Synthesis and preliminary evaluation of pro-RNA 2′- O -masked with biolabile pivaloyloxymethyl groups in an RNA interference assay

Article abstract of DOI:10.1021/jo200826h

The cellular delivery of bioactive nucleic acid-based drugs such as small interfering RNA (siRNA) represents a major technical hurdle for their pharmaceutical application. Prodrug-like approaches provide an attractive concept to address the delivery probl

Full text of DOI:10.1021/jo200826h

ARTICLE
pubs.acs.org/joc
Synthesis and Preliminary Evaluation of pro-RNA 20-O-Masked with
Biolabile Pivaloyloxymethyl Groups in an RNA Interference Assay
Thomas Lavergne,† Carine Baraguey,† Christelle Dupouy,† Nora Parey,† Winfried Wuensche,‡
Georg Sczakiel,‡ Jean-Jacques Vasseur,† and Franc-oise Debart†,*
†IBMM, UMR 5247 CNRS-UM1-UM2, Universitꢀe Montpellier 2, Place Eugꢁene Bataillon, 34095 Montpellier Cedex 05, France
‡Institut f€ur Molekulare Medizin, Universit€atsklinikum Schleswig-Holstein, Universit€at zu L€ubeck, Ratzeburger Allee 160,
D-23538 L€ubeck, Germany
S Supporting Information
b
ABSTRACT: The cellular delivery of bioactive nucleic acid-
based drugs such as small interfering RNA (siRNA) represents a
major technical hurdle for their pharmaceutical application.
Prodrug-like approaches provide an attractive concept to ad-
dress the delivery problem. With the aim to prepare RNA-based
prodrugs bearing biolabile protections which facilitate cellular
uptake and are prone to be removed enzymatically inside cells in
order to release functional RNA, we synthesized pro-RNA
totally or partially masked in 20-OH position with
pivaloyloxymethyl (PivOM) groups. A suitable strategy has
been developed to synthesize and to purify base-sensitive mixed 20-OH/20-O-PivOM oligoribonucleotides, and to include them in
siRNA. In this strategy, the fluoride labile [(triisopropylsilyl)oxy]-benzyloxycarbonyl group (tboc) as nucleobase protection (for A
and C), the TBS group as 20-OH protection and the Q-linker to solid-support were compatible with the PivOM groups masking
some 20-OH. We have taken advantage of the specific stability of the PivOM group to apply selected acidic, basic, and fluoride ions
treatment for the deprotection and release of pro-RNA. This kind of pro-siRNA was studied in a human cell culture-based RNAi
assay and preliminary promising data are discussed.
’ INTRODUCTION
Many authors described the benefits of chemical modifications
of siRNA but no accurate rationale could be ascertained for the
best way to produce efficient and potent modified siRNA.6,7
Most of the existing modifications in RNA are permanent and
may not be introduced in any position to maintain the biological
effect. This drawback would be circumvented with RNA pro-
drugs bearing temporary modifications as biolabile groups.
Indeed, we chemically substituted RNA in 20-OH position with
acetalester groups which should be cleaved by intracellular
esterases to liberate the native RNA inside cells and potentially
lead to RNAi applications (Figure 1). First results demonstrated
that 20-O-acyloxymethyled uridylates fulfilled the criteria of
nuclease resistance, esterase hydrolysis and ability to form stable
dsRNA.8 Among the different evaluated acyloxymethyl groups,
the pivaloyloxymethyl (PivOM) successfully completes the
requirements to functionalize a potential siRNA prodrug.8,9
Moreover, siRNA may exhibit an improved cellular uptake as a
consequence of the lipophilic character of the PivOM group.
In the perspective of in vivo RNA applications, its features
support the ambition of synthesizing mixed-nucleobase RNA (21
nucleotides long) 20-O-masked with PivOM groups. Therefore,
Since 1978 when the first antisense DNA oligonucleotide was
employed to prevent translation of Rous Sarcoma virus,1 syn-
thetic nucleic acids have been used as tools to regulate gene
expression through different mechanisms of action. These meth-
ods include antisense nucleic acids, ribozymes, aptamers, and
more recently small interfering RNAs (siRNA), one of the key
effectors in natural gene regulation processes termed RNA
interference (RNAi), wherein double-stranded RNAs (dsRNA)
interfere with the expression of complementary genes. For more
than one decade from its discovery,2 RNAi has had a broad
impact on biology as attested by the thousands of reports
describing the use of synthetic siRNA to study gene function,
identify drug targets, and develop more specific therapeutics.3,4
Indeed, several siRNA-based drugs are currently under clinical
evaluation.3 However, applications of this gene silencing tech-
nology need to overcome quite a few hurdles. Limitations of
unmodified siRNA are their susceptibility to nuclease degrada-
tion, off-target effects,5 toxicity due to saturation of the endo-
genous RNAi functions, and, most importantly, effective targeted
delivery. To circumvent these problems, chemical modifica-
tions can provide solutions to the challenges facing siRNA
therapeutics.6
Received: April 21, 2011
Published: May 27, 2011
r
2011 American Chemical Society
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Figure 1. Demasking of 20-O-PivOM pro-RNA inside the cells into native RNA.
our goal was to synthesize pro-RNA (for RNA prodrugs) which
were totally or partially 20-modified with PivOM. It is noteworthy
that very recently, L€onnberg et al. reported on the use of the
biolabile PivOM for the synthesis of a 20,50-dinucleoside in a
prodrug strategy to enhance the cellular uptake.10 With the same
objective, Damha et al. developed a new method for the synthesis
of 20-O-acetalester oligonucleotides with levulinic ester or amino
acid moieties to make siRNA prodrugs.11
Generally, RNA oligonucleotides are synthesized on solid
support by the standard phosphoramidite approach following a
coupling-oxidation-capping-detritylation cycle. The deprotec-
tion process is commonly performed by an ammonia treatment
to remove base-labile protective groups from both nucleobases
Figure 2. 20-O-PivOM and 20-O-TBS ribonucleoside phosphoramidites
1aꢀd and 2aꢀe.
’ RESULTS AND DISCUSSION
and phosphates and to release RNA from the solid support.
Obviously, since basic conditions cannot be applied on RNA
bearing base-sensitive modifications such as 20-O-PivOM
groups,12 pro-RNA cannot be obtained through the standard
method. This drove us to work out a different synthetic strategy
avoiding ammonia, the final deprotection of modified RNAs
being achieved with fluoride ions.
In a previous study of pro-RNA uridylates, the fluoride-labile
2-(trimethylsilyl)ethyl group was chosen as the phosphate
protection, but we found that it was prematurely removed during
the RNA assembly by treatment with the solution of iodine
during the normal oxidation protocol leading to low coupling
yields.8 Consequently, we preferred to protect phosphates with
the standard 2-cyanoethyl (CNE) group eliminated by a non-
nucleophilic strong organic base, DBU or piperidine, which does
not affect the PivOM and allows high coupling yields.12 Likewise,
we recently investigated an appropriate linker that could be
cleaved under conditions suitable for the preservation of PivOM.
The accessible hydroquinone-O,O0-diacetic acid (Q-linker)13
was chosen and a fluoride ions treatment was optimized to
efficiently release base-sensitive RNAs from solid supports.14
Thus, removal of nucleobase protections and TBS groups from
20-OH in partially modified 20-O-PivOM pro-RNA, was per-
formed with the same final fluoride treatment.
Synthesis of 20-O-PivOM or 20-O-TBS Ribonucleosides
Amidites 1aꢀd or 2aꢀe. First, our investigations began with
the preparation of both 20-O-PivOM 1aꢀd and 20-O-TBS 2aꢀe
ribonucleoside phosphoramidites appropriate for pro-RNA
synthesis on solid support (Figure 2).
For the incorporation of uridine, we used the regular 20-O-TBS
phosphoramidite 2a and we prepared the 20-O-PivOM uridine
amidite building block 1a as early described.12 For the synthesis
of cytidine and adenosine building blocks 1bꢀc and 2bꢀc, we
protected the exocyclic amino functions of cytosine (C) and
adenine (A) with a silyl group previously used in RNA synthesis
for the preparation of a base-sensitive aminoacylated RNA
sequence.15 This fluoride-labile [(triisopropylsilyl)oxy]-benzy-
loxycarbonyl group (tboc) was introduced differently on C and
A related to the disparity of their amino function reactivity.
Initially, the reagent 2-[(triisopropylsilyl)oxy]-benzyl alcohol 3
was obtained by selective silylation of the phenol function of the
commercially available hydroxybenzylalcohol (Scheme 1).15
In the case of C, the tboc group was introduced via a reactive
carbonochloridate 4 formed on the primary benzyl alcohol by
addition of triphosgene in THF (Scheme 1). This intermediate 4
was directly reacted with 20,30,50-tri-O-acetyl-cytidine 516 in the
presence of N-methylimidazole to give N4-tboc-protected cyti-
dine 6 with 64% yield (Scheme 2). Then the ribose hydroxyls
were deacetylated with a sodium hydroxide solution in EtOH
without loss of the tboc group. For the introduction of the
PivOM group, we chose the site-specific manner involving the di-
tert-butylsilylene group which simultaneously blocks 50-OH and
30-OH of the N4-tboc-cytidine 7 leaving the 20-OH free to accept
the PivOM in compound 8.17 This silyl group takes advantage on
the widely used tetraisopropyldisiloxane protection since it could be
cleaved with fluoride ions under mild conditions (HF/pyridine)
In this work, we describe the first synthesis of pro-RNA
heteropolymers, suited to the preservation of PivOM groups,
involving silyl-based protections on the exocyclic amino func-
tions of the nucleobases combined to CNE on phosphates and
the Q-linker between pro-RNA and the solid support. Several
pro-RNAs (21 nucleotides in length) totally or partially modified
with 20-PivOM groups were successfully prepared and one
of them was evaluated in a human cell culture-based RNA
interference assay.
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Scheme 1. Synthesis of 2-[(Triisopropylsilyl)oxy]-benzyl Carbonochloridate 4
Scheme 2. Synthesis of 20-O-PivOM and 20-O-TBS Cytidine Amidites 1b and 2b
without affecting the fluoride labile tboc group, whereas TBAF or
Et3N-3HF removed it. Furthermore, the regioselective route was
preferred to the direct alkylation in 20-position with PivOM
chloride12 which leads to a mixture of 20- and 30-isomers rather
difficult to separate by chromatography in this case. Thus, com-
pound 8 was converted in its methylthiomethyl ether derivative 9
which was cleaved with sulfuryl chloride to give the chloromethyl
ether.8 This intermediate was reacted with the pivalate sodium salt in
the presence of 15-crown-5 to afford the 20-O-PivOM derivative 10.
Removal of di-tert-butylsilylene protection using HF-pyridine in
DCM at 0 °C furnished 11 in a sufficient purity to proceed to the
tritylation without additional purification. Next, the dimethoxytrityl
(DMTr) group was typically introduced in the 50-OH to give 12. In
the same way, to obtain the 20-O-TBS cytidine derivative 14,
compound 7 was first 50-O-tritylated to give 13 and then 20-O-
protected with TBS following standard procedures. Compound 14
was obtained with 70% yield after several conversions of 30-O-TBS
into 20-O-TBS in MeOH. Finally, subsequent phosphitylation of 12
and 14 was conducted with 2-cyanoethyl N,N-diisopropylchloro-
phosphoramidite under standard conditions to give the N4-tboc
cytidine amidites 1b and 2b, respectively.
tboc following the same procedure as for C. Indeed, the solubility
of tri-O-Ac adenosine in the mixture pyridine/DCM was too low
to give the N6-tboc derivative with good and reproducible yield.
Therefore, 50-O-DMTr-20,30-di-O-Ac adenosine 15 was pre-
ferred as starting material (Scheme 3). It was activated as a N6-
carbamoyl derivative by treatment with carbonyl chloride and
DMAP to react next with 2-[(triisopropylsilyl)oxy]-benzyl alco-
hol 3 in the presence of Et3N resulting in the N6-tboc protected
adenosine 16 with 87% yield. Then, ribose 30-OH and 20-OH
were deacetylated in the same conditions as for C. The PivOM
group was introduced via a 20,30-O-dibutylstannylidene inter-
mediate using the PivOM iodide more efficient than PivOM
chloride with adenosines.18 The 20-O-PivOM derivative 18 was
obtained with 28% yield. The 20-O-TBS N6-tboc adenosine 19
was isolated with 71% yield. Both 20-O-protected N6-tboc
adenosine 18 and 19 were phosphitylated to afford the amidites
1c and 2c with 77% and 73% yield, respectively.
In contrast, several attempts to protect the guanine with tboc
group following the same procedures were unsuccessful, which
led us to consider the use of DMTr as protective group of the 50-
OH and the exocyclic amino function. The two acid-labile groups
will be simultaneously removed during the elongation cycle in
the detritylation step. Indeed, it has been reported that unpro-
tected guanines do not react under coupling conditions because
Concerning the preparation of the 20-O-PivOM and 20-O-TBS
adenosine amidites 1c and 2c, the lack of nucleophilicity of the
exocyclic amino function in adenine prevents its protection by
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Scheme 3. Synthesis of 20-O-PivOM and 20-O-TBS Adenosine Amidites 1c and 2c
Scheme 4. Synthesis of 20-O-PivOM and 20-O-TBS Guanosine Amidites 1d and 2d
of their low nucleophilicity.19ꢀ21 Bis-protection of guanosine
with DMTr was achieved with acceptable yield (66%) in reacting
2.2 equiv of DMTr chloride (Scheme 4).
50-O-DMTr-30-O-amidite-20-O-TBS N2-phenoxyacetyl-guanosine
with MeNH2 gave the unprotected-base derivative 2e (84%).23
Evaluation of the Coupling Efficiency of N2-DMTr Guano-
sine Amidites 1d and 2d, and N2-Unprotected Guanosine
Amidite 2e during Oligonucleotides Synthesis. At first, we
applied the conditions used for standard automated synthesis of
oligonucleotides (ON) to the elongation of a mixed deoxy-
ribonucleotide sequence TGTGTTT using N2-DMTr G ami-
dites 1d and 2d. It was observed that the usual acidic treatment
with 3% di- or trichloroacetic acid (DCA or TCA) in DCM for
60s (6 ꢁ 10s) did not completely remove the trityl group from
the amino function of guanosine at each detritylation step.
Consequently, the incorporation of the deoxythymidine syn-
thon into the growing chain with partially N2-DMTr G at
50-end was achieved with only 95% coupling yield estimated
by DMTr cation assay, possibly because of the steric hin-
drance of the DMTr. Thus the detritylation step was in-
creased to 180s (18 ꢁ 10s) using 3% TCA in DCM to get rid
of the DMTr N-protection. We performed the synthesis of
three small ON with the sequence TGTGTTT incorpora-
ting either N2-DMTr-20-O-PivOM, or N2-DMTr-20-O-TBS,
or N2-unprotected-20-O-TBS guanosine amidites 1d, 2d, 2e,
respectively. The coupling yields of the amidites 1d and 2e
were 97.5% whereas the amidite 2d was incorporated with
96.5% yield within the same 300s coupling time. Although
these yields have been lower than those obtained in standard
RNA synthesis, we considered that the amidites 1d and 2e,
preferred to 2d, could be used to give modified pro-RNA.
The crude ON were analyzed after deprotection with DBU
then aqueous ammonia and their HPLC purity was similar
and estimated at 90%.
PivOM group was not introduced in 20-position of N2,50-O-
bis-DMTr-guanosine 20 in the same conditions as previously
described for N2-tert-butylphenoxyacetyl-50-O-DMTr-guanosine.12
Actually, these conditions were not efficient since the reaction
did not go to completion (50% starting material left) and the
ratio between 20- and 30-O-PivOM isomers was 1/1 instead of
9/1 obtained for the preparation of N2-tert-butylphenoxyacetyl-
50-O-DMTr-20-O-PivOM-guanosine. In order to improve the
PivOM introduction, the equivalents of each reagent (dibutyltin
oxide, TBAB and PivOM-Cl) were increased 2-fold and the
compound N2,50-O-bis-DMTr-20-O-PivOM guanosine 21 could
be obtained with 25% isolated yield. The same difficulty was
observed for the 20-OH protection by TBS of bis-DMTr G. The
standard conditions used for C and A were not convenient for
TBS introduction in guanosine since the reaction progress did
not reach more than 50% after several hours. The addition of
silver nitrate to the reaction mixture allowed the total conversion
of the starting material in the mixture of 20-O- or 30-O-TBS
derivatives without inducing the predominance of the 20-
isomer.22 It is noteworthy that N2-DMTr also slowed down
the TBS isomerization from 30- to 20-position since several days
in a mixture pyridine/MeOH with imidazole were required to
obtain the two isomers in a 1/1 ratio. However, N2,50-O-bis-
DMTr-20-O-TBS guanosine 22 was isolated with 69% yield.
Subsequent phosphitylation of 21 and 22 afforded the guanosine
amidites 1d and 2d in good yields (88% and 71% respectively).
An alternative to this amidite derivative 2d was investigated with
the preparation of the 50-O-DMTr-20-O-TBS guanosine amidite
without nucleobase protection. The treatment of the commercial
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Table 1. Fully or Partially 20-O-PivOM pro-RNA Synthesized by Use of 20-O-PivOM and 20-O-TBS Ribonucleoside Amidites 1aꢀd
and 2aꢀea
Maldi-TOF mass
no.
oligonucleotides sequence 50-30
RNA amidites
1a, 1b, 2a
calcd
found
23
24
25
26
27
28
29
UUCUUCUUTT
3449.41
8696.75
8908.99
7310.95
7223.81
7275.94
7275.94
3450.95
8696.32
8907.43
7311.28
7224.04
7277.85
7273.48
UUUGUAUUCAGCCCAUAUCTT
GAUAUGGGCUGAAUACAAATT
GAUAUGGGCUGAAUACAAATT
GGGUUCUGCUGCCCGUAGCTT
GCUACGGGCAGCAGAACCCTT
GCUACGGGCAGCAGAACCCTT
1aꢀd
1aꢀd
1a, 1cꢀd, 2aꢀc, 2e
1aꢀd, 2aꢀb, 2e
1bꢀc, 2aꢀc, 2e
1aꢀd, 2bꢀc, 2e
a Italic and bold characters: 20-O-PivOM ribonucleosides.
Figure 3. HPLC and MALDI-TOF mass spectrum of the crude pro-RNA 23.
Evaluation of the Deprotection Efficiency of pro-RNA with
the Treatment aq HF-Et3N (1:3) at 65 °C. To check the removal
of tboc groups from A and C in pro-RNA with the final treatment
aq HF-Et3N (1:3),14 we prepared a decamer pro-RNA model
UUCPivOMUPivOMUPivOMCPivOMUUTT 23 with the 20-O-Pi-
vOM and 20-O-TBS uridine building blocks 1a and 2a, and with
the 20-O-PivOM cytidine amidite 1b (Table 1). After assembly,
the CNE were removed with DBU and the fluoride treatment
was applied for 10 h at 65 °C to release deprotected 23 with the
PivOM maintained in the pro-RNA. The crude 23 was passed
through a short reversed-phase silica column to get rid of the salts
and the desired pro-RNA 23 was obtained with 90% purity based
on HPLC analysis (Figure 3). Fifty O.D. (λ = 260 nm) were
recovered corresponding to 55% yield which is comparable with
the amount of ON obtained by RNA standard synthesis using
succinyl linker to the solid support.
50-end of the ON. Interestingly, the yield was higher (97.5%)
when G amidite 1d reacted with 50-OH of cytosine or uridine
than when the coupling occurred between 1d and adenosine
(95.5%) or guanosine (93%). Finally, pro-RNA 24 and 25
were assembled with 65% and 49% overall coupling yield,
respectively. After deprotection by a treatment with DBU for
3 min then with the mixture aq HF-Et3N (1:3) at 65 °C for 10 h,
followed by a desalting through a reverse-phase C8-silica column,
the crude 24 and 25 were purified by C18-RP-HPLC but only few
O.D. were recovered (8 O.D for 25) (Figure 4). The low yield
was due to the high absorption of these 20-O-PivOM 21-mers on
silica and essentially to their precipitation in water because of the
high lipophilicity of completely modified pro-RNA. These data
prompted us to synthesize partially modified 20-O-PivOM pro-
RNA leaving some free 20-OH to circumvent the solubility
problem.
Synthesis of Fully 20-O-PivOM pro-RNA (21-mers). Once
the amidites 1aꢀd and the deprotection method in hands to
achieve the pro-RNA, we synthesized two modified 20-O-PivOM
21-mers with complementary sequences 24 and 25 to evaluate
the RNA duplex forming ability in the silencing of firefly luciferase
gene (Table 1). The stepwise coupling yields during the pro-
RNA assembly varied from 98% (for amidites 1b and 1c) to 99%
(1a). In contrast, the guanosine amidite 1d was incorporated in
RNA chain with lower yields depending on the nucleotide at the
Synthesis of Partially Modified pro-RNA (21-mers) with 20-
O-PivOM. The synthesis of partially 20-O-PivOM modified
oligoribonucleotides was performed with the phosphoramidite
20-O-PivOM 1aꢀd and 20-O-TBS 2aꢀc, e building blocks. It is
noteworthy that the amidites 2b, 2c, and 2e were not soluble in
neat CH3CN and DCM (from 20% to 50%) should be added to
obtain a clear homogeneous solution. The reactivity of such
amidites 2bꢀc bearing both tboc and TBS protections decreased
and the coupling yields were lower (95%). Therefore, to improve
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Figure 4. RP-HPLC of crude (left) and purified (right) fully 20-O-PivOM RNA 25.
Figure 5. RP-HPLC profile of crude partially 20-O-PivOM RNA 26 (top) and RP-HPLC (left) and anion-exchange HPLC (right) profiles of purified
partially 20-O-PivOM RNA 26.
the yield of the elongation, the concentration of the amidites
solutions was increased from 0.1 to 0.15 M, and the coupling time
was up to 500 s in the case of A and G amidites 2c and 1d. With
these changes, several pro-RNA 21-mers 26ꢀ29 were success-
fully synthesized with five PivOM groups located at their 50-or 30-
end. Despite the presence of numerous shorter sequences which
made difficult the RP-HPLC purification of the crude mixture,
the partially 20-O-PivOM pro-RNA 26ꢀ29 were obtained in high
purity (Figure 5 and Supporting Information).
with 13 20-OH ribonucleosides and two thymidines (Figure 6).
Furthermore, a peak of higher retention time in the RP-HPLC
chromatogram was assigned to the hexamer GPivOMAPivOM-
UPivOMAPivOMUPivOMG formed with five 20-O-PivOM ribonu-
cleosides and a guanosine residue. The observation of this 50-fragment
of the pro-RNA strengthened the data already obtained on the
RNA protection by the PivOM modifications against nucleases
degradation.8
Hybridization properties of 20-O-PivOM pro-RNA 25 and
26. Hybridization properties of fully 20-O-PivOM pro-RNA 25
with its complementary strand which is similarly 20-O-PivOM
modified 24, and of pro-RNA 26 containing five PivOM groups
with the complementary unmodified RNA strand were studied in
comparison with the stability of the natural RNA duplex
(Table 2). As a result it turned out that the thermal stability of
the fully 20-O-PivOM modified RNA duplex (Tm 86 °C) was
considerably higher by 19 °C than that of the natural duplex
Digestion of the Partially 20-O-PivOM Modified RNA 26 by
Nuclease P1 and Alkaline Phosphatase. To check the absence
of 20-50 internucleotide linkages and modified nucleobases,
the purified 21-mer 26 was digested by nuclease P1 and alkaline
phosphatase and gave the four natural ribonucleosides and
thymidine. These data indicated the absence of any nucleobase
modification or unnatural internucleoside linkages, such as N-
branched oligonucleotide, in the 30-part of the RNA constituted
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Figure 6. RP-HPLC profile and MALDI-TOF mass spectrum of the digest of 21-mer 26 by nuclease P1 and phosphatase alkaline.
Table 2. Hybridization Properties of 20-O-PivOM pro-RNA
a
no.
sequence 50-30
complementary RNA strand
Tm
ΔTm
ΔTm/mod.
GAUAUGGGCUGAAUACAAATT
GAUAUGGGCUGAAUACAAATT
GAUAUGGGCUGAAUACAAATT
UUUGUAUUCAGCCCAUAUCTT
UUUGUAUUCAGCCCAUAUCTT
UUUGUAUUCAGCCCAUAUCTT
67
86
68
25
26
19.0
1.0
1.0
0.2
a Tm (°C). Conditions: 10 mM sodium cacodylate buffer (pH 7.0), 100 mM NaCl, and 3 μM duplex. Italic and bold characters: 20-O-PivOM
ribonucleosides
Figure 7. (a) Delivery of siRNA to ECV-GL3 cells by electroporation at 10 nM siRNA. (b) ECV-GL3 cells were incubated with 1 μM naked siRNA for
spontaneous uptake (left panel) or transfected with 1 nM siRNAs (right panel). (c) ECV-GL3 cells were incubated with 1 μM naked siRNA at various
cell densities. The expression levels of FLuc (firefly luciferase) were normalized to FDA, an indicator of viable cells. Control siRNA (siscr3) has no cellular
target and was set to 100 in all panels.
(Tm 67 °C). In contrast, stability of the duplex formed between
partially 20-O-modified RNA 26 with five consecutive PivOM at
50-end and the unmodified RNA target (Tm 68 °C), was similar
to the natural one. Although the average stabilizing effect of one
PivOM modification was weak, these data suggested that pro-
RNA would form duplex stable enough to be investigated as
siRNA prodrugs.
cells which bypasses the cellular uptake step. This experiment
indicated an approximately 6% decrease of the potency of siR206-
PivOM versus its unmodified control siR206 (Figure 7a). Similarly,
delivery of siR206-PivOM at 1 nM by Lipofectamine 2000
showed a 1.5 fold decreased target suppression compared to
siR206-PivOM (Figure 7b, right panel). Together both control
experiments indicate a decreased intracellular potency of the 20-
O-PivOM-modified version of siR206. Notably, the addition of
1 μM of siRNA to cell culture medium of ECV304-GL3 cells
showed stronger down-regulation of luciferase expression in the
use of siR206-PivOM versus siR206 approaching 60% of lucifer-
ase expression of untreated cells whereby siR206-PivOM was
more potent than siR206 by 7.5% to 20% (Figure 7b, left panel).
It is important to note that this cell culture-based test system
strongest target suppression at low cell densities of 5.000 to
10.000 cells per dish of 96 well plates. At higher cell density, the
spontaneous cellular uptake of siRNA is substantially reduced
(Figure 7c).
Biological Evaluation. First, we assured that the 20-O-PivOM
modification does not interfere with duplex formation with
complementary RNA to yield siRNA and that this modification
allows extraction of 20-O-PivOM-derived siRNA (26 and its
complementary strand), termed siR206-PivOM, from cell cul-
ture supernatants as well as from cells by phenol/chloroform
extraction (data not shown). In order to investigate the biological
effectiveness of partially 20-O-PivOM-modified siRNA, we used
an established luciferase-based read-out system.24 Briefly, un-
modified or 20-O-PivOM-modified siR206 was delivered to hu-
man ECV304 cells constitutively expressing a chromosomally
integrated firefly luciferase gene (ECV304-GL3). Delivery was
facilitated as described below or cells were incubated with
unmodified naked luciferase-directed siRNAs. First, siRNAs
were delivered by electroporation at 10 nM in ECV304-GL3
In summary, these studies indicate that 20-O-PivOM-derived
siRNA is biologically active and it exerts improved cellular uptake.
Considering the reduced quasi intrinsic potency, increased cellular
delivery is approximately 2-fold over conventional siRNA.
5725
dx.doi.org/10.1021/jo200826h |J. Org. Chem. 2011, 76, 5719–5731

The Journal of Organic Chemistry
ARTICLE
’ CONCLUSIONS
(m, 5H, H ar, H-5), 6.15 (d, J = 4.2 Hz, 1H, H-10), 5.42 (m, 1H, H-20),
5.33 (m, 1H, H-30), 5.29 (s, 2H, OCH2Ar, tboc); 4.41 (m, 3H, H-50,
H-500, H-40), 2.2ꢀ2 (3s, 9H, 3 C(O)CH3), 1.32 (m, 3H, 3 CH(CH3)2),
1.09 (d, J = 7.4 Hz, 18H, CH(CH3)2). 13C NMR (100 MHz, CDCl3) δ
170.1, 169.6, 169.5 (3 OCdO), 162.6 (C4), 154.8 (Cq ar-O), 154.7
(C2), 152.2 (NHCO), 143.5 (C6), 130.6ꢀ118.2 (4 CH, Car), 124.8
This work describes a feasible method to synthesize 20-O-
PivOM-modified siRNA. We have prepared 20-O-PivOM and 20-
O-TBS ribonucleoside amidites bearing a silyl group as the
protecting group of adenine and cytosine and without protection
for G. We were able to synthesize fully 20-O-PivOM RNA
oligomers which were difficult to handle and to isolate because
they were too lipophile and insoluble in water. We succeeded in
applying conditions suitable for selective deprotection of the
phosphates, nucleobases and some 20-OH, and for Q-linker
cleavage while the PivOM groups remained completely intact
to obtain very pure partially 20-O-PivOM modified RNA. One of
them with five PivOM groups at the 50-end was evaluated in a
siRNA assay and it turned out that the delivery of such modified
siRNA was more effective. These preliminary and promising data
provide a proof-of-concept for a prodrug-based approach for the
delivery of siRNA to living human cells. Further studies on the
influence of the number or the position of the PivOM in the RNA
sequence are ongoing.
0
0
0
0
(Cq Car-CH2), 95.4 (C5), 88.8 (C1 ), 79.8 (C4 ), 73.8 (C2 ), 69.7 (C3 ),
0
64.5 (OCH2Ar), 62.6 (C5 ), 20.8ꢀ20.5 (3 OCOCH3), 18.0 (3 CH(CH3)2),
13.0 (3 CH(CH3)2). HRMS (ESIþ) m/z calcd for C32H46N3O11Si (M þ
H)þ 676.2902, found 676.2920.
N4-{{{2-[(Triisopropylsilyl)oxy]benzyl}oxy}carbonyl}-cy-
tidine 7. To a stirred solution of 6 (24 g, 35.6 mmol, 1 equiv) in EtOH
(210 mL) was added a solution of sodium hydroxide 0.1 M in EtOH
(356 mL, 35.6 mmol, 1 equiv). The resulting mixture was stirred at
room temperature for 2 h and neutralized by addition of Dowex 50WX8
under pyridinium form. The resin was removed by filtration and the
solvent was evaporated. The residue was subjected to silica gel column
chromatography with a step gradient with MeOH (0ꢀ6%) in CH2Cl2.
The desired compound 7 was obtained as white foam (17.6 g, 32 mmol,
1
95%). Rf = 0.21 (CH2Cl2/MeOH 90:10 v/v). H NMR (400 MHz,
DMSO-d6) δ 10.81 (br, 1H, NH), 8.42 (d, J = 7.6 Hz, 1H, H-6),
7.37ꢀ6.72 (m, 5H, H ar, H-5), 5.78 (d, J = 2.5 Hz, 1H, H-10), 5.50 (d,
J = 2.8 Hz, 1H, OH-20), 5.18 (m, 4H, OH-50, OH-30, OCH2Ar),
3.99ꢀ3.90 (m, 5H, H-20, H-30, H-40, H-50, H-500), 1.25 (m, 3H, 3
CH(CH3)2), 1,09 (d, J = 7.4 Hz, 18H, CH(CH3)2). 13C NMR
(100 MHz, DMSO-d6) δ 162.7 (C4), 154.4 (Cq ar-O), 153.5 (C2),
153.2 (NHCO), 144.9 (C6), 129.8ꢀ117.8 (4 CH, Car), 125.4 (Cq ar-
’ EXPERIMENTAL SECTION
General Remarks. NMR spectra were obtained on 300 and 400 MHz
1
spectrometers. H and 13C assignments were done using both COSY
1
13
and Hꢀ C HSQC experiments. Flash column chromatography was
performed using silica gel (0.040ꢀ0.063 mm). TLC were performed on
silica gel 60 F254 and compounds were visualized by illumination under
UV light (254 nm) or by spraying with 10% ethanolic sulfuric acid.
General Procedure for Phosphoramidite Synthesis. A nu-
cleoside derivative was dried by 3 coevaporations with anhydrous
CH3CN. Then the residue was dissolved in anhydrous CH2Cl2 and a
mixture of N,N-diisopropylethylamine (1.5 equiv), 2-cyanoethyl N,N-
diisopropylchlorophosphoramidite (1.5 equiv) and CH2Cl2 was added
dropwise. The mixture was stirred under argon, at room temperature for
3 h. After completion, ethyl acetate containing 1% pyridine was added,
the reaction mixture was poured into saturated NaHCO3 solution and
ethyl acetate extractions were carried out. The mixture obtained after
drying of the extract over Na2SO4 and removal of the solvent was
purified by silica gel column chromatography with an isocratic elution of
CH2Cl2/ethyl acetate, 1:1 (v,v) containing 1% pyridine.
0
0
0
0
CH2), 94.1 (C5), 90.1 (C1 ), 84.2 (C4 ), 74.4 (C2 ), 68.7 (C3 ), 62.6
0
(OCH2Ar), 59.9 (C5 ), 17.6 (3 CH(CH3)2), 12.0 (3 CH(CH3)2).
HRMS (ESIþ) m/z calcd for C26H40N3O8Si (M þ H)þ 550.2585,
found 550.2587.
N4-{{{2-[(Triisopropylsilyl)oxy]benzyl}oxy}carbonyl}-
30,50-O-di-tert-butylsilanediyl-cytidine 8. To a solution of 7
(4.00 g, 7.28 mmol, 1eq) and silver nitrate (2.72 g, 16.00 mmol,
2.2eq) in dry DMF (72 mL) at 0 °C, was added dropwise di-tert-
butylchlorosilane (2.6 mL, 8,00 mmol, 1.1 equiv). The mixture was
stirred 10 min at 0 °C, then warmed to room temperature and stirred for
30 min. Et3N (2.25 mL, 16.00 mmol, 2.2eq) was added and the mixture
was stirred for an additional 10 min. The reaction mixture was quenched
with water and extracted with ethyl acetate. The organic layer was
washed with water, then brine, dried (Na2SO4), filtered and concen-
trated. The residue was subjected to silica gel column chromatography
with a step gradient with MeOH (0ꢀ1%) in CH2Cl2. The desired
compound 8 was obtained as white foam (4.48 g, 6.50 mmol, 89%).
Rf = 0.58 (CH2Cl2/MeOH 90:10 v/v).). 1H NMR (400 MHz, DMSO-
d6) δ 10.83 (s, 1H, NH), 8.00 (d, J = 7.5 Hz, 1H, H-6), 7.39ꢀ6.85 (m,
4H, H ar), 7.06 (d, J = 7.5 Hz, 1H, H-5), 5.75 (d, J = 4.5 Hz, 1H, OH-20),
5.68 (s, 1H, H-10), 5.18 (s, 2H, OCH2Ar, tboc), 4.40 (m, 1H, H-50), 4.16
(m, 2H, H-20, H-500), 4.03 (m, 1H, H-30), 3.94 (m, 1H, H-40), 1.30 (m,
3H, 3 CH(CH3)2), 1.08ꢀ0.96 (m, 36H, CH(CH3)2, tbu). 13C NMR
(100 MHz, DMSO-d6) δ 162.9, 162.2, 153.9, 153.5, 153.1, 145.1, 129.8,
129.6, 125.4, 120.8, 117.8, 94.6, 93.0, 75.4, 73.9, 72.7, 66.6, 62.6, 27.2,
26.9, 17.7, 12.3, 11.9. HRMS (ESIþ) m/z calcd for C34H55N3O8Si2 (M
þ H)þ 690.3528, found 690.3599.
The desired phosphoramidite was obtained as white foam after evapora-
tion of the solvent.
Compounds 1a,8,12 3,15 516 and 2025 were synthesized according to
the literature.
N4-{{{2-[(Triisopropylsilyl)oxy]benzyl}oxy}carbonyl}-
20,30,50-tri-O-acetyl-cytidine 6. To a solution of 3 (10.30 g,
36.90 mmol) in dry THF (250 mL), under argon at 0 °C, was added
N,N-diisopropylethylamine (6.4 mL, 36.90 mmol) and triphosgene
(6.8 g, 22.91 mmol) in small portions over 10 min. The resulting
mixture was stirred for 10 min at 0 °C then for 30 min at room
temperature. The mixture was filtered and concentrated. The 2-
[(triisopropylsilyl)oxy]-benzyl carbonochloridate 4 was directly used
in the next step. To a stirred solution of 5 (15 g, 40.7 mmol, 1 equiv) in
dry CH2Cl2 (750 mL), under nitrogen at 20 °C, was added N-
methylimidazole (4.68 mL, 56.9 mmol, 1.4 equiv) and 4 (20 g, 56.9 mmol,
1.4 equiv). The resulting mixture was stirred at room temperature for 2 h
and then diluted with CH2Cl2. The organic layer was quenched with
saturated aqueous NaHCO3, washed with brine, dried (Na2SO4),
filtered and evaporated. The residue was subjected to silica gel column
chromatography with a step gradient with MeOH (0ꢀ4%) in CH2Cl2.
The desired compound 6 was obtained as white foam (17.6 g, 26 mmol,
N4-{{{2-[(Triisopropylsilyl)oxy]benzyl}oxy}carbonyl}-
30,50-O-di-tert-butylsilanediyl-20-O-methylthiomethyl-cyti-
dine 9. To a stirred solution of 8 (4.30 g, 6.24 mmol) in dry DMSO
(9.4 mL), acetic anhydride (9.4 mL) and acetic acid (12.5 mL) were
added. The resulting mixture was stirred at 35 °C until completion
(24 h). Then the mixture was added slowly to a solution of potassium
carbonate (17 g) in water (85 mL) and extracted with ethyl acetate.
The organic layer was washed with water (3 times) then brine, dried
(Na2SO4), filtered and concentrated. The residue was subjected to
silica gel column chromatography with a step gradient with ethyl
1
64%). Rf = 0.85 (CH2Cl2/MeOH 90:10 v/v). H NMR (400 MHz,
CDCl3) δ 7.90 (d, J = 7.6 Hz, 1H, H-6), 7.54 (s, 1H, NH), 7.37ꢀ6.72
5726
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ARTICLE
acetate (10ꢀ20%) in cyclohexane. The desired compound 9 was
obtained as white foam (2.87 g, 3.83 mmol, 61%). Rf = 0.23
(cyclohexane/EtOAc 80:20 v/v). 1H NMR (300 MHz, CDCl3) δ
7.65 (d, J = 7.5 Hz, 1H, H-6), 7.27ꢀ6.76 (m, 5H, H ar, H-5), 5.76 (s,
1H, H-10), 5.21 (s, 2H, OCH2Ar, tboc), 4.95 (J = 11.4 Hz, 2H,
OCH2S), 4.48 (dd, J = 9.3 Hz, J = 4.8 Hz, 1H, H-50), 4.42 (d, J = 4.5 Hz,
1H, H-20), 4.16 (m, 1H, H-40), 3.95 (t, J = 9.3 Hz, 1H, H-500), 3.80 (dd,
J = 9.3 Hz, J = 4.5 Hz, 1H, H-30), 1.28 (m, 3H, 3 CH(CH3)2),
1.18ꢀ1.03 (m, 36H, CH(CH3)2, tbu). 13C NMR (75 MHz, CDCl3) δ
161.3, 153.6, 151.3, 142.1, 129.6, 129.2, 123.9, 119.9, 117.3, 93.8, 90.2,
76.3, 75.3, 74.2, 73.6, 66.6, 63.6, 26.4, 26.0, 25.5, 21.9, 19.4, 17.1, 12.3,
12.0. HRMS (ESIþ) m/z calcd for C36H59N3O8SSi2 (M þ H)þ
750.3561, found 750.3612.
(d, J = 7.5 Hz, 1H, H-6), 7.35 (br, 1H, NH), 7.34ꢀ6.76 (m, 18H, H ar,
H-5), 5.86 (s, 1H, H-10), 5.63 and 5.20 (J = 5.3 Hz, 2H, OCH2O), 5.18
(s, 2H, OCH2Ar, tboc), 4.32 (m, 1H, H-30), 4.21 (d, J = 5.3 Hz, 1H,
H-20), 3.96 (m, 1H, H-40), 3.62 (s, 6H, 2 OCH3), 3.35 (m, H-50, H-500),
2.30 (d, J = 10.9 Hz, 1H, OH-30), 1.28 (m, 3H, 3 CH(CH3)2), 1.09 (m,
27H, CH(CH3)2, C(O)(CH3)3). 13C NMR (100 MHz, CDCl3) δ 178.2
(CdO), 162.2 (C4), 158.7 (Cq, Car), 154.9 (Cq ar-O), 154.5 (C2),
152.1 (NHCO), 144.3 (Cq, Car), 144.1 (C6), 135.6ꢀ113.3 (17 CH,
0
Car), 125.0 (Cq ar-CH2), 94.9 (C5), 89.7 (C1 ), 87.6 (OCH2O), 87.0
0
0
0
(OCq, DMTr), 83.1 (C4 ), 81.6 (C2 ), 67.5 (C3 ), 64.2 (OCH2Ar), 60.7
0
(C5 ), 55.2 (OCH3, DMTr), 38.9 (Cq, OCOC(CH3)3), 26.9
(OCOC(CH3)3), 18.0 (3 CH(CH3)2), 13.0 (3 CH(CH3)2). HRMS
(ESIþ) m/z calcd for C47H58N3O10Si (M þ H)þ 966.4572, found
966.4561.
N4-{{{2-[(Triisopropylsilyl)oxy]benzyl}oxy}carbonyl}-
30,50-O-di-tert-butylsilanediyl-20-O-pivaloyloxymethyl-cyti-
dine 10. To a solution of 9 (500 mg, 0.67 mmol, 1eq) in dry CH2Cl2
(6.7 mL) was added dropwise under argon a 1.0 M sulfuryl chloride
solution in CH2Cl2 (0.8 mL, 0.80 mmol, 1.2eq). The reaction mixture
was stirred at room temperature for 2 h. After completion, the solvent
were removed and the obtained chloromethyl ether derivative was
directly used in the next step. A solution of chloromethyl ether derivative
in dry CH2Cl2 (6.7 mL) was added to a suspension of sodium pivalate
(158 mg, 1.27 mmol, 1.9 equiv) and 15-crown-5 (0.1 mL, 0.50 mmol,
0.75eq) in dry CH2Cl2 (6.7 mL). After stirring at room temperature for
2 h, the mixture was diluted in ethyl acetate and filtered to eliminate the
salt. The organic layer was washed with water, then brine, dried
(Na2SO4), filtered and concentrated. The residue was subjected to silica
gel column chromatography with a step gradient with ethyl acetate
(10ꢀ30%) in cyclohexane. The desired compound 10 was obtained as
white foam (467 mg, 0.58 mmol, 87%). Rf = 0.23 (cyclohexane/EtOAc
N4-{{{2-[(Triisopropylsilyl)oxy]benzyl}oxy}carbonyl}-50-
O-(4,40-dimethoxytrityl)-cytidine 13. Compound 7 (250 mg, 0.46
mmol, 1eq.) was dried by 3 coevaporations with dry pyridine. Then to a
stirred solution of 7 in dry pyridine (4 mL), under nitrogen at 20 °C, was
added dimethoxytrityl chloride (190 mg, 0.55 mmol, 1.2 equiv) in small
portions over 20 min. The resulting mixture was stirred at room
temperature for 2 h. After completion, the mixture was concentrated
and CH2Cl2 was added. The organic layer was washed with brine, dried
(Na2SO4), filtered and evaporated. The residue was subjected to silica
gel column chromatography with a step gradient with MeOH (0ꢀ3%)
in CH2Cl2 containing 1% pyridine. The desired compound 13 was
obtained as white foam (325 mg, 0.38 mmol, 83%). Rf = 0.5 (CH2Cl2/
MeOH 94:6 v/v). 1H NMR (400 MHz, CDCl3) δ 8.10 (d, J = 7.5 Hz,
1H, H-6), 7.67 (br, 1H, NH), 7.26ꢀ6.74 (m, 18H, H ar, H-5), 5.77 (d,
J = 3.8 Hz, 1H, H-10), 5.20 (s, 2H, OCH2Ar, tboc), 4.32 (m, 4H, OH-20,
H-20, H-30, H-40), 3.70 (s, 6H, 2 OCH3), 3.35 (m, 3H, OH-30, H-50,
H-500), 1.28 (m, 3H, 3 CH(CH3)2), 1.09 (d, J = 7.4 Hz, 18H, CH-
(CH3)2). 13C NMR (100 MHz, CDCl3) δ 162.6 (C4), 158.6 (Cq, Car),
156.6 (Cq ar-O), 154.4 (C2), 152.2 (NHCO), 144.3 (Cq, Car), 144.1
(C6), 130.3- 113.3 (17 CH, Car), 125.0 (Cq ar-CH2), 95.1 (C5), 93.4
1
80:20 v/v). H NMR (300 MHz, CDCl3) δ 7.63 (d, J = 7.5 Hz, 1H,
H-6), 7.29 (m, 1H, Har), 7.23 (d, J = 7.5 Hz, 1H, H-5), 7.14 (m, 1H,
Har), 6.86 (m, 1H, Har), 6.77 (m, 1H, Har), 5.69 (s, 1H, H-10), 4.95 (J =
6.3 Hz, 2H, OCH2O), 5.22 (s, 2H, OCH2Ar, tboc), 4.46 (dd, J = 9.3 Hz,
J = 5.1 Hz, 1H, H-50), 4.37 (d, J = 4.5 Hz, 1H, H-20), 4.14 (m, 1H, H-40),
3.99 (t, J = 9.3 Hz, 1H, H-500), 3.80 (dd, J = 9.3 Hz, J = 4.5 Hz, 1H, H-30),
2.15 (s, 3H, CH3S), 1.23 (m, 3H, 3 CH(CH3)2), 1.18ꢀ1.03 (m, 36H,
CH(CH3)2, tbu). 13C NMR (100 MHz, CDCl3) δ 177.9, 162.5, 154.7,
154.3, 152.3, 152.2, 143.0, 130.7, 130.2, 124.9, 118.3, 113.2, 94.9, 92.3,
87, 79.5, 75.7, 74.9, 67.5, 64.5, 27.3, 43.6, 30.3, 27.3, 27.0, 26.5, 22.8, 20.5,
18.1, 13.0. HRMS (ESIþ) m/z calcd for C40H65N3O10Si2 (M þ H)þ
804.4208, found 804.4278.
0
0
0
0
(C1 ), 87.0 (C4 ), 85.8 (OCq, DMTr), 76.7 (C2 ), 69.7 (C3 ), 64.2
0
(OCH2Ar), 62.7 (C5 ), 55.2 (OCH3, DMTr), 18.0 (3 CH(CH3)2), 13.0
(3 CH(CH3)2). HRMS (ESIþ) m/z calcd for C47H58N3O10Si (M þ
H)þ 852.3891, found 852.3893.
N4-{{{2-[(Triisopropylsilyl)oxy]benzyl}oxy}carbonyl}-50-
O-(4,40-dimethoxytrityl)-20-O-(tert-butyldimethylsilyl)-cyti-
dine 14. To a stirred solution of 13 (9.73 g, 11.42 mmol, 1 equiv) in dry
pyridine (70 mL), under nitrogen at 20 °C, was added imidazole (1.74 g,
29.96 mmol, 2.6 equiv) and tert-butyldimethylsilyl chloride (2.28 g,
15.13 mmol, 1.3 equiv). The resulting mixture was stirred at room
temperature for 48 h and then diluted with CH2Cl2. The organic layer
was quenched with saturated aqueous NaHCO3, washed with brine,
dried (Na2SO4), filtered and evaporated. The residue was subjected to
silica gel column chromatography with a step gradient with acetone
(0ꢀ10%) in CH2Cl2 containing 1% pyridine. The compound 14 was
collected and 30-O-TBS derivative was isomerized in MeOH for 24 h.
Purification by chromatography furnished more 14 as white foam, after
three isomerisations, pure 14 is obtained (7.76 g, 8.03 mmol, 70%). Rf =
0.51 (CH2Cl2/MeOH 98:2 v/v). 1H NMR (400 MHz, CDCl3) δ 8.27
(d, J = 7.4 Hz, 1H, H-6), 7.38 (br, 1H, NH), 7.25ꢀ6.64 (m, 18H, H ar,
H-5), 5.70 (s, 1H, H-10), 5.07 (s, 2H, OCH2Ar, tboc), 4.17 (m, 1H, H-30),
4.10 (m, 1H, H-20), 3.92 (m, 1H, H-40), 3.61 (s, 6H, 2 OCH3), 3.47
(m, 2H, H-50, H-500), 2.22 (d, J = 10.9 Hz, 1H, OH-30), 1.14 (m, 3H,
3 CH(CH3)2), 0.95 (m, 27H, C(O)C(CH3)3, CH(CH3)2), 0.74 (s, 9H,
SiC(CH3)3), 0.12, 0.00 (2s, 6H, 2 SiCH3). 13C NMR (100 MHz,
CDCl3) δ 162.3 (C4), 158.7 (Cq, Car), 154.9 (Cq ar-O), 154.5 (C2),
152.2 (NHCO), 144.6 (Cq, Car), 144.1 (C6), 135.6ꢀ113.3 (17 CH,
N4-{{{2-[(Triisopropylsilyl)oxy]benzyl}oxy}carbonyl}-50-
O-(4,40-dimethoxytrityl)-20-O-pivaloyloxymethyl-cytidine
12. To a solution of 10 (467 mg, 0.58 mmol, 1eq) in dry CH2Cl2
(5 mL), under argon at 0 °C, was added dropwise a solution HF
3
pyridine (70%, 60 μL, 2.20 mmol, 3.8 equiv) diluted in dry pyridine
(2.5 mL). The mixture was stirred at 0 °C for 1 h. The reaction was
quenched with saturated aqueous NaHCO3 and extracted with CH2Cl2
(twice). The organic layer was washed with brine, dried (Na2SO4),
filtered and concentrated. The compound 11 was obtained as white
foam and was directly used in the next step. Rf = 0.36 (CH2Cl2/MeOH
95:5 v/v). Compound 11 was dried by 3 coevaporations with dry
pyridine. Then to a stirred solution of 11 in dry pyridine (2 mL), under
nitrogen at 20 °C, was added dimethoxytrityl chloride (180 mg,
0.53 mmol, 1.1 equiv) in small portions over 20 min. The resulting
mixture was stirred at room temperature overnight. After completion,
the reaction was quenched with saturated aqueous NaHCO3 and
extracted with ethyl acetate. The organic layer was washed with brine,
dried (Na2SO4), filtered and evaporated. The residue was subjected to
silica gel column chromatography with a step gradient with ethyl acetate
(20ꢀ40%) in cyclohexane containing 1% pyridine. The desired com-
pound 12 was obtained as white foam (290 mg, 0.30 mmol, 63%). Rf =
0.5 (CH2Cl2/EtOAc 30:70 v/v). 1H NMR (400 MHz, CDCl3) δ 8.41
0
Car), 125.0 (Cq ar-CH2), 94.7 (C5), 90.7 (C1 ), 87.1 (OCq, DMTr),
0
0
0
0
83.1 (C4 ), 76.6 (C2 ), 69.1 (C3 ), 64.1 (OCH2Ar), 61.4 (C5 ),
55.2 (OCH3, DMTr), 25.8 (SiC(CH3)3), 18.0 (3 CH(CH3)2), 13.0
5727
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The Journal of Organic Chemistry
ARTICLE
(3 CH(CH3)2), ꢀ4.3 and ꢀ5.4. (2 SiCH3). HRMS (ESIþ) m/z calcd for
C53H72N3O10Si2 (M þ H)þ 966.4756, found 966.4770.
3 CH(CH3)2), 1.13 (d, J = 7.4 Hz, 18H, CH(CH3)2). 13C NMR
(100 MHz, CDCl3) δ 169.7, 169.3 (OCdO), 158.6 (C, Car), 154.3 (C
ar-O), 153.1 (C2), 151.4 (NHCO), 150.8 (C4), 149.5 (C6), 144.1, 140.8
(Cq, Car), 135.2 (C8), 130.2ꢀ113.3 (17 CH, Car), 125.5 (Cq ar-CH2),
N4-{{{2-[(Triisopropylsilyl)oxy]benzyl}oxy}carbonyl}-50-
O-(4,40-dimethoxytrityl)-20-O-pivaloyloxymethyl-30-O-(2-cy-
anoethyl-N,N-diisopropylphosphoramidite)-cytidine 1b. Using
general procedure starting from 12 (1.7 g, 1.8 mmol) afforded 1b (1.73 g,
1.48 mmol, 82% yield). 31P NMR (121 MHz, CD3CN) δ 150.24, 148.54.
HRMS (ESIþ) m/z calcd for C62H85N5O13PSi (M þ H)þ 1166.5651,
found 1166.5664.
0
0
0
122.1 (C5), 87.1 (OCq, DMTr), 84.8 (C1 ), 82.6 (C4 ), 73.2 (C2 ), 71.7
0
0
(C3 ), 63.8 (OCH2Ar), 63.0 (C5 ), 55.3 (OCH3, DMTr), 20.7, 20.4
(OC(O)CH3), 18.0 (3 CH(CH3)2), 12.7 (3 CH(CH3)2). HRMS
(ESIþ) m/z calcd for C52H62N5O11Si (M þ H)þ 960.4225, found
960.4215.
N6-{{{2-[(Triisopropylsilyl)oxy]benzyl}oxy}carbonyl}-50-
O-(4,40-dimethoxytrityl)-adenosine 17. To a stirred solution of
16 (16 g, 16.7 mmol, 1 equiv) in EtOH (120 mL) was added a solution of
sodium hydroxide 0.1 M in EtOH (200 mL, 20 mmol, 1.2 equiv). The
resulting mixture was stirred at room temperature for 1 h and neutralized
by addition of Dowex 50WX8 under pyridinium form. The resin was
removed by filtration and the solvent was evaporated. The residue was
subjected to silica gel column chromatography with a step gradient with
MeOH (0ꢀ6%) in CH2Cl2. The desired compound 17 was obtained as
white foam (13.46 g, 15.36 mmol, 92%). Rf = 0.45 (CH2Cl2/MeOH
94:6 v/v). 1H NMR (400 MHz, CDCl3) δ 8.58 (s, 1H, H-2), 8.42 (br,
1H, NH), 8.08 (s, 1H, H-8), 7.32ꢀ6.61 (m, 17H, H ar, DMTr-tboc),
5.91 (d, J = 5.8 Hz, 1H, H-10), 5.74 (d, J = 3.1 Hz, 1H, OH-20), 5.29 (s,
2H, OCH2Ar, tboc), 4.77 (m, 1H, H-20), 4.35 (m, 2H, H-40, H-30), 3.65
(s, 6H, 2 OCH3), 3.46 (d, J = 2 Hz, 1H, OH-30), 3.23 (m, 2H, H-50,
H-500), 1.28 (m, 3H, 3 CH(CH3)2), 1.02 (d, J = 7,3 Hz, 18H, CH-
(CH3)2). 13C NMR (100 MHz, CDCl3) δ 158.6 (Cq, Car), 154.3 (Cq
ar-O), 152.4 (C2), 150.8 (NHCO), 150.3 (C4), 149.6 (C6), 144.3, 141.2
(Cq, Car), 135.3 (C8), 130.2ꢀ113.1 (17 CH, Car), 125.5 (Cq ar-CH2),
N4-{{{2-[(Triisopropylsilyl)oxy]benzyl}oxy}carbonyl}-50-
O-(4,40-dimethoxytrityl)-20-O-(tert-butyldimethylsilyl)-30-
O-(2-cyanoethyl-N,N-diisopropylphosphoramidite)-cyti-
dine 2b. Using general procedure starting from 14 (2.58 g, 2.67 mmol)
afforded 2b (2.7 g, 2.31 mmol, 86% yield). 31P NMR (121 MHz, CD3CN)
δ 149.58, 148.88. HRMS (ESIþ) m/z calcd for C62H89N5O11PSi2 (M þ
H)þ 1166.5835, found 1166.5848.
5-O-(4,40-Dimethoxytrityl)-20,30-bis-O-acetyl-adenosine
15. Adenosine (2.7 g, 10 mmol, 1 equiv) was dried by 3 coevaporations
with dry pyridine. Then to a stirred solution of adenosine in dry pyridine
(50 mL), under nitrogen at 20 °C, was added DMAP (60 mg, 0.5 mmol,
0.05 equiv) and dimethoxytrityl chloride (4 g, 12 mmol, 1.2 equiv) in
small portions over 20 min. The mixture was stirred at room tempera-
ture for 3 h. To the resulting solution of 50-O-(4,40-dimethoxytrityl)-
adenosine in pyridine was added DMAP (120 mg, 1 mmol, 0.1 equiv),
Et3N (2.28 mL, 20 mmol, 2 equiv) and acetic anhydride (2.26 mL,
24 mmol, 2.4 equiv). The resulting mixture was stirred at room
temperature for 30 min and then diluted with CH2Cl2. The organic
layer was quenched with saturated aqueous NaHCO3, washed with
brine, dried (Na2SO4), filtered and evaporated. The residue was
subjected to silica gel column chromatography with a step gradient with
MeOH (0ꢀ10%) in CH2Cl2 containing 1% pyridine. The desired
compound 15 was obtained as yellow foam (3.92 g, 6 mmol, 60%). Rf =
0.22 (CH2Cl2/MeOH 94:6 v/v). 1H NMR (400 MHz, CDCl3) δ 8.33
(s, 1H, H-2), 7.99 (s, 1H, H-8), 7.46ꢀ6.81 (m, 13H, H-ar, DMTr), 6.34
(d, J = 6.9 Hz, 1H, H-10), 6.18ꢀ6.08 (m, 3H, H-20,NH2), 5.71 (dd, J =
5.2 Hz; J = 2.8 Hz, 1H, H-30), 4.36 (m, 1H, H-40), 3.79 (s, 6H, 2 OCH3),
3.53 (dd, J = 10.5 Hz, J = 3 Hz, 1H, H-50), 3.46 (dd, J = 10.5 Hz, J = 3.5
Hz, 1H, H-500), 2.16ꢀ2.10 (2s, 6H, C(O)CH3). 13C NMR (100 MHz,
CDCl3) δ 169.6, 169.4 (OCdO), 158.6 (Cq, Car), 155.7 (C6), 153.4
(C2), 150.2 (C4), 144.1, 141.2 (Cq, Car), 138.5 (C8), 130.2, 129, 128.2,
128, 127.1, 113.3 (CH, Car), 119.7 (C5), 87.0 (OCq, DMTr), 86.5
0
0
0
122.2 (C5), 90.7 (C1 ), 86.6 (OCq, DMTr), 86.1 (C4 ), 75.8 (C2 ), 72.7
0
0
(C3 ), 63.9 (OCH2Ar), 63.5 (C5 ), 55.2 (OCH3, DMTr), 18.0 (3
CH(CH3)2), 13.0 (3 CH(CH3)2). HRMS (ESIþ) m/z calcd for
C48H58N5O9Si (M þ H)þ 876.4004, found 876.3996.
N6-{{{2-[(Triisopropylsilyl)oxy]benzyl}oxy}carbonyl}-50-
O-(4,40-dimethoxytrityl)-20-O-pivaloyloxymethyl-adenosine
18. To a solution of 17 (700 mg, 0.8 mmol, 1 equiv) in DCE (4 mL)
were added TBAB (335 mg, 1.04 mmol, 1.3 equiv), dibutyltin oxide
(260 mg, 1.04 mmol, 1.3 equiv) and iodomethyl pivalate (484 μL,
2 mmol, 2.5 equiv). The reaction mixture was stirred under nitrogen at
75 °C for 2 h, cooled and the solvent was evaporated. The reaction
mixture was subjected to silica gel column chromatography with a step
gradient with acetone (0ꢀ30%) in CH2Cl2. The first-eluted isomer was
the desired compound 18 and was obtained as white foam (221 mg,
0.22 mmol, 28%). Rf = 0.6 (CH2Cl2/MeOH 97:3 v/v). 1H NMR (400
MHz, CDCl3) δ 8.59 (s, 1H, H-2), 8.42 (br, 1H, NH), 8.06 (s, 1H, H-8),
7.36ꢀ6.70 (m, 17H, H ar, DMTr-tboc), 6.08 (d, J = 4.6 Hz, 1H, H-10),
5.43 and 5.30 (2d, J = 5.4 Hz, 1H þ 1H, OCH2O), 5.29 (s, 2H, OCH2Ar,
tboc), 4.98 (t, J = 4.6 Hz, 1H, H-20), 4.45 (m, 1H, H-30), 4.18 (m, 1H, H-
40), 3.65 (s, 6H, 2 OCH3), 3.23 (m, 2H, H-50, H-500), 2.54 (d, J = 5.3 Hz,
1H, OH-30), 1.28 (m, 3H, 3 CH(CH3)2), 1.05 (m, 27H, C(O)C(CH3)3,
CH(CH3)2). 13C NMR (100 MHz, CDCl3) δ 177.9 (OCdO), 158.6
(Cq, Car), 154.3 (Cq ar-O), 152.9 (C2), 150.8 (NHCO), 150.8 (C4),
149.5 (C6), 144.4, 141.5 (Cq, Car), 141.3 (C8), 130.2- 113.2 (17 CH,
0
0
0
0
0
(C4 ), 84.5 (C1 ), 73.2 (C2 ), 70.6 (C3 ), 63.1 (C5 ), 55.3 (OCH3,
DMTr), 20.7, 20.5 (OC(O)CH3). HRMS (ESIþ) m/z calcd for
C35H36N5O8 (M þ H)þ 654.2564, found 654.2580.
N6-{{{2-[(Triisopropylsilyl)oxy]benzyl}oxy}carbonyl}-50-
O-(4,40-dimethoxytrityl)-20,30-bis-O-acetyl-adenosine 16. A
solution of 15 (17 g, 26 mmol, 1 equiv) in dry CH2Cl2 (78 mL) was
added during 20 min to a suspension obtained from 1.9 M COCl2 in
toluene (20.6 mL, 39 mmol, 1.5 equiv) and DMAP (317 mg, 2.6 mmol,
0.1 equiv) in dry pyridine (52 mL) and dry CH2Cl2 (104 mL). After
20 min at 20 °C, a solution of 3 (22 g, 78 mmol, 3 equiv) in triethylamine
(14.5 mL, 104 mmol, 4 equiv) was added dropwise. The resulting
mixture was stirred at room temperature for 36 h and then diluted with
CH2Cl2. The organic layer was quenched with saturated aqueous
NaHCO3, washed with brine, dried (Na2SO4), filtered and evaporated.
The residue was subjected to silica gel column chromatography with a
step gradient with MeOH (0ꢀ2%) in CH2Cl2. The desired compound
16 was obtained as white foam (21.72 g, 22.6 mmol, 87%). Rf = 0.19
(CH2Cl2/MeOH 97:3 v/v). 1H NMR (400 MHz, CDCl3) δ 8.73 (s, 1H,
H-2), 8.15 (br, 1H, NH), 8.12 (s, 1H, H-8), 7.45ꢀ6.75 (m, 17H, H ar,
DMTr-tboc), 6.36 (d, J = 6.8 Hz, 1H, H-10), 6.14 (dd, 1H, J = 6.8 Hz, J =
5.5 Hz, H-20), 5.72 (dd, J = 5.2 Hz, J = 2.8 Hz, 1H, H-30), 5.4 (s, 2H,
OCH2Ar, tboc), 4.38 (m, 1H, H-40), 3.79 (s, 6H, 2 OCH3), 3.51 (m, 2H,
H-50, H-500), 2.16 and 2.07 (2s, 6H, C(O)CH3), 1.37 (m, 3H,
0
Car), 125.5 (Cq ar-CH2), 122.8 (C5), 89.0 (OCH2O), 87.3 (C1 ), 86.6
0
0
0
(OCq, DMTr), 84.1 (C4 ), 81.7 (C2 ), 70.5 (C3 ), 63.8 (OCH2Ar), 62.9
0
(C5 ), 55.2 (OCH3, DMTr), 38.9 (Cq, OCOC(CH3)3), 26.9
(OCOC(CH3)3), 18.0 (3 CH(CH3)2), 13.0 (3 CH(CH3)2). HRMS
(ESIþ) m/z calcd for C54H68N5O11Si (M þ H)þ 990.4685, found
990.4678.
N6-{{{2-[(Triisopropylsilyl)oxy]benzyl}oxy}carbonyl}-50-
O-(4,40-dimethoxytrityl)-20-O-(tert-butyldimethylsilyl)-ade-
nosine 19. To a stirred solution of 17 (1.5 g, 1.71 mmol, 1 equiv) in dry
pyridine (10 mL), under nitrogen at 20 °C, were added imidazole
(350 mg, 5.1 mmol, 3 equiv) and tert-butyldimethylsilyl chloride
(370 mg, 5.1 mmol, 3 equiv). The resulting mixture was stirred at room
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The Journal of Organic Chemistry
ARTICLE
temperature for 12 h and then diluted with CH2Cl2. The organic layer
was quenched with saturated aqueous NaHCO3, washed with brine,
dried (Na2SO4), filtered and evaporated. The residue was subjected to
silica gel column chromatography with a step gradient with ethyl acetate
(0ꢀ10%) in cyclohexane containing 1% pyridine. The compound 19
was collected and 30-O-TBS derivative was isomerized in MeOH for
24 h. Purification by chromatography furnished 19 as white foam, after
three isomerisations (1.2 g, 1.21 mmol, 71%). Rf = 0.48 (EtOAc/
Cyclohexane 40:60 v/v). 1H NMR (400 MHz, CDCl3) δ 8.72 (s, 1H,
H-2), 8.18 (s, 1H, H-8), 8.42 (s, 1H, NH), 7.49ꢀ6.81 (m, 17H, H ar,
DMTr-tboc), 6.09 (d, J = 5.4 Hz, 1H, H-10), 5.40 (s, 2H, OCH2Ar, tboc),
5.02 (t, J = 5.2 Hz, 1H, H-20), 4.35 (m, 1H, H-30), 4.25 (m, 1H, H-40),
3.80 (s, 6H, 2 OCH3), 3.45 (m, 2H, H-50, H-500), 2.73 (d, J = 3.9 Hz, 1H,
OH-30), 1.28 (m, 3H, 3 CH(CH3)2), 1.05 (m, 18H, 3 CH(CH3)2), 0.82
(s, 9H, SiC(CH3)3), (ꢀ)0.05-(ꢀ)0.18 (2s, 6H, 2 SiCH3). 13C NMR
(100 MHz, CDCl3) δ 177.9 (OCdO), 158.6 (Cq, Car), 154.2 (Cq ar-
O), 152.9 (C2), 151.1 (NHCO), 150.9 (C4), 149.5 (C6), 144.6, 141.5
(Cq, Car), 141.4 (C8), 130.2- 113.2 (17 CH, Car), 125.6 (Cq ar-CH2),
9.45 mmol, 1.4 equiv) and tert-butyldimethylsilyl chloride (2.55 g, 16.9
mmol, 2.5 equiv). The resulting mixture was stirred at room temperature
for 5 h and then diluted with CH2Cl2. The organic layer was quenched
with saturated aqueous NaHCO3, washed with brine, dried (Na2SO4),
filtered and evaporated. The residue was subjected to silica gel column
chromatography with a step gradient with MeOH (0ꢀ5%) in CH2Cl2
containing 1% pyridine. The compound 22 was collected and 30-O-TBS
derivative was isomerized in MeOH in the presence of imidazole for
24 h. Purification by chromatography furnished 22 as white foam, after
three isomerisations (4.7 g, 4.68 mmol, 69%). Rf = 0.52 (CH2Cl2/
1
MeOH 95:5 v/v). H NMR (400 MHz, DMSO-d6) δ 10.77 (br, 1H,
NH-ar), 7.84 (s, 1H, H-8), 7.58 (br, 1H, NH), 7.44ꢀ6.85 (m, 26H, H-ar,
DMTr), 5.25 (d, J = 4.6 Hz, 1H, H-10), 4.80 (d, J = 5.5 Hz, 1H, OH-30),
4.32 (m, 1H, H-20), 3.94 (m, 2H, H-30, H-40), 3.92ꢀ3.71 (2s, 12H, 4
OCH3), 3.17 (m, 2H, H-50, H-500), 0.82 (s, 9H, SiC(CH3)3), 0-(ꢀ)0.15
(2s, 6H, 2 SiCH3). 13C NMR (100 MHz, DMSO-d6) δ 158.0 (Cq, Car),
157.6 (Cq, Car), 156.5 (C6), 151.1 (C4), 150.1 (C2), 145.1ꢀ112.9 (CH,
0
Cq, Ar), 136.1 (C8), 117.0 (C5), 86.5 (C1 ), 85.5 (OCq, DMTr), 82.6
0
0
0
0
(C3 ), 74.9 (C2 ), 70.2 (C4 ), 69.4 (NHCq, DMTr), 64.1 (C5 ), 54.9
(OCH3, DMTr); 25.7 (SiC(CH3)3), (ꢀ)4.8, (ꢀ)5.2 (2 SiCH3). HRMS
(ESIþ) m/z calcd for C58H64N5O9Si (M þ H)þ 1002.4473, found
1002.4376.
0
0
0
122.3 (C5), 88.4 (C1 ), 86.7 (OCq, DMTr), 84.3 (C4 ), 75.6 (C2 ), 71.6
0
0
(C3 ), 63.8 (OCH2Ar), 63.3 (C5 ), 55.2 (OCH3, DMTr), 25.6
(SiC(CH3)3), 18.0 (3 CH(CH3)2), 13.0 (3 CH(CH3)2), ꢀ5.0 and
ꢀ5.1. (2 SiCH3). HRMS (ESIþ) m/z calcd for C54H72N5O9Si2 (M þ
H)þ 990.4869, found 990.4863.
N2,50-O-Bis-(4,40-dimethoxytrityl)-20-O-pivaloyloxymethyl-30-
O-(2-cyanoethyl-N,N-diisopropylphosphoramidite)-guano-
sine 1d. Using general procedure starting from 21 (1.17 g, 1.17 mmol)
afforded 1d (1.2 g, 1 mmol, 88% yield). 31P NMR (121 MHz, CD3CN)
δ 150.16, 149.90. HRMS (ESIþ) m/z calcd for C67H77N7O12P
(MþH)þ 1202.5368, found 1202.5376.
N6-{{{2-[(Triisopropylsilyl)oxy]benzyl}oxy}carbonyl}-50-
O-(4,40-dimethoxytrityl)-20-O-pivaloyloxymethyl-30-O-(2-cy-
anoethyl-N,N-diisopropylphosphoramidite)-adenosine 1c.
Using general procedure starting from 18 (4.3 g, 4.35 mmol) afforded
1c (4 g, 3.36 mmol, 77% yield). 31P NMR (121 MHz, CD3CN)
δ 150.71, 149.76. HRMS (ESIþ) m/z calcd for C63H85N7O12PSi (M þ
H)þ 1190.5763, found 1190.5771.
N2,50-O-Bis-(4,40-dimethoxytrityl)-20-O-(tert-butyldimeth-
ylsilyl)-30-O-(2-cyanoethyl-N,N-diisopropylphosphorami-
dite)-guanosine 2d. Using general procedure starting from 22 (1.14
g, 1.14 mmol) afforded 2d (1 g, 0.83 mmol, 71% yield). 31P NMR (121
MHz, CD3CN) δ 151.01, 147.96. HRMS (ESIþ) m/z calcd for
C67H81N7O10PSi (M þ H)þ 1202.5552, found 1202.5573.
N6-{{{2-[(Triisopropylsilyl)oxy]benzyl}oxy}carbonyl}-50-
O-(4,40-dimethoxytrityl)-20-O-(tert-butyldimethylsilyl)-30-
O-(2-cyanoethyl-N,N-diisopropylphosphoramidite)-adeno-
sine 2c. Using general procedure starting from 19 (1.5 g, 1.55 mmol)
afforded 2c (1.3 g, 1.09 mmol, 73% yield). 31P NMR (121 MHz, CD3CN)
δ 150.18, 148.63. HRMS (ESIþ) m/z calcd for C63H89N7O10PSi2 (M þ
H)þ 1190.5947, found 1190.5952.
5-O-(4,40-dimethoxytrityl)-20-O-(tert-butyldimethylsilyl)-
30-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite)-gua-
nosine 2e. A solution of the commercial 50-O-(4,40-dimethoxytrityl)-
20-O-TBS-N2-(tert-butylphenoxyacetyl)-guanosine (0.9 g, 0.83 mmol,
1equiv) in 2 M MeNH2 (40% aqueous solution) (0.7 mL) in THF
(10 mL) was stirred at room temperature for 20 min. The reaction
mixture was concentrated and coevaporated twice with CH3CN. The
residue was subjected to silica gel column chromatography with a step
gradient with MeOH (0ꢀ3%) in CH2Cl2 containing 1% pyridine. The
fractions containing pure 2e were pooled and concentrated to give white
foam (0.63 g, 0.70 mmol, 84% yield). 31P NMR (121 MHz, CD3CN) δ
151.03, 149.15. HRMS (ESIþ) m/z calcd for C46H63N7O8PSi (M þ H)þ
900.4245, found 900.4230.
N2,50-O-Bis-(4,40-dimethoxytrityl)-20-O-pivaloyloxymet-
hyl-guanosine 21. To a solution of 20 (2.1 g, 2.34 mmol, 1 equiv)
in DCE (13 mL) were added TBAB (1.88 g, 5.85 mmol, 2.5 equiv),
dibutyltin oxide (1.46 g, 5.85 mmol, 2.5 equiv) and chloromethyl
pivalate (1.76 mL, 11.7 mmol, 5 equiv). The reaction mixture was
stirred under nitrogen at 75 °C for 6 h, cooled and the solvent was
evaporated. The reaction mixture was subjected to silica gel column
chromatography with a step gradient with acetone (0ꢀ30%) in
CH2Cl2. The first-eluted isomer was the desired compound 21 and was
obtained as white foam after evaporation of the solvent (582 mg,
1
Oligonucleotide Synthesis. The oligonucleotides were synthe-
sized at 1-μmol scale on DNA synthesizer (ABI 381A) by using
phosphoramidite building blocks 1aꢀd, 2aꢀe, and thymidine. Synthe-
ses were carried out on thymidine-Q-linker CPG support, 5-benzyl-
mercaptotetrazole (BMT) was used as the activator for coupling, the
capping step was performed with phenoxyacetic anhydride, using a
commercial solution (Cap A: Pac2O/pyridine/THF 10:10:80 v/v/v and
Cap B: 10% N-methylimidazole in THF). Oxidation was performed
with a commercial solution of iodide (0.1 M I2, THF, pyridine/water
90:5:5, v/v/v). Detritylation was performed with 3% TCA in CH2Cl2.
See Supporting Information for detailed oligonucleotides automated
synthesis conditions.
General Procedure for Deprotection of Solid-Supported
Oligonucleotides. CPG beads were treated with an anhydrous 1 M
DBU solution in CH3CN (3 mL) at room temperature for 5 min to get
rid of cyanoethyl groups. The solution was removed by filtration and the
support was washed with CH3CN (5 mL) then dried to be transferred
0.58 mmol, 25%). Rf = 0.45 (CH2Cl2/MeOH 95:5 v/v). H NMR
(400 MHz, DMSO-d6) δ 10.68 (br, 1H, NH-ar), 7.77 (s, 1H, H-8), 7.58
(br, 1H, NH), 7.38ꢀ6.78 (m, 26H, H-ar, DMTr), 5.32 (d, J = 3.9 Hz,
1H, H-10), 5.11 and 4.83 (2d, J = 6.4 Hz, 1H þ 1H, OCH2O), 5.01 (d, J =
7.4 Hz, 1H, OH-30), 4.05 (m, 1H, H-20), 3.81 (m, 1H, H-40), 3.72ꢀ3.62
(2s, 12H, 4 OCH3), 3.58 (m, 1H, H-30), 3.09 (m, 2H, H-50, H-500), 0.96
(s, 9H, C(O)C(CH3)3). 13C NMR (100 MHz, DMSO-d6) δ 177.0
(OCdO), 158.6 (Cq, Car), 158.1 (Cq, Car), 156.9 (C6), 151.6 (C4),
149.7 (C2), 145.3ꢀ113.4 (CH, Cq, Ar), 136.0 (C8), 118.2 (C5), 88.1
0
0
0
(OCH2O), 86.9 (C1 ), 86.1 (OCq, DMTr), 82.5 (C4 ), 79.8 (C2 ), 69.9
0
0
(C3 ), 69.6 (NHCq, DMTr), 63.5 (C5 ), 55.4 (OCH3, DMTr), 38.5 (Cq,
OCOC(CH3)3), 26.8 (OCOC(CH3)3). HRMS (ESIþ) m/z calcd for
C58H60N5O11 (M þ H)þ 1002.4289, found 1002.4308.
N2,50-O-Bis-(4,40-dimethoxytrityl)-20-O-(tert-butyldimeth-
ylsilyl)-guanosine 22. To a stirred solution of 20 (6 g, 6.75 mmol,
1 equiv) in a mixture of dry THF (50 mL) and dry pyridine
(7 mL), under nitrogen at 20 °C, were added silver nitrate (1.6 g,
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The Journal of Organic Chemistry
ARTICLE
into a screw-capped O-ring eppendorf. The support containing 1 μmol
of the oligonucleotide was suspended in a mixture (pH 8.3) of HF 48 wt.%
in H2O (0.5 mL) and Et3N (1.5 mL) at 65 °C for 10 h. After
centrifugation, the supernatant was transferred in a round flask, the
support was washed with water (0.5 mL ꢁ 2). After centrifugation,
the combined supernatants were concentrated under reduced pressure.
The crude material diluted in water (1 mL) was loaded onto a home-
made RP C8 column (3 ꢁ 1 cm) equilibrated in water. The column was
first flushed with water (20 mL) to remove the salts. Then the desalted
ON was eluted with 50% acetonitrile (10 mL). Further purification was
performed using RP-HPLC. ON purity was confirmed by RP-HPLC,
anion-exchange HPLC and MALDI-TOF MS.
Melting Experiments. Tm experiments were performed on an UV
spectrophotometer equipped with a Peltier temperature controller and a
thermal analysis software. The samples were prepared by mixing each
strand together to give 3 μM final concentration in 10 mM sodium
cacodylate, 100 mM NaCl, pH 7. A heatingꢀcooling cycle in the
0ꢀ95 °C temperature range with a gradient of 0.5 °C.minꢀ1 was applied.
Tm values were determined from the maxima of the first derivative plots of
absorbance versus temperature.
naked siRNA was performed as described above in the absence of
lipofectant and using 1 μM instead of 1nM siRNA. For electroporation,
we used 1 ꢁ 106 ECV304-GL3 cells in 200 μL PBS and preincubated
with 10nM siRNA for 1 min on ice. Subsequently, cells were transferred
to electroporation cuvettes with 4 mm interelectrode distance. Cells
were pulsed once at 180 V and 950 μF (t1/2 ≈ 50 ms) and cultured in 12-
well culture dishes. After 24 h, cells were harvested and viability and
firefly luciferase activity were measured as described above.
’ ASSOCIATED CONTENT
Supporting Information. 1H NMR, 13C NMR, and
S
b
31P NMR of all new compounds. HPLC profiles and MALDI-
TOF spectra of synthesized oligonucleotides. This material is
available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*debart@univ-montp2.fr
Enzymatic Digestion. pro-RNA 26 (1 OD unit at 260 nm) was
incubated with nuclease P1 (0.125 units) at 37 °C for 48 h. Then alkaline
phosphatase (1.25 units) and buffer (50 mM Tris-HCl, pH 9.3, contain-
ing 1 mM MgCl2, 0.1 mM ZnCl2 and 1 mM spermidine; final
concentrations) were added to give a total volume of 100 μL and the
mixture was incubated at 37 °C for a further 24 h. The reaction mixture
was then analyzed by HPLC.
’ ACKNOWLEDGMENT
This work was supported by the Association pour la Recherche
contre le Cancer (ARC) and Ministꢁere de l’Enseignement supꢀerieur
et de la Recherche.
Oligonucleotides for Cell Culture Studies. All ON used in this
study were synthesized as described above or purchased from IBA
(G€ottingen, Germany). Before use, the integrity and purity of the single-
stranded siRNAs were checked by electrophoresis on 20% denaturing
polyacrylamide gels. For annealing of complementary RNA strands,
equimolar amounts of sense- and antisense strands respectively were
incubated in 20 mM Tris/HCl pH 7.4 and 100 mM NaCl for 2 min at
95 °C followed by slow cooling to room temperature. Oligoribonucleo-
tides sequences are as follows: siR206, sense strand, 50-GAUAUGGG-
CUGAAUACAAAdTdT-30; antisense strand, 50-UUUGUAUUCA-
GCCCAUAUCdTdT-30; siR206-PivOM, sense strand 26 50-GAU-
AUGGGCUGAAUACAAAdTdT-30 (PivOM-modified nucleotides
indicated in bold and italic); antisense strand, 50-UUUGUAUU-
CAGCCCAUAUCdTdT-30; siscr3, sense strand, 50-CGGACGCA-
CUGGUCUGACCGGdTdT-30; antisense strand, 50-CCGGUCA-
GACCAGUGCGUCCGdTdT-30.
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