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Bioconjugate Chem. 2003, 14, 684−689
TECHNICAL NOTES Synthesis of 3′-3′-Linked Oligonucleotides Branched by a Pentaerythritol Linker and the Thermal Stabilities of the Triplexes with Single-Stranded DNA or RNA Yoshihito Ueno,† Aya Shibata,† Akira Matsuda,‡ and Yukio Kitade*,† Department of Biomolecular Science, Faculty of Engineering, Gifu University, Yanagido, Gifu 501-1193, Japan and Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan. Received August 17, 2002; Revised Manuscript Received February 6, 2003
Synthesis of 3′-3′-linked oligonucleotides branched by a pentaerythritol linker is described. The branched oligonucleotides were synthesized on a DNA/RNA synthesizer using a controlled pore glass (CPG) with a pentaerythritol linker carrying 4,4′-dimethoxytrityl (DMTr) and levulinyl (Lev) groups. The stability of the triplexes between the branched oligonucleotides and the target single-stranded DNA or RNA was studied by thermal denaturation. The oligonucleotides with the pentaerythritol linker formed thermally stable triplexes with the single-stranded DNA and RNA. Furthermore, the branched oligonucleotides containing 2′-O-methylribonucleosides, especially the oligonucleotide composed of 2′-deoxyribonucleosides and 2′-O-methylribonucleosides, stabilized the triplexes with the single-stranded DNA or RNA. Thus, the branched oligonucleotide containing 2′-O-methylribonucleosides may be a candidate for a novel antisense molecule by the triplex formation.
INTRODUCTION
In the antisense strategy for controlling translation of mRNA, an oligonucleotide sequence-specifically binds to the mRNA by the Watson-Crick hydrogen bonds (1,2). On the other hand, in the antigene strategy for controlling gene transcription, an oligonucleotide binds to the major groove of the double-stranded DNA by Hoogsteen or reverse Hoogsteen hydrogen bonds and forms a local triple-helix (triplex) (3). Depending on the orientation of the third strand, two major classes of triplexes are identified. When the third strand mainly consists of pyrimidines, Hoogsteen type Py‚PuPy base triplets (T‚AT and C+•GC, where the first letter represents the pyrimidine Hoogsteen strand, the second is the purine central strand, and the third is the pyrimidine Watson-Crick complementary strand) are formed in which the third strand is parallel to the purine strand of the target duplex (4, 5). When the third strand is predominantly purines, reverse Hoogsteen type Pu‚PuPy base triplets (G‚GC and A‚AT) are formed in which the third strand is antiparallel to the purine strand of the target duplex (6, 7). Much attention is being given to the approach of targeting single-stranded DNA or RNA through the formation of triplexes (8-12). It was demonstrated that a single-stranded oligonucleotide consisting of two oligopyrimidine stretches linked by a poly(ethylene glycol) linker or an oligonucleotide sequence formed a thermally stable triplex with the target single-stranded DNA (8, 9). It was also reported that circular oligonucleotides * To whom reprint requests should be addressed. Phone and fax: +81-58-293-2640. E-mail:
[email protected]. † Gifu University. ‡ Hokkaido University.
formed thermally and thermodynamically stable triplexes with the single-stranded DNA or RNA (10, 11). Triplex structures have been shown to be highly sensitive to DNA versus RNA backbone; for example, the DNA‚DNA‚DNA (D‚DD), RNA‚DNA‚DNA (R‚DD), and R‚DR-type triplexes are stable, while the D‚RD and D‚RR-type triplexes are reported not to be formed (13, 14). Shimizu et al. also showed that the triplex containing the oligonucleotide analogue composed of 2′-O-methylribonucleosides in the third strand was more thermally stable than that formed by DNA oligonucleotides (15). On the other hand, we recently reported the synthesis of branched oligonucleotides with a pentaerythritol at the junction point (16). We found that the oligonucleotides with a four-way junction formed the thermally stable parallel and antiparallel triplexes with the singlestranded DNAs. We also showed that the 3′-3′-linked oligonucleotides with intercalators at the junction point formed thermally stable triplexes with the duplexes composed of oligopurine-oligopyrimidine sequences (17). In the course of the investigation, we planned a synthesis of 3′-3′-linked oligonucleotides containing 2′-O-methylribonucleosides in the third and/or second strand of triplexes (Figure 1). We envisioned that the oligonucleotides would enhance the thermal stability of parallel-type of triplexes with the single-stranded DNA or RNA as compared with the un-linked oligonucleotides composed of 2′-deoxyribonucleosides. In this paper, we report the synthesis of 3′-3′-linked oligonucleotides branched by the pentaerythritol linker and the thermal stability of the triplexes between these branched oligonucleotides and the target single-stranded DNA or RNA.
10.1021/bc020059q CCC: $25.00 © 2003 American Chemical Society Published on Web 04/19/2003
Bioconjugate Chem., Vol. 14, No. 3, 2003 685 Scheme
1a
a (a) (1) N,N′-Carbonyldiimidazole, DMAP, DMF, room temperature; (2) H N(CH ) OH, DMF, room temperature; (b) DMTrCl, 2 2 4 pyridine, room temperature; (c) Lev2O, DMAP, pyridine, room temperature; (d) TBAF, THF, room temperature; (e) BzCl, pyridine, room temperature; (f) succinic anhydride, DMAP, pyridine, room temperature.
Figure 1. Schematic presentation of the triplex composed of the branched oligonucleotide and the single-stranded DNA or RNA. RESULTS AND DISCUSSION
Synthesis. To synthesize the branched oligonucleotides linked at the 3′-ends of each strand on a DNA/RNA synthesizer, we prepared a controlled pore glass (CPG) with pentaerythritol carrying two kinds of protective groups which could be deprotected under orthogonal conditions. We selected 4,4′-dimethoxytrityl (DMTr) and levulinyl (Lev) as such protective groups (18). A strategy for the synthesis of the CPG is shown in Scheme 1. First, 1,3-O-bis(tert-butyldiphenylsilanyl)-2,2-bis(hydroxymethyl)-1,3-propanediol (1) (16) was treated with N,N′-carbonyldiimidazole in the presence of DMAP and then reacted with 4-amino-1-butanol to produce 2 in 75% yield. Compound 2 was treated with 1.2 equiv of DMTrCl to give the mono O-DMTr derivative 3 in 44% yield. After protection of the other hydroxyl group of 3 with a levulinyl group, the tert-butyldiphenylsilanyl groups were removed by treating with tetrabutylammonium fluoride (TBAF) to give 5 in 48% yield. One of two hydroxyl groups of 5 was protected with a benzoyl (Bz) group to produce 6 in 30% yield. Compound 6 was modified to the corresponding 3′-succinate 7, which was reacted with a CPG to give a solid support containing 7 (43 µmol/g). The 3′-3′-linked oligonucleotides 8-11 were synthesized using a CPG on a DNA/RNA synthesizer (Figure 2). The oligonucleotide 8 consists of oligopyrimidine and oligopurine strands which are composed of natural 2′deoxynucleosides. The oligonucleotide 9 is composed of an oligopyrimidine strand comprising thymidine (dT) and 5-methyl-2′-deoxycytidine (dM) and an oligopurine strand comprising 2′-deoxyadenosine (dA) and 2′-deoxyguanosine (dG). The oligonucleotide 10 is composed of an oligopyrimidine strand comprising 2′-O-methylribonucleosides and an oligopurine strand comprising dA and dG. On the other hand, the oligonucleotide 11 consists of oligopyrimidine and oligopurine strands which are composed of 2′-O-methylribonucleosides. The oligonucleotides were synthesized by a phosphoramidite method. One strand was elongated in the usual manner. After capping the 5′-end of the strand and removing the Lev group, the other the strand was elongated. The fully protected oligonucleotides (each 1
Figure 2. Sequences of oligonucleotides used in this study.
µmol) linked to the solid support were treated with concentrated NH4OH at 55 °C for 16 h. The released oligonucleotides were purified by denaturing with 20% polyacrylamide gel electrophoresis (20% PAGE) to give the deprotected oligonucleotides 8, 9, 10, and 11 in 4, 2, 8, and 5 OD260 units, respectively. These oligonucleotides were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). The observed molecular weights supported their structures. Study of Triplex and Duplex Formation by Thermal Denaturation. The stability of the triplexes and the duplexes was studied by thermal denaturation in a buffer of 25 mM Tris-HCl (pH 7.0) containing 100 mM NaCl and 10 mM MgCl2. Sequences of control oligonucleotides 12-16, target DNA 17, and RNA 18 used in this study are shown in Figure 2. Figure 3 shows melting profiles of the triplexes between 12, 14 and 17 (a square), and 8 and 17 (a diamond), and the duplex between 14 and 17 (a circle). The Tm values are listed in Table 1. Two transitions were observed in the melting profile of the complex between 12, 14, and 17: the transition with the lower Tm (29.2 °C) was due to dissociation of the third strand from the triplex, and the transition with the higher Tm (58.2 °C) corresponded to melting of the duplex between 14 and 17. The Tm of the duplex between 14 and 17 (Figure 3; a circle) was 57.8 °C in the same buffer. On the other hand,
686 Bioconjugate Chem., Vol. 14, No. 3, 2003 Table 1. Hybridization Dataa
a
complexes
pH 7.0
pH 6.0
complexes
pH 7.0
duplexes
pH 7.0
pH 6.0
8‚17 9‚17 10‚17 11‚17 8‚18 9‚18 10‚18 11‚18
62.2 67.4 71.8 70.9 51.8 57.8 71.1 77.6
64.1 68.6 72.6 71.1 57.2 61.7 73.6 78.5
12‚14‚17 13‚14‚17 15‚14‚17 15‚16‚17 12‚14‚18 13‚14‚18 15‚14‚18 15‚16‚18
29.3 (58.2) 46.3 (58.2) 58.0 68.0 30.3 (47.1) 37.1 (46.5) 68.3 77.6
14‚17 16‚17 14‚18 16‚18
57.8 68.2 47.8 78.7
57.1 68.1 47.6 79.5
The Tm values of the duplexes are indicated in parentheses. Experimental conditions are described in the Experimental Procedures.
Figure 4. ∆Tms [Tm (at pH 6.0) - Tm (at pH 7.0)].
Figure 3. Melting profiles: the triplex between 12, 14, and 17 (×); the triplex between 8 and 17 (]); the duplex between the 14 and 17 (O).
when the branched oligonucleotide 8 and DNA 17 were used, only one transition was observed in the melting profile. The increment in the absorbance in melting the complex was almost equal to that of the complex between 12, 14, and 17. This means that the thermal denaturation from triplex to complete random coil occurred cooperatively in a single transition. The Tm value of the complex was 62.2 °C. It was 32.9 °C and 4 °C higher than those of dissociation of the third strand from the triplex between 12, 14, and 17 and melting of the duplex between 14 and 17, respectively. Thus, it was found that the triplex was largely thermally stabilized by connecting the second and third strands with an appropriate linker. The thermal stabilities of the complexes targeting the single-stranded DNA 17 were compared. When 2′-deoxy5-methylcytidine was incorporated into the third strand instead of 2′-deoxycytidine, the Tm of the triplex consisting of 13, 14, and 17 increased 17.0 °C as compared with that of the triplex between 12, 14, and 17. On the other hand, when 2′-deoxy-5-methylcythydine was incorporated into the branched oligonucleotide, the Tm of the triplex consisting of 9 and 17 increased 5.2 °C as compared with that of the triplex between 8 and 17. Thus, it was found that 2′-deoxy-5-methylcytidine stabilized the triplex composed of the branched oligonucleotide as well as the triplex composed of the un-branched oligonucleotides, although the increment of the Tm value for the triplex composed of the branched oligonucleotide was smaller than that for the triplex composed of the un-branched oligonucleotides. When the oligonucleotides consisting of 2′-O-methylribonucleosides were used as the second and third strands, a single transition was also observed for the complex between 15, 16, and 17. The Tm (68.0 °C) of the complex was almost equal to that of the duplex between
16 and 17. On the other hand, the Tm of the complex between 11 and 17 was 70.9 °C, which was 2.9 °C higher than that of the complex consisting of 15, 16, and 17. Among the complexes targeting the single-stranded DNA 17, the complex between 10 and 17, which has the oligonucleotide consisting of 2′-O-methylribonucleosides as the third strand, showed the highest Tm value (71.8 °C). This was 13.8 °C higher than that of the complex composed of 15, 14, and 17. Next, we compared the thermal stabilities of the complexes targeting the single-stranded RNA 18. The trend in the relative stabilities of the complexes was similar to that of the complexes targeting the singlestranded DNA 17. When the oligonucleotide 15 consisting of 2′-O-methylribonucleosides was used as the third strand, the triplex was largely stabilized. The Tm of the triplex between 15, 14, and 18 was 20.5 °C higher than that of the duplex between 14 and 18 (47.8 °C). Furthermore, the Tm of the triplex composed of 10 and 18 was 3.4 °C higher than that of the triplex between 15, 14, and 18. Thermal Denaturation Study at pH 6.0. In paralleltype triplexes, the N-3 positions of the cytosine residues of the third strand must be protonated to form two hydrogen bonds with the C/G base pairs in the WatsonCrick duplex (2). It is reported that the parallel-type triplex containing cytosine residues in the third strand is thermally stabilized in slightly acidic solutions as compared with neutral or basic solutions (19-21). Thus, we compared the Tms of triplexes composed of branched oligonucleotides in acidic solutions (pH 6.0) with the Tms in neutral solutions. The complexes that show increased stabilities at the lower pH value are likely to be triple helical in structure, and the complexes that show no pH sensitivity are almost certainly not triple helical. The Tm values in acidic solutions are summarized in Table 1. ∆Tms [Tm (at pH 6.0) - Tm (at pH 7.0)] for the branched oligonucleotides are shown graphically in Figure 4. The ∆Tm value of the triplex between 8 and 17 was 1.9 °C whereas that of the triplex between 9, containing 5-methylcytosine, and 17 was 1.2 °C. The electron-donating methyl substitute at the 5 position of the cytosine residue is thought to stabilize protonation at the N-3 position of the cytosine in the triplex although
Bioconjugate Chem., Vol. 14, No. 3, 2003 687
Figure 5. CD spectra. (a) A 10:17 solution (O); a 15:14:17 solution (b); the normalized sum of an 15 plus 14:17 solutions (+). (b) A 10:18 solution (O); a 15:14:18 solution (b); the normalized sum of an 15 plus 14:18 solutions (+).
this stabilization is modest in the uncomplexed cytosine (pKa values: 2′-deoxycytidine and 2′-deoxy-5-methylcytidine are 4.3 and 4.4, respectively) (22, 23). Thus, the difference in pH sensitivities of these triplexes would reflect the pKa s of N3H+ of the cytosine residues in the triplexes. On the other hand, the ∆Tm value of the triplex between 10, composed of 2′-deoxyribonucleosides and 2′O-methylribonucleosides, and 17 was 0.8 °C while that of the triplex between 11, consisting of 2′-O-methylribonucleosides, and 17 was 0.2 °C. These results indicate that interaction between the third strand and second strand in the complex composed of 11 and 17 is weaker than those of other triplexes. Next, ∆Tms of the triplexes targeting the singlestranded RNA were compared. As shown in Figure 4, although the values were greater than those for the single-stranded DNA, the trend in the relative ∆Tm values was similar to that for DNA 17. Thus, these results suggest that the complexes studied in this experiment are triple helical in structure although the Hoogsteen hydrogen bonds of the complex composed of 11 and 17 are weaker than those of other triplexes. Circular Dichroism. To confirm the triplex formations, the circular dichroism (CD) spectrum of each complex was measured. The CD spectra of the mixtures of a 1:1 molar ratio of the branched oligonucleotide 10: DNA 17 and a 1:1:1 molar ratio of 15:14:DNA 17 in a buffer of 25 mM Tris-HCl (pH 7.0) containing 100 mM NaCl and 10 mM MgCl2 were compared with the appropriately normalized summed spectrum of the DNA 14-DNA 17 duplex and the oligonucleotide 15. As shown in Figure 5a, the CD spectrum of the branched oligonucleotide 10:DNA 17 solution had a positive CD band at 276 nm, which was similar to that of the 15:14:DNA 17 solution and different in intensity from that of the normalized summed spectrum of the DNA 14-DNA 17 duplex. Similarly, the CD spectra of the mixtures of a 1:1 molar ratio of the branched oligonucleotide 10:RNA 18 and a 1:1:1 molar ratio of 15:14:RNA 18 were compared with the appropriately normalized summed spectrum of the DNA 14-RNA 18 duplex and the oligonucleotide 15. As shown in Figure 5b, the CD spectrum of the branched oligonucleotide 10:RNA 18 solution had a positive CD band at 271 nm, which was similar to that of the 15:14: RNA 18 solution and different in intensity from that of the normalized summed spectrum of the DNA14-RNA 18 duplex and 15. Therefore, these CD spectral data also
support the existence of the triplexes between branched oligonucleotide 10 and DNA 17 and between branched oligonucleotide 10 and DNA 18. CONCLUSION
In this paper, we reported the synthesis of 3′-3′-linked oligonucleotides 8-11 branched by the pentaerythritol linker and the thermal stability of the triplexes between these branched oligonucleotides and the target singlestranded DNA 17 or RNA 18. The oligonucleotides with the pentaerythritol linker formed thermally stable triplexes with single-stranded DNA 17 and RNA 18. Furthermore, the branched oligonucleotides containing 2′-O-methylribonucleosides, especially the oligonucleotide 10 composed of 2′-deoxyribonucleosides and 2′-O-methylribonucleosides, stabilized the triplexes (R‚DD and R‚DR-type triplexes) with single-stranded DNA 17 or RNA 18. Thus, the branched oligonucleotide containing 2′-O-methylribonucleosides may be a candidate for a novel antisense molecule by the triplex formation. EXPERIMENTAL PROCEDURES
General Remarks. 1H NMR spectra were recorded at 400 MHz. Chemical shifts (δ) and coupling constants (J) are reported in ppm downfield from TMS and in Hz, respectively. Mass spectra were obtained by fast atom bombardment (FAB) method. Thin-layer chromatography was done on Merck coated plates 60F254. The silica gel used for column chromatography was Wakogel C-300. 1,3-O-Bis(tert-butyldiphenylsilanyl)-2,2-bis[[N-(4hydroxybutyl)carbamoyl]oxymethyl]-1,3-propanediol (2). N,N′-Carbonyldiimidazole (925 mg, 5.70 mmol) and DMAP (36 mg, 0.29 mmol) were added to a solution of 1,3-O-bis(tert-butyldiphenylsilanyl)-2,2-bis(hydroxymethyl)-1,3-propanediol (1) (16) (874 mg, 1.43 mmol) in DMF (4.8 mL), and the mixture was stirred at room temperature. After 2 h, 4-amino-1-butanol (1.97 g, 22.1 mmol) was added to the mixture, which was stirred at room temperature. After 12 h, the mixture was partitioned between EtOAc and H2O. The organic layer was washed with brine, dried (Na2SO4), and concentrated. The residue was purified by column chromatography (SiO2, 0-4% MeOH in CHCl3) to give 2 (905 mg, 75% as a white solid): FAB-MS m/z 843 (MH+); 1H NMR (CDCl3) δ 7.64-7.00 (m, 20H), 4.60 (br, 2H), 4.08 (s, 4H), 3.663.24 (m, 8H), 3.15 (m, 4H), 2.11 (br, 2H), 1.55 (m, 8H), 1.03 (s, 18H). Anal. Calcd for C47H66N2O8Si2‚1/4H2O: C, 66.59; H, 7.91; N, 3.30. Found: C, 66.85; H, 8.20; N, 3.28.
688 Bioconjugate Chem., Vol. 14, No. 3, 2003
1,3-O-Bis(tert-butyldiphenylsilanyl)-2-[N-[(4,4′-dimethoxytrityl)oxybutyl]carbamoyl]oxymethyl-2[N-(4-hydroxybutyl)carbamoyl]oxymethyl-1,3-propanediol (3). A mixture of 2 (848 mg, 1.01 mmol) and DMTrCl (397 mg, 1.17 mmol) in pyridine (10 mL) was stirred at room temperature for 12 h. The mixture was partitioned between EtOAc and H2O. The organic layer was washed with brine, dried (Na2SO4), and evaporated under reduced pressure. The residue was purified by column chromatography (SiO2, 25-100% EtOAc in hexane) to give 3 (512 mg, 44% as a white foam): FAB-MS m/z 1145 (M+); 1H NMR (CDCl3) δ 7.62-6.80 (m, 33H), 4.51 (br, 1H), 4.39 (br, 1H), 4.11 (m, 4H), 3.78 (s, 6H), 3.64 (m, 6H), 3.09 (m, 4H), 1.56 (m, 8H), 1.01 (s, 18H). Anal. Calcd for C68H84N2O10Si2‚3/2H2O: C, 69.65; H, 7.48; N, 2.39. Found: C, 69.68; H, 7.57; N, 2.29. 1,3-O-Bis(tert-butyldiphenylsilanyl)-2-[N-[4-(4,4′dimethoxytrityloxy)butyl]carbamoyl]oxymethyl-2[N-[4-(levulinyloxy)butyl]carbamoyl]oxymethyl-1,3propanediol (4). A mixture of levulinic acid (326 mg, 2.81 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSCI) (275 mg, 1.43 mmol) in DMF (4.8 mL) was stirred at room temperature. After 24 h, the mixture was evaporated under reduced pressure, and the residue was dissolved in pyridine (2.5 mL). To the solution were added 3 (227 mg, 0.198 mmol) and DMAP (17 mg, 0.140 mmol), and the mixture was stirred at room temperature. After 12 h, the mixture was partitioned between EtOAc and H2O. The organic layer was washed with brine, dried (Na2SO4), and evaporated under reduced pressure. The residue was purified by column chromatography (SiO2, 33-50% EtOAc in hexane) to give 4 (148 mg, 60% as a white foam): FAB-MS m/z 1243 (MH+); 1H NMR (CDCl3) δ 7.61-6.80 (m, 33H), 4.46 (br, 1H), 4.39 (br, 1H), 4.11 (m, 6H), 3.78 (s, 6H), 3.63 (s, 4H), 3.15-3.05 (m, 6H), 2.73 (m, 2H), 2.55 (m, 2H), 2.17 (s, 3H), 1.56 (m, 8H), 1.00 (s, 18H). Anal. Calcd for C73H90N2O12Si2‚H2O: C, 69.49; H, 7.35; N, 2.22. Found: C, 69.35; H, 7.51; N, 2.11. 2-[N-[4-(4,4′-Dimethoxytrityloxy)butyl]carbamoyl]oxymethyl-2-[N-[4-(levulinyloxy)butyl]carbamoyl]oxymethyl-1,3-propanediol (5). A mixture of 4 (488 mg, 0.392 mmol) and TBAF (1 M in THF, 1.20 mL, 1.20 mmol) in THF (5 mL) was stirred at room temperature for 12 h. The mixture was evaporated under reduced pressure. The residue was purified by column chromatography (SiO2, 50-100% EtOAc in hexane) to give 5 (145 mg, 48% as an oil): FAB-MS m/z 766 (M+); 1H NMR (CDCl3) δ 7.43-6.81 (m, 13H), 5.14 (br, 1H), 5.08 (br, 1H), 4.10 (m, 6H), 3.79 (s, 6H), 3.47 (m, 4H), 3.21 (m, 4H), 3.08 (t, J ) 5.6, 2H), 2.76 (t, J ) 6.4, 2H), 2.56 (t, J ) 6.2, 2H), 2.18 (s, 3H), 1.59 (m, 8H). Anal. Calcd for C68H84N2O10Si2‚1/2H2O: C, 63.47; H, 7.14; N, 3.61. Found: C, 63.51; H, 7.52; N, 3.43. 1-O-Benzoyl-2-[N-[4-(4,4′-dimethoxytrityloxy)butyl]carbamoyl]oxymethyl-2-[N-[4-(levulinyloxy)butyl]carbamoyl]oxymethyl-1,3-propanediol (6). A mixture of 5 (137 mg, 0.179 mmol) and BzCl (22 µL, 0.188 mmol) in pyridine (1.8 mL) was stirred at room temperature for 12 h. The mixture was partitioned between EtOAc and H2O. The organic layer was washed with aqueous NaHCO3 (saturated) and brine, dried (Na2SO4), and evaporated under reduced pressure. The residue was purified by column chromatography (SiO2, 50-90% EtOAc in hexane) to give 6 (47 mg, 30% as an oil): FAB-MS m/z 871 (MH+); 1H NMR (CDCl3) δ 8.02-6.81 (m, 18H), 4.89 (br, 2H), 4.32 (s, 2H), 4.18 (m, 6H), 3.78 (s, 6H), 3.53 (m, 4H), 3.21 (m, 4H), 3.08 (m, 2H), 2.75 (t, J ) 6.4, 2H), 2.55 (t, J ) 6.4, 2H), 2.18 (s, 3H), 1.59 (m, 8H). Anal.
Calcd for C48H58N2O13‚3H2O: C, 62.32; H, 6.97; N, 3.03. Found: C, 62.59; H, 6.61; N, 2.85. Synthesis of the Controlled Pore Glass Support with 1-O-Benzoyl-2-[N-[4-(4,4′-dimethoxytrityloxy)butyl]carbamoyl]oxymethyl-2-[N-[4-(levulinyloxy)butyl]carbamoyl]oxymethyl-3-O-succinyl-1,3-propanediol (7). A mixture of 6 (66 mg, 75 µmol), succinic anhydride (28 mg, 0.281 mmol), and DMAP (19 mg, 0.154 mmol) in pyridine (1 mL) was stirred at room temperature. After 2 days, the mixture was partitioned between EtOAc and H2O. The organic layer was washed with aqueous NaHCO3 (saturated) and brine, dried (Na2SO4), and concentrated. The residue was dissolved in DMF (2 mL). To the solution were added aminopropyl controlled pore glass (279 mg, 25 µmol, 90.4 µmol/g, CPG Inc.) and WSCI (16 mg, 82 µmol), and the mixture was kept at room temperature for 2 days. After the resin was washed with pyridine, 2 mL of a capping solution (0.1 M DMAP in pyridine:Ac2O ) 9:1, v/v) was added, and the whole was kept at room temperature for 2 days. The resin was washed with pyridine, MeOH, and acetone and was dried under vacuum. The amount of loaded 7 to the solid support is 43 µmol/g from the calculation of released dimethoxytrityl cation by a solution of 70% HClO4:EtOH (3:2, v/v). Synthesis of Oligonucleotides. Branched oligonucleotides were synthesized on a DNA/RNA synthesizer (Applied Biosystems Model Expedite) by the phosphoramidite method. One strand was elongated by the usual manner. The resin was treated with 2 mL of 0.1 M DMAP in pyridine:Ac2O (9:1, v/v) for 1 h and then 2 mL of 0.5 M hydrazine in pyridine:CH3CO2H (4:1, v/v) for 20 min. Other strand was elongated by the usual manner. The oligonucleotides linked to the resins were treated with concentrated NH4OH at 55 °C for 16 h, and the released oligonucleotides were purified by denaturing 20% polyacrylamide gel electrophoresis (20% PAGE) run at 600 V for 8 h. The oligonucleotides were visualized by UV shadowing and eluted from crushed gel slices by incubation at room temperature in 0.1 M triethylammonium acetate (TEAA, pH 7.0) and 1 mM EDTA for 15 h. The oligonucleotides were further purified by a Sep-Pak C18 cartridge to give the deprotected oligonucleotides 8 (4), 9 (2), 10 (8), and 11 (5). The yields are indicated in parentheses as OD units at 260 nm starting from 1 µmol scale. Matrix-Assisted Laser Desorption/Ionization Timeof-Flight Mass Spectrometry. Spectra were obtained on a Voyager Elite reflection time-of-flight mass spectrometry (PerSeptive Biosystems, Inc., Framingham, MA) equipped with a nitrogen laser (337 nm, 3-ns pulse). 3-Hydroxypicolinic acid (HPA), dissolved in H2O to give a saturated solution at room temperature, was used as the matrix. Time-to-mass conversion was achieved by calibration by using the peak representing the C+ cation of the charged derivative to be analyzed. Oligonucleotide 8: calculated mass, 10868.14; observed mass, 10869.00. Oligonucleotide 9: calculated mass, 10952.30; observed mass, 10955.60. Oligonucleotide 10: calculated mass, 11224.29; observed mass, 11227.70. Oligonucleotide 11: calculated mass, 11734.73; observed mass, 11734.90. Thermal Denaturation and CD Spectroscopy. The solution that contained the branched oligonucleotide (2 µM) and the target DNA 17 (2 µM) or RNA 18 (2 µM) in a buffer of 25 mM Tris-HCl (pH 7.0), 100 mM NaCl, and 10 mM MgCl2 or 10 mM PIPES (pH 6.0), 100 mM NaCl, and 10 mM MgCl2 was heated at 90 °C for 5 min, then cooled gradually to an appropriate temperature, and used for the thermal denaturation study. Thermal-induced
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transitions of each mixture were monitored at 260 nm on a Beckman DU 650 spectrophotometer. Sample temperature was increased 0.5 °C/min. Extinction coefficients of the branched oligonucleotides were calculated from those of mononucleotides and dinucleotides according to the nearest-neighbor approximation (24). Samples for CD spectroscopy were prepared by the same procedure used in the thermal denaturation study, and spectra were measured at 20 °C. The ellipticities of triplexes were recorded from 200 to 350 nm in a cuvette with a path length 1 mm. CD data were converted into mdeg‚mol of residues-1‚cm-1. ACKNOWLEDGMENT
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