To date, protocols to use nucleoside H-phosphonates on DNA synthesizers are directed toward the synthesis of unmodified oligonucleotides. These protocols differ from the more common phosphoramidite protocols mainly in that the oxidation step is removed from each cycle and is carried out instead at the end of the synthesis. Despite the clear advantages of this simple, fast synthesis cycle, Hphosphonate chemistry does not appear capable of performing at the level of phosphoramidite chemistry without significant modification. The development of a more hindered activator may increase the coupling efficiency while reducing the number of side reactions, but such an activator may be insufficiently reactive to effect the necessary end-capping. Since nucleoside H-phosphonates are stable in solution, this chemistry may find its place on machines which are used only occasionally for the synthesis of oligonucleotides of less than 50 bases. Contact us for a copy of our review of H-phosphonate chemistry.
The greatest advantage of using nucleoside H-phosphonates may be that they are useful in effecting a variety of backbone modifications of oligonucleotides. These include the preparation of phosphorothioate, phosphoramidate, and phosphotriester linkages. These modifications may be introduced at any position in a growing oligonucleotide while it is being synthesized using phosphoramidite chemistry.
The following protocol describes how this may be accomplished using any DNA synthesizer with columns that have female luer fittings or columns that can be fitted with female luers. Using this procedure, anyone with access to a DNA synthesizer can explore the use of DNA modification to produce novel markers, intercalating reagents, and anti-viral compounds.
1) Nucleoside H-phosphonates (Glen Research items 10-1200-05, 10-1210-05, 10-1220-05, and 10-1230-05 or equivalent).
2) Pivaloyl chloride (Aldrich item T7,260-5 or equivalent).
3) Anhydrous acetonitrile (Glen Research item 40-4050-50 or equivalent).
4) Anhydrous pyridine (Aldrich Gold Label item 27,097-0 or equivalent).
5) Several 10ml glass serum vials with rubber septa.
6) Two 2.5ml and one 10ml gas-tight luer syringes (Hamilton or equivalent).
7) Two 2" syringe needles.
8) Luer adaptors (if necessary).
1) Alcohol or amine (these compounds must be pure and extremely dry) or Sulfur (Aldrich Gold Label item 21,392-2 or equivalent).
2) Anhydrous CCl4 (Aldrich Gold Label item 28,911-6 or equivalent) or Triethylamine (TEA) (Aldrich Gold Label item 23,962-3) and carbon disulfide (Aldrich item 27,066-0).
1) Prepare a 0.1 M solution of the desired nucleoside H-phosphonate in pyridine/acetonitrile 30:70. Transfer the solvents using a dry syringe.
If many syntheses are to be performed within a week, the solution may be prepared in the vial in which the nucleoside H-phosphonate was supplied. (Note: after preparation the solution should be stored cold in a desiccator.) If only a few syntheses are to be performed, it is more economical to weigh an appropriate amount of the nucleoside Hphosphonate as follows:
a. Dry a 10ml serum vial in a 130°C oven for several hours.
b. Cap the vial with a rubber septum, place it in a desiccator containing Drierite® and allow it to cool.
c. Weigh the appropriate amount of nucleoside H-phosphonate. (Note: the vial of nucleoside H-phosphonate must be warmed to room temperature before opening to avoid condensation. Weigh the monomer quickly and under a steady stream of argon or nitrogen to ensure anhydrous conditions.)
d. Open the vial while directing a stream of argon or nitrogen into its mouth and quickly add the nucleoside H-phosphonate; allow the vial to fill with the dry gas and reseal.
e. Add the appropriate amount of solvent mixture using a dry syringe.
2) Prepare a 0.5M solution of pivaloyl chloride in acetonitrile. Handle this preparation as described above to ensure anhydrous conditions.
3) Program the DNA synthesizer to stop just prior to the coupling step (i.e., after detritylation and appropriate washes) at the position where the modification is desired.
4) Coupling Reaction:
a. With a dry syringe draw in an aliquot of the nucleoside H-phosphonate solution. (For a 1 pmole scale synthesis, 0.1 ml of the nucleoside Hphosphonate solution should be sufficient; however, because this step is critical to the success of the experiment, it is recommended that 0.2ml to 0.3ml be used.)
b. Remove any reagent from the syringe needle by drawing in a small amount of gas from the sealed vial (this will prevent contamination of the pivaloyl chloride solution).
c. Draw in a volume of pivaloyl chloride equal to the volume of nucleoside Hphosphonate solution in the syringe. This produces a five-fold molar excess of pivaloyl chloride to the nucleoside H-phosphonate.
d. Invert the syringe quickly two or three times to mix the two solutions.
e. Remove the column from the instrument and attach the syringe to the column using a luer fitting.
f. Attach the second syringe to the other end of the column.
g. Slowly pass the solution back and forth across the support-bound oligonucleotide for two minutes.
h. Remove one of the syringes and draw 2.5ml of air into it. Reconnect this syringe and force the air through the column into the other syringe to remove the coupling solution from the column.
i. Rinse the column and the syringes with anhydrous acetonitrile.
1) Prepare a 0.1 M solution of the desired alcohol or amine in CCl4. (Handle this preparation as described in step A-1)
2) Reaction:
a. Using the same procedure outlined in step A-4, pass ~0.5ml of the solution back and forth across the supporrbound oligonucleotide for ~30 minutes. (Note: the time necessary for quantitative reaction depends on the steric hindrance of the alcohol or amine being used; because this step is crucial to the success of the experiment, a reaction time of 30 minutes is recommended.)
b. Wash the support-bound oligonucleotide by filling one of the syringes with anhydrous acetonitrile and forcing it through the column into the other syringe. Repeat this step several times to thoroughly wash the oligonucleotide.
c. Remove the syringes and reattach the column to the instrument.
1) Prepare a solution of 0.1 M sulfur in TENcarbon disulfide (1 :9).
2) Using the procedure outlined in step A-4, pass ~0.5ml of the solution back and forth across the support-bound oligonucleotide for ~5 minutes.
3) Follow steps B1-2b and 2c.
Restart synthesis by skipping the reaction step and continue through the normal cycle.
D. Deprotection
Deprotection may proceed as normal.
Deoxynucleoside H-Phosphonate Diester Intermediates in the Synthesis of lnternucleotide Phosphate Analogues. B.C. Froehler, Tetrahedron Lett., 1986, 27, 'i575-5578, and references cited therein.
H-Phosphonates have been discontinued.
