An efficient method for large laboratory scale synthesis of dinucleoside (3¢,5¢)-methylphosphonothioates and their methylphosphonoselenoate congeners is presented. Bis-(1,2,4-triazoloyl)methylphosphonite generated in situ from methyldichlorophosphine is used as a phosphitylating agent and the preparations are performed as one-pot-reactions without isolation of the reactive PIII intermediates.Oligonucleotides modified at preselected internucleotide linkages with methylphosphonate functions have become indispensable tools for studies of interactions between DNA and other biomolecules, such as DNA, RNA and proteins. 2 Since the aforementioned modifications by virtue of asymmetry of the phosphorus atom induce polydiastereomerism of the corresponding oligonucleotide constructs, the obvious consequence of their non-stereocontrolled synthesis is the separation of diastereomers and assignment of the sense of P-chirality before application of such modified oligonucleotide constructs in structural studies. An alternative and preferred approach to their synthesis relies upon the preparation of diastereomerically pure dinucleoside (3¢,5¢)-methylphosphonates (1, X = O) or methylphosphonothioates (2, X = S) either methylphosphonoselenoates (3, X = Se) and their use as dimeric building blocks in the solid-phase synthesis of chimeric oligonucleotides. Such an approach in the case of 1 has been successfully used for synthesis of the third generation of antisense agents, 3 as well as for construction of chimeric molecules useful for elucidation of contacts between DNA/RNA and proteins 4 or in the studies of DNA conformation and bending. 5 Dinucleoside methylphosphonoselenoates 3 incorporated into modified oligonucleotides can facilitate crystallization of DNA/RNA or DNA/protein complexes and simplify their X-ray structural analysis due to the MAD effect. 6 However, one of the serious limitations in such applications, despite numerous efforts of different groups, is insufficient availability of diastereomerically pure dinucleotides 1-3, mostly obtained by laborious synthetic procedures. 7 In this communication we present our results on the design of the large-scale synthesis of the aforementioned dinucleotides 2 and 3. The key step in our strategy involves either the sulfurization or selenization of the corresponding PIII intermediates, providing a set of new dinucleotide building blocks that enables the synthesis of chimeric oligonucleotides bearing methylphosphonothioate or methylphosphonoselenoate functions. In addition to the novelty of this phosphonoselenoate modification, both methylphosphonothioates 2 and methylphosphonoselenoate congeners 3 can be stereospecifically and almost quantitatively converted to the parent methylphosphonates 1 by simple treatment with Oxone ® . 6Conventional synthesis of dinucleotides 1 uses MePCl 2 (4) as the phosphitylating agent. 8 However, the use of this bifunctional phosphitylating agent due to its high reactivity and the lack of selectivity of both chlorines with different nucleoside compone...
Chemoselective and stereospecific O-activation of 2'-deoxynucleoside 3'-O-methylphosphonothioates 5 with N-methyl-N-4,6-dimethoxy-1,3,5-triazin-2-yl morpholinium salts results in formation with retention of configuration of 5'-O-DMT-2'-deoxynucleoside 3'-O-(4,6 dimethoxy-1,3,5-triazin-2-yl methylphosphonothioates (7). Active esters 7 are convenient intermediates for hydrolytic interconversion of RP-5 into SP-5 and can be used as monomers for stereoselective synthesis of dinucleoside (3',5')-methyl phosphonothioates.
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