Synthesis and Binding Properties of Oligonucleotides Covalently

Bioconjugate Chem. 1996, 7, 369−379

369

Synthesis and Binding Properties of Oligonucleotides Covalently Linked to an Acridine Derivative: New Study of the Influence of the Dye Attachment Site Ulysse Asseline,† Edwige Bonfils,‡ Daniel Dupret,‡ and Nguyen T. Thuong*,† Centre de Biophysique Molećulaire, CNRS, Rue Charles Sadron, 45071 Orleáns Cedex 02, France, and Appligene-Oncor, Parc d’Innovation, BP, 72, 67407 Illkirch, France. Received November 14, 1995X

2-Methoxy-6-chloro-9-aminoacridine has been coupled via a polymethylene linker to various positions of an oligonucleotide chain: the 3′-position, using a new universal support, the 5′-position, and both 5′- and 3′-positions via a phosphate. The intercalating agent was also linked to the oligonucleotide chain via an internucleotide phosphorothiolate. The mixture of diastereoisomers was obtained as well as each pure Rp and Sp isomer. Finally, the acridine moiety was introduced to the 5-position of the deoxyuridine. The binding properties of these oligonucleotide-acridine conjugates with their DNA counterparts have been studied by absorption spectroscopy.

INTRODUCTION

The base-pairing properties of oligomeric strands of DNA which allow specific recognition of a particular base sequence in a single-stranded RNA target by a complementary base sequence are the foundation for their use as artificial regulators of gene expression via the “antisense” strategy (1). The regulation of gene expression can also be achieved by interaction of an oligonucleotide with a single-stranded DNA target in the opened transcription initiation region (2). More recently, synthetic oligonucleotides were used to regulate gene expression by hybridization with double-stranded DNA (for a review, see ref 3) via Hoogsteen base pairing. The efficiency of these synthetic oligonucleotides in regulating gene expression in living cells depends on the thermodynamic stability of the duplex or triplex structure, their resistance toward nuclease and cellular uptake. In order to increase the stability of the duplexes, intercalating agents such as acridine (4, 5), pyrene (6-9), psoralen (10-12), anthraquinone (13-16), oxazolopyridocarbazole (17), ellipticine (18), phenazinium (19, 20), fagaronine (21), coumarine (22), and ethidium (23) have been covalently coupled to different positions of oligonucleotides 5′ (4, 6, 11-13, 19, 21-23), 3′ (4, 17, 19, 23), and both 5′ and 3′ (4, 19), internucleotidic phosphate (4, 7), the 1′- (8) or 2′(13, 14, 16) position of the sugar moiety, or nucleic bases: at the C5-position of deoxyuridine (20), at the C8position of deoxyadenosine (10), or at the exocyclic amino function of deoxyguanosine (15) and deoxycytidine (9). Only the coupling of the intercalating agent to the 5′- or the 3′-end of the oligonucleotide led to a strong stabilization. In our previous studies carried out with 2-methoxy6-chloro-9-aminoacridine, chosen because of its lack of specificity with respect to base or base pair interactions (24), a polymethylene linker was used to connect the acridine derivative to the oligonucleotide chain (4, 5). These results showed that a linker with five methylene groups between the 9-amino group of the acridine and the 3′-phosphate of the oligonucleotide was long enough to obtain an optimum interaction between the intercalat* To whom correspondance should be addressed. Phone: (33) 38 51 55 97. Fax: (33) 38 63 15 17. E-mail: [email protected]. † CNRS. ‡ Appligene-Oncor. X Abstract published in Advance ACS Abstracts, April 15, 1996.

S1043-1802(96)00024-9 CCC: $12.00

ing agent and the double-stranded structure formed by the oligonucleotide and its complementary sequence (4, 5). In order to increase the stability of the obtained duplex, one intercalating agent was added to both ends of the oligonucleotide via a phosphate. Surprisingly the presence of two intercalating agents led to the same stabilization as did the presence of one (4). The covalent attachment of the intercalating agent at an internucleotidic phosphate was also tested (4). In this case, the stabilizing effect observed was lower than that obtained by linking the intercalating agent at the end (3′ or 5′) of the oligomer. This is probably due to the fact that the phospho triester is a mixture of diastereoisomers having different stability. Other experiments with oligonucleotides involving “phospho triester” groups have shown that one isomer leads to the formation of a duplex having a higher stability than that of the unmodified parent while the second isomer leads to the one with lower stability (25). In order to further explore the possibility of increasing the stability of the complexes by using more than one intercalating molecule per oligomer [to our knowledge, the stabilization of the duplex by more than an intercalating agent has never been described except in one case in which a low additional stabilization occurred (19)], we decided to covalently link the intercalating agent to the oligonucleotide chain via a polymethylene linker using either a terminal (5′, 3′, or both 5′ and 3′) or an internucleotidic phosphorothiolate or the 5-position of a deoxyuridine. In order to investigate the influence of the stereochemistry of the phosphorothiolate triester, the mixture of diastereoisomers as well as each pure Rp and Sp isomer were also synthesized. We report here the preparation of these oligonucleotides covalently linked to 2-methoxy-6-chloro-9-aminoacridine and their binding properties with a complementary single-stranded DNA sequence. EXPERIMENTAL PROCEDURES

General Methods. All solvents used were dried, distilled, and stored as described in ref 26. All chemicals were used as obtained unless otherwise stated. 6,9Dichloro-2-methoxyacridine, phenol, dimethylaminopyridine, acetic anhydride, succinic anhydride, p-nitrophenol, dicyclohexylcarbodiimide, 1,4-diaminobutane, 1,6diaminohexane, 1,7-diaminoheptane, diisopropylethyl© 1996 American Chemical Society

370 Bioconjugate Chem., Vol. 7, No. 3, 1996

amine, 2-(cyanoethyl)-N,N-diisopropylchlorophosphoramidite, dioxane, N-hydroxysuccinimide, and solketal were purchased from Aldrich. Triethylamine and sodium sulfate were from Merck. Pyridine and dichloromethane were from SDS. Acetonitrile was from Labo-Standa and long chain alkylamine CPG from Pierce. 6-Chloro-9phenoxy-2-methoxyacridine synthesis was achieved as described in ref 27. d5′TTTCCTCCTCT3′pS(CH2)6NHAcr was prepared following a previously described procedure (28, 29). Analytical thin-layer chromatography (TLC) was performed on precoated alumina plates (Merck silica gel 60F 254 ref 5554) and preparative TLC on glassbacked plates of Merck silica gel 60 F 254 ref 5717. For flash chromatography, Merck silica gel 60 (70-230 mesh) (ref 7734) and anhydrous solvents were used. All 4,4′dimethoxytrityl-containing substances were identified as orange-colored spots on TLC plates by spraying with a 10% perchloric acid solution. Acridine-containing compounds were directly visualized on the plates. Analysis and purification by ion-exchange chromatography were carried out on a Pharmacia FPLC with a DEAE column (8 µM, 100 mm × 10 mm, Waters) with a linear gradient of NaCl in Tris/HCl (pH 7), 25 mM buffer containing 10% CH3CN. Reversed-phase chromatography analysis was performed on a 600 E instrument (System Controller) equipped with a photodiode array detector (Waters 990) using a Lichrospher 100 RP (5 µM) column (125 mm × 4 mm) from Merck with a linear gradient of CH3CN in 0.1 M aqueous triethylammonium acetate (pH 7) with a flow rate of 1 mL/min. For mass analysis, ion-molecular masses of the oligonucleotides were confirmed by mass spectroscopy using a Lasermat Time-of-Flight instrument (LD-TOF). The average power of the nitrogen laser (337 nm) was about 107 W/cm2. To improve the signal to noise ratio, 10 single shot spectra were accumulated and averaged. All measurements were performed using the negative detection mode. The spectrometer was calibrated with the M - H and 2M - H mass peaks of d5′(Tp)8O(CH2)5NHAcr as reference. The sample preparation was performed by mixing 10 µL of a 0.5 M solution of 2,4,6-trihydroxyacetophenone in ethanol and 5 µL of a 0.1 M aqueous solution of diammonium L-tartrate. To this was added 1 µL of the oligonucleotide solution (10 OD/mL), and the mixture was briefly vortexed. To the probe tip was applied 1 µL of this solution, and the solvents were removed. 1H-NMR spectra were recorded with a Bruker AM 300 MHz instrument. Absorption spectra were recorded with an Uvikon 860 spectrophotometer. Absorption coefficients of one acridine-containing oligomer were chosen to be 8850 M-1 cm-1 at λ ) 425 nm and 16 000 M-1 cm-1 at λ ) 425 nm for oligomers containing two acridine moieties by analogy with our previously described work (30). Nucleases were purchased from Boehringer, Mannheim. Preparation of the Modified Support 3. (3-Aminopropyl)solketal was obtained as described in ref 31. (N-Acridinyl-3-aminopropyl)solketal (1). 2-Methoxy6,9-dichloroacridine (3 g, 10.8 mmol) and (3-aminopropyl)solketal were stirred in phenol (8.6 g) for 90 min at 120 °C. After being cooled, the obtained mixture was poured into a 2 N NaOH aqueous solution. The precipitate was dissolved in ethyl acetate (180 mL) and washed twice with 50 mL of water. After being dried over Na2SO4, the organic layer was concentrated to dryness under reduced pressure. The resultant oil was chromatographed on a silica gel column using CH2Cl2/ CH3OH (9:1 v:v) as eluent. Compound 1 was obtained as a yellow solid with 45% yield (2 g). Rf1 ) 0.23 using CH2Cl2/MeOH (90:10 v:v) as eluent. 1H-NMR (DMSO): δ 1.20 (s, 3H), 1.24 (s, 3H), 1.94 (m, 2H, CH2CH2CH2),

Asseline et al.

3.38 (m, 2H), 3.48 (t, 2H, CH2NH), 3.85 (m, 4H), 3.92 [s, 3H, CH3O (Acr)], 4.03 (m, 1H), 7.24-7.83 (m, 5H, Acr), 8.26-8.31 (d, 1H, H8 Acr). 1-O-(4,4′-Dimethoxytrityl)-3-O-(N-acridinyl-3-aminopropyl)glycerol (2). (N-Acridinyl-3-aminopropyl)solketal (1) (2 g, 4.65 mmol) was dissolved in a mixture of tetrahydrofuran (45 mL) and 1 M hydrochloric acid (45 mL) and left for 30 min, and absolute ethanol (4 mL) was added. The mixture was concentrated, and the residue was redissolved in absolute ethanol (45 mL) and reconcentrated. The obtained residue was dried by coevaporation with anhydrous pyridine and dissolved in pyridine. 4,4′-Dimethoxytrityl chloride (1.56 g, 4.65 mmol) was added, and the mixture was stirred for 2 h at room temperature. Absolute ethanol (20 mL) was added, and the mixture was concentrated under reduced pressure. The residue was dissolved in CH2Cl2 (100 mL) and washed with water (2 × 30 mL) and with a saturated NaCl aqueous solution. The organic layer was dried over Na2SO4 and concentrated to dryness. The resultant oil was chromatographed on a silica gel column eluted with CH2Cl2/MeOH (95:5 v:v) to give compound 2 (2.1 g) with 59% yield. Rf2 ) 0.61 using CH2Cl2/MeOH (90:10 v:v) as eluent. 1H-NMR (DMSO): δ 1.97 (m, 2H, CH2CH2CH2), 2.5 (m, 4H), 3.08 (m, 1H), 3.48 (t, 2H, CH2NH), 3.65 (s, 6H, DMTr), 3.77 (t, 2H, OCH2CH2), 3.90 [s, 3H, CH3O (Acr)], 5.06 (m, 1H, CHOH), 6.56-7.4 (m, 7H, DMTr), 7.40-7.90 (m, 5H, Acr), 8.26-8.88 (d, 1H, H8 Acr). Acridine-Containing Modified Support 3. Compound 2 (1.2 g, 1.6 mmol) and dimethylaminopyridine (DMAP) (200 mg, 0.8 mmol) were dried by coevaporation with anhydrous pyridine (3 × 15 mL) and dissolved in pyridine (15 mL). Succinic anhydride (150 mg, 1.5 mmol) was added, and the solution was stirred overnight at 45 °C. Pyridine was removed by evaporation, and the obtained residue was dissolved in CH2Cl2 (200 mL). The organic layer was washed with an ice-cooled 10% aqueous solution of citric acid (2 × 100 mL) and then with a saturated aqueous NaCl solution. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified on a silica gel column eluted with CH2Cl2/CH3OH (85:15 v:v) to afford 1 g of the succinate derivative. Rf ) 0.38 and 0.35 (stereoisomers) using CH2Cl2/CH3OH (90:10 v:v) as eluent (yield of 77%). The succinate derivative (0.8 g, 1.1 mmol) was dried by coevaporation with anhydrous pyridine (3 × 10 mL) and redissolved in dry dioxane (3 mL). Pyridine (0.8 mL), N-hydroxysuccinimide (136 mg, 1.1 mmol), and DCCI (0.8 g, 2.9 mmol) were added, and the mixture was stirred overnight at room temperature. The precipitate of dicyclohexylurea was filtered, and the filtrate was concentrated under reduced pressure. The residue was chromatographed on a silica gel column eluted with acetone/hexane (6:4 v:v) to afford 0.7 g of the succinimidylated derivative in 77% yield. Rf ) 0.23 using ethyl acetate/MeOH (85:15 v:v) as eluent. Long chain alkylamine CPG (1 g) was added to a solution of the succinimidyl derivative (0.4 g, 0.48 mmol) in 2.5 mL of anhydrous dioxane, 0.5 mL of DMF, and 0.25 mL of NEt3. The suspension was gently stirred for 24 h at room temperature. The supernatant was filtered off, and the support was washed with dioxane (2 × 10 mL), CH2Cl2 (2 × 10 mL), methanol (2 × 10 mL) and ether (2 × 10 mL). Then a capping step was performed and is described in ref 26. The modified support 3 was obtained with a loading of 16 µmol/g. Synthesis of the Oligonucleotides 7, 7a, and 7b Bearing the Acridine at an Internucleotidic Phosphate. Preparation of Oligonucleotide 4 Involving a Central Phosphorothioate Group and Separation of Its

Oligonucleotide−Acridine Conjugates

Diastereoisomers. The oligodeoxyribonucleotide 4 was assembled using classical phosphoramidite chemistry except that at the site selected for the introduction of a phosphorothioate group the oxidation step by iodine was replaced by sulfuration using a 15 min treatment with a 0.5 M tetraethylthiuram disulfide solution in CH3CN (32). The deprotection step was carried out using concentrated ammonia treatment. Purification was performed by reversed-phase chromatography using system A (5 to 42.5% CH3CN in 50 min). Two roughly equivalent peaks with very close retention times (tR ) 20 min 51 s and 21 min 3 s) which correspond to the two diastereoisomers were obtained. These diastereoisomers were separated by using a more flat gradient (system C, see Table 2). Under these conditions, retention times were 23 min 30 s and 24 min 30 s, respectively. Preparation of Dinucleoside d5′Tp(S)C3′. This dinucleoside used as a reference was obtained by solid-phase synthesis using tetraethylthiuram disulfide as the sulfurizing agent. After deprotection and releasing from the support, reversed-phase purification of the dinucleoside gave nearly equivalent peaks (55 and 45%, respectively) which correspond to the diastereoisomers (tR ) 21 min 48 s and 24 min 13 s using the following solvent system: 0% buffer B for 5 min, 0 to 10% in 10 min, and finally 10 to 20% in 30 min). Synthesis of 2-Methoxy-6-chloro-9-(ω-aminooctylamino)acridine 5. 2-Methoxy-6,9-dichloroacridine (0.3 g, 1.2 mmol), phenol (2.5 g), and 1,8-diaminooctane (0.77 g, 5.38 mmol) were reacted for 90 min at 100 °C under magnetic stirring. The mixture was diluted with methanol (5 mL) and added dropwise to a magnetically stirred 2 M sodium hydroxide solution (15 mL) for 10 min. The yellow precipitate was extracted with dichloromethane (3 × 60 mL), and the organic phase was concentrated to dryness. The obtained residue was purified on glassbacked plates of Merck silica gel 60 F 254 ref 5717 using CH2Cl2/MeOH (80:20 v:v) as eluent (twice) and then CH2Cl2/MeOH/DIEA (80:20:4 v:v). Compound 5 was obtained as a yellow powder with 70% yield. The presence of the primary amino group was verified using the ninhydrin test. Rf5 ) 0.48 using CH2Cl2/MeOH/DIEA (80:20:4 v:v) as eluent. Preparation of the Iodoacetamidoacridinyl Derivative 6. Compound 5 (0.15 g, 1.03 mmol) and p-nitrophenyl iodoacetate (0.48 g, 1.55 mmol) were reacted in dry DMF at room temperature for 3 h under magnetic stirring. The solvent was removed by evaporation under vacuum, and the residue was purified on glass-backed plates of Merck silica gel 60 F 254 ref 5717 using CH2Cl2/MeOH (83:17 v:v) as eluent. Compound 6 was obtained as a yellow powder after stirring in hexane with a 75% yield. Rf6 ) 0.40 using CH2Cl2/MeOH (83:17 v:v) as eluent. Synthesis of the Oligonucleotides 7, 7a, and 7b by Coupling of the Acridine Derivative 6 with the Oligonucleotides 4, 4a, and 4b. Oligodeoxyribonucleotide 4, 4a, or 4b (d5′TpTpTpCpCpTp(S)CpCpTpCpT3′) (ammonium salt) (10 OD units) was dissolved in methanol (1 mL) in the presence of crown-6 ether (10 mg), and compound 6 (1 mg) was added. The mixture was kept at 45 °C for 2 h. The reaction mixture was then analyzed on a reversed-phase column using a linear gradient of 15 to 45% of buffer B in 40 min. The coupling yield was higher than 75%. tR4a ) 9 min 12 s, tR4b ) 9 min 30 s, and tR7 ) 23 min 35 s. The two isomers of compounds 7, 7a, and 7b could be better separated using a different gradient, 22 to 24% of buffer B in 25 min. tR7a ) 18 min 5 s, and tR7b ) 19 min 37 s. 5-(ω-Aminohexylamino)-5′-O-(dimethoxytrityl)-2′deoxyuridine (8b). 5-Bromo-5′-O-(dimethoxytrityl)-2′-

Bioconjugate Chem., Vol. 7, No. 3, 1996 371

deoxyuridine (0.3 g, 0.49 mmol) was dried by coevaporation with anhydrous CH3CN (3 × 50 mL), kept in CH3CN (25 mL), and 1,6-diaminohexane (0.56 g, 4.9 mmol) was added. The solution was stirred for 2 days at 65 °C. The solution was then concentrated to dryness, and the residue was dissolved in CH2Cl2 (100 mL) and washed with water (2 × 25 mL). The organic layer was dried over sodium sulfate and then concentrated under reduced pressure. The obtained residue was purified twice on glass-backed plates of silica gel using CH2Cl2/ MeOH (90:10 v:v) as eluent and then CH2Cl2/MeOH/NEt3 (80:15:5 v:v). Compounds 8a and 8c were obtained following the same protocol and using the appropriate diamine, 1,4-diaminobutane and 1,7-diaminoheptane, respectively [Rf8a ) 0 using CH2Cl2/MeOH (90:10 v:v), Rf8b ) 0 using CH2Cl2/MeOH (90:10 v:v), and Rf8c ) 0 using CH2Cl2/MeOH (90/10 v:v)]. 5-(ω-Acridinylaminohexylamino)-5′-O-(dimethoxytrityl)2′-deoxyuridine (9b). 8b (0.19 g, 0.29 mmol) was dried by coevaporation with anhydrous CH3CN and pyridine and redissolved in CH2Cl2 and CH3CN, and 6-chloro-9phenoxy-2-methoxyacridine (0.2 g, 0.58 mmol) was added. The mixture was allowed to react at 65 °C with magnetic stirring. After 1 night, the solvent was removed by evaporation and the yellow residue was purified twice on glass-backed plates of silica gel 60 using CH2Cl2/ MeOH (90:10 v:v) as eluent. Rf9b ) 0.26 using CH2Cl2/ MeOH (87:13) (twice) as eluent. Yield ) 65%. Compounds 9a and 9c were obtained starting from compounds 8a and 8c, respectively, and by using the same protocol. Rf9a ) 0.18 using CH2Cl2/MeOH (87:13 v:v) (twice) as eluent. Yield ) 68%. Rf9c ) 0.30 using CH2Cl2/MeOH (87:13 v:v) (twice) as eluent. Yield ) 70%. 9a. MS (DIC): m/z ) 857 (M•-)CI/NH3. 9a. 1H-NMR (DMSO): δ 1.60-1.90 (m, 4H, NCH2CH2CH2CH2NHAcr), 2.00-2.10 (m, 2H, H-2′′, H-2′); 2.85 (m, 2H, NCH2), 3.55 (m, 2H, H-5′, H-5′′), 3.90 [br s, 5H, CH3O (Acr) + CH2N], 4.25 (m, 1H, H-4′), 4.48 (m, 1H, H-3′), 5.0 (m, 1H, 3′-OH), 5.18 (d, 1H, 5′-OH), 6.24 (t, 1H, H-1′), 6.82 (s, 1H, H-6), 7.29-7.58 (m, 5H, Acr), 8.39-8.42 (d, 1H, H8 Acr), 11.28 (s, 1H, NH). Preparation of the Modified Support 10. This was achieved as described in ref 26 using compounds 9a and 9b instead of 5′-O-(dimethoxytrityl)thymidine. Preparation of 5-(ω-Acridinylaminobutylamino)-5′-O(dimethoxytrityl)-3′-O-[(2-cyanoethyl)-N,N-diisopropylamidophosphite]-2′-deoxyuridine (11a). To a dichloromethane solution (4 mL) of 5-N-[2-methoxy-6-chloro-9(ω-butylamino)acridinyl]-5′-O-(dimethoxytrityl)-2′deoxyuridine (9a) (0.14 g, 0.14 mmol) previously dried by coevaporation with anhydrous pyridine and kept under vacuum overnight was added excess diisopropylethylamine (0.135 mL, 0.093 g, 0.72 mmol) under a nitrogen atmosphere. (2-Cyanoethyl)-N,N-diisopropylchlorophosphite (0.040 g, 0.17 mmol) was added dropwise at room temperature with magnetic stirring. The reaction was monitored by TLC on silica gel with ethyl acetate/triethylamine (90:10 v:v) as eluent. After 30 min, the starting material (R ) 0.40) was fully transformed into a compound having a higher Rf (0.50). After the usual workup, the residue was purified on a silica gel column using ethyl acetate/triethylamine (95:5 v:v) as eluent. The fraction containing the pure product was pooled and the solvent removed under reduced pressure. The obtained residue was precipitated in hexane. Rf11a ) 0.38 using ethyl acetate/NEt3 (90:10 v:v) as eluent. Yield ) 80%. Rf11c ) 0.46 using ethyl acetate/NEt3 (90: 10 v:v) as eluent. Yield ) 76%. Preparation of the Acridine-Containing Oligodeoxynucleotides 12a, 12b, 13-17, and 22. Chain elongation


was performed by the classical phosphoramidite procedure on a modified support 3, 10a, or 10b (at the 0.5 or 1 µmol scale, depending on the loading of the modified support) and/or the acridine-containing phosphoramidites 11a or 11c with increased coupling time (5 min) for the latter. The oligonucleotides, bound to the support, were submitted to an additional detritylation step to deprotect the 5′-hydroxyl function and then treated with a 0.4 M sodium hydroxide solution in H2O/MeOH (50:50 v:v) mixture for 3 h. The pHs of the solutions were then adjusted to 7, and the methanol was removed by evaporation. After extraction with ethyl acetate and filtration using a 0.45 µm disposable filter, the aqueous oligodeoxyribonucleotide solutions were purified by ionexchange or reversed-phase chromatography using the systems described in General Methods.

Asseline et al. Scheme 1. Synthesis of the Universal AcridinylModified Support 3a

RESULTS AND DISCUSSION

Synthesis of the Oligonucleotide-Acridine Conjugates. Synthesis of the Universel Support 3. 3′Acridine-linked oligonucleotides were obtained using a new universal support, while in our previous work, one modified support was needed for each nucleotide at the 3′-position of the sequence (29). The synthesis of the universal support 3 was achieved following a five-step procedure (Scheme 1). Reaction of 3-aminosolketal (obtained as described in ref 31) with 6,9-dichloro-2methoxyacridine in a phenol solution gave the (Nacridinyl-3-aminopropyl)solketal (1) in 45% yield. The acridine derivative 1 was treated with a mixture of 1 M hydrochloric acid and tetrahydrofuran (1:1) to remove the isopropylidene group. Without purification, the product was reacted with 4,4′-dimethoxytrityl chloride in anhydrous pyridine to give 1-O-(4,4′-dimethoxytrityl)-3O-(ω-acridinylaminopropyl)glycerol (2) which was purified by silica column chromatography in a 59% yield. Starting from compound 2, the acridine-containing modified CPG support 3 was obtained following a three-step procedure with a loading of 16 µmol of acridine per gram of support. The oligonucleotides containing the acridine moiety at the 3′-position via a phosphate group were assembled at the micromolar scale using classical phosphoramidite chemistry (26) (Table 1). Attachment of the Acridine Moiety via an Internucleotide Phosphorothiolate. For the introduction of the acridine moiety via an internucleotide phosphorothiolate, we chose a previously described method (33) involving a phosphorothioate group as the site for alkylation by an acridine derivative (Scheme 2). Using this strategy, a phosphoramidite coupling at a preselected position is followed by a sulfuration step instead of oxidation to generate the phosphorothionotriester which is stable in the subsequent oxidation steps necessary to generate the internucleotidic phosphodiester in the remainder of the oligodeoxyribonucleotide. Our previous results (28, 29) showed that the synthesis of a 20-mer oligonucleotide involving a 3′-phosphorothioate group was carried out on a modified support involving a phosphorothiono triester group without significant loss of sulfur. The 11-mer d5′TTTCCTp(S)CCTCT3′ 4 was obtained as a mixture of stereoisomers whose structures are given in Scheme 3. Reversed-phase analysis of compound 4 exhibited two roughly equivalent peaks (56 and 44%, respectively) with very close retention times (Table 1). The two diastereoisomers 4a (fast eluted) and 4b (slowly eluted) were separated by reversed-phase chromatography (see Experimental Procedures for the conditions used) and assigned to be the Rp isomer (fast eluted) and Sp (slowly eluted) after digestion by the nuclease P1 followed by alkaline phosphatase treatment. This led to the full

a (i) phenol, 120 °C; (ii) aqueous HCl, THF; (iii) DMTrCl, Py; (iv) succinic anhydride, DMAP, Py; (v) N-hydroxysuccinimide, Py, DCC, dioxane; (vi) NH2 with cross-hatched circle ) LCAACPG, NEt3, DMF; (vii) acetic anhydride.

degradation of the Sp isomer in dC and dT, while the Rp isomer, under the same conditions, led to dC and dT and to the Rp dinucleoside phosphorothioate d5′Tp(S)C3′ identified by comparison with an authentic sample of the Rp dinucleoside d5′Tp(S)C3′ (34). It must be noted that the ratio obtained for the Rp and Sp dinucleosides d5′Tp(S)C3′ was in accordance with recently reported data (35). Reaction of compound 4 (Scheme 2a) as a mixture of diastereoisomers or as a pure Rp or Sp isomer with the iodoacetamidoacridine derivative AcrN(H)(CH2)8N(H)C(O)CH2I (6) whose preparation is described in Scheme 2b yielded the corresponding mixture of diastereoisomers 7 (Figure 1) of the acridine-labeled 11-mer [d5′TTTCCTp[SCH2C(O)N(H)(CH2)8NHAcr]CCTCT3′] or the pure Rp 7a and Sp 7b isomers whose structures are given in Scheme 3. The acridine derivative 6 was chosen with an 11-atom linker since the CPK model has shown that this arm is better suited than the previously used one involving five methylene groups (4), although this size is long enough when the acridine derivative is attached to the 3′- or 5′end of an oligonucleotide. In these cases, the structures involving a phosphodiester linkage between the linker and the oligonucleotide are less restricted than when the



Table 1. Structures of the Modified Oligodeoxyribonucleotides Synthesized and Reversed-Phase Analysis of the Oligodeoxyribonucleosides Used in This Study Performed on a Lichrospher 100 RP (5 µm) Column (125 mm × 4 mm) from Merck reversed-phase analysis

compounds d5′TTTCCTp(S)CCTCT3′

d5′TTTCCTp(S X)CCTCT3′ d5′TTTCCTCCTCU*3′ d5′TTTCCTCCTCT3′p-Y d5′TTTCCU*CCTCT3′ d5′TTTCCU*CCTCU*3′ Z-O-p-d5′TTTCCTCCTCT3′p-Y Z-O-p-d5′TTTCCU*CCTCT3′ Z-O-p-d5′TTTCCTCCTCT3′ d5′TTTCCTCCTCT3′p-S-Z 5′dAGTAGAGGAGGAAATAG3′ d5′TTTCCTCCTCT3′ 5′dTTTTCTTU*TC3′ 5′TTTAAAAGAAAAGGG3′ 5′dTTTTCTTTTC3′ X ) CH2C(O)NH(CH2)8NHAcr Y ) OCH2CH(OH)CH2O(CH2)3NHAcr Z ) AcrNH(CH2)6

4 (mixture of diastereoisomers) 4a (Rp isomer) 4b (Sp isomer) 7 (mixture of diastereoisomers) 7a (Rp isomer) 7b (Sp isomer) 12a (n ) 4) 12b (n ) 6) 13 14 (n ) 4) 15 (n ) 4) 16 17 (n ) 4) 18 19 20 21 22 23 24 H

H

20 min 52 sb 21 min 2 sb 23 min 30 sc 24 min 30 sc 29 min 36 sb 18 min 5 sd 19 min 37 sd 19 min 6 sa 21 min 14 sa 19 min 26 sa 19 mina 37 min 52 sa 23 min 9 sa 33 minb 21 min 52 sa 21 min 30 sa 17 min 18 sb 16 min 18 sb 34 min 10 sc 16 min 42 sb 17 min 18 sb

O

AcrN(CH2)nN

N

U* = N

O

H

O

O

O a System A: 5 to 42.5% CH CN in 50 min. b System B: 5% CH CN for 5 min and then 5 to 42.5% CH CN in 50 min. c System C: 0 to 3 3 3 40% CH3CN in 50 min. d System D: 21.5 to 23% CH3CN in 40 min.

Scheme 2. Synthesis of the Acridine Linker 6 and of the Oligodeoxyribonucleotides 7, 7a, and 7b Involving an Acridine Attached at an Internucleotidic Phosphorothiolate

acridine is linked to an internucleotidic phosphate via a phosphothiolotriester group. The iodoacetamido derivative 6 was used since coupling with the ω-bromoundecyl derivative Br(CH2)11NHAcr was not possible, probably due to the lower reactivity of an internucleosidic phosphorothioate compared to that of a terminal phosphorothioate. This was achieved in a two-step procedure (Scheme 2b), in which 2-methoxy-6,9-dichloroacridine was reacted with an excess (5 equiv) of 1,8-diaminooctane to yield the ω-amino derivative 5 whose reaction with p-nitrophenyl iodoacetate gave the expected acridine derivative 6.

Attachment of the Acridine Moiety to the 5-Position of the Deoxyuridine. The attachment of the intercalator to the 5-position of deoxyuridine was developed following Scheme 4. First, 5′-O-(4,4′-dimethoxytrityl)-5-bromodeoxyuridine was reacted with 1,4-diaminobutane, 1,6diaminohexane, or 1,7-diaminoheptane to give 5′-O(dimethoxytrityl)deoxyuridine substituted at the 5-position with (4-aminobutyl)amino, (6-aminohexyl)amino, or (7aminoheptyl)amino linker 8a, 8b, or 8c, respectively. Then, the latter were reacted with 6-chloro-9-phenoxy2-methoxyacridine (obtained as described in ref 27) in acetonitrile to give compounds 9a, 9b, and 9c, respec-


Asseline et al. Scheme 4. Synthesis of the Modified Supports 10a,b and of the Phosphoramidites 11a,ba

Figure 1. Reversed-phase analysis of the coupling reaction of the oligonucleotide phosphorothioate d5′TpTpTpCpCpTp(S)CpCpTpCpT3′ 4 (tR ) 21 min) with the acridinyl linker derivative 6. The oligonucleotide-acridine conjugate 7 (tR ) 29 min 26 s) has been obtained with a yield of 75% (left). The insets show the absorption spectra, recorded between λ ) 220 nm and λ ) 550 nm, of the starting oligonucleotide 4 (upper right) and of the oligonucleotide-acridine conjugate d5′TpTpTpCpCpTp[SCH2C(O)NH(CH2)8NHAcr]ICpCpTpCpT3′ 7(lower right). Analysis has been performed on a Lichrospher 100 RP-18 (5 µm) column (125 × 4 mm) using a linear gradient of CH3CN in 0.1 M aqueous triethylammonium acetate buffer (pH 7) with a flow rate of 1 mL/min (0 to 50% buffer B in 50 min). Scheme 3. Structures of the Rp Isomers 4a and 7a and Sp Isomers 4b and 7b of the Oligonucleotides 4 and d5′TpTpTpCpCpTpd5′TTTCCTp(S)CCTCT3′ [SCH2C(O)NH(CH2)8NHAcr]CpCpTpCpT3′ 7

tively, with a yield ranging from 65 to 70%. In order to incorporate acridine-containing modified deoxyuridine into any position of the oligonucleotide chain, both the modified support derivatized with the acridine-modified deoxyuridine and the corresponding 3′-phosphoramidite derivative must be obtained. Starting from the modified nucleosides 9a and 9b, the modified derivatized supports 10a and 10b were obtained following the classical procedure (26) with a loading of 8 and 12 µmol/g, respectively. Phophitylation of compound 9 was carried out using 1.1 equiv of (2-cyanoethyl)(N,N-diisopropylamido)chlorophosphite in dichloromethane in the presence of N,N-diisopropylethylamine. Under these conditions, the desired products 11a and 11c were obtained after silica gel chromatography and precipitation in hexane with 75 and 80% yields, respectively.

a DMTr ) dimethoxytrityl; cross-hatched circle attachment ) LCAA-CPG. (i) DMTrCl, Py; (ii) H2N(CH2)nNH2 (n ) 4, 6, or 7), CH3CN, 65 °C; (iii) 6-chloro-9-phenoxy-2-methoxyacridine, CH3CN; (iv) succinic anhydride, DMAP, Py; (v) p-nitrophenol, Py, DCC, dioxane; (vi) LCA-CPG, NEt3, DMF; (vii) acetic anhydride, Py; (viii) ClPN(iPr2)OCH2CH2CN, DIEA, CH2Cl2.

Synthesis of the Oligonucleotides. Using the abovementioned supports 3, 10a, and 10b and the phosporamidites 11a and 11c, oligonucleotides bearing one or two acridine moieties whose structures are given in Table 1 were assembled. In order to compare the stabilization due to the acridine using the new described linkages with that obtained with previously described acridine-oligonucleotide conjugates, the compounds AcrNH(CH2)6-pd5TTTCCTCCTCT3′ (18) and 5′TTTCCTCCTCT3′pS(CH2)6NHAcr (19) have been prepared by using our previously described procedures (28). After the chain assembly, the removal of the protective group and the releasing from the support of the acridine-containing oligonucleotides were achieved by 0.4 M sodium hydroxide treatment. Using NH4OH with acridine-containing oligonucleotides must be avoided in order to prevent the cleavage of the bond between the acridine ring and the nitrogen atom of the linker (28). After full deblocking of the oligonucleotides, the purification step was achieved either by ionexchange or by reversed-phase chromatography. Attempts to purify an undecamer involving two U acridinemodified bases at internal positions (3 and 9) (d5′TTU*CCTCCU*CT3′) (n ) 4) by either chromatography or polyacrylamide gel electrophoresis under denaturing conditions failed. However, no major problem occurred during the purification of compound 16 with one acridine at each end and compound 15 with one acridine at one end and the second on U at an internal location. After purification, analyses of the oligonucleotide-acridine conjugates were performed on both ion-exchange and reversed-phase columns (Table 1). The structures of oligonucleotide derivatives 7, 7a, 7b, 12a, 12b, and 1319 were confirmed by different methods. Full deprotection and base composition analysis have been ascertained by reversed-phase analysis after nuclease degradation (Figures 2 and 3). The obtained modified oligomers were also analyzed by Maldi mass spectroscopy (36, 37). The results are in accordance with the calculations (see


Figure 2. Reversed-phase HPLC analysis of the hydrolysate of the compounds 12a (d5′TTTCCTCCTCU*) (n ) 4) (upper) and 12b (d5′TTTCCTCCTCU*) (n ) 6) (lower) with nucleases (P1 from Penicilium citrinum and alkaline phosphatase) on a Lichrospher 100 RP-18 (5 µm) column (125 × 4 mm) using a linear gradient of CH3CN in 0.1 M aqueous triethylammonium acetate buffer (pH 7) with a flow rate of 1 mL/min (0% CH3CN for 5 min, then 0 to 20% in 20 min, and then 20 to 50% CH3CN in 15 min). The right parts show the absorption spectra, recorded between λ ) 220 nm and λ ) 550 nm, of the obtained compounds: dC, dT, and dU* (n ) 4) and dU* (n ) 6) from top to bottom, respectively.

Figure 3. Reversed-phase analysis of the hydrolysate of the compounds 12a (upper) and 15 (lower) with nucleases (P1 from P. citrinum and alkaline phosphatase). The conditions are the same as those described in the legend of Figure 2. The ratio of the peaks corresponding to the monomers dC, dT, and U* indicates that the compound 15 (d5′TTTCCU*CCTCU*) (n ) 4) contains two acridinyl-modified U* compared to the compound 12a (d5′TTTCCTCCTCU*) (n ) 4) which contains only one acridine residue.

Experimental Procedures for conditions used). The UV/ visible spectra of the acridine derivatives showed absorption maxima corresponding to the oligonucleotide domain at 260 nm and to the 2-methoxy-6-chloro-9-aminoacridine at 425 nm. Comparison of the spectra of the oligonucleotide and dye with those of the oligonucleotide-acridine conjugates and nuclease degradation analysis confirm both the existence and the number of intercalating residues in the molecular structure (Figures 2 and 3). As expected, the mobility of the oligonucleotide derivatives using a Lichrosorb RP 18 (5 µM) HPLC column (125 × 4 mm) decreases with the introduction of the hydrophobic dye residue. As shown in Table 1, the length of the polymethylene linker also affects the mobility of the acridine derivatives. Ion-exchange analysis performed on a 8 µM DEAE (100 mm × 10 mm) column with a linear gradient of NaCl (0 to 0.9 M in 30 min) in a Tris/ HCl (pH 7) 25 mM buffer containing 10% CH3CN shows very close retention times for all the compounds analyzed.


Figure 4. Difference between the absorption spectrum of a 1:1 mixture (1 µM each strand) of the oligonucleotides 22 (d5′TTTTCTTU*TC3′) and 23 (d5′TTTAAAAGAAAAGGG3′) (- -) or 24 (d5′TTTTCTTTTC3′) and 23 (d5′TTTAAAAGAAAAGGG3′) (s) and the sum of the absorption spectra of the separated components 22 + 23 and 24 + 23, respectively. The spectra were recorded between λ ) 220 nm and λ ) 340 nm at 2 °C in tandem cells. Measurements were carried out in a pH 7 buffer containing 10 mM sodium cacodylate and 0.1 M NaCl.

It must be noted that the acridine is protonated at pH 7 and the retention times are proportional to the total number of negative charges on the molecule. Therefore, compounds 21 (unmodified, 10 phosphate groups) and 13 (11 phosphate groups + 1 acridine) showed the same retention time (11 min 6 s), while compounds 12a, 12b, and 14 (10 phosphate groups + 1 acridine) eluted earlier (10 min 48 s). Interaction with the Complementary Sequence. To determine the stabilizing effect of the acridine residue on the hybridization of the acridine-oligonucleotide conjugates with the complementary sequences, depending on its attachment site, the duplexes formed by the undecanucleotides 7 and 12-19 with an heptadecadeoxynucleotide (5′d-AGTAGAGGAGGAAATAG3′) 20 involving the complementary sequence were studied. The undecadeoxynucleotide (5′dTTTCCTCCTCT3′) 21 was used as a reference. The interaction of the duplexes formed between oligomers 7, 12-19, and 21 and the complementary target DNA sequence 20 was followed by absorption spectroscopy in the visible range where only the acridine ring absorbed light and in the UV range in the absence of acridine. Addition of increasing concentrations of oligonucleotide 20 to acridine-oligonucleotide conjugate solutions at 4 °C led to changes in the absorbance spectra of compounds 12a, 12b, 13, 16, 18, and 19. When absorbance at 425 or 260 nm was plotted versus the target concentration, a sharp break occurred with a nearly 1:1 ratio, indicating a specific interaction between the acridine-oligonucleotide conjugates and their targets. In the case of oligonucleotide-acridine conjugates 12a, 12b, 13, 16, and 19, a red shift of the visible acridine spectrum was observed together with a strong hypochromism, while in the case of the binding of oligomers 14 (d5′TTTCCU*CCTCT3′) and 22 (d5′TTTTCTTU*TC3′) with the internal acridine-modified U, except for a slight hypochromism, no significant change of the visible spectrum of the acridine was observed during the addition of the target sequences 20 (5′dAGTAGAGGAGGAAATAG3′) and 23 (d5′TTTAAAAGAAAAGGG3′), respectively. This probably indicates that different interactions occur between the acridine moiety and the double-stranded duplex when the acridine is linked to uridine located inside the sequence. Nevertheless, a variation of the UV spectrum confirms the formation of a complex between the oligonucleotides 14 and 20 or 22 and 23 (Figure 4). When the temperature of the duplex solutions formed at 4 °C is increased, the changes of the absorbance


Figure 5. Change in absorbance spectrum with the temperature of 1:1 mixtures of oligomer 20 with oligonucleotideacridine conjugates. Compounds 18 [AcrNH(CH2)6p-d5′TTTCCTCCTCT3′] (upper), 16 [AcrNH(CH2)6p-d5′TTTCCTCCTCT3′pOCH2CH(OH)CH2(CH2)3NHAcr] (middle), and 12a (d5TTTCCTCCTCU*3′) (n ) 4) (lower). Measurements were carried out in a pH 7 buffer containing 0.01 mM sodium cacodylate and 0.1 M NaCl. The oligonucleotide concentration was 4 µM (each strand).

spectra observed during the addition of the target sequence 20 (5′dAGTAGAGGAGGAAATAG3′) are reversed (Figure 5). Plotting the difference in absorbance at λ ) 425 nm or λ ) 260 nm of a 1:1 mixture versus temperature gave the melting curves. When the acridine is covalently linked to either the 3′- or the 5′-position of the oligonucleotides, the Tm values (at which half of the duplex dissociation occurred) are not very different [12a, d5′TTTCCTCCTCU*3′ (n ) 4), Tm ) 46.2 ( 0.5 °C; 12b, d5′TTTCCTCCTCU*3′ (n ) 6), Tm ) 46 ( 0.5 °C; 13, d5′TTTCCTCCTCT3′pOCH2CH(OH)CH2(CH2)3NHAcr, Tm ) 45 ( 0.5 °C; 19, d5′TTTCCTCCTCT3′pS(CH2)6NHAcr, Tm ) 48.4 ( 0.5 °C; and 18, AcrNH(CH2)6-p-d5′TTTCCTCCTCT3′, Tm ) 47 ( 0.5 °C]. In any case, a strong stabilization was observed when compared to the Tm value (34.4 ( 0.5 °C) of the duplex formed by oligonucleotide 21 without acridine and the complementary sequence 20 (Table 2). When the acridine is linked to an internal position of the oligonucleotide chain either to the 5-position of U or to an internucleotidic phosphate, different results are observed. The introduction of a single acridine-modified deoxyuridine to the middle of the sequence (compound 14, d5′TTTCCU*CCTCT3′) leads to slight destabilization of the duplex (14 + 20) (Tm ) 32.8 ( 0.5 °C) compared to that of the unmodified parent duplex (20 + 21) (Tm ) 34.4 ( 0.5 °C). Despite the results obtained with oligo-

Asseline et al.

nucleotides 12a (n ) 4) and 12b (n ) 6) showing that the introduction of the acridine residue at the 5-position of deoxyuridine gives good stability of the duplex, we have chosen to use a longer linker between the acridine and the deoxyuridine. Thus, a 7-methylene linker instead of a 4-methylene arm [compound 14 (d5′TTTCCU*CCTCT3′), n ) 4] was chosen. This longer linker arm with a higher degree of freedom should allow the acridine label to intercalate more efficiently. Along with this modification, we also decided to change the neighbor base pair at the possible intercalating site. Although in the sequence 14 the modified labeled deoxyuridine is located between two cytosines, we opted for the preparation of a new sequence in which an internally modified deoxyuridine is surrounded by two thymines (22, d5′TTTTCTTU*TC3′, n ) 7). Once again, the covalent linking of the acridine moiety to the 5-position of an internal deoxyuridine via a heptamethylene linker had a destabilizing effect on the duplex (Tm ) 16 ( 0.5 °C) formed with the complementary sequence 23 (5′dTTTAAAAGAAAAGGG3′) compared to the complex obtained with the unmodified parent 24 (5′dTTTTCTTTTC3′) (Tm ) 19.8 ( 0.5 °C). Reported data (9) have shown that the incorporation of a sole linker with a terminal aminoalkyl group at the 5-position of an internal U in an oligonucleotide had a destabilizing effect. However, at pH 7, the primary amine linker arm was protonated and could thus help counteract the electrostatic barrier to duplex formation by the negatively charged oligomers. A similar effect has been described more recently (38). The incorporation of 5-hexyl-2′deoxyuridine at an internal position of a dodecamer pyrimidine sequence had a strong destabilizing effect on the duplex formation with the complementary sequence (∆Tm ) -4.5 °C in 50 mM NaCl). Consistent with these literature data and with the fact that the linking of the acridine ring to the 5-position of a uracil had a strong stabilizing effect when the modified U is located at a terminal position of an oligonucleotide (compounds 12a and 12b) and a slight destabilizing one when the modified U is located at an internal position (compound 14), one explanation could be that the covalent linking of an acridine to internal positions changes the hydratation spin inside the groove, thus disturbing the duplex stability. Another explanation could be that the best position for the intercalation of the acridine ring is the 9-position in the minor groove while the linker attached to the 5-position of U* protrudes in the major groove. Along these lines, NMR studies (39) have proven that, when linked to an oligonucleotide via a 3′-phosphate, the 2-methoxy-6-chloro-9-aminoacridine intercalates with the 9-amino group in the minor groove. Thus, when the acridine-modified deoxyuridine is located at the 3′-end of the oligonucleotide (compound 12a or 12b), the base can rotate around the glycosidic bond to bring the acridine moiety into the minor groove. In addition, it must be noted that compounds 12a and 12b involving a 4- and 6-methylene linker length, respectively, form duplexes with very similar stability. This probably means that the linkers used have the optimum length to induce maximum stabilization of the duplex when the acridine-modified U* is located at the end of the oligomer. On the contrary, when the acridine-modified U* is located inside the oligonucleotide sequence (compound 14), rotation of the acridine-modified base around the glycosidic bond is more unlikely due to the steric hindrance preventing a favorable position for the acridine moiety to intercalate. Nevertheless, the rotation around the glycosidic bond to allow intercalation of the acridine ring would induce mismatched a base pair and so a strong destabilization for the duplex. The increase of the linker


Oligonucleotide−Acridine Conjugates Table 2. Duplex Melting Temperaturesa

Tm (°C) (+0.5 °C)

duplexes 20 21 19 13 12

d3′GATAAAGGAGGAGATGA5′ d5′TTTCCTCCTCT3′ d5′TTTCCTCCTCT3′p-S-(CH2)6NHAcr d5′TTTCCTCCTCT3′p-OCH2CH(OH)CH2O(CH2)3NHAcr d5′TTTCCTCCTCU*3′

18 16 4

AcrNH(CH2)6O-p-d5′TTTCCTCCTCT3′ AcrNH(CH2)6O-p-d5′TTTCCTCCTCT3′p-OCH2CH(OH)CH2O(CH2)3NHAcr d5′TTTCCTp(S)CCTCT3′

7

O d5′TTTCCTpCCTCT3′ S

CH2 C

H

H

N(CH2)8

N

(target)

12a (n ) 4) 12b (n ) 6) 4a (Rp isomer) 4b (Sp isomer) 7a (Rp isomer) 7b (Sp isomer)

34.4 48.4 45.2 46.2 46 47 52.4 33 33.3 45.2 51.4

Acr

O

14 15 17 23 24 22

d5′TTTCCU*CCTCT3′ d5′TTTCCU*CCTCU*3′ AcrNH(CH2)6Op-d5′TTTCCU*CCTCT3′ d3′GGGAAAAGAAAATTT5′ d5′TTTTCTTTTC3′ d5′TTTTCTTU*TC3′ H

H

(n ) 4) (n ) 4) (n ) 4) (target) (n ) 7)

32.8 no Tm 26 19.8 16

O

AcrN(CH2)nN

N

U* = N

O

H

O

O

O a All duplexes were in T buffer: 0.01 M sodium cacodylate and 0.1 M NaCl (pH 7). The concentration (each strand) were 4 µM except m for duplexes 4a or 4b + 20 (3.6 µM) and for duplexes 14 + 20 and 22 or 24 + 23 (1 µM). Unless stated otherwise in the text absorbance measurements were carried out at 425 nm for the acridine-containing compounds and at 260 nm for the compounds without acridine.

Figure 6. Melting curves at 425 nm of duplex 7 [d5′TpTpTpCpCpTp[SCH2C(O)NH(CH2)8NHAcr]CpCpTpCpT3′] + 20 (+), duplex 7a (Rp isomer) + 20 (2) and duplex 7b (Sp isomer) + 20 (b) at a 4 µM concentration. Measurements were carried out in a pH 7 buffer containing 10 mM sodium cacodylate and 0.1 M Na Cl.

length between the acridine residue and base did not lead to any stabilizing effect (compound 22). On the contrary, when the acridine moiety is attached to an internucleotidic phosphorothiolate 7 (d5′TTTCCTp[SCH2C(O)N(H)(CH2)8NHAcr]CCTCT3′), a strong stabilization is observed when using either the mixture of diastereoisomers (Tm for 7 + 20 ) 48.6 ( 0.5 °C) or each pure Rp isomer 7a (Tm ) 45 ( 0.5 °C) or Sp isomer 7b (Tm ) 51.4 ( 0.5 °C) (Figure 6). These results confirm our hypothesis that in our previous studies the linker used to connect the acridine ring to the internucleotidic phosphate of the oligonucleotide was too short (4). It is important to note that, under very close conditions (3.6 instead of 4 µM duplex concentrations), the Rp and the Sp isomers of the 11-mer phosphorothioate 4 form duplexes with similar Tm values very close to that of the

duplex used as reference (Tm ) 34.4 ( 0.5 °C). Concerning the varying stability obtained with Rp and Sp isomers 7a and 7b, one explanation could be that as observed on the CPK model and in accordance with a literature report (40) when the oligomer 7 is hybridized with its complementary sequence the sulfur of the Rp isomer would direct the covalently attached acridine moiety rather toward the major groove of the double helix in a position unfavorable for intercalation whereas the sulfur of the Sp isomer would direct the intercalating agent in a position allowing its interaction in either the minor or the major groove. When interaction takes place in the major groove, the linker is able to fold back, allowing only partial intercalation of the acridine which can explain the different stability ∆Tm ) 6.2 °C observed for the two isomers. When two acridine moieties are present on the same oligonucleotide, different behaviors are observed. When a second acridine moiety is added to the 5′-end of the conjugate 12a to give the compound 16 (AcrNH(CH2)6pd5′TTTCCTCCTCT3′pOCH2CH(OH)CH2(CH2)3NHAcr), additional stability can be observed (Tm ) 52.4 ( 0.5 °C). On the other hand, the addition of one acridine molecule to the 5′-end of the undecanucleotide 12a to give the bisacridine conjugate 17 (AcrNH(CH2)6p-d5′TTTCCU*CCTCT3′) induces a duplex destabilization (Tm ) 26 ( 0.5 °C). This large destabilization is probably due to the presence of the acridine on a modified U in the middle of the sequence as observed with compound 14 and also to intramolecular interactions. In the case of compound 15 (d5′TTTCCU*CCTCU*3′) involving two acridine residues linked to the 5-position of a modifed deoxyuridine located at the middle and at the 3′-terminal positions of the sequence, respectively, changes in absorbance did not allow us to determine a Tm value. In addition, during


the synthesis, we did not succeed in the purification of an oligonucleotide involving two U modified with acridine at internal positions of the sequence. We have reported the synthesis and binding properties of oligodeoxyribonucleotides covalently linked to an acridine derivative via various positions of the oligonucleotide chain. First, the preparation of a universal modified support 3 which allows the synthesis of oligonucleotides bearing the acridine moiety via a 3′-phosphodiester linkage has been described. The obtained oligonucleotide-acridine conjugate 13 (Tm ) 45 ( 0.5 °C) has binding properties very close to those of compound 19 (Tm ) 48.4 ( 0.5 °C) described previously. The addition of a second acridine moiety via the 5′-phosphate group (compound 16) leads to better stability (Tm ) 52.4 ( 0.5 °C), similar to that obtained by Zarytova et al., who have used the phenazinium intercalating agent. Then, oligodeoyribonucleotides bearing the acridine moiety at the 5-position of the deoxyuridine not involved in the Watson-Crick base pair formation have been synthesized. When the acridine-modified deoxyuridine is located at the 3′-end of the oligonucleotide (oligomers 12a and 12b), strong stabilization of the complexes can be observed (Tm ) 46.2 ( 0.5 and 46 ( 0.5 °C, respectively), very similar to that obtained with the oligomers 13 and 19 bearing the acridine moiety via a 3′-phospho diester or phosphothiolodiester, respectively. Surprisingly, when the acridine-modified deoxyuridine is located inside the oligonucleotide sequence (compounds 14 or 22), a slight destabilization of the complex can be observed (Tm ) 32.8 ( 0.5 and 16 ( 0.5 °C, respectively) compared to the stability of the complexes obtained with the unmodified parent (compounds 21 or 24, Tm ) 34.4 ( 0.5 and 19.8 ( 0.5 °C). The acridine moiety has also been incorporated at the middle position of the sequence via a phosphorothioate group. The mixture of diastereoisomers (compound 7) as well as each pure Rp and Sp isomer (compounds 7a and 7b, respectively) were obtained also. The mixture of diastereoisomers led to a slightly more stable complex (Tm ) 48.6 ( 0.5 °C) than did the 3′-acridinelinked oligonucleotide prepared on the universal support 13 (Tm ) 45 ( 0.5 °C) and similar to that obtained with the oligomer 19 linked to the acridine via a 3′-phosphothiolodiester (Tm ) 48.4 ( 0.5 °C). The Rp isomer 7a (Tm ) 45.2 ( 0.5 °C) led to lower stability than the racemic mixture did while the Sp isomer 7b (Tm ) 51.4 ( 0.5 °C) to the most stable duplex of all those studied. ACKNOWLEDGMENT

This work was supported by the Agence Nationale de Recherches sur le SIDA and Rhoˆne-Poulenc Rorer. We thank Professeur R. L. Inglebert for the use of the mass spectrometer. LITERATURE CITED (1) J. S. Cohen Ed. (1989) Oligodeoxynucleotides. Antisense Inhibitors of Gene Expression. In Topics in Molecular and Structural Biology; Mac Millan Press, London. (2) He´le`ne, C., Montenay-Garestier, T., Saison, T., Takasugi, M., Toulme´, J. J., Asseline, U., Lancelot, G., Maurizot, J. C., Toulme´, F., and Thuong, N. T. (1985) Oligodeoxynucleotides covalently linked to intercalating agents: a new class of gene regulatory substances. Biochimie 67, 773-783. (3) Nguyen, T., Thuong, N. T., and He´le`ne, C. (1993) Sequencespecific recognition and modification of double-helical DNA by oligonucleotides. Angew. Chem., Int. Ed. Engl. 32, 666690. (4) Asseline, U., Toulme´, F., Thuong, N. T., Delarue, M., Montenay-Garestier, T., and He´le`ne, C. (1984) Oligodeoxynucleotides covalently linked to intercalating dyes as base

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BC960024O

Synthesis and Binding Properties of Oligonucleotides Covalently

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