Chemical Phosphorylation

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Applicable Products

2101 Chemical Phosphorylation Reagent (CPR)
2110 CPR II
2127 Solid Chemical Phosphorylation Reagent (solidCPR™)
2279 3'-Phosphate SynBase™ CPG 1000/110
2389 3'-Phosphate SynBase™ Polystyrene
2398 3'-Phosphate SynBase™ CPG 3000/110

Physical & Dilution Data

Dilution volumes (in ml) are for 0.1M solutions in dry acetonitrile (4050). Adjust accordingly for other concentrations. For µmol pack sizes, products should be diluted as 100µmol/ml to achieve 0.1M, regardless of molecular weight.

Item

Mol. Formula

Mol. Wt.

Unit Wt.

250mg

500mg

1g

2101 C34H45N2O7PS 656.77 79.98 3.81 7.61 15.23
2110 C39H51N2O9P 722.82 79.98 3.46 6.92 13.83
2127 C39H49N4O7P 692.79 79.98 3.61 7.22 14.43
2279 - - 79.98 - - -
2389 - - 79.98 - - -
2398 - - 79.98 - - -

Coupling & Deprotection using CPR or the 3’-Phosphate CPGs

Note that 2101 and 2110 must be dissolved in acetonitrile at least 10min prior to placing on the synthesiser.

5’-Phosphorylation

The CPR (2101) is coupled and deprotected using standard instrument conditions. Note that the DMTr-group is lost during ammonium hydroxide deprotection and thus is not available for cartridge purification. For 5’-phosphorylation 2101 is coupled in the final step of the synthesis as with other 5’-modifications. The DMTr group is used to give an indication of the coupling efficiency.

3’-Phosphorylation

In addition, 2101 can also be used for simple phosphorylation of the 3’-terminus. To do this it is introduced as the first addition to the solid support, followed by synthesis of the oligo (any convenient support can be used since the support-bound nucleotide is not incorporated into the sequence). After standard deprotection, the linkage decomposes and is ß-eliminated leaving a phosphate group at the 3’-end. The final DMTr group may be removed on the synthesiser or it may be retained to aid purification. If retained, it can be removed on a purification cartridge or, following purification, by treatment with acetic acid:water (80:20) at room temperature for 1h.

Alternatively, direct preparation of oligos with a 3’-phosphate can be achieved using 3’-Phosphate CPG (2279 and 2398) or polystyrene (2389). These supports are used in a manner identical to standard protected nucleoside supports. Cleavage and deprotection of the oligos synthesised with these modifications is as per the standard bases of the oligo. However, to complete the ß-elimination of the sulphonyl diethanol group to form the phosphate heat is required. The minimum deprotection conditions are AMA for 35min at 55°C.

Compatibility with RNA

The phosphorylation reagents (2101, 2279, 2389 and 2398) are all compatible with TBDMS and TC RNA chemistries, but the DEA wash needs to be eliminated from the cleavage and deprotection step in both cases. The addition of the terminal phosphate adds protection against nucleases to the RNA.

Coupling & Deprotection using solidCPR™ & CPR II

The use of solidCPR™ (2127) or CPR II (2110) has advantages over CPR (2101) and as such have a distinct protocols. Standard CPR protocols cannot be used as this will lead to poor results. 2127 and 2110 are coupled in the final step in the synthesis using a 6min coupling time. If wishing to remove the DMTr group, a second deblock step is recommended.

Deprotection & Cleavage DMT OFF in Solution

The support, after detritylation, is treated using standard deprotection and cleavage conditions. All deprotection and cleavage conditions in current regular use, including fast deprotection, will release the terminal free phosphate. The two most suitable options are either ammonium hydroxide solution for 4h at 55˚C or AMA for 2h at RT. After this cleavage and deprotection step the free 5’ phosphate is obtained.

DMT ON Cartridge Purification and Deprotection

The sequence with a 5’-DMTr group is purified, after synthesis and base deprotection, using a modified protocol for cartridge purification. After detritylation on the cartridge by treatment with 2% aqueous trifluoroacetic acid (TFA) the final phosphorylated sequence is obtained via the following steps:

  1. Prepare a stock solution of either 50mM potassium carbonate, pH 12/1M NaCl or 50mM sodium hydroxide/1M NaCl. Approximately 2ml will be required for a standard size cartridge.
  2. Neutralise the residual TFA on the column by passing approx. 0.5ml of the stock solution through the cartridge fairly rapidly (over ca. 10s).
  3. Then gradually pass the remainder of the stock solution through the column in aliquots over a period of 20min.
  4. Wash the cartridge with water (2ml).
  5. Elute the purified phosphorylated oligo with 20% aqueous acetonitrile (1-3ml depending on the size of the cartridge).

Storage & Stability

Store products in a freezer dry at –10 to -30°C. Use 2101 or 2110 solutions within 24h. 2127 is stable in solution for 2-3 days.

SolidCPRTM is sold by agreement with Glen Research, Corp., Virginia, US under EP Patent No. 0816368.

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