Fluorescent pyrimidine ribonucleotide: Synthesis, enzymatic incorporation, and utilization

Journal of the American Chemical Society

Volumn 129, Issue 7, 2007, Pages 2044-2053

  Srivatsan, Seergazhi G ^a   Tor, Yitzhak ^a  

^a UNIVERSITY OF CALIFORNIA   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

DERIVATIVES; ENZYMES; FLUORESCENCE; MOLECULAR STRUCTURE; RNA; SYNTHESIS (CHEMICAL);

ENZYMATIC PREPARATION; FLUORESCENCE SPECTROSCOPY; PYRIMIDINE RIBONUCLEOTIDE; RNA-LIGAND INTERACTIONS;

NITROGEN COMPOUNDS;

2 AMINOPURINE; AMINOGLYCOSIDE ANTIBIOTIC AGENT; FURAN; HETEROCYCLIC COMPOUND; LIGAND; NUCLEIC ACID BASE; OLIGONUCLEOTIDE; PHOSPHATE; PURINE DERIVATIVE; PYRIMIDINE DERIVATIVE; RNA; RNA POLYMERASE;

ARTICLE; BINDING SITE; CHROMATOPHORE; ENZYME ACTIVE SITE; FLUORESCENCE; FLUORESCENCE SPECTROSCOPY; GENE CONSTRUCT; GENETIC TRANSCRIPTION; MOLECULAR DYNAMICS; MOLECULAR INTERACTION; MOLECULAR RECOGNITION; NONHUMAN; RNA STRUCTURE; RNA SYNTHESIS;

AMINOGLYCOSIDES; BINDING SITES; FLUORESCENT DYES; FURANS; NUCLEIC ACID CONFORMATION; PYRIMIDINE NUCLEOTIDES; RIBONUCLEOTIDES; RNA; RNA, MESSENGER; SPECTROMETRY, FLUORESCENCE; SPECTROMETRY, MASS, MATRIX-ASSISTED LASER DESORPTION-IONIZATION;

EID: 33847298021     PISSN: 00027863     EISSN: None     Source Type: Journal    
DOI: 10.1021/ja066455r     Document Type: Article

References (92)

1. For review articles, see: (a) Millar, D. P. Curr. Opin. Struct. Biol. 1996, 6, 322-326.
2. (b) Wojczewski, C.; Stolze, K.; Engels, J. W. Synlett 1999, 1667-1678.
3. (c) Hawkins, M. E. Cell Biochem. Biophys. 2001, 34, 257-281.
4. (d) Murphy, C. J. Adv. Photochem. 2001, 26, 145-217.
5. (e) Rist, M. J.; Marino, J. P. Curr. Org. Chem. 2002, 6, 775-793.
6. (f) Okamoto, A.; Saito, Y.; Saito, I. J. Photochem. Photobiol. C: Photochem. Rev. 2005, 6, 108-122.
7. (g) Ranasinghe, R. T.; Brown, T. Chem. Commun. 2005, 5487-5502.
8. (h) Silverman, A. P.; Kool, E. T. Chem. Rev. 2006, 106, 3775-3789.
9. (i) Wilson, J. N.; Kool, E. T. Org. Biomol. Chem. 2006, 4, 4265-4274.
10. Most efforts in developing fluorescent nucleosides have focused on DNA modifications. Selected examples include: (a) Coleman, R. S.; Madaras, M. L. J. Org. Chem. 1998, 63, 5700-5703.
11. (b) Nakatani, K.; Dohno, C.; Saito, I. J. Am. Chem. Soc. 1999, 121, 10854-10855.
12. (c) Seela, F.; Zulauf, M.; Sauer, M.; Deimel, M. Helv. Chim. Acta 2000, 83, 910-927.
13. (d) Strässler, C.; Davis, N. E.; Kool, E. T. Helv. Chim. Acta 2000, 83, 2160-2171.
14. (e) Hurley, D. J.; Seaman, S. E.; Mazura, J. C.; Tor, Y. Org. Lett. 2002, 4, 2305-2308.
15. (f) Coleman, R. S.; Mortensen, M. A. Tetrahedron Lett. 2003, 44, 1215-1219.
16. (g) Jiao, G.-S.; Kim, T. G.; Topp, M. R. Burgess, K. Org. Lett. 2004, 6, 1701-1704.
17. (h) Okamoto, A.; Kanatani, K.; Saito, I. J. Am. Chem. Soc. 2004, 126, 4820-4827.
18. (i) Dash, C.; Rausch, J. W.; Le Grice, S. F. J. Nucleic Acids Res. 2004, 32, 1539-1547.
19. (j) Mayer, E.; Valis, L.; Wagner, C.; Rist, M.; Amann, N.; Wagenknecht, H.-A. ChemBioChem 2004, 5, 865-868.
20. (k) Sandin, P.; Wilhelmsson, L. M.; Lincoln, P.; Powers, V. E. C.; Brown, T.; Albinsson, B. Nucleic Acids Res. 2005, 33, 5019-5025.
21. (l) Okamoto, A.; Tainaka, K.; Nishiza, K.-I.; Saito, I. J. Am. Chem. Soc. 2005, 127, 13128-13129.
22. (m) Okamoto, A.; Tainaka, K.; Saito, I. Bioconjugate Chem. 2005, 16, 1105-1111.
23. (n) Gaballah, S. T.; Vaught, J. D.; Eaton, B. E.; Netzel, T. L. J. Phys. Chem. B 2005, 109, 5927-5934.
24. (o) Andreatta, D.; Sen, S.; Perez, L. J. L.; Kovalenko, S. A.; Ernsting, N. P.; Murphy, C. J.; Coleman, R. S.; Berg, M. A. J. Am. Chem. Soc. 2006, 128, 6885-6892.
25. (p) Kim, S. J.; Kool, E. T. J. Am. Chem. Soc. 2006, 128, 6164-6171.
26. (q) Cho, Y.; Kool, E. T. ChemBioChem 2006, 7, 669-672.
27. (a) Ward, D. C.; Reich, E.; Stryer, L. J. Biol. Chem. 1969, 244, 1228-1237.
28. (b) Kawai, M.; Lee, M. J.; Evans, K. O.; Nordlund, T. M. J. Fluorescence 2001, 11, 23-32.
29. (c) Jean, J. M.; Hall, K. B. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 37-41.
30. (d) Rachofsky, E. L.; Osman, R.; Ross, J. B. A. Biochemistry 2001, 40, 946-956.
31. (e) The 7-deaza and 8-aza-7-deaza analogues of 2-AP have been introduced. See: Seela, F.; Becher, G. Helv. Chim. Acta 2000, 83, 928-942.
32. The quantum yield of the free 2-AP nucleoside in aqueous environment is 0.68 (ref 3d).
33. (a) Menger, M.; Tuschl, T.; Eckstein, F.; Porschke, D. Biochemistry 1996, 35, 14710-14716.
34. (b) Kirk, S. R.; Luedtke, N. W.; Tor, Y. Bioorg. Med. Chem. 2001, 9, 2295-2301.
35. Rist, M. J.; Marino, J. P. Nucleic Acids Res. 2001, 29, 2401-2408.
36. Lacourciere, K. A.; Stivers, J. T.; Marino, J. P. Biochemistry 2000, 39, 5630-5641.
37. DeJong, E. S.; Chang, C. E.; Gilson, M. K.; Marino, J. P. Biochemistry 2003, 42, 8035-8046.
38. Kaul, M.; Barbieri, C. M.; Pilch, D. S. J. Am. Chem. Soc. 2004, 126, 3447-3453.
39. Shandrick, S.; Zhao, Q.; Han, Q.; Ayida, B. K.; Takahashi, M.; Winters, G. C.; Simonsen, K. B.; Vourloumis, D.; Hermann, T. Angew. Chem., Int. Ed. 2004, 43, 3177-3182.
40. An isosteric fluorescent dC analogue has been reported: (a) Wut, P.; Nordlund, T. M.; Gildea, B.; McLaughlin, L. W. Biochemistry 1990, 29, 6508-6514.
41. (b) Singleton, S. F.; Shan, F.; Kanan, M. W.; McIntosh, C. M.; Stearman, C. J.; Helm, J. S.; Webb, K. J. Org. Lett. 2001, 3, 3919-3922.
42. For, site-specific fluorescent labeling of RNA molecules by transcription using unnatural base pairs, see: Kawai, R.; Kimoto, M.; Ikeda, S.; Mitsui, T.; Endo, M.; Yokoyama, S.; Hirao, I. J. Am. Chem. Soc. 2005, 127, 17286-17295.
43. Endo, M.; Mitsui, T.; Okuni, T.; Kimoto, M.; Hirao, I.; Yokoyama, S. Bioorg. Med. Chem. Lett. 2004, 14, 2593-2596.
44. To the best of our knowledge, the emission quantum yield for pyrroloC has not been rigorously determined (however, see: Liu, C.; Martin, C. T. J. Mol. Biol. 2001, 308, 465-475). Glen research reports quantum yield values of 0.07 and 0.02 for single- and double-stranded oligonucleotides. respectively.
45. Tinsley, R.; Walter, N. G. RNA 2006, 12, 522-529.
46. Greco, N. J.; Tor, Y. J. Am. Chem. Soc. 2005, 127, 10784-10785.
47. Transcription using a nucleoside triphosphate was chosen as a method for incorporating the fluorescent nucleotide, because of the following: (a) simple synthesis - the triphospate is synthesized in two steps from commercially available precursors via procedures that do not require complicated protecting group chemistry necessary for solid-phase RNA synthesis; and (b) effective incorporation - reasonably large quantities of RNA can be synthesized by transcription reactions with relatively small amounts of the modified triphosphate. This makes the method accessible to almost all laboratories that are not necessarily equipped for complex organic synthesis and solid-phase RNA synthesis. It is important to note, however, that there is no inherent limitation for the synthesis of the corresponding phosphoramidite necessary for the solid-phase synthesis of modified RNA.
48. Ludwig, J. Acta Biochim. Biophys. Acad. Sci. Hung. 1981, 16, 131-133.
49. See Supporting Information for additional details.
50. Reichardt, C. Chem. Rev. 1994, 94, 2319-2358.
51. Slight differences between the absorption and emission of the deoxy furan containing nucleoside and its ribonuclcoside counterpart are summarized in Table S.1. See Supporting information.
52. Milligan, J. F.; Uhlenbeck, O. C. Methods Enzymol. 1989, 180, 51-62.
53. Tor, Y.; Dervan, P. B. J. Am. Chem. Soc. 1993, 115, 4461-4467.
54. Transcription efficiencies are reported with respect to transcription in the presence of natural nucleotides and represent the average of two independent reactions. Errors are ±10%: see Experimental Section.
55. Milligan, J. F.; Groebe, D. R.; Witherell, G. W.; Uhlenbeck, O. C. Nucleic Acids Res. 1987, 15, 8783-8798.
56. Buck, M.; Connick, M.; Ames, B. N. Anal. Biochem. 1983, 129, 1-13.
57. Note this trend (i.e., significantly quenched emission in oligonucleotides when compared to the free nucleoside) is not unique to chromophore 2. 2-AP and pC both qualitatively show similar behaviors, where the incorporated nucleoside when compared to the free nucleoside in aqueous solution is much less emissive. Extensive studies with 2-AP-containing oligonucleotides have suggested that both base stacking and collisions with neighboring bases contribute to quenching. See ref 3d.
58. See Supporting Information for preliminary Stern-Volmer titrations of 2 with all four native nucleotide monophosphates (Figure S.4).
59. Hermann, T.; Tor, Y. Expert Opin. Ther. Pat. 2005, 15, 49-62.
60. (a) Neu, H. C. Science 1992, 257, 1064-1073.
61. (b) Kotra, L. P.; Haddad, J.; Mobashery, S. Antimicrob. Agents Chemother. 2000, 44, 3249-3256.
62. (c) Jansen, W. T. M.; van der Bruggen, J. T.; Verhoef, J.; Fluit, A. C. Drug Resist. Updates 2006, 9, 123-133.
63. Knowles, D. J.; Foloppe, N.; Matassova, N. B.; Murchie, A. I. Curr. Opin. Pharmacol. 2002, 2, 501-506.
64. (a) Fourmy, D.; Recht, M. I.; Blanchard, S. C.; Puglisi, J. D. Science 1996, 274, 1367-1371.
65. (b) Yoshizawa, S.; Fourmy, D.; Puglisi, J. D. EMBO J. 1998, 17, 6437-6448.
66. (a) Vicens, Q.; Westhof, E. Structure 2001, 9, 647-658.
67. (b) Vicens, Q.; Westhof, E. Chem. Biol. 2002, 9, 747-755.
68. (a) Chow, C. S.; Bogdan, F. M. Chem. Rev. 1997, 97, 1489-1513.
69. (b) Tor, Y. Angew. Chem., Int. Ed. 1999, 38, 1579-1582.
70. (c) Hermann, T. Angew. Chem., Int. Ed. 2000, 39, 1890-1905.
71. (d) Hermann, T.; Patel, D. J. Science 2000, 287, 820-825.
72. (e) Sucheck, S. J.; Wone, C.-H. Curr. Opin. Chem. Biol. 2000, 4, 678-686.
73. (f) Wilson, W. D.; Li, K. Curr. Med. Chem. 2000, 7, 73-98.
74. (g) Gallego, J.; Varani, G. Acc. Chem. Res. 2001, 34, 836-843.
75. (h) Tor, Y. Chem. Biol. Chem. 2003, 4, 998-1007.
76. Moazed, D.; Noller, H. F. Nature 1987, 327, 389-394.
77. Purohit, P.; Stern, S. Nature 1994, 370, 659-662.
78. For an A-site construct labeled at its terminus with fluorescein, see: Haddad, J.; Kotra, L. P.; Llano-Sotelo, B.; Kim C.; Azucena, E. F., Jr.; Liu, M.; Vakulenko, S. B.; Chow, C. S.; Mobashery, S. J. Am. Chem. Soc. 2002, 124, 3229-3237.
79. Francois, B.; Russell, R. J. M.; Murray, J. B.; Aboul-ela, F.; Masquida, B.; Vicens, Q.; Westhof, E. Nucleic Acids Res. 2005, 33, 5677-5690.
80. Thermal denaturation studies with singly modified RNA duplexes show no detrimental effect on the furan modification on duplex stability. See Figures S.5 and S.6 in the Supporting Information for data.
81. 50 is the concentration required for 50% response.
82. In certain cases the single-stranded control sequences respond to increasing concentrations of aminoglycosides. Examination of the single-stranded oligonucleotides A1 and A4 shows that they can form a "self-complementary" metastable duplex with eight base pairs and three noncanonical (likely bulged out) A•C and C•C "pairs" (see Figure S.8 in the Supporting Information). The single-stranded oligonucleotide is therefore likely to provide a target for positively charged aminoglycosides, as these antibiotics are known to bind numerous RNA structures in a nonspecific fashion through electrostatic interactions (see ref 33h and Zhao, F.; Zhao, Q.; Blount, K. F.; Han, Q.; Tor, Y.; Hermann, T. Angew. Chem., Int. Ed. 2005, 44, 5329-5334). It is also important to note that the 2-AP-containing single strand, although not apparent from Figures 8E and 9E, is also responding to increasing paromomycin and neomycin concentrations (see Figures S.9 and S.10 in the Supporting Information plotted using an expanded scale).
83. (a) Lynch, S. R.; Puglisi, J. D. J. Mol. Biol. 2001, 306, 1037-1058.
84. (b) Griffey, R. H.; Hofstalder, S. A.; Sannes-Lowery, K. A.; Ecker, D. J.; Crooke, S. T. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 10129-10113.
85. (c) Wong, C. H.; Hendrix, M.; Priestley, E. S.; Greenberg, W. A. Chem. Biol. 1998, 5, 397-406.
86. (d) Recht, M. I.; Fourmy, D.; Blanchard, S. C.; Dahlquist, K. D.; Puglisi, J. D. J. Mol. Biol. 1996, 262, 421-436.
87. Crystallization of the furan-modified RNA constructs with and without aminoglycosides is in progress.
88. (a) Pilch, D. S.; Kaul, M.; Barbieri, C. M.; Kerrigan, J. E. Biopolymers 2003, 70, 58-79.
89. (b) Ryu, D. H.; Rando, R. R. Bioorg. Med. Chem. 2001, 9, 2601-2608.
90. Neomycin B is known to present challenges for 2-AP-labeled A-site constructs. Fluorescent detection can be observed under modified ionic strength conditions, but even then the fluorescence response is significantly weaker than that induced by paromomycin (D. Pilch and T. Hermann, personal communications). Although crystal structures of neomycin and paromomycin complexed with the A-site nearly superimpose each other, at this point there is no satisfactory explanation for the inability of 2-AP model constructs to effectively report on neomycin binding.
91. 1492 is titrated with parmomycin, both fluorophores respond. See Figure S.12 in the Supporting Information.
92. Moffatt, J. G. Can. J. Chem. 1964, 42, 599-604.