1972
Bioconjugate Chem. 2007, 18, 1972–1980
1-Phenylethynylpyrene (1-PEPy) as Refined Excimer Forming Alternative to Pyrene: Case of DNA Major Groove Excimer Irina V. Astakhova,† Andrei D. Malakhov,† Irina A. Stepanova,† Alexey V. Ustinov,† Stanislav L. Bondarev,‡ Alexander S. Paramonov,† and Vladimir A. Korshun*,† Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, Russia, and Institute of Molecular and Atomic Physics, Nezavisimosti av. 70, 220072 Minsk, Belarus. Received July 24, 2007
1-Phenylethynylpyrene fluorochrome was studied as meta- and para-derivatives of arabino-uridine-2′-carbamates in ss and dsDNA. 1-PEPy showed red-shifted emission and increased fluorescence quantum yield compared to pyrene. Although 1-PEPy has very short excited lifetime (10-2 M) is, however, 2–3 orders of magnitude higher compared to pyrene. This correlates with its short excited lifetime (7.72 ns for 1-PEPy in EtOH (52)). The absorbance and fluorescence of 1-PEPy derivatives on DNA are approximately 50 nm and 30 nm red-shifted, respectively, in comparison with pyrene derivatives; the first fluorescence maximum of 1-PEPy in oligonucleotides is within 400–405 nm (48–52). These spectral features reduce 100-fold the 1-PEPy detection limit with respect to that of pyrene (52). 1-PEPy is able to form strong (48, 50, 51) or weak (49, 52) excimers on DNA. With a short excited lifetime taken into account, the pronounced PEPy excimer should be evidence not only for mutual attainability, but rather preassociation of two PEPy molecules. Thus, an excimer-forming 1-PEPy fluorochrome could become a label of choice for some structural studies of biomolecules. However, photophysical properties of 1-PEPy on nucleic acids have not been reported. Our aim was to explore further 1-PEPy on DNA and to elucidate its potential as an excimer-forming fluorescent probe. Pyrene attached to the sugar part of a nucleoside in dsDNA can form interstrand excimers located in +2 zipper (24, 27, 35), +1 zipper (27), or -1 zipper (8) mode (Table 1). We have
10.1021/bc700280h CCC: $37.00 2007 American Chemical Society Published on Web 09/27/2007
1-Phenylethynylpyrene Excimer on DNA Table 1. Reported Examples of Interstrand Pyrene Excimers on DNA from Sugar-Modified Nucleosides
chosen the case of the +2 zipper major groove excimer formed by 1-pyrenemethyl ara-uridine-2′-carbamate (24), replaced pyrene with 1-PEPy, and studied the structural requirements for the 1-PEPy excimer.
EXPERIMENTAL PROCEDURES General. Reagents obtained from commercial suppliers were used as received; 2′-O-(imidazol-1-ylcarbonyl)-3′,5′-O-(tetraisopropyldisiloxan-1,3-diyl)uracil-1-β-D-arabinofuranoside (24), Pd(PPh3)4 (54), 1-ethynylpyrene (47), bis(N,N-diisopropylamino)2-cyanoethoxyphosphine (55), and diisopropylammonium tetrazolide (56) were synthesized as described. HPLC-grade DMF was distilled under reduced pressure and stored over 4 Å molecular sieves under nitrogen. DCM was always used freshly distilled over CaH2. Other solvents were used as received. 500 MHz 1H, 125.7 MHz 13C, and 202.4 MHz 31P NMR spectra were recorded on a Bruker DRX-500 spectrometer at 303 K and referenced to DMSO-d6 (2.50 ppm for 1H and 39.5 ppm for 13C), MeCN-d3 (1.96 ppm for 1H) (57), and 85% aq H3PO4 (0.00 ppm for 31P). 1H–13C gradient-selected HMQC and HMBC spectra were obtained by using 2048 (t2) × 256 (t1) complex point data sets, zero-filled to 2048 (F2) × 1024 (F1) points. The spectral widths were 13 ppm and 200 ppm for 1 H and 13C dimensions, respectively. HMBC spectra were measured with 50 ms delay for evolution of long-range couplings. 1H NMR coupling constants are reported in hertz (Hz) and refer to apparent multiplicities. High-resolution mass spectra were recorded in positive ion mode using IonSpec Fourier Transform ISR mass spectrometer (MALDI) or PE SCIEX QSTAR pulsar mass spectrometer (ESI). Melting points were determined using a Boetius heating table and are uncorrected. Analytical thin-layer chromatography was performed on Kieselgel 60 F254 precoated aluminium plates (Merck). Silica gel column chromatography was performed using Merck Kieselgel 60 0.040–0.063 mm. The oligonucleotide synthesis was carried out on a BiosSet ASM-800 instrument in a 200 nmol scale using standard manufacturer’s protocols. The
Bioconjugate Chem., Vol. 18, No. 6, 2007 1973
coupling step time for modified phosphoramidites was extended to 5 min. Oligonucleotides were isolated using 20% denaturing (7 M urea) PAGE in Tris-borate buffer, pH 8.3, and desalted by gel filtration on a Sephadex G-25 column eluting with saltless buffer. MALDI-TOF mass spectra of oligonucleotides were recorded on Bruker Ultraflex mass spectrometer in positive ion mode; a mixture (1:1 v/v) of 2,6-dihydroxyacetophenone (40 mg/mL in MeOH) and diammonium hydrogen citrate (80 mg/ mL in water) was used as a matrix. Theoretical masses of oligonucleotide conjugates were calculated using the monoisotopic element masses. UV spectra were recorded using Beckman DU520 general purpose UV/vis spectrometer. Thermal denaturation experiments were performed on a Perkin Elmer Lambda 40 UV/vis spectrometer with PTP 6 (Peltier temperature programmer) in the medium salt hybridization buffer containing 100 mM NaCl, 10 mM Na3PO4, 0.1 mM EDTA, pH 7.0 (concentration of duplex 1.0 × 10-6 M). Fluorescence spectra were obtained in same buffer using a Varian Cary Eclipse fluorescence spectrophotometer (λex 360 nm, excitation and emission slits 5 nm, concentrations of each oligonucleotide or duplex 1.0 × 10-7 M). Photophysical studies in the same buffer were performed using a large-aperture apparatus described earlier (58). The fluorescence quantum yields Φf were measured by the relative method using standards of quinine sulfate in 1 N H2SO4 (Φf 0.55 (59)). The measurements were performed at an angle of 90° to the exciting light beam. The optical density of the solutions in 1-cm quartz cuvettes at the excitation wavelength did not exceed 0.1. The values of Φf were corrected with the refractive index of the solvent; the measured refractive indexes of 1 N H2SO4 and phosphate buffer were 1.338 and 1.334 at 20 °C, respectively. Fluorescence lifetimes were determined by the method of time-correlated single-photon counting using a PRA model 3000 ns lifetime fluorometer. The parameters of the fluorescence decays were analyzed using T900 software (Edinburgh Instruments). The samples used in quantum yield and lifetime fluorescence measurements were not degassed; concentrations were 3.0 × 10-6 M. General Procedure for the Preparation of 2′-O-(3(4)-Iodobenzylaminocarbonyl)-3′,5′-O-(tetraisopropyldisiloxan-1,3diyl)uracil-1-β-D-arabinofuranosides (2a,b). To a solution of 2′-O-(imidazol-1-ylcarbonyl)-3′,5′-O-(tetraisopropyldisiloxan1,3-diyl)uracil-1-β-D-arabinofuranoside 1 (2.90 g, 5.0 mmol) in THF–MeCN, 1:1 (v/v) (60 mL) 3- or 4-iodobenzylamine (1.63 g, 7.0 mmol) was added, and the mixture was refluxed for 40 h. After conversion of the starting compound 1 is complete (monitoring by TLC, EtOAc–CHCl3, 2:3 v/v), the mixture was diluted with CHCl3 (150 mL), washed with water (100 mL), 5% citric acid (100 mL), and water (100 mL), then dried over Na2SO4 and evaporated to dryness. The residue was purified by chromatography on a silica gel column (100 g) with a step gradient of EtOAc (0f5%) in CHCl3 (v/v) as the mobile phase to give carbamates 2a,b as white amorphous foams. 2′-O-(3-Iodobenzylaminocarbonyl)-3′,5′-O-(tetraisopropyldisiloxan-1,3-diyl)uracil-1-β-D-arabinofuranoside (2a). Yield 3.50 g (94%), white foam, Rf 0.52 (EtOAc–CHCl3, 2:3 v/v). MALDI-TOF HRMS: m/z ) 768.1642 [M + Na]+, calcd for [C29H44N3O8Si2 + Na]+ 768.1604. 1H NMR (DMSO-d6, δ): 11.33 (s, 1H, H-3), 7.88 (t, 1H, J ) 6.0 Hz, OCONH), 7.58 (m, 2H, H-2″, 4″), 7.45 (d, 1H, J5,6 ) 8.1 Hz, H-6), 7.16–7.05 (m, 2H, H-5″, 6″), 6.12 (m, 1H, H-1′), 5.57 (d, 1H, J5,6 ) 8.1 Hz, H-5), 5.42 (m, 1H, H-2′), 4.38 (m, 1H, H-3′), 4.17–3.90 (m, 4H, H-5′, NCH2), 3.86 (m, 1H, H-4′), 1.10–0.92 (m, 28H, Pri). 13C NMR (DMSO-d6, δ): 163.0 (C4), 154.8 (OCO), 150.0 (C2), 142.1 (C1″), 140.6 (C6), 135.5 (2C, C2″,4″), 130.5 (C5″), 126.2 (C6″), 101.3 (C5), 94.7 (C3″), 81.0 (C1′), 79.0 (C4′), 76.1 (C2′), 72.6 (C3′), 60.6 (C5′), 43.1 (NCH2), 17.3, 17.2 (3C), 16.9, 16.8 (2C), 16.7 (CH3), 12.8, 12.4, 12.2, 11.9 (SiC).
1974 Bioconjugate Chem., Vol. 18, No. 6, 2007
2′-O-(4-Iodobenzylaminocarbonyl)-3′,5′-O-(tetraisopropyldisiloxan-1,3-diyl)uracil-1-β-D-arabinofuranoside (2b). Yield 3.14 g (84%), white foam, Rf 0.50 (EtOAc–CHCl3, 2:3 v/v). MALDI-TOF HRMS: m/z) 768.1631 [M + Na]+, calcd for [C29H44N3O8Si2 + Na]+ 768.1604. 1H NMR (DMSO-d6, δ): 11.38 (s, 1H, H-3), 7.86 (m, 1H, OCONH), 7.64 (d, 2H, J ) 8.2 Hz, H-3″,5″), 7.45 (d, 1H, J5,6 ) 8.1 Hz, H-6), 6.94 (d, 2H, J ) 8.2 Hz, H-2″,6″), 6.14 (m, 1H, H-1′), 5.59 (d, 1H, J5,6 ) 8.1 Hz, H-5), 5.38 (m, 1H, H-2′), 4.39 (m, 1H, H-3′), 4.14 (m, 1H, 2J ) 15.9 Hz, JNHCH ) 6.4 Hz, NCHH), 4.08 (m, 1H, 2J5′a,5′b ) 13.0 Hz, J4′,5′a ) 3.3 Hz, H-5′a), 4.02 (m, 1H, 2J ) 15.9 Hz, JNHCH ) 5.5 Hz, NCHH), 3.94 (m, 1H, 2J5′a,5′b ) 13.0 Hz, J4′,5′b ) 2.7 Hz, H-5′b), 3.86 (m, 1H, H-4′), 1.10–0.93 (m, 28H, Pri). 13 C NMR (DMSO-d6, δ): 163.0 (C4), 154.9 (OCO), 150.0 (C2), 140.9 (C6), 139.3 (C1″), 137.0 (2C, C3″,5″), 129.0 (2C, C2″,6″), 101.3 (C5), 92.4 (C4″), 81.2 (C1′), 78.8 (C4′), 76.1 (C2′), 72.7 (C3′), 60.7 (C5′), 43.2 (NCH2), 17.3, 17.2, 17.1 (2C), 16.9, 16.8 (2C), 16.7 (CH3), 12.8, 12.4, 12.2, 11.9 (SiC). General Procedure for the Preparation of 2′-O-[3(4)(Pyren-1-ylethynyl)phenylmethylaminocarbonyl]-3′,5′-O-(tetraisopropyldisiloxan-1,3-diyl)uracil-1-β-D-arabinofuranosides (3a,b). To a solution of 2′-O-(3(4)-iodobenzylaminocarbonyl)-3′,5′-O-(tetraisopropyldisiloxan-1,3-diyl)uracil1-β-D-arabinofuranoside 2a(2b) (1.49 g, 2.0 mmol), 1-ethynylpyrene (588 mg, 2.4 mmol) and triethylamine (560 µL, 4.0 mmol) in DMF (40 mL) Pd(PPh3)4 (116 mg, 0.10 mmol) and CuI (38 mg, 0.20 mmol) were subsequently added, and the mixture was stirred under argon for 16 h. After completion of conversion of the starting nucleoside 2 (monitoring by TLC in EtOAc), the mixture was poured into CHCl3 (150 mL). The resulting solution was washed with 3% EDTA-Na2 (4 × 100 mL) and water (4 × 100 mL), dried over Na2SO4, and evaporated to dryness. The residue was purified by chromatography on a silica gel column (150 g) with a step gradient of MeOH (0f3%) in CHCl3 (v/v) as the mobile phase to give 1-PEPy derivatives 3a,b as yellow amorphous foams. Compounds were dissolved in DCM (5 mL), and the solution was added dropwise to hexane (50 mL), the solid precipitated was filtered off, washed with hexane, and dried in Vacuo. 2′-O-[3-(Pyren-1-ylethynyl)phenylmethylaminocarbonyl]3′,5′-O-(tetraisopropyldisiloxan-1,3-diyl)uracil-1-β-D-arabinofuranoside (3a). Yield 1.60 g (95%), yellow amorphous solid, Rf 0.46 (EtOAc–toluene, 1:1 v/v). MALDI-TOF HRMS: m/z ) 866.3266 [M + Na]+, calcd for [C47H53N3O8Si2 + Na]+ 866.3263. 1H NMR (DMSO-d6, δ): 11.37 (s, 1H, H-3), 8.61 (d, 1H, J9′″,10′″ ) 9.1 Hz, H-10″), 8.37 (m, 3H, H-6′″,8′″,9′″), 8.33 (m, 1H, J2′″,3′″ ) 8.0 Hz, H-3′″), 8.26 (m, 2H, H-2′″,5′″), 8.23 (m, 1H, J4′″,5′″ ) 8.9 Hz, H-4′″), 8.14 (apparent t, 1H, J6′″,7′″ ) J7′″,8′″ ) 7.6 Hz, H-7′″), 7.97 (m, 1H, OCONH), 7.64 (d, 1H, J ) 7.6 Hz), 7.61 (s, 1H), 7.49 (d, 1H, J ) 8.0 Hz) (H-6, H-2″, H-4″), 7.44 (apparent t, 1H, J ) 7.6 Hz, H-5″), 7.23 (d, 1H, J ) 7.6 Hz, H-6″), 6.15 (br.s, 1H, H-1′), 5.60 (d, 1H, J5,6 ) 8.2 Hz, H-5), 5.46 (m, 1H, H-2′), 4.38 (m, 1H, H-3′), 4.28 (m, 1H, 2 J ) 15.9 Hz, JNHCH ) 6.4 Hz, NCHH), 4.17 (m, 1H, 2J ) 15.9 Hz, JNHCH ) 5.8 Hz, NCHH), 4.07 (m, 1H, 2J5′a,5′b ) 12.8 Hz, J4′,5′a ) 3.0 Hz, H-5′a), 3.92 (m, 1H, 2J5′a,5′b ) 12.8 Hz, J4′,5′b ) 2.4 Hz, H-5′b), 3.86 (m, 1H, H-4′), 1.07–0.87 (m, 28H, Pri). 13C NMR (DMSO-d6, δ): 163.0 (C4), 154.9 (OCO), 150.1 (C2), 140.3 (C1″), 140.2 (C6), 131.2 (C10a′″), 131.1 (C3a′″), 130.9 (C5a′″), 130.6 (C8a′″), 130.0 (2C, C2″, C4″), 129.6 (C2′″), 128.9 (2C, C9′″, C5″), 128.5 (C4′″), 127.4 (C5′″), 127.3 (C6″), 126.8 (C7′″), 126.0 (2C, C6′″, C8′″), 125.0 (C3′″), 124.8 (C10′″), 123.7 (C10b′″), 123.5 (C10c′″), 122.4 (C3″), 116.8 (C1′″), 101.4 (C5), 95.2 (Cb), 88.2 (Ca), 80.4 (C1′), 78.7 (C4′), 75.9 (C2′), 72.5 (C3′), 60.6 (C5′), 43.5 (NCH2), 17.3, 17.2, 17.1 (2C), 16.8 (3C), 16.7 (CH3), 12.8, 12.4, 12.2, 11.8 (SiC).
Astakhova et al.
2′-O-[4-(Pyren-1-ylethynyl)phenylmethylaminocarbonyl]3′,5′-O-(tetraisopropyldisiloxan-1,3-diyl)uracil-1-β-D-arabinofuranoside (3b). Yield 1.58 g (93%), yellow amorphous solid, Rf ) 0.43 (EtOAc–toluene, 1:1 v/v). MALDI-TOF HRMS: m/z) 866.3223 [M + Na]+, calcd for [C47H53N3O8Si2 + Na]+ 866.3263. 1H NMR (DMSO-d6, δ): 11.44 (s, 1H, H-3), 8.63 (d, 1H, J9′″,10′″ ) 9.1 Hz, H-10′″), 8.44–8.20 (m, 7H, H-2′″–6′″,8′″,9′″), 8.14 (apparent t, 1H, J6′″,7′″ ) J7′″,8′″ ) 7.6 Hz, H-7′″), 7.96 (m, 1H, OCONH), 7.71 (d, 2H, J2″,3″) J5″,6″) 8.1 Hz, H-3″, H-5″), 7.48 (d, 1H, J5,6 ) 8.0 Hz, H-6), 7.27 (d, 2H, J2″,3″ ) J5″,6″ ) 8.1 Hz, H-2″, H-6″), 6.17 (m, 1H, H-1′), 5.64 (d, 1H, J5,6 ) 8.0 Hz, H-5), 5.42 (m, 1H, H-2′), 4.43 (m, 1H, H-3′), 4.29 (m, 1H, 2J ) 15.9 Hz, JNHCH ) 6.4 Hz, NCHH), 4.16 (m, 1H, 2J ) 15.9 Hz, JNHCH ) 5.5 Hz, NCHH), 4.10 (m, 1H, 2J5′a,5′b ) 13.1 Hz, J4′,5′a ) 3.4 Hz, H-5′a), 3.95 (m, 1H, 2 J5′a,5′b ) 13.1 Hz, J4′,5′b ) 2.7 Hz, H-5′b), 3.88 (m, 1H, H-4′), 1.12–0.92 (m, 28H, Pri). 13C NMR (DMSO-d6, δ): 163.1 (C4), 155.0 (OCO), 150.1 (C2), 140.4 (C1″), 140.3 (C6), 131.6 (2C, C3″, C5″), 131.1 (C10a′″), 131.0 (C3a′″), 130.9 (C5a′″), 130.6 (C8a′″), 129.6 (C2′″), 128.9 (C9′″), 128.4 (C4′″), 127.3 (C5′″), 127.0 (2C, C2″, C6″), 126.8 (C7′″), 126.0 (2C, C6′″, C8′″), 125.0 (C3′″), 124.9 (C10′″), 123.8 (C10b′″), 123.5 (C10c′″), 120.8 (C4″), 116.9 (C1′″), 101.4 (C5), 95.4 (Cb), 88.0 (Ca), 81.0 (C1′), 78.6 (C4′), 75.9 (C2′), 72.6 (C3′), 60.6 (C5′), 43.6 (NCH2), 17.3, 17.2 (3C), 16.9 (3C), 16.7 (CH3), 12.9, 12.4, 12.2, 11.9 (SiC). General Procedure for the Preparation of 2′-O-[3(4)(Pyren-1-ylethynyl)phenylmethylaminocarbonyl]uracil-1-βD-arabinofuranosides (4a,b). To a solution of 2′-O-[3(4)(pyren-1-ylethynyl)phenylmethylaminocarbonyl]-3′,5′-O(tetraisopropyldisiloxan-1,3-diyl)uracil-1-β-Darabinofuranoside 3a(3b) (1.69 g, 2 mmol) in THF (5 mL), Et3N · 3HF (0.814 mL, 5 mmol) was added, and the mixture was kept at room temperature overnight. The mixture was diluted with CHCl3 (20 mL), and the title compound was precipitated by addition of Et2O (20 mL). The solid was filtered off, washed with CHCl3 (15 mL) and Et2O (2 × 20 mL), and dried in Vacuo to afford the pure nucleoside 4a(4b). 2′-O-[3-(Pyren-1-ylethynyl)phenylmethylaminocarbonyl]uracil-1-β-D-arabinofuranoside (4a). Yield 1.14 g (95%), yellow crystals, Rf 0.32 (MeOH–toluene, 1:9 v/v), mp 118–121 °C (96% aq ethanol). MALDI-TOF HRMS: m/z ) 624.1757 [M + Na]+, calcd for [C35H27N3O7 + Na]+ 624.1741. 1H NMR (DMSO-d6, δ): 11.31 (s, 1H, H-3), 8.63 (d, 1H, J9′″,10′″ ) 9.1 Hz, H-10′″), 8.39 (m, 3H, H-6′″,8′″,9′″), 8.33 (m, 1H, J2′″,3′″ ) 8.2 Hz, H-3′″), 8.29 (m, 1H, H-2′″), 8.27 (m, 1H, H-5′″), 8.23 (m, 1H, J4′″,5′″ ) 8.9 Hz, H-4′″), 8.15 (apparent t, 1H, J6′″,7′″ ) J7′″,8′″ ) 7.6 Hz, H-7′″), 7.94 (m, 1H, OCONH), 7.65 (m, 3H, H-6, H-2″, H-4″), 7.46 (apparent t, 1H, J ) 7.6 Hz, H-5″), 7.25 (d, 1H, J ) 7.6 Hz, H-6″), 6.18 (d, 1H, J ) 4.9 Hz, H-1′), 5.75 (d, 1H, J3′,OH ) 4.9 Hz, 3′-OH), 5.62 (d, 1H, J5,6 ) 8.0 Hz, H-5), 5.09 (m, 1H, H-2′), 5.01 (t, 1H, J5′,OH ) 5.5 Hz, 5′-OH), 4.23 (m, 2H, NHCH2), 4.14 (m, 1H, H-3′), 3.80 (m, 1H, H-4′), 3.73–3.59 (m, 2H, H-5′). 13C NMR (DMSO-d6, δ): 163.1 (C4), 154.9 (OCO), 150.1 (C2), 141.6 (C6), 140.1 (C1″), 131.2 (C10a′″), 131.1 (C3a′″), 130.8 (C5a′″), 130.6 (C8a′″), 130.1 (2C, C2″, C4″), 129.7 (C2′″), 129.0 (2C, C5″, C9′″), 128.5 (C4′″), 127.5 (C5′″), 127.3 (C6″), 126.9 (C7′″), 126.1 (3C, C6′″, C8′″), 125.0 (C3′″), 124.9 (C10′″), 123.7 (C10b′″), 123.5 (C10c′″), 122.4 (C3″), 116.8 (C1′″), 100.9 (C5), 95.2 (Cb), 88.3 (Ca), 83.9 (C4′), 82.9 (C1′), 77.2 (C2′), 73.0 (C3′), 60.2 (C5′), 43.6 (NCH2). 2′-O-[4-(Pyren-1-ylethynyl)phenylmethylaminocarbonyl]uracil-1-β-D-arabinofuranoside (4b). Yield 1.12 g (93%), yellow crystals, Rf 0.30 (MeOH–toluene, 1:9 v/v), mp 114–115 °C (96% aq ethanol). MALDI-TOF HRMS: m/z ) 624.1722 [M + Na]+, calcd for [C35H27N3O7 + Na]+ 624.1741. 1H NMR
1-Phenylethynylpyrene Excimer on DNA
(DMSO-d6, δ): 11.38 (s, 1H, H-3), 8.63 (d, 1H, J9′″,10′″ ) 8.9 Hz, H-10′″), 8.37 (m, 3H, H-6′″,8′″,9′″), 8.33 (m, 1H, J2′″,3′″ ) 8.0 Hz, H-3′″), 8.27 (m, 2H, H-2′″,5′″), 8.23 (m, 1H, J4′″,5′″ ) 8.9 Hz, H-4′″), 8.15 (apparent t, 1H, J6′″,7′″ ) J7′″,8′″ ) 7.6 Hz, H-7′″), 7.94 (m, 1H, OCONH), 7.72 (d, 2H, J2″,3″ ) J5″,6″ ) 8.1 Hz, H-3″, H-5″), 7.65 (d, 1H, J5,6 ) 8.2 Hz, H-6), 7.27 (d, 2H, J2″,3″) J5″,6″) 8.1 Hz, H-2″, H-6″), 6.19 (d, 1H, J ) 5.2 Hz, H-1′), 5.75 (d, 1H, J3′,OH) 4.9 Hz, 3′-OH), 5.65 (d, 1H, J5,6 ) 8.2 Hz, H-5), 5.11 (m, 1H, H-2′), 5.02 (t, 1H, J5′,OH) 5.6 Hz, 5′-OH), 4.32 (m, 1H, 2J ) 10.4 Hz, JNHCH ) 4.8 Hz, NHCHH), 4.19–4.10 (m, 2H, NHCHH, H-3′), 3.80 (m, 1H, H-4′), 3.73–3.59 (m, 2H, H-5′). 13C NMR (DMSO-d6, δ): 163.2 (C4), 155.0 (OCO), 150.1 (C2), 141.6 (C6), 140.4 (C1″), 131.6 (2C, C3″, C5″), 131.1 (C10a′″), 131.0 (C3a′″), 130.9 (C5a′″), 130.6 (C8a′″), 129.6 (C2′″), 128.9 (C9′″), 128.4 (C4′″), 127.3 (C5′″), 127.0 (2C, C2″, C6″), 126.8 (C7′″), 126.0 (2C, C6′″, C8′″), 125.0 (C3′″), 124.9 (C10′″), 123.8 (C10b′″), 123.5 (C10c′″), 120.8 (C4″), 116.8 (C1′″), 101.0 (C5), 95.4 (Cb), 88.0 (Ca), 83.6 (C4′), 82.8 (C1′), 77.2 C2′), 72.8 (C3′), 60.1 (C5′), 43.6 (NCH2). General Procedure for the Preparation of 5′-O-(4,4′-Dimethoxytrityl)-2′-O-[3(4)-(pyren-1-ylethynyl)phenylmethylaminocarbonyl]uracil-1-β-D-arabinofuranosides (5a,b). To a solution of 2′-O-[3(4)-(pyren-1-ylethynyl)phenylmethylaminocarbonyl]uracil-1-β-D-arabinofuranoside 4a(4b) (1.004 g, 1.67 mmol) (twice evaporated with Py (2 × 20 mL), dissolved in dried pyridine (15 mL), and cooled to 0 °C), DmtCl (623 mg, 1.84 mmol) was added, and when TLC showed that the reaction was complete (ca. 6–8 h), the mixture was evaporated, diluted with EtOAc (200 mL), and washed with H2O (200 mL), 5% NaHCO3 (150 mL), H2O (150 mL), dried, and evaporated. The residue was purified by chromatography on a silica gel column (100 g) with a step gradient of acetone (25f35%) in toluene + 1% NEt3 v/v/v as the mobile phase to give 5′-O-Dmtprotected nucleosides 5a(5b). 5′-O-(4,4′-Dimethoxytrityl)-2′-O-[3-(pyren-1-ylethynyl)phenylmethylaminocarbonyl]uracil-1-β-D-arabinofuranoside (5a). Yield 1.28 g (85%), yellow foam, Rf 0.24 (acetone– toluene, 3:7 + 1% Et3N v/v/v). ESI-TOF HRMS: m/z ) 926.3032 [M + Na]+, calcd for [C56H45N3O9 + Na]+ 926.3048. 1 H NMR (DMSO-d6, δ): 11.34 (s, 1H, H-3), 8.62 (d, 1H, J9′″,10′″ ) 9.1 Hz, H-10′″), 8.42–8.21 (m, 7H, H-2′″-H-6′″, H-8′″, H-9′″), 8.15 (t, 1H, J6′″,7′″ ) J7′″,8′″ ) 7.6 Hz, H-7′″), 7.87 (br.t, 1H, J ) 5.8 Hz, OCONH), 7.65 (d, 1H, J ) 7.6 Hz, H-4″), 7.63 (s, 1H, H-2″), 7.54 (d, 1H, J ) 7.9 Hz, H-6), 7.45 (m, 1H, H-5″), 7.41–7.20 (m, 10H, ArH (Dmt), H-6″), 6.88 (m, 4H, ArH (Dmt)), 6.20 (d, 1H, J ) 5.5 Hz, H-1′), 5.81 (d, 1H, J3′,OH ) 5.5 Hz, 3′-OH), 5.43 (d, 1H, J5,6 ) 8.2 Hz, H-5), 5.11 (m, 1H, H-2′), 4.28–4.15 (m, 3H, NHCH2, H-3′), 3.89 (m, 1H, H-4′), 3.72 (s, 6H, CH3), 3.39–3.27 (m, 2H, H-5′). 5′-O-(4,4′-Dimethoxytrityl)-2′-O-[4-(pyren-1-ylethynyl)phenylmethylaminocarbonyl]uracil-1-β-D-arabinofuranoside (5b). Yield 1.28 g (85%), yellow foam, Rf 0.24 (acetone– toluene, 3:7 + 1% Et3N v/v/v). ESI-TOF HRMS: m/z ) 926.3057 [M + Na]+, calcd for [C56H45N3O9 + Na]+ 926.3048. 1 H NMR (DMSO-d6, δ): 11.41 (s, 1H, H-3), 8.63 (d, 1H, J9′″,10′″ ) 9.1 Hz, H-10′″), 8.42–8.21 (m, 7H, H-2′″-H-6′″, H-8′″, H-9′″), 8.15 (t, 1H, J6′″,7′″ ) J7′″,8′″ ) 7.6 Hz, H-7′″), 7.88 (br.t, 1H, J ) 6.0 Hz, OCONH), 7.71 (d, 2H, J2″,3″ ) J5′,6″ ) 8.0 Hz, H-3″, H-5″), 7.55 (d, 1H, J5,6 ) 8.2 Hz, H-6), 7.37–7.26 (m, 11H, ArH (Dmt), H-2″, H-6″), 6.90 (m, 4H, ArH (Dmt)), 6.22 (d, 1H, J ) 5.5 Hz, H-1′), 5.82 (d, 1H, J3′,OH ) 5.5 Hz, 3′-OH), 5.46 (d, 1H, J5,6 ) 8.2 Hz, H-5), 5.12 (m, 1H, H-2′), 4.31 (m, 1H, 2J ) 16.2 Hz, JNHCH ) 6.5 Hz, NHCHH), 4.21–4.10 (m, 2H, NHCHH, H-3′), 3.89 (m, 1H, H-4′), 3.74 (s, 6H, CH3), 3.40–3.28 (m, 2H, H-5′).
Bioconjugate Chem., Vol. 18, No. 6, 2007 1975 Scheme 1a
a Reagents and conditions: (i) 3- or 4-iodobenzylamine, THF, MeCN, reflux 40 h, 94% (2a), 84% (2b); (ii) 1-ethynylpyrene, Pd(PPh3)4, CuI, Et3N, DMF, rt, 16 h, 95% (3a), 93% (3b); (iii) Et3N · 3HF, THF, overnight, 95% (4a), 93% (4b); (iV) DmtCl, pyridine, 0°C, 6–8 h, 85% (5a), 85% (5b); (V) (Pri2N)2POCH2CH2CN, CH2Cl2, 2 h, 92% (6a), 96% (6b).
Figure 1. Sugar part of MHz, 4096 scans).
13
C NMR spectrum of compound 2a (125.7
General Procedure for the Preparation of 3′-O-(N,Ndiisopropylamino-2-cyanoethoxyphosphinyl)-5′-O-(4,4′dimethoxytrityl)-2′-O-[3(4)-(pyren-1-ylethynyl)phenylmethylaminocarbonyl]uracil-1-β-D-arabinofuranosides (6a,b). 5′-O-(4,4′-Dimethoxytrityl)-2′-O-[3-(pyren-1-ylethynyl)phenylmethylaminocarbonyl]uracil-1-β-D-arabinofuranoside 5a(5b) (1.00 g, 1.1 mmol) was evaporated with dried DCM (2 × 20 mL), dissolved in dry DCM (25 mL), and N,N-diisopropylammonium tetrazolide (0.285 g, 1.66 mmol) and bis(N,N-diisopropylamino)-2cyanoethoxyphosphine (0.527 mL, 1.66 mmol) were added under argon. The reaction mixture was stirred for 2 h, and then it was diluted with EtOAc (200 mL), washed with H2O (200 mL), 5% NaHCO3 (150 mL), and H2O (150 mL), dried, and evaporated. The residue was purified by chromatography on a short silica gel column (50 g) with a step gradient of acetone (25f35%) in toluene + 1% NEt3 v/v/v as the mobile phase to give phosphoramidites 6a(6b). 3′-O-(N,N-Diisopropylamino-2-cyanoethoxyphosphinyl)-5′O-(4,4′-dimethoxytrityl)-2′-O-[3-(pyren-1-ylethynyl)phenylmethylaminocarbonyl]uracil-1-β-D-arabinofuranoside (6a). Yield 1.11 g (92%), yellow foam, Rf 0.52, 0.46 (acetone–toluene, 7:13 + 1% Et3N v/v/v). ESI-TOF HRMS: m/z ) 1126.4137 [M + Na]+, calcd for [C65H62N5O10P + Na]+ 1126.4126. 1H NMR (MeCN-d3, δ): 9.05 (br.s, 1H, H-3), 8.71 (m, 1H, H-10′″), 8.37–8.18 (m, 8H, H-2′″–H-6′″, H-8′″, H-9′″), 8.14 (t, 1H, J6′″,7′″ ) J7′″,8′″ ) 7.7 Hz, H-7′″), 7.66 (m, 2H, H-6, H-4″), 7.55 (s, 1H, H-2″), 7.51–7.45 (m, 3H), 7.38–7.28 (m, 10H) (H-6, H-5″, H-6″, OCONH, ArH (Dmt)), 6.90 (m, 4H, ArH (Dmt)), 6.28
1976 Bioconjugate Chem., Vol. 18, No. 6, 2007
Astakhova et al.
Table 2. Oligonucleotides Used in This Studya
a
#
sequence, 5′f3′
MALDI MS
calculated mass, [M + H]+
ON1 ON2 ON3a ON3b ON4a ON4b ON5a ON5b ON6a ON6b ON7a ON7b ON8a ON8b ON9a ON9b ON10a ON10b ON11a ON11b ON12a ON12b
CTCCCAGGCTCAAAT ATTTGAGCCTGGGAG CTCCCAGGCUCAAAT CTCCCAGGCUCAAAT CUCCCAGGCTCAAAT CUCCCAGGCTCAAAT CUCCCAGGCUCAAAT CUCCCAGGCUCAAAT CTCCCAGGCTCAAAUCTGG CTCCCAGGCTCAAAUCTGG AUTTGAGCCTGGGAG AUTTGAGCCTGGGAG ATUTGAGCCTGGGAG ATUTGAGCCTGGGAG ATTUGAGCCTGGGAG ATTUGAGCCTGGGAG CCAGAUTTGAGCCTGGGAG CCAGAUTTGAGCCTGGGAG CCAGATUTGAGCCTGGGAG CCAGATUTGAGCCTGGGAG CCAGATTUGAGCCTGGGAG CCAGATTUGAGCCTGGGAG
4852.1 4857.2 4854.4 4853.4 5212.2 5214.9 6108.7 6107.8 5004.9 5005.5 5007.7 5006.0 5006.0 5004.7 6228.2 6226.7 6228.2 6227.5 6228.6 6229.8
4857.3 4857.3 4857.3 4857.3 5216.7 5216.7 6109.1 6109.1 5008.4 5008.4 5008.4 5008.4 5008.4 5008.4 6229.2 6229.2 6229.2 6229.2 6229.2 6229.2
U - modified nucleoside 4a (for oligonucleotide series a) or 4b (for oligonucleotide series b).
Table 3. Thermal Stabilities of Modified Duplexesa
a
∆Tm (°C)
∆Tm/mod
54.9
-2.7
-2.7
5′-CUC-CCA-GGC-TCA-AAT 3′-GAG-GGT-CCG-AGT-TTA
54.5
-3.1
-3.1
ON5a × ON2
5′-CUC-CCA-GGC-UCA-AAT 3′-GAG-GGT-CCG-AGT-TTA
55.1
-2.5
-1.3
ON3b × ON2
5′-CTC-CCA-GGC-UCA-AAT 3′-GAG-GGT-CCG-AGT-TTA
49.6
-8.0
-8.0
ON4b × ON2
5′-CUC-CCA-GGC-TCA-AAT 3′-GAG-GGT-CCG-AGT-TTA
53.2
-4.4
-4.4
ON5b × ON2
5′-CUC-CCA-GGC-UCA-AAT 3′-GAG-GGT-CCG-AGT-TTA
47.3
-10.3
-5.2
#
duplex
Tm (°C)
ON1 × ON2
5′-CTC-CCA-GGC-TCA-AAT 3′-GAG-GGT-CCG-AGT-TTA
57.6
ON3a × ON2
5′-CTC-CCA-GGC-UCA-AAT 3′-GAG-GGT-CCG-AGT-TTA
ON4a × ON2
U - modified nucleoside 4a (for oligonucleotide series a) or 4b (for oligonucleotide series b).
(m, 1H, H-1′), 5.47 (m, 1H, H-5), 5.41–5.34 (m, 1H, H-2′), 4.63 (m, 0.55H), 4.51 (m, 0.45H) (H-3′, diastereomers), 4.41–4.28 (m, 2H, NCH2), 4.15–4.08 (m, 1H, H-4′), 3.78 (m, 6H, CH3), 3.73–3.42 (m, 6H, POCH2, NCH, H-5′), 2.61 (m, 0.90H), 2.51 (t, 1.10H, J ) 6.0 Hz) (CH2CN, diastereomers), 1.30–1.04 (m, 12H, CHCH3). 31P NMR (CDCl3, δ): 151.70, 150.99 (1:0.8). 3′-O-(N,N-Diisopropylamino-2-cyanoethoxyphosphinyl)-5′O-(4,4′-dimethoxytrityl)-2′-O-[4-(pyren-1-ylethynyl)phenylmethylaminocarbonyl]uracil-1-β-D-arabinofuranoside (6b). Yield 1.16 g (96%), yellow foam, Rf 0.38, 0.26 (acetone–toluene, 7:13 + 1% Et3N v/v/v). ESI-TOF HRMS: m/z ) 1126.4091 [M + Na]+, calcd for [C65H62N5O10P + Na]+ 1126.4126. 1H NMR (MeCN-d3, δ): 9.05 (br.s, 1H, H-3), 8.40–8.19 (m, 7H, H-2′″-H-6′″, H-8′″, H-9′″), 8.15 (t, 1H, J6′″,7′″ ) J7′″,8′″ ) 7.6 Hz, H-7′″), 7.74 (m, 2H, H-3″, H-5″), 7.57–7.48 (m, 3H), 7.41–7.28 (m, 10H) (H-6, H-2″, H-6″, OCONH, ArH (Dmt)), 6.92 (m, 4H, ArH (Dmt)), 6.30 (m, 1H, H-1′), 5.50–5.34 (m, 2H, H-5, H-2′), 4.62 (m, 0.53 H), 4.52 (m, 0.47H) (H-3′, diastereomers), 4.42–4.25 (m, 2H, NCH2), 4.19–4.08 (m, 1H, H-4′), 3.80 (m, 6H, CH3), 3.72–3.40 (m, 6H, POCH2, NCH, H-5′), 2.66 (t, 0.94H, J ) 6.1 Hz), 2.56 (t, 1.06H, J ) 6.0 Hz)
(CH2CN, diastereomers), 1.27–0.96 (m, 12H, CHCH3). NMR (CDCl3, δ): 151.57, 151.10 (1:0.9).
31
P
RESULTS AND DISCUSSION The synthesis of reagents for placement of 1-PEPy in a major groove is shown on Scheme 1. The starting imidazolide 1 (24) was reacted with 3- or 4-iodobenzylamine to give 3′,5′Markiewicz-protected iodobenzyl ara-uridine-2′-carbamates 2a,b. 1-PEPy fluorochrome was assembled using Sonogashira coupling of iodobenzyl compounds with 1-ethynylpyrene (47). Nucleosides 3 contain 1-PEPy unit connected to the sugar part of nucleoside through meta (series a) and para (series b) positions of a phenyl ring. Silyl protection was removed with triethylamine trihydrofluoride to yield fluorescent nuclesides 4a,b as crystalline solids. These were converted into phosphoramidite reagents using standard methods of nucleoside chemistry—5′-O-dimethoxytritylation and 3′-O-phosphitylation. Suddenly, signals from C1′–C5′ and C6 carbons were missing in 13C NMR spectra of 3′,5′-silyl Markiewicz-protected compounds 2a,b and 3a,b. In contrast, nucleosides 4a,b gave normal C1′–C5′ and C6 signals in 13C NMR. Therefore, we repeated
1-Phenylethynylpyrene Excimer on DNA
Bioconjugate Chem., Vol. 18, No. 6, 2007 1977
fl abs Table 4. Spectroscopic (λmax ) and Photophysical (Φf, τf) Properties of Modified Oligonucleotide and Duplexes , λmax
#
abs
λmax, bands I, II (nm)
a
λmax (nm)
Φf
τ1, (ns)
S1, (%)
τ2, (ns)
S2, (%)
χ2
〈τf〉, (ns)
fl
ON3a
392, 372
402
0.22
0.88
27
2.96
73
1.143
2.40
ON3a × ON2
390, 371
406
0.10
0.59
60
2.19
40
1.040
1.23
ON4a
394, 372
404
0.32
0.88
23
2.64
77
1.022
2.23
ON4a × ON2
393, 371
408
0.09
0.50
82
2.39
18
1.045
0.84
ON3b
394, 372
403
0.16
0.60
35
2.50
65
0.996
1.83
ON3b × ON2
397, 375
408
0.23
0.88
44
2.68
56
1.051
1.89
ON4b
396, 374
404
0.28
0.79
25
2.48
75
1.162
2.06
ON4b × ON2
398, 375
409
0.12
0.59
69
1.77
31
1.047
0.96
a
2
S1 and S2, contribution of τ1 and τ2 components in percentages; χ , statistical Pirson criterion associated with the fitted lifetime function and the experimental data; 〈τf〉 ) Σ Siτi/Σ Si, average fluorescence lifetime. Table 5. Fluorescent Properties of meta- and para-1-PEPy Pairs Located in -1, -2, and -3 Zipper Modes
13
C NMR experiments for compounds 2a,b and 3a,b using 4096 scans. Indeed, broad (up to 200 Hz) signals appeared in expected regions (50–100 ppm for sugar carbons and 140 ppm for C6). Typical sugar carbon signals are shown on Figure 1. The signals gave normal cross-peaks in HMQC spectra and can be easily assigned. The nature of the observed effect is not clear. Evidently, two prerequisites—silyl Markiewicz group and aryl carbamate modification—contribute cooperatively in the unusual shielding of six carbon atoms in compounds 2a,b and 3a,b. Phosphoramidites 6a,b were used in automated oligonucleotide synthesis to prepare a series of modified oligonucleotides
Table 6. Fluorescent Properties of meta- and para-1-PEPy Pairs Located in +2, +3, and +4 Zipper Modes
(Table 2) similar to those described for studies of pyrene excimer in DNA major groove (24). It is known that 2′-carbamate modification destabilize dsDNA, but pyrene modification can relieve the negative carbamate influence (20, 24), and para-1-PEPy-ribo-carbamate even displayed some stabilization of the DNA duplex (49). Thermal denaturation studies (Table 3) show that meta-1-PEPy-arabinocarbamate (series a) is similar to pyrene (24) in its ability to neutralize the negative carbamate effect. In contrast, PEPy in para-1-PEPy-arabino-carbamate (series b) has no influence on duplex stability: the resulting destabilization is similar to that from aliphatic carbamate (24). We speculate that, in the case
1978 Bioconjugate Chem., Vol. 18, No. 6, 2007
Astakhova et al.
Figure 2. Molecular models of duplexes ON3b×ON9a (a) and ON3b×ON9b (b). 1-PEPy residues are shown in space-filling mode and marked in green (meta) and yellow (para attachment). Models constructed in HyperChem 7.5 and GaussView 3.0 with use of standard B-DNA parameters.
of meta-1-PEPy-arabino-carbamate modification, the aromatic residue fits the major groove of duplex, and in the case of paramodification, it is exposed in the environment. There are also no direct data for or against intercalation of PEPy residues into the DNA duplex. The intercalation of PEPy in fully matched duplexes is, however, highly unlikely because of excimer formation in +2 and +3 zipper modes (see below). On the other hand, in mismatched sequences, pyrene can probably replace a nonpairing nucleobase (20), and further studies would be useful for 1-PEPy. Next, we determined spectral and photophysical properties of the meta- and para-1-PEPy labels as arabino-2′-carbamates (Table 4). There are minor variations in absorbances between single-stranded oligonucleotides and their duplexes. Hybridization causes a 4 nm bathochromic shift of fluorescence maximum in the a series and a 5 nm shift in the b series. The data show that 1-PEPy fluorescence kinetics in ss and dsDNA always fit to two exponential components with different lifetimes, and the average fluorescence lifetime is between 0.84 and 2.40 ns. This is approximately 2 orders of magnitude less than for pyrene derivatives. Not surpisingly, fluorescence quantum yields (9–32%) are much higher than typical quantum yields of pyrene fluorescence on DNA, except for pyrenoyl on amino-LNA (60). Hybridization causes a 2–3-fold decrease in average excited lifetime and emission quantum yield for conjugates ON3a, ON4a, and ON4b. In contast, Φf and 〈τf〉 of 1-PEPy fluorochrome in ON3b slightly increased in the course of duplex formation. Further, we studied the possibilities of PEPy interstrand excimer formation in DNA duplexes. In cases of excimer formation, the dye showed a broad emission band around 500 nm. We used the excimer-to-monomer fluorescence intensity ratio, I500/I405, as an excimer formation criterion. In the absence of excimer emission, I500/I405 is approximately 0.1. First, we studied a “negative zipper” mode (Table 5). There was no detectable excimer fluorescence in continuous duplexes (ON6×ON10, ON6×ON11, ON6×ON12). In the
case of truncated duplexes, an excimer was observed once, for -2 zipper mode in series b (ON6b×ON8b), probably resulting from duplex distortion. Interestingly, pyrene gave -1 zipper excimer in a similar case (24). Then, we checked +2, +3, and +4 zippers for meta- and para- series of 1-PEPy pairs (Table 6). The intensity of excimer fluorescence always increased within the sequence +4 zipper < +3 zipper < +2 zipper. The weakest +2 excimer forms from two meta-PEPys, and the strongest from two para-compounds. All cases of combinations of a and b series were studied. The configuration b × a is always preferable for excimer formation than a × b. In cotrast to pyrene data (24), not only +2, but +3 and even weak +4 zipper excimers were observed in some cases. This can be easily explained by taking into account that the 1-PEPy molecule is approximately 1 nm longer than the pyrene one. To confirm that the PEPy excimer can be formed in a major groove of a normal B-DNA duplex, molecular models were built (Figure 2). They showed, however, that stacked structures from two spatially accessible PEPy residues are not only located in a major groove, but considerably exposed in a solvent. In addition, fluorescence data revealed that a doubly labeled oligonucleotide can form a metastable hairpin in aq solutions (Table 7). In the hairpin, two para-PEPys are located in close proximity and showed considerable excimer emission. The addition of the same nonlabeled sequence does not decrease the excimer fluorescence. Expectedly, the excimer completely disappears after hybridization with a complementary strand. Thus, the PEPy fluorochrome can be used for preparation of a “molecular beacon”-like excimer-forming probe. To conclude, we report synthesis of meta and para derivatives of 1-phenylethynylpyrene (1-PEPy) arabino-uridine-2′-carbamates, their incorporation into oligonucleotides, spectral and photophysical properties in ss and dsDNA, and stuctural preferences for 1-PEPy major groove excimer. Although the excited lifetime of 1-PEPy derivatives on DNA is ca. 100-fold shorter compared to that of pyrene, PEPy molecules, being
1-Phenylethynylpyrene Excimer on DNA Table 7. Fluorescent Properties of Oligonucleotides Doubly Labeled with meta- or para-1-PEPy Fluorochromes
located in +2, +3, and even in +4 zipper modes, are able to form a major groove or environmentally exposed interstrand excimer on the DNA duplex. The 1-PEPy excimer, more fastidious about the proximity of fluorochromes, could become a useful tool in structural studies of biomolecules.
ACKNOWLEDGMENT We thank Maksim Kvach and Youri Habrus for oligonucleotide synthesis, Dmitry Alexeev for mass spectra, Larisa Grusintseva for technical assistance, and Zakhar Shenkarev for helpful advice with NMR spectra. This work was supported by the Russian Foundation for Basic Research (grant no. 06-0332426).
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