Di(azacrown) Conjugates of 2'-O-Methyl Oligoribonucleotides as

Bioconjugate Chem. , 2007, 18 (5), pp 1583–1592 ... Design, RNA cleavage and antiviral activity of new artificial ribonucleases derived from mono-, ...
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Bioconjugate Chem. 2007, 18, 1583−1592

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Di(azacrown) Conjugates of 2′-O-Methyl Oligoribonucleotides as Sequence-Selective Artificial Ribonucleases Teija Niittyma¨ki, Pasi Virta, Kaisa Ketoma¨ki, and Harri Lo¨nnberg* Department of Chemistry, University of Turku, FIN-20014 Turku, Finland. Received March 2, 2007; Revised Manuscript Received July 5, 2007

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 solidsupport by conventional phosphoramidite chemistry. The branching units employed for the purpose include 2,2bis(3-hydroxypropylaminocarbonyl)propane-1,3-diol, 2-hydroxyethyl 3′-O-(2-hydroxyethyl)-β-D-ribofuranoside, and 2-hydroxyethyl 2′-O-(2-hydroxyethyl)-β-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)β-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.

INTRODUCTION Development of artificial ribonucleases, i.e., agents able to cleave RNA phosphodiester bonds sequence-selectively, has been the subject of numerous studies during the past decade (1-6). Such man-made catalysts are believed to find applications as artificial restriction enzymes with which large RNA molecules could be tailored in a predesign manner in Vitro (7-11) and hopefully also as chemotherapeutic agents capable of silencing an overexpressed gene by recognizing and destroying the respective mRNA (12-16). Although recent advances in gene silencing by short double-stranded RNAs (siRNA) (17-21) has diminished the interest in conventional antisense approaches, the recognition of multiple roles of RNA in gene regulation has simultaneously made RNA an object of extensive studies. Accordingly, development of tools useful in chemical manipulation of RNA, such as artificial ribonucleases, still appears worthwhile. Most of the artificial ribonucleases described so far consist of a metal ion chelate tethered to an oligonucleotide or PNAoligomer that serves as a sequence-recognizing moiety (1-6), although some purely organic conjugates have recently been introduced (22-24). Among the metal-ion-dependent conjugates, those derived from lanthanide ion chelates exhibit the highest cleaving activity (25-31), followed by nucleases based on intracellularly occurring Cu2+ (32-36) and Zn2+ ions (3741). The most efficient lanthanide ion conjugates, Viz. the 5′tethered Dy3+-texaphyrin oligonucleotide conjugates (28), exhibit a half-life of 2 h at pH 7.5 and 37 °C, when used in a stoichiometric amount compared to the oligoribonucleotide target. The best Cu2+- and Zn2+-based cleaving agents, Viz. the Cu2+ (34) and Zn2+ complexes (39) of intrachain 2,9-dimethyl5-aminophenanthroline-oligonucloeotide conjugates, in turn, show under the same conditions half-lives of 8 and 9 h, respectively. A comparable cleaving activity, but at a lower Zn2+ concentration, has been obtained with a dinuclear Zn2+ complex of a conjugate bearing a 5′-terminal 3,5-bis[di(pyridin-2* Corresponding author. E-mail: [email protected]; tel.: +358-2-333 6770; fax: +358-2-333 6776.

ylmethyl)aminomethyl]phenoxy group (38). By simultaneous use of 3′- and 5′-tethered Cu2+-terpyridine conjugates that hybridize to the target leaving no gap between them, the halflife is reduced to 5 h (36) and by covalent 3′,5′-bridging of the two oligonucleotide conjugates to 2.5 h (35). With the most efficient metal-ion-independent oligonucleotide conjugates, the 5′-tris(2-aminobenzimidazole) conjugates, a half-life of 12 h at pH 8 and 37 °C has been achieved (22). We have previously reported on synthesis of 1,5,7-triazacyclododecane conjugates of 2′-O-methyloligoribonucleotides (42) and on studies of the cleaving activity of their 3d transition metal chelates (40). The advantage of such conjugates is that they bind 3d transition metal ions more tightly than 2,9dimethylphenanthroline conjugates, but unfortunately they cleave the target RNA 1 order of magnitude more slowly than the phenanthroline conjugates. We now report on synthesis of difunctionalized oligonucleotides bearing two 3-(3-hydroxypropyl)-1,5,9-triazacyclododecane ligands attached via a phosphodiester linkage to a single non-nucleosidic building block, either to 2,2-bis(3-hydroxypropylaminocarbonyl)propane-1,3diol (1a,b), 2-hydroxyethyl 3′-O-(2-hydroxyethyl)-β-D-ribofuranoside (2a,b), or 2-hydroxyethyl 2′-O-(2-hydroxyethyl)-β-Dribofuranoside (3a,b). Interestingly, the Zn2+ complex of conjugate 3a, bearing the azacrown ligands close to the 3′-terminus, exhibits a 15-fold enhanced catalytic activity over its monofunctionalized counterpart. The catalytic activity of this conjugate is equal to that of the most efficient Zn2+- or Cu2+based artificial ribonucleases described so far.

EXPERIMENTAL PROCEDURES General Methods and Materials. The NMR spectra were recorded at 500 MHz. The chemical shifts are given in ppm from internal TMS and the coupling constants in hertz. The mass spectra of small molecular compounds were recorded by EI ionization and those of oligonucleotides and their conjugates by ESI ionization. For details, see the Supporting Information. The chimeric 2′-O-methylribo/ribo oligonucleotides (4-6) used as targets for the artificial ribonucleases and as reference material for identification of the cleavage site (7-9) were

10.1021/bc070071o CCC: $37.00 © 2007 American Chemical Society Published on Web 08/21/2007

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Chart 1. Structures of Cleaving Agents (1-3), Targets (4-6), and Potential Product Oligonucleotides (7-9)a

a

Bold letters refer to ribonucleotides, the rest to 2′-O-methylribonucleotides.

Scheme 1

a

a Reagents and conditions: (i) levulinic anhydride, DMAP, pyridine, (ii) aq 80% AcOH, (iii) DMTrCl, pyridine, (iv) 2-cyanoethyl N,N,N′,N′tetraisopropylphosphorodiamidite, tetrazole, acetonitrile.

assembled from commercially available 2′-O-methyl- and 2′O-triisopropylsilyloxymethyl-protected 2-cyanoethyl-N,N-diisopropylphosphoramidite building blocks (Glen Research) by conventional phosphoramidite strategy using a 1.0 µmol scale and following the standard RNA-coupling protocol of Applied Biosystems 3400 DNA synthesizer. The artificial ribonucleases 1-3 were prepared as described below. The buffer reagents and salts employed were of reagent grade. All buffer solutions were prepared in sterilized water, and sterilized equipment was used for their handling. N,N′-Bis[3-(levulinoyloxy)propyl]-2-methoxy-1,3-dioxane5,5-dicarboxamide (11). N,N′-Bis(3-hydroxypropyl)-2-methoxy-1,3-dioxane-5,5-dicarboxamide (10, Scheme 1) was synthesized as described earlier (43). A mixture of 10 (4.0 g, 12 mmol), levulinic anhydride (62 mmol) and a catalytic amount of 4-dimethylaminopyridine in pyridine (30 mL) was stirred

overnight at room temperature and evaporated to dryness. The residue was dissolved in dichloromethane and washed with 5% aqueous NaHCO3. The organic phase was dried with Na2SO4 and evaporated to dryness. Purification of the crude product by silica gel chromatography (first 50% EtOAc in dichloromethane and then 5% MeOH in dichloromethane) yielded 5.7 g (86%) of 11 as brownish oil. 1H NMR (CDCl3, 500 MHz): δ 7.69 (br s, 2H), 5.31 (s, 1H), 4.51 (d, 2H, J ) 12.0 Hz), 4.16-4.12 (m, 6H), 3.41 (s, 3H), 3.41-3.33 (m, 4H), 2.77 (m, 4H), 2.63 (t, 4H, J ) 6.8 Hz), 2.20 (s, 6H), 1.87 (m, 4H); 13C NMR (CDCl3, 125 MHz): δ 206.8, 175.9, 169.5, 169.0, 109.6, 65.1, 62.1, 53.1, 50.8, 38.0, 36.9, 35.1, 29.8, 28.4, 28.0; MS (EI): M+ requires 516.2, found 515. 2,2-Bis(hydroxymethyl)-N,N′-bis[3-(levulinoyloxy)propyl]malondiamide (12). A mixture of 11 (2.7 g, 5.3 mmol) in 80% aqueous AcOH (50 mL) was stirred for 2 h at room temperature

Artificial Ribonucleases Scheme 2

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a

a Reagents and conditions: (i) allyl alcohol, BF ‚Et O, dichloromethane, (ii) allyl methyl carbonate, Pd(OAc) , PPh , THF, reflux for 15 min, 3 2 2 3 (iii) (1) NaOMe/MeOH, (2) NaH, 4-methoxybenzyl chloride, DMF, (iv) (1) OsO4, NaIO4, H2O, dioxane, (2) NaBH4, EtOH, dichloromethane, (v) levulinic anhydride, DMAP, pyridine, (vi) H2, Pd/C, EtOH, (vii) DMTrCl, pyridine, (viii) 2-cyanoethyl N,N-diisopropylphosphonamidic chloride, NEt3, dichloromethane.

and evaporated to dryness. The residue was coevaporated several times with water and then subjected to a silica gel column. Chromatographic purification (0 to 10% MeOH in dichloromethane) afforded 1.8 g (70%) of 12 as a colorless oil. 1H NMR (CDCl3, 500 MHz): δ 7.74 (t, 2H, J ) 5.6 Hz), 4.11 (t, 4H, J ) 6.1 Hz), 3.90 (s, 4H), 3.36 (m, 4H), 2.76 (dd, 4H, J ) 6.7 Hz, 6.3 Hz), 2.58 (dd, 4H, J ) 6.7 Hz, 6.3 Hz), 2.19 (s, 6H), 1.85 (m, 4H); 13C NMR (CDCl3, 125 MHz): δ 207.0, 173.0, 171.5, 64.1, 61.8, 58.1, 37.9, 36.4, 35.1, 29.8, 28.3, 27.9; MS (EI): M+ requires 474.2, found 474. 2-(4,4′-Dimethoxytrityloxymethyl)-2-hydroxymethyl-N,N′bis[3-(levulinoyloxy)propyl]malondiamide (13). 4,4′-Dimethoxytrityl chloride (1.3 g, 3.7 mmol) was slowly added to a mixture of 12 (1.8 g, 3.7 mmol) in dry pyridine (10 mL), and the mixture was then stirred overnight at room temperature. After evaporation, the residue was dissolved in dichloromethane and washed with 5% aq NaHCO3. The organic phase was dried with Na2SO4 and evaporated to dryness. The residue was coevaporated with toluene and purified by silica gel chromatography (0 to 10% MeOH in dichloromethane) to give 1.71 g (59%) of 13 as a colorless oil. 1H NMR (CDCl3, 500 MHz): δ 7.51 (t, 2H, J ) 5.8 Hz), 7.40-7.16 (m, 9H), 6.84 (m, 4H), 4.14 (t, 1H, J ) 6.0 Hz), 4.09 (t, 4H, J ) 6.1 Hz), 3.90 (m, 2H), 3.81 (s, 6H), 3.47 (s, 2H), 3.39 (m, 4H), 2.76 (m, 4H), 2.58 (m, 4H,), 2.20 (s, 6H), 1.84 (m, 4H); 13C NMR (CDCl3, 125 MHz): δ 206.7, 173.0, 172.8, 170.6, 158.6, 144.3, 135.2, 130.1, 129.1, 127.7, 126.9, 112.9, 86.4, 65.3, 64.2, 63.9, 62.0, 61.8, 58.5, 57.6, 55.3, 55.2, 37.9, 36.6, 36.3, 29.9, 28.4, 28.3, 27.9; MS (ESI): [M+Na]+ requires 799.3, found 799.7. 2-Cyanoethyl 3-(4,4′-Dimethoxytrityloxy)-2,2-bis[N-(3-levulinoyloxypropyl)carbamoyl]propyl N,N-Diisopropylphosphoramidite (14). Compound 13 (1.6 g, 2.0 mmol) was dried by repeated coevaporations with dry acetonitrile and dissolved in dry acetonitrile (2.0 mL). 2-Cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamidite (0.83 mL, 2.6 mmol) and tetrazole (4.5 mL of 0.45 M solution in acetonitrile, 2.01 mmol) were

added to the mixture, and the reaction was then allowed to stir at room temperature for 2 h. Acetonitrile was removed in vacuo, saturated aq NaHCO3 was added to the residue, and the product was then extracted with dichloromethane. The organic phase was separated, dried with Na2SO4, and evaporated to dryness. The residue was purified by silica gel chromatography (1% triethylamine in EtOAc) to yield 1.6 g (80%) of the product (14) as a colorless oil. 1H NMR (CDCl3, 500 MHz): δ 7.51 (t, 1H, J ) 5.8 Hz), 7.44 (m, 2H), 7.34-7.29 (m, 7H), 7.23 (m, 1H), 4.14-4.02 (m, 4H), 3.81 (s, 6H), 3.75-3.71 (m, 2H), 3.66 (s, 2H), 3.56-3.50 (m, 2H), 3.37-3.24 (m, 4H), 2.77-2.74 (m, 4H), 2.59-2.54 (m, 4H), 2.19 (s, 6H), 1.83-1.77 (m, 4H), 1.18, 1.16, 1.10 and 1.10 (each s, each 3H); 13C NMR (CDCl3, 125 MHz): δ 206.6, 172.7, 169.9, 169.7, 158.6, 144.2, 135.2, 130.0, 128.0, 127.9, 127.0, 113.2, 86.6, 64.9, 64.8, 63.5, 62.0, 61.9, 58.6, 58.5, 55.2, 53.5, 45.4, 45.3, 43.3, 37.9, 36.5, 29.9, 28.4, 27.9, 24.5, 23.5, 23.0, 20.3, 19.3; 31P NMR (CDCl3, 200 MHz): δ 149.2; HRMS (ESI) [M + Na]+ requires 999.4491, found 999.4461. Allyl 2,5-Di-O-acetyl-3-O-allyl-β-D-ribofuranoside (18a) and Allyl 3,5-Di-O-acetyl-2-O-allyl-β-D-ribofuranoside (18b). An equimolar mixture of isomeric allyl di-O-acetyl-β-D-ribofuranosides (16a,b, each 19%) and allyl tri-O-acetyl-β-Dribofuranoside (17; 39%) was obtained by boron trifluoridepromoted glycosidation of 1,2,3,5-tetra-O-acetyl-β-ribofuranose (15) with allyl alcohol in dichloromethane (44) (Scheme 2). The di-O-acetylated products (16a,b; 5.0 g, 18 mmol), allyl methyl carbonate (4.1 mL, 36 mmol), Pd(OAc)2 (80 mg, 0.36 mmol, 0.02 equiv), and Ph3P (0.48 g, 0.18 mmol, 0.1 equiv) were dissolved in THF (50 mL), and the mixture was refluxed for 15 min and then evaporated to dryness (45). The residue was purified by silica gel chromatography (20% EtOAc in petroleum ether) to yield 3.0 g (52%) of allyl 2,5-di-O-acetyl-3-O-allylβ-D-ribofuranoside (18a) and 2.0 g (34%) of allyl 3,5-bis-Oacetyl-2-O-allyl-β-D-ribofuranoside (18b). The isomers were obtained as colorless oils. 18a: 1H NMR (CDCl3, 500 MHz) δ

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5.90-5.81 (m, 2H), 5.28 (m, 2H, 5.25 (d, 1H, J ) 4.4 Hz), 5.01 (s, 1H), 4.86 (dd, 1H, J ) 11.4, 2.9 Hz), 4.24-4.04 (m, 5H), 3.99-3.94 (m, 2H), 2.14 (s, 3H), 2.10 (s, 3H); 13C NMR (CDCl3, 125 MHz) δ 170.8, 169.9, 133.9, 133.6, 118.0, 117.7, 104.3, 78.9, 77.6, 73.8, 72.1, 68.3, 64.8, 20.9, 20.8; HRMS (ESI) [M + Na]+ requires 337.1258, found 337.1257. 18b: 1H NMR (CDCl3, 500 MHz) δ 5.91-5.83 (m, 2H), 5,31-5.27 (m, 2H), 5.22-5.20 (m, 2H), 5.13 (dd, 1H, J ) 6.1 Hz, 5.3 Hz), 5.06 (d, 1H, J ) 0.6 Hz), 4.37-4.32 (m, 2H), 4.23 (m, 1H), 4.13-4.03 (m, 4H), 3.98 (m, 1H), 2.13 (s, 3H), 2.09 (s, 3H); 13C NMR (CDCl3, 125 MHz) δ 170.7, 170.3, 134.0, 133.8, 117.6, 117.3, 104.9, 80.5, 78.8, 73.1, 72.0, 68.5, 64.5, 20.9, 20.8; HRMS (ESI) [M + Na]+ requires 337.1258, found 337.1255. Allyl 2,5-Di-O-(4-methoxybenzyl)-3-O-allyl-β-D-ribofuranoside (19a). Compound 18a (2.3 g, 7.2 mmol) was dissolved in 0.1 mol L-1 methanolic sodium methoxide (25 mL). The mixture was stirred at ambient temperature for 1 h, neutralized by addition of strongly acidic cation-exchange resin, and filtered. The filtrate was evaporated to dryness, and the residue was dissolved in dry DMF (7.0 mL) and cooled to 0 °C. NaH (60% dispersion in mineral oil, 0.63 g, 16 mmol) and 4-methoxybenzyl chloride (2.4 mL, 18 mmol) were added, and the mixture was then stirred overnight at ambient temperature. Water (20 mL) was added, and the product was extracted by diethyl ether (2 × 20 mL). The organic phases were combined, dried with Na2SO4, and evaporated to dryness. The residue was purified by silica gel chromatography (20% EtOAc in petroleum ether) to yield 2.9 g (86%) of the product (19a) as a colorless oil. 1H NMR (CDCl3, 500 MHz) δ 7.33-7.27 (m, 4H), 6.91-6.87 (m, 4H), 5.94-5.82 (m, 2H), 5.28-5.22 (m, 2H), 5.19-5.16 (m, 2H), 5.04 (s, 1H), 4.66 (d, 1H, J ) 11.8 Hz), 4.61 (d, 1H, J ) 11.8 Hz), 4.56 (d, 1H, J ) 11.8 Hz), 4.52 (d, 1H, J ) 11.8 Hz), 4.30 (ddd, 1H, J ) 7.0 Hz, 6.3 Hz, 3.7 Hz), 4.19 (m, 1H), 4.03-3.90 (m, 4H), 3.83 (s, 3H), 3.82 (s, 3H), 3.64 (dd, 1H, J ) 10.6 Hz, 3.6 Hz), 3.54 (dd, 1H, J ) 10.6 Hz, 6.1 Hz); 13C NMR (CDCl3, 125 MHz) δ 159.4, 159.1, 134.5, 134.2, 130.5, 129.9, 129.7, 129.3, 117.4, 117.3, 113.8, 113.7, 104.5, 80.5, 79.2, 78.5, 72.8, 71.9, 71.5, 71.2, 68.2, 55.3, 55.3; HRMS (ESI) [M + Na]+ requires 493.2197, found 493.2200. Allyl 3,5-Di-O-(4-methoxybenzyl)-2-O-allyl-β-D-ribofuranoside (19b). 19b was obtained from 18b (1.0 g, 3.3 mmol) as described for 19a. The product (19b) was a colorless oil in a 75% yield (1.2 g). 1H NMR (CDCl3, 500 MHz) δ 7.29-7.26 (m, 4H), 6.90-6.86 (m, 4H), 5.97-5.83 (m, 2H), 5.35-5.16 (m, 4H), 5.07 (s, 1H), 4.56 (d, 1H, J ) 11.6 Hz), 4.53 (d, 1H, J ) 11.7 Hz), 4.49 (d, 1H, J ) 11.7 Hz), 4.48 (d, 1H, J ) 11.6 Hz), 4.30 (ddd, 1H, J ) 6.3 Hz, 6.3 Hz, 3.9 Hz), 4.20 (m, 1H), 4.17-4.08 (m, 2H), 4.04 (dd, 1H, J ) 7.0 Hz, 4.7 Hz), 3.96 (m, 1H), 3.84 (d, 1H, J ) 4.8 Hz), 3.83 (s, 3H), 3.82 (s, 3H), 3.59 (dd, 1H, J ) 10.7 Hz, 3.8 Hz), 3.49 (dd, 1H, J ) 10.6 Hz, 6.0 Hz); 13C NMR (CDCl3, 125 MHz) δ 159.3, 159.1, 134.5, 134.2, 130.5, 130.0, 129.6, 129.3, 117.6, 117.3, 113.8, 113.7, 104.5, 80.5, 79.9, 78.2, 72.8, 72.1, 71.5, 71.1, 68.3, 55.3, 55.3; HRMS (ESI) [M + Na]+ requires 493.2197, found 493.2182. (2-Hydroxyethyl) 3-O-(2-hydroxyethyl)-2,5-di-O-(4-methoxybenzyl)- β-D-ribofuranoside (20a). Osmium tetroxide (2.5 wt. % solution in 2-methyl-2-propanol, 1.0 mL, 80 µmol) was added to a mixture of 19a (2.9 g, 6.1 mmol) in water-dioxane (1:4 V/V, 20 mL) (46). After 1 h stirring, the mixture was cooled to 0 °C, the solvent volume was increased to 80 mL, and NaIO4 (6.6 g, 31 mmol) was slowly added. The reaction was then stirred at ambient temperature for 5 h. The resulted aldehyde intermediate was extracted with ethyl acetate (3 × 20 mL). The organic phases were combined, dried with Na2SO4, and evaporated to dryness. The residue was dissolved in a mixture of dichloromethane-ethanol (1:2, V/V, 30 mL), and then NaBH4 (1.2 g, 31 mmol) was added. After 5 h stirring, the excess

Niittyma¨ki et al.

NaBH4 was destroyed by addition of saturated ammonium chloride (20 mL) and the product was extracted with dichloromethane (3 × 15 mL). The organic phases were combined, dried with Na2SO4, and evaporated to dryness. The residue was purified by silica gel chromatography (10% MeOH in dichloromethane) to yield 1.8 g (60%) of 20a as a colorless oil. 1H NMR (CDCl3, 500 MHz) δ 7.32-7.27 (m, 4H), 6.92-6.88 (m, 4H), 5.02 (s, 1H), 4.66 (d, 1H, J ) 11.5 Hz), 4.59 (d, 1H, J ) 11.9 Hz), 4.59 (d, 1H, J ) 11.5 Hz), 4.48 (d, 1H, J ) 11.9 Hz), 4.21 (m, 1H), 4.13 (dd, 1H, J ) 7.6 Hz, 4.6 Hz), 3.94 (d, 1H, J ) 4.6 Hz), 3.82 (s, 6H), 3.77-3.63 (m, 7H), 3.58 (m, 1H), 3.54-3.50 (m, 2H), 3.19 (dd, 1H, J ) 6.7 Hz, 5.7 Hz), 2.47 (t, 1H, J ) 6.2 Hz); 13C NMR (CDCl3, 125 MHz) δ 159.5, 159.4, 129.8, 129.6, 129.4, 113.9, 113.8, 105.5, 80.1, 79.6, 78.4, 72.9, 72.1, 71.9, 71.3, 68.9, 61.9, 61.7, 55.3, 55.3; HRMS (ESI) [M + Na]+ requires 501.2095, found 501.2087. (2-Hydroxyethyl) 2-O-(2-Hydroxyethyl)-3,5-di-O-(4-methoxybenzyl)- β-D-ribofuranoside (20b). 20b was synthesized from 19b (1.1 g, 2.4 mmol) as described for 20a. The product (20b) was obtained as a colorless oil in a 46% yield (0.52 g). 1H NMR (CDCl , 500 MHz) β 7.29-7.22 (m, 4H), 6.91-6.87 3 (m, 4H), 5.00 (s, 1H), 4.53 (d, 1H, J ) 11.8 Hz), 4.51 (d, 1H, J ) 11.2 Hz), 4.44 (d, 1H, J ) 11.2 Hz), 4.41 (d, 1H, J ) 11.8 Hz), 4.23 (dd, 1H, J ) 7.6 Hz, 4.3 Hz), 4.19 (m, 1H), 3.82 (s, 3H), 3.81 (s, 3H), 3.82-3.60 (m, 10H), 3.45 (dd, 1H, J ) 10.7 Hz, 3.6 Hz), 3.32 (t, 1H, J ) 6.5 Hz), 2.62 (t, 1H, J ) 6.2 Hz); 13C NMR (CDCl , 125 MHz) β 159.5, 159.3, 129.7, 129.7, 3 129.6, 129.5, 113.9, 113.8, 105.9, 81.4, 80.0, 77.1, 72.9, 72.5, 72.4, 71.5, 68.5, 61.9, 61.8, 55.3, 55.3; HRMS (ESI) [M + Na]+ requires 501.2095, found 501.2072. (2-Levulinoyloxyethyl) 3-O-(2-Levulinoyloxyethyl)-2,5-diO-(4-methoxybenzyl)- β-D-ribofuranoside (21a). Levulinic anhydride (0.90 g, 4.2 mmol) was added to a mixture of 20a (0.56 g, 1.2 mmol) in pyridine (10 mL). After 3 h stirring, saturated NaHCO3 (20 mL) was added to the mixture and then the product was extracted with ethyl acetate (3 × 20 mL). The organic phases were combined, dried with Na2SO4, and evaporated to dryness. The residue was purified by silica gel chromatography to yield 0.73 g (92%) of the product (21a) as a colorless oil. 1H NMR (CDCl3, 500 MHz) δ 7.33-7.26 (m, 4H), 6.91-6.87 (m, 4H), 5.02 (d, 1H, J ) 0.8 Hz), 4.66 (d, 1H, J ) 11.7 Hz), 4.62 (d, 1H, J ) 11.7 Hz), 4.55 (d, 1H, J ) 11.7 Hz), 4.51 (d, 1H, J ) 11.7 Hz), 4.27 (ddd, 1H, J ) 6.9 Hz, 5.9 Hz, 3.8 Hz), 4.23-4.19 (m, 3H), 4.10 (ddd, 1H, J ) 12.0 Hz, 7.0 Hz, 3.4 Hz), 3.98 (dd, 1H, J ) 7.0 Hz, 4.7 Hz), 3.92 (dd, 1H, J ) 4.7 Hz, 0.8 Hz), 3.84 (ddd, 1H, J ) 11.4 Hz, 5.7 Hz, 3.4 Hz), 3.82 (s, 3H), 3.82 (s, 3H), 3.70-3.57 (m, 4H), 3.53 (dd, 1H, J ) 10.7 Hz, 5.8 Hz), 2.76-2.70 (m, 4H), 2.602.53 (m, 4H), 2.20 (s, 3H), 2.18 (s, 3H); 13C NMR (CDCl3, 125 MHz) δ 206.5, 206.5, 172.7, 172.6, 159.4, 159.2, 130.3, 129.9, 129.5, 129.3, 113.8, 113.7, 105.2, 80.5, 79.6, 79.3, 72.8, 72.0, 71.0, 68.4, 65.2, 63.7, 63.5, 55.3, 55.3, 37.8, 37.8, 29.9, 29.8, 27.8, 27.8; HRMS (ESI) [M + Na]+ requires 697.2831, found 697.2860. (2-Levulinoyloxyethyl) 2-O-(2-Levulinoyloxyethyl)-di-O(4-methoxybenzyl)- β- D-ribofuranoside (21b). 21b was synthesized from 20b (0.50 g, 1.0 mmol) as described for 21a. The product (21b) was obtained as a colorless oil in an 81% yield (0.57 g). 1H NMR (CDCl3, 500 MHz) δ 7.29-7.24 (m, 4H), 6.90-6.86 (m, 4H), 5.03 (s, 1H), 4.56 (d, 1H, J ) 11.5 Hz), 4.51 (d, 1H, J ) 11.7 Hz), 4.48 (d, 1H, J ) 11.5 Hz), 4.47 (d, 1H, J ) 11.7 Hz), 4.29-4.20 (m, 4H), 4.10 (ddd, 1H, J ) 11.9 Hz, 7.1 Hz, 3.4 Hz), 4.04 (dd, 1H, J ) 7.1 Hz, 4.7 Hz), 3.86-3.79 (m, 4H), 3.82 (s, 3H), 3.82 (s, 3H), 3.61 (ddd, 1H, J ) 11.3 Hz, 7.0 Hz, 3.4 Hz), 3.59 (dd, 1H, J ) 10.6 Hz, 3.7 Hz), 3.48 (dd, 1H, J ) 10.6 Hz, 5.9 Hz), 2.76-2.73 (m, 4H), 2.60-2.54 (m, 4H), 2.20 (s, 3H), 2.19 (s, 3H); 13C NMR

Bioconjugate Chem., Vol. 18, No. 5, 2007 1587

Artificial Ribonucleases

(CDCl3, 125 MHz) δ 206.5, 206.5, 172.7, 172.6, 159.3, 159.2, 130.3, 130.0, 129.5, 129.3, 113.8, 113.7, 105.2, 81.1, 80.6, 78.2, 72.8, 72.2, 70.9, 68.5, 65.2, 63.8, 63.5, 55.3, 55.3, 37.9, 37.8, 29.9, 29.8, 27.9, 27.9; HRMS (ESI) [M + Na]+ requires 697.2831, found 697.2851. (2-Levulinoyloxyethyl) 5-O-(4,4′-Dimethoxytrityl)-3-O-(2levulinoyloxyethyl)- β-D-ribofuranoside (23a). Compound 21a (0.65 g, 0.96 mmol) was dissolved in ethanol (10 mL). Pd/C (10% Pd/C, 50% wet with water, 0.13 g) was added, and then hydrogen was bubbled through the mixture for 2 h at ambient temperature. The mixture was filtered, the filtrate was evaporated to dryness, and the residue was purified by silica gel chromatography to yield 0.37 g (88%) of the (2-levulinoyloxyethyl) 3-O-(2-levulinoyloxyethyl)-β-D-ribofuranoside (22) as a colorless oil. HRMS (ESI) [M + Na]+ requires 457.1680, found 457.1699. Compound 22 was dissolved in pyridine (5 mL), and then DMTrCl1 (0.29 g, 0.86 mmol) was slowly added. The reaction was stirred overnight at ambient temperature. Saturated NaHCO3 (20 mL) was added to the mixture, and the product was extracted with dichloromethane (3 × 20 mL). The organic phases were combined, dried with Na2SO4, and evaporated to dryness. The residue was purified by silica gel chromatography (80% EtOAc in petroleum ether) to yield 0.54 g (93%, 82% from 21a) of the product (23a) as a colorless oil. 1H NMR (CDCl3, 500 MHz) δ 7.50-7.47 (m, 2H), 7.38-7.35 (m, 4H), 7.32-7.29 (m, 3H), 7.23 (m, 1H), 6.86-6.83 (m, 4H), 5.03 (s, 1H), 4.27 (ddd, 1H, J ) 12.1 Hz, 5.1 Hz, 2.9 Hz), 4.20-4.15 (m, 4H), 4.13-4.08 (m, 2H), 3.83 (ddd, 1H, J ) 11.4 Hz, 5.2 Hz, 3.4 Hz), 3.81 (s, 6H), 3.72 (ddd, 1H, J ) 11.2 Hz, 5.2 Hz, 3.0 Hz), 3.68-3.60 (m, 2H), 3.32 (dd, 1H, J ) 10.0 Hz, 4.5 Hz), 3.19 (dd, 1H, J ) 10.0 Hz, 5.0 Hz), 3.00 (d, 1H, J ) 3.1 Hz), 2.77-2.74 (m, 2H), 2.71-2.69 (m, 2H), 2.57-2.54 (m, 2H), 2.52-2.49 (m, 2H), 2.20 (s, 3H), 2.18 (s, 3H); 13C NMR (CDCl3, 125 MHz) δ 206.7, 206.5, 172.6, 172.6, 158.5, 144.9, 136.1, 136.0, 130.0, 130.0, 128.2, 127.8, 126.8, 113.1, 107.4, 85.9, 80.6, 80.2, 73.4, 68.7, 65.5, 64.2, 63.5, 63.4, 55.2, 37.9, 37.9, 29.8, 29.8; HRMS (ESI) [M + Na]+ requires 759.2987, found 759.3007. (2-Levulinoyloxyethyl) 5-O-(4,4′-Dimethoxytrityl)-2-O-(2levulinoyloxyethyl)- β-D-ribofuranoside (23b). 23b was synthesized from 21b (0.55 g, 0.82 mmol) as described for 23a, except that the 2-levulinoyloxyethyl 2-O-(2-levulinoyloxyethyl)β-D-ribofuranoside intermediate was used without chromatographic purification. The product (23b) was obtained as a colorless oil in a 70% overall yield (0.42 g). 1H NMR (CDCl3, 500 MHz) δ 7.51-7.49 (m, 2H), 7.40-7.36 (m, 4H), 7.317.27 (m, 2H), 7.22 (m, 1H), 6.85-6.82 (m, 4H), 5.08 (d, 1H, J ) 1.4 Hz), 4.34-4.21 (m, 4H), 4.14-4.08 (m, 2H), 3.94 (ddd, 1H, J ) 11.4 Hz, 5.3 Hz, 3.4 Hz), 3.88 (ddd, 1H, J ) 11.3 Hz, 5.6 Hz, 3.3 Hz), 3.86 (dd, 1H, J ) 4.8 Hz, 1.4 Hz), 3.81 (s, 6H), 3.80 (ddd, 1H, J ) 11.3 Hz, 6.6 Hz, 3.4 Hz), 3.66 (ddd, 1H, J ) 11.4 Hz, 7.1 Hz, 3.3 Hz), 3.34 (dd, 1H, J ) 10.0 Hz, 3.6 Hz), 3.15 (dd, 1H, J ) 10.0 Hz, 5.5 Hz), 2.80-2.77 (m, 2H), 2.72 (d, 1H), J ) 8.4 Hz), 2.70-2.67 (m, 2H), 2.63-2.60 (m, 2H), 2.50-2.46 (m, 2H), 2.21 (s, 3H), 2.16 (s, 3H); 13C NMR (CDCl3, 125 MHz) δ 206.7, 206.5, 172.7, 172.6, 158.4, 145.0, 136.1, 136.1, 130.1, 130.1, 128.2, 127.8, 126.7, 113.1, 104.8, 85.9, 83.6, 83.1, 71.6, 68.9, 65.6, 64.7, 63.4, 55.2, 37.9, 37.8, 29.8, 29.8, 27.9, 27.8; HRMS (ESI) [M + Na]+ requires 759.2987, found 759.3015. 2-Cyanoethyl [(2-Levulinoyloxyethyl) 5-O-(4,4′-dimethoxytrityl)-3-O-(2-levulinoyloxyethyl)-β-D-ribofuranoside-2-yl] N,N-Diisopropylphosphoramidite (24). 2-Cyanoethyl N,N1 Abbreviations: All, allyl; CPG, controlled pore glass; DMAP, 4-dimethylaminopyridine; DMTr, 4,4′-dimethoxytrityl; HEPES, 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid; Lev, levulinoyl (4oxopentanoyl); PMB, 4-methoxybenzyl; Tfa, trifluoroacetyl.

Scheme 3

a

a Reagents and conditions: (i) methyl trifluoroacetate, NaOMe, MeOH, (ii) 2-cyanoethyl N,N-diisopropylphosphonamidic chloride, NEt3, dichloromethane.

diisopropylphosphonamidic chloride (91 µL, 0.41 mmol) was added to a mixture of 23a (0.25 g, 0.34 mmol) and triethylamine (0.24 mL, 1.7 mmol) in dichloromethane (3.0 mL) under nitrogen. The mixture was stirred for 1 h at ambient temperature and then subjected directly to a silica gel column. Elution with 1% triethylamine in EtOAc yielded 0.30 g (95%) of the product (24) as a colorless oil. 1H NMR (CDCl3, 500 MHz) δ 7.517.49 (m, 2H), 7.40-7.36 (m, 4H), 7.31-7.28 (m, 2H), 7.247.20 (m, 1H), 6.86-6.82 (m, 4H), 5.15 and 5.01 (s 0.48H and s 0.52H), 4.30-4.10 (m, 7H), 3.96-3.87 (m, 2H), 3.81 and 3.80 (s and s, 6H), 3.82-3.53 (m, 8H), 3.41-3.37 (m, 1H), 3.15-3.12 (m, 1H), 2.73-2.64 (m, 6H), 2.55-2.43 (m, 4H), 2.18 (s, 6H), 2.17 (s, 3H), 2.17 (s, 3H), 1.23-1.19 (m, 12H); 13C NMR (CDCl , 125 MHz) δ 206.5, 206.5, 172.6, 172.6, 3 158.4, 145.0, 145.0, 136.2, 136.1, 130.1, 130.1, 128.2, 128.2, 127.8, 126.7, 117.9, 117.7, 113.1, 106.5, 106.4, 85.7, 85.7, 80.3, 80.3, 79.2, 78.8, 75.0, 74.8, 73.5, 73.4, 68.5, 68.0, 65.5, 65.4, 63.9, 63.7, 63.6, 63.5, 63.4, 59.0, 58.9, 58.0, 57.8, 55.2, 43.4, 43.3, 43.2, 43.0, 37.8, 37.8, 29.8, 27.8, 27.8, 24.7, 24.6, 24.6, 24.5, 20.4, 20.4; 31P NMR (CDCl3, 200 MHz) δ 150.6, 149.8; HRMS (ESI) [M + H]+ requires 937.4246, found 937.4290. 2-Cyanoethyl [(2-Levulinoyloxyethyl) 5-O-(4,4′-dimethoxytrityl)-2-O-(2-levulinoyloxyethyl)-β-D-ribofuranoside-3-yl] N,N-Diisopropylphosphoramidite (25). 25 was synthesized from 23b (0.19 g, 0.26 mmol) as described for 24. The product (25) was obtained as a colorless oil in an 89% yield (0.24 g). 1H NMR (CDCl , 500 MHz) δ 7.54-7.50 (m, 2H), 7.41-7.30 3 (m, 6H), 7.27-7.22 (m, 1H), 6.91-6.88 (m, 4H), 5.05 (m, 1H), 4.43-4.10 (m, 7H), 3.95-3.52 (m, 10H), 3.79 and 3.79 (s and s, 6H), 3.39 (dd, 0.63H, J ) 10.4 Hz, 2.5 Hz), 3.29 (dd, 0.37H, J ) 10.2 Hz, 2.7 Hz), 3.09-3.05 (m, 1H), 2.77-2.74 (m, 2H), 2.69-2.66 (m, 3H), 2.55-2.52 (m, 2H), 2.49-2.46 (m, 1H), 2.44-2.35 (m, 2H), 2.15, 2.14 and 2.11 (each s, 6H), 1.17, 1.16, 1.15, 1.15, 1.14, 1.13, 1.00, 0.99 (each s, 12H); 13C NMR (CD3CN, 125 MHz) δ 206.8, 206.8, 206.7, 172.6, 172.6, 172.5, 172.5, 158.6, 145.3, 136.2, 136.1, 130.1, 130.1, 130.1, 128.1, 128.0, 127.8, 127.8, 126.8, 118.6, 118.3, 113.1, 113.0, 105.6, 105.2, 85.8, 85.7, 82.3, 82.3, 82.2, 82.2, 81.5, 81.5, 81.4, 73.0, 72.9, 72.3, 72.2, 68.7, 68.5, 65.4, 65.4, 64.3, 63.9, 63.7, 63.6, 63.1, 58.6, 58.4, 54.9, 54.9, 43.0, 43.0, 42.9, 42.9, 37.5, 37.4, 29.0, 28.9, 27.7, 27.7, 27.6, 24.1, 24.1, 24.0, 23.9, 23.9, 23.8, 20.0, 20.0, 19.9, 19.9; 31P NMR (CDCl3, 200 MHz) δ 149.6, 149.1; HRMS (ESI) [M + H]+ requires 937.4246, found 937.4283. 1,5,9-Tris(2,2,2-trifluoroacetyl)-3-(3-hydroxypropyl)-1,5,9triazacyclododecane (27). The 3-(3-hydroxypropyl)-1,5,9-triazacyclododecane trihydrochloride (26, Scheme 3) was synthesized as earlier described (42). Freshly distilled methyl trifluoroacetate (7.1 mL, 71 mmol) was added in two portions to a mixture of 26 (1.0 g, 3.0 mmol) and NaOMe (0.96 g, 18 mmol) in MeOH (10 mL). The reaction was stirred at ambient temperature for 48 h, and then ethyl acetate (200 mL) and 1 mol L-1 aqueous hydrogen chloride (20 mL) were added to the

1588 Bioconjugate Chem., Vol. 18, No. 5, 2007 Scheme 4

Niittyma¨ki et al.

a

a Reagents and conditions: (i) (1) hydrazine hydrate, pyridine, AcOH (0.124/4/1, v/v/v, 1h at 25 °C) (2) two consecutive standard phosphoramidite couplings with 28 (600 s coupling time, 0.1 mol L-1 solution of 28 in acetonitrile), (ii) aq NH3, 15 h at 55 °C.

mixture. The organic phase was separated, washed with saturated NaHCO3 and NaCl solutions, dried with Na2SO4, and evaporated to dryness. The residue was purified by silica gel chromatography (70% EtOAc in petroleum ether) to yield 0.98 g (64%) of the product (19) as white foam. 1H NMR (MeOD, 500 MHz) δ 4.10-3.15 (m, 14H), 2.64-1.92 (m, 5H), 1.72-1.28 (m, 4H); HRMS (ESI) [M + H]+ requires 518.1696, found 518.1717. 2-Cyanoethyl 3-[1,5,9-Tris(2,2,2-trifluoroacetyl)-1,5,9-triazacyclododecan-3-yl]propyl N,N-Diisopropylphosphoramidite (28). Cyanoethyl N,N-diisopropylphosphonamidic chloride (0.49 mL, 2.2 mmol) was added to a mixture of 27 (0.94 g, 1.8 mmol) and triethylamine (1.27 mL, 9.0 mmol) in dichloromethane (10 mL) under nitrogen. The mixture was stirred for 1 h at room temperature and then directly applied onto a silica gel column (0.4% triethylamine and 80% EtOAc in petroleum ether as an eluent). The chromatographic purification yielded 1.2 g (91%) of the product (28) as white foam. 1H NMR (CD3CN, 500 MHz) δ 4.05-3.00 (m, 18H), 2.65 (t, 2H, J ) 6.0 Hz), 2.50-1.90 (m, 5H), 1.75-1.25 (m, 4H), 1.19-1.15 (m, 12H); 31P NMR (CDCl3, 200 MHz) δ 148.0, 147.7, 147.6, 147.5, 147.4; HRMS (ESI) [M + Na]+ requires 740.2594, found 740.2594. Synthesis of the 2′-O-Methyl Oligoribonucleotide Conjugates (1a-3a, 1b-3b). The 2′-O-methyl oligoribonucleotide conjugates (1a-3a, 1b-3b, Chart 1) were assembled on an Applied Biosystems 3400 DNA synthesizer in 1.0 µmol scale using conventional phosphoramidite chemistry and following the standard RNA coupling protocol (Scheme 4). Coupling efficiency of branching units 24 and 25 was equal to that of the commercially available 2′-O-methylnucleoside phosphoramidites (0.1 mol L-1 solutions of 24 and 25 in acetonitrile, 600 s coupling time). Building block 1 was somewhat less reactive, requiring two consecutive coupling cycles (0.1 mol L-1 solutions of 1 in acetonitrile, 600 s coupling time, twice) and prolonged detritylation time [three consecutive standard detritylation treatments (3% DCA in dichloromethane)]. Once the 2′-Omethyl oligoribonucleotide chains were assembled to obtain

29a-31a and 29b-31b, the levulinoyl protections were manually removed by 0.5 mol L-1 hydrazine acetate solution (0.124/ 4/1 NH2NH2‚H2O/pyridine/AcOH V/V/V, 1 h at 25 °C). The deprotected resin-bound oligonucleotides were washed with pyridine, MeOH, and acetonitrile, dried, and reset to the DNA synthesizer. The azacrown phosphoramidite reagent (28) was then coupled using two consecutive standard couplings (0.1 mol L-1 solution of 4 in dry acetonitrile, 600 s coupling time, twice). Fully protected resin-bound 2′-O-methyl oligoribonucleotide conjugates (32a-34a, 32b-34b) were released from the support and deprotected by concentrated ammonia (15 h at 55 °C). The crude product mixtures were filtered, and the filtrates were evaporated to dryness, dissolved in water, and then subjected to RP HPLC purification. Purity of the crude conjugates (1a3a, 1b-3b) ranged from 20 to 30% (according to RP HPLC chromatograms). Isolated yields of the conjugates were 10 to 15% [according to UV absorbances of dissolved 1a-3a, 1b3b]. The authenticity of the conjugates was verified by MS(ESI) and MS(MALDI) spectroscopy. Three main multiply charged peaks in the MS(ESI) spectra are listed below. 1a: MS(ESI) [(M - nH)/n]-n, n ) 6, 7, and 8 requires 690.4, 789.1, and 920.8, found 690.2, 789.0, and 920.6, 2a: MS(ESI) {[M + Na - (n + 1)H]/n}-n, n ) 6, 7, and 8 requires 918.2, 786.8 and 688.4, found 920.0, 788.6 and 690.0, 3a: MS(ESI) [(M nH)/n]-n, n ) 7, 8, and 9 requires 783.4, 685.4, and 609.1, found 784.9, 686.4, and 610.3. 1b: MS(ESI) {[M + Na - (n + 1)H]/ n}-n, n ) 7, 8, and 9 requires 879.7, 769.6, and 684.0, found 881.3, 771.2, and 685.5, 2b: MS(ESI) [(M - nH)/n]-n, n ) 7, 8, and 9 requires 870.6, 761.6, and 676.9, found 870.4, 761.6, and 676.9, 3b: MS(MALDI) (M + H)+ requires 6102, found 6103. Kinetic Measurements. The reactions were carried out in Eppendorf tubes immersed in a water bath, and the temperature of which was maintained at 35.0 ( 0.1 °C. pH was adjusted to 7.3 with a HEPES buffer (0.1 mol L-1) prepared in sterilized water. The Zn2+ ion was added as a nitrate salt to give the overall metal ion concentration of 90 or 180 µmol L-1. The

Artificial Ribonucleases

ionic strength was adjusted to 0.1 mol L-1 with sodium nitrate. The concentration of the artificial nuclease (1a-3a, 1b-3b) was 9 or 18 µmol L-1 and that of the target (4-6) 18 or 36 µmol L-1. 4-Nitrobenzenesulfonate ion was used as an internal standard. The total volume of the reaction mixture was 200 µL in each kinetic run. Aliquots of 20 µL were withdrawn at suitable intervals and cooled immediately to 0 °C, and the reaction was quenched by adding aqueous hydrogen chloride (1.0 µL of 1.0 mol L-1 solution). This results in release of the Zn2+ ion from the azacrown ligand and markedly decelerates the reaction, since the metal ion-catalyzed cleavage of RNA is known to be first-order in hydroxide ion concentration at pH < 7 (47). After this, the composition of the samples was quantified by capillary zone elctrophoresis (Beckman Coulter P/ACE MDQ CE System) using a fused silica capillary (inner diameter 50 µm, effective length 50 cm). Inverted polarity, citrate buffer (0.2 mol L-1, pH 3.1) and -30 kV voltage were used. The temperature of the capillary was kept at 25 °C. Hydrodynamic injection at 2 psi for 8 s was applied. Between each analytical run, the capillary was flushed for 3 min with water, 10 mmol L-1 aqueous hydrogen chloride, and the background electrolyte buffer. The quantification of the target and product oligonucleotides was based on comparison of their UV absorption at 254 nm to that of the internal standard. The peak area was first normalized by dividing it by the migration time and then by the similarly normalized peak area of the internal standard. First-order rate constants for the cleavage of the target oligonucleotides (4-6) by the artificial nucleases (1a3a, 1b-3b) were obtained by applying the integrated first-order rate law to the disappearance of the target oligonucleotide. The site of cleavage within target 4 was determined by spiking with reference oligonucleotides 7-9. Each of these was added one after another to a sample containing the cleaved target and the sample was then subjected to capillary electrophoresis. Oligonucleotide 8 comigrated with the product obtained by cleaving 4 with either 1a or 2a, while addition of 7 or 9 resulted in appearance of a new peak in the electropherogram.

RESULTS AND DISCUSSION Synthesis of the Phosphoramidite Reagents (14, 24, 25, 28). Preparation of the artificial nucleases (1a-3a, 1b-3b) bearing two azacrown ligands attached to a single nonnucleosidic building block was based on standard solidsupported phosphoramidite chemistry. For this purpose, three different non-nucleosidic branching units were prepared and converted to 4,4′-dimethoxytritylated 2-cyanoethyl N,N-diisopropylphosphoramidite building blocks (14, 24, 25) as outlined in Schemes 1 and 2. In each building block, the two hydroxy functions aimed at allowing subsequent attachment of the azacrown ligands were protected as levulinic acid esters. As shown previously (43, 48, 49), this group may be conveniently removed on a solid-support by brief treatment with hydrazinium acetate in pyridine. The precursor of the bis(hydroxymethyl)malondiamidederived building-block, N,N′-bis(3-hydroxypropyl)-2-methoxy1,3-dioxane-5,5-dicarboxamide (10 in Scheme 1), was synthesized as described earlier (43). The hydroxypropyl arms of this precursor were esterified with levulinic anhydride (11) and the orthoester protection was removed (12). One of the exposed hydroxy groups was then protected as a DMTr ether (13), and the other one was phosphitylated to obtain 14. The ribose-based branching units (24, 25) were prepared from commercially available 1,2,3,5-tetra-O-acetyl-β-D-ribofuranose (15 in Scheme 2). A mixture of allyl 2,5- and 3,5-di-O-acetylβ-D-ribofuranosides (16a and 16b), together with allyl 2,3,5tri-O-acetyl-β-D-ribofuranoside, was obtained in one step by BF3 promoted glycosidation with allylic alcohol (44). The exposed

Bioconjugate Chem., Vol. 18, No. 5, 2007 1589

hydroxyl group was then allylated under neutral condition with allyl methyl carbonate in the presence of Pd(OAc)2 and triphenylphosphine (45). After this step, the isomers, Viz. allyl 2,5-di-O-acetyl-3-O-allyl-β-D-ribofuranoside (18a) and allyl 3,5di-O-acetyl-2-O-allyl-β-D-ribofuranoside (18b), were separated, and the base-labile acetyl protections were removed and replaced with 4-methoxybenzyl (PMB) groups (19a,b). OsO4 hydroxylation and concomitant NaIO4 oxidation converted the allyl groups to 2-oxoethyl groups, which were subsequently reduced to 2-hydroxyethyl groups with NaBH4 (20a,b) (50). The hydroxyl groups were esterified with levulinic anhydride (21a,b), the PMB protections were removed by Pd/C-catalyzed hydrogenolysis, and the 5-hydroxy group was protected with a DMTr-group (23a,b). Phosphitylation of the remaining secondary hydroxy function gave 24 and 25. Previously prepared (42) 3-(3-hydroxypropyl)-1,5,9-triazacyclododecane (26) was converted to trifluoroacetyl-protected phosphoramidite reagent (28) as outlined in Scheme 3, i.e., by acylation of the nitrogen atoms with methyl trifluoroacetate in the presence sodium methoxide (27) and subsequent phosphitylation of the hydroxypropyl tether (28). Synthesis of the Artificial Ribonucleases (1a-3a, 1b-3b). To verify the applicability of the branching building blocks (14, 24, 25) to the standard automated oligoribonucleotide synthesis, the coupling of these blocks was first followed by DMTr-cation assay and RP HPLC analysis of the released oligonucleotide aliquots. The coupling efficiency of the ribose-based building blocks (24, 25) turned out to be equal to that of the commercially available 2′-O-methylribonucleoside building blocks: the coupling was quantitative in 600 s (standard RNA-coupling protocol, 0.1 mol L-1 phosphoramidite solution used). By contrast, two consecutive coupling cycles (2 × 600 s, 0.1 mol L-1 solution of 14 in acetonitrile) had to be used with the malondiamide-derived block (14) to achieve a satisfactory 95% coupling. For this building block, a prolonged detritylation time (three consecutive standard detritylation cycles) was also needed, consistent with previous observations (43, 51, 52). These exceptions apart, the 2′-O-methyloligoribonucleotides incorporating one of the branching units (14, 24, or 25) close to the 3′-terminus (29a-31a) or in the middle of the chain (29b31b) were assembled in 1 µmol scale, using conventional phosphoramidite chemistry and following the standard RNA coupling protocol (Scheme 4). After chain assembly, the levulinoyl protections were removed by 1 h treatment with 0.5 mol L-1 hydrazinium acetate in pyridine and then the trifluoroacetyl-protected azacrown phosphoramidite (28) was coupled. Two consecutive coupling cycles (2 × 600 s, 0.1 mol L-1 solution of 28 in acetonitrile) were used for this simultaneous phosphorylation of the two hydroxyalkyl branches to obtain the support-anchored conjugates 32a-34a and 32b-34b. These fully protected conjugates were released into solution and deprotected by a slightly prolonged ammonolytic treatment (concd aq NH3, 15 h at 55 °C) and purified by RP HPLC (Figure 1). Purity of the crude conjugates (1a-3a, 1b-3b) ranged from 20 to 30%. The isolated yields varied from 10 to 15%. Authenticity of the conjugates was verified by MS(ESI) and MS(MALDI) spectroscopy. Cleavage by the 3′-Tethered Conjugates (1a-3a). The cleaving activity of conjugates 1a-3a, bearing the two azacrown moieties close to the 3′-terminus of a 15-mer 2′-O-methyl oligoribonucleotide, was determined by using a 20-mer chimeric ribo/2′-O-methylribo oligonucleotide (4) as a target. Within this target, the 3′-terminus consisted of 9 2′-O-methylribonucleotides, ensuring efficient hybridization with the fully complementary artificial nuclease, and the 11 non-methylated nucleotides at the 5′-terminus constituted the actual target sequence incorporating scissile ribonucleoside 3′-phosphodiester bonds. The artificial

1590 Bioconjugate Chem., Vol. 18, No. 5, 2007

Niittyma¨ki et al. Chart 2. Structures of Previously (40) Studied Monofunctionalized Azacrown Conjugates of 2′-O-methyl Oligoribonucleotides

Figure 1. Cleavage of chimeric ribo/2′-O-methylribo oligonucleotide 4 by the Zn2+ complex of artificial nuclease 1a in HEPES buffer (0.1 mol L-1, I ) 0.1 mol L-1 with NaNO3) at pH 7.3 and 35 °C. The concentration of 4 and the initial concentration of 1a (co) was 18 µmol L-1 and that of Zn2+ 90 µmol L-1.

Table 1. First-order Rate Constants for the Cleavage of Target 4 by the Zn2+ Complexes of Oligonucleotide Di(azacrown) Conjugates 1a-3a and Targets 5 and 6 with Conjugates 1b-3b in 0.10 mol L-1 HEPES Buffer at pH 7.3 and 35 °C (I ) 0.1 mol L-1 with NaNO3) cleaving c(cleaving agent), c(target), c(Zn2+), agent target µmol L-1 µmol L-1 µmol L-1 1a 2a 3a 1a 2a 1a 2a 1b 2b 3b 1b 2b 3b

Figure 2. Cleavage of chimeric ribo/2′-O-methylribo oligonucleotide 4 by the Zn2+ complex of artificial nucleases 1a-3a, using the target and cleaving agent at equimolar concentration. The logarithmic concentration of 4 plotted against time. For the conditions, see the legend of Figure 1. Notation: 1a (9), 2a (b), 3a (2).

nuclease and target were incubated at equimolar concentration (18 µmol L-1) and 35 °C in a HEPES buffer (0.1 mol L-1, pH 7.3, I ) 0.1 mol L-1 with NaNO3) containing 90 µmol L-1 Zn(NO3)2. Aliquots were withdrawn at appropriate intervals, and their composition was determined by zone capillary electrophoresis. The progress of the cleavage of 4 by nuclease 1a is shown in Figure 1 as an illustrative example. As seen from Figure 2, the breakdown of target 4 obeyed first-order kinetics with all the cleaving agents studied (1a-3a). Spiking of the product mixture with oligonucleotides 7-9 revealed that 1a-3a all cleaved the target at the 5′-C(6)pA(7)-3′ bond, i.e., at 5′-side of the nucleoside (A7) opposite to the non-nucleosidic unit bearing the azacrown ligands. Interestingly, the catalytic activity of 1a-3a differs considerably in spite of structural similarity of the cleaving agents. We have shown previously (40) that monofunctionalized oligonucleotide conjugate 35 (Chart 2), having the same base sequence as 1a-3a, cleaves target 4 at the 5′-A(7)pG(8)-3′ bond, the first-order rate constant being (1.4 ( 0.1) × 10-6 s-1 under the experimental conditions of the present study. The rate constants obtained now with 1a, 2a, and 3a are (5.9 ( 0.3) × 10-6 s-1, (2.08 ( 0.05) × 10-5 s-1, and (2.5 ( 0.3) × 10-6

4 4 4 4 4 4 4 5 5 5 6 6 6

18 18 18 18 18 9 9 18 18 18 18 18 18

18 18 18 18 18 36 36 18 18 18 18 18 18

90 90 90 180 180 90 90 90 90 90 90 90 90

k, 10-6 s-1 5.9 ( 0.3 20.8 ( 0.5 2.5 ( 0.3 6.3 ( 0.3 20.7 ( 0.9 0.27 ( 0.01 0.76 ( 0.08 0.9 ( 0.3 1.7 ( 0.1 1.1 ( 0.2 1.2 ( 0.1 1.7 ( 0.1 1.0 ( 0.1

s-1, respectively (Table 1). In other words, all the di(azacrown) conjugates are better catalysts than their mono(azacrown) counterpart, the increase in the catalytic activity being most remarkable with 2a, 15-fold compared to 35. The cleaving ability of 2a is equal to that reported (39) for 2,9-dimethyl-5aminophenanthroline-oligonucloeotide conjugates. Compared to the Zn2+ chelate of monomeric 1,5,9-triazacyclododecane at the same concentration (18 µmol L-1), the rate acceleration is 1000-fold (53). The effect of metal ion concentration on the cleavage rate was studied with conjugates 1a and 2a. The concentration of Zn2+ was increased to 180 µmol L-1 keeping the other conditions unchanged. The rate constants obtained were within the limits of experimental errors equal to those obtained at [Zn2+] ) 90 µmol L-1 (Table 1), suggesting that the binding of Zn2+ to 1a and 2a is virtually quantitative. The reaction shows turnover in spite of the fact that the cleavage site is outside the complementary region between the cleaving agent and the target. As seen from Figure 3, the breakdown of target 4 at least roughly obeys first-order kinetics when present in 4-fold excess compared to the cleaving agent. Artificial ribonucleases 1b and 2b, incorporating two azacrown ligands in the middle of the chain, cleaved targets 5 and 6, forming upon hybridization a 3- and 5-nucleotide bulge, respectively, opposite to the azacrown moieties (Table 1). The cleavage was, however, less efficient than with 1a-3a. Again the cleaving agent derived from the 1-O,3-O-funtionalized ribofuranosyl building block (2b) was most efficient, but the catalytic activity was more than 1 order of magnitude lower than with its 3′-terminal counterpart, 2a. In fact, the catalytic

Artificial Ribonucleases

Bioconjugate Chem., Vol. 18, No. 5, 2007 1591 Supporting Information Available: Experimental details and spectral data for phosphoramidites 14, 24, 25, and 28, and the precursor glycosides 23a,b, and the RP HPLC data for the 2′O-methyl oligoriboucleotide conjugates 1a-3a and 1b-3b. The material is available free of charge via the Internet at http:// pubs.acs.org.

LITERATURE CITED

Figure 3. Cleavage of chimeric ribo/2′-O-methylribo oligonucleotide 4 by the Zn2+ complex of artificial nucleases 1a and 2a on using the target in 4-fold excess compared to the cleaving agent. The logarithmic concentration of 4 plotted against time. For the conditions, see Table 1. Notation: 1a (9), 2a (b).

activity was only double compared to that of the monofunctionalized azacrown conjugate 36 (40). The other two intrachain conjugates, 1b and 3b, cleaved 5 and 6 approximately as readily as the monofunctionalized 36. The origin of the high catalytic activity of the 1-O,3-Otethered di(azacrown) conjugate (2a) compared to its 1-O,2-Otethered isomer (3a) or mono(azacrown) counterpart (35) remains obscure. Either the two azacrown moieties of 2a act in a cooperative manner, or the orientation of the 3-O-tethered group is optimal for the cleavage. Of these two alternative explanations, the latter appears more attractive. Cleavage of dinucleoside-3′,5′-monophosphates by a number of dinuclear Zn2+ bis(azacrown) chelates has been previously studied (54). Enhanced catalytic activity over the mononuclear Zn2+ chelate is observed only when one of the chelate moieties anchors the cleaving agent to a neighboring uracil base and, hence, brings the other chelate moiety in the proximity of the phosphodiester bond. This kind of rate-acceleration by additional anchoring to a base moiety in the vicinity of the scissile bond cannot be strictly excluded in the present case, but for the following reasons it appears less probable. First, there is no uracil base in the 5′-terminal ribo sequence of target 4 that would allow efficient anchoring (55). Second, the di(azacrown) unit in 2a is so flexible that binding of one of the chelate moieties to any base or phosphate moiety near the cleavage site hardly increases the local concentration of the catalytic chelate moiety much more than already reached by hybridization. In summary, the present study shows that tethering of two azacrown ligands to the 3′-terminus of a 2′-O-methyl oligoribonucleotide increases the catalytic efficiency by more than 1 order of magnitude compared to the corresponding monoazacrown-derivatized oligonucleotide. Half-lives shorter than 10 h are achieved at equimolar concentration of the target and cleaving agent under physiological conditions. The cleavage shows turnover. The efficiency of the cleaving agent, however, depends on the site of attachment of the azacrown moieties, in spite of marked overall flexibility of such constructs. Di(azacrown)-functionalized intrachain conjugates also cleave their bulged complementary sequences, but the catalytic activity is not much higher than that of their mono(azacrown) counterparts.

ACKNOWLEDGMENT The financial support from the Academy of Finland is gratefully acknowledged.

(1) Niittyma¨ki, T., and Lo¨nnberg, H. (2006) Artificial ribonucleases. Org. Biomol. Chem. 4, 15-25. (2) Morrow, J. R., and Iranzo, O. (2004) Synthetic metallonucleases for RNA cleavage. 8, 192-200. (3) Ha¨ner, R. (2001) Artificial ribonucleases. Chimia 55, 1035-1037. (4) Cowan, J. A. (2001) Chemical nucleases. Curr. Opin. Chem. Biol. 5, 634-642. (5) Komiyama, M., Sumaoka, J., Kuzuya, A., and Yamamoto, Y. (2001) Sequence-selective artificial ribonucleases. Methods Enzymol. 341, 455-468. (6) Travick, B. N., Daniher, A. T., and Bashkin, J. K. (1998) Inorganic mimics of ribonucleases and ribozymes: from random cleavage to sequence-specific chemistry to catalytic antisense drugs. Chem. ReV. 98, 939-960. (7) Whitney, A., Gavory, G., and Balasubramanian, S. (2003) Sitespecific cleavage of human telomerase RNA using PNA-neocuproine‚ Zn(II) derivatives. Chem. Commun., 36-37. (8) Kuzuya, A., Mizoguchi, R., Morisawa, F., and Komiyama, M. (2003) Site-selective RNA scission at two sites for precise genotyping of SNPs by mass spectrometry. Chem. Commun., 770-771. (9) Canaple, L., Hu¨sken, D., and Ha¨ner, R. (2002) Artificial ribonucleases: efficient and specific in vitro cleavage of human c-raf-1 RNA. Bioconjugate Chem. 13, 945-951. (10) Beloglazova, N. G., Fabani, M. M., Zenkova, M. A., Bichenkova, E. V., Polushin, N. N., Silnikov, V. V., Douglas, K. T., and Vlassov, V. V. (2004) Sequence-specific artificial ribonucleases. I. Bisimidazole-containing oligonucleotide conjugates prepared using precursor-based strategy. Nucleic Acids Res. 32, 3887-3897. (11) Mironova, N. L., Pyshnyi, D. V., Ivanova, E. M., Zarytova, V. F., Zenkova, M. A., Gross, H. J., and Vlassov, V. V. (2002) Sequence-specific RNA cleavage by oligonucleotide-peptide conjugates. Russ. Chem. Bull. 51, 1177-1186. (12) Sasaki, S., and Nagatsugi, F. (2006) Application of unnatural oligonucleotides to chemical modification of gene expression. Curr. Opin. Chem. Biol. 10, 615-621. (13) Wilson, C., and Keefe, A. D. (2006) Building oligonucleotide therapeutics using non-natural chemistries. Curr. Opin. Chem. Biol. 10, 607-614. (14) Crooke, S. T. (2004) Progress in antisense technology. Annu. ReV. Med. 55, 61-95. (15) Crooke, S. T. (2004) Antisense strategies. Curr. Mol. Med. 4, 465-487. (16) Manoharan, M. (2002) Oligonucleotide conjugates as potential antisense drugs with improved uptake, biodistribution, targeted delivery, and mechanism of action. Antisense Nucleic Acid Drug DeV. 12, 103-128. (17) Bumcrot, D., Manoharan, M., Koteliansky, V., and Sah, D. W. Y. (2006) RNAi therapeutics: a potential new class of pharmaceutical drugs. Nat. Chem. Biol. 2, 711-719. (18) Dykxhoorn, D. M., Palliser, D., and Lieberman, J. (2006) The silent treatment: siRNA as small molecule drugs. Gene Ther. 13, 541-552. (19) Leung, R. K. M., and Whittaker, P. A. (2005) RNA interference: from gene silencing to gene-specific therapeutics. Pharm. Ther. 107, 222-239. (20) Mittal, V. (2004) Improving the efficiency of RNA interference in mammals. Nat. ReV. Gen. 5, 355-365. (21) Dorsett, Y., and Tuschl, T. (2004) siRNA: applications in functional genomics and potential as therapeutics. Nat. ReV. Drug DiscoVery 3, 318-329. (22) Gnaccarini, C., Peter, S., Scheffer, U., Vonhoff, S., Klussmann, S., and Go¨bel, M. W. (2006) Site-specific cleavage of RNA by a metal-free artificial nuclease attached to antisense oligonucleotides. J. Am. Chem. Soc. 128, 8063-8067.

1592 Bioconjugate Chem., Vol. 18, No. 5, 2007 (23) Riguet, E., Tripathi, S., Chaubey, B., Desire, J., Pandey, V. N., and Decout, J.-L. (2004) A peptide nucleic acidsneamine conjugate that targets and cleaves HIV-1 TAR RNA inhibits viral replication. J. Med. Chem. 47, 4806-4809. (24) Verheijen, J. C., Deiman, B. A. L. M., Yeheskiely, E., van der marel, G. A., and van Boom, J. H. (2000) Efficient hydrolysis of RNA by a PNA-diethylenetriamine adduct. Angew. Chem., Int. Ed. 39, 369-372. (25) Matsumura, K., Endo, M., and Komiyama, M. (1994) Lanthanide complex-oligo-DNA hybrid for sequence-selective hydrolysis of RNA. J. Chem. Soc., Chem. Commun. 2019-2020. (26) Hall, J., Hu¨sken, D., Pieles, U., Moser, H. E., and Ha¨ner, R. (1994) Efficient sequence-specific cleavage of RNA using novel europium complexes conjugated to oligonucleotides. Chem. Biol. 1, 185-190. (27) Magda, D., Miller, R. A., Sessler, J. L., and Iverson, B. L. (1994) Site-specific hydrolysis of RNA by europium(III) texapyrin conjugated to a synthetic oligodeoxyribonucleotide. J. Am. Chem. Soc. 116, 7439-7440. (28) Magda, D., Crofts, S., Lin, A., Miles, M., Wright, M., and Sessler, J. L. (1997) Synthesis and Kinetic Properties of Ribozyme Analogues Prepared Using Phosphoramidite Derivatives of Dysprosium(III) Texaphyrin. J. Am. Chem. Soc. 119, 2293-2294. (29) Magda, D., Wright, M., Crofts, S., Lin, A., and Sessler, J. L. (1997) Metal complex conjugates of antisense DNA which display ribozyme-like activity. J. Am. Chem. Soc. 119, 6947-6948. (30) Hall, J., Hu¨sken, D., and Ha¨ner, R. (1996) Towards artificial ribonucleases: the sequence specific cleavage of RNA in a duplex. Nucleic Acids Res. 24, 3522-3526. (31) Ha¨ner, R., and Hall, J. (1997) The sequence-specific cleavage of RNA by artificial chemical ribonucleases. Antisense Nucleic Acid Drug DeV. 7, 423-430. (32) Bashkin, J. K., Frolova, E. I., and Sampath, U. (1994) Sequencespecific cleavage of HIV mRNA by a ribozyme mimic. J. Am. Chem. Soc. 116, 5981-5982. (33) Trawick, B. N., Osiek, T. A., and Bashkin, J. K. (2001) Enhancing sequence-specific cleavage of RNA within a duplex region: incorporation of 1,3-propanediol linkers into oligonucleotide conjugates of serinol-terpyridine. Bioconjugate Chem. 12, 900-905. (34) Putnam, W. C., Daniher, A. T., Trawick, B. N., and Bashkin, J. K. (2001) Efficient new ribozyme mimics: direct mapping of molecular design principles from small molecules to macromolecular, biomimetic catalysis. Nucleic Acids Res. 29, 2199-2204. (35) Sakamoto, A., Tamura, T., Furukawa, T., Komatsu, Y., Ohtsuka, E., Kitamura, M., and Inoue, H. (2003) Highly efficient catalytic RNA cleavage by the cooperative action of two Cu(II) complexes embodied within an antisense oligonucleotide. Nucleic Acids Res. 31, 1416-1425. (36) Inoue, H., Furukawa, T., Shimizu, M., Tamura, T., Matsui, M., and Ohtsuka, E. (1999) Efficient site-specific cleavage of RNA using a terpyridine-copper(II) complex joined to a 2′-O-methyloligonucleotide by a non-flexible linker. Chem. Commun., 45-46. (37) Hovinen, J., Guzaev, A., Azhayeva, E., Azhayev, A., and Lo¨nnberg, H. (1995) Imidazole tethered oligodeoxyribonucleotides: synthesis and RNA cleaving activity. J. Org. Chem. 60, 2205-2209. (38) Matsuda, S., Ishikubo, A., Kuzuya, A., Yashiro, M., and Komiyama, M. (1998) Conjugates of a dinuclear zinc(II) complexes and DNA oligomers as novel sequence-selective artificial ribonucleases. Angew. Chem., Int. Ed. 37, 3284-3286. (39) Åstro¨m, H., Williams, N. H., and Stro¨mberg, R. (2003) Oligonucleotide based artificial nuclease (OBAN) systems. Bulge size

Niittyma¨ki et al. dependence and positioning of catalytic group in cleavage of RNAbulges. Org. Biomol. Chem. 1, 1461-1465. (40) Niittyma¨ki, T., and Lo¨nnberg, H. (2004) Sequence-selective cleavage of oligoribonucleotides by 3d transition metal complexes of 1,5,9-triazacyclododecane-functionalized 2′-O-methyl oligoribonucleotides. Bioconjugate Chem. 15, 1275-1280. (41) Åstro¨m, H., and Stro¨mberg, R. (2004) Synthesis of new OBAN’s and further studies on positioning of the catalytic group. Org. Biomol. Chem. 2, 1901-1907. (42) Niittyma¨ki, T., Kaukinen, U., Virta, P., Mikkola, S., and Lo¨nnberg, H. (2004) Preparation of azacrown-functionalized 2′-O-methyl oligoribonucleotides, potential artificial RNases. Bioconjugate Chem. 15, 174-184. (43) Katajisto, J., Virta, P., and Lo¨nnberg, H. (2004) Solid-phase synthesis of multiantennary oligonucleotide glycoconjugates utilizing on-support oximation. Bioconjugate Chem. 15, 890-896. (44) Liu, M.-Z., Fan, H.-N., Guo, Z.-W., and Hui, Y.-Z. (1996) Onestep glycosylation and selective deprotection of peracetylated monosaccharides for facile syntheses of allyl glycosides with a free C-2 hydroxyl group. Carbohydrate Res. 290, 233-237. (45) Guibe, F., and Saint M’Leux, Y. (1981) The allyloxycarbonyl group for alcohol protection: quantitative removal or transformation into allyl protecting group via π-allyl complexes of palladium. Tetrahedron Lett. 22, 3591-3594. (46) Buskas, T., So¨derberg, E., Konradson, P., and Fraser-Reid, B. (2000) Use of n-pentenyl glycosides as precursors to various spacer functionalities. J. Org. Chem. 65, 958-963. (47) Kuusela, S., and Lo¨nnberg, H. (1994) Metal-ion-promoted hydrolysis of polyuridylic acid. J. Chem. Soc., Perkin Trans. 2, 23012306. (48) Guzaev, A., Azhayeva, E., Hovinen, J., Azhayev, A., and Lo¨nnberg, H. (1994) Synthesis and primer properties of oligonucleotides containing 3′-deoxypsicothymidine units labeled with fluorescein at the 1′-position. Bioconjugate Chem. 5, 501-503. (49) Azhayev, A., Guzaev, A., Hovinen, J., Azhayeva, E., and Lo¨nnberg, H. (1993) Analogues of oligonucleotides containing 3′deoxy-β-D-psicothymidine. Tetrahedron Lett. 34, 6435-6438. (50) Jin, S., Miduturu, C. V., McKinney, D. C., and Silverman, S. K. (2005) Synthesis of amine- and thiol-modified nucleoside phosphoramidites for site-specific introduction of biophysical probes into RNA. J. Org. Chem. 70, 4284-4299. (51) Katajisto, J., Heinonen, P., and Lo¨nnberg, H. (2004) Solid-phase synthesis of oligonucleotide glycoconjugates bearing three different glycosyl groups: orthogonally protected bis(hydroxymethyl)-N,Nbis(3-hydroxypropyl)malondiamide phosphoramidite as key building block. J. Org. Chem. 69, 7609-76154. (52) Guzaev, A., Salo, H., Azhayev, A., and Lo¨nnberg, H. (1996) Novel non-nucleosidic building blocks for the preparation of multilabeled oligonucleotides. Bioconjugate Chem. 7, 240-248. (53) Kaukinen, U., Bielecki, L., Mikkola, S., Adamiak, R. W., and Lo¨nnberg, H. (2001) The cleavage of phosphodiester bonds within small RNA bulges in the presence and absence of metal ion catalysts. J. Chem. Soc., Perkin Trans. 2, 1024-1031. (54) Wang, Q., and Lo¨nnberg, H. (2006) Simultaneous interaction with base and phosphate moieties modulates the phosphodiester cleavage of dinucleoside-3′,5′-monophosphates by dinuclear Zn2+ complexes of di(azacrown) ligands. J. Am. Chem. Soc. 128, 10716-10728. (55) Aoki, S., and Kimura, E. (2004) Zinc-nucleic acid interaction. Chem. ReV. 104, 769-787. BC070071O