Studies on the synthesis and biochemical properties of phosphonate analogues of 5'-nucleotides

Michael Anthony Bell, Purdue University

Abstract

The present study involves modifications to the nucleic acid backbone in which one or more of the phosphodiester oxygen atoms are substituted with methylene groups. Nucleic acids modified in this manner are expected to be isosteric with their natural counterparts and, due to the chemical stability of the phosphorus-carbon bond, should be resistant to cleavage by nucleases that break the bond between the phosphorus and the oxygen. Generation of nucleic acids containing phosphorus-carbon linkages began with the synthesis of the dTMP analogue, 5′-phosphonomethyl-5 ′-deoxythymidine (pcdT), in which the 5′ oxygen has been substituted with a methylene group. In order to obtain a potential substrate for enzymatic DNA synthesis, pcdT was converted to the corresponding dTTP analogue, (diphospho)-5′-phosphonomethyl-5 ′-deoxythymidine (pppcdT) by chemical pyrophosphorylation. Studies with this analogue using reverse transcriptase (RT) demonstrated that it is an acceptable substrate and capable of completely substituting as the thymidine monomer for cDNA synthesis. Furthermore, reverse transcriptase can be shown to incorporate pppcdT into DNA utilizing both RNA and DNA templates. Single stranded DNA containing the incorporated analogue can also serve as a template for the synthesis of a normal complementary DNA strand with RT. However, RT cannot accept pppcdT as a substrate for the synthesis of a complementary strand when a cDNA containing the incorporated analogue is used as the template. In addition, studies with DNA-dependent DNA polymerases show that these enzymes are unable to use pppcdT as a substrate. In order to study the analogues mentioned above in vivo, 5′-phosphonomethyl analogues of nucleoside phosphate triesters were synthesized bearing lipophilic blocking groups designed to facilitate passive diffusion through cell membranes. These analogues were assayed for cellular cytotoxicity using established tumor cell lines. Data from preliminary assays demonstrated cytotoxicity in the μM range towards a prostate adenocarcinoma cell line (PC-3). We also developed a system of automated oligonucleotide synthesis based on a new support material that can be recycled. This allows an oligonucleotide synthesizer to operate continuously without the necessity for replacing the support cassette between runs. After ten rounds of synthesis on the same support cassette, a hexamer (dC)6 was still being produced in good yields.

Degree

Ph.D.

Advisors

Gilham, Purdue University.

Subject Area

Biochemistry

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