A new approach to the synthesis of the 5′-deoxy-5′-methylphosphonate linked thymidine oligonucleotide analogues

Abstract

A new synthetic method for the preparation of the 5'-deoxy-5'-methylphosphonate linked thymidine oligonucleotides (5'-methylenephosphonate analogues) was developed. The method is based on the use of a phosphonate protecting group, 4-methoxy-1-oxido-2-picolyl, enabling intramolecular nucleophilic catalysis which together with the condensing agent, 2,4,6-triisopropylbenzenesulfonyl chloride, secures fast and efficient formation of the 5'-methylenephosphonate internucleosidic bonds. The produced protected oligomers were treated with thiophenol and triethylamine to remove the phosphonate protecting groups, cleaved from the solid support using concentrated aqueous ammonia, and purified by HPLC. Several thymidine oligonucleotide analogues with the chain length of up to 20 nucleotidic units, in which all internal 5'-oxygen atoms have been replaced by methylene groups directly bound to phosphorus, were synthesised using this methodology.

Novel isosteric, isopolar phosphonate analogs of oligonucleotides: Preparation and properties

“…Monomethyl ester 9a was first transformed into mixed diester 12a on reaction with 4-methoxy-1oxido-2-pyridinemethanol in the presence of 2chloro-5,5-dimethyl-2-oxo-1,3,2-dioxaphosphinane as coupling reagent and 4-methoxypyridine N-oxide as nucleophilic catalyst 13,14 (Scheme 2). The resulting diester 12a was subjected to aqueous pyridine treatment to remove the methyl ester group under formation of 13a, which in turn was desilylated on reaction with TBAF in THF to afford 14a.…”

“…Heinemann 11 reported a crystal structure of a self-complementary octamer d(GCCCG*GGC) containing 2 0 ,3 0 -dideoxyguanosine-3 0 -methylenephosphonate unit 1 located at the G* site. In contrast to this well-tolerated 3 0 -modification of the internucleotide linkage by the duplex structure, the introduction of regioisomeric 5 0 -modification represented by the 2 0 ,5 0 -dideoxythymidine-5 0 -methylenephosphonate unit 2 into oligothymidylates 12,13 had a destabilizing effect on the hybridization. 14 The C3 0 -O-P-CH 2 -CH 2 -C4 00 linkage of these oligomers was found to be resistant toward calf spleen phosphodiesterase but unstable toward snake venom exonuclease.…”

“…14 The C3 0 -O-P-CH 2 -CH 2 -C4 00 linkage of these oligomers was found to be resistant toward calf spleen phosphodiesterase but unstable toward snake venom exonuclease. 13 These phosphonate oligomers did not found a broader biological exploitation but, recently, Liao et al 15 reported on a faster cleavage of a 5 0 -methylene phosphonate unit-containing oligomer by Tetrahymena ribozyme in comparison with unmodified substrate. Another paper showed that the analogue of dTTP and ADP containing likewise a methylene group in place of the 5 0 oxygen atom were substrates for reverse transcriptase 16 and PNPase, 17 respectively.…”

The strand transfer oligonucleotide inhibitors of HIV-integrase

“…For the introduction of the triethylene glycol linker (TEG) we used 8-dimethoxytrityloxy-3,6-dioxaoctyl-(2-cyanoethyl)diisopropylphosphoramidite. Phosphonate unit A (see Figure 1) was attached to the 2-cyanoethanol type of solid support by the phosphotriester method [37,38] using an appropriate 3 0 -phosphonate monomer [39]. The synthesized oligonucleotides were deprotected [37,38] and purified by reverse phase HPLC followed by PAGE under denaturing conditions.…”

Conformational evaluation of labeled C3′‐O‐P‐13CH2‐O‐C4″ phosphonate internucleotide linkage, a phosphodiester isostere

“…However, the study on hybridization of the phosphonate 4 0 -methyl-dT 15 (Type IV and V) showed 18 a strong decrease in the hybridization capability in case of both IV and V modifications, though to a different extent (interestingly, a similar effect was observed in case of another type of isosteric internucleotide linkage III in which the 5 0 -oxygen atom was replaced by methylene group). 20,21 Having excluded any dramatic role of the sugar pucker in influencing the properties of the Type IV (95 % of the C2 0 -endo conformation was found), we reasoned that in all likelihood, it is just the changed conformation of the modified internucleotide linkage due to the 5 0 CH 2 $ 5 0 O transposition which serves as the main factor influencing hybridization properties. Basic description of the likely arrangement of the novel linkages was provided 18 (CD curves, NMR spectra, and an initial modeling study).…”