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RNA mimetics: oligoribonucleotide N3[prime]->P5[prime] phosphoramidates
Introduction
Results And Discussion
Preparation of the monomers
Synthesis of oligoribonucleotide N3[prime]->P5[prime] phosphoramidates
Hydrolytic stability of oligoribonucleotide N3[prime]->P5[prime] phosphoramidites
Thermal stability of the phosphoramidate duplexes and triplexes
Materials And Methods
General methods
3-Azido-1,2-bis-O-acetyl-5-O-benzoyl-3-deoxy-d-ribofuranose (3)
3[prime]-Azido-2[prime]-O-acetyl-5[prime]-O-benzoyl-3[prime]-deoxy-[beta]-d-ribofurano-sylthymine (4t)
3[prime]-Azido-3[prime]-deoxy-[beta]-d-ribofuranosylthymine (5t)
3[prime]-Azido-5[prime]-O-(4,4[prime]-dimethoxytriphenylmethyl)-3[prime]-deoxy-[beta]-d-ribofuranosylthymine (6t)
3[prime]-Amino-5[prime]-O-(4,4[prime]-dimethoxytriphenylmethyl)-3[prime]-deoxy-[beta]-d-ribofuranosylthymine (1t)
3[prime]-Amino-2[prime]-O-(t-butyldimethylsilyl)-3[prime]-deoxy-[beta]-d-ribofurano-sylthymine (7t)
2[prime]-O-(t-Butyldimethylsilyl)-3[prime]-(4-methoxytriphenylmethyl-amino)-3[prime]-deoxy-[beta]-d-ribofuranosylthymine (8t)
2[prime]-O-(t-Butyldimethylsilyl)-3[prime]-(4-methoxytriphenylmethyl)-amino-3[prime]-deoxy-[beta]-d-ribofuranosylthymine 5[prime]-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (2t)
3[prime]-Azido-2[prime]-O-acetyl-5[prime]-O-benzoyl-N6-benzoyl-3[prime]-deoxy-adenosine (4a)
3[prime]-Azido-N6-benzoyl-3[prime]-deoxyadenosine (5a)
3[prime]-Azido-N6-benzoyl-5[prime]-O-(4,4[prime]-dimethoxytriphenylmethyl)-3[prime]-deoxyadenosine (6a)
3[prime]-Amino-N6-benzoyl-5[prime]-O-(4,4[prime]-dimethoxytriphenylmethyl)-3[prime]-deoxyadenosine (1a)
3[prime]-Amino-N6-benzoyl-5[prime]-O-(4,4[prime]-dimethoxytriphenylmethyl)-2[prime]-O-(t-butyldimethylsilyl)-3[prime]-deoxyadenosine (9a)
3[prime]-Amino-N6-benzoyl-2[prime]-O-(t-butyldimethylsilyl)-3[prime]-deoxy-adenosine (7a)
N6-Benzoyl-2[prime]-O-(t-butyldimethylsilyl)-3[prime]-(4-methoxytri-phenylmethylamino)-3[prime]-deoxyadenosine (8a)
N6-Benzoyl-2[prime]-O-(t-butyldimethylsilyl)-3[prime]-(4-methoxytriphenyl-methyl)amino-3[prime]-deoxyadenosine 5[prime]-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (2a)
3[prime]-Azido-3[prime]-deoxyuracil (5u)
3[prime]-Azido-5[prime]-O-(4,4[prime]-dimethoxytriphenylmethyl)-3[prime]-deoxyuracil (6u)
3[prime]-Amino-5[prime]-O-(4,4[prime]-dimethoxytriphenylmethyl)-3[prime]-deoxyuracil (1u)
3[prime]-Amino-5[prime]-O-(4,4[prime]-dimethoxytriphenylmethyl)-2[prime]-O-(t-butyl-dimethylsilyl)-3[prime]-deoxyuracil (9u)
Acknowledgement
References
RNA mimetics: oligoribonucleotide N3[prime]->P5[prime] phosphoramidates
ABSTRACT
INTRODUCTION
Studies involving various RNA molecules have recently attracted significant attention because, in part, of the unique properties of some RNA oligomers. Thus, these compounds can act catalytically as RNA and DNA cleaving agents, as well as ligating enzymes (1-5). Aptamers made from ribonucleosides and their derivatives produced by in vitro selection exhibit high affinity binding and sequence-specific recognition of various ligands (6-9). Natural RNA regulatory elements, recognized by RNA-binding proteins, play an important role in regulation of HIV viral proliferation (10,11). Additionally, RNA molecules have been strongly implicated in the origin of life on Earth (12,13). However, native RNA molecules are chemically unstable and readily degraded by cellular nucleases. Significant effort has been made to prepare hydrolytically stable RNA analogs, which could be of interest for various therapeutic and diagnostic applications (14). Among the prepared compounds are phosphodiester and phosphorothioate RNA oligonucleotide analogs containing 2[prime]-fluoro, 2[prime]-amino and 2[prime]-alkoxy substituents, which are stabilized against hydrolysis by nucleases and functionally similar to native RNA molecules (15,16).
Figure 1. Structure of the oligoribonucleotide N3[prime]->P5[prime] phosphoramidate internucleoside groups. Recently, the synthesis of 2[prime]-deoxyoligonucleotide N3[prime]->P5[prime] phosphoramidate analogs of DNA was described (17). These compounds are resistant to nucleases, form stable duplexes with complementary single-stranded (ss)RNA and DNA, as well as stable triplexes with double-stranded (ds)DNA targets (18,19). Moreover, 2[prime]-deoxy N3[prime]->P5[prime] phosphoramidate duplexes adopt an A-type of helix and these compounds can spatially and functionally mimic isosequential RNA structural elements (20,21). Random size short CG-containing oligoribonucleotide N3[prime]->P5[prime] phosphoramidates were obtained via self-polymerization of preformed dimer blocks in aqueous buffers (22). Ribo N3[prime]->P5[prime] phosphoramidate dimers and trimers were also synthesized in aqueous solutions (23). Here we report the efficient synthesis of RNA mimetic oligoribonucleotide N3[prime]->P5[prime] phosphoramidates (Fig. 
RESULTS AND DISCUSSION
Preparation of the monomers
The synthesis of the oligoribonucleotide N3[prime]->P5[prime]-phosphoramidates was accomplished by using two different types of monomers, 1 and 2 (Schemes 1 and 2), depending on the oligonucleotide assembly protocol. Thus, key 5[prime]-O-DMT-3[prime]-aminoribonucleosides 1a, 1t and 1u were prepared according to Scheme 1. These compounds were incorporated into the oligonucleotide phosphoramidates via the oxidative phosphorylation method (17,24). First, the 3[prime]-azido compounds 4a, 4t and 4u were synthesized by condensing the trimethylsilylated bases with 3-azido-1,2-di-O-acetyl-5-O-benzoyl-3-deoxy-d-ribofuranose 3 in the presence of tin(IV) chloride (25). Removal of the sugar protecting groups with either ammonia/ethanol or sodium hydroxide afforded compounds 5a, 5t and 5u, which were converted into the corresponding 3[prime]-amino-5[prime]-O-dimethoxytrityl nucleosides 1a, 1t and 1u via tritylation with DMT chloride, followed by catalytic hydrogenation. Additionally, 2[prime]-O-protected 9u was obtained through silylation of 1u with t-butyldimethylsilyl chloride (Scheme 1).
Scheme 1. Alternatively, 5[prime]-(2-cyanoethyl-N,N[prime]-diisopropyl)phosphoramidite building blocks 2a and 2t were prepared (Scheme 2). Thus, compounds 1a and 1t were treated with t-butyldimethylsilyl chloride to protect the 2[prime]-hydroxyl group and subsequently detritylated with 80% aqueous acetic acid to afford the 3[prime]-amino-2[prime]-silylated derivatives 7a and 7t. In order to selectively protect the 3[prime]-amino group, the 5[prime]-hydroxyl group was transiently protected with a trimethylsilyl residue (26), followed by addition of monomethoxytrityl chloride and N-methylimidazole. The reaction mixture was stirred at 39°C for 48 h. Anhydrous conditions and temperature control are crucial in order to avoid possible 5[prime]-O-monomethoxytritylation. Removal of the 5[prime]-O-trimethylsilyl group with ammonia and purification on silica gel resulted in the precursors 8a and 8t, which were subsequently phosphitylated to obtain the desired building blocks 2a and 2t. The structure of the synthesized nucleosides was confirmed by 1H, 13C and 31P NMR spectroscopy (Materials and Methods). Scheme 2.
Synthesis of oligoribonucleotide N3[prime]->P5[prime] phosphoramidates
Two different approaches for the synthesis of the title oligonucleotides were employed. We first tried the oxidative phosphorylation method, based on carbon tetrachloride-driven oxidative phosphorylation of the 3[prime]-aminonucleosides in solution by the 5[prime]-H-phosphonate diester groups of the growing oligonucleotide on a solid phase support, as originally described for the preparation of the 2[prime]-deoxynucleotide N3[prime]->P5[prime]phosphoramidates (17,24). Thus, the model dimers r(Unp)dT and r(Anp)dT were prepared using 2[prime]-hydroxyl unprotected compounds 1a and 1u. The products were analyzed and purified by RP HPLC. The 31P NMR and mass spectra of the dimers confirmed the presence of an internucleotide phosphoramidate linkage with a characteristic resonance at ~7.0 p.p.m. and molecular composition of the synthesized compounds (Materials and Methods). Coupling times for 3[prime]-aminoribonucleosides were extended up to 1 h and coupling efficiency was estimated to be in the range 85-91% as judged by RP HPLC analysis. It is important to note that the formation of dinucleosides with 2[prime]-5[prime] phosphodiester links, as a result of coupling of unprotected 2[prime]-hydroxyl rather than 3[prime]-amino groups, was not detected by 31P NMR analysis of the reaction mixtures.
The same synthetic protocol was used to introduce one or two 3[prime]-aminoribonucleosides into longer oligodeoxynucleotide N3[prime]->P5[prime] phosphoramidates. The coupling step of ribonucleoside 1u was followed by capping with acetic or phenoxyacetic anhydride to avoid phosphitylation of the unprotected 2[prime]-hydroxyl group during the next chain elongation cycle. Thus, compound 16 (Table 1) was prepared and isolated by IE HPLC.
One of the main drawbacks of using the 2[prime]-OH unprotected nucleosides 1a and 1u for synthesis of ribophosphoramidates is the accompanying cleavage of the internucleoside phosphoramidate linkage during oligomer deprotection with ammonia. Approximately 20% of the internucleoside ribophosphoramidate groups in compound 16 (Table 1) were cleaved upon treatment with concentrated aqueous ammonia for 1 h at 55°C, as judged by IE HPLC analysis of the reaction mixture. To overcome this problem we synthesized the dimer r(Unp)dT using the 2[prime]-O-TBDMS-protected nucleoside 9u. After deprotecting ammonolysis, the 2[prime]-O-TBDMS group was selectively and cleanly removed with 1 M tetrabutylammonium fluoride in THF, resulting in a fully deprotected dimer. Unfortunately, the reduced observed coupling yields for the 2[prime]-O-TBDMS-3[prime]-aminonucleosides (~60% as was judged by RP and IE HPLC analysis), which is presumably due to steric hindrance by the bulky TBDMS group, limit the utility of this approach.
An alternative chain assembly method, based on a phosphoramidite transfer reaction, has recently been introduced for the synthesis of oligo-2[prime]-fluoro-2[prime]-deoxynucleoside N3[prime]->P5[prime] phosphoramidates and oligo-2[prime]-deoxynucleoside N3[prime]->P5[prime] phosphoramidates, using 3[prime]-(protected)amino 5[prime]-O-(cyanoethyl-N,N[prime]-diisopropylamino)phosphoramidite nucleosides (27-29). Thus, this approach was extended to synthesis of the ribophosphoramidates. The key aminonucleoside building blocks 2a and 2t were prepared according to Scheme 2. The oligoribonucleotide N3[prime]->P5[prime] phosphoramidates were synthesized using these monomers and the assembly protocol outlined in Scheme 3. Thus, the diisopropylamino group of incoming 5[prime]-O-phosphoramidite 2 was exchanged for the 3[prime]-amino group of the solid support-bound 3[prime]-amino-2[prime]-O-TBDMS-ribonucleoside. Coupling time was 10 min using 1H-tetrazole as activator. The newly formed internucleoside phosphoramidite diester group was then oxidized with aqueous iodine to the phosphoramidate one. Following detritylation of the 3[prime]-amino group, the oligonucleotide chain elongation cycle can be repeated until the desired oligomer is assembled. The prepared fully protected oligoribonucleotide phosphoramidate was cleaved from the solid support and base protecting and phosphate protecting groups were removed with ammonia. Finally, 2[prime]-O-TBDMS groups were removed with tetrabutylammonuim fluoride and the completely deprotected oligoribonucleotide N3[prime]->P5[prime] phosphoramidates were analyzed and purified by IE HPLC (Fig.
Scheme 3. Figure 2. Ion exchange HPLC profile of the crude reaction mixture from synthesis of oligonucleotide 12 (Table 1). A Pharmacia MonoQ 5/5 column and a 1%/min gradient of 1.5 M NaCl, 0.01 M NaOH in 0.01 M NaOH were used for the analysis. The stability of the oligoribophosphoramidates toward hydrolysis by a mixture of snake venom phosphodiesterase and alkaline phosphatase was evaluated. Thus, 0.2 OD260 nm oligonucleotide 12 (Table 1) were treated with 0.02 U snake venom phosphodiesterase and 0.8 U alkaline phosphatase in 0.2 ml 10 mM Tris-HCl buffer, pH 7.8, at room temperature. The reaction mixture was analyzed by IE HPLC at several time points. The analysis demonstrated that the half-life of the full-length ribodecanucleotide 12 was 26 h. A similar resistance to enzymatic hydrolysis in vitro and in vivo was observed for the 2[prime]-deoxy N3[prime]->P5[prime] phosphoramidate counterparts, where the presence of a terminal 3[prime]-amino group also significantly increased hydrolytic stability (17,31). For comparison, the isosequential all phosphodiester 2[prime]-deoxydecathymidilate was completely hydrolyzed to the thymidine nucleoside within 20 min under identical reaction conditions. It would be interesting in the future to study stability of oligoribophosphoramidates towards various RNases. In addition, the stability of the ribophosphoramidates under basic and acidic conditions was studied. The ribophosphoramidate dimers r(Anp)dT and r(Unp)dT were relatively stable in concentrated aqueous ammonia. Incubation of r(Unp)dT and r(Anp)dT for 15 h at 55°C in concentrated aqueous ammonia resulted in ~10 and 5% hydrolysis of the starting compounds, respectively, as judged by RP HPLC. Unlike the dimers, oligomer 12 (Table 1) was completely hydrolyzed to multiple products after incubation under similar conditions for 6 h at 55°C. Additionally, treatment of r(Anp)dT dimer with 40% aqueous acetic acid for 20 h at room temperature followed by 2 h at 55°C resulted in 85% cleavage of the 3[prime]-N-P bond and formation of 3[prime]-aminoriboadenosine and 5[prime]-thymidylic acid with 15% of starting material remaining, as determined by RP HPLC analysis. Similarly, 10mer 12 (Table 1) was completely hydrolyzed by 40% acetic acid within 3 h at 55°C. Hydrolysis resulted in formation of 3[prime]-aminoribothymidine and 3[prime]-amino-5[prime]-ribothy-midylic acid at an ~2:3 ratio, along with two minor unidentified products. This indicates that the acid-catalyzed hydrolysis of the internucleoside ribophosphoramidate group in oligomers may proceed via cleavage of both 3[prime]-N-P and P-O-5[prime] bonds, since a significant amount of 3[prime]-aminoribothymidine nucleoside was formed. This acid-catalyzed hydrolysis reaction may involve participation of neighboring phosphates, unlike the dimers, where only the 3[prime]-N-P linkage was cleaved. The ability of oligoribonucleoside N3[prime]->P5[prime] phosphoramidates to form complexes with complementary DNA and RNA strands was evaluated using thermal dissociation experiments. Melting temperatures (Tm) were determined for duplexes and triplexes formed by the ribophosphoramidates under close to physiological salt and buffer conditions. The results are summarized in Table 1. Substitution of the 3[prime]-amino-2[prime]-deoxyfuranose rings by 3[prime]-amino-2[prime]-ribo counterparts in uniformly modified thymidine oligomers resulted in significant stabilization of the duplexes formed with both DNA and RNA complements. The increases in melting temperatures of the duplexes ([Delta]Tm) were up to 14.4 and 4.1°C for the complexes with DNA and RNA strands, respectively (compare experiments 3 and 4 and 6 and 7, Table 1). These correspond to [Delta]Tm of 1.8 and 0.5°C per single nucleoside substitution. The melting temperature of the ribophosphoramidate duplexes was 20.1-30.3°C higher than that for the isosequential 2[prime]-deoxyphosphodiester 10mer 10 (compare experiments 1 and 2 and 6 and 7, Table 1). Additionally, oligoribophosphoramidate 12 forms a very stable triple-stranded complex with the dA10·dT10 duplex region of the hairpin target (experiment 8, Table 1). Melting temperatures for the triplex were 44 and 61.5°C in 150 mM NaCl buffer, pH 7, without or with additional 10 mM MgCl2, respectively. These Tm values were 12.0-14.3°C higher than those for the isosequential 2[prime]-deoxyphosphoramidate counterpart (compare experiments 5 and 8, Table 1). Parent phosphodiester decamer 10 did not form a triplex under identical experimental conditions. Triplex thermal dissociation curves were recorded at both 260 and 282 nm, where change in absorbency is characteristic for dissociation of T·AT triplets. Unlike the oligopyrimidines, oligopurine ribophosphoramidate 14 formed more stable duplexes with DNA and less stable duplexes with RNA complements than the isosequential 2[prime]-deoxyphosphoramidate, with [Delta]Tm values of 4.9 and -13.1°C, respectively (compare experiments 9 and 10 and 11 and 12, Table 1). Detailed NMR and X-ray structural analysis will be needed to explain the difference in thermal stability of duplexes formed by purine- and pyrimidine-containing oligoribophosphoramidates. Interestingly, incorporation of one or two 3[prime]-aminoribonucleosides into 2[prime]-deoxyphosphoramidate oligomers resulted in a 3.5-10°C reduction in duplex thermal stability (compare experiments 15 and 16 and 17 and 18, Table 1). This was unexpected in view of similar sugar puckering of 3[prime]-aminoribo and 3[prime]-amino-2[prime]-deoxynucleosides. Thus, 1H NMR spectra of the synthesized 3[prime]-aminoribonucleosides and 3[prime]-aminoriboadenosine in the r(Anp)dT dimer indicate a C3[prime]-endo or N-type of sugar puckering for the ribofuranose ring: the J3(H1[prime]-H2[prime]) coupling constants for the 3[prime]-aminoribonucleosides and for 3[prime]-aminoriboadenosine within the dimer were <1 Hz and similar to those for 3[prime]-amino-2[prime]-deoxynucleosides (30). Furthermore, the opposite effect was observed for 2[prime]-fluoro N3[prime]->P5[prime] phosphoramidates, where substitution of one or two 2[prime]-deoxynucleosides by 2[prime]-fluoro counterparts resulted in duplex stabilization by ~2°C per substitution (27). Table 1. We think that the increase in the thermal stability of duplexes and triplexes of ribophosphoramidates relative to the 2[prime]-deoxyphosphoramidate counterparts, as well as the parent phosphodiesters, is due to the further increase in the population of N-type sugar conformations for the 3[prime]-amino-2[prime]-hydroxyl nucleosides, which is determined by a cooperative and additive effect of 3[prime]-amino and 2[prime]-hydroxyl groups on furanose puckering. Additionally, improved hydration of the phosphoramidate duplexes, due to the presence of the 3[prime]-amino group as an additional donor and acceptor of hydrogen bonds, as well as 2[prime]-hydroxyl, may contribute to the increase in thermal stability (21). In summary, an efficient method for the synthesis of enzyme-resistant RNA analogs, oligoribonucleotide N3[prime]->P5[prime] phosphoramidates, has been developed. These compounds could be potentially used as hydrolytically stable structural and functional mimetics of native RNA for preparation of ribozymes, aptamers and RNA decoys, as well as other molecules.
Hydrolytic stability of oligoribonucleotide N3[prime]->P5[prime] phosphoramidites
Thermal stability of the phosphoramidate duplexes and triplexes
Experiment
Oligonucleotide
Target
Tm (°C)a
1
d-TTTTTTTTTT, 10
poly(dA)
28.3
2
As experiment 1
poly(A)
26.2
3
d-TnpTnpTnpTnpTnpTnpTnpTnpTnpT, 11
poly(dA)
34.0
4
As experiment 3
poly(A)
50.7
5
As experiment 3
d-A10C4T10
32.0, 47.2b
6
dT-r-TnpTnpTnpTnpTnpTnpTnpTnpTn, 12
poly(dA)
48.4
7
As experiment 6
poly(A)
56.5
8
As experiment 6
d-A10C4T10
44.0, 61.5b
9
d-AnpAnpAnpAnpAnpAnpAnpAnpAnpA, 13
poly(dT)
20.0
10
As experiment 9
poly(U)
50.6
11
As experiment 9
11c
43.4
12
dA-r-AnpAnpAnpAnpAnpAnpAnpAnpAn, 14
poly(dT)
24.9
13
As experiment 12
poly(U)
37.7
14
As experiment 12
12c
52.9
15
d-UnpUnpUnpUnpUnpUnpUnpUnpUnpT, 15
poly(dA)
18.5, 38.2b
16
As experiment 15
poly(A)
38.1, 47.2b
17
d-(Unp)4-r-(Unp)2-d-(Unp)3T, 16
poly(dA)
15.0, 28.9b
18
As experiment 17
poly(A)
30.0, 37.4b
MATERIALS AND METHODS
General methods
Oligonucleotide N3[prime]->P5[prime] phosphoramidates, containing one or two 3[prime]-aminoribonucleosides, were synthesized using the oxidative phosphorylation method on an ABI 394 synthesizer as previously described (18). The coupling step was followed by capping with 10% phenoxyacetic anhydride in the presence of 12.5% N-methylimidazole in THF if 2[prime]-(unprotected)hydroxyl-3[prime]-aminonucleosides were used. Uniformly modified oligoribonucleotide N3[prime]->P5[prime] phosphoramidates were prepared manually in a syringe using the amidite transfer reaction (27,28) and the following protocol: (i) detritylation, 3% dichloroacetic acid in dichloromethane, 1.5 min; (ii) coupling, 0.1 M phosphoramidite and 0.5 M tetrazole in acetonitrile, 10 min; (iii) oxidation, 0.1 M iodine in tetrahydrofuran/pyridine/water, 10/10/1 (v/v/v), 2 min. Chemical steps within the cycle were followed by acetonitrile or dichloromethane washing depending on the reaction solvent. Cleavage from the solid support and deprotection were accomplished with concentrated aqueous ammonia in ethanol, 3/1 (v/v), 1.5 h, 55°C for homothymidine or uracil-containing oligomers and 7.5 h, 55°C for purine-containing compounds. The oligonucleotides were concentrated to dryness in vacuo and the 2[prime]-t-butyldimethylsilyl groups were removed by treatment with 0.5 ml 1 M tetrabutylammonium fluoride in THF for 16 h. The solvent was removed in vacuo and the oligonucleotides were desalted by gel filtration on a Pharmacia NAP-5 column. Oligonucleotides were analyzed and purified by IE HPLC and were desalted by gel filtration immediately after purification and stored frozen at -18°C. Thermal denaturation experiments were performed as described previously (24) in the buffers listed in Table 1.
Thin layer chromatography (TLC) was carried out on DC-Fertigplatten SIL G-25 UV254 silica gel plates (Macherey-Nagel) in dichloromethane/methanol systems. The nucleosides used for oligonucleotide chain assembly were practically pure according to TLC analysis.
The model dimers r(Unp)dT and r(Anp)dT were synthesized on an automated DNA synthesizer on the 1 µmol scale using 2[prime]-unprotected or 2[prime]-O-TBDMS-protected 3[prime]-aminonucleosides. Coupling times were 1 and 2 h for 2[prime]-unprotected and 2[prime]-O-TBDMS-protected nucleosides, respectively. After deblocking with NH3/ethanol (3:1 v/v), the dimers were analyzed and purified by RP HPLC. In the case of nucleoside 9u, the 2[prime]-O-TBDMS group was removed by treatment with 0.5 ml 1 M tetrabutylammoium fluoride in THF at room temperature for 4 h. The observed coupling yields were 85-91 and 55-60% for 2[prime]-unprotected and 2[prime]-protected 3[prime]-aminonucleosides, respectively. 31P NMR: (D2O) [delta] 7.75 p.p.m. for r(Unp)dT and 7.59 p.p.m. for r(Anp)dT. Mass spectrum for r(AnpT): (M-H)-, m/z calculated and observed, 569. Acid hydrolysis of 0.6 OD of the dimer r(Unp)dT was carried out in 0.5 ml 80% AcOH at room temperature, 100 µl stock solution were evaporated and analyzed by RP HPLC. After 24 h, the dimer [retention time (RT) 13.5 min] was hydrolyzed to 3[prime]-aminouridine (RT 3.7 min), 5[prime]-thymidylic acid (RT 7.6 min) and dT (RT 9.3 min). For enzymatic digestion, 0.2 A260 U oligonucleotide were treated with 0.02 U snake venom phosphodiesterase and 0.8 U alkaline phosphatase (Sigma, St Louis, MO) in 0.2 ml 10 mM Tris-HCl, pH 7.8, at room temperature. Aliquots from the reaction were taken at multiple points and analyzed by IE HPLC.
3-Azido-1,2-bis-O-acetyl-5-O-benzoyl-3-deoxy-d-ribofuranose (3)
Compound 3 was synthesized starting from 1,2-O-isopropylidene-[alpha]-d-xylofuranose analogously to the described procedure for 3-azido-1,2-di-O-acetyl-5-O-(4-methylbenzoyl)-3-deoxy-d-ribo-furanose (32).
3[prime]-Azido-2[prime]-O-acetyl-5[prime]-O-benzoyl-3[prime]-deoxy-[beta]-d-ribofurano-sylthymine (4t)
To a solution of 2,4-bis(trimethylsilyl)thymine prepared from 2.0 g thymine and 3 (1.40 g, 3.85 mmol) in 80 ml dry acetonitrile was added 2 ml 1 M solution of stannic chloride in dichloroethane and the stirred reaction mixture was refluxed for 4 h. The solvent was evaporated and 100 ml CH2Cl2 and 100 ml saturated NaHCO3 were added to the reaction mixture. The organic phase was separated and the aqueous phase was back-extracted with 100 ml CH2Cl2. The combined organic phases were dried over Na2SO4, filtered and evaporated to dryness. The residue was purified by silica gel column chromatography eluting with CH2Cl2:MeOH (95:5 v/v) to afford 4t as a white foam (1.39 g, 84.1%). 1H NMR: (CDCl3) [delta] 1.68 (s, 3H), 2.19 (s, 3H), 4.28-4.74 (3 m, 4H), 5.56 (dd, J = 5.7 Hz and J = 4.4), 5.87 (d, J = 4.4 Hz, 1H), 7.28-8.10 (m, 6H), 8.75 (s, 1H).
3[prime]-Azido-3[prime]-deoxy-[beta]-d-ribofuranosylthymine (5t)
Compound 4t (1.35 g, 3.14 mmol) was dissolved in 20 ml ethanol and 40 ml 25% aqueous NH3 was added. The solution was stirred at room temperature for 16 h. The solvent was removed in vacuo and the residue was purified by silica gel column chromatography eluting with CH2Cl2:MeOH (95:5 v/v) to yield 5t as a white foam (650 mg, 73.1%). 1H NMR: (D2O) [delta] 1.76 (s, 3H), 3.70 (m, 1H), 3.80 (m, 1H), 4.01 (m, 2H), 4.46 (dd, J = 4.4 Hz and J = 5.1 Hz, 1H), 5.75 (d, J = 4.4 Hz, 1H), 7.57 (s, 1H). 13C NMR: (D2O) [delta] 11.88, 60.89, 74.43, 82.48, 89.53, 111.68, 137.55, 151.94, 166.66.
3[prime]-Azido-5[prime]-O-(4,4[prime]-dimethoxytriphenylmethyl)-3[prime]-deoxy-[beta]-d-ribofuranosylthymine (6t)
To compound 5t (620 mg, 2.19 mmol), co-evaporated twice from anhydrous pyridine and redissolved in 30 ml pyridine, was added 4,4[prime]-dimethoxytriphenylmethyl chloride (1.44 g, 4.24 mmol) and 4-dimethylaminopyridine (150 mg, 1.22 mmol). The reaction mixture was stirred for 24 h at room temperature, 100 ml 5% aqueous NaHCO3 were added and the reaction mixture extracted with CH2Cl2 (3 × 100 ml). The combined organic phases were dried over Na2SO4, filtered, and evaporated in vacuo. The residue was co-evaporated twice with toluene and then purified by silica gel column chromatography eluting with CH2Cl2:MeOH (95:5 v/v) to yield 6t as a white foam (920 mg, 71.7%). 1H NMR: (CDCl3) [delta] 1.43 (s, 3H), 3.38 (m, 1H), 3.66 (m, 1H ), 3.79 (s, 6H), 4.14 (m, 1H), 4.32 (m, 1H), 4.65 (m, 1H), 5.97 (d, J = 3.3 Hz, 1H), 6.87-7.44 (m, 13H), 7.75 (s, 1H), 10.49 (s, 1H).
3[prime]-Amino-5[prime]-O-(4,4[prime]-dimethoxytriphenylmethyl)-3[prime]-deoxy-[beta]-d-ribofuranosylthymine (1t)
Compound 6t (1.35 mg, 2.31 mmol) was dissolved in 50 ml EtOH, Pd/C (100 mg, 10% Pd) was added and the reaction mixture was hydrogenated at room temperature for 72 h. The catalyst was removed by filtration and the filtrate evaporated. The residue was purified by silica gel column chromatography eluting with CH2Cl2:MeOH (9:1 v/v) to yield 1t as a white foam (1.05 g, 81.5%). 1H NMR: (CDCl3) [delta] 1.42 (s, 3H), 3.41 (m, 1H), 3.65 (m, 2H), 3.79 (s, 6H), 4.04 (m, 1H), 4.26 (d, J = 4.9 Hz, 1H), 5.82 (s, 1H), 6.84-7.47 (m, 13H), 7.79 (s, 1H).
3[prime]-Amino-2[prime]-O-(t-butyldimethylsilyl)-3[prime]-deoxy-[beta]-d-ribofurano-sylthymine (7t)
Compound 1t (1.05 g, 1.88 mmol) was co-evaporated twice with pyridine and redissolved in 20 ml pyridine. To this stirring mixture t-butyldimethylsilyl chloride (345 mg, 2.29 mmol) was added at room temperature and, 24 h later, 25 ml saturated aqueous NaHCO3 was added and the solution was extracted with 3 × 100 ml CH2Cl2. The combined organic phases were dried over Na2SO4, filtered and evaporated. The residue was co-evaporated twice with toluene and dissolved in 100 ml 80% aqueous acetic acid. The solution was stirred at room temperature for 45 min and then 150 ml methanol were added and the solvents were evaporated. The residue was purified by silica gel column chromatography eluting with CH2Cl2:MeOH (95:5 v/v) to yield 7t as a white foam (505 mg, 72.3%). 1H NMR: (CDCl3) [delta] 0.14 (s, 3H), 0.20 (s, 3H), 0.90 (s, 9H), 1.90 (s, 3H), 3.13 (m, 1H), 3.50 (m, 1H,), 3.91-4.22 (3 m, 3H), 5.67 (s, 1H), 7.76 (s, 1H).
2[prime]-O-(t-Butyldimethylsilyl)-3[prime]-(4-methoxytriphenylmethyl-amino)-3[prime]-deoxy-[beta]-d-ribofuranosylthymine (8t)
Compound 7t (250 mg, 0.67 mmol) was co-evaporated twice with pyridine and redissolved in 20 ml pyridine. Trimethylsilyl chloride (0.75 ml, 8.6 mmol) was added and the solution was stirred at room temperature for 2 h. 4-Methoxytriphenylmethyl chloride (492 mg, 1.59 mmol) and N-methylimidazole (0.13 ml, 1.6 mmol) were added and the solution was stirred at 38°C for 48 h. Saturated aqueous NaHCO3 (25 ml) was then added and the solution was extracted with 3 × 100 ml CH2Cl2. The combined organic phases were dried over Na2SO4 and evaporated in vacuo. The residue was dissolved in 50 ml methanol and 5 ml 25% aqueous NH3 was added. The solution was stirred at room temperature for 15 min and then evaporated. The residue was purified by silica gel column chromatography eluting with CH2Cl2:MeOH (95:5 v/v) to yield a light yellow foam. The foam was dissolved in CH2Cl2 and precipitated from hexane to yield 8t as a white solid (270 mg, 62.6%). 1H NMR: (CDCl3) [delta] 0.04 (s, 3H), 0.14 (s, 3H), 0.90 (s, 9H), 1.87 (s, 3H), 2.91-3.07 (m, 3H), 3.79 (s, 3H), 3.95-4.05 (3 m 3H), 5.58 (s, 1H), 6.80-7.53 (m, 14H), 7.77 (s, 1H), 8.85 (s, 1H). LSIMS (MH+) m/z found 644.3; calculated for C36H45N3O6Si 643.3.
2[prime]-O-(t-Butyldimethylsilyl)-3[prime]-(4-methoxytriphenylmethyl)-amino-3[prime]-deoxy-[beta]-d-ribofuranosylthymine 5[prime]-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (2t)
Compound 8t (240 mg, 0.37 mmol) was dissolved in 5 ml anhydrous CH2Cl2. Diisopropylammonium tetrazolide (62.4 mg, 0.38 mmol) and 2-(cyanoethyl)-N,N,N[prime],N[prime]-tetraisopropylphosphorodiamidite (0.38 ml, 1.12 mmol) were added and the solution was stirred at room temperature for 2 h. Saturated aqueous NaHCO3 (5 ml), H2O (10 ml) and CH2Cl2 (30 ml) were added and, following extraction, the organic phase was separated, dried over Na2SO4 and evaporated in vacuo. The residue was purified by silica gel column chromatography eluting with CH2Cl2:Et3N (99:1 v/v) to yield 2t as a white foam (208 mg, 66.6%). 31P NMR: (CDCl3) [delta] 148.45, 149.81.
3[prime]-Azido-2[prime]-O-acetyl-5[prime]-O-benzoyl-N6-benzoyl-3[prime]-deoxy-adenosine (4a)
To a solution of N6-benzoyl-N6,9-bis(trimethylsilyl)adenine prepared from 3.0 g N6-benzoyladenine and 3 (1.95 g, 5.37 mmol) in 150 ml dry 1,2-dichloroethane was added 4 ml 1 M stannic chloride solution in dichloroethane. The stirred reaction mixture was refluxed for 8 h, then cooled to room temperature, diluted with 100 ml CH2Cl2 and 100 ml saturated aqueous NaHCO3 were added. The organic phase was separated and the aqueous phase was back-extracted with an additional 100 ml CH2Cl2. The combined organic phases were dried over Na2SO4, filtered and evaporated. The residue was purified by silica gel column chromatography eluting with CH2Cl2:MeOH (95:5 v/v) to yield 4a as a white foam (2.05 g, 70.5%). 1H NMR: (CDCl3) [delta] 2.19 (s, 3H), 4.39-5.00 (4 m, 4H), 6.13 (m, 2H), 7.28-8.10 (m, 10H), 8.61 (s, 1H), 9.19 (s, 1H).
3[prime]-Azido-N6-benzoyl-3[prime]-deoxyadenosine (5a)
Compound 4a (870 mg, 1.59 mmol) was dissolved in 80 ml methanol:water (1:1 v/v). The solution was cooled to 0°C and 10 ml 2 M NaOH were added. The solution was stirred at 0°C for 30 min, the reaction mixture was neutralized with 2 M HCl and the solvents were evaporated. The residue was extracted with3 × 150 ml CH2Cl2:MeOH (9:1 v/v), the solvent evaporated and the residue purified by silica gel column chromatography eluting with CH2Cl2:MeOH (9:1 v/v) to yield 5a as a white solid (480 mg, 76.2%). 1H NMR: (DMSO) [delta] 3.65 (m, 1H), 3.75 (m, 1H), 4.05 (m, 1H), 4.35 (m, 1H), 5.05 (dd, J = 3.4 Hz andJ = 5.4 Hz, 1H), 6.08 (d, J = 3.4 Hz, 1H), 7.54-8.07 (2 m, 5H), 8.72 (s, 1H), 8.77 (s, 1H), 11.21 (s, 1H).
3[prime]-Azido-N6-benzoyl-5[prime]-O-(4,4[prime]-dimethoxytriphenylmethyl)-3[prime]-deoxyadenosine (6a)
Compound 5a (450 mg, 1.14 mmol) was co-evaporated twice with pyridine and then dissolved in 30 ml pyridine. 4,4[prime]-Dimethoxytriphenylmethyl chloride (0.75 g, 2.20 mmol) and 4-dimethylaminopyridine (75 mg, 0.61 mmol) were added. The reaction mixture was stirred for 24 h at room temperature, 100 ml 5% aqueous NaHCO3 were added and the reaction mixture was extracted with CH2Cl2 (3 × 100 ml). The combined organic phases were dried over Na2SO4 and evaporated in vacuo. The residue was co-evaporated twice with toluene and then purified by silica gel column chromatography eluting with CH2Cl2:MeOH (94:6 v/v) to yield 6a as a white foam (620 mg, 77.8%). 1H NMR: (CDCl3) [delta] 3.35 (m, 1H), 3.52 (m, 1H), 3.79 (s, 6H), 4.25 (m, 1H), 4.39 (m, 1H), 5.22 (dd, J = 5.2 Hz andJ = 5.5 Hz, 1H), 6.08 (d, J = 5.3 Hz, 1H), 6.79-7.98 (m, 18H), 8.20 (s, 1H), 8.60 (s, 1H), 9.31 (s, 1H).
3[prime]-Amino-N6-benzoyl-5[prime]-O-(4,4[prime]-dimethoxytriphenylmethyl)-3[prime]-deoxyadenosine (1a)
Compound 6a (1.80 g, 2.58 mmol) was dissolved in 60 ml ethanol, Pd/C (100 mg, 10% Pd) was added and the reaction mixture was hydrogenated at room temperature for 24 h. The catalyst was removed by filtration, the filtrate was evaporated and the residue was purified by silica gel column chromatography eluting with CH2Cl2:MeOH (9:1, v/v) to yield 2a as a white foam (1.61 g, 92.7%). 1H NMR: (CDCl3) [delta] 3.42 (m, 1H), 3.52 (m, 1H), 3.79 (s, 6H), 3.80 (m, 1H), 4.08 (m, 1H), 4.60 (dd, J = 1.7 Hz and J = 5.3 Hz, 1H), 6.08 (d, J = 1.6 Hz, 1H), 6.80-8.00 (m, 18H), 8.29 (s, 1H), 8.62 (s, 1H). LSIMS (MH+) m/z found 673.3; calculated for C38H36N6O6 672.3.
3[prime]-Amino-N6-benzoyl-5[prime]-O-(4,4[prime]-dimethoxytriphenylmethyl)-2[prime]-O-(t-butyldimethylsilyl)-3[prime]-deoxyadenosine (9a)
To compound 2a (1.61 g, 2.39 mmol), co-evaporated twice from anhydrous pyridine and redissolved in 40 ml pyridine, were added t-butyldimethylsilyl chloride (390 mg, 2.59 mmol) and N-methylimidazole (0.25 ml, 3.03 mmol). The solution was stirred at room temperature for 24 h, followed by addition of 25 ml saturated aqueous NaHCO3. The reaction mixture was extracted with 3 × 100 ml CH2Cl2. The organic phase was dried over Na2SO4, filtered and evaporated. The residue was co-evaporated twice with toluene and purified by silica gel column chromatography eluting with CH2Cl2:MeOH (95:5 v/v) to yield 9a as a white foam (1.71 g, 90.5%).1H NMR: (CDCl3) [delta] 0.15 (s, 3H), 0.21 (s, 3H), 0.90 (s, 9H), 3.48 (m, 1H), 3.60 (m, 1H), 3.66 (m, 1H), 3.79(s, 6H ), 4.08 (m, 1H), 4.65 (d, J = 4.2 Hz, 1H), 6.10 (s, 1H), 6.80-8.00 (m, 18H), 8.36 (s, 1H), 8.79 (s, 1H), 9.29 (s, 1H).
3[prime]-Amino-N6-benzoyl-2[prime]-O-(t-butyldimethylsilyl)-3[prime]-deoxy-adenosine (7a)
Compound 9a (1.68 g, 2.13 mmol) was dissolved in 100 ml 80% aqueous acetic acid and stirred at room temperature for 60 min. Methanol (100 ml) was added and the solvents were evaporated in vacuo. The residue was co-evaporated twice with methanol and then purified by silica gel column chromatography eluting with CH2Cl2:MeOH (95:5 v/v) to yield 7a as a foam (790 mg, 76.7%). 1H NMR: (CDCl3) [delta] 0.07 (s, 6H), 0.90 (s, 3H), 3.48 (m, 1H), 3.60 (m, 1H), 3.80 (m, 2H), 4.51 (m, 1H), 6.09 (d, J = 2.4 Hz), 1H), 7.54-8.07 (m, 5H), 8.76 (s, 1H), 8.81 (s, 1H).
N6-Benzoyl-2[prime]-O-(t-butyldimethylsilyl)-3[prime]-(4-methoxytri-phenylmethylamino)-3[prime]-deoxyadenosine (8a)
To compound 7a (380 mg, 0.78 mmol), co-evaporated twice with anhydrous pyridine and redissolved in 20 ml pyridine, trimethylsilyl chloride (0.5 ml, 4 mmol) was added and the solution was stirred at room temperature for 2 h. Then 4-methoxytriphenylmethyl chloride (310 mg, 1 mmol) and N-methylimidazole (0.08 ml, 1 mmol) were added. The solution was stirred at 38°C for 48 h and then 25 ml saturated aqueous NaHCO3 were added and the solution extracted with CH2Cl2 (3 × 100 ml). The organic phase was dried over Na2SO4, filtered and evaporated. The residue was dissolved in 50 ml methanol and 5 ml 25% aqueous NH3 were added. The solution was stirred at room temperature for 15 min, concentrated in vacuo and the residue was purified by silica gel column chromatography eluting with CH2Cl2:MeOH (95:5 v/v) to yield 8a as a white foam (345 mg, 58.0%). 1H NMR: (CDCl3) [delta] -0.45 (s, 3H), -0.10 (s, 3H), 0.85 (s, 9H), 3.26-3.33 (m, 3H), 3.71(m, 1H), 3.87 (m, 1H), 3.78 (m, 3H), 4.56 (dd, J = 5.3 Hz andJ = 5.5 Hz, 1H), 6.21 (d, J = 5.6 Hz, 1H), 6.80-8.07 (m, 19H), 8.26 (s, 1H), 8.76 (s, 1H), 9.18 (s, 1H). LSIMS (MH+) m/z found 757.3; calculated for C43H48N6O5Si 756.38.
N6-Benzoyl-2[prime]-O-(t-butyldimethylsilyl)-3[prime]-(4-methoxytriphenyl-methyl)amino-3[prime]-deoxyadenosine 5[prime]-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (2a)
Compound 8a (230 mg, 0.31 mmol) was dissolved in 5 ml anhydrous CH2Cl2. Diisopropylammonium tetrazolide (52.7 mg, 0.32 mmol) and 2-(cyanoethyl)-N,N,N[prime],N[prime]-tetraisopropylphosphorodiamidite (0.32 ml, 0.93 mmol) were added and the solution was stirred at room temperature for 2 h. Saturated aqueous NaHCO3 (5 ml), H2O (10 ml) and CH2Cl2 (30 ml) were added and, following extraction, the organic phase was separated, dried over Na2SO4, filtered and evaporated. The residue was purified by silica gel column chromatography eluting with CH2Cl2:Et3N (99:1 v/v) to yield a white foam (205 mg, 77.6%). 31P NMR: (CDCl3) [delta] 149.60, 149.75.
3[prime]-Azido-3[prime]-deoxyuracil (5u)
Compound 5u was synthesized as previously described (24). 13C NMR: (D2O) [delta] 61.54, 61.89, 74.82, 82.75, 88.61, 102.77, 141.30, 151.56, 163.94.
3[prime]-Azido-5[prime]-O-(4,4[prime]-dimethoxytriphenylmethyl)-3[prime]-deoxyuracil (6u)
To compound 5u (800 mg, 20.97 mmol), co-evaporated twice with anhydrous pyridine and redissolved in 20 ml pyridine, 4,4[prime]-dimethoxytriphenylmethyl chloride (1.92 g, 5.65 mmol) and 4-dimethylaminopyridine (181 mg, 1.47 mmol) were added. The reaction mixture was stirred for 24 h at room temperature. To this was added 5% aqueous NaHCO3 (100 ml) and the reaction mixture was extracted with CH2Cl2 (3 × 100 ml). The combined organic phases were dried over Na2SO4, filtered and evaporated. The residue was co-evaporated twice with toluene and then purified by silica gel column chromatography eluting with CH2Cl2:MeOH (95:5 v/v) to yield 6u as a white foam (1.21 g, 71.3%). 1H NMR: (CDCl3) [delta] 3.48 (m, 1H), 3.65 (m, 1H), 3.79(s, 6H), 4.14 (m, 1H), 4.34 (m, 1H), 4.66 (dd, J = 2.2 Hz andJ = 5.0 Hz, 1H), 5.40 (d, J = 8.1 Hz, 1H), 5.94 (d, J = 2.2 Hz, 1H), 6.87-7.44 (m, 13H), 8.03 (d, J = 8.1 Hz, 1H), 8.65 (s, 1H).
3[prime]-Amino-5[prime]-O-(4,4[prime]-dimethoxytriphenylmethyl)-3[prime]-deoxyuracil (1u)
Compound 6u (1.20 g, 2.10 mmol) was dissolved in 30 ml pyridine:triethylamine (85:15 v/v). The solution was cooled to 0°C and H2S was bubbled through the reaction mixture for 30 min. Stirring was continued for another 60 min at 0°C. The solvent was evaporated and the residue co-evaporated with toluene and subsequently dissolved in CH2Cl2 (100 ml). The organic phase was washed with 5% NaHCO3 (100 ml) and H2O (50 ml), dried over Na2SO4, filtered and evaporated. The residue was purified by silica gel column chromatography eluting with CH2Cl2:MeOH (97:3 v/v) to yield 1u as a white foam (825 mg, 71.9%). 1H NMR: (CDCl3) [delta] 3.41 (m, 1H), 3.68 (m, 2H), 3.79(s, 6H), 3.98 (m, 1H), 4.18 (d, J = 3.9 Hz, 1H), 5.35 (d, J = 7.9 Hz, 1H), 5.80 (s, 1H, H-1[prime]), 6.85-7.44 (m, 13H), 8.15 (d, J = 7.9 Hz, 1H).
3[prime]-Amino-5[prime]-O-(4,4[prime]-dimethoxytriphenylmethyl)-2[prime]-O-(t-butyl-dimethylsilyl)-3[prime]-deoxyuracil (9u)
To compound 1u (130 mg, 0.24 mmol), co-evaporated twice with anhydrous pyridine and redissolved in 10 ml pyridine, t-butyldimethylsilyl chloride (72 mg, 0.48 mmol) was added and the reaction mixture was stirred at room temperature for 24 h. Saturated aqueous NaHCO3 (25 ml) was added and the reaction mixture was extracted with CH2Cl2 (3 × 100 ml). The combined organic phases were dried over Na2SO4, filtered and evaporated. The residue was co-evaporated twice with toluene and then purified by silica gel column chromatography eluting with n-hexane:EtOAc (1:1 v/v) to yield 9u as a white foam (115 mg, 73.3%). 1H NMR: (CDCl3) [delta] 0.18 (s, 3H), 0.29 (s, 3H), 0.95(s, 9H), 3.53-3.94 (3 m, 4H), 3.81 (s, 6H), 4.14 (d, J = 3.9 Hz, 1H), 5.32 (d, J = 8.1 Hz, 1H), 5.78 (s, 1H, H-1[prime]), 6.86-7.44 (m, 13H), 8.21 (d, J = 8.1 Hz, 1H).
ACKNOWLEDGEMENT
We would like to thank Krisztina Pongracz for expert assistance in the preparation of this manuscript.
REFERENCES
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