Synthesis of alpha-methylene-deoxynucleotide triphosphates (alpha-m-dNTP) as non-cleavable substrates for polymerases

TOXI 113

Fengting Liang, bcho@uri.edu1, Nidhi Jain, nidhijain@mail.uri.edu1, A. S. Prakasha Gowda2, Thomas E. Spratt, tes13@psu.edu2, and Bongsup Cho, bcho@uri.edu1. (1) Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island, Kingston, RI 02881, (2) Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, PA 17033
The nucleoside 5-triphosphates (NTP) analogs, with modifications in the triphosphate chain, have been widely used as non-cleavable nucleotide probes for crystallographic and spectroscopic studies of various proteins, including polymerases. Here, we report the synthesis and characterization of a set of deoxy NTP analogs (alpha-methylene-dNTP; N= A, C, G, T), in which the alpha-oxygen is replaced by a methylene group. The synthesis entails preparation of the dNDP precursors by coupling 5'-tosyl nucleosides with methylene-diphosphate, followed by ion-exchange FPLC and an enzymatic phosphorylation. The products were characterized by ESI/MS and 1H and 31P NMR. Novel cyclonucleosides were isolated as byproducts in the synthesis of the dG and dT derivatives. The alpha-methylene-dNTPs are potent inhibitors of E. coli DNA polymerase I with a Ki in the micromolar range. [NIH R01CA98296, #P20 RR016457, R01CA75074]
 

Poster Session and Awards
6:00 PM-10:00 PM, Tuesday, August 21, 2007 BCEC -- 204 A/B, Poster

Sci-Mix
8:00 PM-10:00 PM, Monday, August 20, 2007 BCEC -- Exhibit Hall - B2, Sci-Mix

Division of Chemical Toxicology

The 234th ACS National Meeting, Boston, MA, August 19-23, 2007