Oligonucleotides are routinely prepared on supports to which the first nucleoside is attached via a succinate linkage.
Over the years, the succinate linkage has demonstrated stability during the synthesis process but has sufficient lability to be
cleaved quickly in the deprotection step. However, if the cleavage step is carried out with ammonium hydroxide manually
or on the synthesizer, it consumes one hour of precious time while releasing only about 80% of the oligonucleotide.
This step is, therefore, a bottleneck in the productivity of many synthesis groups.
Is it possible to find a replacement to the succinate group which offers good stability to the synthesis reagents while offering
a much faster cleavage step? The oxalate group has been shown to be very labile during cleavage but its stability to the
normal synthesis reagents is not good, requiring changes for successful use. In a practical but elegant study1 of various
bifunctional carboxylic acids, Richard Pon’s group identified hydroquinone-O,O’-diacetic acid as the most satisfactory
alternative to the succinate group. Nucleosides with this linker arm (Q-linker) are attached to supports with the same
ease as the succinyl linker arm.
The cleavage time in ammonium hydroxide at room temperature was found to be 2 minutes, but what about the stability
during synthesis? How significant was premature cleavage of oligonucleotide on the synthesizer because of the basic
reagents in the capping mixes and oxidizer? Pon showed that the Q-linker is stable to the capping reagents but very slightly
labile to the oxidizer (8% cleavage in overnight exposure which would correspond to about 2,000 normal synthesis cycles).
We tested the significance of premature cleavage by preparing sixteen 20mer oligonucleotides on a 0.2μmole scale, eight
with succinate and eight with Q-linkers. The succinate supported oligos were cleaved for 1 hour at room temperature,
while those on the Q-support were cleaved for 2 minutes. Both sets were then deprotected normally with ammonium
hydroxide. The Q-supports actually gave 5% better yields of product than the succinate supports. Oligo purities were
equivalent in both sets.
The Q-linker is absolutely compatible with all hydrolytic cleavage procedures, but especially mild procedures like potassium
carbonate in methanol. Pon also showed that it is preferable for RNA supports, improving the cleavage time for 2’-silyl
protected nucleoside supports from 2 hours (60-65% cleavage) to 5 minutes (95% cleavage).
We are offering Q-linkers of the four regular nucleosides on 500Å CPG in 0.2 and 1μmole scales.
Details
Usage
Coupling: No changes needed from standard method recommended by synthesizer manufacturer.
Deprotection: Deprotect using the protocol required by the nucleobases. Oligo is cleaved from the support in 2 minutes at room temperature.
Specifications
Storage
Refrigerated storage, maximum of 2-8°C, dry
Dilution/Coupling Data
The table below show pack size data and, for solutions, dilution and approximate coupling based on normal priming procedures.