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5'-Amino C12 Modifier (MMT-12-Aminododecyl Amidite)

5'-Amino C12 Modifier (MMT-12-Aminododecyl Amidite)

Phosphoramidite for the incorporation of an amino function at the 5' end of an oligonucleotide.

Key features

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  • Incorporates primary amine to 5' end of an oligonucleotide, for subsequent conjugation.
  • Acid-labile monomethoxytrityl (MMT) protection, used as ‘handle’ in e.g. cartridge purification where the MMT group is removed during the purification process.
  • 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.
  • 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.
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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. 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.

Properties:

  • Formula: C41H60N3O3P
  • Molecular Weight: 673.44
  • Appearance: colorless oil

Product usage:

  • Dilution: 100 µmol/mL
  • Cleavage conditions: The MMT group may be left on or removed prior to cleavage of the oligo depending on the purification method. Flush-pulse 3% TCA in DCM or 3% DCA in DCM through the column for 3- 5 minutes until effluent is clear. For MMT-ON cartridge purification, or solution phase methods, cleavage of the MMT group can be accomplished by using 2.5% TFA in water.
  • Deprotection conditions: Deprotection conditions are dependent upon the type of amidites used for the synthesis of the oligo. If using standard amidites deprotect in concentrated NH4OH for 5 hours at 60 °C. If using fast deprotecting amidites deprotect in concentratied NH4OH for 1 hour at 60 °C.
  • For removal of the MMT group using reversed-phase cartridge purification, see protocol below.Note: Oligo should be cleaved and MMT group left on for this purification procedure.Below is a protocol using reverse phase MicroPure column (MP-1602) for cleanup of 5' MMT oligonucleotides. Each MicroPure column will clean up to a 1 µmol synthesis column. Note that the TFA step cleaves the MMT group resulting in a completely deprotected oligonucleotide. (Detailed instructions for preparation of reagents can be found following protocol)1. Rinse column with CH3CN (2 x 2 mL).2. Activate column with 1 N TEAA

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