Synthesis and Biochemical Evaluation of Phosphonoformate Oligodeoxyribonucleotides

Abstract

Phosphonoformate oligodeoxyribonucleotides were prepared via a solid phase synthesis strategy. The first step in the preparation of appropriate synthons was condensation of bis(N,N-diisopropylamino)phosphine and diphenylmethylsilylethyl chloroformate in the presence of sodium metal to yield formic acid, [bis(N,N-diisopropylamino)phosphino]-beta-(diphenylmethylsilylethyl) ester. The product of this reaction was then condensed with appropriately protected 2'-deoxynucleosides using 4,5-dicyanoimidazole to yield the 3'-O-phosphinoamidite reactive monomers. The exocyclic amines of cytosine, adenine, and guanine were protected with 9-fluorenylmethyloxycarbonyl, and oligodeoxyribonucleotides were synthesized on controlled pore glass using the hydroquinone-O,O'-diacetic acid linker. Synthons were sequentially added to this support using tetrazole as an activator, oxidized to phosphonoformate, and the transient 5'-protecting group was removed with acid. Following total synthesis of an oligomer, protecting groups were removed with TEMED.HF and products purified by HPLC. These analogues were resistant to nucleases, formed duplexes with complementary RNA (A-form), and, as chimeric oligomers containing phosphate at selected sites, stimulated RNase H1 activity.

Chemical Modification of Oligonucleotides for Therapeutic, Bioanalytical and other Applications

“…All three phosphodiester modifications showed very high nuclease resistance when incubated with snake venom phosphodiesterase or DNase I. Phosphonoacetate and thiophosphonoacetate containing oligonucleotides are one of the first examples of chimeras having capped ends with a catalytic PS central region stimulating RNase H1 catalysis at rates faster than native DNA. [2,3] The conformational locking of oligonucleotides has been shown to be favourable for hybridisation with complementary RNA/DNA. Most notable examples include LNA and BNA, which are discussed below.…”

Synthesis of dinucleoside acylphosphonites by phosphonodiamidite chemistry and investigation of phosphorus epimerization

“…However, a minor product with a peak in the 31 P NMR spectrum near 63 ppm was observed that was comparable to that exhibited by 6 at 52 ppm. We repeated Caruthers’ method [ 46 ], which involved a two hour reflux of 4 with sodium, and similarly detected no visible disappearance of the sodium. No reaction of this mixture with acetic anhydride was detected, beyond the occasional appearance of the 63 ppm peak.…”

“…While the deoxygenation of phosphine oxides is known [ 44 – 45 ], deoxygenation of the functionally much more complex phosphonate 3b to the dinucleoside analog of 1 was not attempted. Only one example of an acylphosphonite analog of 1 was found in the literature, namely, a dinucleoside formate reported by Caruthers et al [ 46 ]. As shown in Scheme 1 , the reaction of H -phosphonodiamidite 4 with chloroformate 5 gave acyl diamidite 6 , which by following standard nucleoside coupling protocols gave the formate phosphonite 1a .…”

Synthesis and Evaluation of Modified Oligodeoxynucleotides Containing Diphosphodiester Internucleotide Linkages

“…During the past two decades, chemically modified oligodeoxynucleotides (ODNs) have received much attention in the search for potential therapeutic and diagnostic agents and in the study of numerous biochemical and biological processes 1–5. To a great extent, these modifications have focused on replacing the phosphodiester group by phosphodiester mimics, such as phosphorothioate,6 methylphosphate,7 phosphoramidates,8 boranophosphate,9 alkylphosphotriesters,10 phosphorodithioate,11 phosphonomethyl,12 propynes,13 diene,14 phosphonoformate,15 and other groups 16. The phosphate‐modified ODNs have been used as inhibitors of gene expression,1 viral enzymes,2, 3 in vitro messenger‐RNA translation,4 and sequence‐specific DNA binding proteins 5…”