Quadruplex and Pentaplex Self-Assemblies of Oligonucleotides

Laboratorium für Organische und Bioorganische Chemie, Institut für Chemie, Universität Osnabrück,. Barbarastrasse 7, D-49069 Osnabrück, Germany...
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Bioconjugate Chem. 2001, 12, 1043−1050

1043

Quadruplex and Pentaplex Self-Assemblies of Oligonucleotides Containing Short Runs of 8-Aza-7-deaza-2′-deoxyisoguanosine or 2′-Deoxyisoguanosine Frank Seela* and Rita Kro¨schel Laboratorium fu¨r Organische und Bioorganische Chemie, Institut fu¨r Chemie, Universita¨t Osnabru¨ck, Barbarastrasse 7, D-49069 Osnabru¨ck, Germany. Received June 1, 2001; Revised Manuscript Received July 30, 2001

Oligonucleotides containing 8-aza-7-deaza-2′-deoxyisoguanosine (4) were investigated regarding their self-assembly in aqueous solution. The aggregation of 4 was compared with that of oligonucleotides containing 2′-deoxyisoguanosine (2b) and 2′-deoxyguanosine (1b). For this purpose the phosphoramidite of 4 was synthesized which was protected by a dibutylaminomethylidene residue at the amino group and a diphenylcarbamoyl residue at the 2-oxo function. Solid-phase synthesis furnished oligonucleotide containing short runs of the nucleoside 4. The self-assembly of the oligonucleotide 5′-d(T444T2) was studied by ion-exchange chromatography. The formation of a pentaplex was observed in the presence of Cs+, while a tetraplex is formed when the counter ion is Na+ or Rb+. The cation selectivity of the oligonucleotide 5′-d(T444T2) was found to be different from the parent 5′-d(T4isoG4T2) which was forming the tetraplex as well as a pentaplex in aq RbCl solution.

INTRODUCTION

Guanosine (1a) as well as guanosine derivatives such as 2′-deoxyguanosine (1b) and their oligonucleotides form self-assemblies in the presence of metal ions (1-3). These aggregates can be considered as nucleoside conjugates held together by hydrogen bonds and coordination forces (metal ions) and not by covalent linkages. Oligonucleotides with short runs of guanine, such as d(T4G4), d(T4G4T), or d(T4G4T4), were shown to form tetrameric complexes, and the G-quartet binds cations with a selectivity of K+ (1.33 Å) > Rb+ (1.49 Å) > Na+ (0.98 Å) . Cs+ (1.65 Å), Li+ (0.78 Å) (4-7) following the Hoogsteen base-pair motif. The ions are surrounded by the 6-oxo groups of the hydrogen-bonded guanine quartet. Investigations on the structure of self-assembled nucleosides as well as oligonucleotides have been performed using various techniques such as NMR spectroscopy (8-11), X-ray analyses (12, 13), electrospray ionization mass spectroscopy (14), electrophoresis (5), and energy perturbation studies (15). The X-ray analyses gave a detailed picture of the G-quartet structure showing that the central cation of the complexes is located between two layers of the guanine tetrads. The guanosine-related nucleoside isoguanosine (2a) was found to form also aggregates in aqueous solution. The phenomenon was already observed in 1976 by Golas et al. on poly(isoguanylic acid) (16). Several structures of the aggregated homopolymer have been suggested. The first defined complex formed by an 2′-deoxyisoguanosine (2b) containing oligonucleotide has been reported by our laboratory. The dodecamer d(T4isoG4T4) was found to form a tetrameric assembly in the presence of sodium ions which is structurally different from that formed by 2′-deoxyguanosine (17, 18). Nitrogen-7 is not participat* To whom correspondence should be addressed. Phone +49541-9692791. Fax +49-0541-9692370. E-mail: [email protected].

ing in the hydrogen bonding pattern. Thus, 7-deaza-2′deoxyisoguanosine (3) forms an aggregate structure (19) while 7-deaza-2′-deoxyguanosine prevents guanine-rich oligonucleotides from aggregation (20). Recently pentameric complexes have been reported for an isoguanosine derivative (21) as well as for 2′-deoxyisoguanosine containing oligonucleotides both, in the presence of cesium ions (22).

This manuscript reports on the self-assembly of oligonucleotides containing 8-aza-7-deaza-2′-deoxyisoguanosine (4). Corresponding oligonucleotides are prepared by solid-phase synthesis using the protocol of phosphoramidite chemistry. The self-assembly of those oligonucleotides is investigated for the dependence on the size of the alkali cation, and the aggregate formation of 8-aza-

10.1021/bc010064e CCC: $20.00 © 2001 American Chemical Society Published on Web 09/28/2001

1044 Bioconjugate Chem., Vol. 12, No. 6, 2001

7-deaza-2′-deoxyisoguanosine oligonucleotides is compared to those containing 2′-deoxyisoguanosine (2b) and 2′-deoxyguanosine (1b). EXPERIMENTAL PROCEDURES

General Remarks. Thin-layer chromatography (TLC) was performed on TLC aluminum sheets silica gel 60 F254 (0.2 mm, Merck, Germany). Solvent systems for FC and TLC: CH2Cl2/MeOH 9:1 (A), CH2Cl2/MeOH 95:5 (B), CH2Cl2/MeOH 98:2 (C), CH2Cl2/acetone 9:1 (D), CH2Cl2/ acetone 98:2 (E), CH2Cl2/EtOAc 85:15 (F). Reverse phase HPLC was carried out on a 4 × 250 mm RP-18 (10 µm) LiChrosorb column (Merck) with a Merck-Hitachi HPLC pump (model 655 A-12) connected with a variable wavelength monitor (model 655-A), a controller (model L-5000), and an integrator (model D-2000). UV-spectra were recorded on a U-3200 spectrophotometer (Hitachi, Japan), λmax in nm,  in dm3 mol-1 cm-1. Half-life values (τ) were measured on a U-3200 spectrophotometer (Hitachi, Japan) connected with a temperature controller (Lauda, Germany). NMR spectra were measured on an Avance DPX 250 and an AMX 500 spectrometer (Bruker, Germany); chemical shifts (δ) are in ppm downfield from internal TMS (1H, 13C) or external 85% H3PO4 (31P). The J-values are given in Hz. The solvents were purified and dried according to standard procedures. Ion-Exchange HPLC. The ion-exchange chromatography was performed on a 4.6 × 100 mm Gen-Pak Fax Column (Waters, WAT015490) using a Merck-Hitachi HPLC apparatus with a pump (model L-7100), and a UVdetector (model L-4250) connected with an integrator (model D-2000). A column oven (model L-7350, Merck, Germany) was used to control the temperature of the ionexchange column. The oligonucleotides were prepared as follows. A sample of 0.1 A260-units was dissolved in 20 µL of 1.0 M MCl-buffer (M: Na, K, Rb, Cs). The solution was heated to 60 °C for 5 min, brought to room temperature and kept in a refrigerator (-23 °C) for 15 min up to 16 h (depending on the oligonucleotide). Then, the sample was brought to room temperature, diluted with 100 µL of buffer A, and injected into the system, which had been preheated to 30 °C. The column was eluted using the following systems: 0-40 min with 0-80% B in A with a flow rate of 0.5 mL min-1 (A: 25 mM TrisHCl, 1.0 mM EDTA, pH 8.0; B: 1.0 M MCl (M: Na, K, Rb, Cs) 25 mM Tris-HCl, 1.0 mM EDTA, pH 8.0). Oligonucleotides. The oligonucleotide syntheses were carried out on an ABI 392-08 DNA synthesizer (Applied Biosystems, Weiterstadt, Germany) in a 1 µmol scale using the phosphoramidite 10, those of the regular 2′deoxynucleosides (Applied Biosystems, Weiterstadt, Germany) together with the DPC- and acetamidine-protected phosphoramidite of 2′-deoxyisoguanosine following the synthesis protocol for 3′-β-cyanoethylphosphoramidites. After cleavage from the solid support, the oligonucleotides were deprotected in 25% aqueous ammonia solution for 12-16 h at 60 °C. Purification of the 5′-dimethoxytrityloligomers was performed by reversed-phase HPLC (RP18) with the following solvent gradient system (A: 0.1M (Et3NH)OAc (pH 7.0)/MeCN 95:5; B: MeCN): 3 min 20% B in A with a flow rate of 1.0 mL min-1, 12 min 20-40% B in A with a flow rate of 1 mL min-1. Treatment with 2.5% CHCl2COOH/CH2Cl2 for 5 min at room temperature to remove the 4,4′-dimethoxytrityl residues. The detritylated oligomers were purified by reversed-phase HPLC with the gradient: 20 min 0-20% B in A with a flow rate of 1 mL min-1. The oligomers were desalted and lyophilized on a Speed-Vac evaporator to yield colorless solids.

Seela and Kro¨schel

4-Amino-1-(2-deoxy-β-D-erythro-pentofuranosyl)1H-pyrazolo[3,4-d]pyrimidin-6-one (4). To a solution of 4,6-diamino-1-(2-deoxy-β-D-erythro-pentofuranosyl)1H-pyrazolo[3,4-d]pyrimidine (5) (200 mg, 0.75 mmol) and sodium nitrite (200 mg, 2.9 mmol) in water (6 mL) was added acetic acid (300 µL) dropwise at 60 °C under stirring. Stirring was continued for 8 min, and the pH of the yellowish solution was adjusted to 8.0 with 25% aqueous NH3 solution. The precipitated crude material was filtered, and the residue dissolved in water and applied to a Serdolit AD-4 column (4 × 20 cm, resin 0.10.2 mm, Serva, Germany). The column was washed with water (500 mL), and the product was eluted with H2OPriOH (1:1). Compound 4 was obtained as a yellowish powder (98 mg, 49%). UV, 1H and 13C NMR spectra are identical with the published data (23). 1-(2-Deoxy-β-D-erythro-pentofuranosyl)-4-(N,Ndibutylaminomethylidene)-1H-pyrazolo[3,4-d]pyrimidin-6-one (6). To a stirred suspension of 4 (440 mg, 1.65 mmol) in MeOH (15 mL) was added N,N-dibutylformamide dimethylacetal (800 µL) (24). The mixture was stirred at 40 °C for 2 h and evaporated to dryness. The residue was applied to flash chromatography (FC; silica gel, column 2 × 10 cm, CH2Cl2-MeOH 98:2 to 95:5) and eluted stepwise. Colorless foam of 6 (450 mg, 67%). TLC (A): Rf 0.32; UV (MeOH): 233 (24300); 278 (13000); 338 (15500). 1H NMR (d6-DMSO): 2.20 (1H, m, 2′-HR); 2.65 (1H, m, 2′-Hβ); 3.36 (2H, m, 5′-H2); 3.80 (1H, m, 4′-H); 4.37 (1H, s, 3′-H); 5.21 (2H, m, 3′-OH, 5′-OH); 6.30 (1H, “t”, J ) 6.5, 1′-H); 7.93 (1H, s, 3-H); 8.56 (1H, s, H-Cd N). Anal. Calcd for C19H30N6O4 (406.5): C, 56.14; H, 7.44; N, 20.68. Found: C, 56.08; H, 7.50; N, 20.59. 4-Amino-1-(2-deoxy-β-D-erythro-pentofuranosyl)6-[(diphenylcarbamoyl)oxy]-1H-pyrazolo[3,4-d]pyrimidine (7). Compound 4 (100 mg, 0.37 mmol) was dried by repeated coevaporation with anhydrous pyridine and then suspended in anhydrous pyridine (2 mL). Diphenylcarbamoyl chloride (DPC-Cl)(150 mg, 0.65 mmol) and N,N-diisopropylethylamine (100 µL, 0.57 mmol) were added, and the reaction mixture was stirred for 2 h at room temperature. The excess of DPC-Cl was hydrolyzed with crushed ice. Then, the mixture was poured into a 5% aqueous NaHCO3 (4 mL) and extracted with CH2Cl2 (3 × 5 mL). The combined CH2Cl2 layers were dried (Na2SO4) and filtered. After evaporation of the solvent, the residue was applied to FC (silica gel, column 2 × 15 cm). Elution with CH2Cl2-MeOH (98:2) followed by CH2Cl2MeOH (9:1) furnished as a colorless foam (105 mg, 61%). TLC (A): Rf 0.41. 1H NMR (d6-DMSO): 2.27 (1H, m, 2′HR); 2.80 (1H, m, 2′-Hβ); 3.35-3.57 (2H, m, 5′-H2); 3.81 (1H, m, 4′-H); 4.42 (1H, m, 3′-H); 4.71 (1H, “t”, J ) 5.9, 5′-OH); 5.23 (1H, d, J ) 4.6, 3′-OH); 6.43 (1H, “t”, J ) 6.3, 1′-H); 7.26-7.50 (10H, m, arom. H), 7.80-8.24 (3H, m, NH2, 3-H). Anal. Calcd. for C23H22N6O5 (462.5): C, 59.73; H, 4.79; N, 18.17. Found: C, 59.65; H, 4.79; N, 18.11. 1-(2-Deoxy-β-D-erythro-pentofuranosyl)-4-(N,Ndibutylaminomethylidene)-6-[(diphenylcarbamoyl)oxy]-1H-pyrazolo[3,4-d]pyrimidine (8). To a suspension of compound 6 (290 mg, 0.71 mmol) in dry pyridine (5 mL) were added diphenylcarbamoyl chloride (300 mg, 1.29 mmol) and N,N-diisopropylethylamine (220 µL, 1.26 mmol), and the reaction mixture was stirred for 1 h at room temperature. The mixture was poured into 5% aqueous NaHCO3 (7 mL) and extracted with CH2Cl2 (3 × 5 mL). The combined CH2Cl2 layers were dried (Na2SO4) and filtered. After evaporation of the solvent, the

Quadruplex and Pentaplex Self-Assemblies Table 1.

3J

H,H

Coupling Constants of the Sugar Moieties and Conformer Populations of 2′-Deoxynucleosidesa

3J

4 2b (31) 1b (32) 3b (31) a

Bioconjugate Chem., Vol. 12, No. 6, 2001 1045

1′,2′

6.70 6.95 7.30 7.00

3J 1′,2′′

3J 2′,3′

3J 2′′,3′

3J 3′,4′

3J

4′,5′

3J 4′,5′

%N

%S

%γg+

%γt

%γg-

6.70 6.65 6.50 6.60

6.35 6.45 6.30 6.70

3.80 3.55 3.60 3.40

3.45 3.45 3.20 3.60

3.90 3.60 3.60 3.90

5.60 4.70 4.70 5.00

34 32 29 31

66 68 71 69

39 53 53 46

40 17 30 21

21 30 17 34

RMS e 0.4 for all calculations; |∆Jmax| e 0.5 Hz.

residue was applied to FC (silica gel, column 2 × 15 cm). Elution with CH2Cl2 followed by CH2Cl2-MeOH (stepwise from 98:2 to 9:1) furnished a colorless foam (304 mg, 71%). TLC (A): Rf 0.46. UV (MeOH): 238 (20700), 322 (32000). 1H NMR (d6-DMSO): 0.92 (6H, m, CH3); 1.33, 1.61 (8H, m, CH2); 2.26 (1H, m, 2′-HR); 2.80 (1H, m, 2′Hβ); 3.27-3.67 (6H, m, NCH2, 5′-H2); 3.82 (1H, m, 4′-H); 4.44 (1H, m, 3′-H); 4.76 (1H, “t”, J ) 5.8, 5′-OH); 5.29 (1H, d, J ) 4.6, 3′-H); 6.51 (1H, “t”, J ) 6.3, 1′-H); 7.277.45 (10H, m, arom. H); 8.13 (1H, s, 3-H); 8.85 (1H, s, HCdN). Anal. Calcd for C32H39N7O5 (601.7): C, 63.88; H, 6.53; N, 16.30. Found: C, 64.00; H, 6.78; N, 15.88. 1-[2-Deoxy-5-O-(4,4′-dimethoxytrityl)-β-D-erythropentofuranosyl]-4-(N,N-dibutylaminomethylidene)6-[(diphenylcarbamoyl)oxy]-1H-pyrazolo[3,4-d]pyrimidine (9). Compound 8 (200 mg, 0.33 mmol) was dried by repeated coevaporation with anhydrous pyridine and dissolved in anhydrous pyridine (1 mL). The solution was treated with dimethoxytrityl chloride (163 mg, 0.48 mmol) at 30 °C under stirring (45 min). MeOH (1 mL) was added, and the stirring was continued for 5 min. The mixture was poured into 5% aqueous NaHCO3 solution (5 mL) and extracted with CH2Cl2 (3 × 5 mL). The combined organic layers were dried (Na2SO4). After evaporation of the solvent, the residue was applied to FC (silica gel, column 2 × 15 cm), which was prewashed with CH2Cl2 and eluted with CH2Cl2-acetone (stepwise elution from 98:2 to 9:1) to give a colorless foam (195 mg, 65%). TLC (D): Rf 0.60. UV (MeOH): 236 (31200), 319 (44800). 1H NMR (d6-DMSO): 0.94 (6H, m, CH3); 1.28, 1.99 (8H, m, 4 CH2); 2.31 (1H, m, 2′-HR); 2.77 (1H, m, 2′-Hβ); 3.01, 3.53 (4H, m, NCH2); 3.65-3.67 (8H, m, 5′H2, OCH3); 3.94 (1H, m, 3′-H); 5.32 (1H, d, J ) 5.1, 3′OH); 6.56 (1H, m, 1′-H); 6.68-7.44 (23H, m, arom. H); 8.05 (1H, s, 3-H); 8.85 (1H, s, HCdN). Anal. Calcd. for C53H57N7O7 (904.1): C, 70.41; H, 6.35; N, 10.85. Found: C, 70.08; H, 6.41; N, 10.77. 1-[2-Deoxy-5-O-(4,4′-dimethoxytriphenylmethyl)β-D-erythro-pentofuranosyl]-4-(N,N-dibutylaminomethylidene)-6-[(diphenylcarbamoyl)oxy]-1H-pyrazolo[3,4-d]pyrimidine 3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (10). A solution of compound 9 (200 mg, 0.22 mmol) in CH2Cl2 (2 mL) was preflushed with argon and kept under argon atmosphere. Then, 2-cyanoethyldiisopropylphosphoramidochloridite (233 µL, 1.04 mmol) and N,N-diisopropylethylamine (88 µL, 0.51 mmol) were added at room temperature. Stirring was continued for 45 min. An aqueous solution of 5% NaHCO3 (10 mL) was added. The mixture was shaken, the layers were separated, and the aqueous layer was extracted with CH2Cl2 (3 × 15 mL). The combined organic extracts were dried (Na2SO4), filtered, evaporated, and applied to FC (silica gel, column 2 × 10 cm, CH2Cl2-EtOAc (85:15) containing a few drops of Et3N) yielding a colorless foam (196 mg, 80%). TLC (F): Rf 0.95.31P NMR (CDCl3): 149.6, 149.9. RESULTS AND DISCUSSION

Synthesis of Monomers. Earlier, 8-aza-7-deaza-2′deoxyisoguanosine (4) has been synthesized from 8-aza-

Scheme 1

2-chloro-7-deaza-2′-deoxyadenosine (11) by photochemical substitution of the 2-chloro substituent (23). As this synthetic route is limited to small scale preparation, the nucleoside 4 was now prepared by deamination of the 4,6-diamino-1-(2-deoxy-β-D-erythro-pentofuranosyl)-1Hpyrazolo[3,4-d]pyrimidine 5 (25) as it was reported for the ribonucleoside 2a (26). Selective deamination of compound 5 at position-2 with sodium nitrite and glacial acetic acid furnished the nucleoside 4 (Scheme 1). To investigate the stability of the glycosylic bond of compound 4, it was hydrolyzed in 0.5 M HCl at room temperature, and the kinetic of hydrolysis was followed UV-spectrophotometrically at 268 nm leading to a halflife (τ) of 12.5 min. Compared to the acid-labile purine nucleoside 2b (τ ) 14 min, 0.1 M HCl, 25 °C) (27) it possesses a significantly more stable glycosylic bond. This favorable property makes this compound to a useful substitute for 2′-deoxyisoguanosine as depurination can be avoided during oligonucleotide synthesis or longer storage of oligonucleotides. Next, the conformational analysis of the 2′-deoxyribose moiety of 4 was performed using the program PSEUROT 6.2 (28, 29) which calculates the conformational parameters from 1H,1H coupling constants and relates them to the corresponding mole fractions of the conformers. The populations of the staggered rotamers across the C(4′)C(5′) bond were calculated according to Westhof et al. (30). Table 1 summarizes the coupling constants and the conformational data. For comparison, the data of 2′deoxyisoguanosine (2b) and 2′-deoxyguanosine (1b) as well as of 7-deaza-2′-deoxyisoguanosine (3) are listed in Table 1. While the population of N vs S conformers is similar in all cases, the nucleosides 1b, 2b, and 3 prefer the γg+ (+sc) conformation around the C(4′)-C(5′) bond and for the base-modified compound 4 the +sc-population is reduced to 39%. Earlier, the amino group of 2′-deoxyisoguanosine (2b) was protected with an amidine function, and the 2-oxo group with a diphenylcarbamoyl residue (27). The same protocol was now applied to compound 4. Three different amidine residues were employed which lead on one hand to an increase of the stability of the glycosylic bond and on the other hand to a greater lipophilicity, thus facilitating chromatographic separation (24). Treatment of compound 4 with N,N-dimethylformamide dimethylacetal furnished 12; the isomeric dibutylformamide dimethylacetates gave compounds 6 and 13 while the reaction

1046 Bioconjugate Chem., Vol. 12, No. 6, 2001

Seela and Kro¨schel

Table 2. Half-Life Values (τ) of Deprotection of 8-Aza-7-deaza-2′-deoxyisoguanosine Derivatives in 25% Aqueous Ammonia at 40 ˚Ca compound

τ [min]

compound

τ [min]

12 13

1 82

6 7 8

38 29 45

a

Data observed UV-spectrophotometrically.

Scheme 2a

Figure 1. Structures of (a) Gd-quartet, and (b) isoGd-pentaplex a (i) N,N-Dibutylformamide dimethylacetal/methanol; 40 ˚C; 2 h; (ii) DPC-Cl/pyridine/iPr2EtN; rt; 1 h; (iii) DMT-Cl/pyridine; 30 ˚C; 45 min; (iv) 2-cyanoethyldiisopropylphosphoramidochloridite/iPr2EtN; rt; 45 min.

with diphenylcarbamoyl chloride in pyridine furnished compound 7.

To test the applicability of the protecting groups for oligonucleotide synthesis, their stability was studied under alkaline conditions. The kinetics of deprotection were followed UV-spectrophotometrically in 25% aqueous ammonia (Table 2). As the formamidine 12 was found to be rather labile and the isobutylamidine 13 was too stable to be used, thus the butylamidine 6 was chosen for further reactions. Table 3.

13C

NMR Chemical Shifts of 8-Aza-7-deaza-2′-deoxyisoguanosine Derivatives (d6-DMSO, 25 ˚C)

C(3)a C(7)b 4 (23) 6 7 8 9 a

The resulting amidine 6 was then blocked at the 2-oxo group with the diphenylcarbamoyl residue (33). This protecting group has already been used for the 2-oxoprotection of related purine nucleosides (27, 34-36). From the NMR spectra it was concluded that the DPCgroup is protecting the 2-oxo function as the chemical shift (δ) of C(3a) is remarkably shifted (downfield) about 7 ppm by introduction of a DPC group. An additional change of the C(4)-signal can also be observed. Nevertheless, this is not an unambiguous proof since protection at one of the pyrimidine nitrogens formed by tautomerization cannot be completely ruled out as such reactions have been observed in the case of 2-aza-2′-deoxyinosine by Fernandez-Forner et al. (37). Compound 8 was then converted into the 5′-O-(dimethoxytriphenylmethyl)protected ((MeO)2Tr) derivative 9 under standard conditions (38) (Scheme 2). Finally, the phosphoramidite 10 was prepared from 9 by treatment with 2-cyanoethyldiisopropylphosphoramidochloridite in the presence of N,Ndiisopropylethylamine under argon atmosphere. All compounds were characterized by 1H and 13C NMR spectroscopy (see Table 3 and Experimental Procedures) as well as by elemental analysis. The Self-Assembly of Oligonucleotides Containing Short Runs of 8-Aza-7-deaza-2′-deoxyisoguanosine (4). The formation of oligonucleotide aggregates containing 2′-deoxyisoguanosine is well established

134.7 136.2 133.4 141.5 141.8

C(3a)a C(5)b

C(4)a C(6)b

C(6)a C(2)b

C(7a)a C(4)b

C(1′)

C(2′)

C(3′)

C(4′)

C(5′)

92.6 98.2 99.1 106.1 106.4

157.0d

155.2d

159.2d 155.3 158.5d 158.9d

157.8d 159.3 157.7d 157.9d

e e 141.8 155.6 155.9

84.3 83.7 83.7 83.5 83.2

c c c c c

71.2 71.2 71.0 70.7 74.2

87.6 87.4 87.6 87.3 85.1

62.5 62.6 62.4 62.2 64.4

Systematic numbering. b Purine numbering. c Superimposed by DMSO.

d

Tentative assignment. e Not detected.

HCdN

CO

159.8 151.0 163.6 163.8

151.2

Quadruplex and Pentaplex Self-Assemblies

Bioconjugate Chem., Vol. 12, No. 6, 2001 1047

Figure 2. Ion exchange HPLC elution profile of 5′-d(T444T2) (14) at 30 °C in the presence of Na+ (a), Rb+ (b), and Cs+(c); conditions: see Experimental Procedures.

Figure 3. Ion-exchange HPLC elution profiles of 5′-d(T4G4T2) (16) at 30 °C in the presence of Na+ (a), Rb+ (b), and Cs+ (c); conditions: see Experimental Procedures.

(16, 17, 22). The 12-mer d(T4isoG4T4) forms two wellseparated peaks when applied to an ion-exchange resin (Dionex NucleoPac PA-100) in the presence of sodium ions (18). The fast migrating peak refers to the single strand, while the slow-migrating one represents the aggregate. When the oligomer d(T4G4T4) is analyzed under identical conditions, two peaks are also detected showing similar retention times (7). Again, the first one is the monomer while the second one was assigned to be the tetramer. So far oligonucleotides containing 2′deoxyisoguanosine show a similar tendency to form aggregates as those containing 2′-deoxyguanosine. An equivalent study as performed by our laboratory applying ion-exchange chromatography and using the mobility differences of the monomeric and tetrameric assemblies (17) was performed later by gel electrophoresis (39). The complex formation of d(T4isoG4T4) was also studied in the presence of other monovalent cations indicating that rather stable isoguanine conjugates are formed in the presence of cesium ions, while in the case of the guaninecontaining oligomer d(T4G4T4) fairly unstable complexes did occur. The stabilizing ability of dG-tetrads by Cs+ is ranking in the range of Li+ (40), and in this case K+ has the optimum ionic radius for the formation of the tetraplex (41). The aggregate stability of oligonucleotides

containing 2′-deoxyisoguanosine (2b) was also investigated in dependence of the ion radii of the alkali cations. However, little attention was given by our laboratory to a discontinuity of the complex stabilities between the potassium-, rubidium-, and cesium-ions (7). Chaput and Switzer found that oligonucleotides containing short runs of 2′-deoxyisoguanosine residues such as d(T4iG4T) or d(T8iG4T) form pentameric assemblies in the presence of cesium ions while in the presence of potassium ions tetrameric complexes or mixtures of tetrameric and pentameric structures are observed (22) (Figure 1). As nitrogen-7 is not participating in the hydrogenbonding pattern of the 2′-deoxyisoguanosine assembly (19) the 8-aza-7-deaza-2′-deoxyisoguanosine, which usually forms stronger hydrogen bonds upon base pairing than 2′-deoxyisoguanosine (2b) does, was incorporated in an oligonucleotide containing a short run of compound 4 residues. A decanucleotide 5′-d(T4(c7z8iG)4T2) (14), containing four consecutive nucleoside 4 residues as well as tails of 4 dT’s at the 5′-terminus and two dT’s at the 3′-end, was synthesized. The oligonucleotides 5′-d(T4isoG4T2) (15) and 5′-d(T4G4T2) (16) having a similar structure, but containing either four 2′-deoxyisoguanosine or four 2′-deoxyguanosine residues, were synthesized for comparison.

1048 Bioconjugate Chem., Vol. 12, No. 6, 2001

Figure 4. Structures of (a) c7z8iGd-tetraplex, and (b) c7z8iGdpentaplex.

At first, the self-assembly of 5′-d(T4(c7z8iG)4T2) (14) was studied. The oligonucleotide was stored in 1.0 M MCl solution (M: Na+, Rb+, Cs+) at -23 °C for 16 h. Subsequently, the solution was injected onto an ion-exchange Gen-Pak Fax column thermostated at 30 °C. The ionexchange chromatography of the sodium complex performed in the presence of NaCl is shown in Figure 2a. The peak with the short retention time contains the single-stranded oligomer while the slower migrating peak represents the conjugate. The same experiment (storage and ion exchange chromatography) was then performed with the 2′-deoxyguanosine-containing oligonucleotide 5′d(T4G4T2) (16) showing the same sequence, but c7z8iGd was replaced by dG. This HPLC profile (Figure 3a) shows also two peaks when Na+ is the counterion. Earlier

Seela and Kro¨schel

studies have shown that the faster migrating peak results from the single-stranded oligonucleotide while the slower one represents the tetramer. As the peak of the aggregate formed by oligomer 14 shows a similar retention time as that of 16 (33 min vs 30 min), the complex of the aggregate was also assigned to be a tetraplex. The same protocol was performed with Rb+ as counterion (see Figure 2b); according to this figure a tetraplex is also formed in this case. The chromatographic mobility changed when Cs+ was used. Incubation of 14 in 1.0 M CsCl and chromatography in a running buffer containing CsCl leads to a chromatographic profile shown in Figure 2c. The mobility of the second peak of oligonucleotide 14 shows a significantly increased retention time over that of the sodium complex (37 min vs 33 min) while that of 16 remains unaffected. From this observation it is concluded that the aggregate containing 8-aza-7-deaza2′-deoxyisoguanosine is a tetraplex in the presence of Na+, K+, and Rb+ while a pentaplex is formed in Cs+. This observation is in line with findings on oligonucleotide assemblies containing 2′-deoxyisoguanosine (22). From the series of experiments shown in Figure 2a-c and 3a-c, it is apparent that the Cs+ complex of 14 has a similar stability as the sodium complex while the complex of the oligomer 16 containing 2′-deoxyguanosine becomes less stable in an aqueous CsCl solution. As there was no unambiguous proof whether the lower mobility of this complex formed in the presence of Cs+ results from the formation of a pentameric structure or other phenomena induced by the pyrazolo[3,4-d]pyrimidine system we searched for experimental conditions which allow the detection of both types of complexes in the same solution. The experiments were performed in the presence of a counterion which is smaller than Cs+ (1.65 Å) but significantly larger than sodium (0.98 Å) (Figure 4). For this purpose Rb+ was chosen exhibiting an ion radius of 1.49 Å. However, as shown in Figure 2b, both complexes are not formed in the case of the oligonucletotide 14. Therefore, we went back to the oligonucleotide 5′-d(T4isoG4T2) (15) containing the purine nucleoside 2′-deoxyisoguanosine. This oligomer forms a tetraplex in the presence of Na+ and a pentaplex in the presence of Cs+ (Figure 5a,b). Then, the experiments were performed in the presence of RbCl which was used for incubation and in the chromatographical runs (Figure 5c). In this case two well-separated slow-migrating peaks

Figure 5. Ion-exchange HPLC elution profiles of 5′-d(T4isoG4T2) (15) at 30 °C in the presence of Na+ (a), Cs+ (b), and Rb+ (c); conditions: see Experimental Procedures.

Quadruplex and Pentaplex Self-Assemblies

Bioconjugate Chem., Vol. 12, No. 6, 2001 1049

aggregate peakssthe tetramer of 16 and the pentamer of 15. This is actually the case. Apart from the rather high amount of the monomeric oligonucleotide of 5′d(T4G4T2) (16) and its tetrameric assembly which is present in aqueous CsCl solution the pentameric assembly of 15 is observed. As the mobilities of the selfassembled aggregates of 15 and 16 (Figure 7) look similar to those of Figure 5c it is obvious that the peaks of the tetraplexes migrate significantly faster than those of the pentaplexes, no matter whether the tetraplex is formed by dG or an isoGd quartet. It also confirms the presence of two complexes, the tetraplex and the pentaplex of 15, in the presence of Rb+. The difference of the complex stability observed for the various cations in the case of the oligonucleotide 14 containing 8-aza-7-deaza-2′-deoxyisoguanosine and 15 containing 2′-deoxyisoguanosine was unexpected. It might be traced back to conformational differences of the monomeric pyrazolo[3,4-d]pyrimidine vs the purine nucleoside residues (high-anti vs anti conformation) (42). This can affect the thermodynamic stability of the aggregates and/or the kinetics of complex formation and decay. In this regard it is worth mentioning that the selfaggregation of 2′-deoxyisoguanosine containing oligonucleotides occurs much faster than that of containing 2′-deoxyguanosine or 8-aza-7-deaza-2′-deoxyisoguanosine. CONCLUSION Figure 6. Structure of (a) isoGd-tetraplex, and (b) pentaplex.

Figure 7. Ion-exchange HPLC elution profiles of 5′-d(T4isoG4T2) (15) and 5′-d(T4G4T2) (16) in the presence of Cs+; conditions: see Experimental Procedures.

appeared. The faster migrating one was considered to be the tetraplex while the slower one represents the pentameric structure. The mobility change of the conjugates arises from the increased number of negative charges of the pentameric complex resulting in a stronger binding to the ion-exchange resin than the less charged tetrameric complex. To show that the tetrameric complex can be distinguished from the pentamer another experiment was performed. It is now well established that 2′-deoxyisoguanosine-containing oligomers form pentameric assemblies in the presence of Cs+ ions while 2′-deoxyguanosine forms exclusively tetraplexes even in aqueous CsCl solution. Thus, a chromatographical run of a mixture of the oligonucleotides 5′-d(T4G4T2) (16) and 5′-d(T4isoG4T2) (15) in the presence of CsCl should lead to two

Oligonucleotides containing short runs of 8-aza-7deaza-2′-deoxyisoguanosine (4) form tetrameric as well as pentameric self-assemblies, which can be considered as oligonucleotide conjugates hold together by hydrogen bonds and coordination forces (metal ions) and not by covalent linkages. Those conjugates require the presence of a central cation, as it has been observed for oligomers containing 2′-deoxyisoguanosine (2b) and 2′-deoxyguanosine (1b). In the latter cases it was shown that the metal ion is surrounded by the tetrameric or pentameric assembly of the nucleobases in a sandwich type structure (X-ray) (43). Such a structure is also expected for the selfassemblies of 8-aza-7-deaza-2′-deoxyisoguanosine and its oligonucleotides. The formation of the various complexes (tetraplexes or pentaplexes) depends on the size of the cation. Cs+ generates a pentaplex while the smaller cations (Na+: 0.98 Å, Rb+: 1.49 Å, Cs+: 1.65 Å) can induce a tetraplex structure. In the case of the 2′deoxyisoguanosine containing oligonucleotide 15 a tetraplex as well as a pentaplex can be observed in the same solution when Rb+ is the counterion, while the selfassembly formed by the 8-aza-7-deaza-2′-deoxyisoguanosine containing oligonucleotide 5′-d(T444T2) yields only one species which is expected to be the tetraplex. ACKNOWLEDGMENT

We thank Mr. Yang He and Dr. Helmut Rosemeyer for the NMR spectra, Mr. Harald Debelak for the PSEUROT calculation, and Mrs. Elisabeth Feiling for the oligonucleotide synthesis. Financial support by the Deutsche Forschungsgemeinschaft is gratefully acknowledged. LITERATURE CITED (1) Gellert M., Lipsett M. N., Davies D. R. (1962) Helix Formation by Guanylic Acid. Proc. Natl. Acad. Sci U.S.A. 48, 2013-2018. (2) Zimmerman S. B., Cohen G. H., Davies D. R. (1975) X-ray Fiber Diffraction and Model-Building Study of Polyguanylic Acid and Polyinosinic Acid. J. Mol. Biol. 92, 181-192.

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