Chemical synthesis of oligoribonucleotides: A rebirth of the $o$-nitrobenzyl protecting group

Joseph Allan Hayes, Purdue University

Abstract

In order to carry out studies on RNA structure and synthesis, it is essential to have a ready source of pure defined-sequence oligoribonucleotides. The development of a system for the synthesis of RNA has not kept pace with the notable advances made in the construction of DNA due to the lack of a completely satisfactory protection mode for the ribonucleoside 2$\sp\prime$-hydroxyl. The $o$-nitrobenzyl group has many of the required properties necessary for the protection of this site. However, using the methods recommended in the literature for the photo-deprotection of oligoribonucleotides chemically synthesized utilizing this group results in cleavage of only 79% of the ethers while the rest are photooxidized to an $o$-nitrobenzoate. Formation of the ester is initiated from an enhancement in the acidity of the benzylic hydrogen in the photo-excited singlet state allowing deprotonation. The resulting anion attacks oxygen to produce an alkoxyhydroperoxide. Additional UV light converts this intermediate to the ester product. The $o$-nitrobenzyl groups can be removed quantitatively by buffering the photolysis solution to pH 3.5 and removing oxygen from the system prior to irradiation. The difficult and time-consuming separation of the positional isomers formed during the synthesis of 2$\sp\prime$-$O$-($o$-nitrobenzyl)-nucleosides using published procedures inspired investigation into the development of a method to synthesize these compounds selectively. For uridine and guanosine, the lactam function of their bases are protected as the 3-$N$-($p$-anisoyl) and 6-$O$-($o$-nitrobenzyl) derivatives respectively. These nucleosides, as well as adenosine and cytidine, are reacted with 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane to temporarily protect the 5$\sp\prime$ and 3$\sp\prime$ positions. These intermediates are specifically alkylated at the 2$\sp\prime$-hydroxyl with $o$-nitrobenzyl iodide in the presence of sodium hydride in DMF at $-$42$\sp\circ$C. The exocyclic amino groups of adenosine, cytidine, and guanosine are acylated followed by removal of the silyl protecting group. These newly synthesized compounds, were tritylated at the 5$\sp\prime$-hydroxyl, converted to 3$\sp\prime$-O-($\beta$-cyanoethyl-$N,N$-diisopropyl)phosphoamidites, and used to construct an oligoribonucleotide on solid support. Deprotection using the newly developed methods resulted in isolation of a pure RNA molecule in a high yield. These results have been partially published in Tetrahedron Letters 26, 2407 (1985) and Nucleosides and Nucleotides, in press.

Degree

Ph.D.

Advisors

Gilham, Purdue University.

Subject Area

Biochemistry

COinS