Selective Fluorometric Detection of Guanosine-Containing Sequences by 6-Phenylpyrrolocytidine in DNA

Abstract

The intrinsically fluorescent nucleoside 2′-deoxy-6-­phenylpyrrolocytidine, when incorporated into an oligonucleotide, is selectively quenched by hybridization with match DNA vs. mismatched sequences.

Key words

oligonucleotides - fluorescence detection - 2′-deoxy­pyrrolocytidine - single nucleotide polymorphism analysis

References and Notes

• 2 Rist MJ. Marino JP. Curr. Org. Chem.  2002,  6:  775 
• 3 Miyata K. Tamamushi R. Ohkubo A. Taguchi H. Seio K. Santa T. Sekine M. Org. Lett.  2006,  8:  1545 
• 4 Okamoto A. Tanaka K. Fukuta T. Saito I. J. Am. Chem. Soc.  2003,  125:  9296 
• 5 Martin CT. Liu C. J. Mol. Biol.  2001,  308:  465 
• 6 Martin CT. Liu C. J. Biol. Chem.  2002,  277:  2725 
• 7 Chen P. He C. J. Am. Chem. Soc.  2004,  126:  728 
• 8 Dash C. Raush JW. Le Grice SFJ. Nucleic Acids Res.  2004,  32:  1539 
• 9 Johnson NP. Baase WA. von Hippel PH. Proc. Natl. Acad. Sci. U.S.A.  2005,  102:  7169 
• 10 Marti AA. Jockusch S. Li Z. Ju J. Turro NJ. Nucleic Acids Res.  2006,  34:  e50 ; doi: 10.1093/nar/gkl134
• 13 Hudson RHE. Dambenieks AK. Viirre RD. Synlett  2004,  2400 
• 14 Hudson RHE. Dambenieks AK. Heterocycles  2006,  68:  1325 
• 15 The first synthesis of pyrrolocytidine: Inoue H. Imura A. Ohtsuka E. Nippon Kagaku Kaishi  1987,  7:  1214 
• 16 Berry DA. Jung K.-Y. Wise DS. Sercel AD. Pearson WH. Mackie H. Randolph JB. Somers RL. Tetrahedron Lett.  2004,  45:  2457 
• 17 A previous different synthesis has been reported: Woo J. Meyer RB. Gamper HB. Nucleic Acids Res.  1996,  24:  2470 
• 18 Aucagne V. Amblard F. Agrofoglio LA. Synlett  2004,  13:  2406 
• 20 Thompson KC. Norimune M. J. Phys. Chem. B  2005,  109:  6012 
• 21 Hardman SJO. Thompson KC. Biochemistry  2006,  45:  9145 

1

Current address: Department of Chemistry, Bu-Ali Sina University, Hamadan, 65174, Iran

11

MepC is commerically available from Glen Research, Sterling, Virginia, 20164, USA.

12

Woo et al. (ref. 16) report Tm data for unsubstituted pC, which indicates slight destabilization for each insert, while Berry and co-workers (ref. 15) found MepC to hybridize selectively but do not disclose their Tm data. The same authors indicate that a 19-mer (5′-GCG TAA CTT CXG GAG ATG T-3′, X = pC) with a single, central insert is fluorescent, yet do not report the 19-mer’s response to hybridization.

19

The phosphoramidite reagent was prepared by the following route, selected data are given below.
2′-Deoxy-5′-O-(4,4′-dimethoxytrityl)-5-iodouridine
In a solution of anhyd pyridine (50 mL) and Et₃N (6 mL) was dissolved 2′-deoxy-5-iodouridine (2.124 g, 6 mmol) and then 4,4′-dimethoxytritylchloride (2.642 g, 7.8 mmol). The mixture was stirred at r.t. for 5 h and then the reaction was quenched by the addition of MeOH (2 mL). The reaction mixture was diluted with CH₂Cl₂ and washed with 0.5 M NaHCO₃ (5 × 50 mL). The organic phase was separated and dried over Na₂SO₄ (2 g), then the solvent removed. The residue was purified by silica gel column chromatography using gradient elution with hexane-acetone-Et₃N (80:15:5 to 40:55:5) and 2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-5-iodouridine was isolated as white foam (3.78 g, 96%). ¹H NMR (400 MHz, CDCl₃): δ = 8.14 (s, 1 H), 7.21-7.42 (m, 11 H), 6.84 (m, 4 H), 6.31 (dd, J ₁ = 6.8 Hz, J ₂ = 5.9 Hz, 1 H), 4.55 (m, 1 H), 4.07 (m, 1 H), 3.79 (s, 6 H), 3.78 (m, 1 H), 3.36-3.44 (m, 2 H), 2.46-2.64 (m, 1 H), 2.30-2.33 (m, 1 H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 160.6, 158.9, 150.5, 144.6, 135.7, 135.6, 130.3, 130.3, 128.3, 128.3, 127.3, 113.6, 87.24, 86.9, 85.9, 72.7, 69.0, 63.8, 55.52, 41.7. 32.0 ppm. HRMS (EI) m/z calcd: 656.1019; found: 656.1636.
2′-Deoxy-5′- O -(4,4′-dimethoxytrityl)-6-phenylfurano-uridine A round-bottomed flask was charged with 2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-5-iodouridine (1.3 g, 2 mmol), and phenylacetylene (0.30 g, 3 mmol), Et₃N (0.56 mL, 4 mmol), DMF (15 mL), and was then deoxygenated with N₂. Then Pd(PPh₃)₄ (0.311 g, 0.2 mmol) and CuI (0.076 g, 0.4 mmol) were added and the mixture was stirred for 24 h in the dark and at r.t. After 24 h, with out any workup, additional CuI (0.076 g, 0.4 mmol) was added along with MeOH (20 mL) and Et₃N (10 mL). This mixture was refluxed until the cyclization reaction was complete as monitored by TLC (hexane-acetone, 6:4), 8 h. The reaction mixture was dissolved in CH₂Cl₂ (100 mL) and washed with sat. EDTA (2 × 100 mL), and then organic phase was dried over Na₂SO₄. After filtration the solvent was removed and the residue was further dried under vacuum 2 h. The resulting residue was purified by silica gel column chromatography using gradient elution with hexane-acetone-Et₃N (7.5:2:0.5 to 3:6.5:0.5). Off-white colored foam, 0.99 g (79%). ¹H NMR (400 MH, CDCl₃): δ = 8.94 (s, 1 H), 7.64-7.66 (d, J = 7.1 Hz, 2 H), 7.28-7.45 (m, 12 H), 6.85-6.87 (m, 4 H), 6.33-6.36 (m, 1 H), 5.70 (s, 1 H), 4.67-4.72 (m, 1 H), 4.44-4.46 (m, 1 H), 4.10-4.12 (m, 1 H), 3.77 (s, 3 H), 3.75 (s, 3 H), 3.52-3.66 (m, 2 H), 2.69-2.76 (m, 1 H), 2.45-2.51 (m, 1 H) ppm.
2′-Deoxy-5′- O -(4,4′-dimethoxytrityl)-6-phenylpyrrolo-cytidine
A pressure bottle was charged with one of the 2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-6-phenylfuranouridine (0.95 g, 1.5 mmol), concd aq NH₃ (4 mL) and MeOH (6 mL), and then stirred at 55 °C. Progress of the reaction was followed by TLC (hexane-acetone-MeOH, 4:4:2). After completion of reaction (21 h), the solvent was removed under vacuum and the title compound was isolated by silica gel column chromatography using gradient elution with hexane-acetone-MeOH-Et₃N (7.5:2:0:0.5 to 3:4.5:2:0.5). A yellow foam was isolated, 0.74 g (78%). ¹H NMR (400MHz, CDCl₃): δ = 9.99 (br, 1 H), 8.84 (s, 1 H), 7.59-7.61 (d, J = 7.2 Hz, 2 H), 7.25-7.47 (m, 11 H), 6.83-6.86 (m, 4 H), 6.49 (m, 1 H), 5.69 (s, 1 H), 4.66 (m, 1 H), 4.16 (m, 1 H), 3.77-3.80 (m, 1 H), 3.74 (s, 3 H), 3.75 (s, 3 H), 3.49-3.61 (m, 2 H), 2.82-287 (m, 1 H), 2.41-2.46 (m, 1 H) ppm.
2′-Deoxy-3′-(2-cyanoethyldiisopropylphosphoramidite)-5′- O -(4,4′-dimethoxytrityl)-6-phenylpyrrolocytidine
2-Cyanoethyldiisopropylphosphoramidochloridite (0.473 g, 2 mmol) was added to solution of 2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-6-phenypyrrolcytidine (1 mmol) and Et₃N (2 mL) in dry CH₂Cl₂ (15 mL). Reaction mixture was stirred at r.t. under N₂ for 2 h (reaction followed by TLC). Then reaction was quenched with MeOH (2 mL), washed with 0.5 M NaHCO₃ (50 mL) and the organic phase was dried over Na₂SO₄ (1 g). Residue was purified by column chromatog-raphy using gradient elution with hexane-acetone-Et₃N (7.5:2:0.5 to 4.5:4:0.5) to give a mixture of diastereomers. Yellow foam, 0.661 g (80%). ¹H NMR (400 MHz, CD₃CN): δ = 9.67 (br, 1 H), 8.73 and 8.68 (2 s, 1 H), 6.85-7.61 (m, 14 H), 6.76-6.79 (m, 4 H), 6.32-6.37 (2 m, 1 H), 4.60-4.64 (2 m, 1 H), 4.25 and 4.19 (2 m, 1 H), 3.68 and 3.66 (2 s, 6 H), 3.56-3.64 (m, 2 H), 3.28-3.50 (m, 2 H), 2.61-2.73 (m, 2 H), 2.56-2.60 (m, 1 H), 2.30-2.38 (m, 1 H), 1.05-1.25 (m, 14 H) ppm. ³¹P NMR (161.8 MHz, CD₃CN): δ = 150.02, 149.61 (dr = 0.48:0.52) ppm. ESI-TOF MS: m/z calcd [Na]: 852.3502; found: 852. 3498.