RESUMO
Some commercial batches of dichloroacetic acid (DCA) contain traces of chloral (trichloroacetaldehyde). Using such DCA to effect detritylation during solid-phase oligonucleotide synthesis results in the formation of a family of process impurities in which the atoms of chloral (Cl3CCHO) are incorporated between the 5'-oxygen and phosphorus atoms of an internucleotide linkage. The structure was elucidated by HPLC with UV and MS detection, digestion of the oligonucleotide, synthesis of model compounds, and 1H and 31P NMR spectroscopy. By understanding the chemistry behind its formation, we are now able to limit levels of this impurity in synthetic oligonucleotides by limiting chloral in DCA.
Assuntos
Hidrato de Cloral/análogos & derivados , Oligonucleotídeos/química , Hidrato de Cloral/análise , Hidrato de Cloral/química , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Contaminação de Medicamentos , Espectrometria de Massas , Estrutura Molecular , Oligonucleotídeos/síntese químicaRESUMO
Incomplete sulfurization during solid-phase synthesis of phosphorothioate oligonucleotides using phosphoramidite chemistry was identified as the cause of formation of two new classes of process-related oligonucleotide impurities containing a DMTr-C-phosphonate (DMTr=4,4'-dimethoxytrityl) moiety. Phosphite triester intermediates that failed to oxidize (sulfurize) to the corresponding phosphorothioate triester react during the subsequent acid-induced (dichloroacetic acid) detritylation with the DMTr cation or its equivalent in an Arbuzov-type reaction. This leads to formation of DMTr-C-phosphonate mono- and diesters resulting in oligonucleotides modified with a DMTr-C-phosphonate moiety located internally or at the 5'terminal hydroxy group. DMTr-C-phosphonate derivatives are not detected when optimized sulfurization conditions are employed.
Assuntos
Oligonucleotídeos/síntese química , Organofosfonatos/síntese química , Compostos de Tritil/síntese química , Cromatografia Líquida de Alta Pressão , Oligonucleotídeos/química , Organofosfonatos/química , Fosfitos/química , Relação Estrutura-Atividade , Compostos de Tritil/química , Compostos de Tritil/farmacologiaRESUMO
Multiple phosphorothioate oligonucleotides containing a 3'-terminal negative charge were synthesized and characterized. Influence of the added negative charge on activation of duplexes by RNase H was investigated. No additional help in recruitment of RNase H was observed.
Assuntos
Oligodesoxirribonucleotídeos Antissenso/síntese química , Oligodesoxirribonucleotídeos Antissenso/químicaRESUMO
A phosphorothioate-linked oligonucleotide bearing a 3'-terminal phosphorothioate monoester has been synthesized and characterized. This oligonucleotide has been identified as a process-related impurity formed during synthesis of ISIS 2302. Biological properties of the compound have been determined. Based on these data, it can be concluded that this species (3'-TPT) has biological properties similar to parent drug.
