| 摘要 |
<p>A method for coupling phosphoramidite monomers with nucleophiles, for example, the 5'-hydroxyl group on the growing oligonucleotide chain, using a coupling activator that is less acidic than and at least as nucleophilic as tetrazole and that provides comparable or better coupling efficiency than tetrazole. The pKa of the coupling activator is between 5.0 and 6.0, preferably between 5.0 and 5.5, and, more preferably, between 5.1 and 5.3. Suitable coupling activators include 4,5-dicyanoimidazole (DCI), 4-alkylthioimidazole, 2-alkylthioimidazole, 2-nitroimidazole, 4-nitroimidazole, 4,5-dihaloimidazole, 4-haloimidazole, 2-haloimidazole and 5-alkoxytetrazole. DCI is the most preferred coupling activator. Alternatively, a combination of an acidic coupling activator and a suitable buffer, such as a tertiary amine, can be employed. The tertiary amine can be a tertiary amine with three alkyl groups or a heterocyclic compound containing one or more tertiary amines. Preferably the tertiary amine is less nucleophilic than DMAP, which is known to cause side reactions at the 6-position oxygen of guanosines due to its relatively high nucleophilicity. N-methylimidazole (NMI) is the preferred tertiary amine.</p> |
