New light labile linker for solid phase synthesis of 2′-O-acetalester oligonucleotides and applications to siRNA prodrug development

Article abstract of DOI:10.1016/j.bmcl.2011.04.073

We report on the synthesis and properties of oligonucleotides containing 2′-O-(levulinic acid) and 2′-O-(amino acid) acetalesters. Given that esters serve as promoieties in several therapeutic prodrugs, we believe that these derivatives will have potentia

Full text of DOI:10.1016/j.bmcl.2011.04.073

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3721–3725
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New light labile linker for solid phase synthesis of 20-O-acetalester
oligonucleotides and applications to siRNA prodrug development
⇑
Richard Johnsson , Jeremy G. Lackey , Jovanka J. Bogojeski, Masad J. Damha
Department of Chemistry, McGill University, Montréal, Québec H3A 2K6, Canada
a r t i c l e i n f o
a b s t r a c t
We report on the synthesis and properties of oligonucleotides containing 20-O-(levulinic acid) and 20-O-
(amino acid) acetalesters. Given that esters serve as promoieties in several therapeutic prodrugs, we
believe that these derivatives will have potential use as nucleic acid prodrugs. In addition, we report
on the synthesis of a novel solid support with a photolabile linker that not only allows for the synthesis
of oligonucleotides containing various 20-O-acetalesters, but can be generally adopted to the synthesis of
base-sensitive oligoribonucleotides. The release of oligonucleotides from this support is faster than with
conventional linkers.
Article history:
Received 1 February 2011
Revised 17 April 2011
Accepted 19 April 2011
Available online 27 April 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Harnessing RNA interference (RNAi)1 for therapeutic gene
silencing presents an attractive alternative to small molecule
approaches. Unfortunately, in vivo gene silencing in a therapeutic
setting has proven difficult. The major obstacle of RNAi based ther-
apeutics is the cellular delivery of siRNA since these are generally
too large (ꢀ13 kDa), too negatively charged (ꢀ40 negative charges)
and not hydrophobic enough for efficient intracellular uptake.2
Rapid nuclease degradation and clearance reduces the in vivo
duration of activity of siRNAs, and nonspecific immune responses
pose challenges for siRNA therapeutics. To overcome these
problems, research has mostly focused on the use of delivery
vehicles such as liposomes, lipids, cationic polymer complexes or
lipophilic conjugates,3 while chemically modified siRNAs have
been used to increase both serum and cellular stability and to
prevent the activation of the immune response.4
S-acyloxymethyl8 and the O-(4-acyloxybenzyl)-phosphate(thio-
ate)9 moieties. The 5-nitro-2-furylmethyl and 5-nitro-2-thienylm-
ethyl groups have also shown to increase cell permeability. These
groups are activated by an endogenous nitroreductase, which in
turn starts a cascade reaction releasing the active drug molecule.10
Another very attractive modification, the 2-(N-formyl-N-methyl)-
aminoethyl thiophosphate protecting group, is cleaved by the
temperature of the cellular environment (37 °C).11 Its promoiety
ability was tested in immunomodulatory CpG oligonucleotides
that effectively protected mice from viral infections.11
Other potential sites for modification, the 50- and 30-hydroxyl
positions at the terminal ends of an oligonucleotide, have been
extensively used for covalent attachment of a variety of groups
to aid in cellular uptake, for example, cholesterol and lauric acid,12
but because there are only two positions available for modification,
it has received no attention as a prodrug approach.
Another approach that may be used to overcome the difficulties
experienced in delivery would be the use of siRNA prodrugs. Pro-
drugs are bioreversible derivatives of drug molecules that undergo
an enzymatic and/or chemical transformation in vivo to release the
active parent drug, which can then exert the desired pharmacolog-
ical effect.5
Finally, the ribose 20-hydroxyl position has traditionally been a
very popular position for chemical modification on RNA chains,
particularly to enhance hybridization affinity, lipophilicity and
nuclease stability.13 Surprisingly, it has received little attention
for the prodrug strategy.
Though the prodrug approach has been widely successful for
small molecules, this strategy has been sparingly used for oligonu-
cleotide therapeutics,6 and there are no reports for its use in siRNA
silencing experiments. Nevertheless, there are several positions on
an oligoribonucleotide chain that may be amenable to conjugation
with a promoiety.
Modification of the internucleotide phosphate linkage has re-
ceived the most attention in terms of a prodrug approach due to
its negative charge. Examples involve the S-acylthioethyl,7 the
We have previously reported on the acetal levulinic ester (ALE)
as a promising protecting group for the in situ synthesis of RNA
microarrays.14 Given that esters are involved in several therapeutic
prodrugs, we expected acetalesters to be a promising prodrug moi-
ety. To the best of our knowledge, the 20-O-acetalester of Debart
and co-workers is the only system disclosed thus far.15,16 The
authors used acetyl, propyl, butyl, iso-butyl and pivaloyl acetalest-
ers in a preliminary study of proRNA. They used the Q-linker to-
gether with a polystrene (PS) solid support and released the
oligonucleotides with 3:1 NEt3/48% HF (aq). Notably, the bulkier
pivaloyl acetalester was the only one that remained intact under
these conditions. With this in mind we decided to investigate the
20-O-ALE and various 20-O-(amino acid) acetalesters as prodrug
⇑
Corresponding author.
E-mail address: masad.damha@mcgill.ca (M.J. Damha).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.073

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R. Johnsson et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3721–3725
moieties. We hypothesized that the resulting proRNA molecules,
especially the zwiterionic versions (20-O-amino acids) should have
enhanced binding affinity and cellular uptake by virtue of the pos-
itively charged amino groups. Furthermore, with 20 amino acids to
choose from, it should allow for tuning of the physicochemical
properties and delivery of these RNAs.
HO
Ura
R2
O
a: R1 = O(CH2)2CN
1
R
R
R
R
2 = OCH2C(O)(CH2)2C(O)CH3
O
P
O
1 = O
b:
c:
R1
O
2 = OCH2C(O)(CH2)2C(O)CH3
1 = O
Thy
A 15-nt long DNA-RNA chimeric strand was prepared (50-dt9-
O
R2 = OH
U
ALE-dt5-30) on a Q-linker PS solid support.17 Polystyrene was
chosen as the solid support since it is stable to the final fluoride
treatment required to release the oligonucleotide into solution.
The oligonucleotide was synthesized using both commercially
available thymidine phosphoramidite and our previously disclosed
uridine 20-O-ALE phosphoramidite, both of which were activated
using 5-(ethylthio)-tetrazole (ETT).14 The thymidines were
installed using standard conditions and reaction times while the
20-O-ALE uridine monomer was coupled for 15 min at room
temperature. Standard capping, oxidation and detritylation steps
followed the coupling steps. After the completion of the synthesis,
the solid support was treated with 2:3 NEt3/MeCN for 16 h to re-
move b-cyanoethyl protecting groups. The material was then trea-
ted with 3:1 NEt3/48% HF (aq) for 8 h to release the oligonucleotide
from the polystyrene support. The crude material was analyzed by
HPLC and yielded two major peaks in an approximately 2:1 ratio
(SI). The first eluting peak had the same retention time as an
authentic, fully deprotected oligonucleotide. The major peak eluted
ꢀ2 min later, suggesting that this compound was the desired 20-O-
ALE modified strand. MS analysis confirmed the fully deprotected
and the more retained ALE-modified strand (entry 1, Table 1).
The ALE-modified compound was isolated, lyophilized, redissolved
in water and re-injected into the HPLC and the material eluted
again as two peaks, suggesting that the ALE group undergoes slow
cleavage under the HPLC purification conditions.
OTBDMS
Figure 1. Dimer used in the study of the stability of 20-O-ALE group.
pD 7.2 and monitored in the same manner as above over 7 days.
Again, after 1 day there was approximately 20% cleavage of the
ALE-group and this increased to 30% after 7 days. These results
indicate that while the ALE group is fairly stable in D2O and under
the buffer conditions used during HPLC purification (SI), extended
manipulation in water results in mixtures of compounds.
We therefore turned our attention to photocleavable linkers,
which in principle can be cleaved to release the intact acetalester
modified RNA. An added advantage of this approach is that the oli-
gonucletide can be deprotected while it is attached to the solid
support and be released quickly into anhydrous MeCN, minimizing
extended aqueous deprotection steps and avoiding hazardous HF
solutions. Previously reported photocleavable supports include
those based on an o-nitrophenyl-1,3-propanediol linker,18 as well
as 2 (Fig. 2), designed by Venkatesan and Greenberg.19 The latter
linker, however, requires 1–2 h of irradiation in MeCN/H2O to ob-
tain a satisfactory level of cleavage from oligonucleotides. It was
hypothesized that extending the chain by one carbon atom, and
branching it to generate a tertiary carbon centre, the linker would
resemble the photolabile NPPOC-protecting group20 (3, Fig. 2) and,
hence, it was anticipated to be cleaved in a shorter period of time
relative to 2. The linker, orthogonal to esters and acetalesters, was
conjugated to the nucleoside not through a traditional carbonate to
the 30-end, but through an internucleotide phosphate. In this way,
the use of phosgene, which is highly toxic, or other phosgene alter-
natives, which are difficult to remove, can be avoided. The linker
was prepared according to Scheme 1, by first reacting compound
521 with FmocCl in pyridine to generate 6 in 95% yield. This was
followed by treatment with 80% TFA in CH2Cl2 to liberate the free
acid 7, which then was ready to couple to the solid support. Com-
pound 7 was conjugated to aminomethyl polystyrene (strato-
sphere for DNA synthesis, 1000 Å) or long chain alkyl amine
controlled pore glass (LCAA-CPG, 500 Å) using HATU and DMAP
in pyridine, followed by the capping of unreacted amines with
CAP A and CAP B solutions (Ac2O/Pyridine/N-methyl-imidazole/
THF) to give 8. The Fmoc was removed with 10% 4-methylpiperi-
dine in DMF to give 9. Compounds 1022 and 11 were stirred at
To evaluate the decyanoethylation conditions required for 20-O-
acetalester containing oligonucleotides, a 20-O-ALE containing
dimer (1a, Fig. 1) was synthesized as a model system and the sta-
bility of the dimer was examined by 31P-NMR under the various
conditions. First, compound 1a was treated with 2:3 NEt3/MeCN
at room temperature. To our surprise, under these conditions the
time needed for decyanoethylation was over 24 h. However, when
the mixture was heated to 50 °C under these conditions, the time
for complete decyanoethylation was reduced to one hour with no
trace of ALE cleavage (SI), demonstrating the stability of the ALE
group during deprotection of the phosphate group.
In order to test the stability of the 20-O-acetalester group to
HPLC purification (0.1 M aq TEAA buffer), the model compound
1b, was first dissolved in D2O and monitored by 31P-NMR over a
7-day period. After 1 day there was approximately 20% cleavage
of the ALE-group, with no subsequent decomposition observed
after 7 days. The dimer was also dissolved in 0.1 M TEAA in D2O,
Table 1
MS of oligonucleotides
O
Entry Target sequence and hydrolysis
products
Mass
calculated
Mass
found
O
O
O
O
O
H
N
1a
50-dt9-U-ALE-dt5-30
50-dt9-U-dt5-30
4834.27
4705.21
4778.0
4600.9
4502.9
6961.4
4834.42
4706.19
4778.3
4601.1
4503.2
6963
NO2
NO2
2b
50-dt9-UAPhe-dt5-30-lv
50-dt9-U-dt5-30-lv
50-dt9-U-dt5-30
O
2
3
NO2
O
3b,c
50-dt5-UAPhe-dt4-UAPhe-dt4-UAPhe-dt5-
30-lv
H
N
Two UAPhe inserts
One UAPhe insert
6784.3
6607.2
6783
6606
O
4
a
b
c
Q-linker used with PS solid support. MALDI-TOF results include spermine.
Light labile linker used with CPG solid support. ESI–MS results.
MS done on crude sample.
Figure 2. Structure of the solid support developed by Greenberg and co-workers (2)
of the NPPOC-protectiong group (3) and of the solid support developed in this work
(4).

R. Johnsson et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3721–3725
3723
NO2
NO2
NO2
OFmoc
OH
OFmoc
b
a
tBuO
HO
tBuO
5
O
O
O
6
7
NO2
NO2
OFmoc
OH
c
d
H
N
H
N
Polystyrene or CPG
long chain alkyl amine
O
O
9
8
Scheme 1. Reagent and conditions: (a) Fmoc-Cl, pyridine, rt, 2 h, 95%; (b) 80% TFA in CH2Cl2, rt. 30 min, 93%; (c) Aminomethylated polystyrene or CPG, HATU, DMAP, Pyridine,
Capping (Ac2O, N-Methyl imidazole, pyridine, THF), rt, 18 h; (d) 4-methyl piperidine, DMF, rt, 1.5 h.
The next step to test was the cleavage of an oligonucleotide from
HO
the solid support, thus a 21-nt chimeric DNA/RNA strand (50-dt5-
U-dt4-U-dt4-U-dt5-30-Lv) was synthesized. After cleavage of the
b-cyanoethyl protecting groups with anhydrous 2:3 NEt3/MeCN,
overnight and then at 65 °C for 1 h, the material was dissolved in
anhydrous 1% DIPEA in MeCN and subjected to photolysis in a
transilluminator with an irradiation source in the UV-A region.24
The cleavage was followed over time (Fig. 3A). A 15-nt uridine
DMTrO
Thy
Thy
O
O
+
OLv
(iPr)2N
O
P
10
11
N(iPr)2
a
sequence was also synthesized on a 1 lmol scale, deprotected as
above, and photolyzed at room temperature for 20 min. The oligo-
nucleotide was washed off the support and dried, followed by
de-silylation with 1.5:0.75:1 NEt3-3HF/NMP/NEt3 for 4 h, 65 °C,
DMTrO
Thy
O
yielding 44O.D.s (0.31 lmol) of the crude oligonucleotide. The
NO2
O
recovered crude material eluted as one peak (HPLC; 92% purity;
Fig. 3B) that was confirmed to be the correct oligonucleotide by
MS (Calcd 4526.6; Found 4526.6).
O
P O
H
N
O
Thy
O
OLv
With a fast-working solid support that does not require aque-
ous basic cleavage conditions, our attention turned to amino acid
based acetalesters (AAE). Examination began with alanine, due to
its simplicity; lysine, due to its increased net positive charge;
and phenylalanine, due to its hydrophobicity and increased size.
The synthesis of the monomers is described in Scheme 3. Com-
pound 1325 was activated with sulfuryl chloride and then reacted
with the cesium salt of the desired Fmoc-protected amino acid to
obtain 14a–c in 78–90% yield. The bis-silyl protecting group was
cleaved using NEt3ꢁ3HF in MeCN to give 15a–c in 61–98% yield.
Compound 15a–c was tritylated under standard conditions to give
16a–c in 66–78% yield, followed by phosphitylation to give the fi-
nal phosphoramidites 17a–c in 58–86% yield.
O
12
Scheme 2. Reagents and conditions: (a) (i) DCI in MeCN, rt, 7 h, (ii) 5, rt, 18 h, (iii)
oxidation (I2/H2O/pyridine/THF), rt, 0.5 h, (iv) capping (Ac2O, N-Methyl imidazole,
pyridine, THF), rt, 1.5 h. Loading 35–60 lmol/g
room temperature with 0.5 M DCI in MeCN for 7 h and the resul-
tant mixture was then added to 9 (Scheme 2). The phosphite triest-
er intermediate was then oxidized (I2/H2O/pyridine/THF) and the
unreacted free hydroxyl groups were capped with Ac2O/Pyridine/
N-methyl-imidazole/THF. The support was obtained with a loading
of 35–60 l
mol/g of support, as assessed by the trityl color assay.23
Figure 3. (A) Rate of oligonucleotide (50-dt5-U-dt4-U-dt4-U-dt5-30-Lv) release when solid support 12 is placed in MeCN with 1% DIPEA under a transilluminator. (B) Crude
HPLC of poly uridine synthesized with solid support 12.

3724
R. Johnsson et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3721–3725
O
N
O
O
N
NH
NH
NH
O
Si
O
Si
HO
a
b
O
N
O
O
O
O
O
R
R
O
O
Si
Si
O
O
O
S
O
O
OH
O
O
NHFmoc
NHFmoc
13
14a-c
15a-c
O
O
O
N
O
N
a: R = Me
b: R = (CH2)4NHFmoc
c: R = Bn
NH
NH
DMTrO
DMTrO
O
O
c
d
O
O
R
R
OH
O
O
CNEtO
O
O
O
NHFmoc
P
NHFmoc
16a-c
O
N(iPr)2
17a-c
O
Scheme 3. Reagent and conditions: (a) (i) SO2Cl2, CH2Cl2, 0 °C–rt, 2 h, (ii) Cs-Amino acid-Fmoc, DMF, CH2Cl2, rt, 18 h, 14a 90%, 14b 88%, 14c 78%; (b) NEt3ꢁ3HF, MeCN, rt, 18 h,
15a 87%, 15b 98%, 15c 61%; (c) DMTrCl, AgNO3, Pyridine, THF, rt, 2.5 h, 16a 76%, 16b 66%, 16c 78%; (d) CNEtOP(Cl)N(iPr)2, DIPEA, THF, rt, 5.5 h, 17a 86%, 17b 58%, 17c 62%.
With the amidites in hand, 15-nt chimeras with single acetal
free oligonucleotide was not detected by MS. As anticipated, the
desired product could not be isolated in pure form after attempted
separation by HPLC. Incubation of the crude mixture in phosphate
buffer (pH 7.2) at 37 °C showed that the peak corresponding to 50-
dt5-UAPhe-dt4-UAPhe-dt4-UAPhe-dt5-30-Lv disappeared within 5 h,
with complete conversion of 19 to 50-dt5-U-dt4-U-dt4-U-dt5-30-Lv
occurring within 48 h (SI).
In summary, we have developed and synthesized a new phot-
olabile linker for the solid-supported synthesis of oligoribonucleo-
tides. The photolabile linker is cleaved within 15 min at room
temperature under anhydrous conditions. With the newly devel-
oped solid support, various amino acid conjugated oligonucleo-
tides were synthesized. Different cleavage rates were seen among
various amino acids tested, in which phenylalanine was cleaved
at the slowest rate. The ease of cleavage of the phenylalanine acet-
alester from the parent RNA in the absence of esterases warrant
further studies with amino acid derivatives of siRNAs. Cell uptake
studies are ongoing in our laboratory.
amino ester (AAE) inserts (50-dt9-UAAE-dt5-30-Lv, UAAE = Ala 18a;
Lys 18b; Phe 18c) were synthesized and deprotected on a 1 lmol
scale using the same conditions as described above followed by
RP–HPLC analysis. For the Ala- and Lys-containing oligonucleotides
(18a and 18b), two peaks were observed in the chromatogram, one
major peak corresponding to 50-dt9-U-dt5-30-Lv and a minor, less
retained peak corresponding to the fully deprotected oligonucleo-
tide (50-dt9-U-dt5-30). Therefore, the desired Ala and Lys-containing
oligonuclesotide could not be obtained. For the Phe-containing oli-
gonucleotide (18c), three peaks were observed by HPLC: 50-dt9-U-
dt5-30 and 50-dt9-U-dt5-30-Lv, as observed previously, and a more
retained, prominent peak corresponding to the desired Phe-con-
taining oligonucleotide, 50-dt9-UAPhe-dt5-30-Lv (peak 3; Fig. 4A).
The three oligonucleotides were confirmed by MS (entry 2, Table 1).
The compound was isolated, lyophilized and re-injected into the
HPLC, and the material eluted again as three peaks, indicating that
the Phe-acetalester also hydrolyzed (albeit more slowly relative to
Ala and Lys) during HPLC purification/handling (Fig. 4B).
To further examine the properties of the Phe-containing oligo-
Acknowledgments
nucleotide, a three-insert sequence was prepared (50-dt5-UAPhe
-
dt4-UAPhe-dt4-UAPhe-dt5-30-Lv, 19).
Financial support was provided by NSERC, CIHR, the Swedish
Research Council, the Foundation Blanceflor Boncampgni-Ludovisi,
née Bildt and Sixten Gemzéus Foundation.
Five distinguishable peaks were observed by HPLC, among
which were the desired product and two others with one and
two UAPhe inserts (MS on the crude sample; entry 3, Table 1). The
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.04.073.
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