Synthesis and Properties of AcridineâOligonucleotide Conjugates

Bioconjugate Chem. 2006, 17, 1351−1359

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Directing Quadruplex-Stabilizing Drugs to the Telomere: Synthesis and Properties of Acridine-Oligonucleotide Conjugates Joan Casals,† Laurent Debe´thune,† Karine Alvarez,† Antonina Risitano,‡ Keith R. Fox,*,‡ Anna Grandas,† and Enrique Pedroso*,† Departament de Quı´mica Orga`nica, Facultat de Quı´mica, Universitat de Barcelona, Martı´ i Franque`s 1-11, E-08028 Barcelona, Spain, and School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, U.K. Received July 3, 2006; Revised Manuscript Received August 10, 2006

Conjugates containing quadruplex-stabilizing acridines linked to oligonucleotides that are complementary to the G-rich human telomere sequence were synthesized. Acylation of 3,6-diaminoacridine followed by two Michael reactions provided derivatives suitable for conjugation, which were coupled to resin-linked amine-modified oligonucleotides by activating the carboxyl group with pentafluorophenyl 4-nitrobenzenesulfonate. After deprotection with aqueous ammonia at room temperature, conjugates incorporating different acridines, linkers, and oligonucleotide sequences were obtained. These were tested for their ability to stabilize intramolecular DNA quadruplexes that are based on the human telomeric repeat sequence (GGGTTA)n.

INTRODUCTION Telomerase is an enzyme that extends telomere sequences after DNA replication, and it is active in most cancer cells, but not in somatic cells. The enzyme is a ribonucleoprotein that uses an RNA template to elongate the telomeric single-stranded overhang, which has the sequence (TTAGGG)n in humans (14). It has been hypothesized that folding of this G-rich strand into an intramolecular quadruplex prevents interaction with the complementary RNA of telomerase and consequently interferes with the elongation of telomeres by telomerase in cancer cells (5-11). Several classes of compounds, including anthraquinones, acridines, porphyrins, fluorenones, and other polycyclic aromatic molecules have been reported to bind to and stabilize DNA quadruplexes and possess telomerase-inhibiting properties (1218). However, these aromatic molecules also behave as DNA intercalators and interact with double-stranded DNA as well as with G-quadruplexes. As a result, their potential use as anticancer drugs is limited by their cytotoxicity. The parent aromatic compounds have been extensively modified to favor interaction with quadruplex rather than duplex structures (19-23). An alternative means of increasing the specificity might be to direct the aromatic drug to telomeric DNA by covalently attaching it to an oligonucleotide that is complementary to the G-rich telomere strand. This manuscript describes the preparation of a small library of such acridineoligonucleotide conjugates and examines their interaction with intramolecular DNA quadruplexes.

EXPERIMENTAL PROCEDURES 3,6-Bis(acryloylamide)acridine, 1. A suspension of 2.5 g (10 mmol) of proflavine hydrochloride in acryloyl chloride (70 mL) was refluxed for 4 h. Then, the solid was filtered and washed with ether (4 × 25 mL). Purification of this solid by silica gel column chromatography eluting with acetone and * Corresponding authors. (E.P.) Email: [email protected]. Phone: + 34 93 403 4824. (K.R.F.) Email [email protected]. Phone: +44 (0)23 8059 4374. Fax: +44 (0)23 8059 4459. † Universitat de Barcelona. ‡ University of Southampton.

increasing amounts of MeOH (0-30%) and TEA1 (0-5%) afforded 2.3 g of 3,6-bis(acryloylamide)-acridine (80% yield). Rf (acetone/TEA 8:2) ) 0.66. 1H NMR (DMSO-d6, 200 MHz) δ (ppm): 11.62 (s, 2H, NH), 9.50 (s, 1H, H-9), 8.88 (s, 2H, H-4,5), 8.28 (d, J ) 9.2 Hz, 2H, H-1,8), 7.90 (d, J ) 9.2 Hz, 2H, H-2,7), 6.70 (dd, Jtrans ) 17 Hz, Jcis ) 10 Hz, 2H, COCHAd CHBHC), 6.38 (dd, Jtrans ) 17 Hz, Jgem ) 1 Hz, 2H, COCHAd CHBHC), 5.90 (dd, Jcis ) 10 Hz, Jgem ) 1 Hz, 2H, COCHAd CHBHC). 13C-RMN (DMSO-d6, 63 MHz) δ (ppm): 160.8 (NHCO), 146.6 (C-9), 137.6 (C-11,12), 132.4 (C-3,6), 128.7 (CHd), 126.3 (dCH2), 124.8 (C-1,8), 119.7 (C-10,13), 117.3 (C-2,7), 111.6 (C-4,5). MALDI-TOF MS (positive mode, linear, DHB): m/z 318.72, [M + H]+, calcd mass for C19H15N3O2 317.34. General Procedure for the Synthesis of Derivatives 2. To a stirred solution of 1 in EtOH (250 mL/g of 1) was added dropwise a solution of the cyclic secondary amine (0.5 eq) in the same solvent (4 mL/mmol amine), and the mixture was reacted overnight (room temperature). The solvent was removed at reduced pressure, and the product was purified by silica gel column chromatography eluting with acetone and increasing amounts of MeOH (0-10%) and TEA (0-5%). 2a. 55% yield; Rf (DCM/MeOH 7:3) ) 0.23. 1H NMR (acetone-d6, 200 MHz) δ (ppm): 11.20 (s, 1H, NH), 9.88 (s, 1H, NH), 8.76 (s, 1H, H-9), 8.67 (1H, H-4), 8.54 (s, 1H, H-5), 8.01 (d, J ) 9 Hz, 2H, H-1,8), 7.78 (d, J ) 9 Hz, 1H, H-2), 7.65 (d, J ) 9 Hz, 1H, H-7), 6.47 (m, 2H, CHAdCHBHC), 5.77 1 ACN ) acetonitrile; Bz ) benzoyl; CNE ) 2-cyanoethyl; CPG ) controlled pore glass; DCC ) N,N′-dicyclohexylcarbodiimide; DCM ) dichloromethane; DHB ) dihydroxybenzoic acid; DIEA ) Nethyldiisopropylamine; DMF ) N,N-dimethylformamide; DMSO ) dimethylsulfoxide; DMT ) 4,4′-dimethoxytrityl; Fmoc ) 9-fluorenylmethoxycarbonyl; HATU ) N-[(dimethylamino)-1H-1,2,3-triazolo[4,5b]pyridin-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate N-oxide; HOBT ) 1-hydroxybenzotriazole; MALDI ) matrixassisted laser desorption ionization; MMT ) 4-methoxytrityl; NMP ) N-methylpyrrolidone; PFNB ) 1,2,3,4,5-pentafluorophenyl 4-nitrobenzenesulfonate; PS ) polystyrene-co-1%-divinylbenzene; PyAOP ) (7azabenzotriazol-1-yloxy)trispyrrolidinophosphonium hexafluoroposphate; PyBOP ) (benzotriazol-1-yloxy)trispyrrolidinophosphonium hexafluoroposphate; TCA ) trichloroacetic acid; TEA ) triethylamine; TFA ) trifluoroacetic acid; TOF ) time of flight.

10.1021/bc060194t CCC: $33.50 © 2006 American Chemical Society Published on Web 09/02/2006

1352 Bioconjugate Chem., Vol. 17, No. 5, 2006

(dd, Jcis ) 9 Hz, Jgem ) 1.8 Hz, CHAdCHBHC), 2.8-2.4 (m, 6H, COCH2CH2N(CH2CH2)2CH2), 2.2-1.9 (m, 2H, COCH2CH2N), 1.8-1.4 (m, 6H, N(CH2CH2)2CH2). 13C NMR (DMSOd6, 63 MHz) δ (ppm): 169.7 (NHCO-acryloyl), 164.4 (NHCOCH2), 149.9 (C-11), 141.6 (C-9), 141.3 (C-12), 136.1 (C1,8), 132.2 (C-3), 129.8 (C-6), 129.7 (CHdCH2), 128.2 (C10), 123.1 (CHdCH2), 123.0 (C-13), 122.9 (C-4), 120.7 (C-5), 114.7 (C-2), 113.8 (C-7), 63.7 (CH2N(CH2CH2)2CH2), 29.6 (COCH2), 24.3 (N(CH2CH2)2CH2), 19.8 (N(CH2CH2)2CH2). MALDI-TOF MS (positive mode, linear, DHB): m/z 403.72, [M + H]+, calcd mass for C24H26N4O2 402.49. 2b. 40% yield; Rf (DCM/MeOH 7:3) ) 0.1. 1H NMR (CD3OD, 200 MHz) δ (ppm): 8.78 (s, 1H, H-9), 8.59 (1H, H-4), 8.54 (s, 1H, H-5), 8.03 (d, J ) 9 Hz, 2H, H-1,8), 7.90 (d, J ) 9 Hz, 1H, H-2), 7.73 (d, J ) 9 Hz, 1H, H-7), 7.65 (m, 2H, CHAdCHBHC), 5.77 (dd, Jcis ) 9 Hz, Jgem ) 1.8 Hz, CHAd CHBHC), 3.01 (t, 2H, COCH2CH2N), 2.75 (m, 6H, COCH2CH2N(CH2CH2)2CH2), 1.9 (m, 6H, N(CH2CH2)2CH2). 13C NMR (DMSO-d6, 63 MHz) δ (ppm): 164.8 (NHCO-acryloyl, NHCOCH2), 147.3 (C-9), 139.1 (C-11,12), 135.1 (C-3,6), 129.8 (CHdCH2), 128.3 (CHdCH2), 127.6 (C-1,8), 122.0 (C-10,13), 119.0 (C-2,7), 112.8 (C-4,5), 67.2 (CH2N(CH2CH2)2), 52.8 (CH2N(CH2CH2)2), 50.3 (COCH2CH2N), 22.2 (N(CH2CH2)2). MALDI-TOF MS (positive mode, linear, DHB): m/z 390.13, [M + H]+, calcd mass for C23H24N4O2 388.46. General Procedure for the Synthesis of Asymmetrically Substituted Acridines 3. 2 (250-300 mg) was reacted with a 2- to 3-fold molar excess of either L-proline or isonipecotic acid in MeOH (1 mL/10 mg of 2, approximately) for 6 h at room temperature (rt). Removal of the solvent in vacuo was followed by chromatographic purification. 3a. Purification by silica gel column chromatography eluting with acetone and increasing amounts of MeOH (0-20%) and TEA (0-5%); 62% yield; anal HPLC (C8 column, gradient from 5% to 35% of B in 15 min, A ) 0.045% TFA in H2O, B ) 0.036% of TFA in ACN): tR ) 9.2 min. 1H NMR (CD3OD, 200 MHz) δ (ppm): 8.55 (s, 1H, H-9), 8.29 (s, 1H, H-4), 8.26 (s, 1H, H-5), 7.9-7.7 (m, 2H, H-1,8), 7.7-7.5 (m, 2H, H-2,7), 4.32 (t, J ) 4.4 Hz, 1H, NCHCOOH), 4.08-3.80 (m, 2H, CH2NCH(COOH)CH2), 3.3-2.8 (m, 10H, CH2N). 13C NMR (CD3OD, 63 MHz) δ (ppm): 175.3 (COOH), 171.2 and 170.1 (NHCO), 150.2 (C-9), 141.1 (C-11,12), 140.2 (C-3,6), 129.7 (C-1,8), 123.0 (C-10,13), 120.7 (C-5,4), 114.5 (C-2,7), 68.2 (NCHCOOH), 54.4 (COCH2CH2NCH2), 54.0 (N(CH2)2), 50.6 (CH2NHCOOH), 43.0 (CH2N(CH2)2), 34.2 (COCH2CH2NHCOOH), 29.5 (COCH2CH2N(CH2)2), 25.6 (NCHCOOHCH2), 24.0 (NCH2CH2CH2CHCOOH), 23.9 (N(CH2CH2)2). MALDI-TOF MS (positive mode, linear, DHB): m/z 519.15 [M + H]+, calcd mass for C29H35N5O4 517.62. 3b. Purification by reversed-phase medium-pressure liquid cromatography on a C18-filled column, gradient from 0% to 35% of B (A ) 0.1% TFA in H2O, B ) 0.1% TFA in ACN); 80% yield; anal HPLC (C8 column, gradient from 5% to 35% of B in 15 min, A ) 0.045% TFA in H2O, B ) 0.0036% of TFA in ACN): tR ) 8.0 min. 1H NMR (CD3OD, 200 MHz) δ (ppm): 9.44 (s, 1H, H-9), 8.87 (m, 2H, H-4,5), 8.32 (d, J ) 3.8 Hz, 1H, H-1), 8.27 (d, J ) 3.8 Hz, 1H, H-8), 7.66-7.74 (m, 2H, H2-7), 4.33 (dd, J1 ) 9.4 Hz, J2 ) 6.2 Hz, 1H, NCHCOOH), 3.6-3.94 (m, 10H, CH2N), 3.07-3.15 (m, 4H, COCH2CH2N), 2.48-2.67 (m, 2H, NCH2CH2CH2CHCOOH), 2.00-2.30 (m, 6H, NCH2CH2). 13C NMR (CD3OD, 63 MHz) δ (ppm): 168.1 (COOH), 167.3 and 167.0 (NHCO), 143.9 (C9), 143.6 (C-11,12), 139.9 (C-3,6), 128.8 (C-1,8), 119.9 (C10,13), 118.6 (C-5,4), 102.0 (C-2,7), 65.9 (NCHCOOH), 52.8 (COCH2CH2NCH2), 52.0 (N(CH2)2), 48.3 (CH2N(CH2)2), 47.8 (CH2NHCOOH), 30.2 (COCH2CH2NHCOOH), 30.0 (COCH2CH2N(CH2)2), 26.0 (NCHCOOHCH2), 20.5 (NCH2CH2CH2-

Casals et al.

CHCOOH), 20.4 (N(CH2CH2)2). MALDI-TOF MS (positive mode, linear, DHB): m/z 504.49 [M + H]+, calcd mass for C28H33N5O4 503.59. 3c. Purification by reversed-phase medium-pressure liquid cromatography on a C18-filled column, gradient from 5% to 50% of B (A ) 0.1% TFA in H2O, B ) 0.1% TFA in ACN); 94% yield; anal HPLC (C8 column, gradient from 5% to 35% of B in 15 min, A ) 0.045% TFA in H2O, B ) 0.0036% of TFA in ACN): tR ) 9.1 min. 1H NMR (CD3OD, 200 MHz) δ (ppm): 9.48 (s, 1H, H-9), 8.62 (s, 2H, H-4,5), 8.37 (d, J ) 9.3 Hz, 2H, H-1,8), 7.72 (dd, J1 ) 9.3 Hz, J2 ) 1.5 Hz, 2H, H-2,7), 3.75-3.85 (m, 8H, N(CH2CH2)2), 3.3-3.4 (m, 4H, COCH2CH2N), 3.1-3.3 (m, 4H, COCH2CH2N), 2.9 (m, 1H, CHCOOH), 1.92.5 (m, 8H, N(CH2CH2)2). 13C NMR (D2O, 63 MHz) δ (ppm): 176.5 (COOH), 169.6 and 169.4 (NHCO), 144.3 (C-9), 144.2 (C-11,12), 139.0 (C-3,6), 130.0 (C-1,8), 120.2 (C-10,13), 119.9 (C-5,4), 102.9 (C-2,7), 53.8 (CHCOOH), 51.7 (N(CH2CH2)2CHCOOH), 51.3 (N(CH2CH2)2), 49.5 (CH2N(CH2CH2)2CHCOOH), 49.3 (CH2N(CH2CH2)2), 37.5 (COCH2CH2N), 24.8 (N(CH2CH2)2CHCOOH), 22.0 (N(CH2CH2)2). MALDI-TOF MS (positive mode, linear, DHB): m/z 518.17 [M + H]+, calcd mass for C29H35N5O4 517.62. 3,6-Bis[3-N-(L-prolyl)propionamide]acridine, 3d. To a solution of 250 mg (0.78 mmol) of 1 in EtOH (200 mL) was added a solution of L-proline (907 mg, 10 equiv) in the minimal amount of MeOH, and the mixture was stirred for 24 h at rt. Removal of the solvent was followed by HPLC purification using a preparative C4 column (gradient from 10% to 40% of B in 1 h, A ) 10 mM NH4AcO, B ) ACN). 46% yield; anal HPLC (C8 column, gradient from 10% to 35% of B in 20 min, A ) 0.045% TFA in H2O, B ) 0.036% TFA in ACN): tR ) 11.9 min. 1H NMR (CD3OD, 200 MHz) δ (ppm): 8.73 (s, 1H, H-9), 8.33 (s, 2H, H-4,5), 7.95 (d, J ) 9 Hz, 2H, H-1-8), 7.68 (dd, J1 ) 9 Hz, J2 ) 1.8 Hz, 2H, H-2,7), 4.10 (m, 2H, NCHCOOH), 3.954.01 (m, 2H, COCH2CH2NCH2), 3.70 (t, J ) 6 Hz, 4H, COCH2CH2N), 3.27-3.34 (m, 2H, COCH2CH2NCH2), 3.09 (t, J ) 6 Hz, 4H, COCH2CH2N), 2.00-2.60 (m, 8H, NCH2CH2CH2CHCOOH). 13C NMR (CD3OD, 63 MHz) δ (ppm): 171.0 (COOH), 169.6 (NHCO), 150.0 (C-9), 140.9 (C-11,12), 135.8 (C-3,6), 129.7 (C-1,8), 123.0 (C-5,4), 120.7 (C-10,13), 114.6 (C-2,7), 69.0 (NCHCOOH), 54.8 (COCH2CH2NCH2), 50.8 (COCH2CH2N), 33.4 (COCH2CH2N), 29.5 (NCH(COOH)CH2), 23.9 (NCH2CH2CH2CHCOOH). MALDI-TOF MS (positive mode, linear, DHB): m/z 549.76 [M + H]+, calcd mass for C29H33N5O6 547.60. HO-[protected oligonucleotide]-Resin, 7. The aminesubstituted polystyrene-1%-divinylbenzene support suitable for oligonucleotide synthesis (loading below 0.25 mmol/g) was obtained by partial acylation of the amine groups of pmethylbenzhydrylamine resin (0.70 mmol/g). This was achieved by reaction with 0.35 equiv of Fmoc-alanine and DCC in a DCM/DMF mixture (9:1) for 1 h at rt. The resin was then filtered and washed with DMF, DCM, and MeOH and dried overnight in a desiccator. An aliquot of Fmoc-Ala-resin was treated with 20% piperidine in DMF to remove the Fmoc group, and the amount of 9-fluorenylmethylpiperidine formed was quantified spectrophotometrically. The loading was 0.17 mmol/ g, suitable for oligonucleotide synthesis. The resin was swelled in DCM/DMF, and the remaining free amines were capped by acetylation (reaction with Ac2O for 10 min + 10 additional min after the addition of DIEA, 10 equiv of each reagent). The Fmoc group of the total of Fmoc-Ala-resin was removed by treatment with 20% piperidine/DMF (3 + 10 min, followed by DMF washings). Nucleotide resins were synthesized by reacting 5′-O-DMTT-succinate or 5′-O-DMT-dABz-succinate (3 equiv, previously prepared by reaction between the DMT-protected nucleoside and succinic anhydride in pyridine) (24) with H-Ala-resin in

Directing Quadruplex-Stabilizing Drugs to the Telomere

the presence of DCC (3 equiv) for 4 h at room temperature in a DCM/DMF mixture. The absence of unreacted amines was assessed by the ninhydrin test (25). DMT-T-Succinate. Rf (DCM/MeOH 9:1) ) 0.1. 1H NMR (CDCl3, 200 MHz) δ (ppm): 9.8 (s, 1H, NH), 7.59 (s, 1H, H-6), 6.6-7.4 (m, 14H, Ar-H), 6.38 (t, 1H, H-1′), 5.45 (m, 1H, H-3′), 4.18 (m, 1H, H-4′), 3.77 (s, 6H, Ar-OCH3), 3.44 (m, 2H, H-5′/ 5′′), 2.2-2.8 (m, 6H, OCOCH2CH2COOH, H-2′/2′′), 1.36 (s, 3H, Ar-CH3). DMT-dABz-Succinate. Rf (DCM/MeOH 9:1) ) 0.1. 1H NMR (CDCl3, 200 MHz) δ (ppm): 8.68 (s, 1H, H-8), 8.15 (s, 1H, H-2), 6.98-7.4 (m, 14H, Ar-H), 6.40 (t, 1H, H-1′), 5.42 (m, 1H, H-3′), 4.22 (m, 1H, H-4′), 3.66 (s, 6H, Ar-OCH3), 3.36 (m, 2H, H-5′/5′′), 2.50-3.00 (m, 6H, OCOCH2CH2COOH, H-2′/2′′). Oligonucleotides were automatically assembled (DNA synthesizer: ABI 380B) on aliquots of H-Ala-resin at the 5-µmol scale, using phosphite triester procedures slightly modified for compatibility with polystyrene supports (26, 27) and the 5′-ODMT-3′-O-phosphoramide derivatives of dT, dABz, and dCAc. H2N-Linker-Oligonucleotide-Resin, 8. The 5′ end of the resin-linked oligonucleotide was derivatized by reaction with the commercially available phosphoramidite derivatives of amine-protected linkers L3 (see below), L5, and L6 (MMTNH-linker-O(OCNE)NiPr2; see structures in Scheme 3) using standard phosphite triester methodology. For the preparation of conjugate 4f, linker L18 was first introduced by reaction of the corresponding oligonucleotide resin with commercially available DMT-[O-(CH2)2]6-O-P(OCNE)NiPr2, and this was followed by incorporation of the L5-containing derivative. Assembly of the target linker-oligonucleotide was assessed, in all cases, by treating aliquots of the linker-oligonucleotide-resin with concentrated aqueous ammonia for 6 h at room temperature and analysis of the crude by MALDI-TOF MS (negative mode). MMT-NH-CH2-CH2-CH2-OH. 3-Aminepropan-1-ol (7 mL, 93 mmol) was reacted with MMT-Cl (2 g, 0.1 equiv) in anhydrous pyridine for 3 days (room temperature). The solvent was removed at reduced pressure, and the crude was dissolved in DCM and washed with a 10% aq citric acid solution and brine. Purification was accomplished by silica gel column chromatography, eluting with hexanes and increasing amounts of DCM (5-100%) in the presence of 2% TEA (58% yield). Rf (DCM/hexanes/TEA 79:19:2) ) 0.4. 1H NMR (CDCl3, 200 MHz) δ (ppm): 6.77-7.44 (m, 14H, Ar-H), 3.88 (t, J ) 5.2 Hz, 2H, CH2OH), 3.78 (s, 1H, OH), 2.40 (t, J ) 5.2 Hz, 2H, NHCH2), 1.7 (m, J ) 5.2 Hz, 2H, CH2CH2CH2); anal HPLC tR ) 12.8 min (C18, gradient from 50% to 100% of B in 30 min; A, 10 mM NH4OAc in H2O; B, ACN). MMT-NH-L3-P(OCNE)NiPr2. Phosphitylation of the free hydroxyl group of MMT-NH-CH2-CH2-CH2-OH was accomplished by overnight reaction with P(OCNE)(NiPr2)2 (3 equiv) and tetrazole (0.7 equiv) in anhydrous ACN (room temperature). After removal of the solvent, the product was dissolved in DCM, washed with saturated aq NaHCO3 and brine, dried and evaporated to dryness, and used without further purification (31P NMR (CDCl3, 120 MHz) δ 144.9 ppm); anal HPLC tR ) 28.6 min (C18, gradient from 50% to 100% of B in 30 min; A, 10 mM NH4OAc pH ) 7; B, ACN). Synthesis of the Conjugates 4. 1,2,3,4,5-pentafluorophenyl 4-nitrobenzenesulfonate was prepared as previously described (28). The MMT-NH-linker-oligonucleotide-resin (5 µmol) was transferred from the automatic DNA synthesizer reactor into a 5-mL polypropylene syringe fitted with a polyethylene disc. The MMT group was removed by subsequent treatments with the standard detritylation solution (3% TCA/DCM) until no color corresponding to the monomethoxytrityl cation was observed, which was followed by washing with DCM.

Bioconjugate Chem., Vol. 17, No. 5, 2006 1353

A mixture of the carboxyl-derivatized acridine (3) to be coupled (5 equiv with respect to resin-bound amines), PFNB (5 equiv), DIEA (25 equiv), and a catalytic amount of HOBt was dissolved in 250 µL of a 0.8 M solution of LiCl in DMF/ NMP, and, after 15 min for carboxyl group preactivation, it was added to the linker-oligonucleotide-resin. After 2.5 h at room temperature with stirring, the resin was filtered, washed (DMF, DCM, and MeOH) and dried, and treated with 2 mL of a 1:1 mixture of concentrated aqueous NH3/dioxane for 6 h at room temperature. The filtrate and washings (H2O, MeOH) were collected, and the acridine-oligonucleotide conjugate was purified by reversed-phase medium-pressure liquid chromatography (Vydac C18, 20 × 2 cm column, gradient from 0% to 100% of B, A ) 10 mM NH4AcO, B ) [65% (10 mM NH4AcO) + 35% (ACN/H2O 1:1)], 600 mL of each solvent). Homogeneity of the collected fractions was assessed by reversed-phase HPLC analysis (C18, gradient from 5% to 35% of B in 15 min, A ) 10 mM aq NH4AcO, B ) ACN/H2O 1:1), and characterization was carried out by MALDI-TOF MS (negative mode, reflector, 2,4,6-trihydroxyacetophenone). 4a. 11% yield; tR: 13.7 min, m/z 3007.44 [M - H]- (calcd mass for the neutral molecule, C111H146N32O52P8 3008.32). 4b. 14% yield; tR: 12.1 min, m/z 2980.10 [M - H]- (calcd mass for the neutral molecule, C108H140N32O53P8 2982.24). 4c. 2% yield; tR: 11.2 min, m/z 3023.77 [M - H]- (calcd mass for the neutral molecule, C109H140N32O55P8 3026.25). 4d. 9% yield; tR: 11.4 min, m/z 2993.18 [M - H]- (calcd mass for the neutral molecule, C109H142N32O53P8 2996.27). 4e. 1% yield; tR: 12.4 min, m/z 2964.64 [M - H]- (calcd mass for the neutral molecule, C108H140N32O52P8 2966.24). 4f. 31% yield; tR: 12.6 min, m/z 3319.97 [M - H]- (calcd mass for the neutral molecule, C120H165N32O62P9 3326.54). 4g. 2% yield; tR: 12.5 min, m/z 2994.81 [M - H]- (calcd mass for the neutral molecule, C109H141N31O54P8 2997.25). 4h. 1% yield; tR: 11.3 min, m/z 3027.48 [M - H]- (calcd mass for the neutral molecule, C110H141N37O50P8 3029.31). 4i. 2% yield; tR: 11.9 min, m/z 3013.28 [M - H]- (calcd mass for the neutral molecule, C109H139N37O50P8 3015.28). Fluorescence Melting Experiments. Fluorescence melting curves were determined using a Roche LightCycler as previously described (29-31) with excitation and detection wavelengths of 488 and 520 nm, respectively. The final concentration of the intramolecular quadruplex-forming oligonucleotide was 0.25 µM, in a total volume of 20 µL, dissolved in 50 mM potassium phosphate pH 7.4. Samples were first denatured by heating from 30 to 95 °C at a rate of 0.1 °C.s-1. These were maintained at 95 °C for 5 min and annealed by cooling to 30 °C at 0.1 °C per second. The fluorescence was recorded during both the melting and annealing phases. Although the slowest heating rate for the LightCycler is 0.1 °C.s-1, curves at lower rates of heating were obtained by raising the temperature in 1 °C increments, leaving the samples for 2 min between each temperature rise.

RESULTS AND DISCUSSION Synthesis of the Acridine-Oligonucleotide Conjugates. Conjugates were designed so as to have the general structure shown in Scheme 1a. The aromatic moiety of the conjugates was expected to stabilize G-quadruplex structures and promote their formation, while the oligonucleotide chain was added to impart selectivity for the telomeric region. Acridines were chosen for the aromatic moiety of the conjugate since, when this work started, some of the most effective telomerase inhibitors reported were derivatives of 3,6-diaminoacridine (proflavin) with the two amines acylated with 3-(dialkylamine)propionyl groups. The commercial availability and low price of proflavin also made it an advantageous starting material. We


Casals et al.

Scheme 1. Schematic Representation of Acridine-Oligonucleotide Conjugates (a), and Retrosynthetic Analysis (b)

Scheme 2. Synthesis of the Asymmetrically Substituted Acridine Derivatives

reasoned that it would be convenient to keep the same basic structure as that of the acridine derivatives that have been shown to be telomerase inhibitors (14). Although the two amines of proflavin have to be transformed into 3-(dialkylamino)propionamides, only one of these groups has to be derivatized to allow conjugation to the linker-oligonucleotide (Scheme 1b). Commercially available acridine phosphoramidite derivatives could not be used since their acridine rings are not suitably modified, while acridines that exhibit telomerase-inhibiting properties typically possess alkylamino side chains. The conjugates were synthesized taking advantage of solidphase procedures. Thus, the oligonucleotide was assembled, and its free end was modified with a suitable linker on a solid support, where the key conjugation was also carried out. An amide bond seemed a sensible choice to covalently link the two moieties. Amides are stable and can be obtained by exploiting a variety of reagents developed for peptide synthesis. In addition, amine groups are easy to add to oligonucleotide chains.

The synthesis of the asymmetrically substituted acridines was not straightforward. Attempts based on the selective protection of one of the amines of proflavin either failed or afforded the final product in very low yields. The acridines used in the conjugation reactions were synthesized from 3,6-diacryloylproflavin (1), which was prepared by reacting proflavin with an excess of acryloyl chloride (Scheme 2). The symmetry of the system was broken with the first Michael addition. Reaction of 3,6-diacryloylproflavine with piperidine or pyrrolidine afforded 3-(3-piperidinopropionyl)- or 3-(3-pyrrolidinopropionyl)6-acryloylproflavin (2a or 2b), respectively. The target carboxylderivatized acridines, 3, were obtained after addition of proline or isonipecotic acid (isonipecotic ) 4-piperidinecarboxylic) to 2. Treatment of 3,6-diacryloylproflavin with an excess of proline afforded the symmetrical biscarboxylate 3d. Three oligonucleotide sequences were assembled using the standard phosphite triester methodology (phosphoramidite derivatives of dT, dABz, and dCBz). Two were complementary to



Scheme 3. Side Reactions Taking Place at the Final Deprotection Step when the Ammonia Treatment Was Carried Out at 55 °C, Shown Here for Conjugate 4a

Scheme 4. Synthesis Scheme for the Preparation of the Conjugates

the single-stranded telomeric DNA (dCTAACCCT and dAACCCTAA) and the third, which contained the same bases randomized, was used as a control (dTCACTCAT). The 5′ end of resin-linked oligonucleotides was subsequently derivatized with an amine linker. The linker had to be long and flexible enough to allow the acridine to interact with the quadruplex structure and the oligonucleotide to hybridize with the singlestranded telomeric DNA. Linkers of different lengths containing either alkyl or ethylene glycol chains were used. The latter, more polar linkers were expected to facilitate the approach of the conjugate to the polyanionic DNA chains. In the first conjugation experiments, the carboxyl group of the acridine derivative was activated using either N,N′-diisopropylcarbodiimide and 1-hydroxybenzotriazole, (benzotriazol1-yloxy)trispyrrolidinophosphonium hexafluoroposphate (PyBOP) in the presence of DIEA, or (7-azabenzotriazol-1-yloxy)-

trispyrrolidinophosphonium hexafluoroposphate (PyAOP) and DIEA. Although a large excess of acylating reagent was reacted with the H2N-linker-oligonucleotide-CPG (2 h, rt), coupling yields were either 0% or below 10%. Incorporation of the acridine moiety was controlled by treating an aliquot of conjugate-resin with concentrated aqueous ammonia and analyzing the crude by reversed-phase HPLC and MALDI-TOF MS. Detection of linker-oligonucleotide and/or acetyl-linker-oligonucleotide indicated that the coupling had not gone to completion. The origin of the acetyl-linkeroligonucleotide was not clear, but acetylation of amines has also been found to take place during the elongation of peptides on resin-linked oligonucleotides (32). These experiments also showed that the deprotection conditions had to be fine-tuned. Since standard nucleobase protecting groups (ABz, CBz) had been used for oligonucleotide assembly,


Casals et al.

Table 1.

deprotection was carried out by overnight reaction with concentrated aqueous ammonia at 55 °C. Analysis of the crude product showed that the target conjugate (4a) was accompanied by a conjugate (5) in which the piperidine ring had been replaced by an amine group and proline-linker-oligonucleotide conjugate (6). 5 and 6 derive from the two possible reversed Michael reactions, with subsequent addition of ammonia to the acryloyl group in the case of 5 (Scheme 3). Since we have previously obtained the best results in the synthesis of nucleopeptides and peptide-oligonucleotide conjugates using a polystyrene-1%-divinylbenzene copolymer rather than controlled pore glass (33, 34), the linker-oligonucleotide moiety was resynthesized on a polystyrene matrix, and the PyAOP-mediated acylation reaction was repeated in the same conditions as on controlled pore glass. The yield of the conjugation step was slightly higher (17%) and somewhat better when N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate Noxide (HATU) was used to activate the carboxyl group (28% after 4 h). 1,2,3,4,5-Pentafluorophenyl 4-nitrobenzenesulfonate (28, 35) was the reagent that finally allowed the conjugation reaction to attain acceptable yields (50-55%; for conditions, see below). Finally, the target conjugates (4) were synthesized using the procedure outlined in Scheme 4. Oligonucleotides were assembled on an amine-derivatized polystyrene-1%-divinylbenzene support, replacing the phosphoramidite derivative of CBz by that of CAc. Linkers L3, L5, and L6 (see structures in Table 1) were then introduced by coupling the corresponding monomethoxytritylamine phosphoramidite derivatives to the free 5′-hydroxyl group of the oligonucleotide (7), followed by capping and oxidation of the phosphite to phosphate. The longest linker, L5-L18, was introduced by subsequently coupling a hydroxyl-protected phosphoramidite (L18) and an amineprotected phosphoramidite (L5). The monomethoxytrityl amineprotecting group was removed using the same mild acid reagent commonly used to eliminate the DMT groups, and the carboxylderivatized acridine moiety was coupled to 8 by reaction with PFNB and DIEA (molar ratio with respect to polymer-bound amine groups 10:10:25), in the presence of catalytic amounts of HOBt. A 0.8 M solution of LiCl in a 1:1 mixture of N,Ndimethylformamide and N-methylpyrrolidone was used as the solvent, and the reaction was left to proceed for 4 h at room

temperature. In spite of using these conditions, coupling yields remained below the expected value in some conjugation reactions. Permanent protecting groups were removed by treatment of the conjugate resin with a 1:1 ammonia/dioxane mixture for 6 h at room temperature. In these conditions, the presence of impurities such as 5 and 6 was virtually nonexistant. Conjugates were purified by reversed-phase liquid chromatography and characterized by MALDI-TOF mass spectrometric analysis. Interaction with Intramolecular Quadruplexes. We examined the interaction of these acridine-oligonucleotide conjugates with quadruplex-forming oligonucleotides that contain the human telomeric repeat sequences (GGGTTA)n. These model oligonucleotides had to be designed to allow the formation of quadruplex structures as well as hybridization of the oligonucleotide portion of the conjugate. The quadruplexforming sequence has to contain at least four GGG repeats, so the basic (GGGTTA)3GGG sequence was extended to offer a single-stranded arm suitable for duplex formation with the conjugates. Information on quadruplex stabilization by the conjugates was obtained from thermal denaturation studies, in which an increase in the melting temperature of the quadruplex indicates a stabilizing effect. The thermal stability of the quadruplex was evaluated using a fluorescence-based method (29) which allows melting temperatures to be determined with high sensitivity using a small amount of material. In these experiments, the quadruplex-forming oligonucleotides are labeled at one end (5′) with a fluorophore (F, fluorescein) and at the other (3′) end with a quencher (Q, methyl red). When the quadruplex is folded, these groups are in close proximity, and the fluorescence is quenched. When the quadruplex melts, these groups become separated, and there is a large increase in fluorescence. Conjugate-oligonucleotide interactions were studied using three different model oligonucleotides containing 5 or 6 GGG tracts: F-TGGGTTAGGGTTAGGGTTAGGGTTAGGG-Q (OL1), F-TGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGQ (OL2), and AGGGTTAGTTTT-Q-GGGTTAGGGTTAGGGTTAGGG-Q (OL3), in which F and Q stand for the fluorophore and the quencher, respectively. OL1 and OL2 can adopt different structures, depending on which G tracts are part of the folded quadruplex and which are in the single-stranded overhang (Scheme 5). For OL1, only one of the structures



Figure 1. Fluorescence melting curves showing the effects of oligonucleotide-acridine conjugates (10 µM) on the melting of OL1 (left-hand panel) and OL3 (right-hand panel). The quadruplex concentration was 0.25 µM in each case, and the reactions were performed in 50 mM potassium phosphate pH 7.4. For OL1, the heating rate was 0.1 °C.s-1; this was reduced to 0.5 °C.min-1 for OL3. Scheme 5. Possible Structures for the Intramolecular Quadruplexes Adopted by (a) OL1, (b) OL2, and (c) OL3

(OL1a, Scheme 5a) will allow the oligonucleotide moiety of the conjugates to interact with the complementary telomeric sequence. Two of the conformations of OL2 (OL2a and OL2b, Scheme 5b) are appropriate for interaction with the acridine conjugates. OL3 was designed so that only one quadruplex structure can be formed (Scheme 5c), where the “core” quadruplex system and the single-stranded telomeric sequence are more separated than in the other model oligonucleotides. In these melting experiments, the quadruplex-forming oligonucleotide concentration was 0.25 µM, and the concentration of the conjugates ranged between 2 and 10 µM. Representative melting curves in the presence and absence of different conjugates are shown in Figure 1. The changes in the melting temperatures of the quadruplexes (∆Tm) produced by the conjugates are summarized in Table 2. Examination of the data in Table 2 shows that all the conjugates stabilize quadruplex structures, and the increases in melting temperature (Tm) depend on their concentration, as

Table 2. G-quadruplex-forming oligonucleotides OL1 conjugate 4a 4b 4c 4d 4e 4f 4g 4h 4i

10 µM

OL2 2 µM

5 µM

4.3 3.8

1

2

5.1

0.5

2

4.7 3.2 2.6 2.7

1.5 1

OL3 10 µM

2 µM

5 µM

10 µM

3 1 0.5 4 0.5 4.5 1.5 2 0.5

2 1.5

3.5 3

2

3

2 1.5 1.5

3 2.5 2.5 1.5

4 3.5 2 6.5 0.5 4.5 4 5.5 2

expected. Inspection of the melting profiles of oligonucleotide OL1 (Figure 1) shows that, in some cases, the transition between the folded G-quadruplex and the single-stranded form is accompanied by another transition, with a reversed profile at lower temperatures. This curve corresponds to melting of the


duplex that is formed between the conjugated oligonucleotide and the single-stranded arm of OL1. Its reversed profile is explained by suggesting that hybridization shields the fluorophore and prevents energy transfer to the quencher. This curve was not observed when thermal denaturation experiments were carried out with OL1 either alone, as expected, or in the presence of conjugates 4e, 4f, and 4g. The oligonucleotide sequence in 4g is not complementary to that of the telomere; this conjugate was synthesized as a control, and as expected, it does not display the reverse profile at low temperatures. Duplex formation does not occur with conjugates 4e and 4f. Since the main difference between these and the other conjugates is the length of the spacer linking the aromatic and oligonucleotide moieties of the conjugate, we surmise that duplex formation is hampered by the shortest and longest linkers. Linkers L5 and L6 (8 and 9 atoms long, respectively) thus seem to be the best for allowing duplex formation. Similar results were found with OL2 with respect to duplex formation. In OL3, the relative position of the fluorophore and the quencher is different and is such that melting of the duplex is not observed. Since the main purpose of this work was to add telomerehybridizing properties to quadruplex-stabilizing molecules, these results confirm that our goal has been achieved. Our acridineoligonucleotide conjugates can interact both with quadruplex structures and with the single-stranded telomeric sequence. From a quantitative point of view, the quadruplex stabilization provided by the conjugates is not very high. Nevertheless, it has to be taken into consideration that OL1 and OL2 exist in equilibrium between three or four different structures (see Scheme 5). The Tm data therefore represent averages of the interaction with the different conformations, some of which cannot bind the conjugates. Only OL3 adopts a single quadruplex structure, and the highest ∆Tms were observed between this oligonucleotide and conjugates 4d and 4h. The complexes formed by the other conjugates and OL3 displayed similar or slightly higher than those formed with OL1, which are higher than those formed with OL2. These results suggest that the apparent stabilization of the quadruplex by the acridine conjugate improves if the number of available conformations is lower.

CONCLUSIONS We have developed a methodology for synthesizing a small library of acridine-oligonucleotide conjugates, making use of acylation and Michael reactions to modify the aromatic moiety, and of amide formation for the conjugation reaction. Conditions for the final deprotection of the conjugate were also established. The thermal denaturation experiments demonstrated that the acridine ring of the conjugates interacts with and stabilizes the quadruplex structures. The conjugated oligonucleotide hybridizes with the complementary single-stranded telomeric DNA to impart selectivity to the aromatic moiety. The best results were obtained with conjugate 4d, in which the acridine ring is flanked by pyrrolidine and isonipecotic acid units and linked to oligonucleotide CTAACCCT by a bis(ethylene glycol) linker (L5).

ACKNOWLEDGMENT This work was supported by funds from the European Union (HPRN-CT-2002-00175), the Spanish Ministerio de Educacioń y Ciencia (grant CTQ2004-8275-C02-01), and the Generalitat de Catalunya (2005SGR-693 and Centre de Refere`ncia de Biotecnologia).

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