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Oligonucleotides containing modified cytosines for epigenetic research are synthesized using conventional phosphoramidite solid-phase oligonucleotide synthesis, but replacing the standard cytosine phosphoramidite with modified cytosine phosphoramidites (Figure 2).
Some changes to standard synthesis and deprotection conditions are required for these modified bases. The epigenetic bases (apart from 5-Methylcytosine) have functional groups at the 5-position that must be protected, which dictates the deprotection conditons for oligos containing these modifications, and compatibility with other modifications.
The 5-methylcytosine phosphoramidite monomer (Figure 2a) requires no modification to standard conditions for oligo synthesis and deprotection.
Two 5-hydroxymethylcytosine phosphoramidite monomers are available. The standard 5-hydroxymethylcytosine phosphoramidite monomer (Figure 2b), in which the 5-hydroxymethyl group is protected as a cyanoethyl ether, can be deprotected using ammonium hydroxide, the standard reagent for oligonucleotide deprotection; however, harsh deprotection conditions must be used (65 °C for several days, or 75 °C for 17 h), which may be incompatible with other bases. This monomer cannot be deprotected using either sodium hydroxide or potassium carbonate. The alternative 5-hydroxymethylcytosine phosphoramidite monomer (Figure 2c) is compatible with sodium hydroxide and potassium carbonate deprotection, but urea derivatives are formed when standard ammonium hydroxide deprotection is used.
In the standard formylcytosine phosphoramidite monomer (Figure 2d) the aldehyde is is masked as a protected 1,2-diol. After oligonucleotide synthesis and deprotection (either using ammonium hydroxide, sodium hydroxide or potassium carbonate), the 1,2-diol must be converted to the aldehyde by oxidation with aqueous sodium periodate in a separate step (Figure 3). After the oxidation reaction is complete (in 30 minutes at 4 °C), excess periodate is removed by gel filtration or quenching with ethylene glycol, before HPLC purification.
This standard 5-formylcytosine phosphoramidite has the advantage that it can be used with both standard (ammonium hydroxide) mild (aqueous base) deprotection conditions, which makes it potentially compatible with a wide range of other modifications; the disadvantage is that a separate oxidation step is required after oligo synthesis, which may be incompatible with some other modified bases.
An alternative 5-formylcytosine phosphoramidite (Figure 2e) is in development, in which the aldehyde is not protected, but the 4-amino (amidine) group is protected with as a benzoyl amide (Figure 2). This alternative 5-formylcytosine phosphoramidite monomer cannot be deprotected with potassium carbonate.
In the 5-carboxycytosine phosphoramidite monomer (Figure 2f) the acid is protected as an ethyl ester, and the 4-amino (amidine) group is protected as a benzoyl amide. Standard deprotection with ammonium hydroxide yields the amide as a side product, along with the desired carboxylic acid. Deprotection with sodium hydroxide avoids this problem, although the use of sodium hydroxide in oligonucleotide deprotection prevents the use of dimethylformamidine-protected phosphoramidites in the rest of the oligonucleotide. The 5-carboxycytosine phosphoramidite monomer cannot be deprotected with potassium carbonate.
For further information see the Glen Research epigenetic base report.