Enzymatic Phosphorylation of Unnatural Nucleosides Yiqin Wu, † Ming Fa, Eunju Lee Tae, Peter G. Schultz, and Floyd E. Romesberg* Contribution from the Department of Chemistry, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California 92037 Received August 7, 2002 Abstract: In an effort to expand the genetic alphabet, a number of unnatural, predominantly hydrophobic, nucleoside analogues have been developed which pair selectively in duplex DNA and during enzymatic synthesis. Significant progress has been made toward the efficient in vitro replication of DNA containing these base pairs. However, the in vivo expansion of the genetic alphabet will require that the unnatural nucleoside triphosphates be available within the cell at sufficient concentrations for DNA replication. We report our initial efforts toward the development of an unnatural in vivo nucleoside phosphorylation pathway that is based on nucleoside salvage enzymes. The first step of this pathway is catalyzed by the D. melanogaster nucleoside kinase, which catalyzes the phosphorylation of nucleosides to the corresponding monophosphates. We demonstrate that each unnatural nucleoside is phosphorylated with a rate that should be sufficient for the in vivo replication of DNA. Introduction In an effort to expand the genetic alphabet by supplementing the natural base pairs dA:dT and dG:dC with an unnatural base pair, we have synthesized and characterized a variety of predominantly hydrophobic nucleobases analogues. 1-6 Several of the unnatural base pairs formed between these hydrophobic nucleobases are stable in duplex DNA and are also inserted, proofread, and extended efficiently and selectively by DNA polymerases in vitro. 7 On the basis of these results, as well as pioneering studies from the Kool lab, 8,9 it is apparent that the requirements for duplex stability and replication do not limit the genetic code to hydrogen bonded (H-bonded) base pairs, and hydrophobic interactions may be sufficient to control information storage and retrieval. The in vivo replication of DNA containing these unnatural base pairs requires that the unnatural nucleoside triphosphates be available intracellularly at sufficient concentrations for DNA synthesis. Thus, we have begun to examine different pathways for intracellular activation of unnatural nucleosides. These studies of unnatural nucleoside activation may also aid in the design of pharmacologically active nucleoside analogue drugs that also rely on cellular activation. The simplest strategy to supply E. coli with unnatural nucleoside triphosphates is to supplement the growth media with the unnatural nucleosides. It is possible that these small, hydrophobic molecules will either passively diffuse or be actively transferred across the lipophilic cell membrane and become trapped inside the cell, provided that they are phos- phorylated by cellular kinases of the nucleoside salvage pathway. 10 In this pathway nucleosides are converted to the corresponding triphosphates by the successive action of nucleo- side, monophosphate, and diphosphate kinases. This pathway produces nucleoside triphosphates in sufficient concentrations for a variety of cellular functions. For example, in mammalian cells, DNA repair, mitochondrial DNA synthesis in G1 phase cells, and the activation of antiviral and cytostatic nucleoside analogues, 11,12 all rely on nucleoside triphosphates from the salvage pathway. The first step in the synthesis of nucleoside triphosphates is the nucleoside kinase catalyzed transfer of the γ-phosphate from a donor molecule (ATP) to the nucleoside C5′-OH acceptor to yield the nucleoside monophosphate. In some cases, this is the rate-limiting step in the pharmacological activation of nucleoside drug analogues. 13 Thus, a variety of antiviral therapies are based on the ability of a virus-encoded kinase to phosphorylate a broader range of substrates than the host cellular kinases. For example, acyclovir is selectively activated by herpes simplex virus type-1 thymidine kinase (HSV-1 TK), 14 which * To whom correspondence should be addressed. E-mail: floyd@ scripps.edu. † Present address: Syrrx, Inc. 10450 Science Center Drive, Suite 100, San Diego, CA 92121. (1) McMinn, D. L.; Ogawa, A. K.; Wu, Y.; Liu, J.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc. 1999, 121, 11 585-11 586. (2) Ogawa, A. K.; Wu, Y.; McMinn, D. L.; Liu, J.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc. 2000, 122, 3274-3287. (3) Ogawa, A. K.; Wu, Y.; Berger, M.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc. 2000, 122, 8803-8804. (4) Wu, Y.; Ogawa, A. K.; Berger, M.; McMinn, D. L.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc. 2000, 122, 7621-7632. (5) Berger, M.; Ogawa, A. K.; McMinn, D. L.; Wu, Y.; Schultz, P. G.; Romesberg, F. E. Angew. Chem., Int. Ed. 2000, 39, 2940-2942. (6) Berger, M.; Luzzi, S. D.; Henry, A. A.; Romesberg, F. E. J. Am. Chem. Soc. 2002, 124, 1222-1226. (7) Tae, E. L.; Wu, Y.; Xia, G.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc. 2001, 123, 7439-7440. (8) Matray, T. J.; Kool, E. T. J. Am. Chem. 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