Oligonucleotides Incorporating 7-(Aminoalkynyl)-7-deaza-2

The thermal dissociation/association of the oligomers was measured by ... The sample preparation was performed on Scout MTP MALDI targets (Bruker) as ...
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1274

Bioconjugate Chem. 2002, 13, 1274−1285

Oligonucleotides Incorporating 7-(Aminoalkynyl)-7-deaza-2′-deoxyguanosines: Duplex Stability and Phosphodiester Hydrolysis by Exonucleases† Helmut Rosemeyer, Natalya Ramzaeva, Eva-Maria Becker, Elisabeth Feiling, and Frank Seela* Laboratorium fu¨r Organische und Bioorganische Chemie, Institut fu¨r Chemie, Fachbereich Biologie/Chemie, Universita¨t Osnabru¨ck, Barbarastrasse 7, D-49069 Osnabru¨ck, Germany. Received March 28, 2002; Revised Manuscript Received July 3, 2002

Oligonucleotides containing 7-(ω-aminoalkyn-1-yl)-7-deaza-2′-deoxyguanosines (1a-c) were investigated regarding their thermal stability (Tm values) as well as their phosphodiester hydrolysis catalyzed by exonucleases. Those derivatives are suitable for the labeling of nucleic acid constituents as well as for the postlabeling of DNA. For this, the phosphoramidites 7a,c (obtained from the nucleoside 1a,b), protected by an isobutyryl group at the 2-amino group and a phthaloyl residue at the side-chain amino function, were synthesized. Using compounds 7a,c together with the phosphoramidite of 1c in solidphase synthesis, a series of self-complementary and non-self-complementary oligonucleotides were prepared and characterized by MALDI-TOF mass spectrometry. A comparison of the Tm values of the modified oligomers shows that the thermal stability of the duplexes decreases with the length of the nucleobase 7-(ω-aminoalkyn-1-yl) side chain. Exonucleolytic cleavage of oligonucleotide single strands incorporating either the 7-(3-aminopropyn-1-yl)- or the 7-(4-aminobutyn-1-yl)-substituted nucleosides 1a or 1b, respectively, reveals that 3′ f 5′ specific snake venom phosphodiesterase liberates 1a 5′monophosphate but not the methylene-extended 1b 5′-monophosphate. On the contrary, the 5′ f 3′ specific bovine spleen exonuclease is able to cleave off single 1a and 1b 3′-monophosphate residues; its action is, however, terminated in the case of oligonucleotides containing two consecutive 1a or 1b nucleotide units.

INTRODUCTION

The introduction of aminoalkynyl, aminoalkenyl, and aminoalkyl groups to pyrimidine and purine constituents of DNA has a major impact on its structure and stability. These groups can be used in particular for the conjugation of a DNA with reporter groups (Randolph and Waggoner, 1997; Ramzaeva et al., 2000; Cook et al., 1988; Ramasamy et al., 1994; Takeda et al., 1987; Ono et al., 1994; Haginoya et al., 1997). The incorporation of such cationic groups into a DNA or RNA leads to a partial neutralization of the phosphate negative charge (Hashimoto et al., 1993; Ramzaeva et al., 1997; Seela and Zulauf, 1999; Seela et al., 2001b; Bijapur et al., 1999; Soto et al., 2001). Interestingly, nature has implemented a design of this general type in bacteriophage DNA where half of the thymidine residues are replaced by positively charged R-putrescinylthymidine () 5-{[(4-aminobutyl)amino]methyl}thymidine), resulting in one positively charged base for every eight nucleotides on average (Miller Black et al., 1985; Kropinski et al., 1973). On the basis of the proposition of a neutralization-collaps model by Manning and Rich (Manning, 1978; Manning et al., 1989), Maher and co-workers showed that the introduction of tethered cationic groups into a DNA in phase with the helical turn leads inevitably to an intrinsic curvature of the nucleic acid (Strauss et al. 1996; Williams and Maher, 2000; Hardwidge, 2001 and literature cited therein). This * To whom correspondence should be addressed. Phone +49541-9692791. Fax: +49-541-9692370. E-mail: [email protected]. † Dedicated to Prof. Dr. Dr. h. c. Wolfgang Pfleiderer, Konstanz, Germany, on the occasion of his 75th birthday.

is due to the partial neutralization of the negatively charged sugar-phosphate backbone leading to an asymmetric decrease in phosphate-phosphate charge repulsions. The latter causes a bias of the opposing but normaly balanced forces of compression (e.g. base stacking, van der Waals contacts) and stretching (e.g. interphosphate Coulombic repulsion) rendering an oligonucleotide to a stiff rod with elastic resilience. A further impact concerns the incorporation of charge tags into nucleic acids (DNA and RNA) in order to enhance their detectability in MALDI-TOF mass spectrometry. It has been shown that the incorporation of already one charge into a DNA molecule enhances the sensitivity by which it can be detected by a factor of 100 (Wenzel et al., 2001; Gut and Beck, 1995). This result has recently found application in the development of a novel procedure for efficient genotyping of single nucleotide polymorphism (‘GOOD’ assay) (Sauer et al. 2000). Apart from that, D. M. Williams and co-workers (Lee et al., 2001, Gorlain et al., 2001) used C(7)-modified 7-deaza-dATP derivatives with pendant 3-aminopropyl, (Z)-3-aminopropenyl, and 3-aminopropynyl side chains together with a C(5)-imidazolyl-modified dUTP as substrates for Taq polymerase during PCR and demonstrated the simultaneous incorporation of both functionalities into a DNA molecule. Cleavage of the modified oligonucleotide by restriction endonucleases, however, failed. In this manuscript the synthesis of phosphoramidite building blocks of functionalized 7-deaza-2′-deoxyguanosine derivatives carrying either 7-(3-aminopropyn-1-yl)or 7-(4-aminobutyn-1-yl) side chains is described. These building blocks are used for the preparation of ami-

10.1021/bc020024q CCC: $22.00 © 2002 American Chemical Society Published on Web 11/01/2002

Stability of Base-Modified Oligonucleotides

noalkynyl-functionalized oligodeoxyribonucleotides. The latters are studied with respect to their thermal stability as well as toward their resistance against phosphodiester hydrolysis by two single strand-specific exonucleases. EXPERIMENTAL PROCEDURES

General Remarks. All chemicals were purchased from Aldrich, Sigma, or Fluka (Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany). Snake venom phosphodiesterase (from Crotallus adamanteus) was purchased from USB Amersham Life Science (Cleveland, OH), bovine spleen phosphodiesterase from Sigma (SigmaAldrich Chemie GmbH, Deisenhofen, Germany) and alkaline phosphatase from Roche Diagnostics (Penzberg, Germany). Solvents were of laboratory grade and were distilled before use. Dimethylformamide (DMF) was purified by codistillation with H2O and benzene before use. Compounds 4a, 5a, 6a, and 7a,b were prepared as described (Ramzaeva et al., 1999; Seela et al. 2001b). Thin-layer chromatography (TLC): aluminum sheets, silica gel 60 F254, 0.2 mm layer (Merck, Germany). Column flash chromatography (FC): silica gel 60 (Merck, Germany) at 0.5 bar (4 × 104 Pa); sample collection with an UltroRac II fractions collector (LKB Instruments, Bromma, Sweden). UV spectra: U-3200 spectrometer (Hitachi, Japan). Elemental analyses were performed by Mikroanalytisches Laboratorium Beller (Go¨ttingen, Germany). NMR Spectra were measured on an AMX-500 spectrometer (Bruker, Germany) operating at a proton resonance frequency of 500.14 MHz (125.1 MHz for 13C and 101.3 MHz for 31P nuclei). Chemical shifts are in ppm relative to tetramethylsilane (TMS) as internal standard or external 85% H3PO4. J values are given in hertz. Solid-Phase Synthesis of Oligodeoxyribonucleotides. The synthesis of the oligonucleotides was accomplished on a 1-µmol scale using the 3′-phosphoramidites 7a and 7c, respectively, as well as those of the regular 2′-deoxyribonucleosides being commercially available (Sigma, St. Louis, MO). The synthesis followed the regular protocol of the DNA synthesizer (Model 392 B, Applied Biosystems, Weiterstadt, Germany). The oligonucleotides were recovered from the synthesizer as the 5′-dimethoxytritylated derivatives. After treatment with 25% aq NH3 (12 h, 60 °C) and then with 10% aq methylamine (24 h, room temp) to cleave off the nucleobase protecting groups, the 5′-dimethoxytritylated oligomers were purified by reverse-phase HPLC (see below; RP-18 column, 250 × 4 mm, 7 µm, solvent system I). Detritylation was performed by using 80% HOAc/H2O for 2 min at room temperature. Detritylated oligomers were again purified by RP-18 HPLC (solvent system II). Oligonucleotides were desalted on a 4 × 25 mm HPLC cartridge (RP-18 silica gel). Inorganic material was eluted with H2O (10 mL) while the oligomers were eluted with MeOH/H2O (3:2, v/v). The oligomers were lyophilized on a Speed-Vac evaporator and stored frozen at -23 °C. The enzymatic hydrolysis of the unmodified oligomers using snake-venom phosphodiesterase (EC 3.1.15.1, Crotallus adamanteus) and alkaline phosphatase (EC 3.1.3.1, E. coli) was carried out as described (Seela and Lampe, 1991). The reaction mixture was analyzed on reversedphase HPLC (RP-18, solvent system III). Quantification of the resulting nucleosides was made on the basis of the peak areas which were divided by the extinction coefficients of the nucleoside constituents at λ ) 260 nm: dA 15400, dC 7300, dG 11700, dT 8800 M-1 cm-1. HPLC Separation. HPLC was carried out on a 250 × 4 mm RP-18 column (Merck, Germany) on a MerckHitachi HPLC apparatus with one pump (model 655-A-

Bioconjugate Chem., Vol. 13, No. 6, 2002 1275

12) connected with a proportioning valve, a variable wavelength monitor (model 655 A), a controller (model L-5000), and an integrator (model D-2000). The solvent gradients, consisting of 0.1 M (Et3HN)OAc (pH 7.0)/ MeCN 95:5 (A) and MeCN (B), were used in the following order: gradient I, 3 min 15% B in A, 7 min 15-40% B in A, flow rate 1 mL/min; gradient II, 20 min 0-20% B in A, flow rate 1 mL/min; solvent system III 20 min 100% A, flow rate 0.6 mL/min. Melting Experiments. The thermal dissociation/ association of the oligomers was measured by temperature-dependent UV melting profiles using a Cary 1E UV/ VIS spectrophotometer (Varian, Australia) equipped with a Cary thermoelectrical controller; the actual temperature was measured in the reference cell with a Pt-100 resistor. The thermodynamic data of duplex formation were calculated by curve fitting to a two-state-model using the program MeltWin (McDowell and Turner, 1996) or by concentration-dependent Tm measurements. Mass Spectrometry. MALDI-TOF mass spectra were run on a BIFLEX III instrument (Bruker Saxonia Analytik GmbH, Leipzig, Germany) in the reflector mode. The average power of the nitrogen laser (337.1 nm) at 20 Hz was 3-4 mW (150-200 µJ/pulse) with a delay time of 600 ns. All measurements were performed using the positive detection mode with the following parameters: dwell time: 1.00 ns, delay: 40000 ns, Uis1: 19.00 kV, Uis2: 15.80 kV, Urefl 20.00 kV, Ulens: 9.35 kV. The spectra were obtained by overlaying 500-1000 single pulses with a cutoff mass of 1000 Da. The spectrometer was calibrated using an oligonucleotide calibration standard (Bruker, Part No. 206200) containing a 12-mer (3645.44 Da), a 20-mer (6117.04 Da) and a 30-mer (9191.03 Da). The sample preparation was performed on Scout MTP MALDI targets (Bruker) as follows: 1 µL of the supernatant of a saturated solution of recrystallized 3-hydroxypicolinic acid in doubly distilled H2O containing BioRad microbeads AG 50W-X8 (100-200 mesh, NH4+-form) was spotted on a target well. A suspension (1 µL) containing 15-20 microbeads in H2O was added followed by 1 µL of an aq oligonucleotide solution (concentration: 0.1 A260 units/10 µL H2O). The mixture was carefully dried on the target, and the microbeads were removed mechanically with a tip. Sequencing of Oligonucleotides. The sequencing of oligonucleotides was carried out by time-dependent enzymatic degradation reactions. All reactions were run in H2O without the addition of salts or buffer. Oligonucleotides were degraded from both ends in separate reactions. 5′ f 3′ Degradation. Two A260 units of the oligonucleotide, dissolved in H2O, was mixed with 1 µL (0.1 U) of an aq solution of bovine spleen phosphodiesterase (BSPDE, EC 3.1.16.1). The mixture was diluted to a total volume of 30 µL with H2O and then incubated at 37 °C. Aliquots of 1 µL were taken at different time intervals and analyzed as described above. 3′ f 5′ Degradation. As described for the 5′ f 3′ degradation but using 1 µL (0.1 U) of snake venom phosphodiesterase (SVPDE, EC 3.1.15.1). 3-Phthalimido-1-propyne (3a). Compound 3a was prepared according to Gibson and Benkovic (Gibson and Benkovic, 1987) from 3-amino-1-propyne (20 mL, 0.29 mol) and N-ethoxycarbonylphthalimide (64 g, 0.29 mol). Colorless crystals (45 g, 83%); mp 151-153 °C. 1H NMR (d6-DMSO): 7.84 (4H, m, phthaloyl); 4.36 (2H, d, 4J ) 2.4, CH2); 3.25 (1H, t, 4J ) 2.4, CH). 4-Phthalimido-1-butyne (3b) (Meyer, 1994). To an ice-cold solution of triphenylphosphine (6.5 g, 25 mmol)

1276 Bioconjugate Chem., Vol. 13, No. 6, 2002

in dioxane (50 mL) was added dropwise diisopropylazidodicarboxylate (5 mL, 22.5 mmol) over a period of 30 min. After the addition has been completed, a solid precipitated. Subsequently, a mixture of phthalimide (3.7 g, 25 mmol) and 3-butyn-1-ol (1.75 g, 25 mmol), dissolved in dioxane (12.5 mL), was added in one portion. After stirring overnight at ambient temperature, the reaction mixture was evaporated and the residue crystallized from CH2Cl2 to afford 2.1 g (42%) of 3b as colorless crystals; mp 134-138 °C. 1H NMR (d6-DMSO): 7.80 and 7.70 (2 m, 2 × 2H, phthaloyl); 3.84 (t, 2H, CH2N); 2.55 (‘sextett’, 2H, CH2); 1.93 (t, 1H, 4J ) 2.6, CH). 2-Amino-7-(2-deoxy-β-D-erythro-pentofuranosyl)3,7-dihydro-5-(phthalimido-1-propynyl)-4H-pyrrolo[2,3-d]pyrimidin-4-one (4a). Compound 4a was prepared from 2b (1.2 g, 3.0 mmol) and 3-phthalimido-1propyne (3a, 1.2 g, 6.5 mmol) as described for compound 4b. Yield: 1.1 g (80%) of 4a as a yellowish solid. The crude product was used without additional purification. For elemental analysis the compound was purified by FC (CH2Cl2/MeOH, 9:1) Rf 0.44. 1H NMR (d6-DMSO): 2.07 (1H, m, 2′-HR), 2.29 (1H, m, 2′-Hβ), 3.49 (2H, m, 5′-H), 3.75 (1H, m, 4′-H), 4.27 (1H, m, 3′-H), 4.59 (2H, s, CH2), 4.87 (1H, t, 3J ) 5.4, 5′-OH), 5.18 (1H, d, 3J ) 3.7, 3′OH), 6.26 (1H, t, 3J ) 6.0, 1′-H), 6.31 (2H, s, NH2), 7.26 (1H, s, 6-H), 7.87-7.94 (4H, m, Pht), 10.45 (1H, s, NH). Anal. Calcd for C22H19N5O6 (449.42): C 58.80, H 4.26, N 15.58. Found: C 58.49, H 4.08, N 15.38. 7-(2-Deoxy-β-D-erythro-pentofuranosyl)-3,7-dihydro-2-(isobutyrylamino)-5- (3-phthalimido-1-propynyl)-4H-pyrrolo[2,3-d]pyrimidin-4-one (5a). Compound 5a was prepared from 4a (1.0 g, 2.2 mmol) as described for 5b. Chromatographic workup (silica gel, 5-10% MeOH/CH2Cl2) gave 765 mg (67%) of 5a as a colorless solid. TLC (silica gel, CH2Cl2/MeOH, 9:1): Rf 0.48. 1H NMR (d6-DMSO): 1.11 (6H, m, 2 Me), 2.12 (1H, m, 2′-HR), 2.34 (1H, m, 2′-Hβ), 2.73 (1H, m, CH), 3.49 (2H, m, 5′-H), 3.77 (1H, m, 4′-H), 4.29 (1H, m, 3′-H), 4.60 (2H, s, CH2), 4.88 (1H, t, 3J ) 5.3, 5′-OH), 5.20 (1H, d, 3J ) 3.2, 3′-OH), 6.35 (1H, t, 3J ) 6.2, 1′-H), 7.56 (1H, s, 6-H), 7.86-7.93 (4H, m, Pht), 11.52 (1H, s, NH), 11.78 (1H, s, NH). Anal. Calcd for C26H25N5O7 (519.15): C 60.11, H 4.85, N 13.48. Found: C 59.82, H 4.95, N 13.31. 7-[2-Deoxy-5-O-(4,4′-dimethoxytrityl)-β-D-erythropentofuranosyl]-3,7-dihydro-2-(isobutyrylamino)-5(3-phthalimido-1-propynyl)-4H-pyrrolo[2,3-d]pyrimidin-4-one (6a). Compound 6a was prepared from 5a (519 mg, 1.0 mmol) as described for 6b. FC (silica gel, 10-20% acetone/CH2Cl2) afforded 710 mg (86%) of 6a as a colorless foam. TLC (silica gel, CH2Cl2/acetone, 85:15) Rf 0.30. 1H NMR (d6-DMSO): 1.12 (6H, m, 2 Me), 2.21 (1H, m, 2′-HR), 2.49 (1H, m, 2′-Hβ), 2.77 (1H, m, CH), 3.12 (2H, m, 5′-H), 3.72 (6H, d, 3J ) 3.0, 2 OMe), 3.91 (1H, m, 4′-H), 4.32 (1H, m, 3′-H), 4.62 (2H, s, CH2), 5.28 (1H, d, 3 J ) 3.7, 3′-OH), 6.37 (1H, t, 3J ) 6.5, 1′-H), 6.82-7.35 (13H, m, arom H), 7.88-7.95 (4H, m, Pht), 11.56 (1H, s, NH), 11.83 (1H, s, NH). Anal. Calcd for C47H43N5O9 (821.88): C 68.69, H 5.27, N 8.52. Found: C 68.58, H 4.98, N 8.54. 7-[2-Deoxy-5-O-(4,4′-dimethoxytrityl)-β-D-erythropentofuranosyl]-3,7-dihydro-2-(isobutyrylamino)5-(3-phthalimido-1-propynyl)-4H-pyrrolo[2,3-d]pyrimidin-4-one 3′-[(2-Cyanoethyl)-N,N-(diisopropyl)]phosphoramidite (7a). Compound 7a was prepared from 6a (500 mg, 0.6 mmol) as described for 7b. FC (silica gel, CH2Cl2/EtOAc/Et3N, 69:30:1) gave a mixture of diastereoisomers 7a (520 mg, 84%). TLC (silica gel, CH2Cl2/acetone, 85:15) Rf 0.8. 31P NMR (CDCl3): 148.17, 148.62.

Rosemeyer et al.

7-[2-Deoxy-5-O-(4,4′-dimethoxytrityl)-β-D-erythropentofuranosyl]-3,7-dihydro-2-(isobutyrylamino)-5(3-phthalimido-1-propynyl)-4H-pyrrolo[2,3-d]pyrimidin-4-one 3′-(Triethylammonium Phosphonate) (7b). To a solution of PCl3 (180 µl, 2.0 mmol) and N-methylmorpholine (2.2 mL, 20 mmol) in CH2Cl2 (15 mL) was added 1H-1,2,4-triazole (480 mg, 6.8 mmol). The solution was cooled to 0 °C, 6a (340 mg, 0.4 mmol) in CH2Cl2 (15 mL) was added slowly, and stirring was continued for 30 min at 0 °C. The mixture was poured into 1 M (Et3NH)HCO3 (TBK, pH 8.5, 30 mL). The aq layer was extracted with CH2Cl2 (3 × 40 mL) and the combined organic extract dried (Na2SO4) and evaporated. FC (silica gel, CH2Cl2/Et3N, 98:2, then CH2Cl2/MeOH/ Et3N, 88:10:2) furnished a main zone which was evaporated. The residue was dissolved in CH2Cl2, extracted with aq 0.1 M TBK (6 × 25 mL), dried (Na2SO4) and evaporated to give 7b (270 mg, 69%) as a colorless foam. 1 H NMR (d6-DMSO): δ, 1.14 (15H, m, 5 Me), 2.32 (1H, m, 2′-HR), 2.74 (1H, m, 2′-Hβ), 2.99 (7H, m, 3 CH2 + CH), 3.20 (2H, m, 5′-H), 3.72 (6H, s, 2 OMe), 4.09 (1H, m, 4′H), 4.69 (2H, m, CH2), 4.74 (1H, m, 3′-H), 6.07 (0.5H, s, PH), 6.42 (1H, t, 1′-H), 6.83-7.34 (14.5H, m, arom H + H-6 + PH), 7.89-7.93 (4H, m, Pht), 11.79 (2H, bs, 2 NH). 31 P NMR (d6-DMSO): 0.91 (1J(P,H) ) 581.8; 3J (P,H) ) 8.6). Anal. Calcd for C52H59N6O11P (975.04): C 64.06, H 6.10, N 8.62. Found: C 63.96, H 6.34, N 8.50. 2-Amino-7-(2-deoxy-β-D-erythro-pentofuranosyl)3,7-dihydro-5-(4-phthalimido-1-butynyl)-4H-pyrrolo[2,3-d]pyrimidine-4-one (4b). A suspension of compound 2b (1.2 g, 3.1 mmol), [Pd(0)(PPh3)4] (690 mg, 0.6 mmol), CuI (228 mg, 1.20 mmol), Et3N (0.6 mL, 4.3 mmol), and 4-phthalimido-1-butyne (3b,1.2 g, 6.0 mmol) in anhydrous amine-free DMF (30 mL) was stirred under Ar for 24 h at room temperature. After the reaction was complete, the solvent was evaporated, and the residue precipitated from CH2Cl2, filtered, suspended in CH2Cl2/ MeOH (9:1, v/v), filtered again, and dried. Yield: 1.1 g (78%) of 4b as a brownish foam. TLC (silica gel, CH2Cl2/ MeOH, 9:1): Rf 0.45. 1H NMR (d6-DMSO): 2.10 (1H, m, 2′-HR), 2.26 (1H, m, 2′-Hβ), 2.76 (2H, t, CH2), 3.52 (2H, m, 5′-H), 3.80 (3H, m, 4′-H + CH2), 4.30 (1H, m, 3′-H), 4.70 (1H, t, 5′-OH), 5.04 (1H, d, 3J ) 3.0, 3′-OH), 6.14 (2H, s, NH2), 6.26 (1H, t, 3J ) 6.4, 1′-H), 7.01 (1H, s, 6-H), 7.86-7.88 (4H, m, Pht), 10.25 (1H, s, NH). Anal. Calcd for C23H21N5O6 (463.44): C, 59.61; H, 4.57; N 15.11. Found: C, 59.88; H, 4.37; N, 14.85. 2-Amino-7-(2-deoxy-β-D-erythro-pentofuranosyl)3,7-dihydro-5-(1-aminobut-3-ynyl)-4H-pyrrolo[2,3-d]pyrimidin-4-one (1b). Compound 4b (5 mg) was dissolved in concentrated aq ammonia and stirred at 60 °C for 20 h. The reaction mixture was filtered and the filtrate evaporated to dryness yielding a colorless solid. 1 H NMR (d6-DMSO): 2.00 (1H, m, 2′-HR), 2.14 (1H, m, 2′-Hβ), 3.00 (2H, m, CH2), 3.54 (2H, m, 5′-H), 3.81 (1H, m, 4′-H), 3.88 (2H, m, CH2), 4.33 (1H, m, 3′-H), 4.98 (1H, br, 5′-OH), 5.23 (1H, br., 3′-OH), 6.20 (2H, br., NH2), 6.37 (1H, pt, 3J ) 6.7, 1′-H), 7.62 (1H, s, 6-H). RP-18 HPLC (solvent system III): tR ) 12.8 min. 7-(2-Deoxy-β-D-erythro-pentofuranosyl)-3,7-dihydro-2-(isobutyrylamino)-5-(4-phthalimido-1-butynyl)4H-pyrrolo[2,3-d]pyrimidine-4-one (5b). Compound 4b (380 mg, 0.82 mmol) was dried by coevaporation from anhydrous pyridine. The residue was suspended in pyridine (5 mL), and Me3SiCl (0.53 mL, 4.2 mmol) was added at room temperature. After 15 min of stirring, the solution was treated with isobutyric anhydride (0.67 mL, 4.0 mmol) and maintained at room temperature for 3 h. The mixture was cooled in an ice bath; then H2O (1 mL)

Stability of Base-Modified Oligonucleotides

Bioconjugate Chem., Vol. 13, No. 6, 2002 1277

Chart 1

and 5 min later a 25% aq NH3 solution (1 mL) were added, and stirring was continued for 15 min. The solution was evaporated nearly to dryness and the residue precipitated from H2O. The solid was filtered, washed with Et2O, dried in vacuo, and subjected to flash column chromatography (column: 6 × 10 cm, 5 f 10% of MeOH in CH2Cl2). Yield: 320 mg (73%) of a colorless solid. TLC (silica gel, CH2Cl2/MeOH, 9:1): Rf 0.5. 1H NMR (d6-DMSO): 1.10 (6H, m, 2 Me), 2.13 (1H, m, 2′-HR), 2.32 (1H, m, 2′-Hβ), 2.74 (3H, m, CH2 + CH), 3.49 (2H, m, 5′-H), 3.77 (3H, m, CH2 + 4′-H), 4.29 (1H, m, 3′-H), 4.93 (1H, t, 5′-OH), 5.25 (1H, d, 3′-OH), 6.34 (1H, t, 1′-H), 7.38 (1H, s, 6-H), 7.85 (4H, m, Pht), 11.54 (1H, s, NH), 11.74 (1H, s, NH). Anal. Calcd for C27H27N5O7 (533.53): C, 60.78; H, 5.10; N, 13.13. Found: C, 60.35; H, 5.12; N 12.98. 7-[2-Deoxy-5-O-(4,4′-dimethoxytrityl)-β-D-erythropentofuranosyl]-3,7-dihydro-2-(isobutyrylamino)-5(4-phthalimido-1-butynyl)-4H-pyrrolo[2,3-d]pyrimidin-4-one (6b). Compound 5b (510 mg, 0.96 mmol) was dried by repeated coevaporation from anhydrous pyridine and then dissolved in pyridine (20 mL). After addition of 4.4′-dimethoxytrityl chloride (360 mg, 1.06 mmol), the solution was stirred for 4 h at room temperature. Then, MeOH (3 mL) was added, and stirring was continued for 10 min; subsequently, a 5% aq NaHCO3 solution (30 mL) was added, and the aqueous layer was extracted with CH2Cl2 (3 × 60 mL) affording, after evaporation of the solvent, 520 mg (65%) of a colorless solid. TLC (silica gel, CH2Cl2/acetone, 85:15): Rf 0.3. 1H NMR (d6-DMSO): 1.13 (6H, d, 2 Me), 2.23 (1H, m, 2′-HR), 2.47 (1H, m, 2′-Hβ), 2.76 (3H, m, CH + CH2), 3.18 (2H, m, 5′-H), 3.75 (6H, s, 2 OMe), 3.79 (2H, m, CH2), 3.93 (1H, m, 4′-H), 4.33 (1H, m, 3′-H), 5.32 (1H, d, 3J ) 2.4, 3′-OH), 6.38 (1H, pt, 3J ) 6.2, 1′-H), 6.86-7.39 (14H, m, 6-H + arom H), 7.82-7.85 (4H, m, Pht), 11.54 (1H, s, NH), 11.79 (1H, s, NH). Anal. Calcd for C48H45N5O9 (835.90): C, 68.97; H, 5.43; N, 8.38. Found: C, 68.67; H, 5.32; N. 8.16. 7-[2-Deoxy-5-O-(4,4′-dimethoxytrityl)-β-D-erythropentofuranosyl]-3,7-dihydro-2-(isobutyrylamino)5-(4-phthalimido-1-butynyl)-4H-pyrrolo[2,3-d]pyrimidin-4-one 3′-[(2-Cyanoethyl)-N,N-(diisopropyl)]phosphoramidite (7c). To a solution of 6b (400 mg, 0.48 mmol) in anhydrous CH2Cl2 (15 mL) were added (i-Pr)2EtN (162 µL, 0.93 mmol) and chloro-(2-cyanoethoxy)(diisopropylamino)phosphine (160 µL, 0.72 mmol). After

stirring for 40 min at room temperature, a 5% aq NaHCO3 solution (6 mL) was added, and the mixture was extracted with CH2Cl2 (2 × 15 mL). The organic layer was dried (Na2SO4), filtered, and evaporated. Flash chromatography (column: 6 × 10 cm, CH2Cl2/acetone, 85: 15) gave a mixture of diastereoisomers of 7c (300 mg, 61%). TLC (silica gel, CH2Cl2/acetone, 85:15): Rf 0.6. 31P NMR (CDCl3): 148.79, 149.2. RESULTS AND DISCUSSION

Synthesis of Monomers. The key intermediate for the synthesis of the title compounds is the 7-iodo nucleoside 2b (Chart 1)which has been described earlier (Ramzaeva and Seela, 1995). Pd(0)-catalyzed cross coupling reaction of compound 2b with 3-phthalimido-1-propyne (3a) (Gibson and Benkovic, 1987) gave 4a in 80% yield (Ramzaeva et al., 1999; Seela et al., 2001). Analogously, compound 2b was now reacted with 4-phthalimido-1butyne (3b) (Meyer, 1994) to give 4b in about the same yield. Cleavage of the phthalimido group is unproblematic in concentrated aqueous ammonia at elevated temperature (60 °C) and leads to compound 1b (see Experimental Procedures). Upon the basis of our work on 7-alkynylated 7-deaza2′-deoxyguanosine derivatives (Ramzaeva et al., 1997), we have chosen the isobutyryl group for the protection of the 2-amino function of compounds 4a and 4b (Ti et al., 1982). For comparison, the isobutyryl group of 2-(isobutyrylamino)-7-(hexyn-1-yl)-7-deaza-2′-deoxyguanosine exhibits a half-life value of 80 min (25% aq NH3, 40 °C) (Ramzaeva et al., 1997). Isobutyrylation of compounds 4a and 4b was performed using the protocol of transient protection and furnished compound 5a (67%) and 5b (73%). Subsequently, both compounds were treated with 4,4′dimethoxytriphenyl-methyl chloride (Schaller et al., 1963) in pyridine to give the 5′-protected derivatives 6a (86%) and 6b (65%), respectively (Chart 2). The dimethoxytrityl derivative 6a was phosphitylated with 2-cyanoethyl diisopropylphosphoramidochloridite (Beaucage and Caruthers, 1981) following a standard protocol to yield the phosphoramidite 7a (Ramzaeva et al., 1999; Seela et al., 2001 b). Additionally, from compound 6a the phosphonate 7b was prepared using PCl3/N-methylmorpholine/1H1,2,4-triazole (Ramzaeva et al., 1999; Seela et al., 2001;

1278 Bioconjugate Chem., Vol. 13, No. 6, 2002

Rosemeyer et al.

Chart 2

Froehler et al., 1986). The 7-(4-aminobutyn-1-yl) derivative 6b was phosphitylated as described for 6a to give the phosphoramidite 7c. It should be mentioned that the 5′-triphosphates of compounds 1b and 1c as well as of the phthaloyl-protected 4b have been prepared earlier and used for dye labeling (Seela et al. 2001a). Characterization of the Monomers. The newly synthesized derivatives (Charts 1 and 2) were characterized by elemental analysis as well as by 1H, 13C, and 31P NMR spectroscopy (Tables 1, 2, and Experimental Procedures). Unambiguous assignment of the 13C NMR resonances was made on the basis of gated-decoupled 13C NMR, DEPT-135, and 2D [1H,13C] HETCORR spectra. As can be seen from Table 1, substitution of the iodo atom of 2b by an ω-aminoalkynyl side chain shifts the ipso carbon [C-7] to lower field by about 43 ppm demonstrating the electron-withdrawing effect of the CtC triple bond. Isobutyrylation of the amino function of 4a and 4b results in typical high-field shifts of C-2 (∆δ ) 5.5 ppm), C-4 (∆δ ) 3 ppm), and C-6 (∆δ ) 2 ppm) and in a lowfield shift of C-5 (∆δ ) 4 ppm). Moreover, isobutyrylation affects the chemical shift of the pyrrole carbon C-8: a low-field shift (∆δ ) 2 ppm) proves an electronic longrange coupling between the exocyclic amino group and the pyrrole ring. Table 2 summarizes vicinal 3J(H,H) coupling constants of compounds 4a and 4b as well as of the parent nucleosides 2′-deoxyguanosine (Gd) and 7-deaza-2′-deoxyguanosine (c7Gd , 2a). The data were taken from wellresolved 1H NMR spectra measured in D2O. The sugar conformation in solution (3′T2′ a 3′T2′ ≡ N a S ) was deduced from the pseudorotational parameters P and Ψm by application of the PSEUROT program (van Wijk and Altona, 1993). The rotational equilibrium about the C-4′C5′ bond was calculated according to E. Westhof (Westhof et al., 1975) using the vicinal couplings between H-4′ and H-5′ as well as H-5′′. From these data a slight trend can be deduced: The sugar puckering of the 7-(3-aminopropyn-1-yl) derivative 4a is very similar to that of c7Gd (28 and 27%, respectively) (Rosemeyer and Seela, 1998). Simultaneously, the three-state equilibrium about the C-4′-C-5′ bond of compounds 4a and of c7Gd (γ(+)g a γt a γ(-)g) exhibits very similar populations. On the other hand, the 7-(4-aminobutyn-1-yl) derivative 4b shows slightly different conformational features: The sugar puckering is shifted toward a higher N-type conformer population, and the equilibrium at the C-4′-C-5′ bond is biased away from the γ(+)g conformation. Synthesis and Characterization of Oligonucleotides. Automated solid-phase synthesis of the oligonucleotides 15-20 was performed with the phosphoramidites 7a and 7c (Froehler, 1993). The oligomer 15 has

Figure 1. RP-18 HPLC profile of an enzymatic hydrolysis reaction mixture of the oligomer 5′-d(A-G*-T-A-T-T-G*-A-C-CT-A) after treatment with snake venom phosphodiesterase and alkaline phosphatase (37 °C, 16 h). G*d ) 7-(4-Aminobutyn-1yl)-7-deaza-2′-deoxyguanosine. For details see Experimental Procedures.

been also prepared using the phosphonate 7b. The synthesis followed the standard protocols, and the coupling yields were always higher than 92% for the phosphoramidites and ca. 90% for the phosphonate. Deprotection was performed with 25% aq ammonia (60 °C, overnight) followed by a 24 h-treatment with 10% aq methylamine at room temperature (Seela et al., 2001a) to complete the removal of the phthaloyl residues. The oligonucleotides were detritylated and purified by reversed-phase HPLC (RP-18, see Experimental Procedures). The homogenity of the compounds was established by HPLC (RP-18) as well as by ion-exchange chromatography (NucleoPac-PA100 column, 4 × 50 mm; Dionex, P/N 043018). The modified oligonucleotides were characterized by MALDITOF mass spectra. The detected masses were in good agreement with the calculated values (Table 3). Attempts to determine the composition of the oligonucleotides 19 and 20 by enzymatic tandem hydrolysis with snake venom phosphodiesterase (SVPDE) and alkaline phosphatase (AP) followed by reversed phase HPLC (RP-18) (Seela and Lampe, 1991) turned out to be problematic. After 16 h of treatment with the abovementioned enzymes, RP-18 HPLC indicated incomplete cleavage of the modified oligonucleotides (Figure 1).

Stability of Base-Modified Oligonucleotides Table 1.

13C

NMR Chemical Shifts of 2′-Deoxy-β-D-ribonucleoside Derivativesa

compd

C(2) C(2)

C(4) C(7a)

C(5) C(4a)

C(6) C(4)

C(5)

C(8) C(6)

2 4a 5a 6a 7b 4b 5b 6b

152.7 153.1 147.6 147.6 147.7 153.0 147.6 147.5

150.5 150.2 147.1 147.3 147.4 150.0 147.1 147.1

99.8 99.3 103.3 103.6 103.5 99.3 103.4 103.6

158.0 157.7 155.7 155.7 155.7 157.7 155.9 155.7

54.9 97.6 98.3 98.4 98.5 98.7 99.5 99.5

121.6 122.6 124.8 126.6 126.5 121.3 123.8 123.6

4a 5a 6a 7b 4b 5b 6b

C(1′′)

C(2′′)

CH2

83.5 84.3 84.3 84.4 85.8 85.4 85.5

76.7 75.8 75.6 75.5 76.1 75.3 75.1

27.7 27.7 27.6 27.6 18.7; 36.4 19.0; 36.5 18.8; 36.4

CdO (iBu)

CH3

34.7 34.7 34.8

18.8 18.8 18.7

180.0 180.0 180.0

34.8 34.7

18.9 18.7; 18.8

180.1 180.0

C(2′)

C(3′)

C(4′)

C(5′)

Cquart

82.2 82.1 82.6 82.6 82.9 82.1 82.7 82.6

DMSO DMSO DMSO DMSO 37.9 DMSO DMSO DMSO

70.9 70.9 70.8 70.5 72.5 70.8 70.9 70.5

87.1 87.1 87.8 85.5 85.4 87.0 87.0 86.9

61.8 61.8 61.7 64.1 63.9 61.9 61.9 64.1

113.0 113.0

OCH3 4a 5a 6a 7b 4b 5b 6b

CH

C(1′) 2 4a 5a 6a 7b 4b 5b 6b

a

Bioconjugate Chem., Vol. 13, No. 6, 2002 1279

113.1

phthaloyl (C1′′′)

phthaloyl (C2′′′)

phthaloyl (C3′′′)

CdO (Pht)

131.4 131.4 131.4 131.4 131.5 131.7 131.6

123.3 123.3 123.3 123.3 123.0 123.2 123.0

134.7 134.7 134.7 134.6 134.3 134.5 134.3

166.8 166.7 166.7 166.7 167.5 167.8 167.5

54.9 54.8; 54.9 54.9

Measured in d6-DMSO.

Table 2. 3JH,H Coupling Constants of the Sugar Moieties and Conformer Population of 2′-Deoxy-β-D-ribonucleosides (303 K) as well as Polarizabilities and logP Values of the Corresponding Nucleobasesa 3J H,H

[Hz]

compd

1′,2′

1′,2′′

2′,3′

2′′,3′

3′,4′

4′,5′

4′,5′′

dG c7Gd 4a 4b

7.30 7.25 6.85 6.90

6.50 6.50 6.35 6.60

6.30 6.25 6.00 6.25

3.60 3.00 2.80 3.35

3.20 3.35 2.85 3.50

3.60 4.20 4.30 4.50

4.70 5.00 4.70 5.00

conformation compd dG c7Gd 4a 4b a

Hz.

%N

%S

% γ(+)g

% γt

% γ(-)g

Rm [10-24 cm3]

logPb

29 28 27 32

71 72 73 68

53 43 45 39

30 33 30 33

17 24 25 28

14.1 ( 0.5 14.7 ( 0.5 20.9 ( 0.5 22.7 ( 0.5

-1.28 ( 0.62 -1.26 ( 0.89 -1.10 ( 1.38 -0.93 ( 1.37

Measurements were performed in D2O at a concentration of 5 mg of nucleoside per 0.5 mL; RMS e 0.4 for all calculations; | ∆ | E 0.5 b logP data refer to uncharged molecules as (1)NH tautomers.

Table 3. Molecular Masses (Da) of Selected Oligodeoxynucleotides oligomer 5′-d(TA1a 1aTC AAT ACT) 5′-d(A1aT ATT 1aAC CTA) 5′-d(1a-C)4 5′-d(TA1b 1bTC AAT ACT) 5′-d(A1bT ATT 1bAC CTA) 5′-d(1b-C)4

16 17 15 19 20 18

MH+ calcd

MH+ found

3749 3749 2618 3780 3780 2674

3751 3745 2620 3772 3776 2675

Only after extension of the total reaction time to 6 d were the oligomers hydrolyzed completely. As it is known that oligonucleotides carrying unsubstituted 7-deaza-2′-

deoxyguanosine residues are enzymatically cleaved without problems, it was concluded that the ω-aminoalkynyl side chain of the modified bases causes termination of the phosphodiesterase hydrolysis. In this context it is worth mentioning that it has been reported previously (Seela et al., 1995) that alternating oligonucleotides of the type [5′-d(G*-C)4]2 with G*d being 7-methylated or iodinated 7-deaza-2′-deoxyguanosine are hydrolyzed at a significantly slower rate by both calf spleen PDE as well as SVPDE. This topic will be taken up below. Thermal Stability of Oligonucleotides. For all duplexes shown in Table 4, Tm values were measured (10

1280 Bioconjugate Chem., Vol. 13, No. 6, 2002

Rosemeyer et al.

Table 4. Tm Values and Thermodynamic Data of Duplex Formation of Oligonucleotides Containing 7-(ω-Aminoalk-1-ynyl)-Functionalized 7-Deazaguanine Basesa oligonucleotide

Tm [°C]

∆H° b [kcal/mol]

∆S° b [cal/K mol]

∆G°310 [kcal/mol]

[5′-d(G-C)4]2 5′-d(TAG GTC AAT ACT) 3′-d(ATC CAG TTA TGA) 5′-d(TAG GTC AAT ACT) 3′-r(AUC CAG UUA UGA)

8‚8 9 10 9 11

59 47

-67.0 -86.8

-179.0 -243.7

-11.5 -11.3

45

-92.1

-264.0

-10.2

[5′-d(2a-C)4]2 5′-d(TA2a 2aTC AAT ACT) 3′-d(AT C C AG TTA TGA) 5′-d(TAG GT C AAT ACT) 3′-d(ATC CA2a TTA T2aA) 5′-d(TA2a 2aTC AAT A C T) 3′-d(AT C C A2a TTA T2aA) 5′-d(TA2a 2aTC AAT ACT) 3′-r(AU C C AG UUA UGA)

12 ‚ 12 13 10 9 14 13 14 13 11

53 46

-53.0 -98.3

-162.0 -283.0

-11.5 -10.6

46

-84.2

-239.0

-10.1

43

-85.6

-245.3

-9.6

43

-94.9

-275.0

-9.7

[5′-d(1a-C)4]2 5′-d(TA1a 1aTC AAT ACT) 3′-d(AT C C AG TTA TGA) 5′-d(TA1a 1aTC AAT ACT) 3′-r(AU C C AG UUA UGA) 5′-d(TAG GT C AAT ACT) 3′-d(ATC CA1a TTA T1aA) 5′-d(TA1a 1aTC AAT ACT) 3′-d(AT C CA1a TTA T1aA)

15 ‚ 15 16 10 16 11 9 17 16 17

72 52

-73.8 -96.9

-192.4 -272.2

-14.2 -12.5

48

-80.7

-226.2

-10.5

52

-95.5

-268.4

-12.2

55

-97.5

-271.8

-13.2

[5′-d(1b-C)4]2 5′-d(TA1b 1bTC AAT ACT) 3′-d(AT C C AG TTA TGA) 5′-d(TA1b 1bTC AAT ACT) 3′-r(AU C C AG UUA UGA) 5′-d(TAG GT C AAT A CT) 3′-d(ATC CA1b TTA T1bA) 5′-d(TA1b 1bTC AAT ACT) 3′-d(AT C CA1b TTA T1bA)

18 ‚ 18 19 10 19 11 9 20 19 20

63 50

-62.7 -89.1

-165.5 -250.1

-11.4 -11.5

49

-84.7

-237.6

-11.0

51

-89.9

-251.9

-11.8

52

-83.9

-232.9

-11.6

[5′-d(1c-C)4]2 5′-d(TA1c 1cTC AAT ACT) 3′-d(AT C C AG TTA TGA) 5′-d(TA1c 1cTC AAT ACT) 3′-r(AU C C AG UUA UGA) 5′-d(TAG GT C AAT AC T) 3′-d(ATC CA1c TTA T1cA) 5′-d(TA1c 1cT C AAT AC T) 3′-d(AT C C A1c TTA T1cA)

21 ‚ 21 22 10 22 11 9 23 22 23

ncm 47

-59.2

-158.9

-9.9

45

-82.7

-233.7

-10.2

51

-80.0

-221.8

-11.2

49

-79.4

-220.9

-10.9

a Measured at 260 nm in 10 mM Na-cacodylate, 10 mM MgCl , 100 mM NaCl, pH 7, at 5 µM + 5 µM of single strand concentration; 2 ncm: no cooperative melting. b Data are within 15% of error.

mM Na-cacodylate, 10 mM MgCl2, 100 mM NaCl, pH 7), and the thermodynamic data of duplex formation were calculated from each individual melting profile according to a two-state model (duplex a random coil) model with the program Meltwin (release version 3.0) (McDowell and Turner, 1996) (Table 4). Replacement of all c7Gd residues within the alternating oligomer [5′-d(c7G-C)4]2 (12‚12) (Ramzaeva et al., 1997) by the (3-aminopropyn-1-yl) derivative 1a (f 15‚15) leads to a significant duplex stabilization compared to the parent 12‚12 (∆Tm ) 19 °C; = 5 °C/base pair). This increase of thermal stability is, however, reduced to 2.5 °C/base pair when the ω-aminoalkynyl side chain is extended by one methylene group (18‚18, Tm ) 63°). Further extension of the side chain to a (5-aminopentyn-1-yl) substituent abolishes duplex formation of the alternating oligomer 21 (Ramzaeva et al., 1999; Seela et al., 2001b). These surprising results, summarized in Table 5, are apparently related to the positioning of the protonated amino group within the major groove of the B-DNA helix because the corresponding oligomer carrying four uncharged 7-(hexyn-1-yl)-7-deaza-2′-deoxyguanosines (Ramzaeva et al., 1997) shows a Tm value of 59 °C under analogous conditions. It has been demonstrated earlier

Table 5. Tm Values of [5′-d(G*-C)4]2 as a Function of the Number of Methylene Groups in the 7-(ω-Aminoalk-1-ynyl) Side Chain of G*da number of methylene groups in the ω-aminoalk-1-ynyl side chain 1 2 3 3b

dC(7),N [Å]

Tm [°C]

4.9 6.3 7.3 7.3

72 63