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Synthesis and properties of RNA analogs- oligoribonucleotide N3[prime]->P5[prime] phosphoramidates
Introduction
Materials And Methods
General methods
Synthesis of nucleoside monomers
Results And Discussion
Preparation of monomers
Synthesis of oligoribonucleotide N3[prime]->P5[prime] phosphoramidates
Hydrolytic stability of oligoribonucleotide N3[prime]->N5[prime] phosphoramidate toward RNases T1 and A
Thermal stabilities of oligoribophosphoramidate duplexes
References
Synthesis and properties of RNA analogs- oligoribonucleotide N3[prime]->P5[prime] phosphoramidates
Received July 21, 1999; Revised and Accepted August 23, 1999
ABSTRACT The synthesis and characterization of RNA mimetics, uniformly modified oligoribonucleotide N3[prime]->P5[prime] phosphoramidates containing all four natural bases (uracil, cytosine, adenine and guanine) as well as thymidine and 2,6-diaminopurine, are described. These RNA analogs contain N3[prime]->P5[prime] phosphoramidate internucleotide linkages which replaced natural RNA O3[prime]->P5[prime] phosphodiester groups. These oligonucleotides were constructed from novel monomeric units (2[prime]-t-butyldimethylsilyl)-3[prime]-(monomethoxyltrityl)-aminonucleoside-5[prime]-phosphoramidites, the preparation of which is also presented. Several mixed base 9-13mer oligoribonucleotide phosphoramidates were synthesized with step-wise coupling yields of 96-98%. Thermal denaturation experiments demonstrated that ribo-N3[prime]->P5[prime] phosphoramidates form stable duplexes with a complementary RNA strand. Thus, the melting temperature (Tm) of a duplex formed by a 13mer ribo-N3[prime]->P5[prime] phosphoramidate (84°C) was higher than that observed for the isosequential natural RNA oligomer (64.0°C), or for the 2[prime]-deoxy-N3[prime]->P5[prime] phosphoramidate counterpart (71.7°C). Moreover, substitution of adenine by 2,6-diaminopurine in an oligoribophosphoramidate pentamer resulted in a very significant increase in the duplex melting temperature (~7°C per base substitution). The RNA phosphoramidates also showed similar rates of hydrolysis by both RNase A and RNase T1 as compared to natural RNA oligomers. The data presented indicate that this class of RNA analogs may be used as structural and functional RNA mimetics.
INTRODUCTION
The versatility and biological importance of RNA molecules have made them the subject of numerous research endeavors. The ability of RNA molecules to fold into well-defined secondary structures imparts some RNA compounds with enzyme-like qualities, including phosphodiester backbone cleavage and ligation capabilities (1-4). The RNA cleaving properties of synthetic or endogenously expressed ribozymes have presented a great opportunity for the development of RNA-based therapeutic agents. RNA oligomers can also be selected to fold into special structures, which specifically recognize various proteins or nucleotide triphosphates (5,6).
Work has been directed at preparing a multitude of hydrolytically more stable RNA analogs for a variety of purposes ranging from biological probes to potential therapeutic and diagnostic applications. Many phosphodiester and phosphorothioate RNA derivatives with a host of 2[prime]-modifications have been synthesized with the goal of increasing the hydrolytic stability and hybridization properties of RNA (7-9). Additionally, several pyranosyl-RNA analogs have been successfully prepared by Eschenmoser's group to investigate the `chemical etiology' of nucleic acid structures (10 and references therein).
Other efforts have focused on finding small molecules which can bind to specific sequences of viral RNA and thus disrupt viral replication cycles. Compounds such as the aminoglycoside neomycin B and 2,4,5,6-tetraaminoquinozaline interact with HIV-1 RNA and inhibit the binding of a replication control protein (Tat) to a stem-loop RNA structure (TAR) within the viral RNA (11-13).
Oligodeoxynucleotides with internucleotide N3[prime]->P5[prime] phosphoramidate linkages were recently described (14). These biopolymers are resistant to hydrolysis with nucleases, but they maintain the ability to form stable complexes with both RNA and DNA complementary strands (15). To determine if these desirable characteristics could be imparted to RNA compounds, model homopolymers oligothymidylate and oligoadenylate N3[prime]->P5[prime] ribophosphoramidates were prepared (16). While displaying similar resistance to a phosphodiesterase, the decathymidilate RNA phosphoramidate showed enhanced binding to poly(dA), poly(A), and to oligo(dA):oligo(dT) duplex relative to the 2[prime]-deoxyphosphoramidate counterpart.
Preparation of self-complementary ribo-CG-containing dinucleotides with N3[prime]->P5[prime] phosphoramidate linkages and their self-polymerization into short oligomers was previously described and studied in connection with the evolution and self-replication of nucleic acid polymers (17,18). Also, di- and trinucleotide ribophosphoramidates were prepared in aqueous solutions from 3[prime]-amino-2[prime]-hydroxy-5[prime]-nucleotides (19), and were subsequently used for studying enzymatic aminoacylation processes (20).
In light of these initial literature reports on the interesting properties of this class of compounds, we undertook the preparation of oligo-N3[prime]->P5[prime] ribophosphoramidates containing all major naturally occurring bases, and subsequently investigated both their nucleic acid hybridization properties as well as their susceptibility to hydrolysis by RNases A and T1.
MATERIALS AND METHODS
General methods
1H and 31P NMR spectra were obtained on a Varian 400 MHz spectrometer. 31P NMR spectra were referenced against 85% aqueous phosphoric acid. Anion exchange HPLC was performed using a Dionex DX 500 Chromatography System, with a Pharmacia Biotech Mono Q HR 5/5 or 10/16 ion exchange column. Mass spectral analyses were performed by Mass Consortium (San Diego, CA). MALDI-TOF analysis of oligonucleotides was obtained using a PerSpective Biosystems Voyager Elite mass spectrometer with delayed extraction. Thermal dissociation experiments were conducted on a Cary Bio 100 UV-Vis spectrometer. Thin layer chromatography (TLC) was carried out on Silica Gel 60 F254 0.25 mm precoated plates from EM Science, typically in a hexane/ethyl acetate (1:1 v/v) eluent system.
All reactions were carried out in oven-dried glassware under a nitrogen atmosphere unless otherwise stated. RNases T1 and A were purchased from Boehringer Mannheim Biochemicals. Commercially available DNA synthesis reagents were purchased from Glen Research (Sterling, VA). Anhydrous pyridine, toluene, dichloromethane, diisopropylethyl amine, triethylamine, acetic anhydride, 1,2-dichloroethane, and dioxane were purchased from Aldrich.
All oligonucleotides were synthesized on an ABI 392 or 394 DNA synthesizer using standard protocols for the phosphoramidite-based coupling approach (34). The chain assembly cycle for the synthesis of oligoribonucleotide phosphoramidates was as follows: (i) detritylation, 3% trichloroacetic acid in dichloromethane, 1 min; (ii) coupling, 0.1 M phosphoramidite and 0.45 M tetrazole in acetonitrile, 10 min; (iii) capping, 0.5 M isobutyic anhydride in THF/lutidine, 1:1 v/v (phenoxyacetyl anhydride was used for oligonucleotides containing 2,6-diaminopurine), 15 s; (iv) oxidation, 0.1 M iodine in THF/pyridine/ water, 10:10:1 v/v/v, 30 s.
Chemical steps within the cycle were followed by acetonitrile washing and flushing with dry argon for 20-40 s. Cleavage from the support and removal of base and phosphoramidate protecting groups was achieved by treatment with ammonia/EtOH, 3:1 v/v, for 6-8 h at 55°C. The oligonucleotides were concentrated to dryness in vacuo, after which the 2[prime]-t-butyldimethylsilyl groups were removed by treatment with 1 M TBAF in THF for 4-16 h at 25°C. The reaction mixtures were diluted with water and filtered through a 0.45 nylon acrodisc (Gelman Sciences). Oligonucleotides were then analyzed and purified by ion exchange (IE) HPLC and finally desalted using gel filtration on a Pharmacia NAP-5 or NAP-10 column. Gradient conditions for IE HPLC: solvent A (10 mM NaOH), solvent B (10 mM NaOH and 1.5 M NaCl); solvent A for 3 min then a linear gradient of 0-80% solvent B within 50 min. Thermal denaturation experiments were conducted as described previously using the buffer conditions listed in Table 1 (15).
Table 1. Oligonucleotides and Tm values of their complexes
| Experiment | Oligonucleotide | Target | Tm (°C) |
| 1 | d(CTCTCTGCC) (12) | r(GGCAGAGAG) | 70.7a |
| 2 | dC-r-(UCUCUGCC) (13) | r(GGCAGAGAG) | 74.3a |
| 3 | dC-r-(TCTCTGCC) (14) | r(GGCAGAGAG) | 77.8a |
| 4 | d(GGTTAGGGTTAG) (15) | r(UUGUCUAACCCUAACUG) | 72.1a |
| 5 | dG-r-(GUUAGGGUUAG) (16) | r(UUGUCUAACCCUAACUG) | 80.0a |
| 6 | d(TAGGGTTAGACAA) (17) | r(UUGUCUAACCCUAACUG) | 71.7a |
| 7 | dT-r-(AGGGUUAGACAA) (18) | r(UUGUCUAACCCUAACUG) | 84.7a |
| 8 | r(UAGGGUUAGACAA) (19) | r(UUGUCUAACCCUAACUG) | 64.0a |
| 9 | dA-r-(DDDD) (20) | poly (dT) | <15b |
| 10 | dA-r-(DDDD) | poly (U) | 47.0; 57.4b |
| 11 | d(AAAAA) (21) | poly (dT) | 22.7; 30.3b |
| 12 | d(AAAAA) | poly (U) | 29.8; 44.5b |
| 13 | d(AAAAAAAAAA) (22) | poly (dT) | 30.0a |
| 14 | d(AAAAAAAAAA) | poly (U) | 48.0a |
| 15 | dA-r-(AAAA) (23) | poly (dT) | n.o. |
| 16 | dA-r-(AAAA) | poly (U) | 19.3, 32.3b |
aTm in buffer A.
bTm in buffer B.
For enzymatic digestions, 1.0 OD260 of oligonucleotide 18 and its counterpart RNA oligonucleotide 19 (Table 1) were treated with 100 U of RNase T1 or 10 U of RNase A in 0.1 ml of 10 mM Tris-HCl buffer, pH 7.0, and 5 mM MgCl2 at room temperature. Aliquots from the reaction mixtures were removed at multiple time points and analyzed by IE HPLC under the conditions described above.
Synthesis of nucleoside monomers
3-Azido-1,2-bis-O-acetyl-5-O-toluoyl-3-deoxy-D-ribofuranose (1). Sugar precursor 1 was synthesized starting from 1,2-O-isopropylidene-[alpha]-D-xylofuranose according to the literature procedure (26).
3[prime]-Azido-2[prime]-O-acetyl-5-O-toluoyl-3[prime]-
deoxy-[beta]-D-
ribofuranosyluracil (2u). A solution of 2,4-bis(trimethylsilyl)uracil in 100 ml of acetonitrile was prepared from 2.96 g of uracil and N,O-bistrimethylsilylacetamide (13.7 ml) by heating the mixture at 80°C for 15 min. Then 1 (5.0 g, 13.2 mmol) and stannic chloride (10.3 ml, 88 mmol) were added to the solution of silylated base which had been cooled to -10°C. The reaction mixture was heated to 80°C and stirred for 30 min, cooled to room temperature, and poured into ethyl acetate (400 ml). The resulting solution was washed with saturated NaHCO3 (3 × 100 ml) and saturated NaCl (100 ml). The combined organic phases were dried over Na2SO4 and concentrated in vacuo. The residue was purified by silica gel chromatography eluting with a gradient of EtOAc:hexanes (50:50-60:40 v/v) to afford 2u as a white foam (3.44 g, 60%). 1H NMR: (CDCl3) [delta] 2.18 (s, 3H), 2.40 (s, 3H), 4.22-4.26 (m, 1H), 4.36 (t, J = 6.0 Hz, 1H), 4.51 (dd, J = 3.6 Hz and J = 8.4 Hz, 1H), 4.66 (dd, J = 3.2 Hz and J = 13.2 Hz, 1H), 5.50 (m, 1H), 5.56 (d, J = 8.2 Hz, 1H), 5.80 (d, J = 4.0 Hz, 1H), 7.25 (d, J = 8.0 Hz, 2H), 7.30 (d, J = 8.4 Hz, 1H), 7.90 (d, J = 8.4 Hz, 2H), 8.54 (s, 1H).
2[prime]-O-(t-Butyldimethylsilyl)-3[prime]-(4-methoxytriphenylmethylamino)-3[prime]-deoxy-[beta]-D-
ribofuranosyluracil (8u). Compound 2u (3.4 g, 7.9 mmol) was added to a solution of MeOH in concentrated NH3, 1:1 v/v (125 ml), at -10°C. The solution was stirred for 1 h and then evaporated to give a white foam. The crude material was dissolved in EtOH (80 ml), 10% Pd/C (250 mg) was added and the reaction mixture was hydrogenated under a balloon of hydrogen for 15 h. The catalyst was removed by filtration and washed well with pyridine. The filtrate was concentrated in vacuo and then redissolved in dry pyridine (80 ml). Imidazole (0.9 g, 13.2 mmol) and t-butyldimethylsilyl chloride (1.5 g, 10.0 mmol) were added to the mixture, which was then stirred at room temperature for 15 h. The reaction was concentrated in vacuo and then diluted with CH2Cl2 (150 ml). The organic layer was washed with saturated NaHCO3 (100 ml) and saturated NaCl (100 ml). The combined aqueous phases were back-extracted with additional CH2Cl2 (2 × 50 ml). The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo to give a tan foam. This material was subsequently dissolved in pyridine (50 ml), and 4-methoxytriphenylmethyl chloride (7.8 g, 25.2 mmol) and N-methylimidazole (2.0 ml, 25.2 mmol) were added. The reaction was warmed to 35°C for 15 h and then quenched by addition of MeOH (3 ml). The mixture was concentrated in vacuo to give a foam. To half of this crude material was added 0.1 M NaOH in MeOH (200 ml). The mixture was allowed to stir overnight at room temperature and then concentrated in vacuo. The residue was resuspended in saturated NH4Cl (150 ml) and extracted with CH2Cl2 (3 × 50 ml). The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography eluting with EtOAc:hexanes (50:50 v/v) to yield a white foam (1.3 g, 60% for five steps). 1H NMR: (CDCl3) [delta] -0.12 (s, 3H), 0.06 (s, 3H), 0.83 (s, 9H), 2.50-2.53 (m, 1H), 2.81 (d, J = 8.8 Hz, 1H), 2.82-3.01 (m, 1H), 3.74 (s, 3H), 3.98-4.08 (m, 3H), 5.47 (s, 1H), 5.57 (d, J = 7.8 Hz, 1H), 6.75 (d, J = 8.8 Hz, 2H), 7.18-7.28 (m, 6H), 7.37 (d, J = 8.8 Hz, 2H), 7.42-7.50 (m, 4H), 7.93 (d, J = 8.0 Hz, 1H), 7.97 (s, 1H). Exact mass (HR FAB+) calculated for C35H43N3O6Si (M+Na+) 652.2819, found 652.2838.
2[prime]-O-(t-Butyldimethylsilyl)-3[prime]-(4-methoxytriphenylmethyl-amino)-3[prime]-deoxy-[beta]-D-
ribofuranosyluracil-5[prime]-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (9u). Compound 8u (0.3 g, 0.48 mmol) was dissolved in CH2Cl2 (5 ml), 2-cyanoethyl diisopropychlorophosphoramidite (0.14 ml, 0.62 mmol) and N,N-diisopropylethylamine (0.33 ml, 1.9 mmol) were added. The solution was stirred at room temperature for 2 h and then diluted with CH2Cl2 (50 ml). The resulting solution was washed with saturated NaHCO3 (50 ml) and saturated NaCl (50 ml). The organic layer was dried over Na2SO4 and then concentrated in vacuo. The residue was purified by silica gel chromatography eluting with EtOAc:hexanes (50:50 v/v) containing 1% triethylamine to yield a white foam (0.29 g, 75%). 31P NMR: (CDCl3) [delta] 149.24, 149.44.
3[prime]-Azido-2[prime]-O-acetyl-5-O-toluoyl-3[prime]-deoxy-[beta]-D-
ribofuranosylthymine (2t). Compound 2t was prepared analogously to the described procedure for 3[prime]-azido-2[prime]-O-acetyl-5-O-benzoyl-3[prime]-deoxy-[beta]-D-
ribofuranosylthymine (16). 1H NMR: (CDCl3) [delta] 1.63 (s, 3H), 2.17 (s, 3H), 2.39 (s, 3H), 4.22-4.24 (m, 1H), 4.41-4.47 (m, 2H), 4.68 (dd, J = 12.4 Hz and J = 2.4 Hz, 1H), 4.48-5.50 (m, 1H), 5.85 (d, J = 4.8 Hz, 1H), 7.01 (s, 1H), 7.24 (d, J = 8.4 Hz, 2H), 7.92 (d, J = 8.4, 2H), 8.51 (s, 1H).
2[prime]-O-(t-Butyldimethylsilyl)-3[prime]-(4-methoxytriphenylmethylamino)-3[prime]-deoxy-[beta]-D-
ribofuranosylthymine (8t). Compound 2t (2.0 g, 4.5 mmol) was added to a solution of 50% MeOH in concentrated NH3 (75 ml) at -10°C. The solution was stirred for 1 h and then evaporated to give a white slurry, which was diluted with brine (50 ml). The aqueous mixture was extracted with CH2Cl2 (3 × 50 ml), the combined organics were dried over Na2SO4, filtered, and concentrated in vacuo to give a white foam. The crude material was dissolved in EtOH (80 ml), 10% Pd/C (200 mg) was added, and the reaction mixture was hydrogenated under a balloon of hydrogen at room temperature for 60 h. The catalyst was removed by filtration and washed well with pyridine. The filtrate was concentrated in vacuo and then redissolved in dry pyridine (40 ml). Imidazole (0.33 g, 4.8 mmol) and t-butyldimethylsilyl chloride (0.58 g, 3.8 mmol) were added to the mixture, which was then stirred at room temperature for 3 h. The reaction was concentrated in vacuo and then diluted with CH2Cl2 (100 ml). The organic layer was washed with saturated NaHCO3 (100 ml) which was back-extracted with additional CH2Cl2 (2 × 50 ml). The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography eluting with EtOAc:hexanes (50:50 v/v) to yield a white foam (1.04 g, 48% for three steps). 1H NMR: (CDCl3) [delta] 0.11 (s, 3H), 0.19 (s, 3H), 0.91 (s, 9H), 1.55 (s, 3H), 2.38, (s, 3H), 3.30 (dd, J = 4.8 Hz, and J = 9.2 Hz, 1H), 4.08-4.12 (m, 1H), 4.15 (d, J = 4.8 Hz, 1H), 4.60 (dd, J = 4.0 Hz and J = 12.8 Hz, 1H), 4.72 (dd, J = 2.0 Hz and J = 12.8 Hz, 1H), 5.64 (s, 1H), 7.20 (d, J = 8.4 Hz, 2H), 7.34 (s, 1H), 7.88 (d, J = 8.4 Hz, 2H).
The 2[prime]-O-t-butyldimethylsilyl-3[prime]-amino-5[prime]-O-toluoyl-3[prime]-deoxy-[beta]-D-
ribofuranosylthymine 6t (1.04 g, 2.12 mmol) was then dissolved in pyridine (20 ml), and 4-methoxytriphenylmethyl chloride (2.6 g, 8.5 mmol) and N-methylimidazole (0.68 ml, 8.5 mmol) were added. The reaction mixture was warmed to 35°C for 15 h and then quenched by addition of MeOH (3 ml), after which concentration in vacuo gave a gummy material. To this crude material was added 0.1 M NaOH in MeOH (100 ml). The mixture was stirred overnight at room temperature and then concentrated in vacuo. The residue was resuspended in saturated NH4Cl (100 ml) and extracted with CH2Cl2 (3 × 50 ml). The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography eluting with EtOAc:hexanes (50:50 v/v) to yield a white foam (0.75 g, 56% for two steps). 1H NMR: (CDCl3) [delta] -0.01 (s, 3H), 0.06 (s, 3H), 0.84 (s, 9H), 1.82 (s, 3H), 2.82 (d, J = 3.6 Hz, 1H), 2.88-2.90 (m, 1H), 2.99-3.02 (m, 1H), 3.74 (s, 3H), 3.88-3.99 (m, 3H), 5.51 (s, 1H), 6.75 (d, J = 8.0 Hz, 2H), 7.16-7.25 (m, 6H), 7.36 (d, J = 8.0 Hz, 2H), 7.44-7.47 (m, 4H), 7.72 (s, 1H), 8.31 (s, 1H). Exact mass (HR FAB+) calculated for C36H45N3O6Si (M+Na+) 666.2975, found 666.2963.
2[prime]-O-(t-Butyldimethylsilyl)-3[prime]-(4-methoxytriphenylmethyl-amino)-3[prime]-deoxy-[beta]-D-
ribofuranosylthymine-5[prime]-(2-cyanoethyl-N,N-diiso-propyl)phosphoramidite (9t). Compound 9t was prepared analogously to the described procedure for 9u to yield a white foam (0.3 g, 75%). 31P NMR: (CDCl3) [delta] 148.15, 149.9.
3[prime]-Azido-2[prime]-O-acetyl-5-O-toluoyl-N6-benzoyl-3[prime]-
deoxyadenosine (2a). To a solution of N6-benzoyl-N6,9-bis(trimethylsilyl)-adenine (prepared from 3.15 g of N6-benzoyladenine) and 1 (2.5 g, 6.6 mmol) in dry acetonitrile (100 ml) at -10°C was added stannic chloride (4.7 ml, 39.4 mmol). The reaction was heated to 80°C and stirred for 15 h, after which the mixture was cooled to room temperature, concentrated in vacuo, and diluted with ethyl acetate (400 ml). The resulting solution was washed with saturated NaHCO3 (3 × 100 ml) and saturated NaCl (100 ml). The combined organic phases were dried over Na2SO4 and concentrated in vacuo. The residue was purified by silica gel chromatography eluting with a gradient of EtOAc:hexanes:MeOH (49:49:2-47.5:47.5:5 v/v/v) to afford 2a as a white foam (2.3 g, 63%). 1H NMR: (CDCl3) [delta] 2.18 (s, 3H), 2.37 (s, 3H), 4.36-4.40 (m, 1H), 4.52 (dd, J = 4.0 Hz, and J = 12.4 Hz, 1H), 4.72 (dd, J = 2.8 Hz, and J = 12.8 Hz, 1H), 4.94 (t, J = 5.6 Hz, 1H), 6.07-6.12 (m, 2H), 7.19 (d, J = 8.0 Hz, 2H), 7.48-7.61 (m, 3H), 7.84 (d, J = 8.0 Hz, 2H), 7.98 (d, J = 7.2 Hz, 2H), 8.03 (s, 1H), 8.63 (s, 1H), 8.88 (br s, 1H).
N6-Benzoyl-2[prime]-O-(t-butyldimethylsilyl)-3[prime]-(4-methoxytriphenylmethylamino)-3[prime]-
deoxyadenosine (8a). Compound 2a (1.9 g, 3.4 mmol) was added to a solution of concentrated NH3 in MeOH, 2:5 v/v (80 ml), at 0°C. The solution was stirred for 45 min and then evaporated to give a white foam. The material was dissolved in CH2Cl2 (100 ml) and washed with saturated NaCl (100 ml). The aqueous layer was back-extracted with CH2Cl2 (50 ml), after which the combined organic phases were dried over Na2SO4, filtered, and concentrated in vacuo to give a white foam. The crude material was dissolved in EtOH (80 ml), 10% Pd/C (150 mg) was added, and the reaction mixture was hydrogenated under a balloon of hydrogen at room temperature for 15 h. The catalyst was removed by filtration and washed well with CH2Cl2. The filtrate was concentrated in vacuo and then redissolved in dry pyridine (40 ml). Imidazole (0.34 g, 5.0 mmol) and t-butyldimethylsilyl chloride (0.6 g, 4.0 mmol) were added to the mixture, which was then stirred at room temperature for 15 h. The reaction was concentrated in vacuo and then diluted with CH2Cl2 (100 ml). The organic layer was washed with saturated NaHCO3 (100 ml) and saturated NaCl (100 ml). The combined aqueous phases were back-extracted with additional CH2Cl2 (50 ml). The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo to give a white foam. This material was subsequently dissolved in pyridine (30 ml), and 4-methoxytriphenylmethyl chloride (4.1 g, 13.3 mmol) and N-methylimidazole (1.0 ml, 13.3 mmol) were added. The reaction was warmed to 35°C for 30 h and then quenched by addition of MeOH (3 ml). The mixture was concentrated in vacuo to give a brown foam. To this crude material was added 1.0 M NaOH in 65:30:5 pyridine/MeOH/H2O (70 ml) at 0°C. The mixture was stirred for 8 min and quenched by addition of saturated NH4Cl (300 ml). The solution was extracted with ethyl acetate (2 × 75 ml) and the combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography eluting with EtOAc:hexanes (50:50 v/v) to yield a white foam (1.4 g, 60% for five steps). 1H NMR: (CDCl3) [delta] -0.52 (s, 1H), -0.15 (s, 1H), 0.77 (s, 9H) 3.20-3.29 (m, 3H), 3.64 (d, J = 12.8 Hz, 1H), 3.74 (s, 3H), 3.80 (br s, 1H), 4.55 (t, J = 5.2 Hz, 1H), 6.15 (d, J = 5.2 Hz, 1H), 6.76 (d, J = 8.8 Hz, 2H), 7.13-7.24 (m, 6H), 7.35 (d, J = 8.8 Hz, 2H), 7.43-7.59 (m, 7H), 8.01 (d, J = 7.2 Hz, 2H), 8.21 (s, 1H), 8.72 (s, 1H), 9.07 (br s, 1H). Exact mass (HR FAB+) calculated for C43H48N6O5Si (M+H+) 757.3534, found 757.3514.
N6-Benzoyl-2[prime]-O-(t-butyldimethylsilyl)-3[prime]-(4-methoxytriphenylmethylamino)-3[prime]-
deoxyadenosine-5[prime]-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (9a). Compound 9a was prepared analogously to the described procedure for 9u to yield a white foam (0.34 g, 76%). 31P NMR: (CDCl3) [delta] 149.85.
3[prime]-Azido-2[prime]-O-acetyl-5[prime]-O-toluoyl-N2-isobutryl-O6-diphenylcarbamoyl-3[prime]-
deoxyguanosine (2g). To a solution of N2-isobutryl-O6-diphenylcarbamoyl-N2,9-bis(trimethylsilyl)guanine (prepared from 3.32 g of N2-isobutryl-O6-diphenylcarbamoylguanine) and 1 (2.0 g, 5.3 mmol) in dry toluene (60 ml) was added trimethylsilyl triflate (1.6 ml, 8.0 mmol). The reaction mixture was heated to 80°C and stirred for 4 h, after which the mixture was cooled to room temperature and diluted with ethyl acetate (250 ml). The resulting solution was washed with saturated NaHCO3 (150 ml) and saturated NaCl (150 ml). The aqueous phases were back-extracted with ethyl acetate (100 ml), the combined organic phases were dried over Na2SO4 and concentrated in vacuo. The residue was purified by silica gel chromatography eluting with EtOAc:hexanes (50:50 v/v) to afford 2g as a white foam (2.86 g, 75%). 1H NMR: (CDCl3) [delta] 1.14 (d, J = 9.6 Hz, 3H), 1.17 (d, J = 9.2 Hz, 3H), 2.18 (s, 3H), 2.31, (s, 3H), 2.49 (hept, J = 6.8 Hz, 1H), 4.30-4.35 (m, 1H), 4.55 (dd, J = 5.6 Hz and J = 12.4 Hz, 1H), 4.68 (dd, J = 3.6 Hz and J = 12.0 Hz, 1H), 5.87-5.89 (m, 2H), 6.04-6.08 (m, 1H), 7.10 (d, J = 8.0 Hz, 2H), 7.20-7.43 (m, 10H), 7.75 (d, J = 8.4 Hz, 2H), 7.90 (s, 1H), 8.01 (br s, 1H). Exact mass (HR FAB+) calculated for C37H35N9O8 (M+H+) 734.2687, found 734.2672.
3[prime]-Azido-5[prime]-O-anisoyl-N2-isobutryl-3[prime]-deoxyguanosine (4g). Compound 2g (2.8 g, 3.81 mmol) was dissolved in a 1.0 M NaOH solution (65:30:5 pyridine/MeOH/H2O v/v/v, 40 ml) at 0°C. The mixture was stirred for 10 min, and then quenched by addition of saturated NH4Cl (400 ml). The solution was extracted with CH2Cl2 (5 × 100 ml) and the combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo. The residue was triturated well with Et2O (50 ml) to remove diphenylamine. The resulting material was dissolved in acetonitrile (25 ml), and PPh3 (1.2 g, 4.65 mmol) was added. p-Anisic acid (0.71 g, 4.65 mmol) and diisopropyl azodicarboxylate (0.92 ml, 4.65 mmol) were dissolved in acetonitrile (5 ml) and added dropwise to the reaction mixture. The solution was stirred at room temperature for 1 h and was then quenched by pouring into saturated NaHCO3 (200 ml). The mixture was extracted with ethyl acetate (200 ml) and after separation the organic phase was washed with saturated NaCl (150 ml). The organic phase was then dried over Na2SO4 and concentrated in vacuo. The residue was purified by silica gel chromatography eluting with a gradient of EtOAc:hexanes:MeOH (49:49:2-47.5:47.5:5 v/v/v) to afford 4g as a white foam (0.93 g, 48% for two steps). 1H NMR: (CDCl3) [delta] 1.27 (d, J = 6.8 Hz, 3H), 1.38 (d, J = 6.4 Hz, 3H), 2.81 (hept, J = 6.8 Hz, 1H), 3.81 (s, 3H), 4.31 (d, J = 5.2 Hz, 1H), 4.44-4.49 (m, 2H), 5.13-5.19 (m, 1H), 5.70 (d, J = 7.6 Hz, 1H), 6.89 (d, J = 8.8 Hz, 2H), 7.40 (s, 1H), 7.91 (d, J = 8.8 Hz, 2H), 9.41 (br s, 1H), 11.94 (br s, 1H). Exact mass (HR FAB+) calculated for C22H24N8O7 (M+Na+) 535.1666, found 535.1659.
N2-isobutryl-2[prime]-O-(t-Butyldimethylsilyl)-3[prime]-(4-methoxytriphenylmethylamino)-3[prime]-
deoxyguanosine (8g). Compound 4g (0.9 g, 1.75 mmol) was dissolved in EtOH (50 ml), 10% Pd/C (150 mg) was added, and the reaction mixture was hydrogenated under a balloon of hydrogen at room temperature for 15 h. The catalyst was removed by filtration and washed well with EtOH. The filtrate was concentrated in vacuo and then resuspended in dry pyridine (30 ml). Imidazole (0.24 g, 3.5 mmol) and t-butyldimethylsilyl chloride (0.4 g, 2.6 mmol) were added to the mixture, which was then stirred at room temperature for 1 h, after which more imidazole (0.12 g, 1.75 mmol) and t-butyldimethylsilyl chloride (0.2 g, 1.3 mmol) were added. The reaction mixture was stirred for an additional 2 h and then diluted with CH2Cl2 (200 ml). The organic layer was washed with saturated NaHCO3 (200 ml) and saturated NaCl (50 ml). The combined aqueous phases were back-extracted with additional CH2Cl2 (50 ml). The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo to give a yellowish residue. This material was subsequently dissolved in pyridine (20 ml), and 4-methoxytriphenylmethyl chloride (2.7 g, 8.75 mmol) and N-methylimidazole (0.7 ml, 8.75 mmol) were added. The reaction was warmed to 39°C for 15 h and then quenched by addition of MeOH (3 ml). The mixture was concentrated in vacuo to give an orangish brown viscous residue, which was purified by silica gel chromatography eluting with EtOAc:hexanes:MeOH (47.5:47.5:5 v/v/v) to yield a white foam (1.0 g, 66% for three steps). 1H NMR: (CDCl3) [delta] -0.29 (s, 3H), -0.17 (s, 3H), 0.79 (s, 9H), 1.26 (d, J = 7.2 Hz, 6H), 2.69 (hept, J = 6.4 Hz, 1H), 3.00-3.09 (m, 1H), 3.70 (s, 3H), 3.76-3.87 (m, 4H), 4.07-4.10 (m, 1H), 4.26-4.30 (m, 1H), 4.89-4.39 (m, 1H), 5.92 (d, J = 4.0 Hz, 1H), 6.71 (d, J = 8.8 Hz, 2H), 6.79-6.84 (m, 3H), 7.00-7.49 (m, 11H), 7.67-7.69 (m, 3H), 8.51 (br s, 1H), 11.91 (br s, 1H).
The isolated N2-isobutryl-2[prime]-O-(t-butyldimethylsilyl)-3[prime]- (4-methoxytriphenylmethylamino)-5[prime]-O-anisoyl-3[prime]-
deoxyguanosine (1.0 g, 1.12 mmol) was treated with 1.0 M NaOH in 65:30:5 pyridine/MeOH/H2O v/v/v (20 ml) at 0°C. The mixture was stirred for 12 min, and then quenched by addition of saturated NH4Cl (200 ml). The solution was extracted with CH2Cl2 (2 × 75 ml) and the combined organic layers were washed with saturated NaHCO3 (100 ml). The aqueous layer was back-extracted with CH2Cl2 (25 ml), and then the combined organic phases were dried over Na2SO4, filtered, and concentrated in vacuo to give a yellow foam (0.85 g). By 1H NMR this material was pure except for the presence of residual methyl p-anisate. 1H NMR: (CDCl3) [delta] -0.40 (s, 3H), -0.14 (s, 3H), 0.78 (s, 9H), 1.20 (d, J = 1.6 Hz, 3H), 1.21 (d, J = 2.0 Hz, 3H), 2.57 (hept, J = 6.8 Hz, 1H), 3.20-3.25 (m, 2H), 3.57-3.60 (m, 1H), 3.74 (s, 3H), 3.75-3.76 (m, 1H), 4.19-4.21 (m, 1H), 5.94 (d, J = 5.6 Hz, 1H), 6.76 (d, J = 8.8 Hz), 7.07-7.47 (m, 12H), 7.76 (s, 1H), 8.31 (br s, 1H), 12.01 (br s, 1H). Exact mass (HR FAB+) calculated for C40H50N6O6Si (M+Na+) 761.3459, found 761.3472.
N2-Isobutyrl-2[prime]-O-(t-butyldimethylsilyl)-3[prime]-(4-methoxytriphenylmethylamino)-3[prime]-
deoxyguanosine-5[prime]-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (9g). Compound 9g was prepared analogously to the described procedure for 9u to yield a white foam (0.28 g, 73%). 31P NMR: (CDCl3) [delta] 149.47.
3[prime]-Azido-2[prime]-O-acetyl-5[prime]-O-toluoyl-N2,N6-phenoxyacetyl-3[prime]-deoxydiaminopurine (2d). To a solution of N2,N6-phenoxyacetyl-N2,N6,9-tris(trimethylsilyl)diaminopurine [prepared from 5.0 g of N2,N6-bis(phenoxyacetyl) diaminopurine] and 1 (3.0 g, 8.0 mmol) in dry toluene (100 ml) was added trimethylsilyl triflate (2.4 ml, 12.0 mmol). The reaction was heated to 80°C and stirred for 4 h, cooled to room temperature, and diluted with ethyl acetate (400 ml). The resulting solution was washed with saturated NaHCO3 (200 ml) and saturated NaCl (200 ml). The aqueous phases were back-extracted with ethyl acetate (100 ml), and the combined organic phases were filtered through celite to remove unreacted N2,N6-phenoxyacetyl diaminopurine. The filtrate was dried over Na2SO4 and concentrated in vacuo to give a yellow foam. The residue was purified by silica gel chromatography eluting with a gradient of EtOAc:hexanes (65:35-80:20 v/v) to afford 2d as a yellow foam (3.52 g, 60%). 1H NMR: (CDCl3) [delta] 2.19 (s, 3H), 2.28 (s, 3H), 4.35-4.40 (m, 1H), 4.45-4.76 (m, 4H), 4.80 (s, 2H), 6.89 (s, 1H), 6.95 (s, 2H), 6.98-7.11 (m, 8H), 7.26-7.38 (m, 4H), 7.78 (d, J = 8.8 Hz, 2H), 7.91 (s, 1H), 9.15, (br s, 1H), 9.28 (br s, 1H). Exact mass (HR FAB+) calculated for C36H33N9O9 (M+Na+) 758.2299, found 758.2314.
N2,N6-Phenoxyacetyl-2[prime]-O-(t-butyldimethylsilyl)-3[prime]-(4-methoxytriphenylmethylamino)-3[prime]-
deoxydiaminopurine (8d). Compound 2d (2.8 g, 3.8 mmol) was dissolved in a 1.0 M NaOH solution (65:30:5 pyridine/MeOH/H2O v/v/v, 60 ml) at 0°C. The mixture was stirred for 7 min and quenched by addition of saturated NH4Cl (500 ml). The solution was extracted with CH2Cl2 (4 × 200 ml) and the combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo. The resulting material was dissolved in dimethylformamide (50 ml), and PPh3 (1.5 g, 5.7 mmol) was added. p-Anisic acid (0.87 g, 5.7 mmol) and diisopropyl azodicarboxylate (1.12 ml, 5.7 mmol) were dissolved in dimethylformamide (5 ml) and added dropwise to the reaction mixture. The solution was stirred at room temperature for 3 h and was then quenched by pouring into saturated NaHCO3 (500 ml). The mixture was extracted with ethyl acetate (2 × 200 ml), after which the combined organic phases were washed with saturated NaCl (150 ml). The organic phase was then dried over Na2SO4 and concentrated in vacuo. The residue was passed through a short pad of silica gel eluting with EtOAc:hexanes:MeOH (47.5:47.5:5 v/v/v).
Crude 4d was dissolved in a mixture of EtOH (100 ml) and CH2Cl2 (25 ml), 10% Pd/C (300 mg) was added, and the reaction mixture was hydrogenated under a balloon of hydrogen at room temperature for 15 h. The catalyst was removed by filtration and washed well with pyridine (100 ml). The filtrate was concentrated in vacuo and then resuspended in dry pyridine (40 ml). Imidazole (0.52 g, 7.6 mmol) and t-butyldimethylsilyl chloride (0.86 g, 5.7 mmol) were added to the mixture, which was then stirred at room temperature for 15 h. The reaction was then diluted with CH2Cl2 (200 ml) and washed with saturated NaHCO3 (200 ml) and saturated NaCl (50 ml). The combined aqueous phases were back-extracted with additional CH2Cl2 (50 ml). The organic layers were dried over Na2SO4, filtered, and concentrated in vacuo to give a viscous material. This material was subsequently dissolved in pyridine (40 ml), and 4-methoxytriphenylmethyl chloride (4.7 g, 15.2 mmol) and N-methylimidazole (1.2 ml, 15.2 mmol) were added. The reaction mixture was warmed to 40°C for 60 h and then quenched by MeOH (3 ml). The mixture was concentrated in vacuo to give a brown viscous residue, which was purified by silica gel chromatography eluting with EtOAc:hexanes (50:50 v/v) to yield a white foam (0.96 g, 24% for five steps). 1H NMR: (CDCl3) [delta] -0.09 (s, 3H), 0.11 (s, 3H), 0.87 (s, 9H), 2.81-2.88 (m, 2H), 3.23-3.35 (m, 1H), 3.52 (s, 3H), 3.83 (s, 3H), 4.39-4.42 (m, 1H), 4.67-4.91 (m, 4H), 5.05 (br, s, 2H), 5.61 (s, 1H), 6.57 (d, J = 8.8 Hz, 2H), 6.81-6.89 (m, 4 H), 6.89-7.13 (m, 10H), 7.22-7.38 (m, 10H), 7.62 (d, J = 8.8 Hz, 2H), 8.13 (s, 1H), 8.78 (br s, 1H), 9.39 (br s, 1H). Molecular mass (ESI+) calculated for C60H63N7O10Si (M+Na+) 1092, found 1092.
The isolated N2,N6-phenoxyacetyl-2[prime]-O-(t-butyldimethylsilyl)-3[prime]-(4-methoxytriphenylmethylamino)-5[prime]-O-
anisoyl-3[prime]-deoxydiaminopurine (0.95 g, 0.90 mmol) was treated with 1.0 M NaOH in 65:30:5 pyridine/MeOH/H2O v/v/v (20 ml) at 0°C. The mixture was stirred for 5 min, and quenched by addition of saturated NH4Cl (250 ml). The solution was extracted with CH2Cl2 (2 × 100 ml) and the organic phase was dried over Na2SO4, filtered, and concentrated in vacuo to give a residue, which was purified by silica gel column chromatography eluting with EtOAc:hexanes:MeOH (47.5:47.5:5 v/v/v) to give 8d (0.5 g, 60%) as a white foam. 1H NMR: (CDCl3) [delta] -0.34 (s, 3H), -0.12 (s, 3H), 0.80 (s, 3H), 3.07-3.11 (m, 1H), 3.21-3.24 (m, 1H), 3.48-3.54 (m, 2H), 3.71 (s, 3H), 3.86-3.88 (m, 1H), 4.71 (br s, 2H), 4.93 (br s, 2H), 6.02 (d, J = 5.2 Hz, 1H), 6.72 (d, J = 8.4 Hz, 2H) 6.97-7.46 (m, 22H), 8.19 (s, 1H), 8.93 (br s, 1H), 9.39 (br s, 1H). Exact mass (HR FAB+) calculated for C52H57N7O8Si (M+H+) 936.4114, found 936.4148. N2,N6-Phenoxyacetyl-2[prime]-O-(t-butyldimethylsilyl)-3[prime]-(4-methoxytriphenylmethylamino)-3[prime]-
deoxydiaminopurine-5[prime]-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (9d). Compound 9d was prepared analogously to the described procedure for 9u to yield a white foam (0.45 g, 75%). 31P NMR: (CDCl3) [delta] 149.41, 149.46.
N4-Benzoyl-2[prime]-O-(t-butyldimethylsilyl)-3[prime]-(4-methoxytriphenylmethylamino)-3[prime]-
deoxycytidine (10c). Compound 7u (0.90 g, 1.2 mmol) in dry acetonitrile (5 ml) was added to an ice-cold mixture of triazole (1.66 g, 24.0 mmol), triethylamine (3.6 ml, 26.0 mmol), and phosphorous oxychloride (0.45 ml, 4.8 mmol) in dry acetonitrile (50 ml). The mixture was allowed to warm to room temperature, stirred for 4 h, and then quenched with saturated NaHCO3 (200 ml) and finally extracted with CH2Cl2 (2 × 100 ml). The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo. The residue was dissolved in dioxane (8 ml), and concentrated NH3 (2 ml) was added. After 24 h the reaction mixture was concentrated in vacuo and then redissolved in ethyl acetate (150 ml). The solution was washed with water (2 × 100 ml), dried over Na2SO4, and concentrated in vacuo. Pyridine (15 ml) was added to the crude material and the mixture was cooled to 0°C, and benzoyl chloride (0.84 ml, 7.2 mmol) was added. The solution was then warmed to room temperature and stirred for 15 min. The reaction was quenched with water (5 ml) and then concentrated in vacuo. The resulting material was dissolved in ethyl acetate (150 ml) and washed with water (100 ml) and saturated NaHCO3 (100 ml). The organic layer was dried over Na2SO4, filtered, and concentrated in vacuo to give an off-white foam. This material was treated with 1.0 M NaOH in 65:30:5 pyridine/MeOH/H2O v/v/v (20 ml) at 0°C. The mixture was stirred for 12 min and then quenched by addition of saturated NH4Cl (250 ml). The solution was extracted with ethyl acetate (300 ml) and the organic phase was dried over Na2SO4, filtered, and concentrated in vacuo to give a white foam, which was purified by silica gel column chromatography eluting with EtOAc:hexanes (50:50 v/v) to give 10c (0.61 g, 45% for four steps) as a white foam. 1H NMR: (CDCl3) [delta] -0.07 (s, 3H), 0.15 (s, 3H), 0.85 (s, 9H), 2.65 (d, J = 3.2 Hz, 1H), 2.80-2.85 (m, 1H), 2.98-2.03 (m, 1H), 3.74 (s, 3H), 4.01-4.11 (m, 3H), 5.58 (s, 1H), 6.73 (d, J = 8.4 Hz, 2H), 7.15-7.21 (m, 6H), 7.35 (d, J = 8.4 Hz, 2H), 7.34-7.60 (m, 7h), 7.88 (d, J = 7.6 Hz, 1H), 8.41 (d, J = 7.6 Hz, 1H), 8.63 (br s, 1H). Exact mass (HR FAB+) calculated for C42H48N4O6Si (M+Na+) 755.3241, found 755.3228.
N4-Benzoyl-2[prime]-O-(t-butyldimethylsilyl)-3[prime]-(4-methoxytriphenylmethylamino)-3[prime]-
deoxycytidine-5[prime]-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (11c). Compound 11c was prepared analogously to the described procedure for 9u to yield a white foam (0.24 g, 71%). 31P NMR: (CDCl3) [delta] 149.53, 149.65.
RESULTS AND DISCUSSION
Preparation of monomers
Initial investigations into the assembly of oligoribonucleotide N3[prime]->P5[prime] phosphoramidates determined that a synthetic methodology based on a phosphoramidite transfer reaction was suitable for construction of these biopolymers (16). This approach was previously employed for the synthesis of oligo-2[prime]-fluoro-2[prime]-deoxynucleotide N3[prime]->P5[prime] phosphoramidates as well as for oligo-2[prime]-deoxynucleotide N3[prime]->P5[prime] phosphoramidates (21-23). The synthetic strategy is based on use of 3[prime]-(monomethoxytrityl) amino-5[prime]-O-(cyanoethyl-N,N[prime]-diisopropylamino)phosphoramidite nucleoside monomers (Fig. 1), and assembly of the oligoribonucleotide in the 5[prime]->3[prime] direction. The appropriately protected ribonucleotide monomers were in general synthesized according to the previously reported protocols (16) with some key modifications, which allowed for maximization of overall yields and expedited isolation of the final products (Scheme 1).
Figure 1. Structure of N3[prime]->P5[prime] phosphoramidite monomers. MMT and TBDMS correspond to p-monomethoxytrityl and t-butyldimethylsilyl protecting groups, respectively.
Scheme 1.
The first step of the synthesis involved tin(IV) chloride or trimethylsilyl triflate-mediated glycosylation of trimethylsilylated nucleobases (24,25) to a commonly employed sugar precursor 3-azido-1, 2-di-O-acetyl-5-O-toluoyl-3-deoxy-D-ribofuranose 1, which was prepared according to a literature procedure(26). Glycosylation reactions with ribo-sugar precursor 1 and silylated bases were previously reported to result in formation of only [beta]-isomers (24). Our results also indicate exclusive formation of nucleoside [beta]-isomers, as determined by TLC and 1H NMR analysis. Adenine was protected at N6 with a benzoyl group, while guanine was blocked at N2 with an isobutryl group and at O6 with diphenylcarbamate (27). The protection of O6 with this bulky group allows for selective glycosylation to occur at N9 with very little (<10%) formation of the undesired N7 regioisomer as judged by TLC analysis. 2,6-Diaminopurine was protected at each exocylic amine with a phenoxyacetyl group for all glycosylation reactions with this highly polar purine base analog (28).
Two key synthetic improvements were made to the preparation of the monomers that allowed for rapid access to the final products with improved overall yields. First, experimental conditions were found, which enabled selective removal of the 2[prime]-O-acetyl protecting group in the presence of the 5[prime]-O-toluoyl counterpart (29; Scheme 1). This allowed the omission of a 5[prime]-hydroxyl reprotection step from the synthetic protocol. Also, a low yielding series of steps late in the monomer synthesis, used in the literature procedure (16) to convert a 5[prime]-O-trityl-nucleoside precursor to the 3[prime]-N-trityl-protected amino intermediate, was also averted. Secondly, following the glycosylation reaction, the next five chemical transformations were conducted with very high yields. This eliminated the need for intermediate purification after steps iv-viii (Scheme 1), thus providing rapid and convenient access to compounds 8a, 8t, 8u, 8g and 8d. However, it should be noted that for the guanosine and 2,6-diaminopurine analogs, selective removal of the 2[prime]-O-acetyl protecting group was unsuccessful. Thus, both 2[prime]-O- and 5[prime]-O-protecting groups were removed, after which the 5[prime]-hydroxyl group was selectively reprotected (Scheme 1).
For compounds 2a, 2t and 2u the 2[prime]-O-acetyl group was selectively removed using 50% (v/v) aqueous ammonia in methanol followed by 3[prime]-azido group reduction with hydrogen over palladium on carbon. Each of these reactions proceeded with very high, near quantitative yields as judged by TLC and 1H NMR analysis of the products. The obtained nucleoside precursors were then sequentially protected at the 2[prime]-hydroxyl by treatment with t-butyldimethylsilyl chloride and at the 3[prime]-amino group with 4-monomethoxytrityl chloride to give compounds 7a, 7t and 7u. After work-up (Materials and Methods) the crude mixtures were treated with a 1 M solution of sodium hydroxide in pyridine/methanol/water to remove the 5[prime]-O-toluoyl group and afford nucleosides 8a, 8t and 8u with overall yields of 56-60% based on starting precursors 2a, 2t and 2u (Scheme 1).
The inability to selectively remove the 2[prime]-O-acetyl group from intermediates 2g and 2d necessitated the following synthetic protocol. Both 2[prime]-O- and 5[prime]-O-protecting groups were removed with 1 M sodium hydroxide, after which a 5[prime]-O-anisoyl group was selectively reintroduced under Mitsunobu conditions to give 4g and 4d. It should be noted that the high reactivity of the 2[prime]-hydroxyl group of the 3[prime]-azido-2[prime]-hydroxyl guanosine intermediate prevented selective reprotection of the 5[prime]-hydroxyl group by either benzoyl chloride or benzoyl anhydride. The same series of steps described above was then used to convert 4g and 4d into 8g and 8d, respectively (Scheme 1). The final step for monomer preparation involved phosphitylation of 8a, 8t, 8u, 8g and 8d to give the 5[prime]-(2-cyanoethyl-N,N[prime]-diisopropylamino)nucleoside phosphoramidite building blocks 9a, 9t, 9u, 9g and 9d (Fig. 1).
To minimize the number of overall synthetic transformations, intermediate 7u was converted into an N4-benzoyl-3[prime]-aminocytidine analog (10c). To achieve this, uridine derivative 7u was transformed into a benzoyl-protected cytosine according to literature procedure (Scheme 2; 30). Reaction of 7u with triazole in the presence of phosphorus oxychloride yielded the desired 4-triazolo species, which upon treatment with ammonia generated the 4-amino-unprotected cytosine nucleoside. After work-up (Materials and Methods) the crude reaction mixture was benzoylated and finally deprotected with 1 M sodium hydroxide to give 10c in 45% overall yield from 7u. Phosphitylation of 10c produced the desired 5[prime]-(2-cyanoethyl-N,N[prime]-diisopropylamino)phosphoramidite cytidine monomer (11c) (Scheme 2) used for oligonucleotide construction.
Scheme 2.
Figure 2. Structure of the oligoribonucleotide N3[prime]->P5[prime] phosphoramidite internucleotide linkage.
Synthesis of oligoribonucleotide N3[prime]->P5[prime] phosphoramidates
It was previously reported that homopurine and homopyrimidine oligoribonucleotide N3[prime]->P5[prime] phosphoramidates could be efficiently assembled on a solid phase support using a phosphoramidite transfer reaction (16). We found that this methodology works equally well for the synthesis of heterobased phosphoramidate oligoribonucleotides containing all four natural bases as well as thymidine and 2,4-diaminopurine (Fig. 2). Each of the prepared oligoribonucleotide N3[prime]->P5[prime] phosphoramidites were synthesized starting from the 5[prime]-end using a support-bound 2[prime]-deoxy-3[prime]-aminonucleoside as the 5[prime]-terminal residue. Coupling steps involved exchange of the diisopropylamino group of the approaching 5[prime]-O-phosphoramidite (9a, 9t, 9u, 9g, 9d and 11c) for the 3[prime]-amino group of the support-bound nucleoside. Standard RNA synthesis coupling times (10 min) and activator (1H-tetrazole) were used for each synthetic cycle. Unreacted 3[prime]-amino groups were then capped with isobutyric anhydride, after which oxidation of the internucleotide phosphoramidite diester linkage into the phosphoramidate group was carried out with aqueous iodine. Subsequent detritylation of the 3[prime]-amino group of the added residue enabled additional chain elongation steps to be repeated for the construction of the desired oligoribonucleotide phosphoramidates. The resin-bound compounds were then deprotected and cleaved from the support by treatment with ammonia/ethanol solution. Removal of 2[prime]-O-t-butyldimethylsilyl groups was accomplished using 1 M TBAF in THF, after which the fully deprotected oligoribonucleotide phosphoramidates were analyzed and isolated using IE HPLC (Fig. 3A). Several mixed-base oligoribophosphoramidates were prepared and sequences of some of these compounds are presented in Table 1. Product integrity was confirmed by both 31P NMR and MALDI-TOF mass spectra analysis. Thus, all 31P phosphorous resonances for a model 6mer oligonucleotide 5[prime]-dT-r-UUUUU had characteristic chemical shifts for phosphoramidates, between 7 and 8 p.p.m., while no phosphodiester linkage resonances were detected (Fig. 3B). Electrospray mass spectral analysis of oligomer 18 afforded the correct molecular weight (m/z): calculated 4176, found 4178.
A
 |
B
 |
Figure 3. (A) Ion exchange HPLC profile of the crude reaction mixture from synthesis of oligonucleotide 18 (Table 1). (B) 31P NMR of purified ribophosphoramidate oligonucleotide 5[prime]-dT-r-UUUUU.
Hydrolytic stability of oligoribonucleotide N3[prime]->N5[prime] phosphoramidate toward RNases T1 and A
The stability of oligoribonucleotide N3[prime]->N5[prime] phosphoramidate toward digestion by RNases T1 and A was evaluated. Each of these enzymes is an endonuclease specific for single-stranded RNA: RNase T1 cleaves internucleoside phosphodiester groups 3[prime] to guanosine residues leaving 3[prime]-phosphate products, while RNase A is a pyrimidine-specific nuclease which also hydrolyses 3[prime]-5[prime] phosphodiester bonds, but leaves 2[prime],3[prime]-cyclic phosphates as products. Thus, oligonucleotide 18 and its natural RNA control 19 (Table 1) were treated with RNase T1 or RNase A in Tris-HCl buffer at room temperature. The extent of oligonucleotide degradation was assessed at several time points by IE HPLC (Materials and Methods). Analysis of the reaction mixtures revealed that both oligoribonucleotide N3[prime]->P5[prime] phosphoramidate 18 and its control RNA phosphodiester counterpart 19 are digested with similar rates by RNase T1, and have very similar half-lives of 24 and 26 min, respectively. It should be noted that the final products observed by IE HPLC for the reaction mixtures from digestions of natural RNA and ribophosphoramidates were different. This might indicate that some of the intermediate cleavage products have differing susceptibilities toward further digestion by RNase T1. Moreover, when control RNA oligonucleotide 19 was treated with RNase A its half-life was <2 min, while the phosphoramidate counterpart 18 showed more resistance, having a half-life of ~9 min. As was observed for RNase T1, the final products of digestion were different according to IE HPLC analysis. The higher resistance of ribophosphoramidates to RNase A and not to RNase T1 likely indicates differences in the catalytic mechanism of hydrolysis of RNA internucleotide linkages by these enzymes. It may also be a reflection of an influence by the 3[prime]-amino group on the reactivity of the 2[prime]-hydroxyl moiety involved in the cleavage reaction by RNase A and formation of 2[prime],3[prime]-cyclophosphates.
To examine the alkaline stability of oligoribonucleotide phosphoramidates, a model DNA/RNA chimera was prepared. The N3[prime]->P5[prime] phosphoramidate 9mer (5[prime]-d-GGG-r-G-d-CUAAG-3[prime]) had a single 3[prime]-amino-2[prime]-hydroxyguanosine residue incorporated at the fourth position from the 5[prime]-end. The alkaline lability of this compound under basic conditions was monitored by IE HPLC. When this chimeric oligonucleotide was treated with concentrated aqueous ammonia at 55°C the 9mer was cleaved into two faster eluting fragments, as judged by IE HPLC analysis, presumably a 5mer and a 4mer. Surprisingly, the RNA phosphoramidate linkage proved to be quite robust, with a half-life of 19 h. This indicates the electron donating effect of the 3[prime]-amino group on both the 2[prime]-hydroxyl group and the phosphorous atom, which results in reduction of their reactivities. Oligoribonucleotide phosphoramidate dinucleotides have previously been shown to display similar enhanced stabilities toward alkaline hydrolysis (16).
Thermal stabilities of oligoribophosphoramidate duplexes
In order to assess the ability of oligoribonucleotideN3[prime]->P5[prime] phosphoramidates to form duplexes with complementary RNA and DNA strands, thermal dissociation experiments were performed. The melting temperatures (Tm) for a variety of duplexes were measured under close to physiological conditions and the results are presented in Table 1. We found that all complexes formed with heterobasic oligoribonucleotideN3[prime]->P5[prime] phosphoramidates 13, 14, 16, 18 and their RNA complements have higher Tm values than observed for the isosequential deoxyphosphoramidate counterparts 12, 15, 17 (Table 1). The RNA phosphoramidate Tm values were up to 13.0°C higher than for the complexes containing deoxyphosphoramidate linkages, depending on length of the duplex (compare experiments 2, 3, 5 and 7 with experiments 1, 4 and 6, Table 1). This increase correlates to a [Delta]Tm of 0.5-1.0°C per 2[prime]-hydroxyl-containing residue. Also, a duplex formed by RNA phosphoramidate 18 was noticeably more stable (Tm higher by 20.7°C) than the complex formed by the isosequential phosphodiester RNA oligomer 19 (compare experiments 7 and 8, Table 1).
The obtained experimental data indicate that the ribophosphoramidate oligonucleotides in general form more stable duplexes than their deoxy counterparts. This prompted us to attempt to further increase their binding propensity by incorporating 2,6-diaminopurine residues instead of adenine bases. 2,6-Diaminopurine has an additional amino group at the 2 position which potentially enables this base to form a third hydrogen bond with thymidine or uridine, while at the same time increasing duplex stabilization through stacking interactions (Fig. 4). Results reported in the literature also indicate that the structure of the duplex formed by 2,6-diaminopurine-containing oligomers may enhance or reduce the effects of this base (31,32). Complexes of 2,6-diaminopurine-containing oligomers with RNA appear to be much more stable than with DNA strands, suggesting an influence by the base on sugar pucker. This indicates that oligonucleotides with a higher population of N-type nucleotides, like phosphoramidates, may enhance the stabilizing effects of 2,6-diaminopurine. A model pentamer 20 (Table 1) containing four 2,6-diaminopurines and a 5[prime]-terminal adenosine was synthesized to investigate the binding properties of this base in the context of the ribophosphoramidate sugar-phosphate backbone. We found that this oligonucleotide binds extremely well with complementary RNA, but very poorly to DNA (compare experiments 9 and 10, Table 1). Even with 10 mM magnesium chloride present in the melting buffer, oligonucleotide 20 did not form a complex with poly(dT). However, it formed a very stable duplex with poly(U): Tm values of 47.0 and 57.4°C, in low and high ionic strength buffers, respectively. This corresponds to a remarkable ~6.9°C per residue increase in Tm duplex melting temperature when compared to the adenine-containing RNA phosphoramidate pentamer 23. We are unaware of any other adenine base modification described in the literature that display such an effect on the melting temperature of oligonucleotides. Additional comparisons were made to a deoxyadenylate pentamer phosphoramidate 21, experiments 11 and 12, which showed that duplexes of 2[prime]-deoxyphosphoramidates also have lower melting temperatures than the 2,6-diaminopurine-containing pentamer. Interestingly, a 2[prime]-deoxyadenylate phosphoramidate 10mer 22 has a comparable duplex stability to the pentamer 20 (compare experiments 10 and 14, Table 1).
Figure 4. Base pairing of 2,6-diaminopurine with thymidine and uridine
The enhanced binding properties of pentamer 20 to poly(U) is probably not solely a result of the additional hydrogen bonding capabilities of the base. In aqueous solutions an additional hydrogen bond may contribute ~1°C or 0.4-1.3 kcal/mol to duplex Tm values (33). Thus, taking only hydrogen bonding into account, the net increase in melting temperature for oligomer 20 relative to 23 should be ~5°C, and yet we have observed a 28°C increase. It is possible that a synergistic interaction between the N3[prime]->P5[prime] phosphoramidate backbone and 2,6-diaminopurine bases results in a duplex structure which allows formation of additional interstrand hydrogen bonds, potentially mediated by surrounding water molecules. Additionally, increased [pi]-[pi] stacking interactions might account for a significant fraction of the enhanced binding of 2,6-diaminopurine-containing oligomers. Thorough NMR, X-ray and molecular modeling studies will likely be able to address the structural features of the 2,6-diaminopurine-containing phosphoramidate duplexes, which result in the observed significant stabilization.
In summary, based on the experimental data it appears that N3[prime]->P5[prime] oligoribophosphoramidates offer an attractive scaffold in terms of efficient recognition of RNA targets. We think that this recognition capability is primarily due to an exceptionally high population of C3[prime]-endo sugar conformations of the 3[prime]-amino-2[prime]-hydroxyl nucleotide constituents, where both 3[prime]- and 2[prime]-substituents are able to stabilize this form of sugar puckering. The additional hydration afforded by the 3[prime]-amino and 2[prime]-hydroxyl groups also biases these oligonucleotides into adopting conformations with enhanced RNA binding characteristics. These attributes in conjunction with the developed efficient approach to synthesis of mixed-base N3[prime]->P5[prime] oligoribophosphoramidates make this class of nucleic acid analogs useful as hydrolytically stable structural and functional RNA mimetics.
REFERENCES
*To whom correspondence should be addressed. Tel: +1 650 473 8611; Fax: +1 650 473 7750; Email: sgryaznov{at}geron.com
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