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3'-Amino Modifier C6-dT CPG Column

3'-Amino Modifier C6-dT CPG Column

CPG column for the incorporation of an amino function at the 3' end of an oligonucleotide. 

Key features

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  • Incorporates a primary amine at the 3 end of an oligonucleotide, for subsequent conjugation.
  • Does not block the terminus from any desired enzymatic activity.
  • Base-labile trifluoroacetyl (TFA) protection on the amine, which is removed during cleavage and deprotection.
  • CPG has long-chain alkylamino succinyl linker.
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Product information

Incorporation of a primary amine reactive functional group at specific sites within an oligonucleotide allows for subsequent post-synthesis conjugation of the oligo with a number of different affinity, reporter or protein labels, depending on the application. Such labels need to be reactive towards the incorporated functional group: for example, NHS esters or isothiocyanates will react with primary amines. This approach is often necessary where the desired label or tag is either not available as a phosphoramidite, or is sensitive or unstable to the conditions of oligonucleotide synthesis or deprotection. A common example is the attachment of a rhodamine dye using the TAMRA NHS ester. Functionally-derivatised oligos can also be covalently attached to surfaces such as glass slides or gold microspheres for use in various microarray or nanoelectronic applications.

The most commonly used product for introducing a 3'-amino functionality is Fmoc-protected 3'-amino C7 CPG. Use of an Fmoc-protected amine has both advantages and disadvantages. This is quite stable to oligo synthesis conditions however, if not handled correctly, some loss of Fmoc may occur. This leads to capping of the free amine with acetic anhydride and hence loss of functionality. The main advantage of Fmoc is that it can be removed selectively without cleavage from the support allowing solid-phase conjugation of the desired label. This can be done prior to, or subsequent to, oligonucleotide synthesis.

Alternatives to Fmoc protection have been investigated. Phthalimide (PT) chemistry has been used in the development of 3'-PT-amino-C6 CPG (1), where the nitrogen that will ultimately provide the 3'-amino function is part of the PT group attached to the support through an imide group attached to the aromatic ring. This linkage is stable to all conditions of oligo synthesis and the resulting amino functionality does not add any additional chiral centres/diastereomers to the oligo. A completely analogous C3 product is also available.

Additional products are available for introducing 3'-amino functionality without blocking the terminus from any desired enzymatic activity. There are the C6 dC and dT analogues, plus the mdC TEG product confers additional spacing and hydrophilicity. These products all have base-labile trifluoroacetyl (TFA) protection on the amine, which is removed during cleavage and deprotection.

Ref:

  1. An improved CPG support for the synthesis of 3'-amine-tailed oligonucleotides, C.R. Petrie, M.W. Reed, A.D. Adams and R.B. Meyer, Jr., Bioconjugate Chem., 3, 85-87, 1992.
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

Applicable Products

LK2350 3'-Amino-Modifier-C7 CPG 1000
LK2365 3'-PT-Amino-Modifier-C6 CPG
LK2367 3'-Amino-Modifier-C6-dT CPG
LK2369 3'-Amino-Modifier-C6-dC CPG
LK2371 3'-PT-Amino-Modifier-C3 CPG

Physical Data

Item

Unit Wt.

LK2350 209.18
LK2365 179.15
LK2367 359.24
LK2369 457.42
LK2371 137.07

Coupling

All supports are used as per unmodified nucleoside supports. However, non-nucleosidic modifications are slow to detritylate and require an initial detritylation prior to use in synthesis. In this case it is important not to use a cycle with an initial capping step. AMA deprotection will require dmf-G and Ac-C to be used.

Cleavage & Deprotection

LK2350—Cleavage of the oligonucleotide requires 2h at room temperature with ammonium hydroxide or AMA. It is important to wash the column with 10-20% diisopropylamine in acetonitrile or 10-20% DEA/acetonitrile to prevent blocking with acrylonitrile during deprotection. Alternatively, 20% piperidine in DMF can be used, rather than DEA. In fact this will simultaneously remove Fmoc and cyanoethyl protection, plus remove the vinylfluorene by-product from the Fmoc deprotection reaction.

For the PT-Amino CPGs (LK2371 & LK2365)—Cleavage of the oligonucleotide requires overnight at 55°C with ammonium hydroxide. This treatment will complete the deprotection of the nucleobases for all protection types. It is important to wash the column with 10-20% diisopropylamine in acetonitrile or 10-20% DEA/acetonitrile to prevent blocking with acrylonitrile during deprotection.

LK2369 & LK2367—Use deprotection conditions as required by the nucleobases. It is important to wash the column with 10-20% diisopropylamine in acetonitrile or 10-20% DEA/acetonitrile to prevent blocking with acrylonitrile during deprotection.

Storage & Stability

The supports are stored in a freezer at –10 to –30°C.

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