Chemical Phosphorylating Reagent (CPR)
Chemical Phosphorylating Reagent (CPR)
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- Can be used to phosphorylate at 5' or 3' terminus of an oligonucleotides
- Useful in gene synthesis or to block enzyme activity
- DMT group can be used to monitor phosphorylation efficiency but cannot be used as purification handle
- Product is viscous glass
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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 a Chemical Phosphorylation Reagent (also known as CPR or “Phosphate-ON”). (1) 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. CPR 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 3000 Å 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’”. With CPR II (2) 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 “solid CPR”,(3) that offers all the benefits of CPR II whilst also being a stable solid that permits easy weighing, handling and dissolution (the other phosphate amidites are both viscous glasses). This product also allows the option of DMT ON purification. The presence of the methylamides in this 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 without forming the phosphate moiety until the deprotection step. 3'-Phosphate CPGs allow direct preparation of oligonucleotides with a 3'-phosphate group. CPR II or solid CPR cannot be used for 3'-phosphorylation since the DMTr- protected OH is required to release the phosphate group.
- A chemical 5'-phosphorylation of oligodeoxyribonucleotides that can be monitored by trityl cation release, T. Horn and M. Urdea, Tetrahedron Lett., 27, 4705-4708, 1986.
- A new approach for chemical phosphorylation of oligonucleotides at the 5'-terminus, A. Guzaev, H. Salo, A. Azhayev and H. Lonnberg, Tetrahedron, 51, 9375-9384, 1995.
- Chemical phosphorylation of oligonucleotides and reactants therefor, A. Guzaev, A. Azhayev and H. Lonnberg, US Patent No. 5959090, 1999.