Internal Sequence Modification (Amino, Thiol)

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David Hannah
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Applicable Products

2071 Amino-Modifier-C6-dA-CE Phosphoramidite
2135 Amino-Modifier-C6-dT-CE Phosphoramidite
2141 Amino-Modifier-C6-dC-CE Phosphoramidite
2149 Amino-Modifier-C2-dT-CE Phosphoramidite
2191 Bz-S-dT-CE Phosphoramidite

Physical & Dilution Data

Dilution volumes (in ml) are for 0.1M solutions in dry acetonitrile (4050). Adjust accordingly for other concentrations. For µmol pack sizes, products should be diluted as 100µmol/ml to achieve 0.1M, regardless of molecular weight.

Item

Mol. Formula

Mol. Wt.

Unit Wt.

250mg

500mg

1g
2071 C55H65F3N9O8P 1068.14 427.40 2.34 4.68 9.36
2135 C50H62N6O10F3P 995.05 458.41 2.51 5.02 10.05
2141 C53H68F3N8O9P 1049.14 457.42 2.38 4.77 9.53
2149 C46H54N6O10F3P 938.94 402.30 2.66 5.33 10.65
2191 C58H71N6O11PS 1091.27 546.53 2.29 4.58 9.16

Amino Modifiers (2071, 2141, 2149 & 2135)

Coupling

No changes are required from the standard method recommended by the synthesiser manufacturer. Coupling is as per standard nucleoside amidites.

Cleavage & Deoprotection

The trifluoroacetyl (TFA) protecting group on the primary amine is removed during standard ammonium hydroxide solution deprotection. However, a minor side reaction during deprotection can lead to irreversibly capping 2-5% of the amine. This could be significant if multiple additions of the modifier are made.

To prevent the reaction, synthesise using acetyl-protected C and, prior to cleavage and deprotection, wash the column with 10-20% DEA/acetonitrile. Deprotect in AMA at 65°C for 15min.

Thiol Modifier (2191)

Coupling

BTT or DCI can be used as activators. Do not use ETT. A coupling time of 10min (600s) is recommended. To facilitate AMA deprotection, dmf-G and Ac-C should be used.

Cleavage, Deprotection & Purification

Before carrying out the cleavage and deprotection, wash with a 20% DEA/MeCN solution, then:

  1. Cleave and deprotect using 100mM TCEP in AMA at room temperature for 2h.
  2. Desalt using a G25 column.
  3. Purify if required.
  4. Dry the oligo.
  5. Add 100μl of 87mM TCEP in water.
  6. Leave for 1h at room temperature.
  7. Pass through a G25 column pre-equilibrated with the conjugation buffer.

Conjugation

To conjugate, add the appropriate maleimide, acetamide or equivalent to the above solution and react as appropriate.

Once the reaction is complete, pass through a G25 column pre-equilibrated with 0.1M TEAA or, if conjugation is on a solid surface, thoroughly with water.

All products

Storage & Stability

Store in a freezer below -10°C. Diluted samples must be freshly prepared for use and used within 24h.

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