Synthesis of novel 3 '-C-methylene thymidine and 5-methyluridine/cytidine H-phosphonates and phosphonamidites for new backbone modification of oligonucleotides

Novel 5 ' -O-DMT- and MMT-protected 3 ' -C-methylene-modified thymidine, 5- methyluridine, and 5-methylcytidine H-phosphonates 1-7 with O-methyl, fluor o, hydrogen, and O-(2-methoxyethyl) substituents at the 2 ' -position have been synthesized by a new effective strategy from the corresponding key int ermediates 3 ' -C-iodomethyl nucleosides and intermediate BTSP, prepared in situ through the Arbuzov reaction. The modified reaction conditions for th e Arbuzov reaction prevented the loss of DMT- and MMT-protecting groups, an d directly provided the desired 5 ' -O-DMT- and/or MMT-protected 3 ' -C-met hylene-modified H-phosphonates 1-6 although some of them were also prepared through the manipulation of protecting groups after the P-C bond formation . The modified Arbuzov reaction of 3 ' -C-iodomethyl-5-methylcytidine 53, p repared from its 5-methyluridine derivative 42, with BTSP provided the 5-me thylcytidine H-phosphonate 54, which was further transferred to the corresp onding 4-N-(N-methylpyrrolidin-2-ylidene)-protected H-phosphonate monomer 7 . 5 ' -O-MMT-protected 3 ' -C-methylene-modified H-phosphonates 5, 3, and 7 were converted to the corresponding cyanoethyl H-phosphonates 50, 51, and 56 using DCC as a coupling reagent. One-pot three-step reactions of 50, 51, and 56 provided the desired 3 ' -C-methylene-modified phosphonamidite mono mers 8-10. Some of these new 3 ' -methylene-modified monomers 1-10 have bee n successfully utilized for the synthesis of 3 ' -methylene-modified oligon ucleotides, which have shown superior antisense properties including nuclea se resistance and binding affinity to the target RNA.