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J. Am. Chem. Soc. 1997, 119, 4330-4337
Adenine Derivatives as Phosphate-Activating Groups for the Regioselective Formation of 3′,5′-Linked Oligoadenylates on Montmorillonite: Possible Phosphate-Activating Groups for the Prebiotic Synthesis of RNA K. Joseph Prabahar and James P. Ferris* Contribution from the Department of Chemistry, Rensselaer Polytechnic Institute, Troy, New York 12180-3590 ReceiVed January 9, 1997X
Abstract: Methyladenine and adenine N-phosphoryl derivatives of adenosine 5′-monophosphate (5′-AMP) and uridine 5′-monophosphate (5′-UMP) are synthesized, and their structures are elucidated. The oligomerization reactions of the adenine derivatives of 5′-phosphoramidates of adenosine on montmorillonite are investigated. 1-Methyladenine and 3-methyladenine derivatives on montmorillonite yielded oligoadenylates as long as undecamer, and the 2-methyladenine and adenine derivatives on montmorillonite yielded oligomers up to hexamers and pentamers, respectively. The 1-methyladenine derivative yielded linear, cyclic, and A5′ppA-derived oligonucleotides with a regioselectivity for the 3′,5′-phosphodiester linkages averaging 84%. The effect of pKa and amine structure of phosphate-activating groups on the montmorillonite-catalyzed oligomerization of the 5′-phosphoramidate of adenosine are discussed. The binding and reaction of methyladenine and adenine N-phosphoryl derivatives of adenosine are described.
Introduction The observation that RNA can function as an enzyme and store genetic information revealed that RNA or RNA-like molecules may have played a central role in the first primitive life on earth.1,2 This scenario implies that organic precursors for the RNA-like monomers formed spontaneously and then underwent self-condensation reaction on the minerals catalysts to yield short oligonucleotides. The short oligomers could have been elongated on the mineral surface by reaction with RNA monomers.3 Studies from our laboratory demonstrated that the condensation of 5′-phosphoroimidazolides of nucleosides on montmorillonite in aqueous solution yields oligonucleotides as long as undecamer.4-6 The heterogeneous RNA oligomers formed by mineral catalysis could have served as templates for the formation of their complementary oligomers.7 The condensation of mononucleotides on montmorillonite and the template-directed synthesis of complementary oligonucleotides are successful when the phosphate group of the mononucleotide is activated by imidazole or substituted imidazoles; nucleoside di- and triphosphates react slowly and undergo hydrolysis.8 The effect of structure of the amine phosphateactivating group on the oligomerization of adenosine 5′monophosphate on montmorillonite showed that 4-aminopyridine derivatives are the most effective phosphate-activating groups for oligoadenylate formation on montmorillonite. OliX Abstract published in AdVance ACS Abstracts, May 1, 1997. (1) Been, M. D.; Cech, T. R. Science 1988, 239, 1412-1416. (2) Guerrier-Takoda, C.; Gardiner, K.; Marsh, T.; Pace, N.; Altman, S. Cell 1983, 35, 849-857. (3) Ferris, J. P.; Hill, A. R., Jr.; Liu, R.; Orgel, L. E. Nature 1996, 38, 59-61. (4) Ferris, J. P.; Ertem, G. Science 1992, 257, 1387-1389. (5) Ferris, J. P.; Ertem, G. J. Am. Chem. Soc. 1993, 115, 12270-12275. (6) Ding, Z. P.; Kawamura, K.; Ferris, J. P. Origins Life EVol. Biosphere 1996, 26, 151-171. (7) Ertem, G.; Ferris, J. P. Nature 1996, 379, 238-240. (8) Wieman, B. J.; Lohrmann, R.; Orgel, L. E.; Schneider-Bernloehr, H.; Sulston, J. E. Science 1968, 161, 387.
S0002-7863(97)00076-0 CCC: $14.00
gomers containing up to 12-mers were produced in which the regioselectivity for 3′,5′-phosphodiester bond formation averaged 88%. It was concluded from the study that heterocyclic bases which have 4-aminopyridine type structural units may have been utilized as phosphate-activating groups for the condensation of mononucleotides on the primitive earth.9 Since purines and pyrimidine derivatives have structural units similar to 4-aminopyridine and they were essential for the prebiotic synthesis of RNA monomers, purines and pyrimidines were investigated as phosphate-activating groups for the condensation of adenosine 5′-monophosphate on montmorillonite. Experimental Section General. 1H NMR spectra were recorded on Varian XL-200 and Varian Unity 500 instruments, and 13C NMR spectra were recorded on Varian Unity 500 instrument operating at 125 MHz. NMR spectra were obtained as DMSO and D2O solutions using the following references: tetramethylsilane (TMS) and 3-(trimethylsilyl)-1-propanesulfonic acid (Tsp) for 1H NMR and DMSO-d6 solvent peaks for 13C NMR. The chemical shifts are reported in ppm. UV spectra are recorded on Varian Cary 219 spectrophotometer. High-resolution mass spectra (FAB; matrix of dithiothreitol and dithioerythritol) were obtained at the School of Chemical Sciences, University of Illinois, Urbana, IL. The C18 Bondapak reverse phase gel 100 Å (mesh 15-20 µm) was purchased from Waters for the use in the preparative reverse phase column. The Dowex 50 W-X8 cation exchange resin (mesh 15-20) was purchased from Bio-rad laboratories and activated by the Cooper procedure.10 Adenosine 5′-monophosphate (5′-AMP), uridine 5′monophosphate (5′-UMP), 1-methyladenine, 2-methyladenine hemisulfate, adenine hemisulfate, 3-methyladenine, 1-ethyl-3-((dimethylamino)propyl)carbodiimide (EDAC), ribonuclease T2 (RNase T2), and bacterial alkaline phosphatase (APH) were obtained from Sigma. 2,2′-Dipyridyl disulfide, triphenylphosphine, and 4-(dimethylamino)pyridine were obtained from Aldrich. DMF and DMSO were purchased from Fisher, and ether, acetone, and triethylamine were obtained from Mallinckrodt. (9) Prabahar, K. J.; Cole, T. D.; Ferris, J. P. J. Am. Chem. Soc. 1994, 116, 10914-10920. (10) Cooper, T. G. The Tools of Biochemistry; John Wiley and Sons: New York, 1977.
© 1997 American Chemical Society
Adenine DeriVatiVes as Phosphate-ActiVating Groups Volclay SPV-200 was a gift from the American Colloid Co., Arlington Heights, IL. The homoionic volclay montmorillonite was prepared by titration method.11 HPLC analyses were performed on a Waters HPLC system equipped with Lamda-Max model 481 UV detector, on a µ-Bondapak C18 reverse phase column using a gradient of 0.005 M NaH2PO4 in 5% methanol at pH 3.5 mixed with 0.01 M NaH2PO4 (40% methanol) at pH 4.0 and on a HEMA-IEC BIO Q anion exchange column from Alltech using a gradient of 0-0.4 M NaClO4 at pH 8 with 2 mM Tris buffer. No Tris was used when samples were collected for further analysis, and the column was eluted in an isocratic mode using a buffer A (98%) and buffer B (2%) mixture for 6 min followed by a regular gradient elution mode. General Procedure for the Preparation of Activated Nucleotides (4a, 5a).12 A mixture of 5′-NMP‚H2O (free acid) (0.33 mmol) and 1-methyladenine (0.049, 0.33 mmol) was dissolved in water (2 mL), and the pH of the solution was adjusted to 5. 1-Ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride [EDAC] (0.191 g, 1 mmol) was added to this reaction mixture with stirring. Two additional portions of EDAC (2 × 0.0636 g, 2 × 0.33 mmol) were added at 1 h intervals, and the reaction was allowed to proceed for 4 h. During the reaction, the EDAC was hydrolyzed to the corresponding urea which was separated from the activated nucleotide by passing the reaction mixture through a DOWEX 50 W-X8 cation exchange column and elution with water (150 mL). The collected eluate was lyophilized to yield a colorless solid. General Procedure for the Preparation of Activated Nucleotides (4b-d, 5b-d).13 A mixture of 5′-NMP‚H2O (free acid, 0.5 mmol) and heterocyclic base 3 (0.5 mmol) was dissolved in DMF (10 mL) and DMSO (5 mL) in a 50 mL flask, and the solvents were evaporated to 2 mL at a reduced pressure to remove H2O. The evaporation was repeated twice with DMF (2 × 10 mL). The residue was dissolved in DMF (10 mL) and cooled to -15 °C in an ice-salt mixture. Triethylamine (2 mL) was added to the reaction mixture with stirring followed by a solution of 2,2′-dipyridyl disulfide (0.333 g, 1.5 mmol) and triphenylphosphine (0.393 g, 1.5 mmol) in DMF (5 mL). The stirring was continued for 4 h, and the resulting clear yellow reaction mixture was added dropwise to a 1 L flask containing a cold solution of anhydrous sodium perchlorate (1 g) in a mixture of ether (100 mL), acetone (90 mL), and triethylamine (8 mL) with stirring under argon atmosphere. The stirring was continued for 1 h, and a colorless flocculant solid separated. The stirring was stopped, the solid was allowed to settle for 15 min, the supernatant was drained, and the remaining portion was centrifuged. The resulting colorless pellet was washed twice with 50% acetone-ether mixture, centrifuged, and dried under vacuum. General Procedure for the Purification of Activated Nucleotides (4,5). The activated nucleotides 4 and 5 were purified using a preparative reverse phase column (30 × 300 mm). The column was eluted with water and then a water-acetonitrile mixture. The pH of the eluents was adjusted to 8-9 with a trace amount of triethylamine; 10 µL of triethylamine is sufficient for 1000 mL of water (excess triethylamine leads to the formation of triethylammonium salts of the activated nucleotides). The chromatography was performed at 4 °C, and the column was eluted under 5-10 psi of argon pressure with the flow rate of 7-10 mL/min. Fractions (100 mL) were collected and analyzed by reverse phase HPLC. The fractions which contained the activated nucleotides were pooled and lyophilized to yield the sodium salts of the activated nucleotides as colorless solids. General Procedure for the Analysis of Activated Nucleotides (4-7) by Thin-Layer Chromatography (TLC).14 Thin-layer chromatography was carried out on the precoated 0.25 mm silica gel plates by ascending technique. The solvent system was prepared from (11) Banin, A.; Lawless, J. G.; Mazzurco, J.; Church, F. M.; Margulies, L.; Orenberg, J. B. Origins Life EVol. Biosphere 1985, 15, 89. (12) Ivanovskaya, M. G.; Gottikh, M. B.; Sabarova, Z. A. Nucleosides Nucleotides 1987, 6, 913-934. (13) Mukaiyama, T.; Hashimoto, M. Bull. Chem. Soc. Jpn. 1971, 44, 2284. (14) Verlander, M. S.; Lohrmann, R.; Orgel, L. E. J. Mol. EVol. 1973, 2, 303-316.
J. Am. Chem. Soc., Vol. 119, No. 19, 1997 4331 n-propanol, concentrated ammonia, and water (22:4:14 mL), and the products were identified by cochromatography with the authentic samples. Adenosine 5′-Phosphoro-1-methyladeninium (1-CH3adenpA) (4a). Compound 4a was obtained as a colorless solid (0.130 g, 81.6%) and was shown to be 96% pure by HPLC on a reverse phase column. It showed poor solubility in water and gave a clear solution only if the concentration was
