Glen Research offers a selection of 5’-Amino-Modifiers but which one is appropriate for which application? In this Technical Brief, we detail the pros and cons of each and attempt to direct our customers to the optimal solution. Our most popular 5’-Amino-Modifiers are the C6 versions and this article will focus exclusively on these products but the arguments apply equally well to C3, C12 and hydrophilic TEG spacers.
FIGURE 1: Structures of 5'-Amino-Modifiers C6
If you wish to purify a 5’-amino-modified oligonucleotide, the MMT group is a good choice since the oligo can be easily purified by reverse phase techniques. The MMT group is then removed post purification with aqueous acid. Also, the MMT group can be removed by extended deblocking on the synthesizer, allowing a solid-phase conjugation of a tag containing an activated carboxylic acid. However, the tag must be stable to the subsequent conditions of cleavage and deprotection.
The main problem we have found with this protecting group is that it has a tendency to stay on when it should be removed and fall off when it should stay on. Three key steps can be followed to avoid this problem:
Do not remove the MMT group on the synthesis column unless you plan to conjugate the amine while the oligo is still on the support.
Do not dry down the solution of MMT-on oligo if you plan to do MMT-on purification without first adding a non-volatile base, such as TRIS base, to avoid MMT loss.
Do not remove the MMT group during cartridge purification since it will predominantly reattach. Rather, remove the MMT with aqueous acetic acid after purification. Using 20% glacial acetic acid/80% water allows extraction of MMT-OH using ethyl acetate before drying the solution. Or dry the oligo and desalt by your preferred method.
The DMS(O)MT protecting group is designed to be an improvement over MMT while retaining MMT’s useful characteristics. The DMS(O)MT group is much more reliable in routine use than MMT and is especially useful for trityl-on purification techniques. Unlike MMT, the DMS(O)MT protecting group can be removed using aqueous TFA on the reverse phase purification cartridge without the need for later removal in solution. Although a substantial improvement over MMT in most ways, the following two key points should be remembered:
The base-labile TFA group is used when oligo purification prior to conjugation is not necessary. Since only the amino-modified oligo is full-length, the conjugation reaction will select for the full-length oligo. However, there is one key point to be concerned about when using this product. Since an alkylamine is quickly formed on deprotection, it is possible for side reactions to occur which will reduce the amount of amine available for the desired conjugation reaction. This can be minimized by taking steps to avoid two side reactions:
Treat the newly synthesized oligo with 10% diethylamine (DEA) in acetonitrile while still on the support. A simple 5 minute treatment with 1 mL of 10% DEA in acetonitrile, followed by a rinse with acetonitrile will remove all acrylonitrile.
The oligo is then cleaved and deprotected using AMA (ammonium hydroxide/40% methylamine, 1:1 v/v) in UltraFast conditions which minimizes transamidation.
Phthalic acid diamide (PDA)
We are excited about the potential of 5’-PDA-Amino-Modifiers, which were developed by Stefan Pitsch along with Stefan Berger of ReseaChem in Switzerland, and introduced by Glen Research in the spring of 2012. In contrast to the other protected amino modifiers which are viscous oils, the analogous PDA protected compounds are granular powders. This important property of these compounds allows straightforward handling, storage and aliquoting and leads to a significant increase in stability. This property alone makes 5’-PDA-amino-modifiers the first choice for high throughput DNA modification but their structure also allows a significant cost advantage over their oily counterparts.
Again two key points should be noted:
Oligonucleotides containing PDA-Amino-Modified oligonucleotides must be treated with aqueous methylamine or AMA for complete deprotection.
If ammonium hydroxide must be used, reaction at 55°C will yield around 80% of the deprotected amino group, even with extended deprotection times.
TABLE 1: Comparison of 5'-Amino-Modifiers C6