reaction steps (protecting group manipulation and sulfation) and purification of the CS precursors (as partially protected form, bearing the O-Lev, O-benzoyl, and N-trichloroacetyl groups and methyl esters). Subsequent treatments with aqueous NaOH, concentrated ammonia, and acetic anhydride (i.e., global deprotection and acetylation of the galactosamine units) converted the precursors to final CS structures. The azidopropyl group was exposed to a strain-promoted azide-alkyne cycloaddition (SPAAC) with a dibenzylcyclooctyne-modified carboxyrhodamine dye to give labeled CSs. Conjugation with a 5'-cyclooctyne-modified oligonucleotide was additionally carried out to show the applicability of the precursors for the synthesis of biomolecular hybrids.

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.

A bis(phosphonate) conjugate of 2'-O-methyl oligoribonucleotide (microRNA-21) was synthesized and used as a bone-targeting carrier in the systemic delivery of a (68)Ga-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA)-chelated 2'-O-methyl oligoribonucleotide (anti-microRNA-21). The whole-body biodistribution of the double helical RNA was monitored by positron emission tomography (PET), which verified the expected bis(phosphonate)-induced bone accumulation in healthy rats.

Tetrakis-O-(4-azidomethylphenyl)pentaerythritol, derivatized with 5′-O-(4,4′-dimethoxytrityl)-3′-O-{4-[2-(but-3-yn-1-ylamino)-2-oxoethoxy]phenoxyacetyl}thymidine, was used as a soluble support to assemble fully protected 2′-O-deoxyribonucleotide trimers by the phosphotriester chemistry. After the coupling and detritylation steps, the support-bound construct was purified by precipitation in MeOH. The trimers (TAT, AGT, TTA, CAT, GCT), in fully protected form, were released by a treatment with dilute methanolic K2CO3 and filtered through a short silica gel column in 65–70% overall yield. Two of the trimers (CAT and GCT), prepared in 0.5 mmol scale, were converted to the corresponding phosphoramidites. The entire procedure for the preparation of trimer phosphoramidites proved straightforward and applicable for the large scale synthesis.

Synthesis for (68)Ga-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA)-chelated oligonucleotide hyaluronan (HA) tetra- and hexasaccharide conjugates is described. A solid-supported technique is used to introduce NOTA-chelator into the 3'-terminus of oligonucleotides and a copper-free strain promoted azide alkyne cycloaddition (SPAAC) to HA/oligonucleotide conjugation. Protecting group manipulation, required for the HA-moieties, is carried out after the SPAAC-conjugation. Positron emission tomography (PET) is used (1) in the whole-body distribution kinetic studies of the conjugates in healthy rats and (2) to show the potential of hyaluronan-induced targeting of oligonucleotides into the infarcted area of rats with myocardial infarction.

The preparation of a disulfide-tethered precipitative soluble support and its use for solution-phase synthesis of trimeric oligodeoxyribonucleotide 3´-(2-chlorophenylphosphate) building blocks is described. To obtain the building blocks, N-acyl protected 2´-deoxy-5´-O-(4,4´-dimethoxytrityl)ribonucleosides were phosphorylated with bis(benzotriazol-1-yl) 2-chlorophenyl phosphate. The “outdated” phosphotriester strategy, based on coupling of PV building blocks in conjunction with quantitative precipitation of the oligodeoxyribonucleotide with MeOH is applied. Subsequent release of the resulting phosphate and base-protected oligodeoxyribonucleotide trimer 3’-pTpdCBzpdGibu-5’ as its 3’-(2-chlorophenyl phosphate) was achieved by reductive cleavage of the disulfide bond.

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.

Buffer catalysis of the cleavage and isomerization of uridylyl-3',5'-uridine (UpU) has been studied over a wide pH range in 80% aq. DMSO. The diminished hydroxide ion concentration in this solvent system made catalysis by amine buffers (morpholine, 4-hydroxypiperidine and piperidine) visible even at relatively low buffer concentrations (10-200 mmol L(-1)). The observed catalysis was, however, much weaker than what has been previously reported for the activated RNA model 2-hydroxypropyl 4-nitrophenyl phosphate (HPNP) in the same solvent system. In the case of morpholine, contribution of both the acidic and the basic buffer constituent was significant, whereas with 4-hydroxypiperidine and piperidine participation of the acidic constituent could not be established unambiguously. The results underline the importance of using realistic model compounds, along with activated ones, in the study of the general acid/base catalysis of RNA cleavage.

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.

Nucleic acids that store and transfer biological information are polymeric diesters of phosphoric acid. Cleavage of the phosphodiester linkages by protein enzymes, nucleases, is one of the underlying biological processes. The remarkable catalytic efficiency of nucleases, together with the ability of ribonucleic acids to serve sometimes as nucleases, has made the cleavage of phosphodiesters a subject of intensive mechanistic studies. In addition to studies of nucleases by pH-rate dependency, X-ray crystallography, amino acid/nucleotide substitution and computational approaches, experimental and theoretical studies with small molecular model compounds still play a role. With small molecules, the importance of various elementary processes, such as proton transfer and metal ion binding, for stabilization of transition states may be elucidated and systematic variation of the basicity of the entering or departing nucleophile enables determination of the position of the transition state on the reaction coordinate. Such data is important on analyzing enzyme mechanisms based on synergistic participation of several catalytic entities. Many nucleases are metalloenzymes and small molecular models offer an excellent tool to construct models for their catalytic centers. The present review tends to be an up to date summary of what has been achieved by mechanistic studies with small molecular phosphodiesters.

Appropriately protected structurally modified nucleoside 3′,5′-cyclic monophosphates are known to show antiviral activity. For this reason, a straightforward synthesis of nucleoside 3′,5′-cyclic phosphates protected with three different enzymatically removable groups, viz. 3-acetyloxy-2,2-bis(ethoxycarbonyl)propyl (in 1 and 4), 4-acetylthio-2,2-dimethyl-3-oxobutyl (in 2), and 4-(tert-butyldisulfanyl)-2,2-dimethyl-3-oxobutyl (in 3) groups, is described. Removal of these protecting groups at pH 7.5 and 37 °C was monitored by reverse-phase HPLC.

(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.

Short oligodeoxyribonucleotides have been assembled from appropriately protected nucleoside 3′-(benzotriazol-1-yl 2-chlorophenyl phosphate) derivatives on hundred-milligram scales on a soluble tetrakis-O-(4-azidomethylphenyl)pentaerythritol support bearing four 3′-O-(4-pentynoyl)thymidines. Small-molecule reagents and waste were removed by two precipitations of the support-bound growing oligonucleotides with methanol, the first after removal of the 5′-protecting group and the second after coupling. The building blocks were obtained by one-step phosphorylation of commercially available protected nucleosides with bis(benzotriazol-1-yl) 2-chlorophenyl phosphate. Removal of the phosphate protecting groups from the completed chain with (E)-2-nitrobenzaldoxime followed by conventional ammonolysis allowed precipitation of the oligonucleotide as the sodium salt with ethanol.

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.

The phosphodiester linkage of 3′-O-levulinoylthymidine 5′-methylphosphate (5) has been protected with 2-[(acetyloxy)methyl]-4-(acetylsulfanyl)-2-(ethoxycarbonyl)-3-oxobutyl group (to give 1) to study the potential of this group as an esterase- and thermolabile protecting group. The group turned out to be unexpectedly thermolabile, being removed as ethyl 3-(acetyloxy)-4-(acetylsulfanyl)-2-methylidenebut-3-enoate (10) without accumulation of any intermediates. The half-life of this reaction at pH 7.5 and 37° is 14 min. Hog liver esterase (HLE), in turn, removes the protecting group as ethyl 4-(acetylsulfanyl)-2-methylidene-3-oxobutanoate (12). On using 2.6 units of HLE in 1 ml, the rate of the enzymatic deprotection was still only one third of that of the nonenzymatic reaction. The mechanisms of both reactions have been studied and discussed. The crucial step seems to be removal of the O-bound Ac group, either by esterase or by migration to the neighboring 3-oxo group (nonenzymatic removal). This triggers the removal by retro-aldol condensation/elimination mechanism. No alkylation of glutathione (GSH) upon the deprotection of 1 could be detected.

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.

Novel soluble supports for oligonucleotide synthesis 11a–c have been prepared by immobilizing a 5′-O-protected 3′-O-(hex-5-ynoyl)thymidine (6 or 7) to peracetylated or permethylated 6-deoxy-6-azido-β-cyclodextrins 10a or 10b by Cu(I)-promoted 1,3-dipolar cycloaddition. The applicability of the supports to oligonucleotide synthesis by the phosphoramidite strategy has been demonstrated by assembling a 3′-TTT-5′ trimer from commercially available 5′-O-(4,4′-dimethoxytrityl)thymidine 3′-phosphoramidite. To simplify the coupling cycle, the 5′-O-(4,4′-dimethoxytrityl) protecting group has been replaced with an acetal that upon acidolytic removal yields volatile products. For this purpose, 5′-O-(1-methoxy-1-methylethyl)-protected 3′-(2-cyanoethyl-N,N-diisopropyl-phosphoramidite)s of thymidine (5a), N4-benzoyl-2′-deoxycytidine (5b) and N6-benzoyl-2′-deoxyadenosine (5c) have been synthesized and utilized in synthesis of a pentameric oligonucleotide 3′-TTCAT-5′ on the permethylated cyclodextrin support 11c.

Esterified precursors of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA; 18) and 1,4,7-triazacyclononane-1,4,7-trisacetic acid (NOTA; 17,19) ligands bearing a dimethoxytritylated hydroxyl side arm were prepared and immobilized via an ester linkage to long chain alkyl amine derivatized controlled pore glass (LCAA-CPG). Oligonucleotide chains were then assembled on the hydroxyl function and conjugates were released and deprotected by a two-step cleavage with aqueous alkali and ammonia. The 3'-DOTA and 3'-NOTA conjugated oligonucleotides were converted to (68)Ga chelates by a brief treatment with [(68)Ga]Cl(3) at elevated temperature. Applicability of the conjugates for in vivo imaging with positron emission tomography (PET) was verified.

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.

A method for the synthesis of 7-substituted 3-β-D-ribofuranosyl-3H-imidazo[2,1-i]purines has been devised whereby compounds were prepared in a few steps from a common intermediate, 3-(2′,3′-O-isopropylidene-β-D-ribofuranosyl)-3H-imidazo[2,1-i]purine-7-carbaldehyde, obtained from the reaction of 2′,3′-O-isopropylideneadenosine with bromomalonaldehyde. The formyl group of the carbaldehyde was subsequently reductively aminated and the resulting secondary amines were then further derivatized either by acylation, lactamization or reductive alkylation.

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 (R(P))- and (S(P))-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).

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.

Despite palliative treatments, tumor-induced bone disease (TIBD) remains highly debilitating for many cancer patients and progression typically results in death within two years. Therefore, more effective therapies with enhanced anti-resorptive and cytotoxic characteristics are needed. We developed bisphosphonate-chemotherapeutic conjugates designed to bind bone and hydrolyze, releasing both compounds, thereby targeting both osteoclasts and tumor cells. This study examined the effects of our lead compound, MBC-11 (the anhydride formed between arabinocytidine (AraC)-5'-phosphate and etidronate), on bone tumor burden, bone volume, femur bone mineral density (BMD), and overall survival using two distinct mouse models of TIBD, the 4T1/luc breast cancer and the KAS-6/1-MIP1alpha multiple myeloma models. In mice orthotopically inoculated with 4T1/luc mouse mammary cells, MBC-11 (0.04 microg/day; s.c.) reduced the incidence of bone metastases to 40% (4/10), compared to 90% (9/10; p=0.057) and 100% (5/5; p=0.04) of PBS- or similarly-dosed, zoledronate-treated mice, respectively. MBC-11 also significantly decreased bone tumor burden compared to PBS- or zoledronate-treated mice (p=0.021, p=0.017, respectively). MBC-11 and zoledronate (0.04 microg/day) significantly increased bone volume by two- and four-fold, respectively, compared to PBS-treated mice (p=0.005, p

A method for the synthesis of libraries of 4(5),1',5'-trisubstituted 2,4'-biimidazoles on a solid support was developed. (1) A trivalent scaffold, 2-(5'-amino-4(5)-formyl-1H,1'H-2,4'-biimidazol-1'-yl)acetic acid, was first prepared in solution by a two-step synthesis from ethyl adenin-9-ylacetate and bromomalonaldehyde. The product was coupled to an amino acid loaded Wang resin and the formyl group was subsequently derivatized either by reductive amination, oximation, or hydrazone formation. The 5'-amino group of the resin-bound biimidazole was then acylated and the products were finally released from the resin and purified. 5'-Amino-2,4'-biimidazole offers a scaffold for lead compounds of drug discovery, possibly useful in finding leads for protein kinase inhibitors.

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.

4'-C-Azidomethylthymidine 3'-(H-phosphonate) monomer (10) was synthesized in high yield and three such monomers were incorporated by the H-phosphonate coupling into a 15-mer oligodeoxyribonucleotide. The unmodified 2'-deoxynucleosides could be coupled by either the H-phosphonate or phosphoramidite chemistry, indicating that the Staudinger reaction between the azido group and the phosphoramidite reagent severely hampered the coupling only when it took place intramolecularly. After chain assembly, three alkynyl group bearing ligands, viz., propargyl 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranoside (2), N-{4-[N-(trifluoroacetyl)aminomethyl]benzyl}-4-pentynamide (3) and N (1), N (3), N (2')-tris(trifluoroacetyl)-N (6')-(4-pentynoyl)neamine (4), were conjugated to the azido groups of the oligonucleotide by click chemistry both on a solid support and in solution. The products were deprotected by conventional ammonolysis and purified by HPLC chromatography. Melting temperature studies revealed that the mannose conjugated oligonucleotides formed more stable duplexes with 2'-O-methyl RNA than with DNA strand. With 2'-O-methyl RNA, a slight destabilization compared to an unmodified sequence was observed at low ionic strength, while at high salt content, the manno-conjugation was stabilizing.

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.