Hydrolytic Reactions of Diribonucleoside 3′,5′-(3′-N-Phosphoramidates): Kinetics and Mechanisms for the P-O and P-N Bond Cleavage of 3′-Amino-3′-deoxyuridylyl-3′,5′-uridine Mikko Ora,* ,† Kati Mattila, † Tuomas Lo ¨ nnberg, † Mikko Oivanen, ‡ and Harri Lo ¨ nnberg † Contribution from the Department of Chemistry, UniVersity of Turku, FIN-20014 Turku, Finland, and Laboratory of Organic Chemistry, Department of Chemistry, UniVersity of Helsinki, P.O. Box 55, FIN-00014 Helsinki, Finland Received June 28, 2002 Abstract: Hydrolytic reactions of 3′-amino-3′-deoxyuridylyl-3′,5′-uridine (2a), an analogue of uridylyl-3′,5′- uridine having the 3′-bridging oxygen replaced with nitrogen, have been followed by RP HPLC over a wide pH range. The only reaction taking place under alkaline conditions (pH > 9) is hydroxide ion-catalyzed hydrolysis (first-order in [OH - ]) to a mixture of 3′-amino-3′-deoxyuridine 3′-phosphoramidate (7) and uridine (4). The reaction proceeds without detectable accumulation of any intermediates. At pH 6-8, a pH-independent formation of 3′-amino-3′-deoxyuridine 2′-phosphate (3) competes with the base-catalyzed cleavage. Both 3 and in particular 7 are, however, rather rapidly dephosphorylated under these conditions to 3′-amino-3′-deoxyuridine (5). In all likelihood, both 3 and 7 are formed by an intramolecular nucleophilic attack of the 2′-hydroxy function on the phosphorus atom, giving a phosphorane-like intermediate or transition state. Under moderately acidic conditions (pH 2-6), the predominant reaction is acid-catalyzed cleavage of the P-N3′ bond (first-order in [H + ]) that yields an equimolar mixture of 5 and uridine 5′-phosphate (6). The reaction is proposed to proceed without intramolecular participation of the neighboring 2′-hydroxyl group. Under more acidic conditions (pH < 2), hydrolysis to 3 and 4 starts to compete with the cleavage of the P-N bond, and this reaction is even the fastest one at pH < 1. Formation of 2′-O,3′-N-cyclic phosphoramidate as an intermediate appears probable, although its appearance cannot be experimentally verified. The rate constants for various partial reactions have been determined. The reaction mechanisms and the effect that replacing the 3′-oxygen with nitrogen has on the behavior of the phosphorane intermediate are discussed. Introduction Phosphoramidate analogues of oligodeoxyribonucleotides (1a), having the 3′-oxygen of each internucleosidic phosphodi- ester linkage replaced with nitrogen, have been shown to be resistant toward nucleases, and to form stable duplexes with complementary oligonucleotide sequences 1 and stable triplexes with double-stranded DNA. 2 For this reason, they have received attention as potential antisense oligonucleotides. 3 More recently, similarly modified oligoribonucleotides (1b) have been synthe- sized and shown to be, analogously to their 2′-deoxy counter- parts, so tolerant toward enzymatic cleavage and to hybridize so efficiently that they also may be regarded as viable candidates for antisense purposes. 4,5 Being RNA analogues, they may also be expected to find applications as aptamers, i.e., oligomeric sequences selected to fold into structures exhibiting high affinity for various proteins or low molecular weight ligands. 6 Despite these attractive properties, the intrinsic chemical reactivity of nucleoside 3′-N-phosphoramidates in comparison to their native phosphate ester counterparts has not been studied in detail. Only some semiquantitative data on the stability of 3′-N-bridged phosphoramidate RNA dimers in aqueous acetic acid and ammonia have been reported. 4,7 Besides these, a study on the * Address correspondence to this author. E-mail: mikora@utu.fi. † University of Turku. ‡ University of Helsinki. (1) Chen, L.-K.; Schultz, R. G.; Lloyd, D. H.; Gryaznov, S. M. Nucleic Acids Res. 1995, 23, 2661. (2) Escude, C.; Giovannageli, C.; Sun, J.-S.; Lloyd, D. H.; Chen, J.-K.; Gryaznov, S. M.; Garestier, T.; Helene, C. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 4365. (3) For recent reviews on antisense oligonucleotides, see: (a) Cook, P. D. In Annual Reports in Medicinal Chemistry; Bristol, J. A., Ed.; Academic Press: San Diego, 1998; pp 313-325. (b) Crooke, S. T. In Antisense Research and Application; Crooke, S. T., Ed.; Springer: Berlin 1998; pp 1-50. (c) Bennett, C. F. Expert Opin. InVest. Drugs 1999, 8, 237. (d) Manoharan, M. Antisense Nucleic Acid Drug DeV. 2002, 12, 103. (4) Gryaznov, S. 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