ABSTRACT
Zwitterionic, net neutral oligonucleotides containing alternating negatively charged N3' -> P5' phosphoramidate monoester and positively charged phosphoramidate diester groups were synthesized. The ability of zwitterionic phosphoramidates to form complexes with complementary DNA and RNA was evaluated. Stoichiometry and salt dependency of these complexes were determined as a function of the nature of the heterocyclic bases of the zwitterionic compounds. Unlike the melting temperatures of the natural phosphodiester-containing oligomers, the Tm of the duplexes formed with the zwitterionic oligothymidylates was salt concentration independent. The thermal stability of these duplexes was much higher with [Delta]Tm values of 20-35°C relatively to phosphodiester counterparts at low salt concentrations. The zwitterionic oligoadenylate formed only 2Py:1Pu triplexes with complementary poly(U) or poly(dT) strands. The thermal stability of these complexes was dependent on salt concentration. Also, the Tm values of the complexes formed by the zwitterionic oligoadenylate with poly(U) were 6-41°C higher than for the natural phosphodiester counterpart. Triplexes of this compound with poly(dT) were also more stable with a [Delta]Tm value of 22°C at low salt concentrations. Complexes formed by the zwitterionic oligonucleotides with complementary RNAs were not substrates for RNase H. Surprisingly, the duplex formed by the all anionic alternating N3' -> P5' phosphoramidate-phosphodiester oligothymidylate and poly(A) was a good substrate for RNase H.
Synthetic oligodeoxyribonucleotides represent a class of compounds of major interest for diagnostic as well as for therapeutic applications. These compounds, as therapeutic agents, can be rationally designed to specifically interfere with gene expression via antisense or antigene modes of action (1). Moreover, oligonucleotide aptamers with high affinity to the selected protein can specifically bind and inactivate a target protein either inside or outside the cells (2). Several structural modifications have been introduced to improve the binding properties of oligonucleotides and their resistance to enzymatic hydrolysis (3). Oligonucleotide alkylphosphotriesters (4), methylphosphonates (5) and phosphorothioates (6) were the first oligonucleotide analogues studied. These compounds are more resistant toward cellular nucleases, and they were used in various in vitro and in vivo systems, particularly the phosphorothioates, as antisense agents. Cationic and net-neutrally charged oligonucleotides have been also prepared by the introduction of positively charged substituents onto the nucleosides bases (7) or onto the sugar-backbone (8-10). The cellular uptake of oligonucleotides was significantly improved by conjugation of the positively charged protein poly-l-Lysine to oligonucleotides directed against vesicular stomatitis virus (11) and HIV (12). Additionally, neutral oligonucleotides are somewhat more effective in forming triplexes with double-stranded (ds) targets DNA than the corresponding unmodified compound. Modified oligonucleotides containing positively charged groups at the internucleoside phosphates also form triplexes more efficiently than do natural phosphodiesters (13).
Another new class of interesting compounds, oligonucleotide N3' -> P5' phosphoramidates (14-16), has been recently developed. The oligonucleotide N3' -> P5' phosphoramidates show high-binding affinity to the single-stranded (ss) DNA and RNA as well as to dsDNA (17), and also demonstrate good resistance against enzymatic hydrolysis (18). We sought to combine the two classes of oligonucleotides into one and to prepare the net charged neutral and zwitterionic oligonucleotide N3' -> P5' phosphoramidates.
Thus, oligonucleotides with alternating internucleoside, negatively charged N3' -> P5' monoester amide, and positively charged diester amide groups were synthesized (Fig. 1). These compounds form stable complexes with complementary ssDNA and ssRNA. The nature of the complexes being formed was determined by the base composition of the oligonucleotide. Thus, the zwitterionic pyrimidine phosphoramidate form duplexes with ssRNA and ssDNA polypurines with salt independent thermal stability, while the zwitterionic purines form salt independent triplexes of 2Py:1Pu strand composition with their complementary targets.
Two different strategies were used to assemble the zwitterionic oligonucleotide N3' -> P5' phosphoramidates as shown in Schemes 1 and 2. In the first approach, 3'-aminogroup-containing dimer building blocks with an internucleoside 3-(N,N-dimethylamino)-propylamino group were assembled in solution. This was accomplished viacoupling of the 5'-O-DMTr-nucleoside-3'-H-phosphonate to the 5'-hydroxyl-3'-amino-group protected nucleoside, followed by the oxidative amidation of the internucleoside H-phosphonate with 3-(N,N-dimethylamino)-propylamine (19,20). Then, the 3'-NH-protected group of the dimer, monomethoxytrityl or Fmoc was removed by acid or base treatment respectively, resulting in compounds 5h or 5d (Scheme 1). Removal of the 3'-NH-monomethoxytrityl group was accompanied by the 5'-O-detritylation. Alternatively, a base-driven deprotection of the 3'-NH-Fmoc group resulted in 3'-amino dimer containing a 5'-O-DMTr protective group. The Rp and Sp stereoisomers of the thus prepared phosphoramidate diester dimers were not separable by silica gel column chromatography in a 3'-amino-group deprotected form 5d, or in 5'-O-DMTr-3'-NH-Fmoc protected form 4f. However, the stereoisomers were separated in a fully protected 5'-O-DMTr-3'-NH-Tr form 4t and then completely detritylated with acid. The absolute configuration of the isolated products was not assigned. The prepared 3'-amino-5'-O-DMTr dimer 5d, as a mixture of stereoisomers, or 3'-amino-5'-OH dimer 5h, as separated stereoisomers, were used as dimer building blocks bearing a positively charged group to assemble the oligonucleotides via the oxidative phosphorylation method, previously developed for the 3'-amino nucleosides (14). The 3'-amino nucleosides were also used in the synthetic cycles along with the dimers.
Scheme 1. (i) Pivaloyl chloride/pyridine, (ii) 3-(N,N-dimethylamino)-propylamine/CCl4/CH3CN, (iii)AcOH/H2O or 10% NH3/EtOH.
Coupling efficiency of the dimers 5d, 5h either with or without a 5'-O-DMTr group was within a 60-65% range, which was significantly lower than that for the monomers, where coupling yields were as high as 95-97% per step. Thus, with these types of dimers, incorporation of only one or two positively charged groups into oligonucleotides was practical with the oligonucleotide chain assembly protocol used. All attempts to prepare a fully zwitterionic 11mer via coupling of dimers 5d and 5h failed. Consequently, only partially modified N3' -> P5' phosphoramidates bearing one or two 3-(N,N-dimethylamino)-propylamino groups were synthesized with the described approach. These compounds designated as oligomers 11-14 are presented in Table 3.
The second approach to the zwitterionic phosphoramidates is based on the dimers containing an internucleoside N3' -> P5' phosphoramidate diester group and the 3'-terminal H-phosphonate monoester group, which were prepared according to the Scheme 2, and used for oligonucleotide chain assembly. The compounds 10a,t, Scheme 2, were synthesized similarly to the published procedure (14) and as follows. The 3'-O-levulinyl protected nucleoside 5'-H-phosphonate diester 8 was oxidatively coupled to the 5'-O-DMTr-3'-amino nucleoside. The 3'-phosphitylation (21) following selective removal of 3'-levulinyl group with hydrazine produced the desired H-phosphonate dimer building block 10 with an internucleoside N3' -> P5' phosphoramidate link.
Scheme 2. (i) Levulinic acid/DCC, (ii) AcOH/H2O, (iii) ClP(NiPr)2OCE; tetrazole + H2O, (iv) 5'-ODMT-3'-NH2dT CCl4, (v)N2H4/pyridine/AcOH, (vi) PCl3/imidazole.
The ability of the synthesized zwitterionic and alternative oligonucleotide phosphoramidates to form duplexes and triplexes with complementary DNA and RNA strands was evaluated using thermal denaturation experiments. Melting curves for the duplexes were recorded at three different salt concentrations to evaluate the effects of net charge of the oligonucleotides on the complexe stability. Results from these studies are summarized in Table 1 and 2 for pyrimidine and purine containing oligomers respectively.
It is important to point out that, in all tested buffers, zwitterionic net neutral phosphormidate 18 formed duplexes with thermal stability independent of salt concentrations which was in contrast to its negatively charged conterparts, 15-17 (experiment 4, Table 1). It is also interesting that for the zwitterionic phosphoramidate 18, thermal stability of the duplexes with its ssDNA and ssRNA complements were practically identical, unlike complexes formed by the uniform anionic N3' -> P5' phosphoramidate 16, which preferred RNA over DNA strands (compare experiments 2 and 4, Table 1). At low salt concentration, the zwitterionic oligothymidilate 18 formed the most stable duplex with both DNA and RNA complements, (experiment 4, Table 1). The increase in thermal stability was ~20 and 35°C relative to the parent phosphodiester compound 15 for the complexes formed with poly(dA) and poly(A) respectively (compare experiments 1 and 4, Table 1). Similarly, stability of the complexes was noticeably increased relative to the uniformly modified N3' -> P5' phosphoramidate 16, especially for the complexes formed with poly(dA) (compare experiments 2 and 4, Table 1). Near the physiological value of 150 mM NaCl, the majority of the oligonucleotides formed complexes with similar stability, with Tm values around 35°C. The exceptions are the duplexes formed by the uniformly modified N3' -> P5' phosphoramidate 16 with poly(A) where the duplex was much more stable, Tm = 53.6°C, and by the alternative phosphodiester N3' -> P5' phosphoramidate 17 with poly(dA), where the duplex was somewhat less stable, Tm = 23.6°C. At 1.0 M NaCl, the zwitterionic oligomer 18 formed less stable duplexes with Tm [approx] 37°C than did the anionic counterparts. All other complexes formed by the negatively charged oligonucleotides were stabilized by 10-23°C relative to 150 mM NaCl. The highest salt stabilizing effect was seen for the N3' -> P5' phosphoramidate 16 complex with poly(dA), [Delta]Tm was 23.2°C. It is important to mention that this compound formed a 2T:1A triplex, but not a 1T:1A duplex with poly(dA), even when the ratio between purine and pyrimidine strands was kept one to one. Thus, the sigmoidal melting transitions for the triplexes formed by the oligomer 16 with poly(dA) were observed when absorbance changes were monitored at 260 nm and at 282 nm. This finding correlates well with the recently reported propensity of the N3' -> P5' oligothymidilates to form triplex rather than duplex with oligo(dA) complement (23,24). No melting transitions at 282 nm were observed for the same oligomer duplexes with poly(A) as well as for the compounds 15, 17 and 18 under all conditions tested, whereas at 260 nm classical shape sigmoidal melting curves were always recorded. Presumably, the nature of the complexes formed by 16 with poly(dA) determines relative high salt dependency of its Tm values; the triplexes have higher charge density than the duplexes do. Additionally, the oligonucleotides 16-18 formed triplexes with dsDNA hairpin target d(A10C4T10), containing d(A10:T10) duplex region, as was judged by the UV melting transitions at 282 nm, as well as by the mixing curve experiments. Melting temperature for the dissociation of the modified third strand from the duplex was 27.1, 28.4 and 56.5°C for oligomers 17, 18 and 16, respectively, in the buffer containing 150 mM NaCl and 10 mM MgCl2 (see Materials and Methods). Under identical conditions, triplex formation by the phosphodiester counterpart 15 was not observed.
Table 1.
Table 2
In contrast to the zwitterionic oligothymidylate 18, the zwitterionic net charge neutral oligoadenylate 21 forms complexes with significant salt dependency in their thermal stability, which is indicative of different stoechiometry of the complexes being formed (experiment 3, Table 2). A similar trend was observed for the negatively charged oligomers 19 and 20 (experiments 1 and 2, Table 2). The zwitterionic phosphoramidate 21 formed the most stable complex compared to the parent phosphodiester 19 or with the alternative phosphodiester, N3' -> P5' phosphoramidate 20 (compare experiments 1, 2 and 3, Table 2), with the exception for the complex of phosphodiester 19 at 1.0 M NaCl concentration. It is noteworthy that neither the natural phosphodiester 19 nor the alternating phosphodiester, N3' -> P5' phosphoramidate 20 formed complexes with poly(U) at low ionic strength conditions, whereas zwitterionic phosphoramidate 21 gave a Tm value of 41.2°C (experiment 3, Table 2). The stoichiometry of the complexes formed by the zwitterionic phosphoramidate 21 with both poly(dT) and poly(U) was determined using thermal dissociation experiments with melting curves recorded at 282 nm as well as 260 nm (Fig. 4A and B), and also using mixing curves and hypochromicity measurements (Fig. 4C). The data demonstrate the following. All melting curves recorded at 260 nm or at 282 nm for oligonucleotides 19-21 were monophasic under all buffer conditions used. Zwitterionic oligoadenylate 21 formed only 2Py:1Pu triplexes with either poly(dT) or poly(U) strands in all buffers even for the 1:1 ratio between purine and pyrimidine strands in the melting mixtures. Natural phosphodiester counterpart 19 formed only duplexes with both DNA and RNA complements. Alternative phosphodiester, N3' -> P5' phosphoramidate oligonucleotide 20, (Table 2) formed duplex with poly(dT) at the lowest salt concentration and 2Py:1Pu triplexes under all other conditions tested with either poly(dT) or poly(U) strands. Apparently, the net neutral charge of the zwitterionic oligoadenylate 21 significantly eases the formation of 2Py:1Pu triplexes by this compound by reducing electrostatic repulsion between the strands.
Complementary zwitterionic homopurine and zwitterionic homopyrimidine oligonucleotides 18 and 21 can also form a duplex with each other with melting temperature 25.4°C, which was again independent of the buffer salt concentrations. The duplex is much more stable than one formed by the isosequential natural phosphodiester counterparts in the buffer A, but less stable at higher salt concentrations. Melting temperatures for the all phosphodiester dA11:dT11 duplexes was <0, 29.4 and 39.5°C in buffers A, B and C respectively.
We analysed the ability of RNase H to cleave the RNA strand in the duplexes formed by oligothymidylates 15-18 (Table 1 and poly(A). The analysis by IE-HPLC under denaturing conditions and thermal dissociation experiments were used to analyze the reaction mixtures (for conditions see Materials and Methods). First, we validated an assay using the phosphodiester oligothymydilate 15, which is known to be a substrate for RNase H. Thus, after 1 h of incubation, neither the starting RNA target nor the starting duplex were found in solution as was judged by IE HPLC and by thermal dissociation experiments. The fully modified N3' -> P5' phosphoramidate 16, as was expected based on previous results (25), and also the zwitterionic phosphoramidate 18 did not induced hydrolysis of poly(A) with RNase H. The presence of the positively charged groups attached to the sugar-phosphate backbone apparently inhibits hydrolysis by RNase H. Similar observations were recently reported for the phosphodiester oligonucleotides containing alkylamino groups (26). Interestingly, the duplex formed with the alternating anionic phosphodiester, N3' -> P5' phosphoramidate 17, and poly(A) was recognized by the RNase H and the poly(A) strand was cleaved. In a control experiment, without the enzyme, no hydrolysis of the RNA strand was observed by IE HPLC analysis and the intact poly(A):alternating decathymidilate 17 duplex had the same melting curve as the starting compounds. It was demonstrated that the 2'-deoxy-3'-amino nucleosides adapt C3'-endo sugar ring conformation in contrast to the 2'-deoxy-3'-hydroxyl counterparts, which are in C2'-endo conformations (27). The different sugar puckers of RNA-like 3'-amino nucleosides and consequently different structures of the oligonucleotide N3' -> P5' phosphoramidates presumably resulted in a lack of recognition by RNase H of the duplexes formed by these compounds and RNA strands (28). Recognition by RNase H of the complex formed by the oligonucleotide containing alternating 3'-amino and 3'-hydroxy nucleosides indicates that RNase H may well tolerate some structural variations in a negatively charged substrate duplex.
In conclusion, the zwitterionic net neutral oligonucleotide N3' -> P5' phosphoramidates containing the alternating negatively and positively charged internucleoside groups were synthesized and studied. It was found that these compounds form stable complexes with complementary nucleic acids at the low ionic strengths used for hybridization, with melting temperatures significantly higher than that for the natural phosphodiester counterparts. This property as well as an increased hydrolytic stability mark these oligonucleotides as a good candidates for various diagnostic and possibly other applications.
TLC analysis was performed on 0.2 mm thick pre-coated silica gel plates from Merck with a fluorescent indicator, using either 5 or 10% CH3OH in CH2Cl2 as eluent. Flash silica gel chromatography was performed with 230-400 mesh 60 Å silica from Merck.
1H and 31P NMR spectra were recorded on a Bruker Avance DRX-400 spectrometer.
Ion exchange IE HPLC was performed on a Dionex DX 500 system. For IE analysis and purification either a Pharmacia MonoQ 10/10 or a Pharmacia MonoQ 5/5 columns were used with a 1% per min gradient of 1.5 M NaCl in 10 mM NaOH, pH 12. Oligonucleotides were desalted on a Sephadex gel filtration columns NAP-5 from Pharmacia and lyophlized in vacuo or precipitated with EtOH. Thermal dissociation experiments were performed on a Cary-1E spectrophotometer, equipped with a temperature controller and data processor. Absorbance values at 260 nm were collected at 1 min intervals at a heating rate of 1.0°C/min. Oligonucleotide concentrations were ~5 µM in 10 mM sodium phosphate buffer, pH 7.0, and in the same buffer containing 0.15 M NaCl or 1.0 M NaCl corresponding to buffers A, B and C respectively.
Oligodeoxyribonucleotides were prepared manually in syringe using standard DNA assembly protocols via H-phosphonate method (21). Syntheses were performed on a 1 µmol scale using 9a and 9b as building block and according to the following procedures: (i) detritylation 5% dichloroacetic acid in CH2Cl2, 1.5 min; (ii) washing CH2Cl2, CH3CN; (iii) coupling 3'-H-phosphonate dimer, 0.03M in CH3CN/pyridine 1/1, v/v, in presence of pivaloyl chloride, 0.1M in CH3CN, 3 min; (iv) washing CH3CN. This cycle was repeated 5 times and than oxidation was accomplished with either 0.1 M I2/pyridine/water to lead to the oligonucleotides 16 and 19 or with 3-(N,N-dimethylamino)-propylamine/CCl4/CH3CN, 1/4/5, v/v, to form zwitterionic oligonucleotides 17 and 20. The oxidation step was followed by the treatment with concentrated aqueous ammonia, 5 h at 55°C.
RNase H mediated study was done with ~5 µM of oligothymidylate and poly(A) solutions in 10 mM Tris-HCl buffer, pH 7.0, containing 10 mM MgCl2. RNase H (5 U/µl) from Pharmacia was added to the oligonucleotide complexes and course of hydrolysis was followed by either IE-HPLC or by UV spectrophotometry, by mesuring changes in UV absorbance and by recording the duplexes melting curves.
3'-NH-Fmoc-deoxythymidine, 2f. 5'-O-DMTr-3'-amino-deoxythymidine (1 g, 1.8 mmol) was stirred for 2 h at room temperature in presence of 9-fluorenylmethylchloroformate (570 mg, 2.2 mmol) and N,N-diisopropylethylamine (385 µl, 2.2 mmol) in 5 ml of CH2Cl2. The organic layer was washed with saturated aqueous NaHCO3 and then concentrated in vacuo to give 1.3 g of a yellow foam. The reaction mixture was dissolved in 80% aqueous acetic acid and stirred for 30 min at room temperature. The mixture was then concentrated in vacuo and purified by silica gel column chromatography, 0-5% gradient of CH3OH in CH2Cl2 to yield 460 mg of 2, 55% based on starting 3-aminothymidine. 1H NMR: (CDCl3) [delta] 11.00 (s, 1H), 7.80 (m, 3H), 7.64 (m, 3H), 7.43 (t, 2H), 7.35 (t, 2H), 6.30 (t, J = 6.12 Hz, 1H), 4.89 (br s, 1H), 4.41 (d, 2H), 4.30 (t, 2H), 3.93 (br s, 1H), 3.70 (m, 2H), 2.30 (m, 2H), 2.24 (s, 3H).
O-[5'-O-DMTr-thymidinyl-3'-yl] O-[3'-NH2-thymidinyl-5'yl] dimethylaminopropylamino phosphoramidate, 5d. Compound 2 (450 mg, 0.97 mmol) and O-[5'-O-DMTr-thymidinyl-3'-yl] H-phosphonate monoester (590 mg, 0.97 mmol) were dissolved in 15 ml of pyridine and 295 µl (2.42 mmol) of pivaloyl chloride was added dropwise under argon. After 45 min stirring at room temperature 3 ml of 0.5 M triethylammonium acetate buffer was added. Then the organic layer was washed with water to give after concentration in vacuo 970 mg of compound 3 (95%), which was used without further purification. 31P NMR: (CDCl3) [delta] 9.80, 8.46, J = 217.2 Hz. Then 3 was dissolved in a mixture of 15 ml 3-(N,N-dimethylamino)-propylamine, 6 ml of anhydrous carbon tetrachloride and 7.5 ml of acetonitrile. Amidative oxidation proceeded within 15 min, as judged by TLC control. Then saturated aqueous NaHCO3 was added and the organic phase was washed three times. After evaporation in vacuo of all solvents the product was precipitated from hexane (690 mg, 65%). 31P NMR: (CDCl3) [delta] 10.31, 10.20, J = 10.5 Hz. Then the precipitate was dissolved in 15 ml of 10% concentrated aqueous NH3 in EtOH and the solution was heated for 2 h at 55 °C, concentrated in vacuo and precipitated from hexane to give 530 mg of a white solid 5d (90% yield). Mass spectrometry (M + H)+: 931.9 calculated, 932.4 observed. 31P NMR: (CDCl3) [delta] 11.17, 11.06, J = 17.1 Hz. 1H NMR: (CDCl3) [delta] 7.56 (m, 1H), 7.39 (m, 2H), 7.27 (m, 9H), 6.85 (d, 4H), 6.45 (q, 1H), 6.13 (q, 1H), 5.15 (m,1H), 4.30 (m, 2H), 4.15 (m, 2H), 3.80 (s, 8H), 3.50 (m, 2H), 3.20 (q, 1H), 2.92 (q, 1H), 2.40 (m, 2H), 2.24 (s, 3H), 2.19 (s, 3H), 1.92 (s, 3H), 1.70 (q, J = 12.4 Hz, 1H), 1.57 (q, J = 12.3 Hz, 1H), 1.38 (d, 3H), 1.25 (s, 2H).
O-[5'-OH-thymidinyl-3'-yl] O-[3'-OH-thymidinyl-5'-yl] dimethylaminopropylamino phosphoramidate, 5h. The compound 5h was synthetized analogously to 4f starting from 3t; it was then treated with 80% aqueous acetic acid. 31 P NMR: (D2O) [delta] 5h (slow isomer): 10.166 p.p.m.; (fast isomer): 9.927 p.p.m.
3'-O-levulinylthymidine, 7t. Levulinic acid (531 µl, 5.4 mmol) and dicyclohexylcarbodiimide (1.1 g, 5.4 mmol) were dissolved in 5 ml of pyridine and stirred for 2 h at room temperature. Then the reaction mixture was filtered under argon and 5'-O-DMTr-thymidine 6t was added (1 g, 1.8 mmol). The mixture was left overnight at room temperature and then the organic layer was washed with saturated aqueous NaHCO3. The crude mixture was dissolved in 10 ml of 80% aqueous acetic acid and after 30 min reaction mixtures was evaporated and co-evaporated with toluene. The product was purified by silica gel chromatography in 0-5% gradient of CH3OH in CH2Cl2 to give 460 mg pure 7t with 75% (based on 6t); 1H NMR: (CDCl3) [delta] 8.60 (s, 1H), 7.54 (s, 1H), 7.30 (s, 1H), 6.25 (t, J = 6.4 Hz, 1H), 5.36 (m, 1H), 4.11 (br s, 1H), 3.91 (m, 2H), 2.80 (t, J = 6.0 Hz, 2H), 2.59 (t, J = 6.1 Hz, 2H), 2.39 (m, 2H), 2.20 (s, 3H), 1.91 (s, 3H).
3'-O-levulinyl N6-benzoyl adenosine, 7a. This compound was synthesized analogously to 7t;1H NMR: (CDCl3) [delta] 9.20 (br s, 1H), 8.80 (s, 1H), 8.15 (s, 1H), 8.05 (m, 2H), 7.60 (m, 1H), 7.50 (m, 2H), 6.39 (dd, J = 3.4, 5.6 Hz, 1H), 5.90 (br s, 1H), 5.56 (t, 1H), 4.29 (m, 1H), 3.94 (q, 2H), 3.18 (m, 1H), 2.82 (t, 2H), 2.61 (t, 2H), 2.50 (m, 1H), 2.15 (m, 3H).
N-[5'-O-DMTr-thymidinyl-3'-yl] O-[3'-OH-thymidinyl-5'-yl] O- cyanoethyl phosphoramidate, 9t. The nucleoside 7t (460 mg, 1.35 mmol) was dried overnight in dessicator over P2O5 and then dissolved in 10 ml of anhydrous CH2Cl2 and N,N-diisopropyl-O-cyanoethyl chlorophosphite (380 µl, 1.7 mmol) and N,N-diisopropylethylamine (295 µl, 1.7 mmol) were added simultaneously. TLC analysis showed that the reaction was complete within 15 min. Then 0.5 M solution of tetrazole in 10% H2O in CH3CN was added and the reaction mixture was stirred for an additional 30 min. Organic layer was washed with aqueous NaCl to give 530 mg, 92%, of 5'-H-phosphonate-3'-O-levulinyl thymidine 8t. 31P NMR: (CDCl3) [delta] 9.94, 8.56, J = 224.8 Hz. This compound (530 mg, 1.05 mmol) after being dried over P2O5 in a dessicator was coupled with 5'-O-DMTr-3'-amino-thymidine (570 mg, 1.05 mmol) in presence of 7 ml CCl4, 9 mL CH3CN and 0.5 mL NEt3. After 30 min of stirring at room temperature, the reaction was stopped and the organic layer was washed with saturated aqueous NaCl. 31P NMR: (CDCl3) [delta] 9.26, 9.02, J = 38.7 Hz. Then the crude reaction mixture, 940 mg (0.9 mmol), was dissolved in a mixture containing 1 mL of hydrazine, 24 ml of pyridine and 6 ml of glacial acetic acid. After 5 min, organic phase was washed with aqueous NaCl and then the compound formed was purified by silica gel chromatography in 3-10% gradient of CH3OH in CH2Cl2 containing 0.8% of pyridine; 9t was isolated with 60% yield, 490 mg; Mass spectrometry (M + H)+, 900.8 calculated, 900.3 observed. 31P NMR: (CDCl3) [delta] 9.80, 9.46, J = 54.5 Hz. 1H NMR: (CDCl3) [delta] 10.29 (2s, 2H), 7.56 (d, J = 7.5 Hz, 1H), 7.35 (s, 1H), 7.28 (m, 11H), 6.80 (d, 4H), 6.35 (br s, 1H), 6.10 (br d, 1H), 5.10 (m, 1H), 4.50 (m, 1H), 4.05 (m, 6H), 3.70 (s, 6H), 3.40 (m, 2H), 2.30 (m, 6H), 1.80 (s, 3H), 1.40 (d, 3H).
N-[5'-O-DMTr-N6-benzoyladenosine-3'-yl] O-[3'-OH-N6benzoyladenosine-5'-yl] O-cyanoethyl phosphoramidate, 9a. This compound was obtained analogously to 9t with 96.5% yield. Compound 8a was precipitated from hexane and isolated with 99.7% yield. 31P NMR: (CDCl3) [delta] 9.27, 9.05, J = 37.0 Hz; 9a: 31P NMR: (CDCl3) [delta] 10.49, 10.23, J = 42.4 Hz. 1H NMR: (CDCl3) [delta] 9.28 (m, 1H), 8.62 (d, 2H), 8.20 (m, 2H), 8.02 (m, 2H), 7.43 (m, 3H), 7.15 (m, 12H), 6.77 (m, 4H), 6.39 (m, 2H), 4.78 (m, 1H), 4.09 (m, 5H), 3.73 (s, 8H), 3.41 (m, 2H), 2.85 (m, 2H), 2.53 (m, 2H), 1.13 (m, 2H).
N-[5'-O-DMTr-thymidinyl-3'-yl] O-[3'-O-H-Phosphonate-thymidinyl-5'-yl] O-cyanoethyl phosphoramidate, 10t. To a stirring solution of phosphorus trichloride (230 µl, 2.7 mmol) and N,N-diisopropylethylamine (4.6 ml, 27 mmol) in 25 ml of CH2Cl2, 590 mg (8.6 mmol) of imidazole was added. After stirring for 30 min the reaction mixture was cooled to 0°C and 490 mg (0.54 mmol) of dimer 9t in 5 ml CH2Cl2 was added dropwise. The mixture was stirred for another 15 min and then poured into 150 ml of 1.0 M triethylammonium acetate buffer. The product was purified by silica gel chromatography in a gradient 5-15% of CH3OH in CH2Cl2, yield 51%; 31P NMR 1H decoupled: (CDCl3) [delta] 10.57, 9.53, J = 168 Hz; 3.61, 2.74, J = 141 Hz. 31P NMR 1H coupled: (CDCl3) [delta] 10.57, 9.53, 5.55, 4.67, 1.69, 0.81, J = 168 Hz, 142 Hz, 624 Hz.
N-[5'-O-DMTr-N6benzoyladenosine-3'-yl] O-[3'-O-H-Phosphonate-N6-benzoyladenosine-5'-yl] O-cyanoethyl phosphoramidate, 10a. 31P NMR 1H decoupled: (CDCl3) [delta] 10.47, 9.61, J = 149.8 Hz; 2.65, 2.27, J = 62.1 Hz. 31P NMR 1H coupled: (CDCl3) [delta] 10.48, 9.60, 4.59, 4.20, 0.76, 0.36, J = 141.6, 62.36, 621.6 Hz. Mass spectrometry (M + H)+, 1189.3 calculated, 1189.3 observed31P NMR analysis of the oligonucleotides. Data given are the average of the two groups of peaks observed: compound 17 Table 1 31P NMR: (D2O) [delta] 7.56; 0.36; Coumpound 18 Table 1,31P NMR: (D2O) [delta] 11.82; 7.82; Coumpound 20 Table 2, 31P NMR: (D2O) [delta] 7.70; 0.04; Coumpound 21 Table 2, 31P NMR: (D2O) [delta] 11.62; 7.60.
Nucleic Acids Research
Pages
Introduction
Results And Discussion
Oligonucleotides synthesis
Hybridization properties of the zwitterionic oligonucleotides
RNase H-mediated cleavage experiments
Materials And Methods
Synthesis of the oligonucleotides and their building blocks
References
Expt
Oligodeoxyribonucleotide
Complement
POLY(dT)
POLY(U)
A
B
C
A
B
C
1
d(AAAAAAAAAAA) 19
22.0
51.2
72.2
< 0
41.4
61.5
2
d(AnpA-AnpA-AnpA-AnpA-AnpA-A) 20
22.2
42.9
63.2
< 0
44.9
64.5
3
d(AnpA+AnpA+AnpA+AnpA+AnpA+A) 21
45.4
55.2
64.7
41.2
54.3
67.2
Expt
Oligodeoxyribonucleotide
Complement
POLY(dT)
POLY(U)
A
B
C
A
B
C
1
d(AAAAAAAAAAA) 19
22.0
51.2
72.2
< 0
41.4
61.5
2
d(AnpA-AnpA-AnpA-AnpA-AnpA-A) 20
22.2
42.9
63.2
< 0
44.9
64.5
3
d(AnpA+AnpA+AnpA+AnpA+AnpA+A) 21
45.4
55.2
64.7
41.2
54.3
67.2
REFERENCES
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