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DMT-Phosphate-Suc-CPG Column (O-DMT-2,2'-sulfonyldiethanol-Suc-CPG)

DMT-Phosphate-Suc-CPG Column (O-DMT-2,2'-sulfonyldiethanol-Suc-CPG)

CPG column for 3' phosphate modification of an oligonucleotide.

Key features

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  • Allows direct 3' phosphorylation of an oligonucleotide
  • Useful in gene synthesis or to block enzyme activity
  • Available in different column formats
  • 1000 Å CPG suitable for highly modified oligonucleotides (> 20mers)
Option 1: Select a Pore Size
Option 2: Select a Column Type
Option 3: Select a Scale
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Product information

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.

Ref:

  1. 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.
  2. 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.
  3. Chemical phosphorylation of oligonucleotides and reactants therefor, A. Guzaev, A. Azhayev and H. Lonnberg, US Patent No. 5959090, 1999.
Synthesizer
Column
Type/Description
Notes
MerMade 6,12
MerMade, syringe (all scales)
Pipette type column
A MerMade column is also known as a Supercolumn
MerMade 4, 48X, 96E, 192E, 192X
MerMade, Syringe (up to 1.3 mL)
Pipette type column
A MerMade column is also known as a Supercolumn
ABI 384 / 394
Luer
Barrel column with luer fitting at either end
Also known as ALL-FIT or Standard
Expedite 8909
Luer
Barrel column with luer fitting at either end
Also known as ALL-FIT or Standard
ABI3900
MerMade
Pipette type column
A MerMade column is also known as a Supercolumn
K&A H4, H8, H8SE, H2, H32, H64
Luer
Barrel column with luer fitting at either end
Also known as ALL-FIT or Standard
K&A S4CL/S8CL
Luer
  Barrel column with luer fitting at either end 
 Also known as Standard. For this instrument, we recommend the Luer (Standard) column as the ALL-FIT columns have a wider barrel.
Dr Oligo 48
MerMade
Pipette type column
A MerMade column is also known as a Supercolumn
 Dr Oligo 192XLc, 768XLc just plates 
 MerMade, Syringe (up to 1.3 mL) 
Pipette type column
A MerMade column is also known as a Supercolumn
 OligoMaker X12, 48, 192, X192, X96 
MerMade, Syringe (up to 1.3 mL)
Pipette type column
A MerMade column is also known as a Supercolumn

Properties:

  • Appearance: White Powder

Product usage:

  • Cleavage conditions: Use concentrated ammonia for 90 minutes at 25 °C when using standard amidites or 1:1 ammonia:methylamine (AMA) for 25 minutes at 25 °C when using fast deprotecting amidites.
  • Deprotection conditions: When using fast deprotecting amidites (eg. C-Ac, G-DMF, G-PAC) use concentrated ammonia for 1 hour or AMA for 30 minutes at 60 °C. When using standard amidites (eg. C-Bz, G-iBu) use concentrated ammonia for 5 hours at 60 °C.
  • ABI 3900 users: Columns should be compatible with standard ABI protocols. However, when using 50 nmol Super Columns, the following changes are recommended: 1) Change the head pressure/purge pressure/chamber pressure to 3.5 psi.2) An additional oxidation step3) OPTIONAL (but may help): extension of the coupling time to 20 secImage of cleaved and deprotected structure:
  • The mass this product adds after conjugation and work-up (the additional mass seen by mass spectrometry) is: 79.98

Storage and handling:

  • Shipping conditions: Ambient
  • Storage conditions: -15 to -30 °C in sealed container

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