Skip to content Skip to navigation menu

3'-Palmitate CPG

3'-Palmitate CPG

CPG used to incorporate a lipophilic palmitoyl-modifier at the 3' end of an oligonucleotide.
  • Useful modification to aid cell delivery of nucleic acid therapeutics.
  • Similar use as cholesterol.
  • C6 spacer alleviates steric effects of large palmitate group.
  • dR structure compatible with sugar-phosphate backbone.
  • 1000 Å CPG suitable for highly modified oligonucleotides (> 20mers).
Option 1: Select a Pore Size
Option 2: Select a Functional Loading
Option 3: Select a Size
TBD
Add to basket to request a quote

Product information

The introduction of hydrophobic (lipophilic) residues into oligonucleotides with a view to improving their penetration into cells has met with some success.(1)

Cholesteryl-conjugated oligonucleotides have in particular been the subject of substantial interest in antisense and other studies due to the lipophilicity and good availability of cholesterol.

Of similar application to cholesterol, but comparatively less studied to date, is the incorporation of the palmitoyl moiety into oligonucleotides. One such use employs an oligonucleotide conjugate with a 5'-palmitoyl group attached through an amide bond.(2) This has been used to modify GRN163, a thio-phosphoramidate oligonucleotide, to enhance the potency of telomerase inhibition. We offer both 5'-Palmitate-C6-CE Phosphoramidite and 3'-Palmitate CPG for direct incorporation of a palmitoyl group during oligo synthesis, at the 5’ and 3’ end respectively.

Ref:

  1. See for example: Cholesterol conjugated oligonucleotide and LNA: A comparison of cellular and nuclear uptake by Hep2 cells enhanced by Streptolysin-O, Š. Holasová, M. Mojžíšek, M. Bunček, D. Vokurková, H. Radilová, M. Šafářová, M. Červinka and R. Haluza, Molecular and Cellular Biochem., 276, 61-69, 2005.
  2. Lipid modification of GNR163, an N3’ P5’ thio-phosphoramidate oligonucleotide, enhances the potency of telomerase inhibition, B.-S. Herbert, G.C. Gellert, A. Hochreiter, K. Pongracz, W.E. Wright, D. Zielinska, A.C. Chin, C.B. Harley, J.W. Shay and S.M. Gryaznov, Oncogene, 24, 5262-5268, 2005.

Applicable Products

LK2163 5'-Tocopherol-CE Phosphoramidite
LK2170 5'-Cholesterol-CE Phosphoramidite
LK2189 5'-Cholesterol-TEG-CE Phosphoramidite
LK2194 5'-Octyltocopherol-CE Phosphoramidite
LK2199 5'-Palmitate-C6-CE Phosphoramidite
LK2393 3'-Palmitate SynBase™ CPG 1000/110
LK2394 3'-Cholesterol SynBase™ CPG 1000/110

Physical & Dilution Data

Dilution volumes (in ml) are for 0.1M solutions in dry, alcohol-free DCM, except for LK2189 which is dissolved 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.

Item

Mol. Formula

Mol. Wt.

Unit Wt.

250mg

500mg

1g

LK2163 C38H67N2O3P 630.94 492.68 3.96 7.92 15.85
LK2170 C43H76N3O4P 730.07 591.81 3.42 6.85 13.70
LK2189 C46H82N3O7P 820.15 682.90 3.05 6.10 12.19
LK2194 C46H83N2O4P 759.15 620.89 3.29 6.59 13.17
LK2199 C31H62N3O3P 555.83 417.57 4.50 9.00 17.99
LK2393 - - 533.69 - - -
LK2394 - - 707.93 - - -

Dissolution

LK2163, LK2170, LK2194 and LK2199 - Dilute in anhydrous, alcohol-free DCM to a concentration of 0.1M.

LK2189 - Use anhydrous acetonitrile to a concentration of 0.1M.

Prepare the amidite solution 5-10min before placing on the synthesiser to ensure complete dissolution.

Coupling

LK2163, LK2170 and LK2194 - An increased coupling time of 15min is recommended for the phosphoramidites. Contrary to MacKellar et al1, we have found that when using LK2170, column washes with DCM before and after coupling are unnecessary. In our hands, omitting the DCM washes gave the highest final coupling results and there was no evidence of reagent precipitation in the lines.

LK2199 - 3-5min (3min up to 1umol).

LK2393 and LK2394 - Both CPG supports are used as any standard nucleoside support as per instrument instructions. However, non-nucleosidic modifications are slow to detritylate and require an initial detritylation prior to use in synthesis. In this case it is important not to use a cycle with an initial capping step.

It is recommmended that the oligonucleotide is synthesised DMT OFF when using the 3’ modifications (CPGs), otherwise the presence of the DMTr and hydrophobic group can result in difficult purification and solubility issues. None of the 5’ modifiers have DMTr blocking therefore this is not an issue with them.

Cleavage & Deprotection

For the amidites no changes are required from your standard method, however the optimum conditions are AMA for 2h at RT. The amidites - except for LK2189 - are stable to most common deprotection methods e.g. AMA, 10mins, 65oC (cholesterol-TEG has a tendency to cleave through the carbamate at elevated temperatures).

The CPG supports use the succinyl linker which will cleave under most ammonium hydroxide solution and AMA deprotection conditions (typically 1-2h at room temperature with ammonium hydroxide solution, and a few minutes at 65°C with AMA). The linker will also cleave with potassium carbonate at room temperature (>90% after 4h). Therefore cleavage and deprotection of the oligo is carried out according to the deprotection protocols required by the nucleobases and other modifiers (if present). When synthesising short oligos (<15 bases) it is, however, advantageous to add 20% EtOH to the cleavage and deprotection solution to ensure complete removal of the oligo from the resin.

Purification

Purification by RP-HPLC is recommended for oligonucleotides modified with hydrophobic labels. Where 3’-modifiers are used DMT ON purification is possible but makes the oligonucleotide extremely hydrophobic. As a result short oligos often have solubility issues. Also, removing the DMTr group in aqueous acetic acid can cleave the cholesterol label from the oligo through the carbamate bond.

Where modification is incorporated at the 5’ end, there is a significant difference in the retention time between the labelled and unlabelled oligo, making purification simple. In general the HPLC gradient must reach at least 95% MeCN to elute the product.

Whilst this removes the unlabelled failures from the labelled oligo very efficiently, if there is a requirement to remove any labelled deletion sequences IE-HPLC or PAGE is the preferred choice. Similarly, where the modifier is incorporated at the 3’ end, the latter is the preferred choice of purification. RP-HPLC gives limited separation in this case since the full-length and failure sequences are all labelled with the hydrophobic group.

Storage & Stability

All products are stored dry in a freezer at –10 to –30°C and are stable under these conditions for over 12 months. Diluted samples must be used within 24h.

Reference

  1. Synthesis and physical properties of anti-HIV antisense oligonucleotides bearing terminal lipophilic groups, C. MacKellar, D. Graham, D.W. Will, S. Burgess and T. Brown, Nucleic Acids Research, 20, 3411-3417, 1992.

Access support

Need some support with placing an order, setting up an account, or finding the right protocol?

Contact us