Bioconjugate Chem. 2009, 20, 1673–1682
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TECHNICAL NOTES Practical Synthesis of Isomerically Pure 5- and 6-Carboxytetramethylrhodamines, Useful Dyes for DNA Probes Maksim V. Kvach,† Irina A. Stepanova,‡ Igor A. Prokhorenko,‡ Aleksander P. Stupak,§ Dmitry A. Bolibrukh,| Vladimir A. Korshun,*,‡ and Vadim V. Shmanai*,† Institute of Physical Organic Chemistry, Surganova 13, 220072 Minsk, Belarus, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, Russia, Institute of Physics, Nezavisimosti av. 70, 220072 Minsk, Belarus, and Institute of Bioorganic Chemistry, Kuprevicha 5/2, 220141, Minsk, Belarus. Received January 29, 2009; Revised Manuscript Received June 21, 2009
Simple and scalable synthesis of 5- and 6-carboxytetramethylrhodamines (TAMRAs) is reported. Acylation of 3-dimethylaminophenol with 1,2,4-benzenetricarboxylic anhydride afforded a mixture of 4-dimethylamino-2hydroxy-2′,4′(5′)-dicarboxybenzophenones, which can be easily separated into individual compounds upon recrystallization from methanol and acetic acid. Individual benzophenones were reacted with 3-dimethylaminophenol to give 5- or 6-carboxytetramethylrhodamines. The dyes were converted into hydroxyprolinol-based phosphoramidite reagents suitable for oligonucleotide synthesis. 5- and 6-TAMRA isomers on oligonucleotides showed similar absorption and emission spectra. Fluorescence quantum yield of the dyes correlates with the presence of dG nucleosides in the adjacent region of oligonucleotide sequence. Several energy transfer primers containing on their 5′-termini (6-FAM)dTn(6-TAMRA) dye system (n ) 0, 2, 4, 6, 8, 10, 12, 14) were prepared, and their spectral properties were studied.
INTRODUCTION Xanthene fluorescent dye tetramethylrhodamine (TMR, TAMRA) is extremely popular for covalent labeling of various types of biomolecules (1-3), especially oligonucleotides for various formats of DNA analysis (4-6). The most convenient compounds for bioconjugation are 5- and 6-carboxy derivatives of tetramethylrhodamine. The mixture of isomers, easily prepared from 3-dimethylaminophenol and trimellitic anhydride (7), can be separated by chromatography (8) to give individual 5and 6-isomers suitable for labeling of biomolecules, e.g., DNA (9), PNA (10), and peptides (11). TAMRA-labeled peptide substrates were used for determination of caspase-3 (12) and protein kinase (13) activity. Recent study showed that TAMRA conjugate with one of the sodium-dependent glucose cotransporter 2 (SGLT2) inhibitors is a 10-fold more potent agent than phlorizin (14). However, more diverse applications have tetramethylrhodamine dye in the field of nucleic acids. Fluorescent properties of TAMRA-labeled oligonucleotides were thoroughly studied (15-18). The dye attached to the terminal position of an oligonucleotide showed little influence on the duplex stability (19). Changes in fluorescence polarization and intensity of singly TAMRA-labeled oligonucleotide probes were used for the monitoring of DNA-protein interactions (20-25). Fluorescence * Corresponding authors. V.A.K.: E-mail:
[email protected], Tel/Fax: +7-495-330-6738. V.V.S.: E-mail:
[email protected], Tel: +375-17-284-2055, Fax: +375-17-284-2539. † Institute of Physical Organic Chemistry. ‡ Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry. § Institute of Physics. | Institute of Bioorganic Chemistry.
resonance energy transfer (FRET) dye pair fluorescein-tetramethylrhodamine on oligonucleotides can be used for the measurement of ribonuclease activity (26), hydroxyl radical detection (27), and in DNA-protein binding studies (28, 29). In DNA-related FRET investigations and applications, TAMRA was used as donor (30-32), acceptor (33-38), or energy bridge (39-42). The dye is an important label in four-color DNA sequencing (43-48). Many real-time PCR platforms have spectral channels suitable for TAMRA (49), and it is a standard reporter (50-52) or quencher (53, 54) dye for real-time PCR probes. The widespread popularity of tetramethylrhodamine dye requires highly producible and easy scalable methods for the preparation of isomerically pure carboxytetramethylrhodamines. These would be suitable for the preparation of phosphoramidite reagents that can be automatically incorporated into oligonucleotides. Although several 5′-TAMRA and TAMRA-dT phosphoramidites for terminal and internal labeling are commercially available (55, 56), only two TAMRA phosphoramidite reagents have been described in the literature in detail (57, 58). Their disadvantage is that they are terminal, i.e., can be attached exclusively to the 5′-position of an oligonucleotide. On the other hand, phosphoramidites and solid supports derived from (3R,5S)5-hydroxymethyl-3-hydroxypyrrolidine (trans-L-4-hydroxyprolinol) allow one to introduce modification into any position of an oligonucleotide (59-72). Hence, the aims of the present work are the development of a simple and easily scalable procedure for the synthesis of 5- and 6-carboxytetramethylrhodamines, the synthesis of phosphoramidite reagents from both isomers using
10.1021/bc900037b CCC: $40.75 2009 American Chemical Society Published on Web 07/16/2009
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trans-L-4-hydroxyprolinol as a scaffold, and the study of spectral and photophysical properties of TAMRA-labeled oligonucleotides.
EXPERIMENTAL PROCEDURES General. Reagents and solvents obtained from commercial suppliers were used without further purification. CH2Cl2 was used freshly distilled from CaH2. HPLC-grade DMF was distilled under reduced pressure and stored over 4 Å molecular sieves under argon. Other solvents were used as received. N,N-Diisopropylamino-2-cyanoethoxychlorophosphine (73, 74), trimethylsilyl polyphosphate solution in chloroform (75), and (3R,5S)-5-[(4,4′-dimethoxytrityl)oxymethyl]-3-hydroxypyrrolidine (69) were prepared as described. 500 MHz 1H, 125.7 MHz 13 C, and 202.4 MHz 31P NMR spectra were recorded on a Bruker DRX-500 or Bruker Avance 500 spectrometers and referenced to CDCl3 (7.26 ppm for 1H and 77.1 ppm for 13C), DMSO-d6 (2.50 ppm for 1H and 39.5 ppm for 13C), CD3CN (1.94 ppm for 1 H and 118.3 ppm for 13C) (76), and 85% aq H3PO4 (0.00 ppm for 31P). 1H NMR coupling constants are reported in hertz (Hz) and refer to apparent multiplicities. High-resolution mass spectra (HRMS) were recorded in positive-ion mode using an IonSpec FT 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 aluminum plates (Merck); spots were visualized under UV light (254 nm). Silica gel column chromatography was performed using Merck Kieselgel 60 0.040-0.063 mm. The oligonucleotide synthesis was carried out on a BiosSet ASM800 instrument on a 200 nmol scale using standard manufacturer’s protocols. Oligonucleotides were isolated using electrophoresis in 20% denaturing (7 M urea) PAGE in Tris-borate buffer, pH 8.3, and desalted by gel filtration on a Sephadex G-25 column in 1 × 10-5 M Tris-HCl buffer, pH 7.5 (“saltless” buffer). MALDI-TOF mass spectra of oligonucleotide conjugates were recorded in positive-ion mode on a Bruker Ultraflex spectrometer. UV absorption spectra were recorded using a Varian Cary 500 spectrophotometer in 0.1 M bicarbonate buffer (pH 8.5). Fluorescence studies in the same buffer were performed using a Solar CM 2203 spectrofluorometer. Rhodamine 6G (R6G) in ethanol was used as a quantum yield standard. The fluorescence quantum yields (Φf) were calculated using the relative method according to the equation Φf ) Φf(R6G) ×
I IR6G
×
AR6G n2 × A nR6G2
where Φf (R6G) is rhodamine 6G quantum yield in ethanol (Φf (R6G) ) 0.94 (77)); I and IR6G are integrated intensities of sample and R6G dye solutions, respectively; A and AR6G are optical densities of sample and R6G dye solutions, respectively; n and nR6G are refractive indexes of ethanol and 0.1 M bicarbonate buffer, 1.3591 and 1.3340 at 20 °C, respectively. The measurements were performed at an angle of 90° to the exciting light beam. The optical density of the solutions in the 1 cm quartz cell at the excitation wavelength did not exceed 0.05. The samples used in quantum yield measurements were not degassed. 4-Dimethylamino-2-hydroxy-2′,4′(5′)-dicarboxy-benzophenones (1a,b). Freshly distilled 3-dimethylaminophenol (13.70 g, 0.10 mol) was dissolved in toluene (300 mL), heated to 60 °C, and pounded trimellitic anhydride (23.04 g, 0.12 mol) was added with magnetic stirring. The mixture was refluxed for 24 h and cooled. The residue was filtered off, washed with toluene (3 × 50 mL), dissolved in MeOH (300 mL), and refluxed for 10 min. Then, acetic acid (100 mL) was added and the mixture was evaporated to dryness. Crystalline solid was refluxed with
Kvach et al.
MeOH (200 mL) for 2 h and kept at 4 °C overnight. The crystals formed were filtered off and washed with MeOH (50 mL) to give pure 1a as brown crystalline solid (5.16 g, 16%). The mother liquid was evaporated to dryness, refluxed 1 h with acetic acid (150 mL), and stored at room temperature overnight. Crystals were filtered off and dried in Vacuo at 115-120 °C to give mixture 1a,b (9.66 g). This was recrystallized twice from acetic acid to give pure 1b as a yellow crystalline solid (3.35 g, 10%). Filtered solutions can be combined, evaporated, and recrystallized succesively from MeOH and AcOH as above to give further amounts of individual 1a and 1b. 4-Dimethylamino-2-hydroxy-2′,4′-dicarboxy-benzophenone (1a). Rf 0.34 (Et3N-MeOH-CHCl3 5:20:75 v/v/v); dec. >250 °C (MeOH). 1H NMR (DMSO-d6) δ ) 13.44 (br s, 2H, CO2H), 12.38 (s, 1H, OH), 8.49 (d, 1H, 4J3′,5′ ) 1.7 Hz, H-3′), 8.20 (dd, 1H, J5′,6′ ) 7.9 Hz, 4J3′,5′ ) 1.7 Hz, H-5′), 7.52 (d, 1H, J5′,6′ ) 7.9 Hz, H-6′), 6.80 (d, 1H, J5,6 ) 9.2 Hz, H-6), 6.20 (dd, 1H, J5,6 ) 9.2 Hz, 4J3,5 ) 2.5 Hz, H-5), 6.11 (d, 1H, 4 J3,5 ) 2.5 Hz, H-3), 3.00 (s, 6H, CH3). 13C NMR (DMSO-d6) δ ) 197.66 (CO), 166.14 (2C, CO2H), 164.24 (C2), 155.83 (C4), 143.76 (C1′), 133.82 (C6), 132.71 (C5′), 131.66 (C2′ or C4′), 130.73 (C3′), 130.03 (C2′ or C4′), 128.28 (C6′), 109.46 (C1), 104.52 (C5), 97.03 (C3), 39.63 (2C, CH3). HRMS (MALDI+): m/z [M+H]+ calcd for C17H16NO6+ 330.0972; found 330.0975. 4-Dimethylamino-2-hydroxy-2′,5′-dicarboxy-benzophenone (1b). Rf 0.52 (Et3N-MeOH-CHCl3 5:20:75 v/v/v); mp 244-245 °C (AcOH). 1H NMR (DMSO-d6) δ ) 13.46 (br s, 2H, CO2H), 12.41 (s, 1H, OH), 8.14 (dd, 1H, J3′,4′ ) 8.3 Hz, 4 J4′,6′ ) 1.7 Hz, H-4′), 8.06 (d, 1H, J3′4′ ) 8.3 Hz, H-3′), 7.83 (d, 1H, 4J4′,6′ ) 1.7 Hz, H-6′), 6.86 (d, 1H, J5,6 ) 9.3 Hz, H-6), 6.22 (dd, 1H, J5,6 ) 9.3 Hz, 4J3,5 ) 2.5 Hz, H-5), 6.11 (d, 1H, 4 J3,5 ) 2.5 Hz, H-3), 3.01 (s, 6H, CH3). 13C NMR (DMSO-d6) δ ) 197.40 (CO), 166.53 (2′-CO2H), 166.10 (5′-CO2H), 164.40 (C2), 155.87 (C4), 140.10 (C1′), 133.93 (C6), 133.73 (C2′), 133.65 (C5′), 130.42 (C3′), 130.23 (C4′), 128.25 (C6′), 109.43 (C1), 104.56 (C5), 97.08 (C3), 39.62 (2C, CH3). HRMS (MALDI+): m/z [M+H]+ calcd for C17H16NO6+ 330.0972; found 330.0970. Carboxytetramethylrhodamines (2a,b); General Procedure. Benzophenone 1a(1b) (3.29 g, 10.00 mmol) was mixed with 3-dimethylaminophenol (1.78 g, 13.00 mmol) and dissolved in DMF (80 mL). Trimethylsilylpolyphosphate solution in chloroform (20 mL) was added, and the mixture was refluxed for 3 h. Solvents were removed in Vacuo; the residue was dissolved in 5% NaOH (70 mL) and stirred at room temperature overnight. Then, the solution was diluted with water (150 mL), and rhodamine was precipitated with concentrated hydrochloric acid (ca. 5 mL). The solid was filtered off, washed with water (50 mL), and dried in Vacuo at 150 °C. Yields: 4.25 g, 99% of 2a, or 4.27 g, 99% of 2b. Carboxytetramethylrhodamines 2a,b were used in the next step without additional purification. Analytical samples were purified on preparative silica gel TLC in Et3N-MeOH-CHCl3 5:15:80 v/v/v as a mobile phase and characterized as triethylammonium salts. 3,6-Bis(dimethylamino)-9-[2,4-dicarboxylatephenyl]xanthylium, Triethylammonium Salt (4-Carboxytetramethylrhodamine, Triethylammonium Salt) (2a · Et3N). Rf 0.14 (Et3N-MeOHCHCl3 5:20:75 v/v/v). 1H NMR (CD3OD), open chain numbering, δ ) 8.73 (br s, 1H, H-3′), 8.18 (d, 1H, J5′,6′ ) 7.5 Hz, H-5′), 7.28 (d, 1H, J5′,6′ ) 7.5 Hz, H-6′), 7.25 (d, 2H, J1,2 ) J7,8 ) 9.3 Hz, H-1,8), 6.98 (dd, 2H, J1,2 ) J7,8 ) 9.3 Hz, 4J2,4 ) 4 J5,7 ) 1.7 Hz, H-2,7), 6.83 (d, 2H, 4J2,4 ) 4J5,7 ) 1.7 Hz, H-4,5), 3.23 (br s, 12H, NCH3), 3.13 (q, 6H, J ) 7.4 Hz, NCH2), 1.25 (t, 9H, J ) 7.4 Hz, CH2CH3). 13C NMR (CD3OD) δ ) 173.42 (4′-CO2-), 173.06 (2′-CO2-), 160.64 (C9), 158.75 (2C, C4a,10a), 158.40 (2C, C3,6), 140.82 (C2′ or 4′), 140.47 (C2′ or 4′), 136.30 (C1′), 132.66 (2C, C1,8), 131.81 (C3′), 131.38 (C5′), 129.96
Technical Notes
(C6′), 114.73 (2C, C2,7), 114.65 (2C, C8a,9a), 97.35 (2C, C4,5), 47.33 (3C, CH2CH3), 40.81 (4C, NCH3), 9.07 (3C, CH2CH3). HRMS (MALDI+): m/z [M+H]+ calcd for C25H23N2O5+ 431.1601; found 431.1597. 3,6-Bis(dimethylamino)-9-[2,5-dicarboxylatephenyl]xanthylium, Triethylammonium Salt (5-Carboxytetramethylrhodamine, Triethylammonium Salt) (2b · Et3N). Rf 0.25 (Et3N-MeOHCHCl3 5:20:75 v/v/v). 1H NMR (CD3OD), open chain numbering, δ ) 8.23 (d, 1H, J3′,4′ ) 8.0 Hz, H-4′), 8.11 (d, 1H, J3′,4′ ) 8.0 Hz, H-3′), 7.97 (br s, 1H, H-6′), 7.27 (d, 2H, J1,2 ) J7,8 ) 9.3 Hz, H-1,8), 6.96 (dd, 2H, J1,2 ) J7,8 ) 9.3 Hz, 4J2,4 ) 4J5,7 ) 2.4 Hz, H-2,7), 6.84 (d, 2H, 4J2,4 ) 4J5,7 ) 2.4 Hz, H-4,5), 3.22 (br s, 12H, NCH3), 3.09 (q, 6H, J ) 7.4 Hz, NCH2), 1.21 (t, 9H, J ) 7.4 Hz, CH2CH3). 13C NMR (CD3OD) δ ) 173.02 (2C, 2′-CO2-, 4′-CO2-), 162.71 (C9), 158.85 (2C, C4a,10a), 158.49 (2C, C3,6), 142.85 (C2′), 140.21 (C5′), 133.49 (C1′), 132.84 (2C, C1,8), 131.51 (C4′), 131.38 (C6′), 130.57 (C3′), 114.95 (2C, C8a,9a), 114.78 (2C, C2,7), 97.38 (2C, C4,5), 47.39 (3C, CH2CH3), 40.82 (4C, NCH3), 9.09 (3C, CH2CH3). HRMS (MALDI+): m/z [M+H]+ calcd for C25H23N2O5+ 431.1601; found 431.1595. Amides 3a,b; General Procedure. To a solution of carboxytetramethylrhodamine 2a(2b) (1.72 g, 4.00 mmol) in DMF (22 mL), benzotriazol-1-yloxy-tris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP, 2.08 g, 4.00 mmol) was added, followed by DIEA (8.00 mmol, 1.39 mL), and the mixture was stirred for 5 min. A solution of (3R,5S)-5-[(4,4′-dimethoxytrityl)oxymethyl]-3-hydroxypyrrolidine (1.68 g, 4.00 mmol) in DMF (8 mL) was added in one portion, and the stirring was continued for 2 h. The mixture was diluted with ethyl acetate (350 mL), washed with 5% NaHCO3 (350 mL) and brine (200 mL), dried over MgSO4, evaporated, and chromatographed on silica gel column in 1% Et3N in acetone as a mobile phase to give the dark red amorphous foam. 1-{3′,6′-Bis(dimethylamino)-3-oxo-spiro[isobenzofuran-1(3H),9′[9H]xanthene]-5-yl-carbonyl}-(3R,5S)-5-[(4,4′-dimethoxytrityl)oxymethyl]-3-hydroxypyrrolidine (3a). Yield 1.98 g (60%). Rf 0.25 (Et3N-MeOH-acetone 2:20:78 v/v/v). 1H NMR (CDCl3), spirolactone form numbering, δ ) 8.14 (br s, 1H, H-4′), 7.79 (d, 1H, J6′,7′ ) 7.8 Hz, H-6′), 7.43 (d, 2H, J2′′′′,3′′′′ ) J5′′′′,6′′′′ ) 7.4 Hz, H-2′′′′,6′′′′), 7.31 (m, 4H, H-2′′′,6′′′), 7.27 (m, 2H, H-3′′′′,5′′′′), 7.18 (m, 2H, H-7′,4′′′′), 6.81 (m, 4H, H-3′′′,5′′′), 6.75-6.66 (m, 2H, H-1′′,8′′), 6.49 (m, 2H, H-4′′,5′′), 6.44 (m, 2H, H-2′′,7′′), 4.72 (m, 1H, H-3), 4.49 (m, 1H, H-5), 3.82-3.54 (m, 9H, OCH3, OCH2, H-2a), 3.30 (m, 1H, H-2b), 2.98 (m, 12H, NCH3), 2.34-2.26 (m, 1H, H-4a), 2.15-2.08 (m, 1H, H-4b). 13C NMR (CDCl3) δ ) 169.20 (CO), 169.06 (CO), 158.50 (2C, C4′′′), 153.77 (2C, C4a′′,10a′′), 153.04 (2C, C3′′,6′′), 150.94 (C7a′), 145.10 (C1′′′′), 138.34 (C3a′ or C5′), 136.24 (C1′′′), 136.16 (C1′′′), 133.21 (C6′), 130.08 (5C, C2′′′,6′′′, C5′ or C3a′), 129.38 (2C, C1′′,8′′), 128.16 (2C, C3′′′′,5′′′′), 127.92 (2C, C2′′′′,6′′′′), 126.87 (C4′′′′), 125.34 (C7′), 124.91 (C4′), 113.21 (4C, C3′′′,5′′′), 109.67 (2C, C2′′,7′′), 107.55 (2C, C8a′′,9a′′), 98.16 (C4′′ or C5′′), 98.10 (C4′′ or C5′′), 85.94 (Ar3C (Dmt)), 70.59 (C5), 63.49 (C2), 59.08 (OCH2), 56.19 (C3), 55.27 (2C, OCH3), 40.37 (4C, NCH3), 36.75 (C4). HRMS (MALDI+): m/z [M+H]+ calcd for C51H50N3O8+ 832.3592; found 832.3595. 1-{3′,6′-Bis(dimethylamino)-3-oxo-spiro[isobenzofuran-1(3H),9′[9H]xanthene]-6-yl-carbonyl}-(3R,5S)-5-[(4,4′-dimethoxytrityl)oxymethyl]-3-hydroxypyrrolidine (3b). Yield 1.52 g (46%). Rf 0.16 (Et3N-MeOH-acetone 2:20:78 v/v/v). 1H NMR, spirolactone form numbering, (CDCl3) δ ) 8.01 (d, 1H, J4′,5′ ) 8.0 Hz, H-4′), 7.76 (d, 1H, J4′,5′ ) 8.0 Hz, H-5′), 7.34 (m, 2H, H-2′′′′,6′′′′), 7.27 (br s, 1H, H-7′), 7.26-7.19 (m, 4H, H-2′′′,6′′′), 7.15-7.05 (m, 3H, H-3′′′′,4′′′′,5′′′′), 6.80-6.70 (m, 6H, H-1′′,8′′,3′′′,5′′′), 6.51-6.41 (m, 4H, H-2′′,4′′,5′′,7′′), 4.60 (m,
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1H, H-3), 4.39 (m, 1H, H-5), 3.78-3.40 (m, 9H, OCH3, OCH2, H-2a), 3.21 (m, 1H, H-2b), 2.96 (m, 12H, NCH3), 2.16 (m, 1H, H-4a), 2.04 (m, 1H, H-4b). 13C NMR (CDCl3) δ ) 169.30 (OCO), 169.07 (NCO), 158.45 (C4′′′), 158.39 (C4′′′), 154.29 (C4a′′ or C10a′′), 154.24 (C4a′′ or C10a′′), 153.43 (2C, C3′′,6′′), 147.40 (C7a′), 144.99 (C1′′′′), 141.58 (C6′), 136.44 (C1′′′), 135.93 (C1′′′), 130.15 (4C, C2′′′,6′′′), 130.08 (C3a′), 129.88 (2C, C1′′,8′′), 129.03 (C5′), 128.05 (2C, C3′′′′,5′′′′), 127.82 (2C, C2′′′′,6′′′′), 126.77 (C4′′′′), 126.68 (C4′), 124.59 (C7′), 113.11 (4C, C3′′′,5′′′), 110.32 (C2′′ or C7′′), 110.24 (C2′′ or C7′′), 108.38 (C8a′′ or C9a′′), 108.31 (C8a′′ or C9a′′), 97.91 (2C, C4′′,5′′), 85.78 (Ar3C (Dmt)), 70.35 (C5), 63.58 (C2), 58.90 (OCH2), 56.21 (C3), 55.22 (2C, OCH3), 40.36 (4C, NCH3), 36.53 (C4). HRMS (MALDI+): m/z [M+H]+ calcd for C51H50N3O8+ 832.3592; found 832.3594. Phosphoramidites 4a,b; General Procedure. Amide 3a(3b) (0.83 g, 1.0 mmol) was evaporated with dry CH2Cl2 (2 × 50 mL), dissolved in dry CH2Cl2 (100 mL), and diisopropylammonium tetrazolide (0.26 g, 1.5 mmol) was added, followed by addition of bis(N,N-diisopropylamino)-2-cyanoethoxyphosphine (476 µL, 1.5 mmol). The mixture was half-evaporated and stirred under argon for 1 h. After conversion of the starting compound was complete (monitoring by TLC, 2% Et3N in Me2CO v/v), the mixture was diluted with CH2Cl2 (100 mL) and washed with 5% NaHCO3 (100 mL) and brine (100 mL). Organic layer was dried over Na2SO4 during 2 h, evaporated to dryness, and the residue was purified by chromatography on a silica gel column with 1% Et3N in acetone (v/v) as the mobile phase. Fractions containing product were combined and evaporated. The resulting phosphoramidite was dissolved in CH2Cl2 (2 mL) and precipitated into hexane (100 mL). The solid was filtered off, dissolved in dry CH2Cl2 (20 mL), and evaporated to dryness to afford compounds 4a(4b) as red amorphous solids. 1-{3′,6′-Bis(dimethylamino)-3-oxo-spiro[isobenzofuran-1(3H),9′[9H]xanthene]-5-yl-carbonyl}-(3R,5S)-5-[(4,4′-dimethoxytrityl)oxymethyl]-3-(N,N-diisopropylamino-2-cyanoethoxyphosphinyloxy)pyrrolidine (4a). Yield 0.75 g (73%). Rf 0.60 (1% Et3N in acetone). 1H NMR (MeCN-d3), spirolactone form numbering, δ ) 8.05 (br s, 1H, H-4′), 7.82 (m, 1H, H-6′), 7.46 (m, 2H, H-2′′′′,6′′′′), 7.36-7.18 (m, 8H, H-7′,2′′′,6′′′,3′′′′,4′′′′,5′′′′), 6.84 (m, 4H, H-3′′′,5′′′), 6.75-6.62 (m, 2H, H-1′′,8′′), 6.51 (m, 4H, H-2′′,4′′,5′′,7′′), 4.55 (m, 2H, H-3,5), 4.15-3.45 (m, 13H, OCH3, POCH2, DmtOCH2, NCH, H-2a), 3.21 (m, 1H, H-2b), 2.98 (m, 12H, NCH3), 2.75 (t, 1H, J ) 5.9 Hz, CH2CN), 2.63 (m, 1H, CH2CN), 2.37-2.20 (m, 2H, H-4), 1.17-1.04 (m, 12H, CHCH3). 13C NMR (MeCN-d3) δ ) 169.56 (CO), 169.50 (CO), 159.55 (2C, C4′′′), 154.14 (2C, C4a′′,10a′′), 153.85 (2C, C3′′,6′′), 153.09 (C7a′), 146.33 (C1′′′′), 139.54 (0.5C, C5′ or C3a′), 139.37 (0.5C, C5′ or C3a′), 137.09 (2C, C1′′′), 134.45 (m, C6′), 130.90 (m, 4C, C2′′′,6′′′), 129.86 (m, 3C, C3a′ or C5′, C1′′,8′′), 128.90 (2C, C2′′′′,6′′′′), 128.78 (2C, C3′′′′,5′′′′), 127.74 (C4′′′′), 125.63 (C7′), 125.08 (m, C4′), 119.44 (m, CN), 113.99 (4C, C3′′′,5′′′), 110.30 (2C, C2′′,7′′), 107.75 (2C, C8a′′,9a′′), 98.77 (2C, C4′′,5′′), 86.56 (Ar3C (Dmt)), 73.64 (m, C5), 64.12 (m, C2), 59.50-58.60 (m, 2C, CH2ODmt, POCH2), 56.91 (m, C3), 55.82 (2C, OCH3), 43.96 (m, 2C, CHCH3), 40.49 (4C, NCH3), 36.35 (C4), 23.09 (m, 4C, CHCH3), 20.94 (m, 2C, CH2CN). 31P NMR (MeCN-d3) δ ) 147.35, 147.22 (diastereomers). HRMS (ESI+): m/z [M+Na]+ calcd for C60H66N5NaO9P+ 1054.4490; found 1054.4492. 1-{3′,6′-Bis(dimethylamino)-3-oxo-spiro[isobenzofuran-1(3H),9′[9H]xanthene]-6-yl-carbonyl}-(3R,5S)-5-[(4,4′-dimethoxytrityl)oxymethyl]-3-(N,N-diisopropylamino-2-cyanoethoxyphosphinyloxy)pyrrolidine (4b). Yield 0.48 g (47%). Rf 0.35 (1% Et3N in acetone). 1H NMR (MeCN-d3), spirolactone form numbering, δ ) 8.01 (m, 1H, H-4′), 7.74 (m, 1H, H-5′), 7.40-7.04 (m, 10H, H-7′,2′′′,6′′′,2′′′′,3′′′′,4′′′′,5′′′′,6′′′′), 6.86-6.62 (m, 6H,
1676 Bioconjugate Chem., Vol. 20, No. 8, 2009 Chart 1. Tetramethylrhodamine Dye in Zwitterionic Open-Chain Form (left) and Spirolactone Form (right)
H-1′′,8′′,3′′′,5′′′), 6.51-6.41 (m, 4H, H-2′′,4′′,5′′,7′′), 4.54-4.39 (m, 2H, H-3,5), 3.79-3.39 (m, 14H, OCH3, POCH2, DmtOCH2, NCH, H-2), 3.03-2.86 (m, 13H, NCH3, CH2CN), 2.57 (t, 1H, J ) 5.9 Hz, CH2CN), 2.36-2.19 (m, 2H, H-4), 1.21-0.86 (m, 12H, CHCH3). 13C NMR (MeCN-d3) δ ) 169.59 (CO), 169.20 (CO), 159.50 (C4′′′), 159.43 (C4′′′), 154.00-153.00 (m, 4C, C3′′,4a′′,10a′′), 146.03 (C1′′′′), 144.28 (C6′), 137.26 (C1′′′), 136.66 (C1′′′), 130.96 (2C), 130.67 (2C) (C2′′′′,6′′′′), 129.93 (C5′),129.51(m,3C,C3a′,1′′,8′′),128.70(m,4C,C2′′′′,3′′′′,5′′′′,6′′′′), 127.62 (C4′′′′), 125.92 (m, C4′), 123.73 (0.5C), 123.57 (0.5C) (C7′), 113.85 (4C, C3′′′,5′′′), 109.85 (m, 2C, C2′′,7′′), 107.05 (2C, C8a′′,9a′′), 98.97 (m, 2C, C4′′,5′′), 86.37 (Ar3C (Dmt)), 73.50-73.00 (m, C5), 63.54 (m, C2), 59.16 (m, POCH2), 58.45 (m, CH2ODmt), 56.79 (m, C3), 55.79 (2C, OCH3), 43.84 (m, 2C, CHCH3), 40.39 (4C, NCH3), 35.89 (C4), 24.75 (m, 4C, CHCH3), 20.86 (m, 2C, CH2CN). 31P NMR (MeCN-d3) δ ) 147.39, 146.55 (diastereomers). HRMS (ESI+): m/z [M+Na]+ calcd for C60H66N5NaO9P+ 1054.4490; found 1054.4494.
RESULTS AND DISCUSSION Like others rhodamine dyes, tetramethylrhodamine can exist in two tautomeric open-chain and lactone forms (Chart 1). The fluorescent open-chain structure prevails in polar protic media, whereas in aprotic solvents, the dye becomes nonfluorescent switching to spirolactone form. It is worth noting that the alteration in tautomeric structure changes the numbering system. The major ring system in the open-chain form is xanthene changing to isobenzofuran in the lactone form (Chart 1). Thus, carboxyrhodamines derived from trimellitic anhydride are usually known as 5(6)-derivatives corresponding to spirolactone form. Actually, in cases these dyes exist in open-chain form: their correct name is 4′(5′)-derivatives. Carboxytetramethylrhodamines can be easily obtained as a mixture of 5- and 6- isomers. However, in many analytical applications individual isomers are preferred. In a previous communication (78), we described an easy method for chromatographic separation of multigram quantities of 5(6)-carboxyfluorescein derivatives. The chromatographic technique (8) for the separation of 5(6)-carboxytetramethylrhodamines in our hands turned out to be ineffective: we were able to separate less than 1 g of isomeric mixture in one run. We found the method hardly scalable and not applicable for multigram synthesis of single TAMRA isomers. In the present paper, we report a convenient procedure for the synthesis of isomerically pure carboxytetramethylrhodamines using a two-step approach (Scheme 1) with crystallization as a key stage leading to separation of isomers. Benzophenones 1a,b are easily available from trimellitic anhydride and 3-dimethylaminophenol by refluxing in toluene (79). After filtration, the crude mixture of benzophenones was obtained as an amorphous solid. Dissolving it in methanol and evaporation with addition of acetic acid promoted crystallization. The solubilities of benzophenones 1a and 1b in methanol differ significantly: 1a is slightly soluble, whereas 1b dissolves well. In that way, the major part of benzophenone 1a was separated from the mixture by simple extraction 1b with methanol.
Kvach et al.
Evaporation of extract enriched with 1b followed by double crystallization of the residue from acetic acid afforded pure 5′carboxy substituted benzophenone 1b. The simple, direct method using inexpensive starting compounds gives 16% of 1a and 10% of 1b together with mixture 1a+1b (ca. 20%), suitable for further fractional crystallization. The procedure can be scaled up several times without difficulty. The structures of isomeric benzophenones 1a,b were determined using 2D 1H-13C HSQC and HMBC NMR techniques. Here, we consider signal assignment for benzophenone 1b (the resulting 13C NMR shifts assignment is shown in Chart 2). There are signals of two 1,2,4-trisubstituted benzene systems seen in the proton NMR spectrum of this compound (Figure 1A): one has signals at 6.11, 6.22, and 6.86 ppm with constants 3J ) 9.3 Hz and 4J ) 2.5 Hz; another one has shifts 7.83, 8.03, and 8.15 ppm and constants 3J ) 8.3 Hz and 4J ) 1.7 Hz. The direct correlation HSQC (Figure 1B) shows carbons attached to protons. The next step is the assignment of phenolic ring carbons. In the HMBC spectrum, the phenolic proton OH at 12.41 displays cross-peaks with carbon signals at 97.08, 109.43, and 164.40, which apparently belong to carbons 1, 2, and 3 (Figure 1C). Only the carbon at 97.08 is bound to a proton, evidently being C3. Thus, the high field proton system belongs to the phenolic ring, and the low field system is protons of the phthalic acid. From HSQC (Figure 1B), the assignment of C5 and C6 is clear. HMBC correlation of protons 3 and 5 shows that the signal at 109.43 originates from C1 (Figure 1D). Therefore, the signal at 164.40 belongs to C2. HMBC correlation of methyl protons (data not shown) allows assignment of C4. The assignment of C2 and C4 is confirmed by cross-peaks from H6 (Figure 1E). Thus, the assignment of carbon signals of the phenolic ring is complete. The carbonyl signal at 197.40 in HMBC spectrum gives cross-peaks with H6 and H6′ (Figure 1F). The assignment of C3′ and C4′ arises from the HSQC spectrum (Figure 1B). The assignment of closely located signals at 133.65 and 133.73 is carried out using the HMBC spectrum (Figure 1G). The signal at 133.73 displays cross-peaks with protons H4′ and H6′ thus coming from C2′. The signal at 133.65 gives a cross-peak with H3′ and is therefore C5′. Hence, the substitution pattern in the benzene ring is unambiguous: carboxyl groups are located in 2′- and 5′-positions. The assignment of carboxylic carbons is performed using cross-peaks in HMBC spectrum (Figure 1E): the carboxyl signal at 166.10 gives crosspeaks with H4′ and H6′ thus arising from 5′-carboxyl. The signal at 166.53 shows a cross-peak with H3′ and is therefore assigned as 2′-carboxyl. The core of tetramethylrhodamine was assembled by refluxing 1a or 1b with an additional equivalent of 3-dimethylaminophenol in DMF using trimethylsilylpolyphosphate as a dehydrating agent and weak acid catalyst (80, 81). The resulting pure isomers of tetramethylrhodamine were obtained in almost quantitative yields and were used without additional purification. Thus, the two-step assembly method of tetramethylrhodamines allows us to obtain multigram quantities of isomerically pure dye in one run avoiding column chromatography. Carboxytetramethylrhodamines can be converted further into various useful derivatives: oxysuccinimide, maleimide, biotin, acetylene, azide, and so forth. To demonstrate the utility of isomerically pure tetramethylrhodamines, two phosphoramidite reagents for the labeling of oligonucleotides in automated solid-phase synthesis were prepared. Acids 2a,b were reacted with (3R,5S)-5-[(4,4′-dimethoxytrityl)oxymethyl]-3-hydroxypyrrolidine (69) using benzotriazol1-yloxy-tris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP) as a coupling reagent to afford amides 3a,b in satisfactory yields. The phosphitylation of secondary alcohols 3a,b with bis(N,N-diisopropylamino)-2-cyanoethoxyphosphine
Technical Notes
Bioconjugate Chem., Vol. 20, No. 8, 2009 1677
Scheme 1a
a Reagents and conditions. i, PhMe, reflux, 24 h, 16% (1a), 10% (1b); ii, 3-dimethylaminophenol, trimethylsilylpolyphosphate, DMF, reflux, 3 h, 99% (2a), 99% (2b); iii, (3R,5S)-5-[(4,4′-dimethoxytrityl)oxymethyl]-3-hydroxypyrrolidine, PyBOP, DIEA, DMF, 2 h, 60% (3a), 46% (3b); iV, bis(N,N-diisopropylamino)-2-cyanoethoxyphosphine, diisopropylammonium tetrazolide, CH2Cl2, 1 h, 73% (4a), 47% (4b). For compounds 3a,b and 4a,b, the shown numbering is used for NMR assignments.
Chart 2.
13
C NMR Shifts in Benzophenone 1b (solvent DMSO-d6)
in the presence of diisopropylammonium tetrazolide gave the desired phosphoramidites 4a,b. The structures of the compounds were confirmed by 1H, 13C, and 31P NMR spectra and by highresolution mass spectra (HRMS). For the tetramethylrhodamine derivatives 2-4, the assignment of 1H and 13C signals in NMR spectra was accomplished using 2D 1H-1H COSY and 1H-13C HSQC and HMBC techniques. In 13C spectra, we observed the signal of C1-atom in compounds 2a,b at ∼160 ppm (when dissolved in polar d4-MeOD); however, it completely disappears (in compounds 3 and 4) when dissolving in aprotic solvents CDCl3, CD3CN, or DMSO-d6 due to the fast equilibrium process between lactone and open-chain forms (Chart 1). Reagents 4a,b were tested in automated oligonucleotide synthesis. TAMRA phosphoramidites 4a,b are readily soluble in dry acetonitrile and were used as 0.1 M solutions. They reacted well with hydroxy groups of a growing oligonucleotide in tetrazoleassisted coupling conditions. The reaction time for 4a,b was increased from 20 s to 7.5 min to achieve maximum coupling yield. After capping and oxidizing steps, the last dimethoxytrityl group was removed and coupling yield was estimated to be >95%. Using phosphoramidites 4a,b oligonucleotides ON1-ON4 carrying tetramethylrhodamines on 5′-terminus (Table 1) were synthesized. Reagent 4b was also applied for the synthesis of the oligomer ETand energy transfer oligonucleotides ET0-ET14 carrying 6-TAMRA label in internal position and 6-FAM label (78) on the 5′
terminus (Chart 3). Similar energy transfer probes with poly-T linker between donor and acceptor dyes can be applied for construction of wavelength-shifting molecular beacons (82, 83). Oligonucleotides were cleaved from solid support and deprotected by “TAMRA-cocktail”stBuNH2/MeOH/H2O 1:1:2 mixture (84). Conjugates obtained were purified by PAGE and characterized by MALDI-TOF, UV-vis, and fluorescence spectra (Tables 1, 2, and Figures 2, 3). Previous study of fluorescein-labeled oligonucleotides (78) showed that absorption spectra of 5- and 6-FAM-labeled oligonucleotides do not differ significantly, whereas in fluorescence spectra, the 5-isomer displays a 3-5 nm red shift against 6-isomer and emission band in the spectrum of 5-FAM is substantially broader. Like FAM-labeled oligonucleotides, different isomers of TAMRA show differences in fluorescence spectra but they are not so evident. 5-TAMRA on an oligonucleotide displays only 1-3 nm red shift vs 6-isomer. The emission band in the spectrum of 5-TAMRA is slightly broader (Figure 2). Fluorescence quantum yield of the dye in TAMRA-labeled oligonucleotides is sequence dependent (Table 1). It is wellknown that guanine residues quench fluorescence if they are near fluorescent label (85, 86), this is also the case for TAMRA (17, 18). Data show that quantum yield of TAMRAlabeled oligonucleotides ON1-ON4 correlates with the presence or absence of guanine residues in proximity to the TAMRA label. The highest quantum yield (0.69) is observed for conjugate ON1b with no neighbor guanine. The conjugate ON2b with three closely located adjacent guanines shows decreased quantum yield (0.29). There are no significant differences in quantum yields between 5- and 6-isomers of carboxytetramethylrhodamines on oligonucleotides. The synthesis of energy transfer primers ET0-ET14 (Chart 3) demonstrates the internal labeling of oligonucleotides. The conjugates are 5′-labeled with the combined fluorophore (6-FAM)(dT)n(6-TAMRA) and their formal Stokes shift is ca. 85 nm (Table
1678 Bioconjugate Chem., Vol. 20, No. 8, 2009
Kvach et al.
Figure 1. NMR spectra of benzophenone 1b. (A) Proton spectrum of aromatic region; (B) 1H-13C HSQC spectrum of the aromatic region; (C-G) fragments of 1H-13C HMBC spectrum.
2). For detection purposes, FAM dye is usually excited at 480 nm; thus, the difference between FAM excitation and TAMRA emission (580-583 nm) for ET conjugates is about 100 nm.
The efficiency of energy transfer from donor to acceptor depends on the spacing between them (37, 87, 88). Increasing the length of the oligo-dT spacer decreases the quenching of the FAM label,
Technical Notes
Bioconjugate Chem., Vol. 20, No. 8, 2009 1679
Table 1. Spectroscopic and Photophysical Properties of TAMRA-Labeled Oligonucleotidesa #
MALDI-TOF, [M+H]+, found/calcd
sequence, 5′ f 3′
ON1a ON1b ON2a ON2b ON3a ON3b ON4a ON4b
b
5-TAMRA-aataatcagtatgtgacttggattga 6-TAMRA-aataatcagtatgtgacttggattga 5-TAMRA-tgggctctgtaaagaatagtg 6-TAMRA-tgggctctgtaaagaatagtg 5-TAMRA-aaataaaattaggcatatttacaagc 6-TAMRA-aaataaaattaggcatatttacaagc 5-TAMRA-aggcttgaggccaaccatcag 6-TAMRA-aggcttgaggccaaccatcag
8640.4/8639.8 8638.5/8639.8b 7115.8/7114.3c 7116.5/7114.3c 8584.1/8585.8b 8584.9/8585.8b 7032.3/7029.3c 7031.4/7029.3c
abs λmax , nm
fl λmax , nm
Φf
561 561 562 561 561 560 561 562
588 586 589 585 588 586 586 585
0.66 0.69 0.32 0.29 0.63 0.62 0.65 0.58
a 5-TAMRA-modified oligonucleotides were prepared using phosphoramidite 4a; 6-TAMRA-modified oligonucleotides were prepared using phosphoramidite 4b; absorption and fluorescence spectra were measured in 0.1 M bicarbonate buffer (pH 8.5) using rhodamine 6G as a reference (see Experimental Procedures). b Average mass. c Monoisotopic mass.
Chart 3. Structures of Energy Transfer Oligonucleotides
Table 2. Spectroscopic Properties of 6-FAM-6-TAMRA-Labeled Oligonucleotides ETa #
n
ETET0 ET2 ET4 ET6 ET8 ET10 ET12 ET14
0 2 4 6 8 10 12 14
d
MALDI-TOF, [M+H]+, found/calcd d
4490.6/4490.9 5024.8/5026.0d 5432.0/5634.1d 6241.2/6242.2d 6848.8/6850.3d 7459.9/7458.4d 8071.1/8069.5e 8679.0/8677.9e 9289.1/9286.3e
donor (6-FAM) λmaxabs, nm
acceptor (6-TAMRA) λmaxfl, nm
I(TAMRA)/IET-(TAMRA)b
I(TAMRA)/I(FAM)c
497 493 494 495 495 495 495 495
583 583 583 581 581 581 581 580 580
1.0 2.9 11.8 14.9 16.5 16.3 16.3 14.5 14.6
5.2 7.9 4.2 2.8 2.0 1.7 1.2 1.0
a Structures of energy transfer oligonucleotides ET are shown in Chart 3; for conditions, see Figure 3a. Monoisotopic mass. e Average mass.
and its emission at 519 nm rises nearly linearly from ET0 to ET14 (Figure 3a). The behavior of the emission intensity of TAMRA label is more complicated. The spacer is very important for the intensity of TAMRA fluorescence: the insertion of (dT)2 between dyes increases the acceptor emission of ET2 four times in comparison to ET0. Maximal values of TAMRA emission are observed for conjugates ET6, ET8, and ET10stheir emission is 16 times more intense as compared to single TAMRA label excited at 480 nm in conjugate ET- (Table 2, Figure 3a). The values are higher than expected, because the combined chromophore absor-
b
See Figure 3a.
c
See Figure 3b.
bance at the excitation wavelength 480 nm increases only ca. 10 times (ε480(FAM) + ε480(TAMRA)/ε480(TAMRA) ) 10.5). The highest TAMRA/FAM (581 nm/519 nm) emission intensity ratio was observed for ET2, not for ET0 (7.9 vs 5.2). The same behavior of the acceptor intensity was mentioned in other investigations when donor-acceptor distances were too close (37, 87). When two dyes appear in proximity, a contact quenching occurs, and the emission of both the donor and the acceptor diminishes. However, the insertion of a few nucleosides between two dyes prevents such a contact quenching; even with a spacer of two
1680 Bioconjugate Chem., Vol. 20, No. 8, 2009
Figure 2. Normalized absorption (left) and fluorescence (right) spectra of conjugates ON3a (straight lines) and ON3b (dashed lines) in 0.1 M bicarbonate buffer (pH 8.5), excitation at λ ) 525 nm.
Kvach et al.
band (550-650 nm), and 10% in the FAM band (500-550 nm). However, the overall emission efficiency of the absorbed light (FAM emission + TAMRA emission) decreases in the series ET14 > ET12 ≈ ET10 > ET8 > ET6 > ET4 > ET2 . ET0 (Figure 3a). Thus, conjugates ET6-ET10 showed highest TAMRA emission, ET14 the highest FAM+TAMRA overall emission, and ET2 the highest TAMRA/FAM emission intensity ratio. The data may appear useful for design of energy transfer primers and probes. To conclude, we have developed a simple and scalable procedure for the preparation of individual isomers of carboxytetramethylrhodamines, described hydroxyprolinol-based TAMRA phosphoramidite reagents containing individual dye isomers, prepared a series of oligonucleotides 5′- and internal labeled with carboxytetramethylrhodamines, and compared spectral and photophysical spectra of TAMRA-oligonucleotide conjugates. We also showed that in energy transfer primers the optimal spacer between donor (FAM) and acceptor (TAMRA) dyes for maximum acceptor fluorescence should contain 6-10 dT nucleotides.
ACKNOWLEDGMENT The research was supported by BFBR (project B06R-004) and RFBR (projects 06-04-81019 and 08-03-90900). We thank Primetech ltd. (Minsk, Belarus) for the synthesis of labeled oligonucleotides, Alexander Peregudov for NMR spectra, Alexander Baranovsky and Alexey Ustinov for helpful discussion, and anonymous referees for their useful comments.
LITERATURE CITED
Figure 3. (a) Fluorescence spectra of oligonucleotides ET- and ET0...ET14 in 0.1 M bicarbonate buffer (pH 8.5), excitation at λ ) 480 nm, concentration 6 × 10-7 M; (b) fluorescence spectra of oligonucleotides ET0...ET14 normalized to 6-FAM fluorescence maximum (519 nm).
nucleosides, no other interaction than dipole-dipole was observed within the donor-acceptor dye pair (88). The TAMRA/FAM intensity ratio decreases rapidly in the series ET2...ET14 (Table 2, Figure 3b). Conjugate ET2 emits 90% of the integral light energy in the TAMRA fluorescence
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