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  6. Formylindole Modifier CE-Phosphoramidite

Formylindole Modifier CE-Phosphoramidite

Formylindole Modifier CE-Phosphoramidite, 100 μmol, ABI (8 mL / 20 mm Septum)

Formylindole Modifier CE-Phosphoramidite

Formylindole Modifier CE-Phosphoramidite, 100 μmol, ABI (8 mL / 20 mm Septum)

Phosphoramidite for the incorporation of an aldehyde function internally or at the 5' end of an oligonucleotide.

Key features

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  • Introduces reactive aldehyde functionality into oligo for conjugation.
  • Sugar unit of the pseudo nucleoside is unmodified allowing multiple incorporations of dR-formylindole.
  • Modification is stable to most cleavage and deprotection conditions.
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Item ID LK2056-F100
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Product information

The aldehyde function is often used to conjugate biopolymers to other molecules by processes such as reductive amination or adduct formation with hydroxylamines, hydrazines and semicarbazides.

Aldehydes have also been used as a means of immobilising oligonucleotides onto solid surfaces. (1)

The use of this functionality has been hampered by the complexity of existing routes such as post synthetic periodate oxidation of a diol to produce the aldehyde, and the lack of conveniently available ready-made phosphoramidites or supports to incorporate an aldehyde functionality into an oligonucleotide.

This formylindole modifier (2) can be placed either in the centre of or at the 5'-end of an oligonucleotide, but an extended coupling time of 15min for this modifier is recommended to provide a coupling efficiency of >95%.

Since the sugar unit of the pseudo nucleoside is unmodified, multiple incorporations of dR- formylindole are possible. This not only provides multiple conjugation sites but formylindole is known to act as a universal base resulting in destabilisation of the duplex by 7-10 ºC per addition when compared with the natural duplex. This modification is stable to most cleavage and deprotection conditions.

Post-synthetic modification of oligonucleotides bearing this moiety, and still bound to the solid support, has also been achieved. In essence, the options for post-synthetic modification of the aldehyde functionalised oligonucleotide are limited only by the reactive nature of aldehydes and the conditions to which the conjugate is stable.

We have used the aldehyde function to conveniently attach molecules such as O-benzylhydroxylamine and diphenylhydrazine. The use of DMT ON oligonucleotides produced extremely hydrophobic material with these substituents, however, DMT OFF oligonucleotides reacted in a mixture of acetate buffer (pH 4.7) and DMSO (1:1) at 37 °C overnight to give conjugation yields in excess of 70% when modified in the centre and in excess of 80% when modified at the 5'-end of the oligonucleotide.

Ref:

  1. 134 For a review of this area see: Use of carbonyl group addition-elimination reactions for synthesis of nucleic acid conjugates, T.S. Zatsepin, D.A. Stetsenko, M.J. Gait and T.S. Oretskaya, Bioconjugate Chemistry, 16, 471-489, 2005.
  2. A facile incorporation of the aldehyde function into DNA: 3-formylindole nucleoside as an aldehyde containing universal nucleoside, A. Okamoto, K.Tainaka and I. Saito, Tetrahedron Lett., 43, 4581-4583, 2002.

Applicable Products

LK2056 Formylindole-Modifier-CE Phosphoramidite

Physical & Dilution Data

Dilution volumes (in ml) are for 0.1M solutions in dry acetonitrile (LK4050). Adjust accordingly for other concentrations. For µmol pack sizes, dilute as 100µmol/ml to achieve 0.1M.

Item

Mol. Formula

Mol. Wt.

Unit Wt.

250mg

500mg

1g
LK2056 C44H50N3O7P 763.87 323.24 3.27 6.55 13.09

Coupling

An extended coupling time of 15min is recommended.

Cleavage & Deprotection

Deprotection is dictated by other modifiers and nucleobase protection. This is stable to all commonly used deprotection strategies.

Storage & Stability

Stable in solutions for up to 4 days; store at -20oC.

Example conjugation using LK2056 with a functionalised hydrazine1

  1. Synthesise the 5’- or mid-sequence aldehyde-modified oligo as per notes above. Retain on the support.
  2. Prepare a solution of the reagents used for the conjugation (10mM N,N-diphenylhydrazine hydrochloride and 10mM sodium acetate in 1.5ml ethanol).
  3. To the solution add the support-bound oligo (ca. 0.5μmol) then shake at 60°C for 24h. Alternatively the solution from Step 2 can be passed through the column using two syringes.
  4. Evaporate the solution under vacuum, or (if using syringes), remove the solution from the column and wash the resin in the column with MeCN. Dry the resin by passing argon through the column.
  5. The conjugate can then be deprotected and cleaved from the solid support using deprotection conditions suitable for the modified oligo.
  6. Pass through a G25 column to remove the deprotection solution.
  7. The modified oligo is now ready for purification.

Further examples of the use of an alternative aldehyde modifier2 (whose protocols could be extended to LK2056) are available in the literature.

References

  1. A facile incorporation of the aldehyde function into DNA: 3-formylindole nucleoside as an aldehyde containing universal nucleoside, A. Okamoto, K.Tainaka and I. Saito, Tetrahedron Lett., 43, 4581-4583, 2002.
  2. Synthesis of peptide-oligonucleotide conjugates with single and multiple peptides attached to 2’-aldehydes through thiazolidine, oxime, and hydrazine linkages, T.S. Zatsepin, D.A. Stetsenko, A.A. Arzumanov, E.A. Romanova, M.J. Gait and T.S. Oretskaya, Bioconjugate Chemistry, 13, 822-830, 2002.

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