dC (Ac) Me-Phosphonamidite
dC (Ac) Me-Phosphonamidite
Key featuresShow Hide
- Me-phosphonate linkages are uncharged and nuclease resistant.
- Can be used for targeted cellular delivery of antisense therapeutic agents. Among the first modified oligonucleotides shown to inhibit protein synthesis via an antisense mechanism.
- Synthesis using these monomers requires a low water content oxidiser and changes are necessary from commonly used deprotection procedures because the linkages are more base-labile.
- Should be used in conjunction with standard cyanoethyl phosphoramidites.
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Since methyl (Me) phosphonate linkages are uncharged and nuclease resistant, oligonucleotides containing these have many applications, particularly in developing novel strategies for targeted cellular delivery of antisense therapeutic agents.(1) These were among the first modified oligonucleotides shown to inhibit protein synthesis via an antisense mechanism.
Synthesis using these monomers requires a low water content oxidiser and changes are necessary from commonly used deprotection procedures because the linkages are more base-labile. EDA in 95% EtOH (1:1) is typically used, but other methods have been reported.(2) To help in purification and isolation of these oligos, it is best to incorporate as many phosphodiester linkages (prepared from standard ß-cyanoethyl phosphoramidites) into each oligo as possible.
- See for example: (a) Comparative hybrid arrest by tandem antisense oligodeoxyribonucleotides or oligodeoxyribonucleoside methylphosphonates in a cell-free system, L.J. Maher, III and B.J. Dolnick, Nucleic Acids Research, 16, 3341-3358, 1988; (b) Solid-phase synthesis of oligo-2-pyrimidinone-2'-deoxyribonucleotides and oligo-2-pyrimidinone-2'-deoxyribose methylphosphonates, Y. Zhou and P.O.P. Ts’o, Nucleic Acids Research, 24, 2652-2659, 1996; and (c) Nuclear antisense effects of neutral, anionic and cationic oligonucleotide analogs, P. Sazani, S.-H. Kang, M.A. Maier, C. Wei, J. Dillman, J. Summerton, M. Manoharan and R. Kole, Nucleic Acids Research, 19, 3965-3974, 2001.
- Deprotection of methyiphosphonate oligonucleotides using a novel one-pot procedure, R.l. Hogrefe, M.M. Vaghefi, M.A. Reynolds, K.M. Young and L. Arnold Jr, Nucleic Acids Research, 21, 2031-2038, 1993.
Physical & Dilution Data
Dilution volumes (in ml) are for 0.1M solutions in dry acetonitrile (LK4050), except for LK2074 which is diluted in dry THF. Adjust accordingly for other concentrations. For µmol pack sizes, products should be diluted as 100µmol/ml to achieve 0.1M, regardless of molecular weight.
dG-Me Phosphonamidite (LK2074) is dissolved in anhydrous THF prior to use in synthesis. Other monomers are dissolved in anhydrous acetonitrile.
A coupling time of 6min is recommended for syntheses of 1μmol and below. The use of the Ac-dC (LK2077) monomer is preferred to avoid base modification of dC residues during deprotection with ethylenediamine.1 Trityl monitors may understate the coupling efficiency, presumably due to a difference in the rate of release of the trityl group.
Cleavage & Deprotection
A one-pot procedure for cleavage and deprotection has been described.2 This is preferred to the procedure3 used in the past since it leads to less cleavage of the methyl phosphonate backbone during the ammonium hydroxide solution cleavage step and evaporation of the ethylenediamine.
- Air-dry the support in the synthesis column, open the column and transfer the support to a deprotection vial.
- Add 0.5ml of an ammonium hydroxide solution consisting of acetonitrile/ethanol/ammonium hydroxide (45:45:10) to the support. Seal the vial and leave it at room temperature for 0.5h.
- Add 0.5ml of ethylenediamine to the vial and reseal it. Leave it at room temperature for a further 6h.
- Filter in a microfilter tube then desalt on a G25 sephadex column and wash the support twice with 0.5ml acetonitrile/water (1:1).
- Dilute the combined supernatant and washes to 15ml with water.
- Adjust the pH to 7 with 6M hydrochloric acid (1 part) in a 1:1 mix of acetonitrile/water (9 parts) (ca. 2ml).
- Desalt using standard procedures.
Regular purification procedures can be used if the oligo contains several phosphodiester linkages. If the oligo contains a very high percentage of Me phosphonate linkages, RP-HPLC purification may work. However, these oligos have poor solubility characteristics and may precipitate in the sample loop, HPLC column, or other surfaces. More detailed descriptions of HPLC purification have been published.2
Note: These procedures have been shown to work well for small scale synthesis (1μmol or below). For larger synthesis scales, changes may be required in the capping and oxidation steps. See reference 3 for more details.
Storage & Stability
All phosphonamidites are stored refrigerated at a maximum of 2-8°C. They are stable in solution for 24h.
- (a) Elimination of transamination side product by the use of dC(Ac) methylphosphonamidite in the synthesis of oligonucleotide methylphosphonates, M.P. Reddy, F. Farooqui and N.B. Hanna, Tetrahedron Lett., 37, 8691-8694, 1996; (b) Correction to above, M.P. Reddy, F. Farooqui and N.B. Hanna, Tetrahedron Lett., 38, 1101-1101, 1997.
- (a) Deprotection of methylphosphonate oligonucleotides using a novel one- pot procedure, R.I. Hogrefe, M.M. Vaghefi, M.A. Reynolds, K.M. Young and L.J. Arnold, Nucleic Acids Research, 21, 2031-2038, 1993; (b) R.I. Hogrefe, M.A. Reynolds, M.M. Vaghefi, K.M. Young, T.A. Riley, R.E. Klem and L.J. Arnold, Protocols for Oligonucleotides and Analogs, Vol. 20 in the series Methods in Molecular Biology, 143-164, 1993, S. Agrawal, editor, Humana Press Inc., Totowa, NJ.
- Oligodeoxynucleoside methylphosphonates–synthesis and enzymatic degradation, S. Agrawal and J. Goodchild, Tetrahedron Lett., 28, 3539-3542, 1987.