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Furthermore, lower phosphorylated byproducts as mono- and
diphosphorylated oligonucleotides may be formed during the
synthesis, which are often difficult to separate from the target
triphosphorylated product by chromatographic means.
In previous reports we synthesized various nucleotide bio-
conjugates applying the cycloSal-method[17,18] using activated
cycloSaligenylchlorophosphites of the general structure
1 (Figure 1).
found to be stable (Cl/H !Ac<NO2). In addition, the chemical
stability of the 5-acetyl- and 5-nitro-cycloSal compounds with
regard to hydrolysis was markedly low which made the purifi-
cation difficult. Therefore, we decided to focus on 5-chloro-sali-
genyl-N,N-diisopropylphosphoramidite (2c) for further studies
because it is the most stable compound of the cycloSal-phos-
phoramidites 2a–c and the starting materials used for the syn-
thesis are readily available. The 5-unsubstituted phosphorami-
dite 2d was found to be only poorly reactive towards the
phosphorylation with pyrophosphate and thus has not been
further considered.
The synthesis of 5-chloro-saligenyl-N,N-diisopropylphosphor-
amidite (2c) was achieved in 3 steps in an overall yield of 53%
(Scheme 1).
Figure 1. General structure of cycloSal-chlorophosphites 1, cycloSal-phos-
phoramidites 2 and their Arbuzov isomerized form 3.
With regard to an automated 5’-pppDNA/RNA synthesis
these compounds cannot be used in commercially available
synthesizers because of their high reactivity. Nevertheless, al-
though the difference to the salicyl phosphorochloridite
method reported by Ludwig/Eckstein to the cycloSaligenyl-
chlorophosphite seems to be small, the latter compounds
showed in the past already clear advantages.[17,18]
Scheme 1. Synthesis of the cycloSal-phosphoramidite 2c used for the syn-
thesis of DNA- and RNA-5’-triphosphates. DIPA = diisopropylamine.
Therefore, our aim was to develop a method that is compat-
ible with reagents used in the standard phosphoramidite
chemistry protocol and a protocol that allows that the whole
process can be used in an automated oligonucleotide synthe-
sizer based on the cycloSal-method. We focused our attention
on the preparation of cycloSal-phosphoramidites that have
a lot of advantages compared to the currently published meth-
ods (compounds 2, Figure 1): 1) cycloSal-phosphoramidites
may be coupled and oxidized with the same reagents used in
oligonucleotide synthesis, 2) in principle the same excellent
coupling efficiencies can be expected as with standard DNA
phosphoramidites, and 3) the subsequent phosphorylation re-
action proceeds in a short reaction time at room temperature
due to the increased reactivity caused by an electron-with-
drawing substituent in the cycloSal-moiety.[17]
First, 5-chlorosalicylic acid 4 was reduced with lithium alumi-
numhydride to give 5-chlorosaligenol, which was then reacted
with phosphorus trichloride in the presence of pyridine to give
5-chloro-cycloSal-chlorophosphite 2. After purification by Ku-
gelrohr distillation, compound 2 was reacted with 2.2 equiva-
lents of diisopropylamine. The resulting 5-chloro-saligenyl-N,N-
diisopropylphosphoramidite (2c) was purified by silica gel fil-
tration and was obtained as a colorless solid after evaporation
of the solvent. This material was found to be suitable to be
used in a DNA/RNA synthesizer.
CycloSal-phosphoramidite 2c showed comparable stability
to normal nucleoside 3’-O-phosphoramidites and it can be
stored in the refrigerator for months without degradation.
When dissolved in acetonitrile cycloSal-phosphoramidite 2c
was stable for at least a few days when kept under dry condi-
tions and no formation of the Arbuzov product was observed.
In a separate control experiment, 5-chloro-cycloSal-phosphon-
amidate 3c was used together with the cycloSal-phosphorami-
dite 2c in the coupling reaction but 3c did not couple to the
5’-end of the oligonucleotides. Thus, even in the case that
some degree of rearrangement of 2c to give 3c occured, no
formation of side products during the coupling reaction has to
be considered. In contrast to the cycloSal-phosphoramidites
one might consider to convert the Ludwig/Eckstein phosphity-
lation reagent into its phosphoramidite form in order to obtain
a more selective phosphitylation reaction. To the best of our
knowledge, this compound hasn’t been described before. Con-
sequently, we synthesized and purified it on a route compara-
Results and Discussion
After the preparation and evaluation of cycloSal-phosphorami-
dites 2 with different electron-withdrawing groups at the 5-po-
sition, compound 2c bearing a 5-chloro substituent was select-
ed to be used in all further coupling reactions. In principle, for
a fast phosphorylation reaction with pyrophosphate it is desir-
able to have a strong electron-withdrawing group attached to
the 5-position of the cycloSal-ring because of the high electro-
philicity at the phosphorus atom.[17,21] However, we observed
that strong electron-withdrawing substituents, for example,
the 5-acetyl- and 5-nitro-group in the cycloSal derivatives led
to a rapid isomerization to give the thermodynamically more
stable Arbuzov products 3 (Figure 1).[22] In contrast, the 5-un-
substituted and 5-chloro-cycloSal-phosphoramidites were
Chem. Eur. J. 2015, 21, 16421 – 16426
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