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*****Glen Research Glen Report*****
DEPROTECTION – VOLUME 4 – ALTERNATIVES TO AMMONIUM HYDROXIDE
Back in the 1990s, deprotection of DNA oligos was carried out using ammonium hydroxide overnight at 55 °C. The only option to increase the speed was to raise the deprotection temperature to 80 °C (and even above!) with the time being halved for every 10 °C the temperature was increased. But in those days, the most common application for oligos was as sequencing primers so a small percentage of unprotected ibu-dG was never noticed. The first attempt to increase the speed of deprotection was the introduction1 of dmf-dG (and dmf-dA) as “Fastphoramidites” since dmf-dG is deprotected at about twice the rate of ibu-dG. In our view, there was no downside to the adoption of dmf-dG but dmf-dA proved to be rather too labile for routine use and was discontinued. However, ammonium hydroxide was still the only deprotection method at this time.
Although ammonium hydroxide is still immensely popular for deprotection of DNA oligos, the advent of high throughput synthesis, labile bases and fluorescent tags has led to the adoption of a variety of newer procedures. In this article, Deprotection - Volume 4, we will describe some of the most popular deprotection procedures and will note when they may be most applicable.
As usual, when reviewing the variety of procedures available to deprotect any modified or unmodified oligonucleotide, you must heed the primary consideration: First, Do No Harm. You can then proceed with confidence to Deprotect to Completion.
First, Do No Harm!
As we have stated in the past, determination of the appropriate deprotection scheme should start with a review of the components of the oligonucleotide to ascertain if any group is sensitive to base and requires a mild deprotection or if there are any pretreatment requirements. Sensitive products are defined as such on the Analytical Report, Certificate of Analysis, or Technical Bulletin. Occasionally, some products require a special pretreatment to prevent unwanted side reactions. If the oligo has several unusual components, you must follow the mildest procedure recommended. As you might expect, some highly modified oligos can become VERY challenging.
The use of dmf-dG to speed up deprotection with ammonium hydroxide was only an incremental improvement in speed. However, UltraFast deprotection quickly became a commercial reality with the introduction2 of deprotection using using ammonium hydroxide/methylamine (AMA).
By adding an equal volume of 40% aqueous methylamine solution to ammonium hydroxide to form AMA, it is possible to speed up the deprotection of oligonucleotides enormously.2 Deprotection can be completed in 5 minutes at 65 °C, thereby allowing oligonucleotides to be delivered to customers on the same day of manufacture. The only change required in the synthesis strategy is the substitution of Ac-dC for Bz-dC to avoid transamination of dC by displacement of benzamide by methylamine to form the mutant N4-Me-dC.3 This modification is well tolerated and probably codes perfectly as dC in any case. However, as with dmf-dG described above, we see no downside to the use of Ac-dC and recommend it at all times. UltraFast deprotection has found favor with groups processing many oligonucleotides where the decreased processing time, and, therefore, cost savings, becomes highly significant.
Options for UltraFast deprotection, where the removal of the dG protecting group is the rate determining step, are shown in Table 1.
Note: UltraFAST system requires acetyl (Ac) protected dC to avoid base modification at the C base.
The consequences of fast but incomplete deprotection are illustrated in Figure 1 on the following page. RP HPLC traces show the location of incompletely deprotected oligonucleotides relative to the main component in DMT-ON and DMT-Off situations.
As an aside, we have found that AMA deprotection is also the optimal procedure for RNA deprotection.
Deprotection using sodium hydroxide in aqueous alcoholic solvents is a very mild (and fast) alternative to ammonium hydroxide. For a mild deprotection scheme, you can deprotect DNA oligos with 0.4M sodium hydroxide in methanol/water (4:1). For example, we recommend this method for oligos containing acridine. This technique is necessary for oligos where esters are hydrolyzed to carboxylates, such as Carboxy-dT and EDTA-dT, where deprotection with amine-containing reagents would lead to undesired amide formation. You can also deprotect DNA oligos in a few minutes at 80 °C with no concern about vials popping since the mixture contains no volatile gas. The resulting deprotected oligo can be isolated by precipitation or by dilution with water followed by desalting or DMT-ON purification. In all cases, the oligos are isolated as desirable sodium salts. The downside is that oligos cannot be isolated simply by evaporation and a desalting step is mandatory.
Many years ago, we were confronted with the reality that some DNA bases that we wanted to introduce for DNA damage and repair studies simply were not compatible with ammonium hydroxide deprotection. Although deprotection with ammonium hydroxide at room temperature did allow oligos containing these bases to be isolated, it was not optimal and clearly a new deprotection scheme was needed.
In the early 1990s, we were prompted to look at an alternative DNA protecting group, acetoxymethylbenzoyl (AMB), which could be removed using potassium carbonate in methanol.4 Unfortunately, the AMB-protected monomers proved to be too unstable to store for long periods. But we found that the use of a combination of Pac-dA, Ac-dC and iPr-Pac-dG allowed complete deprotection with potassium carbonate in methanol at room temperature for four hours as long as capping was carried out using phenoxyacetic anhydride rather than acetic anhydride. (With UltraMild reagents, ammonium hydroxide at room temperature for two hours was also effective.) If acetic anhydride was used, a small amount of transamidation occurred at dG residues and overnight treatment with potassium carbonate or ammonium hydroxide was required to deprotect formed Ac-dG residues.
An even milder deprotection scheme has been described5 for the synthesis of highly base labile nucleoside adducts. In this UltraMild variation, Q-supports must be used since the succinate linkages of normal supports are virtually untouched under the deprotection conditions. Normal yields are achieved with Q-supports. The reagent for this Ultra UltraMild deprotection procedure is 10% diisopropylamine/0.25M ß-mercaptoethanol in methanol overnight at 55 °C. This is a method which has not been tested in very many facilities but it is surely worthy of consideration when challenged with the preparation of oligos with very labile bases.
TAMRA-containing oligonucleotides remain popular as single and dual labelled probes. Unfortunately, the stability of TAMRA to the conditions of oligonucleotide deprotection is really marginal. In the past, we have recommended the use of UltraMild monomers and deprotection and this procedure does indeed work well. An alternative approach has been described6 using t-butylamine/methanol/water, which does allow the use of regular monomers. We have evaluated a simpler t-butylamine/water (1:3) mix (4 hours at 60 °C), described by Biosearch Technologies, and, in model studies, this generates TAMRA-oligos with the highest purity and with negligible degradation detected.
Although gas phase deprotection does require specialist equipment, this technique is excellent for high throughput synthesis. Columns and plates can be placed in the reactor without concern for cross contamination since the product oligos will remain adsorbed to the synthesis support. This is doubly advantageous since the product can be eluted from the columns and plates in such a way that the organic debris can be removed. Also, using anhydrous ammonia gas and using UltraMild monomers, the cleavage and deprotection processes can be completed in less than 1 hour.7 However, methylamine gas has proved to be more popular for routine synthesis and is in common use in our industry. Please note that deprotection times and temperatures vary with the equipment and number of columns and will need to be optimized.
Oligonucleotide deprotection has come a long way since the early days when ammonium hydroxide was the only option. Now a variety of procedures are available to fit a variety of circumstances. Each synthesis should be reviewed to ensure that the deprotection conditions are compatible with the components of the oligo. Special deprotection requirements can be found on our website: http://www.glenresearch.com.
1. H. Vu, et al., Tetrahedron Lett., 1990, 31, 7269-7272.
2. M.P. Reddy, N.B. Hanna, and F. Farooqui, Nucleos Nucleot, 1997, 16, 1589-1598.
3. M.P. Reddy, N.B. Hanna, and F. Farooqui, Tetrahedron Letters, 1994, 35, 4311-4314.
4. W.H.A. Kuijpers, E. Kuylyeheskiely, J.H. Vanboom, and C.A.A. Vanboeckel, Nucleic Acids Res., 1993, 21, 3493-3500.
5. L.C.J. Gillet, J. Alzeer, and O.D. Scharer, Nucleic Acid Res., 2005, 33, 1961-1969.
6. B. Mullah, and A. Andrus, Tetrahedron Lett., 1997, 38, 5751-5754.
7. J.H. Boal, et al., Nucleic Acids Res., 1996, 24, 3115-3117.
Please contact Glen Research if you have any questions or comments!