Key featuresShow Hide
- Introduces a carboxylate functionality for conjugation to amines.
- Incorporates a dT nucleobase internally.
- Methyl ester is hydrolysed during deprotection and can be coupled directly to a molecule containing a primary amino group by via a peptide coupling reaction.
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The use of the 5'-carboxylate modifier (LK2057) (1) allows the introduction of a carboxylic acid function at the 5'-end of an oligonucleotide that is available for conjugation to amines whilst still on the solid support. This strategy avoids the problems of low yields, long reaction times and the need for excess reactants often encountered by other post-cleavage solution methods.
The 2'-chlorotrityl protecting group is stable during coupling, capping and oxidation or sulphurisation but is easily removed during the deblock step; typically 3% TCA in DCM. At this stage the 5'-carboxylate oligonucleotide can be cleaved and deprotected or further modified by on-column conjugation. Alternatively the 2-chlorotrityl protection can be retained until after cleavage and deprotection.
If the conjugation step has already been completed, provided the label is stable, most deprotection conditions are applicable. Otherwise it is best to use 0.4 M NaOH in methanol/water (4:1) overnight at room temperature. This will avoid the formation of an amide as would be the case using ammonium hydroxide or AMA deprotection conditions.
Subsequent conjugation of the 5'-carboxylic acid function to a range of primary and secondary aliphatic amines can be achieved through amide bond formation on the solid support. We have demonstrated the flexibility of this modifier through reactions with the diene furfurylamine, an aminocaproic ester spacer and ß-casomorphin-5-amide, the latter providing a simple and expedient synthesis of an oligonucleotide-peptide conjugate.
The analogous 5'-Carboxy-C10 CE-Phosphoramidite product (LK2531) is used in the same way as LK2057, but offers the flexibility of an elongated C10 spacer.
Internal carboxylate functions can be achieved using Carboxy-dT-CE Phosphoramidite (LK2142). The methyl ester is hydrolysed during deprotection and can be coupled directly to a molecule containing a primary amino group by via a peptide coupling reaction.
- A new and efficient method for the synthesis of 5'-conjugates of oligonucleotides through amide-bond formation on solid phase, A.V. Kachalova, D.A. Stetsenko, E.A. Romanova, V.N. Tashlitsky, M.J. Gait and T.S. Oretskaya, Helvetica Chimica Acta, 85, 2409-2416, 2002.
Physical & Dilution Data
Dilution volumes (in ml) are for 0.1M (LK2142) and 0.15M (LK2057, LK2531) solutions in dry acetonitrile (LK4050). Adjust accordingly for other concentrations. For µmol pack sizes, 2142 should be diluted as 100µmol/ml to achieve 0.1M, and LK2057/LK2531 as 150µmol/1ml to achieve 0.15M.
Phosphoramidites are best dissolved in anhydrous acetonitrile, although for LK2057 and LK2531 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).
LK2057 & LK2531 - An extended coupling time of 15min is recommended. For LK2142 use 25-60s.
Cleavage & Deprotection
For LK2057 and LK2531, 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.
LK2142 - 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 LK2057 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 LK20571 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.