*****Glen Research Glen Report*****
DEPROTECTION – VOLUME 5 – On-Column Deprotection of Oligonucleotides in Organic Solvents
The deprotection of oligonucleotides, especially for high-throughput syntheses, can be the rate-limiting step during the production of oligos and is often difficult to automate due to issues with liquid handling. To streamline the deprotection process, gas phase deprotection using ammonia or methylamine gas is often employed1. After the removal of the protecting groups is complete, the oligo is conveniently eluted directly in water or the buffer of choice. However, the equipment necessary to safely handle a pressurized, corrosive gas is expensive and the additional cost is not worthwhile for many smaller production facilities and research labs.
Volume 5: Deprotect to completion in organic solvents
1) When should I use on-column deprotection?
When using high throughput synthesis.
2) Do I need special monomers?
On-column deprotection requires the use of Ac-dC.
3) Do I need to desalt after on-column deprotection?
No, the organic by-products remain on the synthesis column.
An alternative method that incorporates much of the convenience of gas-phase deprotection but still utilizes low-cost and simple equipment is On-Column deprotection. In this case, the nucleophilic amine used to remove the protecting groups is dissolved in a non-polar solvent, such as toluene, in which the deprotected oligonucleotide is insoluble. After the deprotection of the oligonucleotide is complete, the column is rinsed, allowed to briefly dry and the oligo, still bound to the support, is eluted in the aqueous buffer of choice, as described by Kempe2. A similar strategy was used by Damha for the deprotection of RNA on glass slides3. Based upon a protocol used to deprotect TC RNA monomers4, we have developed a procedure for the deprotection of standard DNA as shown in the Procedure below:
Procedure : Deprotection using EDA/Toluene (1:1)
- After the synthesis is complete, treat the support with 10% diethylamine in acetonitrile, slowly pushing the solution through the column to waste over a 3-5 minute period. This will remove the cyanoethyl protecting groups from the phosphate backbone. This initial treatment is critical to the success of the protocol.
- Rinse the column with acetonitrile.
- Briefly dry the CPG under vacuum.
- Treat the column with Ethylenediamine (EDA)/Toluene solution 1:1 (v/v), pulling the EDA solution into the column so that the support is completely wetted. Use approximately 500 μL per μmole for small-scale syntheses.
- Let the solution sit over the support for 2 hours at room temperature. Apply vacuum and remove the deprotection solution.
- Rinse the column with Toluene (3x).
- Briefly dry the support under vacuum.
- Elute the oligo from the support in aqueous buffer of choice.
In Figure 1, we show the results of an oligonucleotide synthesis that was split, with half being deprotected in standard aqueous AMA and the other half in an EDA/toluene solution. Both product oligos had the same molecular weight as determined by electrospray mass spectrometry. We also found there was no drop in yield from the On-Column deprotection compared with the standard aqueous deprotection.
Figure 1: rP hPlc oF An oligo DeProtecteD with 1. AmA AnD 2. eDA/toluene
When oligos of the same length but different molecular weights were synthesized on Glen UnySupport Frits and deprotected in the same EDA solution, we found there was no indication of any cross-contamination of oligos between the frits by mass spec analysis. This means that an entire 96 well plate can be conveniently deprotected in a single vessel. It should be noted, however, that the Glen UnySupport required 2 hours at 65 °C to be fully eliminated from the 3' terminus of the oligo in the EDA solution.
We have found that this method is compatible with PS supports as well as CPG supports. However, if there are hydrophobic labels on the product oligo, e.g., DMT or CyDyes, some oligo (10-20%) may be retained on a PS support. In this case, we recommend eluting the oligo in buffer containing 10-20% acetonitrile.
1. J.H. Boal, et al., Nucleic Acids Res., 1996, 24, 3115-3117.
2. T. Kempe, Anhydrous amine cleavage of oligonucleotides. United States Patent 5750672.
3. J.G. Lackey, D. Mitra, M.M. Somoza, F. Cerrina, and M.J. Damha, J. Amer. Chem. Soc., 2009, 131, 8496-502.
4. D.J. Dellinger, personal communication.