Synthesis and properties of 2′-O-neopentyl modified oligonucleotides

Article abstract of DOI:10.1039/c2ob26871j

2′-O-Neopentyldeoxyuridine (Un) was synthesized and incorporated into a series of oligodeoxyribonucleotides. Single and triple incorporations in various arrangements were performed. The Watson and Crick pairing properties with complementary DNA and RNA were investigated by UV melting curves, CD spectroscopy, and molecular dynamic simulations. The results were compared to those obtained with DNA-DNA and DNA-RNA duplexes involving dU at the same positions. Oligonucleotides containing Un clearly demonstrated their ability to form duplexes with both complementary DNA and RNA but with higher stabilities for the DNA-RNA duplexes similar to the one of the parent DNA-RNA duplex. Investigations into the thermodynamic properties of these 17-base-pair duplexes revealed ΔG values (37 °C) that are in line with the measured T _m values for both the DNA-DNA and DNA-RNA duplexes. CD spectroscopic structural investigations indicated that the conformations of the DNA-DNA and DNA-RNA duplexes involving Un are similar to those of the dT-rA and dU-rA containing duplexes. Only small changes in intensities and weak blue shifts were observed when three Uns were incorporated into the duplexes. The results of the molecular dynamic simulations showed, for the six duplexes involving the modified nucleoside Un, calculated curvatures similar to those of the corresponding unmodified duplexes without base-pair disruption. The neopentyl group is able to be accommodated in the minor grooves of both the DNA-DNA and RNA-DNA duplexes. However, molecular dynamic simulations indicated that the Uns adopt a C2′-exo sugar pucker conformation close to an A-helix type without perturbing the C2′-endo sugar pucker conformations of their 2′-deoxynucleoside neighbours. These results confirm the potential of 2′-O-neopentyldeoxyuridine as a nucleoside surrogate for oligonucleotide based therapeutic strategies.

Full text of DOI:10.1039/c2ob26871j

Organic &
Biomolecular Chemistry
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Synthesis and properties of 2’-O-neopentyl modified
oligonucleotides†
Cite this: Org. Biomol. Chem., 2013, 11,
1345
Gérald Mathis,a,b Stéphane Bourg,a,c Samia Aci-Sèche,a Jean-Christophe Truffertb
and Ulysse Asseline*a
2’-O-Neopentyldeoxyuridine (Un) was synthesized and incorporated into a series of oligodeoxyribo-
nucleotides. Single and triple incorporations in various arrangements were performed. The Watson and
Crick pairing properties with complementary DNA and RNA were investigated by UV melting curves,
CD spectroscopy, and molecular dynamic simulations. The results were compared to those obtained with
DNA–DNA and DNA–RNA duplexes involving dU at the same positions. Oligonucleotides containing Un
clearly demonstrated their ability to form duplexes with both complementary DNA and RNA but with
higher stabilities for the DNA–RNA duplexes similar to the one of the parent DNA–RNA duplex. Investi-
gations into the thermodynamic properties of these 17-base-pair duplexes revealed ΔG values (37 °C)
that are in line with the measured Tm values for both the DNA–DNA and DNA–RNA duplexes. CD spectro-
scopic structural investigations indicated that the conformations of the DNA–DNA and DNA–RNA
duplexes involving Un are similar to those of the dT–rA and dU–rA containing duplexes. Only small
changes in intensities and weak blue shifts were observed when three Uns were incorporated into the
duplexes. The results of the molecular dynamic simulations showed, for the six duplexes involving the
modified nucleoside Un, calculated curvatures similar to those of the corresponding unmodified duplexes
without base-pair disruption. The neopentyl group is able to be accommodated in the minor grooves of
both the DNA–DNA and RNA–DNA duplexes. However, molecular dynamic simulations indicated that the
Uns adopt a C2’-exo sugar pucker conformation close to an A-helix type without perturbing the C2’-
endo sugar pucker conformations of their 2’-deoxynucleoside neighbours. These results confirm the
potential of 2’-O-neopentyldeoxyuridine as a nucleoside surrogate for oligonucleotide based therapeutic
strategies.
Received 23rd September 2012,
Accepted 10th December 2012
DOI: 10.1039/c2ob26871j
www.rsc.org/obc
double-stranded oligoribonucleotides via the interference
Introduction
(RNAi) pathway.15 RNA interference involves complex biologi-
Chemically modified oligonucleotides (ONs) are powerful cal gene-silencing mechanisms and homogeneously increased
research tools for gene structure, regulation and function stability of chemically modified oligoribonucleotides to their
analysis as well as promising therapeutic agents.1–13 It Has target may reduce their efficiency by impairing guide strand
been known for decades that modulation of gene expression dissociation. In addition to the common properties (resistance
can be achieved by targeting messenger RNAs with single- to nucleases, cell-specific delivery, efficient uptake and ade-
stranded chemically modified ONs (antisense-strategy).14 More quate intracellular distribution), efficient double-stranded
recently, it has been demonstrated that inhibition of gene chemically modified siRNAs require fine tuning of their
expression in mammalian cells can be efficiently achieved by binding affinity along the sequences. The rapid development
of this strategy has benefited from the different structural
modifications of ONs previously selected for the antisense
aCentre de Biophysique Moléculaire UPR 4301 CNRS, Conventionnée avec
strategy and potent siRNA have been selected.16–33 However,
l’Université d’Orléans, Rue Charles Sadron, 45071 Orléans Cedex 02, France.
this strategy triggers unintended gene silencing due to the
E-mail: ulysse.asseline@cnrs-orleans.fr; Fax: +33-2-38-63-15-17
bNucleoSyn. Pépinières d’entreprises – Centre Innovation 16, Rue Léonard de Vinci,
inherent miRNA-like behaviour of double-stranded RNAs. It
has been recently shown that chemical modifications of
siRNAs at specific positions can reduce off-targeting.8,22–27,30,32
The most efficient is the strongly destabilizing unlocked
nucleic acid (UNA).26 A few modifications have been tested. We
do believe that other new chemically modified nucleosides, to
45074 Orléans Cedex 2, France
cInstitut de Chimie Organique et Analytique, Université d’Orléans, UMR 7311 CNRS,
rue de Chartres, 45067 Orléans Cedex 2, France
†Electronic supplementary information (ESI) available: 1H NMR, 13C NMR,
31P NMR spectra. HPLC and MS characterisations for ONs and Fig. 6–8. See DOI:
10.1039/c2ob26871j
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be identified, may be used to finely tune the hybridization
properties of chemically modified siRNA. Previous studies
indicated that 2′-modified ONs appear to be promising due
to their favourable hybridization with complementary
RNA.6,16,17,30–37 More often, such modifications force the ONs
to adopt a higher proportion of the northern conformation in
a helical structure, which is one of the contributing factors of
the stronger hybridization property of these modified ONs.
The binding affinity of the modified ONs is also dependent on
the size of the alkoxy group. We focused on the neopentyl
group that to our knowledge has never been reported for
2′-substitution of nucleosides. We made the hypothesis that
this bulky group could be accommodated in the minor groove
upon hybridization of the ON with its RNA target.
Here, we report the synthesis of 2′-O-neopentyldeoxyuridine
(Un) and study the effect of its single and multiple incorpor-
ations, in different arrangements, into a series of 17-mer oligo-
deoxyribonucleotides on their binding properties with DNA
and RNA targets. The thermal and thermodynamic transition
profiles of duplexes with complementary DNA and RNA were
evaluated. Owing to the structural differences between Un and
dT, in order to assess the contribution of the 2′-O-neopentyl
group to duplex stability, a series of ONs involving dU in the
same positions was also studied. A study of the helix confor-
mation of duplexes with different Un contents was carried out
by CD spectroscopy and molecular dynamic simulations.
Fig. 2 Sequences of the DNA and RNA targets 1 and 2, the reference ON 3
and the modified ONs 4–11.
nucleoside, the binding properties of ONs involving either one
(ONs 8 and 9) or three dU incorporations (ONs 10 and 11) at
the same positions along with the unmodified ON (ON 3) used
as a reference were also studied. 23-mer DNA (ON 1) and RNA
(ON 2) targets were chosen so that, upon hybridization, three
nucleotides would overhang on each side of the duplexes in
order to mimic the interaction of ONs with full length
targets.38,39 The structures of the modified nucleosides Un and
dU used in this study are shown in Fig. 1 and those of the ONs
1–11 are depicted in Fig. 2.
Results and discussion
Structures and experimental design
The neopentyl group was linked to the 2′-position of 2′-deoxy-
uridine and the modified nucleoside was incorporated in
various arrangements into a 17-mer sequence (Fig. 1 and 2).
A single incorporation was performed at either the 5′-end or
the internal position of the sequence (ONs 4 and 5). Multiple
insertions (three) were performed either in a continuous row
(ON 6) or spaced by natural nucleosides (ON 7). For a compre-
hensive comparison of the properties of the new modified
Synthesis
Synthesis of the 2′-O-neopentyldeoxyuridine and its phos-
phoramidite building block. Several methods for the 2′-alkyl-
ation of uridine have been reported previously. Most of them
include the protection of 5′- and 3′-hydroxyl groups as well as
the N-3 position of the nucleic base to make the alkylation of
the 2′-position specific.40,41 We chose to proceed without pro-
tection of the nucleoside following a strategy adapted from
literature reports (Scheme 1).42,43 2,2′-Anhydro-1-β-D-arabino-
furanosyluracil 13 was obtained by reaction of uridine (12)
with diphenylcarbonate44 and reacted with the trimethylsilyl
ether of neopentyl 1545 in the presence of BF3–Et2O as the
Lewis acid for the activation of the cyclic ether bond
(Scheme 1). By using two-fold excess of BF3–Et2O and five-fold
excess of the trimethylsilyl ether in DMF at 120 °C for 5 days
the Un 16 was obtained with an 18% yield after purification.
The phosphoramidite building block 18 was obtained by selec-
tive 5′-O-dimethoxytritylation of the unprotected modified
nucleoside 16 with DMTCl in pyridine (compound 17) followed
by reaction with 2-cyanoethyltetraisopropylphosphorodi-
amidite in the presence of 4,5-dicyanoimidazole with a 50%
yield after purification. (Experimental procedures are given
vide infra.)
Fig. 1 Chemical structures of 2’-O-neopentyldeoxyuridine (Un), 2’-deoxy-
uridine (dU) and thymidine (dT).
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Scheme 1 Synthesis of the modified nucleoside 16 and the phosphoramidite derivative 18. Reagents and conditions: (i) diphenylcarbonate, DMF, 85 °C;
(ii) NaHCO3, 120 °C, 4 h; (iii) TMS-Cl, TEA, Et2O, 0 °C, 30 min; (iv) rt, 72 h; (v) BF3–Et2O, DMF, 120 °C, 120 h; (vi) DMTrCl, Py; (vii): 2-cyanoethyltetraisopropylphosphoro-
diamidite, 4,5-dicyanoimidazole, CH3CN, CH2Cl2.
Synthesis of the oligonucleotides 4–7. The structures of the
ONs synthesized are given in Fig. 2. The coupling of 2′-O-neo-
pentyldeoxyuridine phosphoramidite 18 was performed using
standard procedures. The main change to the typical synthesis
was the use of longer coupling times (10 min). The coupling
yields were around 97–98% based on a trityl cation assay. The
deprotection step was performed using standard conditions
(see the Experimental section). After purification by reversed-
phase chromatography, the purity of all oligomers described
was verified by reversed-phase analysis. Retention times of
modified ONs are higher than that of the unmodified ON and
increased with the number of Un incorporations (ESI†). These
results indicated the presence of more lipophilic compounds.
The retention times are also dependent on the position of
incorporation of the modified nucleoside. As previously
reported with other nucleoside analogues46 the presence of
the modified nucleoside at the 5′-end (ON 4) induces an
increased retention time as compared to its incorporation at
the internal position of the sequence (ON 5). The ON 6 invol-
ving three contiguous modified nucleosides is also largely
more retained than ON 7 containing also three modified
nucleosides but separated by at least two intervening natural
nucleosides. The integrity of all ONs was confirmed by MALDI-
TOF mass spectrometry (ESI†).
Thermal denaturation studies
The influence of dT replacement (one or three) by 2′-O-neo-
pentyldeoxyuridine (Un) on duplex stability was investigated by
thermal denaturation studies, followed by absorption spectro-
scopy, of DNA–DNA and RNA–DNA duplexes obtained by
mixing modified ONs 4–7 with 23-mer single-stranded DNA
(ON 1) and RNA (ON 2) target sequences (X = A) (Fig. 2). For a
better understanding of the properties of the new modified
nucleoside, the binding properties of ONs 8–11 involving dU
incorporations at the same positions were also studied. The
Fig. 3 UV melting curves (260 nm) for DNA–DNA duplexes (a): 1 [5’d-(TAC ACT
purity of ONs 1–3, 8–11 (from commercial sources) has been
verified by reversed-phase chromatography and MALDI-TOF
spectrometry before use (data not shown). The melting curves
AAA ACT GGT AAC AAC TC)3’ + ONs 3–7, and DNA–RNA duplexes (b): 2 [5’r-
(UAC ACU AAA ACU GGU AAC AAC UC)3’ + ONs 3 (diamond), 4 (up triangle),
5 (square), 6 (circle) and 7 (cross) in a 10 mM sodium phosphate, pH 7, buffer
of the DNA–DNA and RNA–DNA duplexes containing the modi-
containing 150 mM NaCl and 1 mM EDTA. Concentrations were 1 μM for each
fied ONs 4–7 and the parent duplexes are shown in Fig. 3. strand. Tm values are reported in Table 1.
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Table 1
Tm and thermodynamic parameter data for the duplexes
1 5′ d-(TAC ACT AAA ACT GGT AAC AAC TC) 3′
2 5′ r-(UAC ACU AAA ACU GGU AAC AAC UC)3′
ΔTm/mod
(°C)
−ΔH0
−ΔG037°C
ΔTm/mod
(°C)
−ΔH0
−ΔG037°C
a
b
a
b
ONs
Tm a (°C)
(kcal mol−1
)
−ΔS0 (eu)
(kcal mol−1
)
Tm a (°C)
(kcal mol−1
)
−ΔS0 (eu)
(kcal mol−1
)
3
4
5
6
7
8
9
10
11
49.5
49.4
46.3
44.1
40.6
49.0
49.0
47.4
48.2
—
−132.6
—
−122.0
−111.6
−106.7
—
−130.7
−126.2
−126.4
−381.6
—
−352.5
−322.9
−311.2
—
−378.1
−364.2
−364.5
−14.2
—
−12.7
−11.5
−10.2
—
−13.9
−13.25
−13.35
50.5
50.6
48.7
50.4
47.8
50.1
49.7
48.1
48.9
—
−137.3
—
−127.0
−135.3
−132.9
—
−135.3
−130.0
−130.3
−395.1
—
−365.8
−389.2
−385.0
—
−390.3
−375.5
−375.6
−14.7
−0.1
−3.2
−1.8
−2.9
−0.5
−0.5
−0.7
−0.4
+0.1
−1.8
—
−0.9
−0.4
−0.8
−0.8
−0.5
−13.6
−14.6
−13.5
—
−14.3
−13.5
−13.8
Experiments were performed in a 10 mM sodium phosphate, pH 7, buffer containing 150 mM NaCl and 1 mM EDTA.a Concentrations were 1 μM
for each strand and the uncertainty of the Tm value was estimated to be 1 °C. b The thermodynamic data of duplex formation for the modified
ONs (5–7 and 9–11) and the reference (ON 3) with the DNA (ON 1) and RNA (ON 2) targets were determined from melting curves by the
concentration variation method (see the Experimental section). The uncertainty of the ΔG37°C values can be estimated to be 10%.
The melting temperatures (Tm) for all duplexes are reported in In the presence of the RNA target, the incorporation of the
Table 1. The results are an average of at least two series of three Uns in a continuous way (ON 6) led to a Tm value similar
independent experiments.
to that of the reference (ΔTm = −0.1 °C) while the incorpor-
ation of the three Un residues in a discontinuous way (ON 7)
had a destabilizing effect (ΔTm = −0.9 °C per modification). In
the same conditions, the incorporation of the three dUs either
in a row (ON 10) or in a discontinuous way (ON 11) had a
slight destabilizing effect (ΔTm = −0.8 °C and −0.5 °C per
modification, respectively).
It should be noted that Tm values for the DNA–DNA
(ONs 1 + 2) and DNA–RNA (ONs 1 + 3) duplexes used as refer-
ences are very close (ΔTm = 1 °C). These results are not
surprising since Tm values are largely dependent on
sequences.47–49 They are in accordance with the literature
data dealing with similar pyrimidine/purine ratios. The incor-
poration of dU in place of thymidine led to a weak Tm value
decrease similar to the literature report.50 DNA–DNA duplexes
containing Un are clearly less stable than the corresponding
duplexes involving dU. Conversely, the stability of DNA–RNA
duplexes with three Uns in a row is slightly superior to that of
the corresponding duplexes involving dU (ΔTm = +0.8 °C per
modification) while the incorporation of Uns in a discontinu-
ous way led to a slight destabilisation (ΔTm = −0.4 °C per
modification). These results show clearly a binding prefer-
ence for the RNA target that can be tuned by the number and
the positions of incorporation of the Un nucleoside.
Duplexes with single nucleotide change
Analysis of the data showed different results that were depen-
dent on both the position of the modified nucleoside inside
the duplexes (terminal versus internal position) and the target
considered (DNA versus RNA). The presence of the modified
nucleoside Un at the 5′-end of the duplex had a less destabiliz-
ing effect than its incorporation at the internal position. In the
presence of the DNA target (ON 1), single modifications at the
5′-end of the sequence (ONs 4 and 8) induced very weak devi-
ations in Tm compared to that of the unmodified duplex (ΔTm
=
−0.1 °C for Un and ΔTm = −0.5 °C for dU). A more pronounced
effect was observed (ΔTm = −3.2 °C) when Un was located at
the internal position of the sequence (ONs 5), while the desta-
bilization induced by the presence of dU (ON 9) at the same
position was weaker (ΔTm = −0.5 °C). In the presence of the
RNA target (ON 2), the 5′-end modification led to a weak stabil-
ization for Un (ΔTm = +0.1 °C) and a weak destabilization
for dU (ΔTm = −0.4 °C) while a more pronounced effect
was observed for the internal nucleotide substitution (ΔTm
−1.8 °C for Un and −0.8 °C for dU).
=
Duplexes with three nucleotide changes
The Tm values were also dependent on both the positions of
the modified nucleosides and the target considered (DNA
versus RNA). In the presence of the DNA target, the incorpor-
Mismatch discrimination for DNA–DNA and DNA–RNA
duplexes
ation of three Uns in a continuous way (ON 6) was slightly less To determine the base-pairing selectivity in DNA and RNA rec-
destabilizing (ΔTm = −1.8 °C per modification) than a single ognition by Un, ON 5 involving modification at the 11th pos-
incorporation (ON 5) (ΔTm = −3.2 °C), while the incorporation ition was also hybridized with the targets DNA (ON 1) and RNA
of the three Un residues in a discontinuous way (ON 7) had a (ON 2) involving cytosine (X = C), guanine (X = G) or thymine
much larger destabilizing effect (ΔTm = −2.9 °C per modifi- (X = T) in the position opposite the modified nucleoside
cation). In the same conditions, the incorporation of three dUs (Table 2). The same experiments were also performed with the
either in a continuous (ON 10) or in a discontinuous way ONs 3 and 9, involving respectively dT and dU, used as refer-
(ON 11) had a similar destabilizing effect (ΔTm = −0.7 °C per ences. Observation of the results indicated that in any case the
modification and ΔTm = −0.5 °C per modification, respectively). presence of mismatched base-pairs resulted in a dramatic
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Table 2
T
m and ΔTm values for mismatched versus matched duplexes
1 5′d-(TAC ACT AAA XCT GGT AAC AAC TC)3′
2 5′r-(UAC ACU AAA XCU GGU AAC AAC UC)3′
X = T
X = C
X = G
X = U
X = C
X = G
X = A
Tm
ONs (°C)
X = A
Tm
(°C)
Tm
(°C)
ΔTm
(°C)
Tm
(°C)
ΔTm
(°C)
Tm
(°C)
ΔTm
(°C)
Tm
(°C)
ΔTm
(°C)
Tm
(°C)
ΔTm
(°C)
Tm
(°C)
ΔTm
(°C)
3
5
9
49.5
46.3
49.0
39.2
39.2
38.6
−10.3
−7.1
−10.4
38.8
37.3
37.3
−10.7
−9.0
−11.7
43.3
41.4
41.9
−6.2
−4.9
−7.1
50.5
48.7
49.7
41.0
41.2
40.1
−9.5
−7.5
−9.6
40.6
39.0
39.6
−9.9
47.7
45.7
46.9
−2.8
−3.0
−2.8
−9.7
−10.1
Left: DNA–DNA duplexes, Right: DNA–RNA duplexes. 3: [5′TTGTTACCAGTTTTAGT3′], 5: [5′TTGTTACCAGUnTTTAGT3′], and 9:
[5′TTGTTACCAGdUTTTAGT3′]. Concentrations were 1 μM each strand in a 10 mM sodium phosphate, pH 7, buffer containing 150 mM NaCl and
1 mM EDTA.
decrease of Tm values. However, several differences can be three modifications (ONs 6, 7, 10 and 11) in the presence of
pointed out. When incorporated in DNA–DNA duplexes Un either their complementary DNA or RNA target sequences
was slightly less efficient than dT and dU at discriminating the (ONs 1 and 2). The unmodified duplexes (ONs 1 + 3 and 2 + 3)
mismatches. Conversely, in RNA–DNA duplexes the ΔTm were used as references. The results are shown in Fig. 4. In the
observed were nearly equivalent in the three series except presence of DNA as a complement (Fig. 4a), and compared
when rU was the opposite nucleoside. Importantly, the ΔTm with the unmodified duplex, the introduction of a single Un
observed for the less destabilizing wobble base pair Un–dG (ONs 4 and 5) resulted in unchanged CD spectra. However,
was similar to those obtained for dU–dG and dT–dG. From spectra modifications were observed for the duplexes involving
these experiments, it appears that for the sequence studied three Un nucleosides (ONs 6 and 7). The presence of three con-
ONs involving Un recognize the RNA target with selectivity secutive Uns (ON 6) resulted in a 30% intensity increase of the
equivalent to those obtained with the natural dT and the base- positive peak around 275 nm together with a blue shift of
modified nucleoside dU.
about 5 nm (between 270 and 215 nm). The intensity of the
negative peak at 245 nm was also slightly reduced. The CD
spectra of the DNA–DNA duplex involving the three Uns at
non-consecutive positions was also changed as compared to
that of the unmodified duplex. A blue shift was also observed
as well as a reduced negative peak at 245 nm slightly more pro-
nounced than for the duplex involving the contiguous Uns.
These changes reflect differences in the duplex structures due
to the presence of the neopentyl group at the 2′-position of the
2′-deoxyribose. The CD spectra of the duplexes involving dU
are identical to that of the unmodified DNA–DNA duplex
(Fig. 4c).
As observed for the DNA–DNA duplexes the presence of a
single Un in the DNA–RNA duplexes did not result in modifi-
cations in the CD spectra (Fig. 4b). But changes were observed
for the duplexes involving three Uns. Once again, a slight blue
shift and also intensity changes were observed for both
duplexes. A small intensity increase at 260 nm and an impor-
tant decrease at 220 nm were observed for the duplex with
three contiguous Uns, and only a very weak intensity decrease
at 270 nm for the duplex with three non-contiguous Uns. The
CD spectra of the DNA–RNA duplexes containing dU were
identical to that of the DNA–RNA unmodified duplex except in
the case of the presence of three contiguous dUs for which a
small blue shift was observed between 255 and 215 nm
(Fig. 4d). Comparison with the data obtained for the DNA–
RNA duplexes involving dU incorporations in the same pos-
itions allowed the conclusion that the observed weak spectra
modifications reflect small structural changes of the duplexes
that are mainly due to the presence of the neopentyl group.
Thermodynamic data of duplex formation
The thermodynamic data of duplex formation for the modified
ONs (5–7 and 9–11) and the reference (ON 3) with both the
DNA (ON 1) and RNA (ON 2) targets were determined from
melting curves by the concentration variation method. The
thermodynamic data obtained from 1/Tm versus log [Cm]
plots51,52 are summarized in Table 1. In the presence of the
DNA target, an important decrease of the enthalpy of duplex
formation was observed for the three ONs involving Un (4–7)
as compared to the unmodified ON (3) and to the ONs con-
taining dU (9–11). This was to some extent counterbalanced by
entropy compensation leading to standard enthalpies of
duplex formation ΔG37°C in agreement with the observed
thermal stabilities of the different series of duplexes. In the
presence of the RNA target, except for ON 5 involving a single
Un incorporation, the enthalpy of duplex formation for ONs 6
and 7 involving three Uns was only slightly inferior to that of
the reference and slightly superior to those obtained for the
corresponding ONs involving the dU nucleosides (9–10). For
ONs 6 and 7 involving three Uns the changes in entropy terms
were also weak as compared to the reference values. The calcu-
lated standard enthalpies of DNA–RNA duplex formation
ΔG37°C are in line with the observed thermal stabilities.
CD spectra of modified duplexes
In order to investigate the influence of the incorporation of Un
on the duplex structures, we recorded CD spectra of modified
ONs involving either one modification (ONs 4, 5, 8 and 9) or
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Fig. 4 CD spectra of the unmodified and modified duplexes [4 μM solutions (each strand) in a 10 mM sodium phosphate, pH 7, buffer containing 150 mM NaCl
and 1 mM EDTA] were recorded at 10 °C between λ = 215 and 315 nm. (a): DNA target 1 and ONs 3 (diamond), 4 (up triangle), 5 (square), 6 (circle) and 7 (cross);
(b): RNA target 2 and ONs 3 (diamond), 4 (up triangle), 5 (square), 6 (circle) and 7 (cross); (c): DNA target 1 and ONs 3 (diamond), 8 (up triangle), 9 (square),
10 (circle) and 11 (cross); (d): RNA target 2 and ONs 3 (diamond), 8 (up triangle), 9 (square), 10 (circle) and 11 (cross).
Although weak spectra changes were observed for the duplexes average value of their calculated curvature comparable to the
involving three Uns, their general shapes were similar to those reference. The RMSD times-series were quickly stabilized
of the B-type DNA–DNA and A/B type RNA–DNA unmodified around values from 2.4 Å to 3.4 Å. For the three duplexes con-
duplexes.
taining the modified nucleoside Un, we observed that the neo-
pentyl group remained stable and localized in the minor
groove. The positioning of the neopentyl group is shown in
Fig. 5a. The variations of the sugar pucker conformation of
each residue during the simulation in the form of a heat map
for the DNA–DNA duplex involving a single Un are shown in
ESI (Fig. 6a†). We observed that all residues conserved a C2′-
endo sugar pucker conformation, B-helix type, during the
entire simulation time except the Un residue that adopted a
C2′-exo sugar pucker conformation, close to an A-helix type.
The same result was observed for the DNA–DNA duplexes con-
taining three Uns where all residues adopted a C2′-endo sugar
pucker conformation (B-helix) except the modified nucleoside
Uns that adopted a C2′-exo sugar pucker conformation (close
to an A-helix) (data not shown). For the three simulations
involving dU nucleosides, all the residues adopt a C2′-endo
sugar pucker conformation and the heat maps obtained
cannot be distinguished from the reference (data not shown).
Molecular dynamic simulations
In order to correlate the thermal and thermodynamics proper-
ties of the different series of duplexes with structures on a
more detailed level, we performed molecular dynamic simu-
lations of 5 ns on the DNA–DNA and DNA–RNA duplexes invol-
ving a single internal incorporation of Un as well as those
involving three Un incorporations. The structures of the corre-
sponding duplexes involving dU were also investigated. Simu-
lations of the unmodified DNA–DNA and DNA–RNA duplexes
were used as references (see the Experimental section for
conditions).
DNA–DNA duplexes
The six DNA–DNA duplexes containing Un and dU modified
nucleosides showed a stable helical structure during the 5 ns
of simulation without any base-pair disruption and showed an
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stabilized around higher values than the DNA–DNA duplexes,
from 2.7 Å to 5.0 Å, which was probably caused by a longer
adaptation time for the DNA–RNA hybridization. However, for
the three duplexes containing the modified nucleoside Un, the
neopentyl group was stable and well-localized in the minor
groove. The positioning of the neopentyl group is shown in
Fig. 5b. Heat maps of the sugar pucker conformation vari-
ations of paired residues during the simulation showed two
distinct areas. The RNA strand showed the characteristic sugar
pucker conformations of an A-helix whereas those obtained for
the DNA strand were characteristic of a B-helix. As for DNA–
DNA duplexes, the Un modifications inserted on the DNA
strand adopted the characteristic sugar pucker conformations
of an A-helix without disturbing their neighboring sugar
pucker conformations. The heat map of the sugar pucker con-
formation variations of paired residues for the DNA–RNA
duplex containing a single modified nucleoside Un is shown
in Fig. 6b in ESI.† The A-tract influence on the minor groove
width is less visible on the DNA–RNA hybrids than on the
DNA–DNA hybrids (Fig. 7 in ESI†). A slight effect is however
still discernible on the minor groove of the unmodified DNA–
RNA duplex (Fig. 7c†) and the modified DNA–RNA duplex
involving a single Un that is not observed for the DNA–RNA
duplexes involving three Un modifications, contiguous or not.
The heat map for the DNA–RNA duplex involving three non-
contiguous Uns is shown in Fig. 7d in ESI.† Finally, the solvent
exposure of the Un nucleosides was globally higher in DNA–
RNA duplexes than in DNA–DNA duplexes, which could be
explained by the widening of the A-tract minor groove in DNA–
RNA duplexes compared to DNA–DNA duplexes. However, we
always observed a decrease of solvent exposure for the three
contiguous Uns. So we noticed a slight increase of the solvent
exposure dependent on the number and positioning of the Un
nucleosides. The most reduced solvent exposure occurred with
the insertion of three contiguous Uns (∼255 Å2) followed by
the insertion of one Un modification (∼270 Å2) which is com-
parable with the insertion in the A-tract of three discontinuous
Un modifications (∼275 Å2).
Fig. 5 Structure visualization of duplexes: DNA–DNA (a) and DNA–RNA (b),
containing a single modified nucleoside Un. Standard nucleoside atoms are rep-
resented as surface and neopentyl group atoms as van der Walls spheres.
We also studied the evolution of major and minor groove
width of each duplex during the simulation. The results
obtained for the minor groove width measured on unmodified
DNA–DNA duplex are shown in Fig. 7a (ESI†). A narrowing of
the minor groove width (around 8–10 Å in blue, instead of
12–15 Å in green) due to the presence of an A-tract in the
target sequence was observed. This phenomenon has been
largely described in the literature.53–57 The incorporation of a
single Un nucleotide in the T-tract sequence also caused a nar-
rowing of the minor groove (data not shown). However, the
presence of three Un nucleosides, contiguous or not, pre-
vented the narrowing of the minor groove probably due to the
presence of the neopentyl groups localized in the minor
groove. Fig. 7b in ESI† shows the heat map for the DNA–DNA
duplex involving three non-contiguous Uns. Finally, we esti-
mated the average solvent-accessible surface area (SASA) of the
neopentyl residues for the three duplexes containing the modi-
fied nucleoside Un in order to correlate their exposure to the
solvent with their number and positioning in the sequence.
We observed a lower neopentyl exposure to the solvent
(∼240 Å2) for a single and three contiguous modifications. In
the first case, the low exposure was probably due to the groove
narrowness that tends to bury the neopentyl group while in
the second case, the decrease of exposure was due to the proxi-
mity of the three neopentyl groups. On the contrary, the intro-
duction of three non-contiguous Un nucleosides increased the
solvent exposure of the neopentyl groups (∼260 Å2). This is
consistent with the fact that the presence of a second Un
nucleoside within the T tract prevents the narrowing of the
minor groove.
The results of the melting studies and CD experiments are
consistent with those of the molecular simulation studies indi-
cating that either a single or three neopentyl groups, in
different arrangements, were able to be accommodated in the
minor groove of both the DNA–DNA and RNA–DNA duplexes.
However, an important difference was observed between the
structures involving dU and Un modified nucleosides in the
simulations. While a C2′-endo sugar pucker was conserved in
all the dU residues, all the Un residues adopted a C2′-exo
sugar pucker conformation, close to an A-helix type. The
results of the melting studies indicated a binding preference
of the ONs 5, 6 and 7, involving Un residues, for the RNA over
the DNA complement, whatever the number and positions of
modifications. The selectivity of Un base-pairing in RNA–DNA
RNA–DNA duplexes
We still observed that the six DNA–RNA duplexes containing duplexes was equivalent to those of dT and dU and slightly
modifications remained stable with a conserved helical struc- weaker in the presence of the DNA targets. Another obser-
ture during the 5 ns of simulation without any base-pair dis- vation concerns the non-additive effect of three Un incorpor-
ruption or significant curvature. The RMSD times-series were ations. While a single Un incorporation at the internal
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position of the sequence (ON 5) had a destabilizing effect (rela- chain increased. It has also been shown that the binding
tive to dU) (ΔTm = −1 °C per modification for the RNA–DNA affinities of these modified ONs were largely sequence
duplex and ΔTm = −2.7 °C per modification for the DNA–DNA dependent and also dependent on the number and positions
duplex), the presence of three consecutive Uns (ON 6) stabil- of the modified nucleosides inside the sequence. So the com-
ized the RNA–DNA duplex (ΔTm = +0.8 °C per modification) parison of the ΔTm per modification cannot be quantitative.
and destabilized the DNA–DNA duplex (ΔTm = −1.1 °C per However, it has been reported that substitutions smaller
modification). When the three Un insertions were spaced by at than 2′-O-propyl stabilized the RNA–DNA duplexes. The litera-
least two natural 2′-deoxynucleosides both the RNA–DNA and ture report on modified ONs involving 2′-O-modified adeno-
DNA–DNA duplexes were destabilized (ΔTm = −0.4 °C per sines indicated that the incorporation of five 2′-O-alkylated
modification and ΔTm = −2.5 °C per modification, respecti- adenosines involving methyl, ethyl, propyl, butyl or pentyl
vely). Although a single Un incorporation in both the DNA– groups in purine-rich ONs did not change the CD spectra of
DNA and RNA–DNA duplexes was destabilizing, their CD the RNA–DNA duplexes compared to the parent while a
spectra were indistinguishable from those reported for the bulkier group induced important changes.35 In addition,
unmodified duplexes and those containing dU at the same similarly to our finding, the effects of several incorporations
position. Only a small blue shift at 275 nm and reduced inten- were not additives. It has been reported that for almost
sity at 220 nm were observed for the CD spectra of the most every modification (including O-ethyl) the ΔTm per modifi-
stable RNA–DNA duplex involving three contiguous Uns. The cation was negative in ONs that contained a single substitution
most important changes in CD spectra were observed for the and three consecutive 2′-substitutions were less destabilizing
DNA–DNA duplexes involving three non-consecutive Uns (cor- (or more stabilizing) than isolated substitutions spaced by
responding to the less stable duplex). They consisted of a unmodified deoxynucleosides. As observed with our modified
slight blue shift associated with 30% increase at 275 nm when ONs, 2′-O-substitutions on adenosine have been reported
Uns were consecutive and a reduced negative peak at 245 nm to induce a binding preference for their RNA over DNA
in the presence of non-consecutive Uns.
complements.
On the basis of these results and the literature reports on
The RNA binding preference of ONs involving Un nucleo-
modified oligonucleotides involving different series of 2′-modi- sides is likely due to the tendency of the 2′-modified nucleo-
fied nucleosides several observations can be made and hypoth- sides to adopt a 2′-exo geometry inconsistent with B-form
eses suggested to explain the different binding abilities DNA–DNA geometry. Furthermore, the presence of the neopen-
observed for our modified ONs involving the modified 2′-O- tyl groups could induce more perturbations of the hydration in
neopentyldeoxyuridine. Different classes of 2′-modified nucleo- the minor groove of the DNA–DNA duplex than in the minor
sides have been incorporated into ONs.6,16,18,24,30,37,40,58–66 groove of the RNA–DNA duplex. Concerning the variations in
Among them, those involving O-methyl,34,40 O-2′-O-methoxy- ΔTm per modification, although the same trend was observed
ethyl,31,36 2′-O-[2-(methyl-amino)-2-oxoethyl],36 2′-O-(3-amino- in the presence of both the DNA and the RNA targets, several
propyl),60,61 2′-O-[2-(guanidinium)ethyl]62 and 2′-fluor18,24,58,59 observations can be made. A single incorporation of Un (ON 5)
substituents have been reported to improve the biophysical was performed at the end of a tract of four consecutive A–T
properties of ONs. A specific mention must be made of the base-pairs. It is known that this structure facilitates formation
2′-O,4′-C-methylene
bicyclonucleosides6,25,26,28,64,65
being of a water spine in the minor groove of the B-DNA stabilizing
among the most useful modified nucleosides to date exhibit- the duplex. Furthermore the minor groove width of the unmo-
ing unique binding properties on the basis of their use in a dified dA–dT tract was reduced.53–57 The results of molecular
great variety of applications. The improved binding affinities simulation studies indicated that the incorporation of a single
of these different classes of modified ONs have been reported Un nucleotide in the T-tract sequence also caused a narrowing
to rely mainly on their tendency to adopt the 2′-exo pucker of the minor groove. However, the presence of three Un nucleo-
conformation. The binding affinities of the 2′-modified ONs sides, contiguous or not, prevented the contraction of the
are also influenced by other different parameters: the size minor groove. It should be noted that in ON 7 involving three
of the 2′-O-substituent, the number and positions of the modi- non-contiguous Uns, both of them were located in the T-tract
fied nucleosides, the sequence effect, the hydration changes in and separated by two natural 2′-deoxynucleosides. In the
the minor groove and potential additional interactions RNA–DNA duplexes the A-tract influence on the minor
between heteroatoms present in the substituents and the ON groove width was still present but less visible than on the
backbone.
DNA–DNA hybrids. In addition, the decrease of solvent
More specifically, comparison of our results with the litera- exposure observed when three Uns were consecutive suggested
ture reports on ONs involving a few nucleosides modified at that interactions between them were possible on the surface of
the 2′-position with aliphatic alkyl groups in a background of the minor groove which could also be a source of additional
2′-deoxynucleosides showed similar behaviors.16,35 These stabilization.
modified ONs were able to form DNA–DNA and RNA–DNA
We have reported the ability of modified ONs, involving
hybrids with stabilities dependent on the size and structure of either one or three modified 2′-O-neopentyluridines, to hybri-
the pendent groups at the 2′-position. The thermal stability of dize preferentially with their RNA target sequences with a stab-
the complexes was reduced as the length of the 2′-O-alkyl ility increase of +0.8 °C per modification when the modified
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nucleosides are consecutive. This affinity increase is nearly dynamic simulations. The results showed for all duplexes
similar to that observed for fully modified 2′-O-methyl and stable helical structures during the 5 ns of simulation, similar
2′-O-propyl ONs.36 It is likely that the incorporation of to the reference, without any base-pair disruption. For the
2′-O-neopentyluridines in different numbers and arrange- duplexes containing the modified nucleoside Un, the neo-
ments in other sequences could lead to slightly different pentyl group remained stable and localized in the minor
binding properties. However, the incorporation of a greater groove. However, an important difference was observed
number of 2′-O-neopentyluridines introducing more A-charac- between the structures involving dU and Un modified nucleo-
ter for the duplex formed with the RNA complement could sides. While in the modified ONs containing the dU modified
contribute to an increased stabilization of the duplex as pre- nucleosides all the residues adopted a C2′-endo sugar pucker
viously reported for ONs involving other 2′-O-alkyled nucleo- conformation, in the modified ONs involving the new modi-
sides.36 In addition, Un nucleosides could be used in fied Un nucleoside all residues conserved a C2′-endo sugar
association with other modified nucleosides able to adopt a pucker conformation, except the Un residues which adopted a
2′-exo geometry to obtain a modulation of the binding affinity C2′-exo sugar pucker conformation, close to an A-helix type.
of modified ONs.
The differences in stability observed for the DNA–DNA
and RNA–DNA duplexes, involving three Uns in different
arrangements, can be explained by the sequence context, the
local minor structure changes as well as by the hydration
changes. On the basis of these results, Un represents an inter-
Conclusion
A 2′-deoxyuridine analogue involving a neopentyl group at the esting dU analogue that could be used for tuning the hybridiz-
2′-position Un was synthetized and incorporated into a series ation properties of ONs and due to this is worthy of further
of 17-mer ONs. The modified ONs involved either one (at the exploration for applications in oligonucleotide therapeutic
5′-end or the internal position) or three (either in a contiguous based strategies.
fashion or separated by at least two natural nucleosides) modi-
fied Un nucleosides. The corresponding series of 17-mer ONs
involving dU at the same positions was also used for compari-
son. The pairing ability of these two series of ONs with their
DNA and RNA complements was investigated by thermal dena-
Experimental
General methods
turation and circular dichroism measurements. The results All solvents used were of the highest purity and did not
showed clearly the ability of all modified ONs to specifically contain more than 10 ppm H2O. All chemicals were used as
bind their DNA and RNA targets with the formation of duplex obtained unless otherwise stated. Analytical TLC was per-
structures similar to those of the B-type DNA–DNA and A–B formed on pre-coated alumina plates (Merck silica gel 60F 254,
type RNA–DNA references. Small changes in the CD spectra ref. 5554), and preparative TLC on glass-backed silica plates
were observed only for the duplexes containing three Un (60F 254, ref. 5717). For flash chromatography, Merck silica gel
nucleosides. Different duplex stabilities were observed. The 60 (70–230 mesh, ref. 7734) was used. Compounds were
presence of Un at the terminal position of the duplexes had no directly visualized on the plates by UV-shadowing. ODN synth-
effect on their stability. However, a single incorporation at the eses were performed on an Expedite Nucleic Acid Synthesis
internal position had a destabilizing effect in the presence of system 8909 from PerSeptive Biosystems. Reversed-phase
both the DNA and RNA complements (ΔTm = −2.7 °C and chromatography was performed on a 600E (System Controller)
ΔTm = −1 °C, respectively relative to the corresponding duplexes equipped with a photodiode array detector (Waters 990) using
containing dU). The presence of three Uns led to different duplex a Lichrospher 100 RP 18 (5 mm) column (125 mm × 0.4 mm)
stability changes dependent on both their arrangements and from Merck with a linear gradient of CH3CN in 0.1 M aqueous
the target considered. In all cases, the presence of three Uns in triethylammonium acetate, pH 7, with a flow rate of 1 cm3
DNA–DNA duplexes had a destabilizing effect. However, the min−1. Oligonucleotides containing dU were from Eurogentec
presence of three contiguous Uns had a less important effect and RNA sequence from Biomers. 1H NMR, 13C NMR and
(ΔTm = −1.1 °C per modification relative to dU) as compared 31P NMR were recorded at 400 MHz, 100 MHz and 162 MHz,
1
to their incorporation in a non-contiguous way (ΔTm = −2.5 °C respectively. H chemical shifts are reported in ppm relative to
per modification relative to dU). Three consecutive Un incor- either residual solvent peak DMSO (2.54 ppm) or Me4Si.
porations led to the RNA–DNA duplex with a stability slightly 31P chemical shifts were referenced to H3PO4 (external refer-
superior to that of the RNA–DNA duplex containing dU–rA ence). The following abbreviations are used for the proton
pairs (ΔTm = +0.8 °C per modification) while a slight destabili- spectra multiplicities: s: singlet, d: doublet, t: triplet, q:
zation was observed (ΔTm = −0.4 °C per modification relative quartet, qt: quintuplet, m: multiplet, br: broad, dd: doublet of
to dU) when the three Uns were inserted in a non-contiguous doublet, dt: doublet of triplet. Coupling constants (J) are
fashion. This trend was confirmed by the results of the thermo- reported in Hz. Absorption spectra were recorded with an
dynamic investigations. To rationalize these differences in Uvikon 860 spectrophotometer. The molar extinction coeffi-
stability, the behaviour of the modified nucleosides Un and cient of the modified nucleosides was considered to be equi-
dU in the different duplexes was evaluated by molecular valent to that of the 2′-deoxyuridine. Consequently, the molar
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extinction coefficients of ONs were determined according to H–C(4′)]; 4.07 [1H, q, J = 5.2 Hz, H–C(3′)]; 4.97 [1H, d, J = 6 Hz,
the literature.67
HO–C(3′)]; 5.11 [1H, t, J = 4.8 Hz, HO–C(5′)]; 5.63 [1H, d, J = 8.0
Hz, H–C(5)]; 5.83 [1H, d, J = 4.8 Hz, H–C(1′)]; 7.83 [d, J = 8.0
Hz, H–C(6)]; 11.30 (1H, s, –NH). δC (100 MHz, DMSO-d6), 26.2
Synthesis
2,2′-Anhydrouridine 13.44 To a suspension of uridine 12 [1q, –CH2C(CH3)3]; 31.9 [1s, –CH2C(CH3)3]; 60.3 [1t, C(5′)]; 68.3
(40 g, 0.16 mol) into dry DMF (44 cm3) was added diphenyl [1d, C(3′)]; 79.8 [1t, –CH2C(CH3)3]; 82.4 [1d, C(2′)]; 84.9 [1d,
carbonate (38.6 g, 0.18 mol) under argon at room temperature. C(4′)]; 86.1 [1d, C(1′)]; 101.6 [1d, C(5)]; 140.3 [1d, C(6)]; 150.3
The mixture was stirred at 85 °C until the slurry became clear, [1s, C(2)), 163.0 (1s, C(4)]. HRMS (ESI+-TOF): m/z, found
then sodium hydrogen carbonate (0.21 g, 2.50 mmol) was 337.1360 ([M + Na]+, C14H22N2O6Na+ calc. 337.1376).
added. After stirring at 120 °C for 4 hours, the reaction was
5′-O-Dimethoxytrityl-2′-O-neopentyluridine 17. To a solution
diluted with MeOH (60 cm3) and then filtered through a of 2′-O-neopentyluridine 16 (1.59 g, 5.05 mmol) into dry pyri-
bunker funnel and the solid washed with MeOH. The solid dine (40 cm3) was added dropwise at 0 °C a solution of
was re-suspended into MeOH (60 cm3) and stirred for 30 min dimethoxytrityl chloride (2.05 g, 6.06 mmol) into dry pyridine
at room temperature. The suspension was again filtered to (10 cm3) over 1 hour. After stirring for 5 hours at room temp-
yield 13 (27.43 g, 0.12 mol, 74%) as white solid. Rf13 0.12 erature, the reaction was quenched with MeOH (0.5 cm3). The
(CHCl3–MeOH, 95 : 5, v/v). δH (400 MHz, DMSO-d6), 3.18 [1H, reaction was again stirred for 30 minutes and then diluted
m, H–C(5′)]; 3.28 [1H, m, H–C(5′) ]; 4.06 [1H, dt, J = 0.8, 4.8 Hz, with CH2Cl2 (100 cm3). The mixture was washed successively
H–C(4′)]; 4.38 [1H, s, H–C(3′)]; 4.97 [1H, s, HO–C(5′)]; 5.19 [1H, with cold water (3 × 50 cm3), cold 5% aq. NaHCO3 (2 × 50 cm3)
d, J = 6.0 Hz, H–C(2′)]; 5.83 [1H, d, J = 7.6 Hz, H–C(5)]; 5.88 and cold brine (50 cm3). The organic phase was dried over
[1H, d, J = 7.2 Hz, HO–C(3′)]; 6.30 [1H, d, J = 5.6 Hz, H–C(1′)]; Na2SO4 and concentrated to dryness under reduced pressure.
7.83 [1H, d, J = 8.4 Hz, H–C(6)]. δC (100 MHz, DMSO-d6), 60.7 The residue was co-evaporated with toluene (2 × 25 cm3) and
[1t, C(5′)]; 74.6 [1d, C(3′)]; 88.6 [1d, C(2′)]; 89.1 [1d, C(4′)]; 89.9 purified by silica gel chromatography (the column was packed
[1d, C(1′)]; 108.5 [1d, C(5)]; 136.7 [1d, C(6)]; 159.7 [1s, C(4)]; with eluent containing 5% TEA to prevent detritylation, eluent:
171.1 [1s, C(2)]. HRMS (ESI+-TOF): m/z, found 227.0664 hexane–EtOAc–TEA, 80 : 19 : 1 v/v/v to hexane–EtOAc–TEA,
([M + H]+, C9H11N2O5 calc. 227.0662).
50 : 49 : 1, v/v/v) to yield 17 (1.78 g, 2.88 mmol, 57%) as a white
1-Trimethylsilyloxy-2,2-dimethylpropane 15.45 To a solution foam. Rf17 = 0.59 (CH2Cl2–MeOH, 9 : 1, v/v). δH (400 MHz,
of neopentyl alcohol 14 (20 g, 0.23 mol) into dry Et2O CDCl3), 0.95 [9H, s, –CH2C(CH3)3]; 2.62 [1H, d, J = 9.6 Hz, HO–
(150 cm3) was added dropwise a solution of TMS-Cl (35 cm3, C(3′)]; 3.29 [1H, d, J = 9.2 Hz, –OCH2C(CH3)3]; 3.53 [1H, dd, J =
0.28 mol) and TEA (39 cm3, 0.28 mol) into dry Et2O (50 cm3) 2.8, 11.2 Hz, H–C(5′)]; 3.58 [1H, dd, J = 2.0, 11.2 Hz, H–C(5′)];
over 30 minutes at 0 °C. The reaction was stirred for 3 days at 3.66 [1H, d, J = 9.2 Hz, –OCH2C(CH3)3]; 3.79, 3.80 (6H, 2s,–
room temperature and then filtered. The precipitate was OCH3); 3.87 [1H, d, J = 5.2 Hz, H–C(2′)]; 4.02 [1H, m, H–C(4′)];
washed with Et2O then the filtrate was concentrated under 4.47 [1H, dt, J = 5.2, 8.8 Hz, H–C(3′)]; 5.28 [1H, d, J = 8.0 Hz,
reduced pressure. The crude product was distilled under H–C(5)]; 5.93 [1H, s, H–C(1′)]; 6.83–6.86 (4H, m, arom. H);
atmospheric pressure to yield 15 (25.31 g, 0.16 mol, 69%) as a 7.22–7.32 (7H, m, arom. H); 7.38–7.41 (2H, m, arom. H); 8.04
colourless liquid. bp 130 °C–760 mmHg (lit.,45 bp 122 °C– [1H, d, J = 8.4 Hz, H–C(6)]; 9.30 (1H, br s, –NH). δC (100 MHz,
734 mmHg). δH (400 MHz, CDCl3), 0.09 [9H, s, –Si(CH3)3]; 0.86 CDCl3), 26.5 [q, –C(CH3)3]; 32.2 [s, –C(CH3)3]; 55.2 (q, –OCH3);
[9H, s, –C(CH3)3]; 3.20 (2H, s, CH2). δC (100 MHz, CDCl3), −0.5 61.1 [t, –OCH2C(CH3)3]; 68.4, 80.9, 82.7, 83.3, 87.1 [4d, 1t,
[q, Si(CH3)3]; 26.6 [q, –C(CH3)3]; 32.6 [s, –C(CH3)3]; 72.8 C(1′), C(2′), C(3′), C(4′), C(5′)]; 87.4 [s, –C(Ar)3]; 101.9 [d, C(5)];
(d, CH2). MS characterization has been unsuccessful.
113.3 (d, arom. CH), 127.1, 127.9, 128.1, 130.1, 130.2 (5d,
2′-O-Neopentyluridine 16. To a suspension of 2,2′-anhydro- arom. CH), 135.0, 135.3 (2s, arom. Cq), 140.0 [d, C(6)]; 144.3
uridine 13 (3.50 g, 15.5 mmol) and 1-trimethylsilyloxy-2,2- (s, arom. Cq), 150.0 [s, C(2)]; 158.6, 158.7 (2s, arom. Cq); 163.4
dimethylpropane 15 (12.4 cm3, 77.3 mmol) into 31 cm3 of dry [s, C(4)]. HRMS (ESI+-TOF): m/z, found 639.2675 ([M + Na]+,
DMF was added BF3–Et2O (4.8 cm3, 38.8 mmol) under argon. calc. 639.2682).
The mixture was stirred at 120 °C for 5 days and then cooled
5′-O-Dimethoxytrityl-3′-O-(2-cyanoethyl-N,N′-diisopropylphos-
down at room temperature. The precipitate was filtered phoramidite)-2′-O-neopentyluridine 18. To a mixture of 5′-O-
through a bunker funnel and washed with EtOAc. The filtrate dimethoxytrityl-2′-O-neopentyluridine 17 (2.10 g, 3.41 mmol)
was diluted with EtOAc (250 cm3) and washed with brine (4 × and 4,5-dicyanoimidazole (0.27 g, 2.28 mmol, 0.25 M in
150 cm3). The organic phase was dried over Na2SO4, filtered CH3CN) into dry CH2Cl2 (43 cm3) was added dropwise 2-cyano-
and concentrated to dryness under reduced pressure. The ethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (1.6 cm3,
crude product was purified by silica gel chromatography 5.12 mmol) over 10 minutes under argon. The reaction was
(eluent: CH2Cl2–MeOH 100 : 0, v/v to CH2Cl2–MeOH 95 : 5, v/v) stirred for 1 hour at room temperature and then diluted with
to yield 16 (0.88 g, 2.8 mmol, 18%) as a white solid. Rf16 0.40 CH2Cl2 (100 cm3). The mixture was washed with cold 5% aq.
(CHCl3–MeOH, 85 : 15, v/v). δH (400 MHz, DMSO-d6), 0.85 [9H, NaHCO3 (2 × 50 cm3) and cold brine (50 cm3). The organic
s, –CH2C(CH3)3]; 3.12 [1H, d, J = 8.8 Hz, –CH2C(CH3)3]; 3.30 phase was dried over Na2SO4 and evaporated to dryness under
[1H, d, J = 8.8 Hz, –CH2C(CH3)3]; 3.56 [1H, m, H–C(5′)]; 3.66 reduced pressure. The residue was purified by silica gel chrom-
[1H, m, H–C(5′)]; 3.81 [1H, dt, J = 4.8 Hz, H–C(2′)]; 3.88 [1H, m, atography (the column was packed with eluent containing 5%
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TEA to prevent detritylation, eluent: CH2Cl2–TEA (99 : 1, v/v) to TOF mass spectrometry. The observed single-product ions
CH2Cl2–EtOAc–TEA, 70 : 29 : 1, v/v/v) to yield 18 (1.81 g, were all within 0.1% of the calculated mass.
2.22 mmol, 65%) as a white foam. Rf18 = 0.35, 0.42 (CH2Cl2–
Physical studies
EtOAc, 8 : 2, v/v). δH (400 MHz, CDCl3), 0.91, 0.93 [9H, 2s,
–CH2C(CH3)3]; 1.04–1.30 [6H, m, 3H, –CH(CH3)2]; 2.43 (1H, t,
UV-melting curves. Solutions of duplexes (1 μM each strand)
J = 6.0 Hz, –OCH2CH2CN); 2.61 (1H, t, J = 6.0 Hz, –OCH2CH2CN); were prepared in a 10 mM NaH2PO4 buffer (pH 7.0) containing
3.32–3.99 [7H, m, –OCH2C(CH3)3, –CH2(5′), –OCH2CH2CN, 150 mM NaCl and 1 mM EDTA. Thermal melting experiments
–NCH(CH3)2], 3.78, 3.79, 3.80, 3.81 (6H, 4s, –OCH3); 3.97, 3.98 were performed on an Uvikon 941 UV/VIS spectrophotometer
[1H, 2m, H–C(2′)], 4.23, 4.28 [1H, 2m, H–C(4′)], 4.39–4.56 [1H, equipped with a Ministat with a heating rate of 0.5 °C min−1
.
2m, H–C(3′)], 5.20, 5.22 [1H, 2d, J = 8.2 Hz, H–C(5)]; 5.91, 5.94 Tm values were obtained by the baseline method.68 The uncer-
[1H, 2d, J = 2 Hz, H–C(1′)]; 6.82–6.86 (4H, m, arom. H); tainty of the Tm values reported was 1 °C based on indepen-
7.33–7.86 (7H, m, arom. H); 7.38–7.43 (2H, m, arom. H); 8.01, dent series of experiments.
8.08 [1H, 2d, J = 8 Hz, H–C(6)]; 8.67 (br s, 1H, –NH). δC
Thermodynamic parameters. The thermodynamic para-
(100 MHz, CDCl3), 20.2, 20.4 (2dt, J = 6.9 Hz, –OCH2CH2CN); meters of the duplex formation for the modified ONs involving
24.5–24.8 [m q, –CH(CH3)2]; 26.5, 26.6 [2q, –CH2C(CH3)3]; 32.1, either a single (ONs 5 and 9) or three modifications (ONs 6, 7,
32.2 [2s, –CH2C(CH3)3]; 43.1, 43.2 [2dd, J = 12.7 Hz, –CH- 10 and 11) with both the DNA (ON 1) and the RNA (ON 2)
(CH3)2]; 55.2, 55.3 (2q, –OCH3); 57.8, 58.1 (2dt, J = 18.3 Hz, targets were determined by a plot of 1/Tm versus log [Cm].51,52
–OCH2CH2CN); 60.7, 61.1 [2t, –CH2C(CH3)3]; 69.9–87.1 [1t, 4d, Cm is the concentration of free ONs 5–7 and 9–11 at the
C(1′), C(2′), C(3′), C(4′), C(5′)]; 87.9, 88.1 [2s, –C(Ar)3]; 101.7, melting temperature. ON concentrations were 10−5 M, 5 × 10−6
101.8 [2d, C(5)]; 113.2 (d, arom. CH), 117.4, 117.5 (2s, –CN), M, 2.5 × 10−6 M, and 10−6 M in a 10 mM NaH2PO4 buffer
127.1, 127.9, 128.0, 128.2, 128.3, 130.3 (6 d, arom. CH); 134.9, (pH 7.0) containing 150 mM NaCl and 1 mM EDTA. On the
135.1, 135.2, 135.3 (4s, arom. Cq); 140.1, 140.2 [2d, C(6)]; basis of the Tm values observed for different independent
144.2, 144.3 (2s, arom. Cq), 149.9, 150.0 [2s, C(2)]; 158.6, 158.7 experiments, the uncertainty of the ΔG values can be esti-
(2s, arom. Cq); 163.2, 163.3 [2s, C(4)]. 31P NMR (162 MHz, mated to be 10%.
CDCl3), δ = 149.6, 149.8. C44H57N4O9P (816.92). HRMS (ESI+-
TOF): m/z, found 839.3773 ([M + Na]+, C44H57N4O9NaP+, calc. carried out on a Jasco J-810 spectropolarimeter. An optical cell
839.3761). with a path length of 0.1 cm was used. The temperature of the
Circular dichroism spectroscopy. CD measurements were
Oligonucleotide synthesis, purification and characterization. cell was adjusted with a Jasco PTC-4325 temperature controller
All ONs were prepared from modified nucleoside phosphor- at 10 0.5 °C. Solutions of duplexes (4 μM each strand) were
amidite 18 and commercially available dA, dC, dG, and dT prepared in a 10 mM NaH2PO4 buffer (pH 7.0) containing
phosphoramidites (Biosolve) and deoxynucleoside-CPG 150 mM NaCl and 1 mM EDTA. The CD spectra were recorded
(1 μmol, Glen Research) on an Expedite Nucleic Acid automated between 215 and 315 nm. The reported spectra correspond to
synthesizer by standard solid-phase phosphoramidite chem- the average of three scans.
istry except for the modified phosphoramidite 18. The coup-
Molecular dynamic simulations. Initial structures of the
ling time increased to 10 min. Tetrazole (0.45 M in MeCN, DNA–DNA duplexes were generated with the nucleic acid
Biosolve) was used as a coupling reagent and 3% dichloro- builder (NAB) module of AMBER1169 into a uniform ideal
acetic acid in 1,2-dichloroethane for the detritylation step. Coup- Watson/Crick B-form DNA duplex. Regarding the RNA–DNA
ling efficiency was estimated from a trityl assay and was >95%. duplex of the unmodified sequence, each strand was built fol-
After chain elongation and final detritylation, the oligomers lowing its ideal geometry (A-helix and B-helix for the RNA and
were cleaved from the resin and deprotected by treatment with DNA strand respectively), then both strands were combined to
1 cm3 of conc. aq. ammonia solution (25%) at 55 °C during form a hybrid A–B duplex. The initial structures of the modi-
16 h and filtered through syringe filters with a 0.45 μm GHP fied RNA–DNA duplexes were extracted from the MD simu-
membrane (Pall). The ammonia solution was then removed by lation of the parent RNA–DNA duplex to allow the adjustment
evaporation. The crude oligonucleotides were purified by of the two strands together before inserting modifications.
reversed-phase HPLC with a Lichrocart column (250 × 10 mm) Geometry and charge distribution of modified nucleotides (Un
packed with Lichrospher RP 18 (10 μm) from Merck on a HPLC and dU) were computed using the Jaguar program70 at the
system using a linear gradient of acetonitrile from 0 to 30% HF/6-31G* level and fitted with the Resp program for
over 30 min, in 0.1 M aq. triethylammonium acetate buffer, compatibility with the Wang et al. force field71 including
pH 7, with a flow rate of 4 cm3 min−1 and detection at λ = parmbsc0 modifications.72
260 nm. After purification, the purity of all oligomers
The SANDER module of the AMBER11 package was used
described was verified by reverse-phase analysis using a for the simulations. All systems were neutralized with Na+
Lichrocart system (125 × 4 mm) packed with Lichrospher counter-ions and solvated with TIP3P water molecules. After
RP 18 (5 μm) from Merck eluted with a linear gradient of aceto- 4000 steps of energy minimization, the system was heated
nitrile from 0 to 27.5% over 30 min in a 0.1 M aq. triethylam- from 0 K to 300 K with restraints applied on atomic positions
monium acetate buffer, pH 7, with a flow rate of 1 cm3 min−1
.
of the oligomer and the counter-ions, then relaxed during
The integrity of all oligonucleotides was confirmed by MALDI- 250 ps with gradual removal of the atomic restraints. A final
This journal is © The Royal Society of Chemistry 2013
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Organic & Biomolecular Chemistry
relaxation step of 10 ps was performed without any restraint, 17 M. Egli and P. S. Pellan, Annu. Rev. Biophys. Biomol. Struct.,
followed by 5 ns of free MD simulation in an NPT ensemble 2007, 36, 281.
with periodic boundary conditions. The conformational 18 G. F. Deleavey, J. K. Watts, T. Alain, F. Robert, A. Kalota,
parameters of nucleic acids were analyzed as time series using
the program CURVES+73 and the PTRAJ module of the
V. Aishwarya, J. Pelletier, A. M. Gewirtz, N. Sonenberg and
M. J. Damha, Nucleic Acids Res., 2010, 38, 4547.
AMBER11 package. To follow the evolution of the neopentyl 19 J. D. Moulton and S. Jiang, Molecules, 2009, 14, 1304.
group positioning during the simulation, we defined the Ω 20 J. K. Watts, G. F. Deleavey and M. J. Damha, Drug Discovery
angle as the torsion angle {PA, PUn, O3′Un, CUn} (Fig. 8 in ESI†).
Today, 2008, 13, 842.
21 D. Ittig, S. Luisier, J. Weiler, D. Schumperli and
C. Leumann, Artif. DNA PNA XNA, 2010, 1, 9.
22 A. L. Jackson, J. Burchard, D. Leake, A. Reynolds,
J. Schelter, J. Guo, J. M. Johnson, L. Lim, J. Karpilow,
K. Nichols, W. Marshall, A. Khvorova and P. S. Linsley,
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Acknowledgements
A grant and post-doctoral funding (G. M.) from the Départe-
ment du Loiret and post-doc co-funding (G. M.) from Nucleo-
Syn are gratefully acknowledged. Analyses have been 23 K. Ui-Tei, Y. Naito, S. Zenno, K. Nishi, K. Yamato,
performed by the CBM and ICOA spectrometry facilities and
Centre de Mesures physiques, Univ. B.P Clermont-Ferrand. We
F. Takahashi, A. Juni and K. Saigo, Nucleic Acids Res., 2008,
36, 2136.
thank G. Gaband (CBM), Dr C. Colas (FR 2708 CBM/ICOA) and 24 M. Manoharan, A. Akinc, R. K. Pandey, J. Qin, P. Hadwiger,
Dr B. Legeret (Univ BP, CF) for recording MS spectra,
H. Meudal (CBM) and Dr M. Lefoix (NucleoSyn) for recording
NMR spectra and Dr S. Villette (CBM) for her help in the Tm
and CD experiments.
M. John, K. Mills, K. Charisse, M. A. Maeir, L. Nechev,
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25 J. B. Bramsen, M. B. Laursen, A. F. Nielsen, T. B. Hansen,
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