Efficient Synthesis of [2′-18O]Uridine and Its Incorporation into Oligonucleotides: A New Tool for Mechanistic Study of Nucleotidyl Transfer Reactions by Isotope Effect Analysis Qing Dai,† John K. Frederiksen,† Vernon E. Anderson,¶ Michael E. Harris,*,# and Joseph A. Piccirilli*,†,‡,§ Department of Biochemistry & Molecular Biology, Department of Chemistry, and Howard Hughes Medical Institute, The UniVersity of Chicago, 929 East 57th Street, MC 1028, Chicago, Illinois 60637, and Department of Biochemistry and Center for RNA Molecular Biology, School of Medicine, Case Western ReserVe UniVersity, CleVeland, Ohio 44106
[email protected];
[email protected] ReceiVed August 8, 2007
Lack of sufficient quantities of isotopically labeled materials has precluded the use of heavy atom isotope effects to investigate mechanisms of nucleotidyl transfer reactions in nucleic acids. Here we achieve regioselective opening of 2,2′cyclouridine with [18O2]benzoic acid/potassium hydride, allowing an efficient “one-pot” synthesis of [2′-18O]uridine in 88% yield. Conversion to the corresponding phosphoramidite enables solid-phase synthesis of [2′-18O] RNA substrates for isotope effect studies with nucleotidyl transferases and hydrolases. A wide range of enzymes, including both protein and RNA catalysts, promotes cleavage of RNA phosphodiester bonds by attack of the adjacent 2′-OH and displacement of the 5′-O leaving group. In solution, this reaction is catalyzed by both acid and base, and both stepwise and concerted reaction mechanisms are observed.1 Despite significant effort, little direct experimental evidence exists that determines which specific mechanism is followed for enzyme-catalyzed RNA cleavage. The effect of isotopic substitution on chemical reaction kinetics and equilibria provides an especially powerful way to investigate enzymatic transition states and their active site interactions. Isotopic substitution represents the smallest possible chemical perturbation of a catalytic system but can nonetheless have important influences on chemical reactivity.2 Interpretation of † Department of Biochemistry & Molecular Biology, The University of Chicago. ‡ Department of Chemistry, The University of Chicago. § Howard Hughes Medical Institute, The University of Chicago. ¶ Department of Biochemistry, Case Western Reserve University. # Center for RNA Molecular Biology, Case Western Reserve University.
(1) Oivanen, M.; Kuusela, S.; Lonnberg, H. Chem. ReV. 1998, 98, 961.
these isotope effects on reactivity can provide essential information for defining chemical mechanism. For nucleotidyl transfer reactions, the nucleophile isotope effect on the attacking 2′-O in particular offers the opportunity to distinguish unambiguously between stepwise and concerted reaction mechanisms.3 Technical challenges severely limit the use of isotope effects to study phosphoryl transferase enzymes that operate on nucleic acids. One such limitation involves access to nucleic acid substrates bearing the desired site-specific isotope enrichment. Here we describe an efficient synthesis of [2′-18O]uridine, which enables synthesis of RNA substrates for isotope effect analyses on protein and RNA enzymes that catalyze nucleophilic attack by the 2′-OH. Few strategies exist for the synthesis of isotope-enriched nucleosides, particularly sugar isotopomers.4 Previously, Pang et al. obtained [3′-18O]uridine in low yield via reversible hydration (HCl/H218O) of a 3′-ketouridine derivative followed by reduction and separation from the predominant xylo epimer.5 Recently, Wnuk et al. reported access to [3′-18O]-1-(β-Darabinofuranosyl)uracil through a six-step synthesis from 2,3′cyclouridine involving a Fox thermal rearrangement as the key step.6 However, this strategy generated arabinofuranosyl derivatives and could not be used to prepare [2′-18O]uridine. Another strategy reported in the literature to prepare 18O-labeled sugar isotopomers makes use of SN2-substituted reaction of triflate with 18O-labeled nucleophiles.7 Unfortunately, the 2′-β triflate derivative of uridine failed to undergo the analogous reaction to generate [2′-18O]uridine. Commercially available 2,2′-cyclouridine (1) could provide direct access to [2′-18O]uridine (2) if an 18O-enriched oxygen nucleophile can be made to attack the ribose C-2′ regioselectively. Initial attempts were directed toward hydrolysis of 1 under alkaline conditions (1 N NaOH, MeOH, rt, overnight). Consistent with previous observations,6 the reaction gave only 1-(β-D-arabinofuranosyl)uracil, indicating that hydroxide attacks exclusively at the nucleobase C-2 position. Therefore, to gain access to 2 from 1, we sought to identify an oxygen nucleophile with altered regioselectivity. Ueda has summarized the reactions of 2,2′-cyclouridine with various nucleophiles.8 Unfortunately, none of the reported examples (Table 1) can be used to synthesize [2′-18O]uridine (2). In general, reactions of 2,2′-cyclouridine with nucleophiles have two possible regiochemical outcomes: (1) attack at the (2) (a) Northrop, D. B. Methods 2001, 24, 117. (b) Hengge, A. C. FEBS Lett. 2001, 501, 99. (3) Anderson, V. E.; Ruszczycky, M. W.; Harris, M. E. Chem. ReV. 2006, 106, 3236. (4) (a) Follman, H.; Hogencamp, H. P. C. J. Am. Chem. Soc. 1970, 92, 671. (b) Solsten, R. T.; McCloskey, J. A.; Schram, K. H. Nucleosides Nucleotides 1982, 1, 57. (c) Schubert, E. M.; Schram, K. H. J. Labelled Compd. Radiopharm. 1982, 19, 929. (d) Schwartz, H. M.; MacCoss, M.; Danyluk, S. S. J. Am. Chem. Soc. 1983, 105, 5901. (e) Schwartz, H. M.; MacCoss, M.; Danyluk, S. S. Magn. Reson. Chem. 1985, 23, 885. (5) Pang, H.; Schram, K. H.; Smith, D. L.; Gupta, S. P.; Townsend, L. B.; McCloskey, J. A. J. Org. Chem. 1982, 47, 3923. (6) Wnuk, S. F.; Chowdhury, S. M.; Garcia, P. I., Jr.; Robins, M. J. J. Org. Chem. 2002, 67, 1816. (7) (a) Jiang, C.; Suhadolnik, R. J.; Baker, D. C. Nucleosides Nucleotides 1988, 7, 271. (b) D’Souza, F. W.; Lowary, T. L. J. Org. Chem. 1998, 63, 3166. (8) Ueda, T. Synthesis and Reaction of Pyrimidine Nucleosides. In Chemistry of Nucleosides and Nucleotides, 1st ed.; Townsend L. B., Ed.; Plenum Press: New York and London, 1988; pp 1-112.
10.1021/jo701727h CCC: $40.75 © 2008 American Chemical Society
Published on Web 12/04/2007
J. Org. Chem. 2008, 73, 309-311
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TABLE 1. Reaction of 2,2′-Cyclouridine with Various Nucleophiles
a
entry
nucleophile
solvent
T (°C)
time (h)
product
yielda (%)
16 211 312 413 514 615 716 816 917 1018 1119 1220 1321 1422
NaOH Mg(OMe)2 B(OMe)3 NH3 NaN3+BzOH H2S+Et3N EtSH+reagent 1b t-BuSH+reagent 1b AcSH PhSe-SePh+NaBH4 HF HCl LiBr NaI+TsOH‚H2O
MeOH/H2O MeOH MeOH+CH(OMe)3 MeOH HMPA DMF DMF DMF dioxane EtOH dioxane dioxane dioxane acetone
rt 65 150 37 150 20 60 100 110 78 100-110 75-80 60 50
16 5 42 48-168
