Steric effects in RNA interference: Probing the influence of nucleobase size and shape

Article abstract of DOI:10.1002/chem.200800837

Nonpolar nucleosides with varying size and shape have been used to study the hydrogen-bonding stabilization and steric effects on RNA interference. The uracil and adenine residues of siRNA guide strands have been replaced by nonpolar isosteres of uracil a

Full text of DOI:10.1002/chem.200800837

DOI: 10.1002/chem.200800837
Steric Effects in RNA Interference: Probing the Influence of Nucleobase Size
and Shape
Alvaro Somoza, Adam P. Silverman, Rand M. Miller, Jijumon Chelliserrykattil, and
Eric T. Kool*[a]
Abstract: Nonpolar nucleosides with
varying size and shape have been used
to study the hydrogen-bonding stabili-
zation and steric effects on RNA inter-
ference. The uracil and adenine resi-
dues of siRNA guide strands have
been replaced by nonpolar isosteres of
uracil and adenine and by steric var-
iants. RNAi experiments targeting Re-
nilla luciferase mRNA have shown
close correlation between siRNA ther-
mal stability and gene suppression. In-
terestingly, siRNA modified at posi-
tion 7 on the guide strand does not
follow this correlation, having substan-
tial RNAi activity despite low thermal
stability. Sequence-selectivity studies
were carried out at this position with
mutated target mRNAs and nucleobase
analogues with varied size (2,4-di-
fluoro- and 2,4-dichlorobenzene) and
different shape (2,3-dichlorobenzene,
4-methylbenzimidazole). The results
point out the importance of nucleobase
shape and steric effects in RNA inter-
ference.
Keywords: base pairs · nucleobases ·
RNA recognition · RNA structures
Introduction
way, the guide strand is incorporated into the active RISC
complex; meanwhile the passenger is cleaved and released
to the cytoplasm.[7] The guide strand in the RISC complex
serves as template to localize the complementary mRNA in
the cytoplasm. Once the guide strand binds the targeted
mRNA, the target is cleaved due to the RNAse activity
present on the PIWI domain of the RISC complex.[8] The
mRNA fragments are released to the cytoplasm and the
RISC complex repeats this process, cleaving more target
mRNAs.
The catalytic mechanism involved and the high specificity
achieved by sequence complementarity make this process a
powerful tool for the control of gene expression. For these
reasons it has been considered a potential strategy for tar-
geted therapy, since the expression of proteins involved in
diseases can be selectively inhibited. However, there are still
several drawbacks to overcome, such as off-target effects,[9]
stability in serum,[10] and biodistribution.[11] In this context, a
variety of chemically modified siRNAs has been studied in
an effort to address these limitations.[12] Most of the modifi-
cations have been incorporated or attached on the passenger
strand, since this strand is degraded during RISC activation
and does not interfere in the process. These modifications
are intended to provide higher stability in serum and better
biodistribution, such as the use of cholesterol on the 3’ end
reported by Soutschek et al.[13] Modifications on the guide
strand are more problematic, since they may directly inhibit
the RNAi activity. However, selected modifications at spe-
RNA interference (RNAi) has become an essential tool for
studying gene function in biological and biomedical research
by specifically silencing a targeted gene.[1] This process is
commonly triggered by double-stranded RNA of 21–23 nu-
cleotides with two nucleotides overhanging (DT),[2] although
different length and structures, such as hairpins[3] or blunt-
end duplexes,[4] can be employed as well. These short RNA
duplexes are known as small-interfering RNAs (siRNAs).
The two strands of the duplexes have different significance:
one is responsible for recognizing the target mRNA se-
quence (the guide strand), while the complementary strand
has no key role in the recognition and is known as the pas-
senger strand.
The RNAi pathway is initiated when a siRNA reaches the
cytoplasm and a protein complex known as RISC (RNA-in-
duced silencing complex) binds the siRNA duplex.[5] The
RISC complex selects the strand 5’ end with lowest thermal
stability in the duplex by using the RD2D2 protein.[6] In this
[a] Dr. A. Somoza, Dr. A. P. Silverman, R. M. Miller,
Dr. J. Chelliserrykattil, Prof. Dr. E. T. Kool
Department of Chemistry, Stanford University
Stanford, CA 94305–5080 (USA)
Fax : (+1)650-725-0259
E-mail: kool@stanford.edu
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200800837.
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FULL PAPER
cific positions of the guide strand can have interesting ef-
fects. Modifications on the sugar moiety, particularly on the
2’-hydroxyl groups, provide enhanced resistance to nucle-
ACHTREUNG
ases[14] and can reduce off-target effects.[12c,15] Moreover, the
use of chemically modified nucleotides has been essential
for the study of the mechanism of RNAi,[16] providing funda-
mental knowledge for the efficient design of siRNAs.[17]
Despite the significant contributions focused on under-
standing the RNAi process and developing therapies based
on RNAi, the RISC-mediated siRNA-target interaction is
not completely understood yet, and simple nucleic acid hy-
bridization does not explain target recognition.[18] The inter-
actions that take place inside the RISC complex (protein–
siRNA–mRNA) are key in target recognition, and better
understanding of these interactions is required for optimal
design of siRNAs.
In connection with our program devoted to studying the
role of electrostatics and sterics in biological systems,[19] and
due to the significance of the RNA interactions inside the
RISC complex, we have undertaken studies to evaluate the
roles of these molecular properties on the RNAi process. In
an early study we previously reported the effect on RNAi
activity of uracil replacement by its nonpolar isostere 2,4-di-
fluorobenzene (rF).[20] In that case, the gene supression ac-
tivity was retained in several modified siRNAs despite the
lack of hydrogen bonding stabilization. In particular, posi-
tion 7 of the guide strand was surprisingly insensitive to this
substitution, yielding significant RNAi activity despite low
thermal stability compared with the wild-type duplex. Simul-
taneous studies by Manoharan also found a similar effect.[21]
In our study, selectivity studies at this position employing
singly mutated target mRNAs showed that the nonpolar iso-
stere retained the selectivity of uracil. This led to the hy-
pothesis that sequence selectivity might arise from protein-
enforced steric effects.
In this new work, we test this hypothesis by studying non-
polar nucleobase analogues of varied size and shape. We
report the synthesis of modified phosphoramidites and their
use in RNAi experiments, evaluating biological activity and
sequence specificity. In addition we have tested effects in a
second target mRNA sequence to evaluate generality. The
results provide new insights on the influences of sterics and
hydrogen bonding stabilization on RNAi, and they confirm
that nucleobase shape and size can play strong roles in the
sequence specificity of RNAi.
Scheme 1. Synthesis of nonpolar C-ribonucleosides. Bn=benzyl.
The free nucleosides 5–7 were then functionalized for
RNA synthesis on an automated synthesizer. The 5’-hydrox-
yl group of the nonpolar nucleosides was protected as a di-
methoxytrityl ether (DMTr) under the standard conditions.
Further protection of the 2’-hydroxyl groups was carried out
using the [(triisopropylsilyl)oxy]methyl group (TOM) in a
standard two-step process.[22] As is commonly observed, ad-
dition of the protecting reagent TOMCl gave rise to a mix-
ture of 2’- and 3’-TOM derivatives, in which the desired 2’-
O-TOM was the major compound (Scheme 2).
The 4-methylbenzimidazole free ribonucleoside 8 was ob-
tained as reported[23] and functionalized in the same way as
the halogenated analogues. In this case the TOM protection
gave rise to a 1:1 mixture of 2’- and 3’-derivatives.
Results and Discussion
In the case of the dichloro analogues, the 2’-O-TOM de-
rivative was easily isolated by silica column chromatography.
In contrast, the difluoro analogue required further manipu-
lations to obtain the 2’-TOM-protected nucleoside in a pure
form. The previously obtained mixture was treated with
acetic anhydride and triethylamine, leading to the corre-
sponding mixture of acetylated derivatives. In this case, the
mixture could be easily purified by silica chromatography.
The desired 2’-TOM nucleoside was finally obtained by de-
Synthesis of modified nucleosides and RNAs: The halogen-
ated nucleosides (rF, rL, rZ, and 2,3-rL) were prepared by
addition of the corresponding aryllithium species to the
benzyl-protected ribonolactone 1. Stereoselective reduction
was performed with triethylsilyl hydride in the presence of
BF3·OEt2, giving rise to the corresponding b-anomer deriva-
tives (2–4). Deprotection of the benzyl groups by using BBr3
at ꢀ788C afforded the free nucleosides (Scheme 1).
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E. T. Kool et al.
Figure 1. Wild-type siRNA duplex used to target the Renilla luciferase
mRNA. Positions 7 (guide) and 13 (passenger) (at which substitutions
were made) are numbered.
signed to target the fragment 501–519 of Renilla luciferase,
which was efficiently inhibited by an unsubstituted
siRNA.[24]
Duplex stability: First we investigated the effect of altered
nucleobase shapes and sizes on siRNA duplex stability. The
three halogenated benzenes were evaluated as a closely re-
lated set. The thermal stability of the duplexes was mea-
sured by using the wild-type and singly mismatched passen-
ger strands, allowing selectivity to be evaluated. The adenine
at position 13 (opposite the modified bases) was replaced by
C, G, and U in the mismatched strands. The results of the
studies are graphically depicted in Figure 2.
Scheme 2. Protection of free nucleosides. DMTr=4,4’-dimethoxytrityl;
Py=pyridine; DIPEA=diisopropylethylamine; DCE=1,2-dichloro-
ethane; TOM=[(triisopropylsilyl)oxy] methyl.
protection of the acetyl group by using ammonia in metha-
nol. The isolation of the 2’-TOM-protected benzimidazole
derivative was also difficult, but in this case the derivatiza-
tion of the remaining hydroxyl groups to acetyl or isobutyl
groups did not provide significant improvement in the pu-
rification. The desired isomer was ultimately obtained by re-
peated silica column chromatography.
The final step was the activation of the 3’-hydroxyl group
as the cyanoethylphosphoramidite, required for the forma-
tion of the phosphodiester internucleotide bond during the
RNA synthesis. The addition of 2-cyanoethyl-N,N-diisoprop-
yl-phosphonamidic chloride and diisopropylethylamine af-
forded the corresponding phosphoramidite derivatives,
which were utilized on an automated synthesizer for their
incorporation into RNA (Scheme 3).
Figure 2. Thermal stability of matched and mismatched duplexes. The
mismatches are at position 13 on the passenger strand. The guide strands
are natural (“U”) and modified at position 7 with nonpolar derivatives
(“rF”, “rL” and “2,3-rL”). WT=wild-type.
Overall, the nonpolar analogues at position 7 were desta-
bilizing and showed little if any selectivity, in contrast to
natural uracil. The wild-type guide strand (U at position 7)
showed strong destabilization for the C:U and U:U mis-
matches, but in the case of the G:U mismatch the thermal
stability was close to that of the canonical base pair A:U, as
expected from wobble pairing.[25] In the modified duplexes
(rF, rL, and 2,3-rL) the thermal stability dropped compared
with the A–U paired case, reflecting the loss hydrogen
bonding groups at position 7. Among the modified duplexes,
the four cases modified with 2,3-rL showed similar thermal
stabilities (63–648C), but the duplexes modified with rF and
rL showed some differences, giving a weak preference for
pairing opposite A and G. This small difference in stability
could be due to nucleobase stacking stabilization, since
purine nucleobases contribute more than pyrimidines.[26]
Similar effects have been seen in DNA with dihalogenated
deoxyribosides,[27] and Egli and co-workers have reported
analogous behavior in RNA utilizing the related 2,4-
Scheme 3. Synthesis of nonpolar nucleoside phosphoramidites. Ac=
acetyl, DMTr=4,4’-dimethoxytrityl; DIPEA=diisopropylethylamine;
TOM=[(triisopropylsilyl)oxy] methyl.
The modified nucleosides rF, rL, 2,3-rL, and rZ were suc-
cessfully incorporated at position 7 of a 21 mer RNA guide
strand of the siRNA duplex utilized in our early report
(Figure 1) and characterized by MALDI-TOF mass spec-
trometry after PAGE purification. The siRNAs were de-
toluene analogue.[28] In summary, the steric differen-
difluoroCAHTREUNG
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ces in the halogenated compounds yielded only small
changes in selectivity in the absence of the biological RISC
complex.
contrasting effects by the varying shapes of the modified nu-
cleobases. The two highly active analogues retain the shape
of uracil, and are thus complementary in shape to the ade-
nine target. However, the poorly active ones do not have
the shape of uracil: 2,3-rL has a steric projection (chlorine)
at position 7, which is expected to clash sterically with ade-
nine. Analogue rZ is a nucleobase analogue shaped similarly
to adenine, and thus would also clash with an adenine part-
ner. Interestingly, a subtle change in size (from rF to rL,
which represents a 0.3 Š increase in bond lengths at the hal-
ogens) had no strong effect on activity.
The changes in nucleobase shapes at position 7 had sub-
stantial effects on sequence selectivity of gene suppression.
The two uracil-shaped analogues (rF and rL) showed a simi-
lar selectivity profile as U, but with greater selectivity
against mismatches. Both analogues showed a preference
for the A target, with a secondary preference for a U mis-
match, thus behaving similarly to natural uracil. However,
both showed a strong selectivity against activity at the G
and C mismatches, while the uracil-containing RNA showed
only moderate selectivity against C. Thus the two analogues
displayed greater sequence selectivity than the natural base.
We tentatively attribute the selectivity of the analogues as
reflecting a similar shape selectivity of the natural base, but
with the addition of stronger destabilization against the
most polar partners (C and G), due to the cost of desolva-
tion. We note that this selectivity profile in RNAi activity is
markedly different from the pairing selectivity of the RNAs
alone (Figure 2). This suggests a pronounced role of the
RISC-associated protein(s) in enforcing steric effects that
appear to be modest with the RNAs alone. Moreover, it ap-
pears that the RISC complex reduces the importance of hy-
drogen bonding at position 7 that strongly affects the RNAs
in the absence of protein.
The nucleobase shape differences also had a substantial
effect on the selectivity of the analogues that were not
shaped like uracil. Significantly, both of these analogues
showed a preference for activity when paired opposite uracil
in the target gene, thus reversing the selectivity of the
uracil-shaped analogues, which preferred adenine. For the
analogue rZ, this can be explained by its steric resemblance
to adenine, thus being complementary to uracil in forming a
Watson–Crick base-pair shape. Even more interesting is 2,3-
rL, which also preferred uracil as a partner in the RISC
complex, and showed large differences in activity with dif-
ferent partners even though on its own it shows essentially
no pairing selectivity (see Figure 2). A simple steric compar-
ison of 2,3-rL with the four natural nucleobases shows that
it most resembles adenine in size and steric projection
toward the opposite strand (see Supporting Information).
Also significant is the fact that a close analogue of 2,3-rL is
paired selectively with thymine by polymerases.[30] Thus the
selectivity of analogues rZ and 2,3-rL in cellular RNA inter-
ference can also be explained by steric effects that are en-
forced inside the RISC protein-RNA complex.
Shape effects on RNAi activity: For the RNAi experiments
at this initial target site we employed a commercial wild-
type Renilla luciferase plasmid, and to evaluate sequence se-
lectivity, three mutant targets were generated by site-direct-
ed mutagenesis of the plasmid. These mutants encode luci-
ferase mRNA with C, G, and U mutations at the position
corresponding to position 13 of the passenger RNA (and
positioned to be paired opposite position 7 of the modified
guide RNAs). The mutations are genetically silent in the Re-
nilla luciferase mRNA, as they occur in the third position of
the gly171 codon. Thus the reporter protein is unchanged.
We carried out the RNAi experiments employing the
wild-type and mutated plasmids to express the desired
RNAs in HeLa cells. The results, showing Renilla luciferase
activity normalized to nontargeted firefly luciferase, are rep-
resented in Figure 3. The wild-type guide strand (U at posi-
Figure 3. Luciferase suppression activity of natural (WT) and modified
(rF, rL, 2,3-rL, rZ) siRNAs at position 7 of the guide strands for singly
mismatched mRNA targets. Data were normalized by internal control of
a noncomplementary firefly luciferase gene. Data are for the 21 ng RNA
amount and are the average of three replicates; error bars reflect stan-
dard deviations.
tion 7) showed the highest activity when no mismatches
were present in the target (WT, with A at the target posi-
tion). When G and U mutants were targeted, the inhibition
of the gene was also strong, as observed previously.[20] Of
the three mismatches, only a U:C mismatch at position 7
showed a significant decrease of RNAi activity (C mutant).
This relatively low sequence specificity for single mismatch-
es has been observed earlier in a broad study of mutant
RNA targets.[29]
Importantly, two of the modified siRNAs displayed activi-
ty nearly the same as the natural siRNA, and most showed
sequence selectivity that was better than the wild-type
RNA. The siRNAs modified with rF and rL showed strong
gene suppression activity against the wild-type Renilla luci-
ferase mRNA despite the absence of hydrogen-bonding sta-
bilization at this position. In contrast, the analogues 2,3-rL
and rZ showed relatively poor activity. We explain these
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E. T. Kool et al.
Evaluating target generality of size and shape effects: The
preceding results provide evidence that the protein/guide
RNA/mRNA complex enhances steric effects in RNA–RNA
recognition and lowers the importance of hydrogen-bond-
ing-dependent stabilization at some positions. However,
these effects were only observed at a single site in an
mRNA target. Since RNA forms varied secondary and terti-
ary structure in different sequence contexts, one may well
ask whether such effects would be general (i.e., arising
chiefly from the generic RISC structure) or variable with
each new RNA target.
similar to that of a mismatch (entry 7A in Figure 5). Similar
position-dependent destabilizations were seen for rF and rZ
substitutions in the passenger strand as well. Overall, the
strongest destabilizations were seen at positions 7–16, while
positions 1–4 and 19 were little affected.
The RNAi activity of the modified siRNAs in this second
context was evaluated targeting the luciferase gene in HeLa
cells as before. This provides a test of the importance of hy-
drogen-bonded stabilization at ten positions along the
siRNA duplex in this new sequence context. The results are
shown in Figure 6. The duplexes modified on the guide
To begin to test this we evaluated hydrogen bonding and
steric effects at a second target sequence in the Renilla luci-
ferase gene, employing the rF and rZ nucleoside analogues
as probes in the guide and passenger strands. The siRNAs in
this case targeted the site 690–708 of the luciferase gene,
189 nucleotides distant from the previous target (see
Figure 4). We substituted uracil residues of the siRNA with
rF, and adenine monomers with rZ; in all, ten of the nine-
teen guide-strand pairing positions were replaced in sepa-
rate experiments, and two passenger-strand bases were also
replaced.
Figure 6. Gene suppression activity of modified siRNAs. Modifications at
the guide strand are denoted with the position number and the modifica-
tion. Modifications at the passenger strand are denoted as PS. Data were
normalized by internal control of a noncomplementary firefly luciferase
gene. Data are for the 21 ng RNA amount and are the average of three
replicates ꢁ standard deviation. WT=wild-type.
Figure 4. Wild-type siRNA duplex targeted to the 690–708 segment of
the Renilla luciferase gene.
First we measured the effects of single nonpolar analogue
substitutions on thermal stability of the siRNA duplex. The
results are given in Figure 5. The data show that nonpolar
modification at inner positions of the duplex resulted in
lower thermal stability, while modifications near the ends
had only small effects. This is essentially the same as was ob-
served for the 501–519 target site, as probed with rF in the
previous report.[20] The destabilization near the center was
strand at position 1 and 19 with rZ and rF, respectively,
showed a gene inhibition activity close to wild-type siRNA
(WT). The modified duplexes substituted at positions 3–7
presented moderate activity, while the duplexes substituted
at positions 8–16 had no activity in suppressing the lucifer-
ase gene.
A comparison with the duplex stabilities
(Figure 5) shows a general correlation of higher stability
with greater RNAi activity. However, position 7 is anoma-
lous, showing stronger activity than expected from its ther-
mal stability. These results are in agreement with our previ-
ously reported findings, that is, the nonpolar isostere modifi-
cations at the end of the duplexes do not affect the RNAi
activity, but near the middle of the strand have significant
negative effect. Moreover, the new data are also in agree-
ment with our earlier observation that the position 7 substi-
tution shows higher-than-expected activity despite its inner
position in the guide strand and its low thermal stability.
This position 7 effect was also observed by Manoharan at an
entirely different gene target;[21] thus we believe it to be
general to the human RISC complex at many if not all
mRNA targets.
The mismatched siRNA 7A has an adenosine at position 7
of the guide strand and shows very low RNAi activity de-
spite its moderate thermal stability. The activity is substan-
tially lower than the 7F nonpolar uracil analogue case. This
result further underscores the importance of steric effects at
Figure 5. Thermal stability of modified siRNA duplexes targeted to the
Renilla luciferase 690–708 site. Modifications at the guide strand are de-
noted with the position number and the modification. F stands for the rF,
Z for rZ and A for adenosine. Modifications at the passenger strand are
denoted as PS. WT=wild-type.
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RNA Recognition
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that position, as the adenine–adenine mismatch may not fit
as well as the difluorobenzene–adenine pair inside the
active RISC complex.
amples, the rF:U mismatch was again the best tolerated.
Thus in this second target site, the selectivity of a natural
nucleobase is well mimicked by an analogue that copies its
shape accurately.
Modifications to nucleobases might have indirect effects
on biological activity of siRNAs; for example, they may sta-
bilize the siRNA duplex against degradation, or enhance its
entry into the RISC complex. However, our data suggest
that is not the case for the rF and rZ substitutions. The
siRNA PS13Z has an rZ modification at position 13 of the
passenger strand, pairing it opposite natural rU at position 7
of the guide strand. This duplex has a thermal stability simi-
lar to the 7F duplex, but the RNAi activity is close to wild-
type. This result supports the idea that the RNAi activity
shown by the siRNA 7F is only due to the interaction of the
guide strand inside the RISC complex with the messenger
RNA.
Our data at position 7 show that steric effects may be
largely if not entirely responsible for base selectivity of the
RISC complex. Moreover, some of the analogues yield im-
proved selectivity over natural nucleobases, which may be
useful in practical application for avoiding “off-target” ef-
fects. In the future it would be of interest to determine
steric versus hydrogen-bonding contributions to selectivity
at other positions in the complex. Although nonpolar ana-
logues are not well tolerated at some of the other central
positions, work is underway to develop new compounds that
may be able to contribute information about these other
positions.
Finally, we carried out selectivity studies at position 7, em-
ploying the wild-type and rF-modified siRNAs. This allowed
a second test of whether the selectivity of uracil arises from
its hydrogen-bonding ability or its shape. Once again we
prepared plasmids encoding three mutated target mRNAs
at the complementary position. Similarly to the previous
target, the nucleotide required for mutation in the Renilla
luciferase mRNA is the third one of the val234 codon,
which is universally variable without changing the composi-
tion of the Renilla luciferase protein.
Figure 7 shows the results of the selectivity experiments.
As expected, the unmodified siRNA (wt) showed strongest
activity against the complementary target mRNA. The U:C
and U:G mismatches at position 7 were less well tolerated
than the corresponding U:U mismatch, showing a selectivity
profile quite similar to that in the previous target site
(Figure 3). In the case of the modified siRNA (rF) the selec-
tivity pattern was the same as the wt siRNA, but with de-
creased activity. The best activity was achieved against the
wild-type mRNA and conversely, the C and G mutants
showed the lowest RNAi activity. Finally, as in previous ex-
Conclusion
A set of nonpolar ribonucleotides, of varied shapes and
sizes, has been incorporated into synthetic RNAs for RNAi
experiments. The results have shown that hydrogen-bonding
stabilization is most important generally at positions 10–16
of the guide RNA, but that hydrogen-bonding deficient nu-
cleobases are well tolerated at positions 1–4, 7, and 19. We
find that RNAi activity generally correlates with thermal
stability of these substitutions. However, position 7 substitu-
tion shows good activity at multiple targets despite its low
thermal stability. Further experiments at this position with
mutant target mRNAs have pointed out the importance of
steric effects on selectivity. Wild-type selectivity for adenine
can be achieved or improved utilizing nonpolar nucleosides
of similar size and shape to rU, and selectivity for uracil can
be evinced by nonpolar adenine shape mimics. Such ana-
logues may be generally useful both as tools for evaluating
electrostatic and steric effects in RNA biology.
ExperimentalSection
Generalexperimentalmethods : Reagents were purchased from Aldrich
and used without further purification. All water-sensitive reactions were
carried out in oven-dried glassware with a stirring bar under argon at-
mosphere. Pyridine and CH2Cl2 were dried using a Solvent Purification
System (Innovative Technology, USA). All other anhydrous solvents
were used directly without further distillation. Thin-layer chromatogra-
phy was carried out by using Silica Gel 60 F254 plates. Column chroma-
tography was performed with Silica Gel (60 Š, 230”400 mesh). All
NMR spectra were recorded on 400 or 500 MHz instruments as solutions
in the deuterated solvent indicated, and the chemical shifts are reported
in ppm. Coupling constants are reported in Hz.
Synthesis of C-arylsubstituted ribonucelosides (Procedure A) : nBuLi
(1,6m in hexanes, 3 equiv) was added dropwise to a solution of the corre-
sponding bromo benzene derivative (3 equiv) in Et2O (0.2m) at ꢀ788C
under Ar atmosphere. After 30 min a solution of ribonolactone in Et2O
(0.2m) was added and the resulting solution was stirred for 2 h at ꢀ788C.
The mixture was quenched by addition of NH4Cl solution, extracted with
AcOEt, washed with brine, and dried over MgSO4. The evaporation of
the organic solvent afforded a yellowish oil that was used in the next
Figure 7. RNAi activity of natural (“WT”) and modified (“rF”) siRNAs
at position 7 of the guide strands for singly mismatched RNA targets.
The mismatch is opposite position 7 of the guide strand and involves mu-
tating the corresponding nucleotide in the complementary RNA. Data
were normalized by internal control of a noncomplementary firefly luci-
ferase gene. Data are for the 21 ng RNA amount and are the average of
three replicates ꢁ standard deviation. WT=wild-type.
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E. T. Kool et al.
step. It was dissolved in CH2Cl2 (0.2m) and cooled at ꢀ788C, then
Et3SiH (3 equiv) and BF3OEt2 (3 equiv) were added. The mixture was
stirred for 12 h at the same temperature and quenched with a saturated
solution of NaHCO3. The aqueous layer was extracted with CH2Cl2, and
the organic fractions were combined and dried over MgSO4. The residue
obtained after evaporation of the solvent was purified by silica chroma-
tography (hexanes/AcOEt 4:1).
DMT and TOM protection of nucleosides (Procedure C): The corre-
sponding nucleoside was co-evaporated with anhydrous pyridine (2”)
then dissolved in anhydrous pyridine (0.1m). Diisopropylethylamine
(2 equiv) and 4,4’-dimethoxytrityl chloride (DMT-CL; 2 equiv) were
added and the mixture was stirred for 4 h at room temperature. The reac-
tion was quenched with methanol and concentrated in vacuo to yield a
yellow oil, which was eluted down a short silica gel column (pretreated
with 2% NEt3, run with CH2Cl2 then 20:1 CH2Cl2/MeOH) to remove the
excess of DMT-Cl. The crude 5’-O-DMT-protected material was dis-
solved in 1,2-dichloroethane (0.26m) and diisopropylethylamine
(3.5 equiv) and Bu2SnCl2 (1.1 equiv) were added. The reaction was stirred
under Ar at room temperature for 1 h, then at 858C for 20 min. (Triiso-
propylsiloxy)methyl chloride (TOMCl; 1.3 equiv) was added. After
20 min at the same temperature, the reaction was quenched by addition
of 5% NaHCO3 and cooled to room temperature. The mixture was
stirred for 1 h and then extracted with dichloromethane. The organic
layers were dried over MgSO4, filtered, and concentrated in vacuo. The
residue was purified by column chromatography (silica gel pretreated
with 2% NEt3, hexanes/ethyl acetate 20:1!10:1). The faster eluting 2’-
TOM isomers were isolated as foams.
2’,3’,5’-Tri-O-benzyl-1’-deoxy-b-1’-(2,4-difluorophenyl)-d-ribofuranose
(2):[31] Compound 2 was obtained as a white solid in 69% yield by follow-
ing Procedure A. 1H NMR (400 MHz, CDCl3): d=7.61 (dd, J=15.2,
8.5 Hz, 1H), 7.40–7.27 (m, 15H), 6.79 (dd, J=8.8, 2.4 Hz 1H), 6.70 (dt,
J=8.4, 2.4 Hz, 1H), 5.39 (d, J=3.8 Hz, 1H), 4.71 and 4.47 (AB system,
J=12.2 Hz, 2H), 4.65–4.55 (m, 4H), 4.39 (td, J=6.6, 3.3 Hz, 1H), 4.07
(dd, J=6.5, 5.0 Hz, 1H), 3.96 (t, J=4.4 Hz, 1H), 3.86 (dd, J=10.7,
3.1 Hz, 1H), 3.70 ppm (dd, J=10.7, 3.5 Hz, 1H).
2’,3’,5’-Tri-O-benzyl-1’-deoxy-b-1’-(2,3-dichlorophenyl)-d-ribofuranose
(3): Compound 3 was obtained as a white solid in 41% yield by following
Procedure A. 1H NMR (400 MHz, CDCl3): d=7.74 (dd, J=7.9, 1.5 Hz,
1H), 7.36–7.26 (m, 16H), 6.97 (t, J=7.9 Hz, 1H), 5.49 (d, J=2.5 Hz,
1H), 4.76 (AB system, J=11.8 Hz, 2H), 4.62 (d, J=11.8 Hz, 1H), 4.56
(dd, J=13.5, 11.8 Hz, 2H), 4.42 (d, J=11.8, 1H), 4.39 (dt, J=7.9, 2.8 Hz,
2H), 4.08 (dd, J=7.9, 4.4 Hz, 1H), 3.95 (ddd, J=13.3, 7.6, 2.5 Hz, 2H),
3.72 ppm (dd, J=10.8, 3.2 Hz, 1H). 13C NMR (126 MHz, CDCl3): d=
140.6, 138.2, 138.0, 137.7, 132.7, 129.4, 128.34, 128.33, 128.30, 127.83,
127.78, 127.73, 127.67, 127.60, 127.58, 127.3, 126.5, 81.8, 81.5, 80.0, 77.2,
73.3, 72.4, 72.3, 68.9 ppm. HRMS: m/z calcd for C26H26NaO5 [M+Na]+:
571.1413; found: 571.1435.
1’-Deoxy-b-1’-(2,4-difluorophenyl)-5-O-(4,4’-dimethoxytrityl)-2’-O-{[(tri-
AHCTREiGNU sopropylsilyl)oxy]methyl}-d-ribofuranose (9): The isolation of this deriv-
ative was more easily achieved by using the additional manipulations ex-
plained below. 1H NMR (400 MHz, CDCl3): d=7.64 (q, J=8.4 Hz, 1H),
7.50 (d, J=7.1 Hz, 2H), 7.39 (dd, J=8.9, 1.3 Hz, 4H), 7.28 (t, J=7.1 Hz,
2H), 7.21 (tt, J=7.3, 1.2 Hz, 1H), 6.82 (d, J=8.9 Hz, 4H), 6.79 (q, J=
8.5 Hz, 2H), 5.23 (d, J=5.3 Hz, 1H; C1-H,), 5.04 (AB system, J=4.7 Hz,
2H; O-CH2-O), 4.28 (q, J=5.1 Hz, 1H; C4-H), 4.15 (m, 1H; C3-H)),
4.11 (t, J=5.3 Hz, 1H; C2-H), 3.79 (s, 6H; 2CH3O), 3.48 (dd, J=10.3
and 2.9 Hz, 1H), 3.35 (dd, J=10.3, 4.2 Hz, 1H), 3.14 (d, J=4.8 Hz, 1H;
2’,3’,5’-Tri-O-benzyl-1’-deoxy-b-1’-(2,4-dichlorophenyl)-d-ribofuranose
(4): Compound 4 was obtained as a white solid in 58% yield by following
Procedure A. 1H NMR (500 MHz, CDCl3): d=7.72 (dd, J=8.5, 0.4 Hz,
1H; ArH), 7.27–7.37 (m, 18H; ArH), 6.99 (ddd, J=5.8, 2.1 Hz, 0.5, 1H;
ArH), 5.44 (d, J=3.0 Hz, 1H; H1’), 4.72 (dd, J=24.2, 11.9 Hz, 2H;
CH2OPh), 4.57 (dd, J=37.8, 11.7 Hz, 2H; CH2OPh), 4.50 (dd, J=71.9,
11.9 Hz, 2H; CH2OPh), 4.36 (dt, J=7.5, 2.8 Hz, 1H; H4’), 4.03 (dd, J=
7.5, 4.6 Hz, 1H; H3’), 3.88–3.92 (m, 2H; H2’, H5’a), 3.70 ppm (dd, J=
10.8, 3.7 Hz, 1H; H5’b); 13C NMR (126 MHz, CDCl3): d=133.8, 132.5,
130.5, 129.6, 129.0, 128.55, 128.52, 128.47, 128.44, 128.39, 127.9, 127.88,
127.85, 127.77, 127.69, 127.35, 127.32, 81.9, 80.6, 80.2, 77.7, 73.4, 72.4,
72.3, 69.1 ppm; HRMS: m/z calcd for C26H26NaO5 [M+Na]+: 571.1413;
found: 571.1422.
(iPr)3); 13C NMR (101 MHz, CDCl3): d=
OH), 1.07–1.03 ppm (m, 21H; SiCAHTREUNG
162.5 (dd, J=227.4, 11.9 Hz), 160.0 (dd, J=228.8, 11.9 Hz), 158.3 (2C),
144.8, 136.0, 135.9, 130.0 (4C), 129.2 (dd, J=9.6, 5.7 Hz), 128.2 (2C),
127.7 (2C), 126.7, 123.2 (dd, J=12.9, 3.6 Hz), 113.0 (4C), 111.2 (dd, J=
21.0, 3.5 Hz), 103.6 (t, J=25.4 Hz), 90.5, 86.1, 85.3, 83.2, 71.1, 63.5, 55.1
(2C), 46.1, 17.7 (6), 11.7 ppm (3C); 19F NMR (376 MHz, CDCl3):
ꢀ111.93 (td, J=15.1, 7.3 Hz), ꢀ114.87 ppm (16.9, 8.4 Hz); HRMS
(MALDI): m/z calcd for C42H52O7F2NaSi [M+Na+]: 757.3343; found:
757.3307.
1’-Deoxy-b-1’-(2,3-dichlorophenyl)-5-O-(4,4’-dimethoxytrityl)-2’-O-{[(tri-
Removalof the benzylprotecting groups (Procedure B) : BBr3 was added
(1m in dichloromethane, 3.5 equiv) to a solution of tribenzylated nucleo-
side in dichloromethane (0.1m) at ꢀ788C. The mixture was stirred for
3 h, then quenched with methanol at ꢀ788C and allowed to reach room
temperature. After concentration in vacuo, the resulting oil was purified
by silica column chromatography (CH2Cl2/MeOH 20:1).
AHCTREiGNU sopropylsilyl)oxy]methyl}-d-ribofuranose (10): Compound 10 was ob-
1
tained as a white foam in 33% yield by following Procedure C. H NMR
(400 MHz, CDCl3): d=7.80 (d, J=7.8 Hz, 1H), 7.52 (d, J=7.1 Hz, 2H),
7.41 (d, J=8.9 Hz, 4H), 7.40–7.36 (m, 1H) 7.29 (t, J=7.1 Hz, 2H), 7.22
(t, J=7.2 Hz, 1H), 7.06 (t, J=7.9 Hz, 1H), 6.83 (d, J=8.9 Hz, 4H), 5.43
(d, J=2.9 Hz, 1H), 5.26 (d, J=4.8 Hz, 1H), 4.98 (d, J=4.9 Hz, 1H),
4.25–4.18 (m, 1H), 4.17–4.12 (m, 1H), 4.10–4.06 (m, 1H), 3.79 (s, 6H),
3.49 (ddd , J=14.8, 10.5, 3.3 Hz, 2.), 3.48 (d, J=6.6 Hz, 1H), 1.06 ppm (
s, 21H); 13C RMN (101 MHz, CDCl3): d=158.4 (2C), 144.9, 140.5, 136.1,
135.9, 132.8, 130.2 (4C), 130.1, 129.4, 128.3 (2C), 127.8 (2C), 127.5, 126.7,
126.3, 113.1 (4C), 90.8, 86.6, 86.2, 82.4, 81.4, 70.1, 63.1, 55.2 (2C), 17.8
(4C), 17.7 (2C), 12.2, 11.8 ppm (2C); HRMS: m/z calcd for
C42H52Cl2O7NaSi [M+Na]+: 789.2757; found: 789.2747.
1’-Deoxy-b-1’-(2,4-difluorophenyl)-d-ribofuranose (rF nucleoside; 5):
Compound 5 was obtained as a white solid in 74% yield by following
Procedure B. 1H NMR (400 MHz, CDCl3): d=7.72 (dd, J=15.3, 8.2 Hz,
1H), 6.98 (t, J=8.8 Hz, 2H), 4.98 (d, J=5.8 Hz, 1H), 4.28 (AB system,
1H), 4.13 (dd, J=10.2, 5.1 Hz, 1H), 4.01 (td, J=11.3, 5.7 Hz, 1H), 3.94
(dd, J=8.3, 4.1 Hz, 1H), 3.85–3.70 ppm (m, 2H).
1’-Deoxy-b-1’-(2,3-dichlorophenyl)-d-ribofuranose (2,3-rL nucleoside; 6):
Compound 6 was obtained as a white solid in 63% yield by following
Procedure B. 1H NMR (400 MHz, CDCl3): d=7.70 (dd, J=7.8, 1.0 Hz,
1H), 7.52 (dd, J=7.9, 1.4 Hz, 1H), 7.35 ppm (t, J=7.9 Hz, 1H); 13C
RMN (101 MHz, CDCl3): d=141.3, 132.7, 130.4, 130.0, 128.4, 126.9, 87.3,
85.7, 83.1, 81.7, 62.1 ppm; HRMS: m/z calcd for C11H12Cl2NaO4
[M+Na]+: 301.0011; found: 301.0014.
1’-Deoxy-b-1’-(2,4-dichlorophenyl)-5-O-(4,4’-dimethoxytrityl)-2’-O-{[(tri-
AHCTREiGNU sopropylsilyl)oxy]methyl}-d-ribofuranose (11): Compound 11 was ob-
1
tained as a white foam in 43% yield by following Procedure C. H NMR
(500 MHz, CDCl3): d=7.79 (d, J=8.5 Hz, 1H; ArH), 7.52 (d, J=7.3 Hz,
1H; ArH), 7.39–7.42 (m, 5H; ArH), 7.29–7.32 (m, 3H; ArH), 7.25 (t, J=
7.3 Hz, 1H; ArH), 7.10 (dd, J=8.5, 2.0 Hz, 1H; ArH), 6.85 (d, J=8.8 Hz,
4H; ArH), 5.37 (d, J=3.4 Hz, 1H; H1’), 5.11 (dd, J=134, 4.8 Hz, 2H;
CH2OSi), 4.24 (app dd, J=11.9, 6.5 Hz, 1H; H4’), 4.13–4.16 (m, 1H;
H3’), 4.08 (app t, J=4.1 Hz, 1H; H2’), 3.82 (s, 6H; OCH3), 3.54 (dd, J=
10.5, 2.3 Hz, 1H; H5’a), 3.42 (dd, J=10.5, 4.3 Hz, 1H; H5’b), 3.40 (d, J=
1’-Deoxy-b-1’-(2,4-dichlorophenyl)-d-ribofuranose (rL nucleoside; 7):
Compound 7 was obtained as a white solid in 56% yield by following
Procedure B. 1H NMR (500 MHz, CD3OD): d=7.74 (d, J=8.5 Hz, 1H;
H3), 7.44 (d, J=2.1 Hz, 1H; H5), 7.32 (dd, J=8.4, 2.0 Hz, 1H; H6), 5.18
(d, J=3.9 Hz, 1H; H1’), 3.93–3.98 (m, 2H; H3’, H4’), 3.88–3.91 (m, 1H;
H5’a), 3.75–3.78 ppm (m,1H; H5’b); 13C NMR (126 MHz, CD3OD): d=
138.9, 134.9, 134.3, 130.8, 129.9, 128.5, 97.0, 85.2, 82.9, 79.0, 71.8,
62.8 ppm; HRMS: m/z calcd for C11H12Cl2NaO4 [M+Na]+: 301.0005;
found: 301.0006.
(CH3)2); 13C NMR
3.4 Hz, 1H; 3’-OH), 1.06–1.15 ppm (m, 21H; 3CHACHTREUNG
(126 MHz, CDCl3): d=158.7, 136.9, 136.2, 136.0, 133.9, 132.8, 130.4,
130.31, 130.27, 129.3, 129.1, 128.4, 127.9, 127.4, 126.8, 113.2, 90.8, 86.6,
82.8, 80.4, 70.5, 63.2, 55.1, 17.9, 11.9 ppm; HRMS: m/z calcd for
[M+Na]+: 789.2752; found: 789.2743.
C42H52Cl2O7NaSiACHTREUNG
7984
www.chemeurj.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 7978 –7987

RNA Recognition
FULL PAPER
Procedure for 2’-O-TOM-rF purification: The crude product from the
TOM protection reaction was dissolved in dichloromethane (0.2m), and
diisopropylethylamine (4 equiv), Ac2O (2 equiv) and DMAP (cat) were
added at 08C. After 15 min the mixture was allowed to reach room tem-
perature and stirred for 2 h. The reaction was quenched with NaHCO3,
extracted with CH2Cl2, dried over MgSO4, and concentrated. Alter flash
chromatography purification (hexanes/EtOAc 12:1), the 2’- and 3’-TOM-
protected isomers were isolated in 51 and 28% yield, respectively.
3.0 Hz), 111.0 (dd, J=9.9, 2.8 Hz), 103.6 (dt, J=25.5, 5.7 Hz), 90.0, 88.9,
88.7, 86.3, 86.2, 83.2 (m), 80.5, 79.8, 79.7, 76.2, 72.2, 72.1, 71.8, 71.7, 63.6,
63.1, 58.9, 58.8, 57.9, 57.8, 55.2, 55.1, 43.3, 43.2, 43.0, 42.9, 24.5 (m), 20.6,
20.3, 20.2, 20.0, 19.9, 17.7, 11.8 ppm; 19F NMR (376 MHz, CDCl3): d=
ꢀ112.19 (td, J=15.4, 7.7 Hz; major isomer), ꢀ114.10 (td, J=15.6, 7.9 Hz;
minor isomer), ꢀ114.38 ppm (dd, J=17.7, 8.0 Hz); 31P NMR (162 MHz,
CDCl3): d=148.34 (s; major isomer), 148.11 ppm (s; minor isomer);
HRMS (MALDI): m/z calcd for C51H69N2O8F2NaSiP [M+Na]+:
957.4421; found: 957.4389.
3’-O-Acetyl-1-deoxy-1-(2,4-difluorophenyl)-5’-O-(4,4’-dimethoxytrityl)-2’-
O-{[(triisopropylsilyl)oxy]methyl}-b-d-ribofuranose: 1H NMR (400 MHz,
CDCl3): d=7.68 (q, J=14.7, 8.0 Hz, 1H), 7.54 (d, J=7.3 Hz, 2H), 7.43
(dd, J=8.6, 1.6 Hz, 4H), 7.32 (t, J=7.7 Hz, 2H), 7.25 (t, J=7.4 Hz, 1H),
6.87 (d, J=8.8 Hz,4H), 6.90–6.80 (m, 2H), 5.40 (dd, J=5.0, 4.0 Hz, 1H),
5.27 (d, J=7.2 Hz, 1H), 4.93 (s, 2H), 4.54 (dd, J=7.1, 5.4 Hz, 1H), 4.30
(q, J=3.5 Hz, 1H), 3.79 (s, 6H), 3.54 (dd, J=10.4, 3.3 Hz, 1H), 3.41 (dd,
J=10.4, 3.7 Hz, 1H), 2.14 (s, 3H), 1.01 ppm (s, 21H); 13C NMR
1’-Deoxy-b-1’-(2,3-dichlorophenyl)-5-O-(4,4’-dimethoxytrityl)-2’-O-{[tri-
AHCTREiUGN sopropylsilyl)oxy]methyl}-d-ribofuranose-3’-O-cyanoethyl-N,N-diisopro-
pylphosphoramidite (14): Compound 14 was obtained as white foam in
70% yield by following Procedure D. 1H NMR (400 MHz, CDCl3): 7.79
and 7.72 (dd, J=7.8, 1.5 Hz, 1H), 7.49 (t, J=7.0 Hz, 2H), 7.38 (dt, J=
8.9, 2.4 Hz, 5H), 7.32–7.18 (m, 3H), 7.08–7.00 (m, 1H), 6.82 (t, J=
8.4 Hz, 4H), 5.52–5.59 (m, 1H), 5.05–5.92 (AB systems, 2H), 4.41–4.18
(m, 2H), 3.79 and 3.78 (2s, 6H), 3.71–3.46 (m, 5H), 3.31 (dt, J=8.9,
3.9 Hz, 1H), 2.71–2.56 (m, 2H), 2.30 (t, J=6.7 Hz, 1H), 1.17–1.07 (m,
12H), 0.98 and 0.97 ppm (2s, 21H); 13C NMR (126 MHz, CDCl3): 158.4,
144.6, 140.4, 140.2, 135.9–135.8, 132.8, 132.8, 131.3, 131.0, 130.2, 130.1,
129.5, 129.4, 128.4, 128.3, 127.8, 127.3, 127.3, 126.8, 126.7, 126.5, 126.4,
117.6, 117.4, 113.0, 89.2, 86.3, 82.7, 81.6, 80.3, 79.7, 71.8, 71.5, 63.2, 62.7,
58.8, 58.7, 57.9, 57.8, 57.2, 56.9, 55.2, 43.3, 42.2, 29.7, 24.5, 20.3, 20.0, 17.7,
12.6, 11.9 ppm; 31P NMR (162 MHz, CDCl3): d=150.75 (s; major
isomer), 150.58 ppm (s; minor isomer).
ꢀ
(100 MHz, CDCl3): d=170.1 (C=O), 162.9 (C F, dd, J=182.9, 12.0 Hz),
ꢀ
ꢀ
160.4 (C F, dd, J=184.5, 11.9 Hz), 158.4 (2C, C OMe), 144.6, 135.8,
135.7, 130.0 (2C), 129.9 (2C), 129.5 (dd, J=9.7, 5.2 Hz), 128.1 (2C), 127.7
(2C), 126.7, 122.3 (dd, J=12.7, 3.7 Hz) 113.0 (4C), 111.2 (dd, J=20.9,
3.4 Hz), 103.5 (t, J=25.6 Hz), 88.9, 86.3, 81.8, 79.4, 76.0, 75.9, 72.4, 63.2,
54.9 (2C), 20.8, 17.6 (6C), 11.6 ppm (3C); 19F NMR (376 MHz, CDCl3):
ꢀ111.74 (td, J=15.6, 8.0 Hz), ꢀ114.65 ppm (dd, J=15.6, 7.9 Hz); HRMS
(MALDI +): m/z calcd for C44H54O8F2NaSi [M+Na+]: 799.3448; found:
799.3461.
2’-O-Acetyl-1-deoxy-1-(2,4-difluorophenyl)-5’-O-(4,4’-dimethoxytrityl)-3’-
O-{[(triisopropylsilyl)oxy] methyl}-b-d-ribofuranose: H NMR (400 MHz,
1’-Deoxy-b-1’-(2,4-dichlorophenyl)-5-O-(4,4’-dimethoxytrityl)-2’-O-{[tri-
AHCTREiUGN sopropylsilyl)oxy]methyl}-d-ribofuranose-3’-O-cyanoethyl-N,N-diisopro-
1
CDCl3): d=7.57 (dd, J=15.5, 7.5 Hz, 1H), 7.50 (d, J=8.0 Hz, 2H), 7.38
(d, J=8.8 Hz, 4H), 7.28 (t, J=8.0 Hz, 2H), 7.21 (t, J=7.5H; 1H), 6.82
(d, J=8.8 Hz, 4H), 6.81–6.35 (m, 2H), 5.29 (d, J=5.4 Hz, 1H), 5.21 (t,
J=5.4 Hz, 1H), 4.89 (d, J=4.8 Hz, 1H), 4.76 (d, J=4.8 Hz, 1H), 4.42–
4.37 (m, 1H), 4.36–4.32 (m, 1H), 3.79 (s, 6H), 3.54 (d, J=10.7 Hz, 1H),
3.35 (dd, J=10.4, 4.8 Hz, 1H), 2.12 (s, 3H), 1.15 (s, 3H), 0.96 ppm (s,
18H); 13C NMR (100 MHz, CDCl3): d=169.9, 162.6 (dd, J=238.3,
11.8 Hz), 160.2 (dd, J=240.4, 12.1 Hz), 158.4 (2C), 144.7, 135.9, 135.8,
130.2 (4C), 128.9 (dd, J=10.7, 6.7 Hz), 128.3 (2C), 127.7 (2C), 126.7,
122.5 (dd, J=13.1, 3.7 Hz), 113.0 (4C), 111.3 (dd, J=20.9, 3.5 Hz), 103.7
(t, J=25.4 Hz), 89.4, 86.2, 81.9, 76.6, 76.3, 74.7, 63.4, 54.1 (2C), 20.8, 17.7
(6C), 11.7 ppm (3C); 19F NMR (376 MHz, CDCl3) : ꢀ111.54 (td, J=15.3,
7. 7 Hz), ꢀ114.59 (dd, J=17.6, 8.2 Hz); HRMS (MALDI+): m/z calcd
for C44H54O8F2NaSi [M+Na]+: 799.3448; found: 799.3450.
pylphosphoramidite (15): Compound 15 was obtained as white foam in
90% yield by following Procedure D. 1H NMR (500 MHz, CDCl3): d=
7.76 (d, J=8.4 Hz, 1H; ArH), 7.52–7.46 (m, 2H; ArH), 7.39–7.20 (m,
8H; ArH), 7.09–7.05 (m, 1H; ArH), 6.83 (d, J=8.9 Hz, 4H; ArH), 5.42
(d, J=5.16 Hz, 1H; H1’), 5.02–4.92 (m, 2H; OCH2OSi), 4.40–4.38 (m,
3H; H2’, H3’, H4’), 3.80 (s, 6H; OCH3), 3.55 (m, 2H; CH2CN), 2.31 (t,
J=6.62 Hz, 2H; OCH2), 1.13 (m, 14H; CH
ACHTRE(UNG CH3)2), 0.97 ppm (s, 21H;
SiCH
AHCTREUNG
130.5–126.8, 113.1, 86.5, 80.8, 63.0, 58.2, 55.2, 43.3, 24.7, 17.7, 11.8 ppm;
31P NMR (126 MHz, CDCl3): d=150.80, 150.75 ppm; HRMS: m/z calcd
for C51H70N2O8SiPCl2 [M+H]+: 967.4016; found: 967.4037.
1-[5-O-(4,4’-Dimethoxytriphenylmethyl)-b-d-erythropentofuranosyl]-4-
methyl-1H-benzimidazole: Diisopropylethylamine (1.9 mL, 10.9 mMol)
and 4,4’-dimethoxytrityl chloride (3.6 g, 10.6 mMol) were added to a solu-
tion of benzimidazole free nucleoside (1.4 g, 5.30 mmol) in pyridine
(50 mL) at 08C. The mixture was stirred at room temperature for 4 h,
then it was quenched with methanol, concentrated in vacuo and purified
by silica column chromatography (CH2Cl2/MeOH, 20:1; NEt3 2%) to
yield a white foam (50%). 1H NMR (400 MHz, CDCl3): d=7.68 (d, J=
2.1 Hz, 1H), 7.50 (d, J=8.0 Hz, 1H), 7.43 (dd, J=8.1, 1.3 Hz, 2H), 7.31
(dd, J=8.9, 2.1 Hz, 4H), 7.25–7.16 (m, 3H), 6.86 (d, J=7.3 Hz, 1H), 6.81
(d, J=7.8 Hz, 1H), 6.76 (dd, J=8.9, 5.5 Hz, 4H), 5.81 (d, J=6.4 Hz, 1H),
4.72 (t, J=6.1 Hz, 1H), 4.50 (dd, J=5.8, 3.1 Hz, 1H), 4.27 (q, J=3.2 Hz,
1H), 3.76 and 3.75 (2 s, 6H), 3.46 (d, J=3.3 Hz, 2H), 2.43 ppm (s, 3H);
13C NMR (100 MHz, CDCl3): d=158.5 (2C), 144.5, 142.1, 140.6, 135.6,
135.4, 131.7, 130.1 (4C), 129.1, 128.1 (2C), 127.9 (2C), 126.9, 123.5, 123.4,
113.1 (4C), 109.7, 90.1, 86.6, 84.0, 72.8, 71.1, 63.5, 55.2 (2C), 16.6 ppm;
HRMS: m/z calcd for C34H35N2O6 [M+H]+: 567.2495; found: 567.2508.
The 2’-TOM-protected isomer was dissolved in CH2Cl2 (0.6m) and a solu-
tion of NH3 (in MeOH 7m) was added to make a 0.04m solution. The
mixture was stirred for 10 h at room temperature, then it was concentrat-
ed in vacuo and purified by silica column chromatography (hexanes/
EtOAc 10:1) affording the alcohol 9 as a white foam in 86% yield.
Synthesis of phosphoramidites (Procedure D): Diisopropylethylamine
(3 equiv)
and
2-cyanoethyl-N,N-diisopropylchlorophosphoramidite
(1.5 equiv) were added to a solution of the corresponding 5’-O-DMT-2’-
O-TOM-protected nucleoside in dichloromethane (0.2m) at 08C. The re-
action mixture was stirred at room temperature for 3 h, then the mixture
was loaded directly onto a silica gel column for purification (hexanes/
ethyl acetate 6:1, +2% NEt3).
1’-Deoxy-b-1’-(2,4-difluorophenyl)-5-O-(4,4’-dimethoxytrityl)-2’-O-{[tri-
ACHTREUNGisopropylsilyl)oxy]methyl}-d-ribofuranose-3’-O-cyanoethyl-N,N-diisopro-
1-Deoxy-5-O-(4,4’-dimethoxytrityl)-1’-(4-methyl-1H-benzimidazolyl)-2’-
O-{[(triisopropylsilyl)oxy]methyl}-b-d-erythropentofuranose (12): Diiso-
propylethylamine (1 mL, 5.74 mMol) and Bu2SnCl2 (550 mg, 1.81 mMol)
were added to a solution of the DMT-protected benzimidazole derivative
(927 mg, 1.64 mMol) in dichloromethane (6.6 mL). The mixture was
stirred at room temperature for 2 h. Then it was heated to 858C for
20 min and (Triisopropylsiloxy)methyl chloride (494 mL, 2.13 mMol) was
added. The mixture was stirred at the same temperature for 20 min,
quenched by addition of 5% NaHCO3 and cooled to room temperature.
The mixture was stirred for 1 h, then extracted with dichloromethane.
The organic layers were dried over MgSO4, filtered and concentrated in
vacuo. The residue was purified by column chromatography (silica gel
pretreated with 2% NEt3, hexanes/ethyl acetate 2:1). The faster eluting
pylphosphoramidite (13): Compound 13 was obtained as white foam in
91% yield by following Procedure D. 1H NMR (400 MHz, CDCl3): d=
7.67–7.55 (m, 1H), 7.48 (t, J=6.9 Hz, 2H), 7.37 (dt, J=8.9, 1.1 Hz, 4H),
7.31–7.18 (m, 3H), 6.81 (t, J=8.4 Hz, 4H), 6.78–6.71 (m, 2H), 5.23 (d,
J=6.3 Hz, 1H), 4.96 (d, J=5.1 Hz, 1H), 4.92 (dd, J=7.8, 5.9 Hz, 2H),
5.89 (d, J=5.1 Hz, 1H), 4.37 (ddd, J=14.1, 4.4, 4.4 Hz, 1H), 4.28 (m,
1H), 3.97–3.80 (m, 1H), 3.78 and 3.77 (2 s, 6H), 3.62–3.47 (m, 2H), 3.44
(dd, J=10.3, 3.3 Hz, 1H), 3.26 (ddd, J=10.6, 6.6, 4.3 Hz, 1H), 2.64 (dd,
J=12.6, 6.4 Hz, 1H), 2.30 (t, J=6.7 Hz, 1H), 1.14 (d, J=6.7 Hz, 12H),
0.95 ppm (s, 21H); 13C NMR (126 MHz, CDCl3): d=163.5, 161.5 and
169.7 (3m), 158.4, 144.8, 144.7, 136.0, 135.9, 135.9, 135.8, 130.2, 130.1,
129.5 (m), 128.4, 128.3, 127.7, 126.8, 126.7, 124.8, 123.2 (dd, J=12.9,
3.7 Hz), 123.0 (dd, J=12.9, 3.0 Hz), 117.7, 117.4, 113.0, 111.2 (dd, J=9.3,
Chem. Eur. J. 2008, 14, 7978 –7987
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
7985

E. T. Kool et al.
2’-TOM isomer was isolated as foam (9%). 1H NMR (400 MHz,
[D6]acetone): d=8.25 (s, 1H), 7.54 (d, J=6.8 Hz, 1H), 7.49 (d, J=8.2 Hz,
2H), 7.37 (d, J=8.4 Hz, 4H), 7.29 (tt, J=8.3, 1.7 Hz, 2H), 7.22 (tt, J=
6.2, 1.3 Hz, 1H), 7.05–6.99 (m, 2H), 6.85 (dd, J=9.0, 1.2 Hz, 4H), 6.13
(d, J=5.9 Hz, 1H), 5.09 (AB system, J=5.2 Hz, 2H), 4.82 (t, J=5.6 Hz,
1H), 4.61 (dd, J=9.3, 4.7 Hz, 1H), 4.27 (q, J=3.8 Hz, 1H), 4.14 (d, J=
4.6 Hz, 1H), 3.77 and 3.76 (2 s, 6H), 3.44 (d, J=3.6 Hz, 2H), 2.58 (s,
3H), 1.03–0.95 ppm (m, 21H); 13C NMR (100 MHz, [D6]acetone): d=
160.6 (2C), 146.9, 145.7, 142.6, 137.6, 137.5, 134.6, 132.0 (2C), 131.9 (2C),
131.6, 129.9 (2C), 129.6 (2C), 128.6, 124.5, 124.2, 114.9 (4C) 110.9, 91.8,
89.6, 88.2, 85.8, 81.6, 71.9, 65.4, 56.4 (2C), 19.1 (6C), 17.7, 13.5 ppm (3C);
HRMS: m/z calcd for C44H57N2O7Si [M+H]+: 753.3935; found: 753.3941.
rently two-state transitions. The data were analyzed by the denaturation
curve-processing program, MeltWin v. 3.0. Melting temperatures (Tm)
were determined by computer-fit of the first derivative of absorbance
with respect to 1/T.
Preparation of mutant luciferase plasmid vectors: The luciferase gene in
the pRL CMV vector (Promega, Madison, WI) was mutated at position
1580 and 1769 to create three different mutant constructs at both posi-
tions that differ from the wild type by single nucleotide substitutions.
These substitutions do not create a corresponding change in amino acid
residue in the luciferase gene, but retain the same amino acid residue,
glycine, as in the wild-type construct. By using three pairs of designed
primers (Supplementary), one pair for each mutant (A to T, A to C, and
A to G), mutant gene constructs were made with the site-directed muta-
genesis kit, (Stratagene, La Jolla, CA) following the protocol provided by
the manufacturer. The resulting constructs were sequenced to confirm
the required mutations in the gene (Supporting Information).
1-Deoxy-5-O-(4,4’-dimethoxytrityl)-1’-(4-methyl-1H-benzimidazolyl)-2’-
O-{[(triisopropylsilyl)oxy]methyl}-b-d-erythropentofuranose-3’-O-cyano-
A
(16):
Diisopropylethylamine
A
RNA interference methods: Hela cells were grown at 378C, 5% CO2 in
Dulbeccoꢁs modified Eaglesꢁs medium (DMEM, GIBCO) supplemented
with 10% fetal bovine serum (FBS), 100 UmLꢀ1 penicillin and
100 mgmLꢀ1 streptomycin. The cells were maintained in exponential
growth. The cells were plated in 24-well plates (0.5 mL medium per well)
to reach about 50% confluence at transfection. The cells were grown for
24 h and the culture medium was changed to OPTIMEM 1 (GIBCO),
0.5 mL per well. Two luciferase plasmids, Renilla luciferase (pRL-CMV)
and firefly luciferase (pGL3) from Promega, were used as reporter and
control, respectively. Co-transfection of plasmids and siRNAs was carried
out with GeneSilencer (GTS) as described by the manufacturer for ad-
herent cell lines. Per well, 0.17 mg pGL3, 0.017 mg pRL-CMV and 2.1 ng
siRNAs, formulated into liposomes, were applied. The final volume was
500 mL per well. The cells were harvested 22 h after transfection, and
lysed by using passive lysis buffer (PLB), 100 mL per well, according to
the instructions of the Dual-Luciferase Reporter Assay System (Prom-
ega, USA). The luciferase activities of the samples were measured using
a Fluoroskan Ascent FL Luminometer (Thermo Electron Corporation,
USA) with a delay time of 2 s and an integrate time of 10 s. The volumes
used were: 20 mL of sample and 30 mL of each reagent (luciferase assay
reagent II and Stop & Glo Reagent). The inhibitory effects generated by
siRNAs were expressed as normalized ratios between the activities of the
reporter (Renilla) luciferase gene and the control (firefly) luciferase
gene.
(15 mL) at 08C. The mixture was stirred for 2 h at room temperature, the
solvent was evaporated, and the crude reaction was purified by silica
column chromatography (hexanes/AcOEt 2:1, Et3N 2%). The corre-
1
sponding phosphoramidite was isolated as a white foam (76%). H NMR
(400 MHz, CDCl3): d=8.14 and 8.12 (2 s, 1H), 7.51–7.46 (m, 3H), 7.34 (t,
J=8.9 Hz, 4H), 7.30–7.17 (m, 3H), 7.07–6.98 (m, 2H), 6.81 (t, J=8.7 Hz,
4H), 6.09 and 6.05 (d, J=6.0 Hz, 1H), 4.96–4.88 (m, 2H), 4.76 and 4.72
(t, J=5.6 Hz, 1H), 4.58–4.48 (m, 1H), 4.39 and 4.35 (q, J=3.4 Hz, 1H),
3.96–3.80 (m, 2H), 3.78, 3.77, 3.76 and 3.75 (4 s, 6H), 3.66–3.52 (m, 3H),
3.49 and 3.46 (d, J=3.1 Hz, 1H), 3.40–3.44 (m, 1H), 2.66 (s, 3H), 2.65–
2.60 (m, 1H), 2.31 (dt, J=6.5, 1.8 Hz, 1H), 1.18 (t, J=6.0, 8H), 1.03 (d,
J=6.7 Hz, 4H), 0.92 and 0.89 ppm (2s, 20H); 13C NMR (100 MHz,
CDCl3): d=158.4, 144.4, 144.3, 143.5, 143.4, 140.2, 140.1, 135.6, 135.5,
135.4, 132.4, 132.4, 132.3, 130.1, 130.0, 128.2, 128.1, 127.8, 126.9, 126.8,
123.0, 122.9, 122.8, 122.7, 117.5, 117.2, 113.1, 108.9, 108.7, 89.6, 89.5, 89.2,
89.1, 88.1, 87.9, 86.6, 86.5, 83.7, 83.6, 83.3, 83.3, 78.1, 78.0, 77.3, 77.2, 71.4,
71.3, 71.1, 70.9, 63.2, 62.8, 58.9, 58.7, 57.9, 57.7, 55.1, 43.4, 43.2, 43.1, 43.0,
24.6, 24.5, 24.4, 17.6, 17.5, 16.6, 11.7 ppm; 31P NMR (126 MHz, CDCl3):
d=151.98, 151.35 ppm; HRMS: m/z calcd for C53H74N4O9SiP
[M+H+O]+ (oxidized): 969.4963; found: 969.4970.
RNA synthesis and purification methods: RNA oligonucleotides were
synthesized BY using 2’-TOM phosphoramidites. Acetonitrile (synthesis
grade), the standard 2’-TOM phosphoramidites for A, C, G, and U, 5-eth-
ylthio-1H-tetrazole, and other standard solutions were obtained from
commercial suppliers. RNA oligonucleotides were synthesized on the
1 mmol scale. The standard coupling time of 6 min was used for the four
standard phosphoramidites, and an increased coupling time of 10 min
was used for the modified phosphoramidites. After the solid-phase syn-
thesis, the solid support was transferred to a screw-cap glass vial and in-
cubated at room temperature for 16 h with 1.5 mL of methylamine solu-
tion, prepared by mixing equal volumes of 40% aqueous methylamine
and 33% methylamine in ethanol. After the vial was cooled briefly on
ice, the supernatant was transferred by pipette into 2 mL Eppendorf
tubes; the solid support and vial were rinsed with 50% ethanol (2”
0.25 mL). The combined solutions were evaporated to dryness using an
evaporating centrifuge. The residue was dissolved in a total volume of
1.0 mL of TBAF (1m) in THF and rocked at 378C for 12 h. Tris·HCl
(1 mL, 1m), pH 7.5, was added to the solution, and the oligonucleotide
was desalted on a NAP-10 column by using water as the eluent and
evaporated to dryness. The oligonucleotides were purified by 20% dena-
turing PAGE, isolated by the crush and soak method and quantitated by
absorbance at 260 nm. Mutated passenger strands (C, G and U) were
synthesized by Integrated DNA Technologies (Coralville, IA).
Acknowledgements
This work was supported by the US National Institutes of Health
(GM072705). A.S. acknowledges support from Marie Curie OIF Fellow-
ship (6th Framework Program, UE). A.P.S. acknowledges support from a
National Science Foundation Graduate Fellowship and a Lieberman Fel-
lowship. R.M.M. acknowledges the Bing Program for a fellowship.
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Received: May 2, 2008
Published online: July 15, 2008
Chem. Eur. J. 2008, 14, 7978 –7987
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
7987