Frequently Asked Technical Question
QUESTION: How does this work? What would happen if I used the AMA reagent with Bz protected dC?
RESPONSE:dC when proected as an amide linkage is susceptible to transamination or displacement of the amide with the deprotecting amine. Normally, the deprotecting amine is ammonia and dispacement leads to the same product as hydrolysis. However, if methylamine is used for the deprotection step with a benzoyl protected dC, approximately 10% of the product is from the displacement reaction to give N-methyl-dC residues, while the remaining 90% is the desired product formed by hydrolysis. A more labile protecting group on dC is isobutyryl which is also commercially available. This group is displaced by methylamine to about 5% - an improvement but still a mutation. Ac-dC, on the other hand, is hydrolyzed virtually instantaneously by the AMA reagent and none of the slower displacement reaction gets a chance to occur.
M.P. Reddy, Beckman Instruments, personal communication.
QUESTION: Can the AMA reagent be made up and stored? for how long? in what kind of container?
RESPONSE:Yes. Store tightly sealed in a glass bottle in the refrigerator. The reagent does not decompose but routine opening will lose some of the volatile bases. Dispose properly after 4-6 weeks.
Candy Lee, Dartmouth
QUESTION: What is the procedure for deprotecting with AMA?
RESPONSE:The AMA reagent is a 50:50 mixture of aqueous Ammonium
hydroxide and aqueous MethylAmine. With AMA the cleavage of the
oligonucleotide from the support is accomplished in 5 minutes at room
temperature. The deprotection step is carried out at 65°C for a
further 5 minutes. Deprotection can also be carried out at lower
temperatures as shown in Table 1. In all cases, no base modification
has been observed.
TABLE 1: DEPROTECTION WITH AMA
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