Key featuresShow Hide
- Creates phosphorothioate (PS) linkage in oligonucleotide backbone
- Soluble in acetonitrile
- Stable in solution for several months (a silanised bottle is not required)
- Exhibits high sulphurisation efficiency with both DNA and RNA
- Compatible with labile (fast) deprotection chemistry
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During synthesis using the phosphoramidite approach, the backbone of either DNA or RNA can be modified by sulphurisation (or sulphur-transfer) reagents to replace one non-bridging oxygen atom in the phosphodiester, thus creating a phosphorothioate (PS) linkage. This makes this method more suitable than H-phosphonate chemistry for controlling the state of each linkage [P=O versus P=S] in a site-specific manner.
EDITH has emerged as the reagent of choice for sulphurisation due to its all-round capability and ease of use. This product is soluble in acetonitrile, stable in solution for several months (a silanised bottle is not required), and exhibits high sulphurisation efficiency with both DNA and RNA. Its high efficiency in RNA synthesis, often unobtainable with other reagents, is of particular benefit, giving >99% sulphurisation efficiency.
EDITH also shows compatibility with labile (fast) deprotection chemistry. Some deleterious G modification has been observed, however this can be eliminated by using a modified coupling-capthio-cap cycle. This allows the preparation of certain phosphorothioates that may be sensitive to prolonged ammonium hydroxide solution treatment. However, it should be noted that capping prior to sulphurisation can lead to some oxidation of the PIII species.
Physical & Dilution Data
Dilution volumes (in ml) are for 0.05M solutions in dry acetonitrile (LK4050). Adjust accordingly for other concentrations.
Beaucage Reagent (LK0023) is supplied in solid form in 1g and 2g packs, together with a silanised bottle suitable for direct attachment to an automated DNA synthesiser. Simply pour the reagent into the silanised bottle and add anhydrous acetonitrile (100ml/g) to obtain a 0.05M solution. For smaller scale syntheses, smaller quantities can be prepared (e.g. 0.5g/50ml). After use, the silanised bottle may be rinsed with acetonitrile, dried and stored for future use. EDITH (2171) is available in millimolar pack sizes ready for dilution with convenient amounts of anhydrous acetonitrile to a 0.05M concentration, according to the table above.
Routinely sulphurisation reagents are used on an automated DNA synthesiser, utilising the dedicated thio port where available, following the instrument manufacturer’s recommendations for sulphurisation cycles. Depending on the type of synthesiser, there may be a cycle for sulphurising reagent available. If a dedicated port and line is not available, the oxidiser port and line may be used. In this case, the line must first be flushed extensively with acetonitrile followed by liberal flushing with the sulphurising reagent. After use, remove the reagent immediately from the instrument and flush the line with acetonitrile.
When using EDITH (LK2171) a sulphurisation time of 30s is recommended, although longer times will not have any adverse affect on the oligo if reducing the sulphurisation time in the cycle is not possible.
For optimum results, it is recommended that the sulphurisation is carried out prior to the capping step in order to minimise competitive oxidation.
Some deleterious G modification has been observed when using EDITH with certain labile (fast) deprotection chemistry.1 This can be eliminated by using a modified coupling-cap- thio-cap cycle, thus allowing preparation of phosphorothioate oligos without the need for prolonged ammonium hydroxide deprotection. The cap-thio-cap cycle is possible using EDITH due to the short sulphurisation time required.
Cleavage & Deprotection
Phosphorothioate oligos can be cleaved from the support and deprotected using ammonium hydroxide, therefore follow any necessary guidelines according to the nucleobases used.
Phosphorothioate purification is routinely carried out by RP-HPLC or cartridge methods. SAX2 and WAX3 HPLC has also been described, however the former requires high salt concentrations. FPLC at high pH is not recommended.
Note that high purity or 2’-OMe phosphorothioates are best purified by PAGE.
The best means of identifying the presence of oxidised linkages in the oligo is by MS. Either ESI or MALDI are effective for this purpose.
Storage & Stability
LK2171 - Store the solid in a glass container in the freezer at –10 to –30°C. Although EDITH is reportedly stable in solution for several months, we recommend, as with all other oligonucleotide synthesis reagents, that solutions are freshly prepared as close to use as possible. It can be used on the synthesiser for several days, however, without any drop in performance provided no precipitate is observed.
LK0023 - Store the solid reagent in a freezer at –10 to –30 ̊C, and dry. In solution in acetonitrile, it must be stored in plastic or silanised glass containers. It will remain stable under these conditions for several months (precipitation will accompany decomposition). Where the solution has been used on an oxidiser port, the reagent will decompose over 1-2 days. Where a spare port is routinely used for this reagent, the solution will be stable for up to a month.
- Evaluation of 3-ethoxy-1,2,4-dithiazoline- 5-one (EDITH) as a new sulfurizing reagent in combination with labile exocyclic amino protecting groups for solid-phase oligonucleotide synthesis, M.Y.-X. Ma, J.C. Dignam, G.W. Fong, L. Li, S.H. Gray, B. Jacob- Samuel and S.T. George, Nucleic Acids Research, 25, 3590-3593, 1997.
- Separation of synthetic phosphorothioate oligonucleotides from phosphodiester-defect species by strong-anion exchange HPLC, B.J. Bergot and G. Zon, Annals of the New York Academy of Sciences, 660, 310-312, 1992.
- Ion-exchange high-performance liquid chromatography analysis of oligodeoxyribonucleotide phosphorothioates, V. Metelev and S. Agrawal, Anal. Biochem., 200, 342-346, 1992.
The use of EDITH in the US is protected under US Patent No. 5,852,168 and licence for such use must be sought from The University of Minneapolis.