Phosphonate Oligos |
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H-Phosphonate Chemistry H-phosphonate monomers are useful for the preparation of internucleotide linkages that are not attainable by standard phosphoramidite chemistry. Using this method, the monomer that is able to be activated is a 5'-DMT-base-protected, nucleoside 3'-hydrogen phosphonate. The presence of the H- phosphonate moiety on these monomers renders phosphate protection unnecessary. The same base protecting groups are used in phosphite triester chemistry. The H-phosphate synthesis cycle is very similar to that of the phosphoramidite method. Slight differences result from the properties of the monomers utilized. For instance, a different activating agent is used. In addition, the H-phosphonate diesteres generated by the coupling reactions are stable to the normal reaction conditions, so oxidation at every step is unnecessary. Instead, a single oxidation step can be performed at the end of the chain elongation. This single oxidation step makes it easy to produce modified DNA. For instance, if a sulfur containing compound is used as the oxidizing agent, all of the internucleotide bonds will then contain sulphur instead if oxygen attached to the phosphorous atom. The most popular use of this chemistry is for the radioactive labeling of all phosphodiester linkages to phosphorothioate by using radioactive sulfur. | ||||||||||||||||||||||||
The Support H-Phosphonate synthesis uses the same supports as does the β-cyanoethyl phosphoramidite chemistry. | ||||||||||||||||||||||||
The Monomers The H-Phosphonate monomers are protected at the 5'-hydroxyl position with dimethoxytrityl groups. The exocyclic amines on the bases are protected by the same groups as in the β-cyanoethyl phosphoramidite analogues. The phosphonate group is attached to the 3' position. | ||||||||||||||||||||||||
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Detritylation This process is the same as in the ß-cyanoethyl phosphoramidite procedure. | ||||||||||||||||||||||||
Neutralization Residual DCA from the detritylation step is neutralized with a solution of pyridine in acetonitrile. This step also provides the proper solvent medium for the coupling reaction. | ||||||||||||||||||||||||
Coupling
After detritylation and neutralization, the next protected H-phosphonate is delivered to the reaction column.
Adamantoyl carboxyl acid chloride is used to activate the phosphonate by the addition of the adamantoyl moiety to the free oxygen of the H-phosphonate. | ||||||||||||||||||||||||
Cycling Following the coupling step, the cycle of reactions is repeated, beginning with the detritylation step, until chain elongation is complete. | ||||||||||||||||||||||||
Oxidation
Oxidation step is normally omitted during cyclic chain elongation and a single oxidation is performed later with the required oxidizer. | ||||||||||||||||||||||||
Final Detritylation If the oligomer is to be purified by Oligo-Pak column methods or by reverse phase HPLC, the 5'-end of the oligomer should not be detritylated after the last coupling cycle is completed. | ||||||||||||||||||||||||
Purification Purification by gel electrophoresis is recommended. | ||||||||||||||||||||||||
References:
1. Hall,R.H.,Todd,A.R., and Webb,R.F. (1957). Mixed anhydrides as intermediates in the synthesis of dinucleoside phosphates. J. Chem. Soc., 3291-3296. |