Synthesis of Twisted Intercalating Nucleic Acids Possessing Acridine

Twisted intercalating nucleic acids (TINA) possessing acridine derivatives have been ... postsynthetic modifications of oligonucleotides possessing in...
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Bioconjugate Chem. 2006, 17, 950−957

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Synthesis of Twisted Intercalating Nucleic Acids Possessing Acridine Derivatives. Thermal Stability Studies Imrich Ge´ci, Vyacheslav V. Filichev, and Erik B. Pedersen* Nucleic Acid Center, Department of Chemistry, University of Southern Denmark, Campusvej 55, DK-5230, Odense M, Denmark. Received March 7, 2006; Revised Manuscript Received May 3, 2006

Twisted intercalating nucleic acids (TINA) possessing acridine derivatives have been synthesized via the postsynthetic modifications of oligonucleotides possessing insertions of (R)-1-O-(4-iodobenzyl)glycerol (8) or (R)-1-O-(4-ethynylbenzyl)glycerol (9) at the 5′-end or in the middle as a bulge. In the first postsynthetic step, oligonucleotides 8 and 9 on the CPG support were treated with a Sonogashira coupling reaction mixture containing 9-chloro-2-ethynylacridine or 9-chloro-2-iodoacridine, respectively. After the postsynthetic step, treatment of the oligonucleotides with 32% aq ammonia or 50% ethanolic solution of tris(2-aminoethyl)amine led to the substitution of chloride on acridine concurrent with deprotection of the bases and cleavage of the oligonucleotides from CPG. Molecular modeling of the parallel triplex with a bulged insertion of the monomer (R)-3-O-[4-(9-aminoacridin2-ylethynyl)benzyl]glycerol in the triplex-forming oligonucleotide (TFO) showed that the acridine moiety was stacking between the bases of the duplex, while phenyl was placed between the bases of the TFO. Thermal denaturation studies and fluorescence properties of TINA-acridine oligonucleotide duplexes and triplexes are discussed.

INTRODUCTION Although double-stranded DNA (dsDNA) has long been known to form a triple-helical structure, only recently, new strategies of using triplexes in molecular biology became largely investigated. The sequence-specific recognition of dsDNA by triplex-forming oligonucleotide (TFO) has led to the transcriptional control, sequence selective treatment of genomic DNA aiming for mutated or recombined genes (1). Because of low stability of triplexes at physiological pH, much effort has been devoted to modifying the TFO to achieve better triplex stability. Derivatives of 9-aminoacridine are well-known due to their broad biological activities. They are ascribed to the ability of planar acridine to stack between the nucleic acids bases (2). Derivatives of 6-chloro-2-methoxy-acridin-9-ylamine have been used for triplex stabilization as oligonucleotide conjugates, when covalently linked to the TFO in the middle or at the end of the sequence (3-8). In the case of 3′-3′ alternate-strand triple helix complexes, the stabilization effect has been observed, when the acridine moiety was placed on the nucleotide base flanking the 3′-3′ junction (9). 9-Aminoacridine conjugates stabilized Hoogsteen type triplexes with a single base pair inversion, when inserted to the oligonucleotide via a hexyl linker as a bulge. On the other hand, the absence of base pair inversion, or introduction of double base pair inversion, led to destabilization (10, 11). Recently, we observed the extraordinary stable Hoogsteen type triplexes and duplexes, when the monomer (R)-1-O-[4-(1pyrenylethynyl)benzyl]glycerol (1, Figure 1) was inserted as a bulge in the middle of the TFO (12). At the same time, WatsonCrick duplexes were destabilized. Moreover, TINA was able to discriminate between matched and mismatched sequences. It was supposed that twisting around the triple bond could help the intercalator to fit properly inside of the dsDNA part of the triplex. Because of this property, this type of oligonucleotide was called a twisted intercalating nucleic acid (TINA). * To whom correspondence should be addressed. Phone: (+45) 6550-2555; fax: (+45) 6615-8780; e-mail: [email protected].

Figure 1. TINA monomer.

In our present study, we focused on the synthesis of TINA bearing acridine derivatives instead of a pyrene-1-yl moiety in the structure of 1. We suppose that this replacement should also lead to a high thermal stability of Hoogsteen type triplexes, as we have already seen for the naphthalene and pyrene systems (12). Acridine is also an attractive substituent for the incorporation of functional groups in different positions of the aromatic ring. This can be further exploited in the construction of peptide-oligonucleotide conjugates for delivery of DNA into the cell nucleus (13). Here, we report the postsynthetic synthesis of (R)-3-O-[4(9-aminoacridin-2-ylethynyl)benzyl]glycerol (X) and (R)-3-O[4-(9-{2-[bis-(2-aminoethyl)amino]ethylamino}acridin-2-ylethynyl)benzyl]glycerol (Y) for the synthesis of the oligonucleotides 11 and 12, respectively. Such incorporations led to an increased stability of parallel triplexes in comparison with unmodified triplexes, which were also correlated with molecular modeling and fluorescence studies.

EXPERIMENTAL PROCEDURES Materials. Solvents were purchased from Bie and Berenstein Co., and chemicals were purchased from Aldrich Chemical Co. Solvents were sufficiently dried before they were used, and chemicals were used as purchased. Thin-layer chromatography (TLC) analyses were carried out with use of TLC plates 60 F254 purchased from Merck and were visualized in an UV light (254 nm). The silica gel (0.040-0.063 mm) used for column chromatography was purchased from Merck. Solvents used for column chromatography were distilled prior to use.

10.1021/bc060058o CCC: $33.50 © 2006 American Chemical Society Published on Web 06/21/2006

Synthesis of TINA-Acridine

Spectroscopy. NMR spectra were measured on a Varian Gemini 2000 spectrometer at 300 MHz for 1H using TMS (δ: 0.00) as an internal standard and at 75 MHz for 13C using CDCl3 (δ: 77.0) as an internal standard. Mass spectra of the synthesized compounds were determined on the Ionspec 4.7 T HiResMALDI Ultima Fourier transform (FT) mass spectrometer (Ion Spec, Irvine, CA). The [M + Na]+ ions were peak matched using ions derived from the 2,5-dihydroxybenzoic acid matrix. Electrospray ionization mass spectra (ESI-MS) were performed on 4.7 T HiResESI Ultima (FT) mass spectrometer. Both spectrometers are controlled by the OMEGA Data System. MALDITOF mass spectra of isolated oligodeoxynucleotides (ONs) were determined on a Voyager Elite biospectrometry research station (PerSeptive Biosystems). Molecular Modeling. Molecular modeling experiments and the construction of the modified triplex were performed using the program MacroModel 8.0. 2-Phenylethynyl-9-aminoacridine was energy-minimized with MM3 method before being inserted to the triplex. The starting 14-mer parallel triplex was built via consecutive superimposition of triples (CGC) and (TAT), which were generated with Insight II v9.72 from MSI and transported to MacroModel. The insertion of the acridine intercalator was done in several steps. TFO was disconnected by deletion of the phosphate group in place of the predicted bulged insertion. Afterward, 2-phenylethynyl-9-aminoacridine was placed between the bases of the triplex, and minimization was performed to extend the triplex and create enough space for the intercalator. The triplex was reconnected by addition of the two phosphate groups with an ethyleneglycol linker between, which were supposed to be a part of the linker. The structure was again minimized. The intercalator was connected to the TFO backbone by creating the site chain, taking into consideration the stereochemistry of the linker and the whole structure possessing (R)-3-O-[4-(9aminoacridin-2-ylethynyl)benzyl]glycerol as a bulge was again energy-minimized. All molecular dynamics simulations were achieved with the AMBER* force field. The stochastic dynamics calculation generating 250 structures was performed using a water solvent model and extended cutoff potential. The simulation temperature was 300 K, the simulation time was 400 ps, and the equilibration time was 100 ps. In the next step, all 250 structures were minimized at the same time by a multiple minimization method. For classification of founded conformations, the XCluster program was used, in which the lowest energy representative structure was selected. Measurement of Melting Temperatures (Tm). Melting profiles were measured on a Perkin-Elmer UV/vis spectrometer Lambda 35 fitted with a PTP-6 temperature programmer. The parallel triplexes were formed by mixing the two strands of the Watson-Crick duplex each at a concentration of 1.0 µM and the TFO at a concentration of 1.5 µM in the corresponding buffer solution. The solution was heated to 80 °C for 5 min and afterward cooled to 15 °C for 30 min. The antiparallel duplexes were formed by mixing the two strands, each at a concentration of 1.0 µM in the corresponding buffer solution followed by heating to 70 °C for 5 min and then cooling to room temperature. The absorbance of both triplexes and duplexes was measured at 260 nm from 10 to 70 °C with a heating rate of 1.0 °C/min. The melting temperatures (Tm, °C) were determined as the maximum of the first derivative plots of the melting curves. Fluorescence Measurements. Fluorescence measurements were carried out on a Perkin-Elmer luminescence spectrometer LS-55 fitted with a Julabo F25 temperature controller. The spectra were recorded at 10 °C in the same buffer solution as for Tm studies using a 1.0 µM concentration of each ON. The

Bioconjugate Chem., Vol. 17, No. 4, 2006 951

excitation slit was set to 4 nm, and the excitation wavelength was 322 nm. The emission slit for ON11 and ON12 was set to 2.5 nm and for ON4 and ON5 was set to 0 nm. Synthesis of 9-Chloro-2-ethynylacridine (3). 9-Chloro-2iodoacridine (2, ref 17) (0.34 g, 1 mmol), PdCl2(PPh3)2 (14 mg, 0.02 mmol), and CuI (8 mg, 0.04 mmol) were added to a roundbottomed flask flushed with Ar. The flask was again flushed for the next 5 min with Ar. Afterward, THF (3 mL) and Et3N (1 mL) were added, and Ar was bubbled through the mixture for 10 min. Trimethylsilylacetylene (TMSA, 0.155 mL, 1.1 mmol) was added, and the reaction was completed after 30 min at room temperature under Ar. The solvent was evaporated in vacuo. The residue was dissolved in CH2Cl2 (15 mL) and washed with H2O (3 × 20 mL). The organic layer was dried (Na2SO4), filtered, and evaporated in vacuo. The crude product was purified using silica gel dry column vacuum chromatography with cyclohexan/acetone (1%, v/v). 9-Chloro-2-trimethylsilylethynylacridine was obtained in 98% yield (0.15 g) as a yellow powder. 1H NMR (DMSO-d6) δ 0.36 (s, 9H, 3 × CH3), 7.63 (m, 1H, Ar), 7.80 (m, 2H, Ar), 8.17 (m, 2H, Ar), 8.38 (d, 1H, J ) 9.2 Hz, Ar), 8.56 (d, 1H, J ) 2.4 Hz, Ar); 13C NMR (DMSO-d6) 0.2 (3 × CH3), 97.4, 104.6 (CtC), 121.7, 123.8, 124.7, 127.2, 128.5, 129.8, 130.6, 130.9, 133.1, 140.8, 148.1, 149.2 (Ar); HR-MALDI-MS: m/z calcd for C18H17NClSi [M + H]+ 310.0813, found 310.0809. The trimethylsilyl compound (0.15 g, 0.48 mmol) was dissolved in THF (6 mL), and Et3N‚3HF (26 µL, 0.48 mmol) was added. The reaction mixture was stirred at room temperature overnight. The solvent was evaporated in vacuo, and acridine 3 was purified using silica gel dry column vacuum flash chromatography with acetone (1%, v/v)/cyclohexane in quantitative yield (0.115 g). 1H NMR (DMSO-d6) δ 2.21 (s, 1H, CtCH), 7.24-7.86 (m, 5H, Ar), 8.32 (d, 1H, J ) 8.7 Hz, Ar), 8.62 (d, 1H, J ) 2.2 Hz, Ar); 13C NMR (DMSO-d6) 82.3 (Ct CH), 97.9 (CtCH), 116.2, 118.4, 119.8, 125.0, 132.2, 131.1, 133.6, 138.9, 139.6, 143.4, 147.5, (Ar); HR-MALDI-MS: m/z calcd for C15H9NCl [M + H]+ 238.0418, found 238.0427. (S)-1-O-(4,4′-Dimethoxytriphenylmethyl)-3-O-(4-ethynylbenzyl)glycerol (5). To the solution of (R)-3-O-(4-iodobenzyl)glycerol (4, 1.18 g, 4.2 mmol) in DMF (40 mL) was added Et3N (5.8 mL), and Ar was bubbled through the solution for 30 min. Afterward, TMSA (0.67 g, 4.62 mmol) was dissolved under Ar, and CuI (48 mg, 0.25 mmol) and Pd(PPh3)4 (96 mg, 0.08 mmol) were added to the mixture. This was stirred at room temperature under Ar overnight, followed by the addition of Et3N‚3HF (0.38 mL, 2.38 mmol). After 30 min, CH2Cl2 (100 mL) was added, and the mixture was extracted with a 0.3 M aq solution of ammonium salt of EDTA (2 × 75 mL). The organic layer was washed with H2O (3 × 100 mL), dried (Na2SO4), filtered, and evaporated in vacuo to dryness. The residue was coevaporated twice with toluene/EtOH (50 mL, 3:1, v/v) affording (R)-1-O-(4-ethynylbenzyl)glycerol as an oil. The oil was coevaporated with pyridine (20 mL) and then dissolved in anhydrous pyridine (75 mL) and cooled by an ice water bath, and 4,4′-dimethoxytrityl chloride (1.45 g, 4.41 mmol) was added under Ar. The mixture was stirred at room temperature for 16 h, and then an extra portion of 4,4′-dimethoxytrityl chloride (0.7 g, 2.1 mmol) was added. After 24 h, TLC showed no more starting material, and the reaction mixture was quenched by MeOH (2 mL), diluted with EtOAc (150 mL), and extracted with saturated aq NaHCO3 (2 × 100 mL). The water phase was extracted with EtOAc (2 × 50 mL). The combined organic layers were dried (Na2SO4), filtered, and evaporated under diminished pressure. The residue was coevaporated twice with toluene/EtOH (25 mL, 1:1, v/v). The residue was dissolved in EtOAc (50 mL) and adsorbed on silica gel (2.5 g) by evaporation in vacuo and subsequently purified using dry column vacuum

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chromatography with Et3N (0.5%, v/v)/EtOAc (0-50%)/cyclohexane to afford (S)-1-O-(4,4′-dimethoxytriphenylmethyl)-3-O(4-ethynylbenzyl)glycerol (5, 58%, 1.23 g) as a yellow foam. 1H NMR (CDCl ) δ 2.48 (br. s, 1H, OH), 3.06 (s, 1H, CtCH), 3 3.21 (dd, 2H, J ) 5.1 and 7.7 Hz, CH(OH)CH2OCH2), 3.57 (m, 2H, CH2ODMT), 3.78 (s, 6H, 2 × OCH3), 3.95 (m, 1H, CHOH), 4.52 (s, 2H, CH2Ph), 6.80 (d, 4H, J ) 8.8 Hz, DMT), 7.20-7.50 (m, 13H, CH2-Ph and DMT); 13C NMR (CDCl3), 55.2 (OCH3), 64.3 (CHOH), 69.9 (CH(OH)CH2OCH2), 71.6 (CH2ODMT), 72.8 (CH2-phenyl), 76.5 (CtCH), 83.4 (CtCH), 86.1 [C(Ar)3], 121.3, 128.8, 132.1, 138.9 (phenyl), 113.1, 126.7-129.9, 135.9, 144.8, 158.5 (DMT); HR-MALDI-MS: m/z calcd for C33H32Na+O5 [M + Na]+ 531.2142, found 531.2143. (S)-2-O-[2-Cyanoethoxy(diisopropylamino)phosphino]-1O-(4,4′-dimethoxy triphenylmethyl)-3-O-(4-ethynylbenzyl)glycerol (6). (S)-1-O-(4,4′-Dimethoxytriphenylmethyl)-3-O-(4ethynylbenzyl)glycerol (5, 1.2 g, 2.4 mmol) was dissolved under nitrogen in anhydrous CH2Cl2 (50 mL). N,N-Diisopropylammonium tetrazolide (0.62 g, 3.6 mmol) was added followed by dropwise addition of 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphordiamidite (0.95 g, 3.2 mmol) under external cooling with an ice water bath. After 24 h, analytical TLC showed no more starting material, and the reaction was quenched with H2O (30 mL). Layers were separated, and the organic phase was washed with H2O (30 mL). Combined water layers were washed with CH2Cl2 (25 mL). Combined organic phase was dried (Na2SO4) and filtered, and silica gel (1.5 g) and pyridine (0.5 mL) were added. The solvents were removed under reduced pressure. The residue was purified using silica gel dry column vacuum chromatography with Et3N (0.5%, v/v)/EtOAc (0-25%)/cyclohexane. Combined UV-active fractions were evaporated in vacuo affording the final compound 6 (1.45 g, 85%) as a foam that was used in DNA synthesis. 31P NMR (CDCl3) δ 150.3, 150.4 in a ratio of 3:2; HR-MALDI-MS: m/z calcd for C42H49N2Na+O6P [M + Na]+ 731.3220, found 731.3246. Solid-Phase Synthesis of TINA-Acridine Oligonucleotides. DMT-on oligodeoxynucleotides were synthesized in a 0.2 µmol scale on CPG supports using Expedite Nucleic Acid Synthesis System Model 8909 (Applied Biosystems), using 4,5-dicyanoimidazole as an activator and a 0.075 M solution of the corresponding phosphoramidites 6 and 7 in a 1:1 mixture of dry MeCN/CH2Cl2. For insertion of the monomer 6 or (S)-2O-[2-cyanoethoxy(diisopropylamino)phosphino]-1-O-(4,4′-dimethoxytriphen ylmethyl)-3-O-(4-iodobenzyl)glycerol (7, ref 12), the increased coupling (2 min) and deprotection (100 s) time were used. DMT-on oligonucleotides 8 and 9 on a CPG supports were treated twice with a freshly prepared Sonogashira coupling reagent mixture consisting of Pd(PPh3)4 (8.7 mg, 0.08 mmol), CuI (1.4 mg, 0.25 mmol), DMF/Et3N (3.5:1.5, 500 µL), and iodoacridine 2 (5.4 mg, 22.5 µmol) in the case of sequence 9 (Method B) or ethynylacridine 3 (7.6 mg, 22.5 µmol) in the case of sequence 8 (Method A, Scheme 3). The reaction mixture was prepared by weighing all the reagents in a 1 mL plastic syringe followed by flushing with Ar and the addition of DMF and Et3N. The syringe was connected to the one side of the column with the oligonucleotide on a CPG support. This system was thoroughly shaken to dissolve reagents, and another syringe was connected to another end of the column. The reaction mixture was thoroughly flushed from one syringe to the other through the column several times, and this operation was repeated every 45 min for 3 h. Obtained DMT-on oligonucleotide 10 bound to CPG supports was treated with 32% aq ammonia (1 mL) or with a freshly prepared 50% ethanolic solution of tris(2-aminoethyl)amine (1 mL) at room temperature for 2 h and then at 55 °C overnight. Purification of 5′-O-DMTon ONs was accomplished using a reverse-phase semipreparative

Ge´ci et al. Scheme 1. Synthesis of Ethynylacridinea

a (i) Trimethylsilylacetylene (TMSA), Pd2Cl2(PPh3)2, CuI, Et3N/THF, room temperature, 99% and (ii) Et3N‚3HF, THF, room temperature, 98%.

HPLC on a Waters Xterra MS C18 column. DMT groups were cleaved with 80% AcOH (100 µL) for 20 min. Afterward, H2O (100 µL) and 3 M aq AcONa (50 µL) were added, and ONs were precipitated from 99% EtOH (550 µL). Their purity was found to be over 80% by ion-exchange chromatography on a LaChrom system (Merck Hitachi) using a GenPak-Fax column (Waters).

RESULTS AND DISCUSSION Synthesis of TINA-Acridine. We decided to use 9-chloro2-ethynylacridine (3, Scheme 1), which can be coupled by a Sonogashira reaction to the ONs possessing an insertion of the previously described monomer 7 (12). Afterward, the chloride in the nine position of the acridine can be substituted by primary amines. Free amino groups can be protonated under physiological conditions and therefore reduce the electrostatic repulsion between the phosphates of the nucleic acid backbones. This may stabilize nucleic acid duplexes and triplexes. Oligonucleotides possessing 2-methoxy-2-oxoethyl (14), methoxyoxalamido (15), and benzyl thioester (16) precursor groups have been successfully derivatized using different primary amines in high yields. Simultaneously, the base and phosphate protective groups were removed, and the desired oligonucleotides were cleaved from the solid support. The use of secondary amines (40% aq dimethylamine) resulted in a considerably lower conversion of the oligonucleotides (14). The starting 9-chloro-2-iodoacridine (2, ref 17) was prepared by Ullmann condensation from o-chlorobenzoic acid and p-iodoaniline in the presence of Cu and K2CO3 in refluxing i-amyl alcohol in 20% yield followed by cyclization with POCl3 in 75% yield. The yield of the Ullmann condensation was increased to 48%, when the reaction was done in refluxing 2-methoxyethanol (18). In the next step, the Sonogashira reaction with TMSA took place selectively on iodine. When PdCl2(PPh3)2 instead of Pd(PPh3)4 was used as a catalyst (2 mol %), the reaction was finished in 30 min in 99% yield as compared to 24 h and 20% yield for Pd(PPh3)4. In the latter case, the addition of the fresh portion of the catalyst did not increase the yield. To obtain the high yield of the product, it was also necessary to remove the oxygen from the reaction environment with argon. The Sonogashira reaction has been demonstrated on 9-chloroacridine (19), but the substitution of the iodine atom in compound 2 by TMSA was confirmed by the characteristic signals in 13C NMR and by mass spectrometry. Thus, the shift from 93.2 ppm (compound 2) to 121.7 ppm (compound 3) in 13C NMR was observed for the C-2 atom of the acridine ring, which corresponded to the substitution of iodine. At the same time, the chemical shift in 13C NMR of the C-9 atom of the acridine bearing chlorine remained in the same region: 130.0 and 130.6 ppm for compounds 2 and 3, respectively. The mass spectra (HiResMALDI) of 3 confirmed the presence of the chlorine atom in the structure. The protective trimethylsilyl group was removed using Et3N‚3HF in THF at room temperature, and acridine 3 was obtained in quantitative yield. The required TINA precursor (R)-1-O-(4-iodobenzyl)glycerol phosphoramidite 7 was synthesized in four steps as previously described (12), and it was used in the synthesis of ON 8 (Method A).

Synthesis of TINA-Acridine Scheme 2. amiditea

Synthesis of (4-Ethynylbenzyl)glycerol Phosphor-

Bioconjugate Chem., Vol. 17, No. 4, 2006 953 Scheme 3. Postsynthetic Modification of Oligonucleotidesa

a (i) TMSA, CuI, Pd(PPh3)4, Et3N/DMF, room temperature, Ar, overnight. (ii) Et3N‚3HF, CH2Cl2. (iii) DMTCl, pyridine, 0 °C, Ar, 24 h. (iv) NC(CH2)2OP(NPri2)2, diisopropylammonium tetrazolide, CH2Cl2, 0 °C to room temperature, 24 h.

Because of the selectivity observed in the Sonogashira reaction between TMSA and acridine 2, we decided to explore an alternative route using compound 2 for the conversion ONs into TINA-acridine oligonucleotides (Method B). For this purpose, the (R)-1-O-(4-ethynylbenzyl)glycerol phosphoramidite 6 was synthesized in four steps from (R)-3-O-(4-iodobenzyl)glycerol (4) in 49% overall yield (Scheme 2). The latter compound was treated with the Sonogashira reaction mixture having TMSA followed by cleavage of the silyl protective group with Et3N‚3HF, which was done in one pot affording (R)-3-O(4-ethynylbenzyl)glycerol. This compound was used in the next step without chromatographic purification. Compound 5 was obtained in 56% overall yield from 4 after protection of the primary alcohol with DMTCl in dry pyridine. Treatment of compound 5 with 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphordiamidite in the presence of diisopropylammonium tetrazolide in dry CH2Cl2 overnight followed by silica gel purification gave the phosphoramidite 6, which was used in DNA synthesis. Corresponding ONs 9 were prepared, applying the same conditions as for 8 (12). To avoid the transamination during the treatment with amines, dCAc phosphoramidite was used for the incorporation of 2′-deoxycytidine into ONs. Standard phosphoramidites for the incorporation of A, T, and G and commercial CPG solid supports were used. For the postsynthetic Sonogashira type modification on a solid phase, ONs with an insertion of (R)-1-O-(4-iodobenzyl)glycerol (8) or (R)-1-O-(4-ethynylbenzyl)glycerol (9) in the middle and at the 5′-end were used. CPG supports with DMT-on ONs 8 or 9 were treated with a freshly prepared Sonogashira coupling reagent mixture possessing ethynylacridine 3 or iodoacridine 2, respectively (Scheme 3). In both cases, the same product 10 on a solid support was obtained. However, we preferred to use Method B because the preparation of compound 9 is easy. In a previous study, it has been determined that double treatment of the CPG-bound oligonucleotides with a freshly prepared Sonogashira mixture increased the coupling efficiency (12). In our case, it was also obligatory due to the low solubility of acridines 2 and 3 in DMF/Et3N at room temperature. We have now shown that the previously reported postsynthetic Sonogashira coupling on ONs can be extended to aromatic heterocyclic systems (12). Furthermore, this route was an advantage in our case because the required phosphoramidite for the synthesis of ONs 8 and 9 was readily available in our laboratory. In the second postsynthetic step, the treatment of ONs 10 with 32% aq ammonia or a 50% ethanolic solution of tris(2aminoethyl)amine at 55 °C overnight led to the substitution of the chlorine atom on acridine. The cleavage of ONs from the CPG support and deprotection of the bases and phosphates were performed in the same step. The reaction mixture was purified

a (i) Reaction of 8 with 3 or reaction of 9 with 2 both using Pd(PPh3)4, CuI, DMF/Et3N, Ar. (ii) 32% NH4OH or 50% ethanolic solution of tris(2-aminoethyl)amine, room temperature for 2 h then 55 °C overnight. (iii) RP-HPLC; 80% aq AcOH, 3 M aq AcONa, 99% EtOH.

Figure 2. Reverse-phase HPLC profiles of oligonucleotides 11 (a) after treatment of ON 10 using 32% aq ammonia (isolated ON4 at 22.4 min and ON8 at 25.2 min). (b) After treatment of ON 10 using a 50% ethanolic solution of tris(2-aminoethyl)amine (isolated ON13 at 16.4 min).

by semipreparative HPLC on a C18 column. UV-active fractions after evaporation of the solvent were treated with 80% aq AcOH to remove DMT protective groups followed by precipitation from EtOH. ONs were characterized by MALDI-TOF MS. Treatment of ONs 10 with secondary amines (N,N-dimethylethylenediamine or piperazin) gave no desired product after reaction at 55 °C for several days. In the case of treatment of ON 10 with 32% aq ammonia, two peaks at 22.4 and 25.2 min were collected during HPLC purification (Figure 2a), which gave the same m/z values in MALDI-TOF. More accurate determination was performed using ESI mass spectrometry. For the fraction that appeared at 22.4 min, the m/z peak was detected at 4580.98 Da. For the second fraction, m/z was found to be 4581.99 Da. According

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Table 1. Tm (°C) Data for Parallel Triplex1 Melting, Taken from UV-Melting Curves at 260 nm and Mass Spectra of Synthesized ONsa

a 1c ) 1.5 µM TFO and 1.0 µM each strand of dsDNA in 20 mM sodium cacodylate, 100 mM NaCl, 10 mM MgCl , pH 5.0, 6.0, and 7.2; duplex T ) 2 m 56.5 °C (pH 5.0), 58.5 °C (pH 6.0), and 57.0 °C (pH 7.2). 2The Tm values for parallel triplex were taken from a previous study (12). 3Third strand and duplex melting overlaid. 4Not determined. 5Measured by ESI-MS. 6Measured by MALDI-TOF MS.

Table 2. Tm (°C) Dataa for Antiparallel Duplex1 Melting, Taken from UV-Melting Curves at 260 nm and Mass Spectra of Synthesized ONs

a1 c ) 1.0 µM each oligonucleotide in 140 mM NaCl, 10 mM sodium phosphate buffer, 1 mM EDTA, pH 7.0. 2The T values were taken from a previous m study (12). 3Not determined. 4Measured by MALDI-TOF MS.

to calculated mass, the fraction appearing at 22.4 min corresponded to 11 (ON4) containing the X monomer with 9-aminoacridine. One dalton difference m/z for the peak at 25.2 min means that the use of 32% aq ammonia for the modification of 10 resulted also in the formation of 13 (ON8) containing the Z monomer with 9-oxoacridine. It has been shown previously that in the reaction of 9-chloroacridine with amines, 9-oxoacridine also can be observed as a side product (20). From RPHPLC profiles in Figure 2a,b, we observed a high conversion of ONs during the two postsynthetic steps. The lowest amounts of ONs were obtained in the case of treatment with a 50% ethanolic solution of tris(2-aminoethyl)amine. Thermal Stability and Fluorescence Studies. Melting temperatures (Tm) of parallel triplexes and antiparallel duplexes consisting of ON1-ON13 were estimated as the first derivative of melting curves and are shown in Tables 1 and 2. For comparison, previously published data for TINA possessing monomer 1 are also listed in Tables 1 and 2. Stabilization of parallel triplexes was found in all cases when compared with the wild-type ON1 at pH 5.0, 6.0, and 7.2 (Table 1). TINA-acridine intercalators X-Z gave less stable parallel triplexes, when inserted as a bulge in the middle of TFO as compared to ON2 possessing a pyrene derivative 1 (12). In the

case of ON5 bearing 9-aminoacridine X at the 5′-end, the triplex stabilization was found to be similar as for TINA-pyrene (ON3). The similarity in Tm values for ON4 and ON8 means that the contribution of the amino group connected directly to the acridine ring (monomer X) into the triplex stabilization is negligible. The introduction of the 2-[bis-(2-aminoethyl)amino]ethylamino group by reaction of 10 with tris(2-aminoethyl)amine (monomer Y) in the middle of the TFO (ON6) led to lower triplex stabilization as compared with ON4. Moreover, the incorporation of the monomer Y at the 5′-end of the TFO (ON7) led to the least stabilized modified triplexes studied here. We suppose that the latter effect is due to electrostatic interactions between the positively charged cytosine of TFO and the protonated amino groups in the monomer Y. When monomers X and Y were incorporated as a bulge in the middle of the oligodeoxynucleotides (ON11 and ON13) (Table 2), destabilization of antiparallel DNA/DNA and DNA/ RNA duplexes was observed when compared with the wildtype duplexes ON9/ON14 and ON9/ON15. This destabilization effect was found to be smaller as compared with the TINAcontaining pyrenyl moiety (ON10) (TmON11/ON14 - TmON10/ON14 ) 5.0 °C). Such a difference can be explained by a lower number of fused benzene rings in acridine than in pyrene and

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Figure 3. Fluorescence emission spectra of TINA bearing 9-aminoacridine inserted in the middle (a) and at the 5′-end (b) of the duplex and at the middle (c) and at the 5′-end (d) of the triplex.

by the presence of amino groups in X and Y. When the intercalator X was inserted at the 5′-end (ON12) as a dangling end, stabilization of the DNA/DNA (∆Tm ) +6.0) and DNA/ RNA (∆Tm ) +5.0) duplexes was observed. We studied fluorescence properties of TINA bearing 9-aminoacridine (X) in the middle and at the 5′-end of ONs in the structure of Hoogsteen type triplexes and Watson-Crick duplexes. When the modification was placed in the middle of the strand (ON11, Figure 3a), no quenching of the fluorescence intensity was observed for DNA/DNA and DNA/RNA duplexes, when compared with the single-strand ON11. However, the fluorescence intensity of ON4 at the same excitation wavelength was quenched considerably, when a parallel triplex was formed (Figure 3c). Quenching was also observed upon both duplex and triplex formation with the oligonucleotides ON12 and ON5, respectively, upon insertion of the acridine moiety at the 5′end (Figure 3b,d). This can be explained by a partial quenching of the monomer fluorescence as a result of communication of the acridine with neighboring bases upon its insertion in the middle of the sequence. These results indicate that the acridine ring in the structure of TINA intercalates better, when inserted in the TFO strand of the parallel triplexes, than insertion of the TINA-acridine moiety in one of the strands of the antiparallel duplexes. This is also in agreement with the melting studies from the present work. Molecular Modeling. Modeling studies were carried out to provide better structural understanding of binding of the acridine derivative in the parallel triplex mode used in triplex experiments (ON4 + D1). Such a triplex structure was modified by the insertion of (R)-3-O-[4-(9-aminoacridin-2-ylethynyl)benzyl]glycerol (X) as a bulge in the middle of the 14-mer sequence (Figure 4a,b). According to the structure minimized by the AMBER* force field, 9-aminoacridine was stacked between the bases of the dsDNA, while the phenyl was placed in the TFO strand.

Twisting around the triple bond of 19.3° was also observed for structure X, which is shown on the detailed view in Figure 4b. As it can be seen in Figure 4a, the amino group in the ninth position of the acridine was directed into the major groove of dsDNA. This can explain why the presence of both the amino group and the 2-[bis-(2-aminoethyl)amino]ethylamino group at position 9 of the acridine did not give a great contribution in thermal stabilization of the parallel triplex. With this orientation, these groups cannot reach the phosphates of the nucleic acid backbone and neutralize the negative charge. According to modeling, directing the amino group into the minor groove gave more energy-rich structures. It can be proposed that substitutions at positions 4 and 5 of the acridine ring or at position 10 of the acridone should direct aminoalkyl groups in the minor groove of dsDNA and point amines closer to phosphates by the appropriately chosen alkyl linker. To elucidate the destabilization of duplexes, we simulated the intercalation of X in the modeling of the antiparallel duplex ON11/ON14. The amino group was directed into the minor or into the major groove, as is shown in Figure 4c,d, respectively. After the comparison of potential energies of the leading conformations obtained from AMBER* calculations, it was found that the preferable position of the acridine is when the amino group is directed into the minor groove. A twisting around the triple bond of 34.9° was observed. However, in both cases, the stacking of the intercalator between the bases was imperfect. These results are in agreement with the lower Tm values of the duplexes, and they explain also the negligible fluorescence quenching of the acridine upon duplex formation.

CONCLUSION In this paper, we describe the postsynthetic synthesis of twisted intercalating nucleic acids possessing acridine derivatives. Using the Sonogashira reaction, 9-chloroacridine was

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Molecular modeling calculations showed that the triplex stabilization was due to the intercalation of the acridine moiety between the bases of the dsDNA and via the interaction of the phenyl ring with the bases of TFO. 9-Chloroacridine was found to be a good precursor for the incorporation of primary amines to the oligonucleotides. We believe that parallel triplex stabilization can be further improved by the appropriately chosen substitution on the acridine in the structure of TINA monomers.

ACKNOWLEDGMENT This work was supported by the Sixth Framework Program Marie Curie Host Fellowships for Early Stage Research Training under Contract MEST-CT-2004-504018 and by the Nucleic Acid Center, which is funded by The Danish National Research Foundation for studies on nucleic acid chemical biology. Kim F. Haselmann is gratefully acknowledged for measuring the ESIMS spectra. Molecular graphics images (Figure 4c,d) were produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIH P41 RR01081).

LITERATURE CITED

Figure 4. (a) Energy-minimized averaged AMBER* representative structure of the parallel triplex-containing (R)-3-O-[4-(9-aminoacridin2-ylethynyl)benzyl]glycerol (X) (dsDNA displayed in CPK form in white, TFO in stick form as well as the TINA-acridine intercalator marked with green). (b) Detailed view of the monomer X placed between the two triples showing the torsion between the acridinyl and the phenyl ring connected via acetylene. (c) Representative structure of the antiparallel duplex-containing monomer X directed into the minor groove. (d) Representative structure of the antiparallel duplex-containing monomer X directed into the major groove.

introduced into ONs possessing TINA precursor monomers 8 and 9. In the second postsynthetic step, treatment of ONs 10 with aq ammonia or ethanolic tris(2-aminoethyl)amine allowed substitution of the chlorine atom on acridine, cleavage from the CPG support, and deprotection of the bases in one pot. Thermal denaturating studies showed that the introduction of acridine into TINA led to the stabilization of parallel triplexes. However, as compared with TINA possessing pyrene 1, the stabilization effect of acridines X-Z was not as significant with the exception of the ON5-containing TINA-acridine monomer X at the 5′-end. It was supposed that the triplex thermal stability can be further increased by the introduction of primary amines into the acridine via an alkyl linker, which should reduce the electrostatic repulsion between phosphates. Nevertheless, the introduction of TINA-acridine Y possessing a tris(2-aminoethyl)amine moiety gave the least thermally stable parallel triplex. In the case of antiparallel duplexes, introduction of TINA acridines X and Y to the dsDNA structures resulted in less destabilized DNA/DNA and DNA/RNA Watson-Crick duplexes as compared with TINA 1. The stabilization effect in the case of ON12/ON14 and ON12/ON15 was found when 9-aminoacridine X was inserted at the 5′-end of the ON.

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