Physical & Dilution Data
Dilution volumes (in ml) are for 0.1M (2142) and 0.15M (2057, 2531) solutions in dry acetonitrile (4050). Adjust accordingly for other concentrations. For µmol pack sizes, 2142 should be diluted as 100µmol/ml to achieve 0.1M, and 2057/2531 as 150µmol/1ml to achieve 0.15M.
Phosphoramidites are best dissolved in anhydrous acetonitrile, although for 2057 and 2531 increased concentrations of 0.15M and 0.1M solutions are recommended for ABI/MerMade and Expedite synthesisers respectively (hence the provision of a 150μmol pack).
2057 & 2531 - An extended coupling time of 15min is recommended. For 2142 use 25-60s.
Cleavage & Deprotection
For 2057 and 2531, the 2’-chlorotrityl protecting group is stable to oligonucleotide coupling conditions, but is easily removed by acidic detritylating conditions (3% w/v trichloroacetic acid in DCM). Deprotection with 0.4M NaOH in methanol/water (4:1) overnight is recommended to ensure the free carboxylate is released. Ammonium hydroxide or AMA deprotection can result in amide formation.
2142 - Cleavage and deprotection is carried out using a mild deprotection: 0.4M methanolic sodium hydroxide (methanol:water 4:1) for 17h at room temperature. Remove the support and neutralise with 2M TEAA. Note: the methyl-ester is hydrolysed during this deprotection and can therefore be coupled directly to a molecule containing a primary amino group by standard peptide coupling or via the intermediate N-hydroxy-succinimide (NHS) ester. Use of ammonium hydroxide or AMA must be avoided, otherwise the amide or methylamide derivative will be formed in preference to the free acid.
Storage & Stability
Store in a freezer below -10oC. Diluted samples must be freshly prepared for use within 24h.
Example conjugation using 2057 to conjugate an amine
- Synthesise the 5’-carboxylate-modified oligo as per notes above. Retain on the support.
- Activate the carboxylic acid by treating the support-bound oligo with HATU (100 eq.), HOBt (100 eq.), and dry DMF (100μl).
- Warm the reaction to 35°C and shake for 35min.
- After activation of the acid is complete, add triethylamine (100 eq.) and the amine (100 eq.).
- Warm the conjugation mixture to 35°C and shake for 1h.
- The unbound amine can easily be removed from the solid support by washing successively with DMF (2 x 100μl), ethanol (2 x 200μl), and distilled water (2x 200μl).
- The conjugate can then be deprotected and removed from the solid support using deprotection conditions suitable for the modified oligo.
- The conjugate is now ready for purification.
Further examples of the use of 20571 are available in the literature.
- A new and efficient method for synthesis of 5’-conjugates of oligonucleotides through amide-bond formation on solid phase, A.V. Kachalov, D.A. Stetsenko, E.A. Romanova, V.N. Tashlitsky, M.J. Gait, and T.S. Oretskaya, Helvetica Chimica Acta, 85, 2409-16, 2002.