5'-TFA-Amino Modifier C12 CE-Phosphoramidite
5'-TFA-Amino Modifier C12 CE-Phosphoramidite
Key featuresShow Hide
- Incorporates primary amine to 5' end of an oligonucleotide, for subsequent conjugation.
- Base labile trifluoroacetate (TFA) group useful where purifcation "handle" is not necessary.
- C12 spacer useful in e.g. affinity chromatography, where the oligo must be sufficiently distanced from the surface, and in some cases labelling with fluorescent tags, where close interaction may lead to partially quenched fluorescence.
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.
One of the most common modifications is the incorporation of a primary amine at the 5'-terminus of the oligonucleotide using an ‘amino-linker’ phosphoramidite, protected with either the base labile trifluoroacetate (TFA) or the acid-labile monomethoxytrityl (MMT) groups.
The choice between the MMT and TFA-protected amino modifiers is dependent on the purification strategy used on the oligo, or whether on-column or solution-phase conjugation is required. If the oligo is purified, the MMT protection is preferable since the trityl group, stable to the basic cleavage and deprotection conditions, can be used as a ‘handle’ in e.g. cartridge purification where the MMT group is removed during the purification process. Otherwise, the TFA protection is perfectly suitable.
The MMT group can also be removed by extended deblocking on the synthesizer, allowing a solid-phase conjugation of a label containing e.g. an activated carboxylic acid. However, in this case it is important to remember that the conjugate must be stable to the subsequent cleavage and deprotection conditions.
The shorter C5 or C6 carbon chain linkers may be used to attach compounds where proximity to the oligonucleotide causes no problem. The longer C12 analogue has specific applications in e.g. affinity chromatography, where the oligo must be sufficiently distanced from the surface, and in some cases labelling with fluorescent tags, where close interaction may lead to partially quenched fluorescence.
|5'-TFA-Amino-Modifier 11 CE Phosphoramidite
|5'-MMT-Amino-Modifier 11 CE Phosphoramidite
Physical & Dilution Data
Dilution volumes (in ml) are for 0.1M solutions in dry acetonitrile (LK4050). Adjust accordingly for other concentrations. For µmol pack sizes, products should be diluted as 100µmol/ml to achieve 0.1M, regardless of molecular weight.
For all, prepare the amidite solution 5-10min before placing on the synthesiser to ensure complete dissolution.
A 5min coupling time is recommended.
LK2193 - A 2min (120s) coupling time is recommended.
For TFA-protected 5’-amino modifiers (LK2124, LK2182, LK2532 and LK2534) this final step is not required. For MMTr 5’-modifiers (LK2123, LK2133 and LK2193) it is better to retain the MMTr protection and remove this during or after purification.
Cleavage, Deprotection & Purification
For all amino modifiers it is recommended that the synthesis column is washed with 10-20% DEA/acetonitrile for 3min prior to cleavage from the support. This will remove the cyanoethyl groups that can react with the free amino or thio groups during deprotection, blocking these site with acrylonitrile. The resin must then be thoroughly washed (acetonitrile) and dried (Ar, He, N2, air) prior to cleavage and base deprotection.
If fast deprotection on standard amidites (G, C) is used, e.g. Ac-dC and/or dmf-dG, cleavage and deprotection can be carried out in either AMA (35min at 65˚C or 2h at RT) or ammonium hydroxide solution (2h at 55˚C). If Bz-dC and iBu-dG are used then cleavage and deprotection is done using ammonium hydroxide solution (2.5h at 70˚C, 4h at 55˚C, or overnight at 55˚C).
For TFA-protected modifiers the TFA group is lost during deprotection. If the intended use is post-synthesis labelling the oligo with an active ester (e.g. TAMRA, LK0251), it is recommended that the oligo is desalted prior to the labelling reaction.
MMTr protection is stable to base treatment and this can be used to aid purification in e.g. “DMT ON” HPLC or cartridge purification. If, after purification or desalting, the MMTr group still needs to be removed this is typically carried out using 80% acetic acid/water for 30min and the oligo is immediately desalted.
Determination of coupling efficiency for MMTr Modifiers
This procedure is used solely for the purpose of determining coupling efficiency; it should not be used for routine deprotection. The calculation is based on a comparison of the absorbance of the MMTr cation at 472nm versus the absorbance of the DMTr cation at 497nm. The ratio factor (RF) of the absorbance of a standard solution is:
RF = A-DMTr(497)/A-MMTr(472) = 1.33
- Carry out the synthesis (1μmol) in the DMT ON mode.
- Collect the last DMTr solution in a 100ml volumetric flask and make up to the mark with 0.1M toluenesulphonic acid in anhydrous acetonitrile (TSA). Zero the instrument with TSA at 497nm and then measure the absorbance (A1) at 497nm.
- After the synthesis is complete, remove the synthesis column and manually deblock the MMTr group with aliquots of the normal deblocking mix until all the yellow colour has eluted. This process takes up to 15min. Collect the solution in a 100ml volumetric flask and make to 100ml with TSA. Measure the absorbance (A2) at 472nm.
- Calculate the coupling efficiency using the formula:
Coupling (%) = A2/A1 x 1.33 x 100
Note that, due to incomplete deblocking of the MMTr group, this procedure yields a coupling efficiency determination about 5% below the actual coupling efficiency.
The modified oligonucleotide may be purified using a cartridge, HPLC, or gel electrophoresis. Cartridge purification is accomplished using the DMT ON procedure. MMTr removal on the cartridge is not reliable with a standard 2% aqueous trifluoroacetic acid (TFA) wash, therefore 2 additional TFA washes are required.
Alternatively, treatment of the purified DMT ON oligo (see below) with acetic acid:water (80:20) in solution at room temperature for 1h will remove the MMTr group. Precipitate the oligo with an ethanol/2.5M sodium acetate precipitation. Again, complete removal is not achieved therefore additional HPLC is required.
RP-HPLC may be performed either before or after attachment of the label. If purification is desired prior to a label attachment, the MMTr group should not be removed from the oligo as the lipophilic nature of the group aids in HPLC purification. RP-HPLC is best carried out using a C18 or equivalent column. The MMTr group can be removed in cartridge purification or by treatment with acetic acid in solution as above.
Storage & Stability
The oils are stored dry in a freezer at –10 to –30°C. Acetonitrile solutions must be used within 24h.