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Phosphonate Oligos

Phosphonate Oligos. $/base*
1 micromolar Scale $40/base
*minimum charges for 20 mer applies
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.

dA-H-Phosphonate

dC-H-Phosphonate

dG-H-Phosphonate

dT-H-Phosphonate

Detritylation

This process is the same as in the -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.
The coupling mechanism is a nucleophilic attack by the free 5'-hydroxyl group on the phosphorous of the incoming activated monomer. For this reason, it is important that the environment be hydroxyl-free. The phosphonate diester linkages generated by this coupling are stable. Therefore, oxidation is performed at the end of the synthesis.

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.
In the oxidation step, the hydrogen atom which is bound to the phosphorous is replaced with an oxygen atom. Oxidation is accomplished with a mixture of two solutions. One solution contains iodine in tetrahydrofuran (THF), and the other N-methylmorpholine in water and THF.
If required, the DNA can be modified at this step. For instance, phosphorothioates are generated by oxidizing with a sulphur-containing solution.

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.
2. Garegg,P.J., Regberg,T., Stawinski,J. and Stromberg,R. (1985). Formation of internucleotidic bonds via phosphonate intermediates. Chem. Scripta 25, 280-282.
3. Garegg,P.J., Regberg,T., Stawinski,J. and Stromberg,R. (1986). Nucleoside hydrogenphosphonates in oligonucleotide synthesis. Chem. Scripta 26, 59-62.
4. Garegg,P.J., Lidh,I.,Regberg,T., Stawinski,J. and Stromberg,R. (1986). Nucleoside H-phosphonates. Chemical synthesis of oligodeoxyribonucleotides by the hydrogenphosphonate approach. Tetrahedron Lett. 27, 4051-4054.
5. Froehler,B.C., Ng,P.G., and Matteucci,M.D. (1986). Synthesis of DNA via deoxynucleoside H-phosphonate intermediates. Nucleic Acids Res. 14, 5399-5407.
6. Froehler,B.C., and Matteucci,M.D. (1986). Nucleoside H-phosphonates : Valuable intermediates in the synthesis of oligonucleotides. Tetrahedron Lett. 27, 469-472.
7. Froehler,B.C. (1986). Nucleoside H-phosphonates : Deoxyoligonucleotide H-phosphonate diester intermediates in the synthesis of internucleotide phosphate analogues. Tetrahedron Lett. 27, 5575-5578.
8. Andrus,A.,Efcavitch,J.W.,McBride,L.T., and Giusti,B. (1988). Novel activating and capping reagents for improved hydrogenphosphonate DNA synthesis. Tetrahedron Lett. 29, 861-864.
9. Seliger,H. and Roesch,R. (1990). Simultaneous synthesis of multiple oligonucleotides using nucleoside H-phosphonate intermediates. DNA and Cell Biol., 9, 691-696.
10. Stawinski,J.,Stromberg,R.,Thelin,M., and Westman,E.. (1988). Studies on the t-butyldimethyl-silyl group as 2'-O- protection in oligoribonucleotide synthesis via the H-phosphonate approach. Nucl. Acids Res. 16, 9285-9288.
11. Stein,C.A.,Iversen,C.A.,Subashinge,C.,Cohen,J.S.,Stec,W.J. and Zon,G. (1990). Preparation of 35S-labeled polyphosphorothioate oligodeoxyribonucleotides by the use of H-phosphonate chemistry. Analytical Biochem. 188, 11-16.

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