Synthesis and Binding Properties of Oligo-2'-deoxyribonucleotides

Bioconjugate Chem. 2001, 12, 757−769

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Synthesis and Binding Properties of Oligo-2′-deoxyribonucleotides Covalently Linked to a Thiazole Orange Derivative Eric Privat and Ulysse Asseline* Centre de Biophysique Molećulaire, UPR 4301 CNRS. Affiliated with the University of Orleáns and with INSERM, Rue Charles Sadron, 45071 Orleáns Cedex 02, France. Received January 22, 2001; Revised Manuscript Received July 5, 2001

Thiazole orange label was coupled to the eighth phosphate of a pentadeca-2′-deoxyriboadenylate via a phosphoramidate linkage using different linkers. The stereoisomers were separated, and their absolute configurations were determined. Finally, the thiazole orange moiety was also linked to the tenth phosphate of icosathymidylates in both the R and the β series via a phosphoramidate linkage. Once again, the thiazole orange-icosathymidylate conjugates were obtained as pure stereoisomers. The binding properties of these oligo-2′-deoxyribonucleotide-thiazole orange conjugates with their complementary sequences were studied by absorption spectroscopy. The covalent attachment of the thiazole orange derivatives to the oligoadenylates stabilizes the complexes formed with both the DNA and RNA targets. On the contrary, when the thiazole orange is tethered to the oligo-R-thymidylate or oligo-β-thymidylate, no significant stabilization of the duplexes formed with poly r(A) can be observed.

INTRODUCTION

The rapidly increasing rate of sequencing has opened the way to greater progress in various research areas such as gene organization and function including research on the underlying genetic causes of inherited and acquired diseases with possible applications for their prevention and treatment, the detection of pathogens for clinical diagnoses, and finally food technology, agricultural, environmental, and forensic sciences. Base-pairing properties of nucleic acids are used, in most methods which involve the hybridization of a single-stranded oligonucleotide labeled probe, to detect a complementary target sequence in a complex nucleic acid mixture. The most commonly used hybridization assays employ a solid phase in order to facilitate the separation of bound from unbound analytes (1, 2). In contrast, assays that process in a homogeneous solution avoid problems such as nonspecific adsorption associated with the heterogeneous assays, but the separation of hybridized and free probes is required. The separation step can be eliminated by a probe whose signal changes upon hybridization (3, 4). Such probes could be used inside cells to detect their complementary sequences in situ directly on their RNA targets. The development of fluorescent or luminescent labeled oligonucleotides that display a modified signal upon hybridization with their target sequences has been the subject of intense research. Among the reported systems, molecular beacons are based on the quenching/ dequenching of fluorescence. This system involves stemloop-forming oligo-2′-deoxyribonucleotides bearing a fluorescent dye at one end and a quencher at the other end (5). Upon hybridization to the target sequence, the stem is dissociated and the distance between the fluorescent dye and the quencher is largely increased, generating an increase in fluorescence. Other methods utilize a change in the fluorescent emission wavelength, rather than in * Address correspondence to this author. Phone: (33) 02 38 25 55 97. Fax: (33) 02 38 63 15 17. E-mail: [email protected].

the intensity alone. Among these are the fluorescence resonance energy transfer (FRET) between a donor and an acceptor dye (6, 7), and the luminescence resonance energy transfer (LRET) (8) between a sensitized lanthanide metal and an acceptor dye. Another example of the color-changing strategy involving a two-chromophore system is the utilization of excimer-forming chromophores such as pyrene (9, 10). The last strategy is based on modification of the signal generated by a single fluorescent or luminescent DNA binding agent linked to oligonucleotides upon hybridization with their target sequences. A minor groove binder, Hoechst 33258 (11, 12), and intercalating agents such as ruthenium complex (13) and oxazole orange (14, 15) have been covalently tethered to oligonucleotides, and fluorescent or luminescent, in the case of the ruthenium complex, signal increases were observed upon hyridization of these labeled probes. Following our previous work (16) and one report (17) concerning the fluorescent properties of thiazole orangelinker derivatives in interaction with single-stranded and double-stranded targets, we now describe the synthesis and binding properties of oligo-2′-deoxyribonucleotides covalently linked to thiazole orange. [During the course of this work, results on the fluorescent properties of thiazole orange-peptide nucleic acid conjugates were published (18-20).] Since the homodimeric thiazole orange (TOTO) was reported to intercalate in doublestranded DNA (21) and our previous work on oligo-2′deoxyribonucleotide-acridine conjugates showed stronger stabilization of the duplex when the acridine moiety, an intercalator, is linked to an internucleotidic phosphate (22), we chose to tether the thiazole orange residue to an internucleotidic position of the oligonucleotide sequence. EXPERIMENTAL PROCEDURES

General Methods. All solvents used were dried, distilled, and stored as described in ref 23. All chemicals were used as obtained unless otherwise stated. Cystamine dihydrochloride, 3-methylbenzothiazole-2-thione,

10.1021/bc0100051 CCC: $20.00 © 2001 American Chemical Society Published on Web 08/29/2001

758 Bioconjugate Chem., Vol. 12, No. 5, 2001

Privat and Asseline

Scheme 1. Synthesis of the Thiazole Orange-Linker Derivatives 5 and 6

methyl iodide, lepidine, 1,10-diiododecane, dimethylaminopyridine, acetic anhydride, tetraethylene glycol, triphenylphosphine, BrCCl3, pivaloyl chloride, and tris(2carboxyethyl)phosphine were purchased from Aldrich. Triethylamine and sodium sulfate were purchased from Merck, 2-chloro-5,6-benzo-1,3,2-dioxaphosphorin-4-one was from Fluka, pyridine and dichloromethane were from SDS, and acetonitrile was from Labo-Standa. Analytical thin-layer chromatography (TLC) was performed on precoated alumina plates (Merck silica gel 60F 254 ref. 5554). For flash chromatography, Merck silica gel 60 (70230 mesh) (ref. 7734) or Lichroprep Si 60, 15-25 µM (ref. 109336), was used. All 4,4′-dimethoxytrityl-containing substances were identified as orange-colored spots on TLC plates by spraying with a 10% perchloric acid solution. Amino-containing substances were identified as purple-colored spots on TLC plates by spraying with a 0.1% ninhydrin solution in 1-butanol. Tetraethylene glycol derivatives were identified as blue-green-colored spots on TLC plates by spraying with a 5% phosphomolybdic acid solution in ethanol. Thiazole orange containing compounds were directly visualized on the plates as red-colored spots. Oligonucleotide syntheses were performed on Expedite Nucleic Acid Synthesis System 8909 from Perseptive Biosystems. Analyses and purifications 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 25 mM Tris-HCl, pH 7, buffer containing 10% CH3CN. Reversed-phase chromatography analysis was performed on a 600 E (System Controller) equipped with a Waters 990 photodiode array detector 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. Mass analysis ion-molecular weights of the oligonucleotides were confirmed by electrospray mass spectroscopy using a Quattro II (Micromas) instrument. 1H NMR spectra were recorded on a Bruker AM 300WB or on a Varian Unity 500 spectrometer. Absorption spectra were recorded with a Uvikon 860 spectrophotometer. The

concentrations were determined using the following molar absorption coefficients: 260 ) 183 500 M-1 cm-1 for 5′d-(A15)3′ (24), 260 ) 175 900 M-1 cm-1 for 5′d-CACT15CAC3′ (25), 260 ) 10 000 M-1 cm-1 for poly r(A) (24), 260 ) 9400 M-1 cm-1 for poly r(U), 260 ) 192 500 M-1 cm-1 for pentadeca-2′-deoxyriboadenylate-thiazole orange conjugates [)260(d-5′(A15)3′) + 260(thiazole orange) (26)], 260 ) 176 000 M-1 cm-1 for RdT20 and βdT20 (25) (we assumed that the 260 value for RT was the same as for βT), and 260 ) 192 600 M-1 cm-1 [)260(T20) + 260(thiazole orange)]for R- and β-icosathymidylate-thiazole orange conjugates. Nucleases were purchased from Roche Biochemicals. Synthesis of Thiazole Orange-Linker Derivatives 5 and 6 (Scheme 1). Synthesis of N-(11-Iodo-3,6,9trioxaundecyl)-thiazole Orange 5. Synthesis of 1,11-Dibromo-3,6,9-trioxaundecane 1. A triphenylphosphine solution (10.8 g, 40 mmol) in dry dichloro-1,2-ethane (10 mL) was added dropwise, over 4 h, to a stirred mixture of tetraethylene glycol (2 g, 10 mmol) and BrCCl3 (12.25 g, 60 mmol) in dichloro-1,2-ethane (6 mL). After the addition of triphenylphosphine was completed, stirring was maintained for an additional hour. After addition of n-pentane (100 mL), the triphenylphosphine oxide precipitated and was removed by filtration. The filtrate was concentrated by evaporation, and the residue was purified on a silica gel chromatography column using a gradient of MeOH in CH2Cl2 (0-3%) to give a pale yellow oil: yield 35% (1.12 g, 3.5 mmol); Rf1 ) 0.85 using CH2Cl2/MeOH (95:5, v/v) as eluent. The reaction product was identified as a pale blue-green-colored spot on a TLC plate by spraying with a 5% phosphomolybdic acid solution in ethanol. In these conditions, the starting material was eluted with Rf ) 0.35 and appeared as a dark blue-green-colored spot on TLC: 1H NMR (CDCl3) δ 3.46 [t, 4H, J ) 6.34 Hz, CH2 (1, 11)], 3.66 [s, 8H, CH2 (4, 5, 7, 8)], 3.80 [t, 4H, J ) 6.33 Hz, CH2 (2, 10)]; mass analysis, ESI, polarity positive; calculated for C8H16O3Br2: M+H ) 321, found 321. Synthesis of N-(11-Bromo-3,6,9-trioxaundecyl)-lepidine 2. Lepidine (0.162 g, 1.17 mmol) was added dropwise,

Oligo-2′-deoxyribonucleotide−Thiazole Orange Conjugates

over 30 min, to a stirred solution of 1,11-dibromo-3,6,9trioxaundecane 1 (1 g, 2.84 mmol) in anhydrous (fresly distilled) refluxing dioxane (3 mL) contained in a twonecked round-bottomed flask equipped with a reflux condenser and a dropping funnel and protected from the light. After 3 h of heating, the mixture was left under stirring for 16 h at room temperature, and then concentrated by evaporation. The residue was dissolved with a CH2Cl2/MeOH mixture (97:3, v/v) (1 mL) and purified on a silica gel column using a CH2Cl2/MeOH mixture (97:3, v/v, to 93:7, v/v) to give a colorless oil: yield 60% (0.315 g, 0.68 mmol); Rf 2 ) 0.31 using CH2Cl2/MeOH (95:5, v/v) as eluent; 1H NMR (CDCl3) δ 2.98 (s, 3H, CH3), 3.403.79 (m, 12H, CH2O), 4.18 (t, 2H, J ) 4.77 Hz, CH2Br), 5.58 (t, 2H, J ) 4.75 Hz, NCH2), 7.90-7.95 (m, 2H, Ar), 8.16-8.18 (m, 1H, Ar), 8.30 (d, 1H, Ar, J ) 7.62 Hz), 8.66 (d, 1H, Ar, J ) 9.03 Hz), 10.2-10.5 (m, 1H, Ar); mass analysis, ESI, polarity positive; calculated for C18H25O3NBr: M+H ) 384.3, found 384.4. Synthesis of N-(10-Iododecyl)-lepidine 3. This compound was obtained following the procedure used for the preparation of N-(11-bromo-3,6,9-trioxaundecyl)-lepidine 2 except that 1,11-dibromo-3,6,9-trioxaudecane 1 was replaced by 1,10-diiododecane. Starting from lepidine (0.162 g, 1.17 mmol) and 1,10-diiododecane (1.12 g, 2.84 mmol), N-(10-iododecyl)-lepidine 3 was obtained with 65% yield (0.304 g, 0.74 mmol): Rf3 ) 0.23 using CH2Cl2/MeOH (95:5, v/v) as eluent; 1H NMR (CDCl3) δ 1.221.28 [m, 8H, (CH2)4], 1.29-1.33 (m, 2H, CH2), 1.42-1.50 (m, 2H, CH2), 1.73-1.79 (m, 2H, CH2), 2.03-2.09 (m, 2H, CH2), 2.99 (s, 3H, CH3), 3.40-3.79 (m, 12H, CH2O), 3.17 (t, 2H, J ) 7.5 Hz, CH2I), 5.24 (t, 2H, J ) 7.5 Hz, NCH2), 7.94-8.0 (m, 2H, Ar), 8.16-8.19 (m, 1H, Ar), 8.34 (d, 1H, Ar, J ) 8 Hz, J ) 9.03 Hz), 10.15 (d, 1H, Ar, J ) 6 Hz); mass analysis, ESI, polarity positive; calculated for C20H29NI: M+H ) 410, found 410.3. Synthesis of S,3-Dimethylbenzothiazole-2-thiol 4. S,3Dimethylbenzothiazole-2-thiol 4 was obtained as reported in ref 27: yield 92% (5.97 g, 18.48 mmol); Rf4 ) 0.51 using CH2Cl2/MeOH (80:20, v/v) as eluent; mp ) 152-154 °C; 1 H NMR (DMSO-d6) δ 3.12 (s, 3H, SCH3), 4.10 (s, 3H, NCH3), 7.71 (t, 1H, J ) 7.8 Hz), 7.82 (t, 1H, J ) 7.8 Hz), 8.18 (d, J ) 8 Hz, 1H), 8.40 (d, J ) 8 Hz, 1H); mass analysis, ESI, polarity positive; calculated for C9H10NS2: M+H ) 197, found 196.6. Synthesis of N-(11-Iodo-3,6,9-trioxaundecyl)-thiazole Orange 5. N-(11-Bromo-3,6,9-trioxaundecyl)-lepidine 2 (0.300 g, 0.65 mmol) and S,3-dimethylbenzothiazole-2thiol 4 (0.303 g, 0.94 mmol) were dissolved in warm ethanol (4 mL) under stirring, and then triethylamine (200 µL, 1.5 mmol) was added to the solution. The redcolored mixture was protected from the light and left under stirring for 1 h at room temperature. The solvent was removed by evaporation, and the residue was purified on a silica gel column using a gradient of MeOH (03%) in CH2Cl2 to give a red powder: yield 35% (0.120 g, 0.22 mmol); Rf5 ) 0.77 using CH2Cl2/MeOH (80:20, v/v) as eluent; 1H NMR (CDCl3) δ 3.43-3.67 (m, 12H, CH2O ), 3.98 (t, 2H, J ) 5.16 Hz, CH2I), 4.00 (s, 3H, NCH3), 4.71 (m, 2H, NCH2), 6.72 (s, 1H, CH), 7.24-7.35 (m, 4H, Ar), 7.40-7.50 (m, 1H, Ar), 7.64-7.86 (m, 4H, Ar), 8.64 (d, J ) 7.5 Hz, 1H, Ar); mass analysis, ESI, polarity positive; calculated for expected product C26H30BrN2O3S: M+H ) 530, found 577.2, corresponds to C26H30IN2O3S. (These results indicate that halogen exchange occurred. This can be explained by the fact that the reaction between 2 and 4 to give 5 is performed in basic conditions in the presence of triethylamine.) mp ) 117-118 °C. Synthesis of N-(10-Iododecyl)-thiazole Orange 6. This

Bioconjugate Chem., Vol. 12, No. 5, 2001 759

compound was obtained following the procedure used for the preparation of N-(11-iodo-3,6,9-trioxaundecyl)-thiazole orange 5 except N-(11-bromo-3,6,9-trioxaundecyl)lepidine 2 was replaced by N-(10-iododecyl)-lepidine 3. Starting from 0.383 g (1 mmol) of N-(10-iododecyl)lepidine 3 and 0.325 g (1 mmol) of S,3-dimethylbenzothiazole-2-thiol 4, 0.208 g of 10-iododecylthiazole orange 6 was obtained: yield 62% (0.208 g, 0.29 mmol); Rf6 ) 0.46 using CH2Cl2/MeOH (90:10, v/v) as eluent; 1H NMR (CD3OD) δ 1.22-1.45 (m, 12H, (CH2)6), 1.75-1.78 (m, 2H, CH2), 1.84-1.95 (m, 2H, CH2), 3.15 (t, 2H, J ) 4.2 Hz, CH2I), 3.98 (s, 3H, NCH3), 4.52 (t, 2H, J ) 4.5 Hz, NCH2), 6.72 (s, 1H, CH), 7.21-7.32 (m, 2H, Ar), 7.38-7.40 (m, 1H, Ar), 7.41-7.48 (m, 1H, Ar), 7.57-7.61 (m, 1H, Ar), 7.65-7.70 (m, 2H, Ar), 7.75-7.78 (m, 1H, Ar), 8.65 (d, J ) 4.8 Hz, 1H, Ar), 8.90 (d, J ) 4.2 Hz, 1H, Ar); mass analysis, ESI, polarity positive; calculated for C28H34IN2S: M+H ) 558.1, found 557.1; mp ) 164-165 °C. Synthesis of Pentadeca-2′-deoxyriboadenylates 19a, 19b, 20a, and 20b Bearing a Thiazole Orange Derivative at the Eighth Internucleotidic Phosphate and Icosa-r-thymidylates 21a and 21b and Icosa-β-thymidylates 22a and 22b Bearing a Thiazole Orange Derivative at the Tenth Internucleotidic Phosphate (Scheme 2). Preparation of 5′-O-(4,4′Dimethoxytrityl)-R-thymidine-3′-H-phosphonate 11. This synthesis was adapted from ref 28. To a stirred solution of 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one (1.39 g, 6.86 mmol) in a pyridine/ClCH2CH2Cl (90:10, v/v) mixture cooled to 0 °C was added slowly a solution of 5′-O(4,4′-dimethoxytrityl)-R-thymidine, previously dried by coevaporation with anhydrous pyridine and kept under vacuum overnight (2.5 g, 4.6 mmol) in an anhydrous pyridine/ClCH2CH2Cl (50:50, v/v) mixture. The reaction was monitored by TLC on silica gel with CH2Cl2/MeOH/ NEt3 (90:9.5:0.5, v/v/v) as eluent. After 15 min, the starting material (Rf ) 0.54) was fully transformed into a new compound having a lower Rf (0.31). The reaction mixture was poured into an NEt3/H2O (20:80, v/v) mixture (100 mL) under vigorous stirring for 5 min. The reaction mixture was then extracted with CH2Cl2 (50 mL), twice. The organic phase was then washed with a saturated NaCl solution (15 mL), and the latter was backextracted with CH2Cl2 (50 mL). The organic phases were pooled, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified on a silica gel column using a gradient of methanol in dichloromethane in the presence of triethylamine, CH2Cl2/NEt3 (99.5:0.5, v/v) to CH2Cl2/MeOH/NEt3 (94.5:5:0.5, v/v/v), as eluent to give a pale yellow solid: Rf11 ) 0.62 using CH2Cl2/MeOH/NEt3 (79.5:20:0.5, v/v/v) as eluent; yield 40%, 1.3 g, 8.4 mmol; mass analysis, ESI, polarity negative; calculated for C31H32N2O9P: M+H ) 608, found 607.3. Preparation of Oligonucleotides 16, 17, and 18 Bearing a Linker at the Central Phosphate Group and Separation of Their Diastereoisomers. The oligo-2′-deoxyribonucleotides were assembled using classical phosphoramidite chemistry on a CPG support at a micromolar scale except that at the site selected for the introduction of the linker arm, a coupling step using H-phosphonate chemistry was manually performed. The CPG support involving R-thymidine, the phosphoramidite, and the H-phosphonate derivatives of R-thymidine was not commercially available. The first two were available in the laboratory, while the last one (compound 11) was obtained as described above. The syntheses were performed as follows. At the end of the chain assembly of the hepta-2′-deoxyriboad-


Privat and Asseline

Scheme 2. Synthesis of the Oligo-2′-deoxyribonucleotide-Thiazole Orange Conjugates 19a, 19b, 20a, 20b, 21a, 21b, 22a, and 22b

enylate 7, the deca-R-thymidylate 8, and the deca-βthymidylate 9, an additional detritylation step was

performed. N6-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenosine-3′-H-phosphonate 10 (0.033 g, 0.04 mmol) and


5′-O-(4,4′-dimethoxytrityl)-R-thymidine-3′-H-phosphonate 11 [or 5′-O-(4,4′-dimethoxytrityl)-β-thymidine-3′-Hphosphonate 12] (0.04 g, 0.056 mmol) in a pyridine/ CH3CN (50:50, v/v) mixture (0.4 mL) and pivaloyl chloride (0.02 g, 0.166 mmol) in a pyridine/CH3CN (50:50, v/v) mixture (0.5 mL) were then added simultaneously to the hepta-2′-deoxyriboadenylate, the deca-R-thymidylate, or the deca-β-thymidylate bound to the support. After a 2.5 min reaction, the solution was removed and the support washed with an anhydrous pyridine/CH3CN (50:50, v/v) mixture (1 mL, 3 times). Then cystamine (0.165 g, 0.87 mmol) solubilized by a pyridine/CCl4 (50:50, v/v) mixture (1 mL) was added. After 1 h, the solution was removed and the support washed with an anhydrous pyridine/CH3CN (50:50, v/v) mixture (1 mL) 3 times and then with anhydrous CH3CN (1 mL) 3 times. The support was treated with a mixture of capping solutions used on the synthesizer (0.5 mL each) for 10 min, washed with CH3CN (1 mL, 4 times), and dried. The oligonucleotide chain assemblies were completed via phosporamidite chemistry to give the fully protected pentadeca-2′-deoxyriboadenylate 13, icosa-R-thymidylate 14, and icosa-β-thymidylate 15 bound to the support bearing the linker at the central phosphates of the oligonucleotide chains. At the end of the chain assembly, an additional detritylation step was performed to deblock the 5′-terminal hydroxyl function. The deprotection step was completed by overnight concentrated ammonia treatment at 50 °C in the case of the oligo-2′-deoxyriboadenylates and at room temperature in the case of the oligothymidylates (in both cases, the support was discarded after a 2 h treatment). Purification was performed by ion exchange chromatography on a DEAE column (8 µM, 100 mm × 10 mm, Waters) using a linear gradient of NaCl (0.075-0.525 M over 30 min) in 25 mM Tris-HCl, pH 7, buffer containing 10% MeOH. After the desalting steps, reversed-phase chromatography analyses were performed on a Lichrospher 100 RP (5 µM) column (125 mm × 4 mm) from Merck with a linear gradient of CH3CN (17.4-18.4% over 15 min) in 0.1 M aqueous triethylammonium acetate, pH 7, with a flow rate of 1 mL/min. Two roughly equivalent peaks with very close retention times (Rt) were obtained for both oligo-2′-deoxyribonucleotides with Rt16a ) 8 min, 24 s and Rt16b ) 9 min in the case of the oligo-2′-deoxyriboadenylates. In the case of oligothymidylate isomers, analyses were performed on the same column with the same buffer and flow rate but with different gradients of CH3CN: 5-35% over 50 min for oligo-R-thymidylate isomers (Rt17a ) 19 min, 46 s; Rt17b ) 21 min, 40 s) and 12.5-20% over 30 min for oligo-β-thymidylate isomers (Rt18a ) 18 min, 52 s; Rt18b ) 19 min, 40 s). Synthesis of Oligonucleotides 19a, 19b, 20a, 20b, 21a, 21b, 22a, and 22b by Coupling Thiazole Orange-Linker Derivatives 5 and 6 with the Oligonucleotides Bearing a Thiol Function Obtained after Treatment of Oligo-2′deoxyribonucleotides 16a, 16b, 17, and 18 with a Reducing Agent. Solutions of 16a and 16b, d5′(Ap)7Ap*[NH(CH2)2S-S-(CH2)2NHCOCH3]A(pA)63′ (15 OD each), in 5% aqueous bicarbonate buffer, pH 9 (800 µL), were degassed by bubbling argon. An aqueous tris(2-carboxyethyl)phosphine (TCEP) solution (0.06 mg in 5 µL of H2O) was then added, and the solutions were vortexed for a few minutes. Finally, a DMF solution of thiazole orangelinker derivative 5 or 6 (1.5 mg in 800 µL) was added. The solutions were vortexed and heated at 35-40 °C. The coupling reactions were controlled by reversed-phase analyses as follows. The oligonucleotides were recovered by centrifugation of an aliquot of the reaction mixture. The pellets were dissolved in water, and the red solutions


Figure 1. HPLC reversed-phase analysis of the coupling reaction between the pentadeca-2′-deoxyriboadenylate 16 (isomer mixture) and the thiazole orange-linker derivative 5 after 2 h incubation. Pentadeca-2′-deoxyriboadenylate-thiazole orange conjugates 19a and 19b are eluted with Rt19a ) 26 min, 48 s and Rt19b ) 28 min, 10 s, respectively. The inset shows the absorption spectrum, recorded between λ ) 240 and λ ) 600 nm, of the conjugate 19a. The analysis was performed on a Lichrospher 100RP-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 (12-36% over 45 min).

were analyzed by reversed-phase chromatography on the Lichrospher column described above with a linear gradient of CH3CN (12-36% over 45 min) in 0.1 M aqueous triethylammonium acetate, pH 7, with a flow rate of 1 mL/min (Rt19a ) 19 min, 1 s; Rt19b ) 20 min, 5 s; Rt20a ) 30 min; Rt20b ) 31 min). The coupling reaction could be performed on the mixture of isomers of oligo-2′-deoxyriboadenylate (16a + 16b), but separate coupling reactions on each isomer are required for determination of the stereochemistry of oligo-2′-deoxyriboadenylate-thiazole orange conjugates. Reversed-phase analysis of the coupling reaction between the oligo-2′-deoxyriboadenylate 16 and the thiazole orange-linker derivative 5, after 2 h of incubation, is shown in Figure 1. The inset shows the absorption spectra, recorded between λ ) 240 nm and λ ) 600 nm, of the oligo-2′-deoxyriboadenylate-thiazole orange conjugates with a hydrophilic linker, 19a. Usually the reaction was completed after 5-6 h of incubation: yield 50% after purification; mass analysis, ESI, polarity negative. Pentadeca-2′-deoxyriboadenylate-thiazole orange conjugates with a polymethylene linker: compounds 20a and 20b: calculated mass for C180H219O72N78S2P17: M-H ) 5125.08, found 5125.26 ( 0.63 for compound 20a and 5125.23 ( 0.63 for compound 20b. Solution of icosa-R-thymidylate 17 (20 OD, 0.11 µmol) in 5% aqueous NaHCO3, pH 9, buffer (800 µL) was degassed by bubbling argon. Then an aqueous tris(2carboxyethyl)phosphine (TCEP) solution (0.06 mg, 0.22 µmol in 1 µL of H2O) was added, and the solutions were vortexed for a few minutes. After verification of the cleavage of the disulfide bridge by reversed-phase chromatography [Rt17a ) 17 min, 22 s; Rt17b ) 17 min, 42 s; and for the thiol derivatives, Rt ) 17 min, 9 s; Rt ) 17 min, 22 s on the Lichrospher column described above with a linear gradient of CH3CN (0-24% over 15 min) in 0.1 M aqueous triethylammonium acetate, pH 7, with a flow rate of 1 mL/min], a DMF solution (1 mL) of thiazole orange-linker derivative 6 (1.5 mg, 2.2 × 10-6 mol) was added. The solutions were vortexed and heated at 3540 °C. The coupling reactions were controlled by reversed-


Privat and Asseline

Scheme 3. Isomer Identification

phase analyses as follows. The oligonucleotides were recovered by centrifugation. The pellets were dissolved in water, and the solutions were analyzed by reversedphase chromatography on a Lichrospher column (the same as above) with a linear gradient of CH3CN (0-40% over 50 min) in 0.1 M aqueous triethylammonium acetate, pH 7, with a flow rate of 1 mL/min. Two peaks corresponding to the Rp and Sp isomers were obtained (Rt21a ) 34 min, 40 s; Rt21b ) 36 min, 2 s). The coupling reactions were nearly complete after 5-6 h of reaction. The oligonucleotide-thiazole orange isomers 21a and 21b were purified by using a more flat gradient: yield 50% after purification; mass analysis, ESI, polarity negative. Icosa-R-thymidylate-thiazole orange conjugates 21a and 21b: calculated mass for C230H298 O167N43S2P19: M-H ) 6509.74, found mass 6509.64 ( 1.93. The icosa-β-thymidylate-thiazole orange conjugates 22a and 22b were obtained by using the isomer mixture of the icosa-β-thymidylate-linker derivative 18 and the thiazole orange-linker derivative 6, and proceeding as reported for the preparation of the icosa-R-thymidylatethiazole orange conjugates 21a and 21b. HPLC reversedphase analyses were performed on a Lichrospher column

(the same as above) with a linear gradient of CH3CN (2030% over 30 min) in 0.1 M aqueous triethylammonium acetate, pH 7, with a flow rate of 1 mL/min. Two peaks corresponding to the Rp and Sp isomers were obtained (Rt22a ) 12 min, 42 s; Rt22b ) 19 min, 50 s). Isomer Identification (Scheme 3). Preparation of Dinucleosides d-5′DMTrABzp*(H)ABzBz3′ 24a and 24b. To an anhydrous stirred pyridine solution (5 mL) of a mixture of N6-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyriboadenosine-3′-H-phosphonate 10 (0.412 g, 0.5 mmol) and N6-benzoyl-3′-O-benzoyl-2′-deoxyriboadenosine 23 (0.230 g, 0.5 mmol), previously dried by coevaporation with anhydrous pyridine (20 mL, 3 times) and kept under vacuum overnight, was added pivaloyl chloride (0.180 g, 1.5 mmol) at room temperature. After 15 min of stirring, the reaction mixture was diluted with CH2Cl2 (50 mL), and the organic phase was washed with a 5% aqueous NaHCO3 solution (50 mL). The aqueous phase was backextracted with CH2Cl2 (50 mL), twice. The organic phases were then pooled, dried over Na2SO4, filtered off, and concentrated under reduced pressure. The residue was solubilized with acetone and coevaporated with toluene (10 mL), 3 times, and then purified on a silica gel column


(ref. 109336 from Merck ) using an ethyl acetate/acetone mixture (60:40, v/v) containing 0.2% acetic acid as eluent until the first isomer was eluted and then an ethyl acetate/acetone mixture (50:50, v/v) containing 0.2% acetic acid as eluent for the elution of the second isomer. After removal of the solvents by evaporation, the residual acetic acid was removed by co-evaporation with CH3CN (3 mL), 3 times. Products corresponding to each pure dinucleoside-H-phosphonate isomer (0.2 g each) were obtained as pale yellow solids: Rf24a ) 0.61 and Rf24b ) 0.53 using CH2Cl2/iPrOH (90:10, v/v) as eluent; Rf24a ) 0.56 and Rf24b ) 0.53 using CH2Cl2/MeOH (90:10, v/v) as eluent; mass analysis, ESI, polarity positive; calculated mass for C62H55O12N10P19: M+H ) 1163.4, found mass 1163.3. Preparation of Dinucleosides d-5′Ap*(S)A3′ 25a and 25b. To pyridine solutions (1 mL) of each pure dinucleosideH-phosphonate (24a and 24b) (0.02 g, 17.2 mmol), previously dried by co-evaporation with anhydrous pyridine (5 mL, 3 times), was added, at room temperature, under stirring, and in an argon atmosphere, a pyridine/ CS2 (50:50, v/v) solution of S8 (4.25 mg/mL) (2 mL) (32). After 1 h of stirring, the reaction mixtures were evaporated to dryness, and the residues were co-evaporated with toluene (5 mL), 3 times. The residues were solubilized with ethyl acetate (6-7 mL). The reaction mixtures were filtered and then washed with water (1-2 mL). The organic phases were concentrated to dryness, and an 80% solution of acetic acid in water (5 mL) was added. After 30 min, acetic acid was removed by evaporation. The residues obtained were solubilized with a 28% aqueous ammonia solution (4-5 mL) and left overnight at 50 °C. The reaction mixtures were evaporated to dryness. The residues were solubilized with water (7-8 mL), and the aqueous phases were extracted with ethyl acetate (3 × 1 mL): Rf25a ) 0.53 and Rf25b ) 0.61 using iPrOH/NH4OH/ H2O (70:20:10, v/v/v) as eluent; Rf25a ) 0.17 and Rf25b ) 0.31 using CH2Cl2/MeOH (90:10, v/v) as eluent. The presence of the sulfur atom was confirmed by the obtention of pink-colored spots on TLC plates by spraying with a 2,6-dibromo-4-benzoquinone-N-chloroimine (DBPNC) solution in ethanol (2 mg/mL) and heating: mass analysis, ESI, polarity positive; calculated for C20H25N10O7SP: M+H ) 580.52, found 581.1. Preparation of Dinucleoside d-5′Ap*[NH(CH2)2S-S(CH2)2NHCOCH3]A3′ 26a and 26b. Both pure isomers 24a and 24b of the dinucleoside-H-phosphonate N6-benzoyl-5′-O(4,4′-dimethoxytrityl)-2′-deoxyriboadenoylyl-N6-benzoyl3′-O-benzoyl-2′-deoxyriboadenosine-3′-H-phosphonate(0.035 g each, 0.03 mmol) were treated, separately, with cystamine (0.09 g, 0.6 mmol) solubilized by an anhydrous pyridine/CCl4 (50:50, v/v) mixture (1 mL) for 10 min over an argon atmosphere. The reaction mixture was concentrated to dryness and solubilized with CH2Cl2 (7-8 mL). The organic phase was washed with water (1-2 mL), dried over Na2SO4, and concentrated to dryness. The residue was co-evaporated with anhydrous pyridine (3 mL, 3 times), and during the last evaporation, 1 mL of pyridine was kept in the flask. Acetic anhydride (0.93 g, 0.9 mmol) was added under stirring at room temperature. The reaction was monitored by TLC analysis using CH2Cl2/MeOH (90:10, v/v) as eluent. When the reaction was completed, the reaction mixture was evaporated to dryness, co-evaporated with toluene, and purified by flash chromatography using a gradient of MeOH in CH2Cl2 (020%). An 80% acetic acid solution in water (3 mL) was then added. After 30 min of reaction at room temperature, the solution was evaporated to dryness and coevaporated with toluene (3 mL), twice. The residue was


then treated with a 28% solution of aqueous ammonia in a MeOH/H2O (80:20, v/v) mixture at 50 °C overnight. After removal of the ammonia solution by evaporation, the residue was solubilized with water (3 mL), and the aqueous phase was extracted with ethyl acetate (0.5 mL), 3 times. Purification by reversed-phase chromatography was performed on a Lichrospher 100 RP (5 µM) column (125 mm × 4 mm) from Merck with a linear gradient of CH3CN (10-35% over 50 min) in 0.1 M aqueous triethylammonium acetate, pH 7, with a flow rate of 1 mL/min: Rt26a ) 19 min, 30 s; Rt26b ) 19 min, 55 s; mass analysis, ESI, polarity positive; calculated for C26H37N12O8S2P: M+H ) 741.76, found 741.1. RESULTS AND DISCUSSION

Synthesis of the Oligonucleotide-Thiazole Orange Conjugates. One report shows that the homodimeric thiazole orange TOTO bisintercalates in DNA with the linker in the minor groove (21). Since our previous work on oligo-2′-deoxyribonucleotide-acridine conjugates showed that stronger stabilization (therefore better intercalation) was observed when the intercalating residue was linked to an internucleotidic phosphate (22), we chose the same position for the linking of the thiazole orange moiety. Substitution at an internucleotidic position introduces a chirality at the phosphorus atom with the formation of two diastereoisomers (Figures 2 and 3). These oligonucleotides can be separated as pure isomers. When these oligonucleotides are hybridized with their complementary sequences, the substituent of one isomer protrudes in the direction of the minor groove, while the substituent of the other isomer points in the direction of the major groove. The CPK model indicates that a linker involving 14 atoms fixed on an internal phosphorous atom and on the nitrogen atom of the quinolinium ring of the thiazole orange residue would be suitable for the intercalation of the thiazole orange residue by either the minor or the major groove. We chose to attach the linker via a phosphoramide linkage at the central phosphate of a pentadeca-2′-deoxyriboadenylate (Figure 2), an icosaR-thymidylate, and an icosa-β-thymidylate (Figure 3) and to separate both isomers in order to study the influence of the stereochemistry at the phosphorus atom on the interactions of the oligonucleotide-thiazole orange conjugates with their complementary sequences. A linker involving a masked thiol function such as cystamine that can withstand the conditions required for the deprotection and purification steps of the oligonucleotide was chosen (29). After in situ cleavage of the disulfide bridge, the released thiol function was selectively reacted with a halogenoalkyl group linked at the nitrogen atom of the quinolinium ring of the thiazole orange. Of the 2 different linkers fixed on the thiazole orange, 1 was hydrophilic, derived from tetraethylene glycol (compound 5), and the other was hydrophobic with 10 methylene groups (compound 6). These thiazole orange-linker derivatives, 5 and 6, were reacted with each pure Sp and Rp isomer of the pentadeca-2′-deoxyriboadenylate 16 while only the thiazole orange derivative involving a polymethylene linker, 6, was reacted with the mixture of the Sp and Rp isomers of icosa-R-thymidylate 17 and with the mixture of the Sp and Rp isomers of icosa-β-thymidylate 18. Sp and Rp isomers of the icosa-R-thymidylate-thiazole orange conjugates and of the icosa-β-thymidylate-thiazole orange conjugates were then separated by reversed-phase chromatography. Preparation of Thiazole Orange-Linker Derivatives 5 and 6. The preparation of thiazole orange-linker derivatives 5 and 6 (Scheme 1) was achieved by a


Privat and Asseline

Figure 2. Structures of the pentadeca-2′-deoxyriboadenylate-thiazole orange conjugates 19a, 19b, 20a, and 20b.

procedure adapted from the literature (27, 30). The lepidine-linker derivatives 2 and 3 were obtained by reaction of lepidine with 1,11-dibromo-3,6,9-trioxaudecane 1 and 1,10-diiododecane, respectively. 1,11-Dibromo3,6,9-trioxaudecane 1 was obtained by treatment of tetraethylene glycol with triphenylphosphine and BrCCl3. Reaction of the lepidine-linker derivatives 2 and 3 with S,3-dimethylbenzothiazole-2-thiol 4 (obtained by reaction of 3-methylbenzothiazole-2-thione with methyl iodide) led to the expected thiazole orange-linker derivatives 5 and 6, respectively. Synthesis of Oligo-2′-deoxyribonucleotide-Thiazole Orange Conjugates 19a, 19b, 20a, 20b, 21a, 21b, 22a, and 22b. This was achieved following Scheme 2. At the end of the chain assembly of hepta-2′-deoxyriboadenylate 7, deca-R-thymidylate 8, and deca-β-thymidylate 9, an additional detritylation step was performed, and the fixation of the cystamine on the central phosphate of the oligo-2′-deoxyribonucleotide was achieved by performing an H-phosphonate coupling step, manually, including an oxidation step using a mixture of CCl4 and cystamine in pyridine, at a position preselected for the attachment of the linker (31). Acetylation of the terminal primary amino function of the linker was performed in

order to prevent its phosphitilation during the assembling of the second parts of the oligonucleotide chains via phosphoramidite chemistry. This led to the fully protected pentadeca-2′-deoxyriboadenylate 13, icosa-Rthymidylate 14, and icosa-β-thymidylate 15 bound to the support and bearing the linker at the central phosphates. After deprotection and purification by ion-exchange chromatography, reversed-phase analyses of pentadeca-2′deoxyriboadenylate 16, icosa-R-thymidylate 17, and icosaβ-thymidylate 18 bearing the linker at their central phosphate via a phosphoramidate linkage exhibited two roughly equivalent peaks with very close retention times (see Experimental Procedures). The pentadeca-2′-deoxyriboadenylate isomers 16a (fast eluted) and 16b (slowly eluted) were separated by reversed-phase chromatography (see Experimental Procedures for the conditions used) while the mixture of isomers was used in the case of icosa-R-thymidylate 17 and icosa-β-thymidylate 18 for the coupling step with the thiazole orange derivative 6. Determination of the configuration was made for pentadeca-2′-deoxyriboadenylates 16a and 16b after their digestion by the nuclease P1 or snake venom phosphodiesterase followed by alkaline phosphatase treatment. Each hydrolysate was then compared with pure Sp or Rp



Figure 3. Structures of the icosa-R-thymidylate-thiazole orange conjugates 21 and 21b, and the icosa-β-thymidylate-thiazole orange conjugates 22a and 22b.

isomer samples of the dinucleoside d-5′Ap*[NH(CH2)2SS(CH2)2NHCOCH3]A3′, whose stereochemistry was determined following the method reported in Scheme 3 and discussed in the next paragraph. The thiazole orange derivatives 5 and 6 were coupled with the released thiol function obtained after in situ treatment of pure isomers 16a and 16b of the pentadeca-2′-deoxyriboadenylate with a reducing agent (TCEP) to give the pentadeca-2′deoxyriboadenylate-thiazole orange conjugates 19a and 19b, 20a and 20b, respectively. The same reaction was performed with the thiazole orange derivative 6 and a mixture of the isomers of icosa-R-thymidylate 17 or icosaβ-thymidylate 18 to give a mixture of the isomers of icosaR-thymidylate-thiazole orange conjugates 21a and 21b and of icosa-β-thymidylate-thiazole orange conjugates 22a and 22b, respectively. The latter were then separated by chromatography. Determination of the Configuration of Isomers 19a, 19b, 20a, and 20b of the Pentadeca-2′-deoxyriboadenylate-Thiazole Orange Conjugates. This was achieved as described in Scheme 3. The fully protected dinucleoside-H-phosphonate 24 was obtained following a procedure adapted from the literature (32) except that the benzoyl group was used for the protection of the 3′-

hydroxyl function. Dinucleoside isomers 24a and 24b were purified and separated by silica gel chromatography. A part of both pure dinucleoside-H-phosphonate isomers 24a and 24b was separately transformed (with retention of configuration at the phosphorus center) into pure dinucleoside phosphorothioates by treatment with S8 in a pyridine/CS2 mixture. After deprotection and purification by reversed-phase chromatography, aliquots of both pure dinucleoside phosphorothioate isomers 25a and 25b were treated separately with P1 endonuclease and snake venom phosphodiesterase (SVP) and then with phosphatase alkaline (PA). Treatment with P1 + PA led to the full degradation of the Sp isomer into dA while the Rp isomer remained unchanged. On the contrary, treatment with SVP + PA led to full degradation of the Rp isomer into dA while the Sp isomer remained unchanged (33). These results showed that only dinucleoside phosphorothioate 25b which comes from dinucleoside-Hphosphonate 24b was cleaved by P1 + PA treatment while only dinucleoside phosphorothioate 25a which comes from dinucleoside-H-phosphonate 24a was cleaved by SVP + PA treatment. The remaining part of both pure dinucleoside-H-phosphonate isomers 24a and 24b was separately transformed (with inversion of configuration


at the phosphorus center) (34) into the dinucleoside d5′ Ap*[NH(CH2)2S-S(CH2)2NHCOCH3]A3′ 25a and 25b by treatment with cystamine in a CCl4/pyridine mixture followed by an acetylation step. After deprotection, reversed-phase purification of the dinucleoside gave nearly equivalent peaks which correspond to diastereoisomers 25a and 25b. Both pure dinucleoside isomers 25a and 25b d-5′Ap*[NH(CH2)2S-S(CH2)2NHCOCH3]A3′ were used as a reference during analysis of the mixture obtained after nuclease degradation by either P1 + PA or SVP + PA of each pure isomer of the pentadecamers d-5′(Ap)7Ap[NH(CH2)2S-S(CH2)2NHCOCH3](Ap)6A3′ 16a and 16b, thus allowing the determination of their configuration and subsequently those of all the pentadeca2′-deoxyriboadenylate-thiazole orange conjugates described (16a gives dA + 25a, and 16b gives dA + 25b). We then verified that the elution order for pentadeca2′-deoxyriboadenylate-thiazole orange conjugates 19a and 19b was the same as that for oligonucleotides 16a and 16b. The same result was observed for oligo-2′deoxyriboadenylates 20a and 20b with the hydrophobic linker. The coupling of 16a with 5 and 6 gave 19a and 20a, respectively, while the coupling of 16b with 5 and 6 gave 19b and 20b, respectively. From these results and in accordance with a literature report (35), we were able to conclude that the fast-eluted pentadeca-2′-deoxyriboadenylate-thiazole orange conjugates 19a and 20a have the Rp configuration while the slow-eluted conjugates 19b and 20b have the Sp configuration. When the conjugates with the Rp configuration are hybridized with their target sequences, the linker is directed toward the minor groove of the duplex while in the case of the conjugates with the Sp configuration the linker is directed toward the major groove. Interaction with the Complementary Sequence. The stabilizing effect of the thiazole orange residue on the hybridization of the thiazole orange-oligonucleotide conjugates with their complementary sequences was determined by absorption spectroscopy studies carried out between λ ) 240 nm and λ ) 600 nm in the case of the thiazole orange-oligonucleotide conjugates and between λ ) 240 nm and λ ) 350 nm in the case of the corresponding unmodified oligonucleotides used as references. Pentadeca-2′-deoxyriboadenylate-Thiazole Orange Conjugates 19a, 19b, 20a, and 20b. To determine the influence of the nature of the linker and of the stereochemistry at the phosphorus atom bearing the thiazole orange residue, modified oligo-2′-deoxyriboadenylates 19a, 19b, 20a, and 20b were hybridized with an oligo-2′-deoxyribonucleotide, 5′d-CACT15CAC 3′, involving the complementary sequence. The unmodified 5′d(A15)3′ was used as a reference. The spectra of all four oligo-2′-deoxyriboadenylate-thiazole orange conjugates (19a, 19b, 20a, and 20b) were almost identical. For a typical UV-visible spectrum of a pentadeca-2′-deoxyriboadenylate-thiazole orange conjugate, see the inset in Figure 3. The spectra contain one absorption band in the visible region between λ ) 400 nm and λ ) 600 nm (where only the thiazole orange absorbs light) with λvismax ≈ 511 nm and a shoulder at λ ) 485 nm. The other absorption band in the UV range corresponds to the absorbance of the pentadeca-2′-deoxyriboadenylate and the thiazole orange with a λuvmax ≈ 257 nm. A small additional band was also present at λ ≈ 305 nm. Adding increasing concentrations of 5′d-CACT15CAC 3′ to a 0.1 µM solution of 5′d-(A15)3′ in 10 mM sodium cacodylate, pH 7, buffer containing 0.1 M NaCl at 3 °C led to a sharp break when the absorbance at 260, 270,

Privat and Asseline Table 1. Tm Values for Oligo-2′-deoxyribonucleotides β-5′d-(A15)3′, r-5′d-(T20)3′, and β-5′d-(T20)3′ and Oligo-2′-deoxyribonucleotide-Thiazole Orange Conjugates 19a, 19b, 20a, 20b, 21a, 21b, 22a, and 22b Hybridized with Their Complementary Sequences

β-5′d-(A15)3′ 19a 19b 20a 20b

β-5′d-(CACA15CAC)3′

poly r(U)

32.5a 41a 40a 40.5a 38a -

26.4b 35b 33b -

poly r(A) R-5′d-(T20)3′ 21a 21b β-5′d-(T20)3′ 22a 22b

49.5c 50c 49c 34d 36d 35.5d

a Oligonucleotide concentrations are 0.1 µM, each strand, in 10 mM sodium cacodylate, pH 7, buffer containing 0.1 M NaCl. b Oligonucleotide concentrations are 1 µM (with U/A ) 2) in 10 mM sodium cacodylate, pH 7, buffer containing 0.1 M NaCl. c Oligonucleotide concentrations are 1 µM (with T/A ) 1) in 66.5 mM, pH 7, phosphate buffer. d Oligonucleotide concentrations are 1 µM (with T/A ) 1) in 10 mM sodium cacodylate, pH 7, buffer containing 0.1 M NaCl.

or 280 nm was plotted versus the A/T concentration ratio (data not shown). The break occurred at a 1:1 ratio, indicating the formation of a double helix. We considered that the presence of thiazole orange in the conjugates should not have changed the stoichiometry of their binding with their complementary targets. For all the oligo-2′-deoxyriboadenylate-thiazole orange conjugates (19a, 19b, 20a, and 20b) studied, the addition of 1 equiv of the complementary sequences at 3 °C induced a change of the absorption spectra between λ ) 240 and λ ) 600 nm (data not shown). Only changes observed between λ ) 400 and λ ) 600 nm will be discussed. Addition of 1 equiv of 5′d-CACT15CAC3′ to oligo-2′-deoxyriboadenylatethiazole orange conjugates 19a, 19b, 20a, and 20b induces a blue shift of the λmax of the visible spectra of thiazole orange 3, 4, 4, and 4 nm, respectively, without a significant change in intensity. In all cases, the melting temperatures of the duplexes formed by the oligo-2′-deoxyriboadenylates and their complementary sequences increased when the thiazole orange derivative was linked to the oligonucleotides with similar ∆Tm ) +8.5 and +7.5 °C for oligonucleotidethiazole orange conjugates 19a and 19b with the hydrophilic linker, respectively (Table 1). In the case of oligonucleotide-thiazole orange conjugates 20a and 20b involving the polymethylene linker, ∆Tm differences of +8 and +5.5 °C were observed, respectively. The greatest stabilization was observed for oligonucleotide-thiazole orange conjugate 20a (with the Rp configuration) with the linker directed toward the minor groove. This result is consistent with literature data indicating that the homodimeric thiazole orange TOTO intercalates with the nitrogen atom of the quinolinium ring in the minor groove (36). The lower stabilization observed with the second isomer 20b could be explained by the fact that the linker was directed toward the major groove and intercalation of thiazole orange is only possible if the linker fold-back is in the direction of the minor groove. Being so, the linker may be too short to permit full intercalation. In the case of conjugates 19a and 19b, the linker length is a little longer than it is for conjugates 20a and 20b. Therefore, in both cases full intercalation is possible, giving nearly the same Tm values. To test the ability of the thiazole orange moiety to intercalate into DNA/RNA hybrids, we have studied the interaction of the conjugates 20a and 20b involving the polymethylene linker with poly r(U). Adding increasing concentrations of poly r(U) to a 0.1 µM solution of 5′d-


(A15)3′ or 20b in 10 mM sodium cacodylate, pH 7, buffer containing 0.1 M NaCl at 3 °C led to a sharp break when the absorbance at 260 nm was plotted versus the A/U concentration ratio (data not shown). The break occurred at a 1:2 ratio, indicating the formation of a triple helix. These results indicating the formation of a triplex with both the unmodified 5′d-(A15)3′ and the conjugate 20b are consistent with the intercalation of the thiazole orange. Thus, when the isomer 20b (Rp configuration) is hybridized with poly r(U) forming a duplex, unless being intercalated, the thiazole orange is directed toward the major groove and should prevent the binding of a second strand of poly r(U) in the major groove to form a triplex. Adition of 2 equiv of poly r(U) to conjugates 20a and 20b induces a red shift of the λvismax value (≈5 nm) of the visible spectra of thiazole orange in both cases. No significant change in intensity of the spectra was observed, but the general shape was less broad on the left part of the visible band of the spectra. These changes are reversed when increasing the temperature. The melting of the triple helices formed between the conjugates 20a or 20b (or the unmodified pentadecadeoxyriboadenylate) and poly r(U) showed only one transition. Once again, the presence of the thiazole orange stabilized the duplexes (Table 1) with ∆Tm ) +8.6 and +6.6 °C for oligonucleotide-thiazole orange conjugates 20a and 20b, respectively. These results are in accordance with intercalation of the thiazole orange in DNA/RNA complexes. Icosa-R-thymidylate-Thiazole Orange Conjugates 21a and 21b and Icosa-β-thymidylate-Thiazole Orange Conjugates 22a and 22b. In the same way, the stability of the complexes formed by the thiazole orange-icosa-R-thymidylate conjugate isomers 21a and 21b and the thiazole orange-icosa-β-thymidylate conjugate isomers 22a and 22b with their target poly r(A) was compared to those of the complexes formed between the corresponding icosathymidylates and poly r(A) (Table 1). In the case of the thiazole orange-icosa-R-thymidylate conjugates 21a and 21b, the studies were performed in the buffer used for microinjections in cells for in situ hybridization experiments, whose results will be discussed elsewhere, while in the case of thiazole orangeicosa-β-thymidylate conjugates 22a and 22b the buffer used was the same as for the studies carried out with oligoadenylates. The spectra of the two icosa-R-thymidylate-thiazole orange conjugates 21a and 21b were almost identical. Adding increasing concentrations of R-T20 to a poly r(A) solution (20 µM) in 66.5 mM phosphate, pH 7, buffer at 3 °C led to a sharp break when the absorbance at 260, 270, or 280 nm was plotted versus the A/T concentration ratio (data not shown). The break occurred at a 1:1 ratio, indicating the formation of a double helix. As in the case of the oligo-2′-deoxyriboadenylate-thiazole orange conjugates 19a, 19b, 20a, and 20b, we considered that the presence of thiazole orange in the conjugates should not have changed the stoichiometry of their binding with their complementary targets. In the presence of 1 equiv of the complementary sequence poly r(A), differences could be observed for the two isomers. While no change of the λvismax value (508 nm) was observed for compound 21a, the general shape of the spectrum was less broad as compared to that for compound 21a alone. In the case of the other isomer (compound 21b) in the presence of 1 equiv of the target sequence, the spectrum was also less broad as compared to that for the conjugate alone and also there was a small red shift of the λvismax value (≈2 nm). But in the latter case the change could be observed on the left part of the


visible band of the spectrum, while for the first isomer (21a) changes were observed on both the left and right sides of the spectrum. No notable variations in intensity were observed in the spectra of compounds 21a and 21b upon hybridization with the target sequence. Spectral modifications observed between λ ) 400 and 600 nm when 1 equiv of conjugates 21a or 21b was mixed with 1 equiv of poly r(A) at 3 °C were, however, reversed upon a temperature increase to 70 °C. In the case of duplexes formed between icosa-Rthymidylate-thiazole orange conjugates 21a and 21b with poly r(A), the presence of thiazole orange did not induce any stabilizing effect (Table 1). These results indicate that intercalation of thiazole orange is unlikely to occur. This may probably be due to the difference in the structure of the duplex formed between the icosa-Rthymidylate and the poly r(A). Oligo-R-thymidylates have been reported to hybridize with poly r(A) in the antiparallel orientation, forming a type A duplex (37), but to our knowledge examples of intercalation in such duplexes have never been reported. To address this point, we have synthesized and studied the hybridization properties of the icosa-β-thymidylate-thiazole orange conjugate isomers 22a and 22b. The unmodified icosa-β-thymidylate was used as reference. The spectra of the icosa-βthymidylate-thiazole orange conjugate isomers 22a and 22b were almost identical. The stoichiometry for the binding with poly r(A) was also equal to 1. But once again, no significant change of the visible absorption spectra of the thiazole orange was observed upon the mixing of conjugates 22a and 22b with 1 equiv of poly r(A), and melting studies of the duplexes indicated that hardly no stabilization (∆Tm ) +2 and +1.5 °C for oligonucleotide-thiazole orange conjugates 22a and 22b, respectively) was induced by the presence of the thiazole orange (Table 1). Another explanation for this absence of stabilization could be that intercalation of TOTO was reported to occur with the quinolinium ring placed between purine bases and the thiazole ring between pyrimidine bases (36). Linkage of the thiazole orange on the pyrimidine strand (via the quinolinium ring) compared to the purine strand should change possible intercalation interactions. We have reported the synthesis and binding properties of oligo-2′-deoxyribonucleotide-thiazole orange conjugates. The thiazole orange label was coupled to the eighth phosphate of a pentadeca-2′-deoxyriboadenylate via a phosphoramidate linkage. Two different linkers, either hydrophilic or more hydrophobic, were used to tether the oligonucleotide to the nitrogen atom of the quinolinium ring of the thiazole orange moiety. Substitution at the internucleotidic position introduces a chirality at the phosphorus atom with the formation of two stereoisomers. The latter were separated, and their absolute configurations were determined. Finally, the thiazole orange moiety was also tethered to the tenth phosphate of an icosa-R-thymidylate using the hydrophobic polymethylene linker. Once again, the thiazole orange-icosaR-thymidylate conjugates were obtained as pure isomers. The binding properties of these oligo-2′-deoxyribonucleotide-thiazole orange conjugates with their complementary sequences were studied by absorption spectroscopy. In the case of the pentadeca-2′-deoxyriboadenylate series, the presence of the label induces a stabilization of the complexes formed both with the DNA and with the RNA targets. This is consistent with the intercalation of the thiazole orange moiety. In contrast, the stability of the duplexes formed between the icosa-R-thymidylate derivatives and poly r(A) is similar to that of the corresponding


unmodified duplex, indicating the absence of intercalation of the thiazole orange. Studies carried out with the corresponding icosa-β-thymidylate derivatives indicated also a lack of stabilization by covalent linking of the thiazole orange in the presence of poly r(A). These results could be explained by the fact that in this case the thiazole orange is linked to the pyrimidine strand via the quinolinium ring when intercalation of the TOTO was reported to take place with the benzothiazole ring between the pyrimidine bases. Work to address this question will be developed in the near future. ACKNOWLEDGMENT

We thank the CNRS and the Region Centre for a fellowship to E.P., C. Bure´ for running the electrospray mass spectrometer, and H. Labbe´ for recording the NMR spectra. We also thank M. Chassignol for the preparation of the compound 6 sample used in this work and for providing the modified support involving R-thymidine and V. Roig for providing the phosphoramidite derivative of R-thymidine. LITERATURE CITED (1) Meinkoth, J., and Wahl, G. (1984) Hybridization of nucleic acids immobilized on solid supports. Anal. Biochem. 168, 267-284. (2) Niemeyer, C. M., and Blohm, D. (1999) DNA microarrays. Angew. Chem., Int. Ed. Engl. 38, 2865-2869. (3) Morrison, L. E. (1999) Homogeneous detection of specific DNA sequences by fluorescence quenching and energy transfer. J. Fluoresc. 9, 187-196. (4) Clegg, R. M. (1992) Fluorescence resonance energy transfer and nucleic acids. Methods Enzymol. 211, 353-388. (5) Tyagi, S., and Kramer, F. R. (1996) Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. 17, 303-308. (6) Cardullo, R. A., Agrawal, S., Flores, C., Zamecnik, P. C., and Wolf, D. E. (1988) Detection of nucleic acid hybridization by nonradiative fluorescence resonance energy transfer. Proc. Natl. Acad. Sci. U.S.A. 85, 8790-8794. (7) Mergny, J.-L., Boutorine, A. S., Garestier, T., Belloc, F., Rougeé, M., Bulychev, N. V., Koshkin, A. A., Bourson, J., Lebedev, A. V., Valeur, B., Nguyen, T., Thuong, and He´le`ne, C. (1994) Fluorescence energy transfer as a probe for nucleic acid structures and sequences. Nucleic Acids Res. 22, 920928. (8) Selvin, P. R., and Hearst, J. E. (1994) Luminescence energy transfer using a terbium chelate: improvements on fluorescence energy transfer. Proc. Natl. Acad. Sci. U.S.A. 91, 10024-10028. (9) Ebata, K., Masuko, M., Ohtani, H., and Kashiwasake-Jibu, M. (1995) Nucleic acid hybridization accompagnied with excimer formation from two pyrene-labeled probes. Photochem. Photobiol. 62, 836-839. (10) Paris, P. L., Langenhan, J. M., and Kool, E. T. (1998) Probing DNA sequences in solution with a monomer-excimer fluorescence color change. Nucleic Acids Res. 26, 3789-3793. (11) Wiederholt, K., Rajur, S. B., Giuliano, J., Jr., O’Donnell, M. J., and McLaughlin, L. W. (1996) DNA-tethered Hoechst groove-binding agents: duplex stabilization and fluorescence characteristics. J. Am. Chem. Soc. 118, 7055-7062. (12) Rajur, S. B., Robles, J., Wiederholt, K., Kuimelis, R. G., and McLaughlin, L. W. (1997) Hoechst 33258 tethered by a hexa(ethylene glycol) linker to the 5′-termini of oligodeoxynucleotide 15-mers: Duplex stabilization and fluorescence properties. J. Org. Chem. 62, 523-529. (13) Jenkins, Y., and Barton, J. K. (1992) A sequence-specific molecule light switch: tethering of an oligonucleotide to a dipyridophenazine complex of ruthenium II. J. Am. Chem. Soc. 117, 8736-8738. (14) Ishiguro, T., Saitoh, J., Yawata, H., Otsuka, M., Inoue, T., and Sugiura, Y. (1996) Fluorescence detection of specific sequence of nucleic acids by oxazole yellow-linked oligonucle-

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BC0100051

Synthesis and Binding Properties of Oligo-2'-deoxyribonucleotides

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