Chemical studies on the synthesis of nucleic acids and nucleic acid analogues

Tod Jeffrey Miller, Purdue University

Abstract

In studies aimed at improving the automated solid-phase synthesis of RNA, a new protecting group, 4-nitrobenzyloxymethyl, was developed to block the 2$\sp\prime$ hydroxyl of ribonucleoside monomers during construction and isolation of oligoribonucleotides. The required monomers, 5$\sp\prime$-O-dimethoxytrityl-2$\sp\prime$-O-(4-nitrobenzyloxymethyl) derivatives of uridine, 4-N-benzoylcytidine, 6-N-benzoyladenosine and 2-N-isobutyrylguanosine have been synthesized. These nucleosides have been converted into their 3$\sp\prime$-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidites for use in the phosphite-triester method of RNA synthesis. The use of this new blocking group results in RNA synthesis that requires coupling times of only 3 minutes, and, as such, the efficiency of synthesis and the quality of the products are markedly increased. The new protecting group also has the advantage that it can be readily removed under mild conditions (fluoride ion). This lability to fluoride ion (a new reaction) has thus led to the development of this group as a new method for general hydroxyl protection. The strategy involves reaction of an alcohol with 4-nitrobenzyl chloromethyl ether and the resulting 4-nitrobenzyloxymethyl derivatives are formed in high yield. Removal of this group is effected by treatment with tetrabutylammonium fluoride in tetrahydrofuran to afford the recovered alcohol in near quantitative yield. In a separate project aimed at the development of new nucleotide therapeutic agents, the isosteric nucleotide analogues of GMP, CMP, AMP and dAMP that contain a methylene group substituted for the 5$\sp\prime$ oxygen were synthesized in order to investigate their potential for enzymatic incorporation into RNA or DNA. For this purpose the nucleotide monophosphate analogues may be converted into their triphosphate derivatives and tested in transcription reactions catalyzed by polymerases. In the case of bacteriophage T3 RNA polymerase, the ATP analogue (diphospho)-5$\sp\prime$-deoxy-5$\sp\prime$-phosphonomethyladenosine, along with CTP, GTP and UTP were used to prepare RNA-like products. These products were degraded with snake venom phosphodiesterase to the 5$\sp\prime$-mononucleotides CMP, GMP, UMP and the corresponding analogue of AMP. Additionally, by using the appropriate DNA template and promoter strands, the AMP analogue was specifically incorporated into the hammerhead ribozyme substrate 5$\sp\prime$-GCUGUCACCGCG-3$\sp\prime$. While the dodecamer containing the normal C-A linkage is cleaved into two hexamers, the dodecamer containing the analogue linkage at this position is completely resistant to cleavage.

Degree

Ph.D.

Advisors

Gilham, Purdue University.

Subject Area

Biochemistry|Organic chemistry

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