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Chemical phosphorylation is a cost-effective alternative to enzymatic methods (using T4 polynucleotide kinase and ATP), allowing efficient introduction of terminal phosphate groups. Oligonucleotides containing a 5'-phosphate group have various applications, being most widely used as a means of ligating one oligo to another, e.g. as linkers and adapters, in cloning, gene construction, and ligation in general. This is still the most common method of gene synthesis. 3'-Phosphorylations, however, are used to block enzyme activity. For example, this is an efficient and commonly used PCR blocking technique.

Phosphorylation of the 5'-terminus on oligonucleotides is routinely achieved, with higher yields than using kinase, using Phosphate-ON (2101) (also known as Chemical Phosphorylation Reagent (CPR)). Aside from its inherent convenience, CPR also has the advantage over enzymatic methods in allowing determination of the phosphorylation efficiency due to the presence of the DMTr protecting group. However, the trityl group cannot be used as a purification handle. It is eliminated along with the sulphonyl ethyl group to produce the 5'-phosphate during the ammonium hydroxide deprotection. 2101 can also be used at the 3'-end to incorporate a 3'-phosphate by addition to any support (e.g. a dT column). This is particularly useful for labelling long oligos where higher pore
sized resins for modification are not available. It is for this reason we introduced the 3000Å phosphate support. This technique is not only limited to phosphate modification, since any modifying phosphoramidite can be added to the phosphate-ON-T. In this case the oligo will be terminated at the 3'-end with "modifier-phosphate-3'".

Another phosphorylation reagent, known as CPR II (2110), has been described by researchers at the University of Turku in Finland. With this product, conventional ammonium hydroxide cleavage gives rise to an oligonucleotide protected at the 5'-phosphate with a DMTr-ether. At this stage, the oligo may be easily separated from truncated impurities by e.g. RP-HPLC or cartridge-purification. The DMTr-group is then removed by aqueous acid and brief ammonium hydroxide treatment yields the 5'-phosphate. Alternatively, the yield of the last coupling may be quantified by detritylation of the oligo whilst still on the support. Deprotection then leads to the 5'-phosphorylated oligo. This CPR II reagent has been further refined (by substituting the ethyl esters for methyl amides) to provide a product, solidCPRTM (2127, also known as CPR IIa), that offers all the benefits of CPR II whilst also being a stable solid that permits easy weighing, handling and dissolution (2101 and 2110 are both viscous glasses). This product also allows the option of DMT ON purification. Although 2101 can be used in 3'-phosphorylations, 3'-Phosphate SynBaseTM CPG 1000/110 and 3000/110 (2279 and 2398), allow direct preparation of oligonucleotides with a 3'-phosphate group. 2127 or 2110 cannot be used for 3'-phosphorylation since the DMTr-protected OH is required to release the phosphate group.

The presence of the methylamides in 2127 protects the modification from ß-elimination reactions until the base hydrolysis during deprotection. This can therefore be used in conjunction with Fmoc or levulinyl protected branching monomers (e.g. 2150) without forming the phosphate moiety until the deprotection step. 2101 is not protected against ß-elimination and would form the phosphate moiety during deprotection of the branching point. Hence if this is used at the 3' end it would cleave the oligo from the support (as would 2279/2389).

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  2. CPR II
  3. View Product Details > Customers in the US should purchase this product from Glen Research (view).
      Pack Size Catalogue No Price Quantity
      100µmol 2110-F100 £22.00
      250mg 2110-B250 £61.00
     
  4. Chemical Phosphorylation Reagent (CPR) (5'-Phosphate/Phosphate-ON)
  5. View Product Details >

    Customers in the US should purchase this product from Glen Research (view).

    Where different unit prices are given for a pack size, please refer to the minimum quantity required to order at that price (e.g. X5, X10). These are not multi-packs, therefore enter the actual quantity of vials you wish to order.
      Pack Size Catalogue No Price Quantity
      100µmol 2101-F100X1 £22.00
      100µmol (Min. Qty. 5) 2101-F100X5 £18.70
      100µmol (Min. Qty. 10) 2101-F100X10 £17.60
      100µmol (Min. Qty. 25) 2101-F100X25 £15.40
      100µmol (Min. Qty. 50) 2101-F100X50 £14.30
      250mg 2101-B250X1 £50.00
      250mg (Min. Qty. 5) 2101-B250X5 £42.50
      250mg (Min. Qty. 10) 2101-B250X10 £40.00
      250mg (Min. Qty. 25) 2101-B250X25 £35.00
      250mg (Min. Qty. 50) 2101-B250X50 £32.50
     
  6. Solid Chemical Phosphorylation Reagent (solidCPR™) (5'-Phosphate)
  7. View Product Details > Customers in the US should purchase this product from Glen Research (view).
      Pack Size Catalogue No Price Quantity
      100µmol 2127-F100 £22.00
      250mg 2127-B250 £61.00
     
  8. 3'-Phosphate SynBase™ CPG 1000/110
  9. View Product Details >

    Loading: 35-50μmol/g.

    Customers in the US should purchase this product from Glen Research (view).

      Pack Size Catalogue No Price Quantity
      4x0.2µmol 2279-P001 £30.00
      10x0.2µmol 2279-P002 £60.00
      4x1µmol 2279-P010 £37.00
      10x1µmol 2279-P008 £74.00
      4x0.2µmol MerMade 2279-P015 £30.00
      10x0.2µmol MerMade 2279-P016 £60.00
      4x1µmol MerMade 2279-P026 £37.00
      10x1µmol MerMade 2279-P022 £74.00
      100mg 2279-B100 £20.00
      1g 2279-C001 £130.00
     
  10. 3'-Phosphate SynBase™ CPG 3000/110
  11. View Product Details >

    This product is not normally held in stock. Please contact us for batch availability.

    Customers in the US should purchase this product from Glen Research (view).

      Pack Size Catalogue No Price Quantity
      4x0.2µmol 2398-P001 £60.00
      10x0.2µmol 2398-P002 £120.00
      4x0.2µmol MerMade 2398-P015 £60.00
      10x0.2µmol MerMade 2398-P016 £120.00
      100mg 2398-B100 £30.00
      1g 2398-C001 £190.00