Synthesis, binding and cellular uptake properties of oligodeoxynucleotides containing cationic bicyclo-thymidine residues

Article abstract of DOI:10.1016/j.bmc.2011.08.022

The synthesis and incorporation into oligodeoxynucleotides of two novel derivatives of bicyclothymidine carrying a cationic diaminopropyl or lysine unit in the C(6′)-β position is described. Compared to unmodified DNA these oligonucleotides show T_m

Full text of DOI:10.1016/j.bmc.2011.08.022

Bioorganic & Medicinal Chemistry 19 (2011) 5869–5875
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, binding and cellular uptake properties of oligodeoxynucleotides
containing cationic bicyclo-thymidine residues
⇑
Jory Lietard, Damian Ittig, Christian J. Leumann
Department of Chemistry & Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and incorporation into oligodeoxynucleotides of two novel derivatives of bicyclothymidine
carrying a cationic diaminopropyl or lysine unit in the C(60)-b position is described. Compared to unmod-
ified DNA these oligonucleotides show Tm-neutral behavior when paired against complementary DNA
and are destabilizing when paired against RNA. Unaided uptake experiments of a decamer containing five
lys-bcT units into HeLa and HEK293T cells showed substantial internalization with mostly cytosolic
distribution which was not observed in the case of an unmodified control oligonucleotide.
Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
Received 22 May 2011
Revised 29 July 2011
Accepted 9 August 2011
Available online 16 August 2011
Keywords:
Oligonucleotides
Nucleosides
Antisense
DNA diagnostics
Bicyclo-DNA
1. Introduction
of an oligonucleotide, the latter technology frequently suffers from
low loading of oligonucleotide per average molecular weight of the
Oligonucleotide based gene silencing, discovered in the late sev-
enties,1,2 has evolved over the years into oligonucleotide based
therapeutic strategies, the two most important ones being anti-
sense and RNA interference.3,4 In the therapeutic context it is
believed that chemically modified oligonucleotides have superior
properties compared to natural DNA or RNA as they allow to
address and improve on critical parameters as RNA target affinity,
nuclease resistance and cellular uptake and distribution. As a con-
sequence, a large variety of chemically modified oligonucleotide
analogues have been synthesized in the past and their biological
properties evaluated. One successful concept applied to the design
of oligonucleotide analogues is that of conformational restriction,
resulting in structures such as locked-nucleic acids (LNA),5,6 hexi-
tol nucleic acids (HNA),7 and tricyclo-DNA (tc-DNA, Fig. 1).8,9 These
analogues typically exhibit increased affinity to RNA and feature
higher nuclease resistance.
While in this way the challenges linked to target affinity and
nuclease resistance have largely been met, other requirements
such as improving cellular uptake and distribution are yet elusive.
Promising strategies to ameliorate cellular delivery include bio-
conjugation of therapeutic oligonucleotides to specific or unspe-
cific cell targeting molecular entities,10,11 or packaging into
cationic and/or lipophilic complexes or nanoparticles.12 While in
the former case, covalent conjugation is often limited to the ends
particles.
For cellular transfection experiments, oligonucleotides are typ-
ically delivered in complex with cationic lipids such as lipofect-
amine. Due to the toxic properties of these lipids this is,
however, no method for therapeutic delivery. An alternative there-
fore may be to covalently add cationic lipid-like groups throughout
O
P
O-
O
O
6'
P
O-
4'
O
P
O-
O
O
5'
O
O
O
O
R
Base
Base
Base
1'
3'
7'
2'
O
O
bcOalk-DNA, R = O-alk
bcalk-DNA, R = alk
bc-DNA
tc-DNA
O
P
O-
O
P
O-
O
O
R = H
O
O
R = (CH2)3NH2
R = Lys
RHN
Base
Base
N
BnO
O
O
bcox-DNA
6'-amino-bc-DNA
⇑
Corresponding author. Tel.: +41 31 631 4355; fax: +41 31 631 3422.
E-mail address: leumann@ioc.unibe.ch (C.J. Leumann).
Figure 1. Selected derivatives of the bi- and tricyclo-DNA family.
0968-0896/$ - see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.08.022

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J. Lietard et al. / Bioorg. Med. Chem. 19 (2011) 5869–5875
the sequence of an oligonucleotide to emulate such complexes. Gi-
ven the structure of the nucleic acids, two points of attachment
seem to be exquisitely suited for such manipulations, namely the
internucleosidic phosphate and the 20-OH group (in RNA).
Substitution of non-bridging phosphate oxygens by alkylamino
or alkylguanidinium groups leading to uncharged or positively
charged phosphoramidates,13–15 or complete replacement of the
phosphate by guanidine groups16,17 have been explored in the past
in this context and have proven successful in increasing RNA affin-
ity due to reduced electrostatic repulsion and in some cases also in
promoting cellular delivery. Also the 20-OH function in oligoribo-
nucleotides has been modified with cationic side chains in the past,
and increased RNA binding and improved antisense effects were
reported for selected cases.18–21
skeleton, started from the already known nucleoside 1 (2:1 mixture
of b- and
-anomers, Scheme 1).25 Removal of the N-protecting
a
group by ammonolysis lead to aminobicyclothymidines 2 that could
be separated into the pure anomeric forms by column chromatogra-
phy. The b-anomer of 2 was subsequently converted to 3 by reduc-
tive amination with Fmoc protected b-amino propanal. The basic
amino function in 3 was again protected as a trifluoroacetamide
(? 4) and the synthesis of building block 5 completed by standard
phosphitylation. Alternatively, 2 was converted into the lysine-
derived nucleoside 6 by carbodiimide mediated condensation of
N,N-bis-Fmoc protected lysine in excellent yield. Again, standard
transformation to the phosphoramidite 7 concluded the synthesis
of this building block.
In the context of oligonucleotide therapeutics our laboratory
has developed over the years the bicyclo-DNA scaffold ( Fig. 1).
The five-membered carbocyclic ring in bicyclo-DNA offers unique
possibilities for further chemical modification, specifically at C(60)
and C(70), to introduce additional functional groups for various
purposes. In recent work we reported on the DNA and RNA affinity
of 6-alkyl or -oxyalkyl modified bc-DNA,22,23 and particularly on
C(60)-oxime modified bc-DNA (bcox-DNA), where the uncharged
lipophilic benzyl group was shown to improve cellular uptake rel-
ative to unmodified oligonucleotides when delivered with lipofect-
amine as carrier.24 In extension of these investigations we became
interested in positioning positively charged residues at C(60) in
b-configuration. Model building suggests such residues to be lo-
cated at the outer rim of the grooves pointing towards the solvent
with a slight bias towards the major groove (Fig. 2). Here we report
on the synthesis and incorporation into oligodeoxynucleotides of
two novel C(60)-amino-modified bicyclonucleosides carrying either
a doubly cationic diaminopropyl or lysin group. We found that
complementary base-pairing to DNA is associated with essentially
no loss or gain in affinity against DNA while RNA affinity is com-
promised with respect to unmodified oligonucleotides. More
importantly, a decamer with five lys-bcT modifications was shown
to undergo unaided cellular uptake into HeLa and HEK293T cells, a
property that was not observed in the case of the unmodified con-
trol oligonucleotide.
2.2. Oligonucleotide synthesis and deprotection
Oligodeoxyribonucleotides ON1–9 (Table 1) containing single
to triple substitutions in different nearest neighbor contexts were
synthesized on a 1.3 lmol scale by standard automated phospho-
ramidite chemistry. For incorporation of the modified building
blocks 5 and 7 the coupling time was extended to 9 min. No further
changes to the synthesis cycle were necessary. Coupling yields of 5
were between 90% and 95%. For the lysine containing building
block 7 coupling yields were in the same range (P90%) with the
exception for ON6, containing two consecutive modifications,
where the yield dropped to 68% for the second coupling. This is
most likely due to increased steric crowding around O(50) by the
double Fmoc protected lysine unit. The 30fluorescein labeled
ON10 containing five lys-bcT residues was synthesized analo-
gously for the purpose of following cellular uptake by fluorescence
microscopy. Deprotection after chain assembly was carried out by
standard treatment with concd NH3 (55 °C, 16 h). The crude oligo-
nucleotides were purified by ion-exchange HPLC and obtained in
>95% purity. Table 1 gives an overview over the sequences
ON1–10 obtained in this way as well as a confirmation of their
respective structures by ESIꢀ mass spectrometry.
2.2.1. Tm measurements
UV-melting curves were measured at 260 nm in standard saline
buffer (10 mM Na-phosphate, 150 mM NaCl, pH 7.0). The Tm data
(Table 1) of singly and doubly substituted oligonucleotides
(ON1–8) against complementary DNA indicated almost negligible
thermal affinity changes compared to an unmodified DNA unit in
2. Results and discussion
2.1. Synthesis of nucleosides and building blocks
the respective positions.
DTm values per modification range be-
tween ꢀ1.2 and +1.5 °C. This clearly demonstrates that neither
an aminopropyl nor a lysyl residue at the C(60)-b-amino function
sterically interferes with duplex formation, indicating smooth
accommodation within the major groove. On the other hand,
increasing the number of positive charges from one (ON1) to two
(ON2/3), does not lead to coherent additional stability, indicating
that the electrostatic shielding of phosphate groups is limited in
these cases. This could be a consequence of the relative positioning
and the conformational flexibility of the cationic alkyl groups. We
further note that consecutive modifications as in ON4–6 have a
The synthesis of the two building blocks 5 and 7, containing
either a diaminopropyl or a lysine unit at C(60) of the bicyclo-DNA
stronger destabilizing effect (
D
Tm/mod ca. ꢀ1.0 °C) relative to sin-
gle modifications. It is not excluded that this is the consequence of
local electrostatic repulsion of the positive charges or, also less
likely, to steric interference of the alkyl chains.
Complexes of ON1–9 with complementary RNA were in all
cases destabilizing with
D
Tm/mod of ꢀ1.5 to ꢀ5.6 °C. Again, con-
secutive substitutions are less well tolerated than single or multi-
ple, discontinuous substitutions. Also, there is no significant
difference between the aminopropyl or the lysyl unit relative to
the primary amine (R = H) indicating that changing the steric de-
mand near to C(60) does not influence the affinity. There seems
to be a trend that destabilization diminishes with increasing the
Figure 2. Stereoview of an energy minimized DNA duplex containing a lys-bcT
residue. The lysyl group points from the rim of the grooves into the solvent.

J. Lietard et al. / Bioorg. Med. Chem. 19 (2011) 5869–5875
5871
ODMTr
O
ODMTr
ODMTr
O
O
O
e)
H
N
a)
FmocHN
N
O
O
N
O
H2N
N
H
N
O
O
F3C
93%
NH
NH
NH
66%
OH
OH
2 (
OH
6
O
O
1
β:α 2:1)
NHFmoc
d)
85% b)
80%
ODMTr
O
ODMTr
O
ODMTr
O
O
O
c)
86%
FmocHN
N
N
O
N
O
O
HN
N
O
O
N
F3C
H
NH
NH
NH
O
P
OH
O
OH
(iPr)2N
OCH2CH2CN
FmocHN
4
3
FmocHN
NHFmoc
7
d)
70%
ODMTr
O
O
N
O
O
N
F3C
NH
O
P
(iPr)2N
OCH2CH2CN
FmocHN
5
Scheme 1. Synthesis of building blocks 5 and 7. Reagents and conditions: (a) NH3 concd in MeOH, 55 °C; (b) 3-(Fmoc-NH(CH2)2CHO, NaBH3CN, MeOH, rt, 5 h; (c) TFAA,
a
x
pyridine, 0 °C, 1 h; (d) (iPr)2NP(Cl)CH2CH2CN, iPr2NEt, THF, rt, 45 min; (e) N ,N -Fmoc-Lys-OH, EDC, DMAP, CH2Cl2, rt, 1 h;
Table 1
Tm data from UV-melting curves (260 nm) of modified dodecamer duplexes with complementary DNA and RNA
ODNA
O
Tm (°C) vs DNAa,b
Tm/mod)
Tm (°C) vs RNAa,c
Tm/mod)
ESI--MS m/z calc
ESIꢀ-MS m/z found
t =
RHN
N
O
O
Sequence
(
D
(D
NH
ODNA
ON1
ON2
ON3
ON4
ON5
ON6
ON7
ON8
ON9
ON10
d(GGATGTTCtCGA)
d(GGATGttGTCGA)
d(GGAtGTTGtCGA)
R = H
R = 3-aminopropyl
R = Lys
R = H
R = 3-aminopropyl
R = Lys
R = 3-aminopropyl
R = Lys
R = Lys
3718.5
3774.6
3845.7
3760.6
3872.8
4014.9
3872.8
4014.9
4184.2
4424.7
3718.0
3774.4
3846.0
3759.0
3873.0
4015.0
3872.4
4015.0
4182.0
4423.0
48.3 (+0.8)
47.3 (ꢀ0.2)
49.0 (+1.5)
45.5 (ꢀ1.0)
45.1 (ꢀ1.2)
45.5 (ꢀ1.0)
46.1 (ꢀ0.7)
47.3 (ꢀ0.1)
46.5 (ꢀ0.3)
—
46.5 (ꢀ3.0)
45.7 (ꢀ3.8)
46.0 (ꢀ3.5)
38.8 (ꢀ5.4)
38.3 (ꢀ5.6)
38.5 (ꢀ5.5)
43.3 (ꢀ3.1)
46.0 (ꢀ1.7)
40.1 (ꢀ3.1)
—
d(GGAtGtTGtCGA)
d(tTtTtTtTtT)-FAM
R = Lys
a
b
c
Duplex concn 2 lM in 10 mM NaH2PO4, 150 mM NaCl, pH 7.0. estimated error in Tm = 0.5 °C.
Tm of unmodified duplex: 47.5 °C.
Tm of unmodified duplex: 49.5 °C.
number of discontinuous modifications (ON7–9). This suggests
that in these cases the global reduction of negative charge of the
oligonucleotide becomes beneficial. This is important as only 50%
modification of an oligonucleotide is needed to render it mono
cationic.
There are clear structural differences between a pure DNA and a
DNA/RNA double helix, the former being of B-conformation and
the latter being an intermediate between A and B conformation.
An unsubstituted bcT unit in the same sequence context has been
shown previously to be nearly Tm neutral.23 Thus the relative
differences in affinity between complementary RNA and DNA
observed here are not a consequence of the bicyclic core structure
but that of the substituents. Previous C(60) substitutions on the
the solvent with a-substituents showing a slight bias towards
the minor groove and b-substituents towards the major groove.
Both configurations seem therefore to be well suited to present
aminoalkyl groups towards a potential nucleic acid binding inter-
face, but obviously the linker lengths still need to be optimized
to take full advantage of nucleic acid charge screening.
2.3. Cellular uptake
One of the main aims of this work was to investigate whether
multiple positive charges change the cellular uptake properties.
We therefore prepared the fluorescein labeled 10-mer model oligo-
nucleotide ON10, containing five alternately positioned lys-bcT
units. The sequence was specifically designed to be short in order
to exclude antisense effects potentially obstructing cellular metab-
olism and/or cellular distribution of the oligonucleotide. Two
bicyclo-skeleton in
found to be slightly less destabilizing when paired against RNA.23
In both the C(60)
- and b-cases the substituents point towards
a-configuration with aminoalkoxyl chains were
a

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J. Lietard et al. / Bioorg. Med. Chem. 19 (2011) 5869–5875
in the absence of any transfection agent. After 48 h cells were
washed fixed and analyzed by fluorescence microscopy (Fig. 3).
As can be seen from Figure 3 (left column), the natural control
oligonucleotide showed no visible internalization. In contrast
ON10 showed considerable uptake after the same exposure time
into both cell lines (Fig. 3, right column). The cellular distribution
was predominantly cytosolic and less nuclear. We quantified the
fraction of cells associated with fluorescein fluorescence by FACS
(Fig. 4) and found that in the case of ON10, 95% of the cells shifted
towards higher intensity in both cell lines. In the case of the control
d(T10)-FAM only 50–75% of the cells were shifted towards higher
fluorescence. The absence of cytosolic and nuclear fluorescence
spots in these cases suggests that the residual fluorescence is asso-
ciated with surface bound, labeled material. The fact that no inter-
nalization is observed with d(T10)-FAM proves that cellular uptake
of ON10 is not an artifact of the fluorescent label.
It has been shown previously that fixation of cells influences the
uptake and distribution of cationic peptide-PNA conjugates, lead-
ing to artifactual results.26 In a more recent study with oligonu-
cleotide phosphorohioates, however, such effects were absent.27
We therefore assume that fixation does not alter uptake and distri-
bution in the cases reported here, but we cannot rule it out
completely.
Figure 3. Fluorescence microscopic images (overlay of contour, green and blue
fluorescence) of HeLa cells (top row) and HEK293T cells (bottom row) treated with
d(T10)-FAM (left column) or ODN10 (right column) 48 h after transfection with
oligonucleotides (10 lM) without any transfection agent. Cells were incubated live
and only later fixed with formaldehyde for imaging. Nuclei (blue) were DAPI
stained.
3. Conclusions
We successfully prepared the two phosphoramidite building
blocks 5 and 7 containing a b-60-diaminopropyl or -lysyl substitu-
ent on the bcT skeleton and incorporated them into oligodeoxynu-
cleotides. Both modifications were essentially Tm neutral when
paired against DNA but were destabilizing when paired against
RNA. Cellular uptake studies in two cell lines with a decamer car-
rying five lys-bcT units showed consistent, unaided cellular inter-
nalization which was absent in the case of the corresponding
unmodified decamer. From these results we conclude that amino-
alkyl side chains are indeed a measure to increase cellular uptake
of oligonucleotides. We further conclude that position C(60), which
localizes the substituent in a unique and for natural nucleosides
inaccessible position relative to the double helix, is well suited
for this. The decrease of Tm against complementary RNA (which
is largely absent if DNA is the complement) is most likely a conse-
quence of inefficient screening of the negative phosphate charges
in the specific duplex conformation of an RNA/DNA hybrid. To rem-
edy this it will be necessary to further vary chain length and posi-
tion of the positive charges within the substituent.
Our experiments clearly demonstrate that the lysine unit at
C(60) of bcT improves unaided cellular uptake. These results are
in line with earlier findings on the internalization of fully modified
cationic phosphoramidate
a
-oligonucleotides,14 and with im-
proved antisense or siRNA activity of oligoribonucleotides carrying
cationic residues (including lysine units) attached to the 20-OH
group.19,20
4. Experimental
4.1. General
All reactions were performed under Ar in dried glassware.
Anhydrous solvents for reactions were obtained by filtration
through activated aluminum oxide, or by storage over 3 Å molecu-
lar sieves. Column chromatography (CC) was performed on silica
Figure 4. Histograms representing HeLa cells (top) and HEK293T cells (bottom)
treated with ON10 (orange) or d(T10)-FAM (green). Control: untreated cells (red).
20,000 cells were counted in each case.
gel (Fluka) with an average particle size of 40 lm. All solvents
for CC were of technical grade and distilled prior to use. Thin layer
chromatography (TLC) was performed on silica gel plates (Mache-
rey–Nagel, 0.25 mm, UV254). Visualization was performed either
different cell lines, HeLa and HEK293T, were transfected with
ON10 (10 lM) and with the unmodified d(T10)-FAM as a control,

J. Lietard et al. / Bioorg. Med. Chem. 19 (2011) 5869–5875
5873
by UV or by staining in dip solution (10.5 g Cer(IV)-sulfate, 21 g
phosphormolybdenic acid, 60 ml concd sulfuric acid, 900 ml H2O)
followed by heating with a heat gun. NMR spectra were recorded
on a Bruker DRX 400 or a Bruker AC 300 spectrometer at
400 MHz or 300 MHz (1H NMR) or 100 MHz (13C NMR) in either
CDCl3, CD3OD or DMSO-d6. d in ppm relative to residual undeuter-
ated solvent [CHCl3: 7.26 ppm (1H) and 77.0 ppm (13C); CHD2OD:
3.35 ppm (1H) and 49.3 ppm (13C); (CHD2)2SO: 2.54 ppm (1H) and
40.45 ppm (13C)], J in Hz. Signal assignments are based on DEPT
and on 1H–1H and 1H–13C correlation experiments (COSY/HSQC).
High resolution electrospray ionization (ESIꢀ) mass spectra (MS,
m/z) were recorded on an Applied Biosystems Sciex QSTAR Pulsar
instrument.
(CH3O–C-arom), 151.04 (C(2)), 145.50, 144.14, 144.11, 141.53,
136.83, 136.54 (C-arom), 137.62 (C(6)), 129.87, 127.97, 127.89,
125.17, 120.17 (C-arom(Fmoc)), 128.32, 127.33, 127.23, 113.66,
113.61 (C-arom(DMTr)), 111.05 (C(5)), 89.45 (C(40)), 87.53, 86.23
(C(30), C(Ph)3), 85.51 (C(10)), 74.22 (C(50)), 66.77 (CH2-Fmoc),
61.63 (C(60)), 55.48 (CH3O-DMTr), 48.54 (C(20)), 47.46 (CH-Fmoc),
45.86 (C(80)), 39.38 (C(100)), 38.25 (C(70)), 31.25 (C(90)), 12.86
(CH3–C(5)). ESI+-HRMS: calcd for C51H53O9N4 ([M+H]+) 865.3807,
found 865.3806.
4.1.3. 1-[(30S,50R,60R)-50-O-[(4,40-dimethoxytriphenyl)methyl]-
60-[300-(N-Fmoc-aminopropyl)]trifluoroacetylamino-20-deoxy-
30,50-ethano-b-
D-ribofuranosyl]thymine 4
Trifluoroacetic anhydride (0.161 ml, 1.15 mmol, 5 equiv) was
carefully added to a mixture of nucleoside 3 (200 mg, 0,231 mmol)
in dry pyridine (3.6 ml) at 0 °C. The solution immediately turned
yellow and the reaction was complete within 1 h. After dilution
with EtOAc (30 ml) the mixture was washed with sat aq NaHCO3
(2 ꢁ 25 ml) and the aqueous phases were extracted with EtOAc
(2 ꢁ 50 ml). The combined organic phases were dried over MgSO4,
filtered and evaporated. Purification by CC (EtOAc hexane/EtOAc,
60%?100%) afforded the title compound 4 (190 mg; 86%) as a yel-
low foam. TLC (CH2Cl2/EtOH 9:1) R = 0.67. 1H NMR (300 MHz,
DMSO-d6) d: 7.88–7.86 (m, 2H, H-arom(Fmoc)), 7.67 (d, J = 7.3 Hz,
2H, H-arom(Fmoc)), 7.39–7.11 (m, 14H, H-arom(Fmoc), H-ar-
om(DMTr), H–C(6)), 6.80–6.74 (m, 4H, H-arom(DMTr)), 5.74–5.65
(m, 1H, H–C(10)), 4.39–4.18 (m, 4H, H–C(50), CH2-Fmoc, CH-Fmoc),
3.71, 3.70, 3.68 (3s, 6H, MeO-DMTr), 3.76 (m, 1H, H–C(100)), 3.57 (m,
1H, H–C(100)), 3.04–2.96 (m, 2.3H, H–C(300), H–C(40)), 2.89 (d,
J = 4.2 Hz, 0.7H, H–C(40)), 2.60–2.53 (m, 1H, H–C(70)), 2.27 (dd,
J = 13.4, 7.9 Hz, 1H, H–C(20)), 2.09 (dd, J = 13.5, 7.2 Hz, 1H, H–
C(20)), 2.02 (dd, J = 12.7, 5.5 Hz, 1H, H–C(70)), 1.79, 1.70 (2s, 3H,
Me-C(5)), 1.75–1.59 (m, 2H, H–C(200)). 13C NMR (75 MHz, DMSO-
d6) d: 163.59 (C(4)), 158.19 (C-arom), 156.17, 156.13 (COCF3),
150.12 (C(2)), 143.77, 140.66 (C-arom), 136.47, 136.08 (C(6)),
135.68, 135.38 (C-arom), 130.61, 130.41, 128.26, 128.17, 127.54,
127.44, 126.98, 125.01, 120.05 (CH-arom), 112.70 (C-arom(DMTr)),
109.32 (C(5)), 87.85 (C(Ph)3), 87.31, 86.56 & 86.02 (C(40)), 84.58
(C(10)), 81.12 (C(30)), 74.11 (C(50)), 65.37 (CH2-Fmoc), 58.09
(C(60)), 54.92 (MeO-DMTr), 46.68 (CH-Fmoc), 44.84 (C(100)), 43.05
(C(20)), 41.22 (C(70)), 38.29 (C(300)), 27.86 (C(200)), 11.98, 11.88
(Me-C(5)).19F NMR (376 MHz, DMSO-d6) d ꢀ67.49, ꢀ67.51. ESI+-
HRMS: calcd for C53H51O10N4F3Na ([M+Na]+) 983.3449, found
983.3461.
4.1.1. 1-[(30S,50R,60R)-50-O-[(4,40-dimethoxytriphenyl)methyl]-
60-amino-20-deoxy-30,50-ethano-b-
D
-ribofuranosyl]thymine 2
To a solution of 60-aminobicyclothymidine 1 (3.37 g, 4.9 mmol,
2:1 mixture of b/ anomers) in MeOH (18 ml) was added concd
a
NH3 (150 ml) at rt. The mixture slowly turned purple and was stir-
red for 1 h at rt, then at 55 °C overnight. After evaporation of the
solvents the white solid was suspended in EtOH (150 ml), adsorbed
on silica gel (30 g) and purified by CC (CH2Cl2/EtOH, 2%?10%) to
give 2b (1.28 g, 44%) and 2a-anomer (632 mg, 22%) both as a white
foams. Analytical data of 2b: TLC (CH2Cl2/EtOH 9:1) Rf = 0.25. 1H
NMR (300 MHz, CD3OD) d: 7.96 (d, J = 0.8 Hz, 1H, H–C(6)), 7.56–
7.54 (m, 2H, H-arom), 7.46–7.42 (m, 4H, H-arom), 7.29–7.17 (m,
3H, H-arom), 6.85 (dd, J = 8.9, 1.3 Hz, 4H, H-arom), 5.93 (dd,
J = 9.0, 6.5 Hz, 1H, H–C(10)), 4.08 (t, J = 5.6 Hz, 1H, H-C(50)), 3.75
(s, 6H, MeO-DMTr), 3.69 (d, J = 5.3 Hz, 1H, H–C(40)), 2.84 (dd,
J = 13.5, 9.2 Hz, 1H, H–C(20)), 2.57 (t, J = 5.9 Hz, 1H, H–C(60)), 2.33
(dd, J = 13.5, 6.5 Hz, 1H, H–C(20)), 2.08 (d, J = 14.8 Hz, 1H,
H–C(70)), 1.88 (s, 3H, Me-C(5)), 1.65 (dd, J = 14.9, 6.4 Hz, 1H,
H–C(70)). 13C NMR (100 MHz, CD3OD) d: 167.58 (C(4)), 160.61
(C-arom), 153.53 (C(2)), 146.91 (C-arom), 141.45 (C(6)), 137.76,
137.53, 131.44, 131.41, 129.24, 129.14, 128.24, 114.54, 114.52
(C-arom), 111.91 (C(5)), 90.02, 89.93 (C(40), C(10)), 89.18, 85.08
(C(30), C(Ph)3), 75.62 (C(50)), 55.92 (MeO), 55.56 (C(60)), 47.63
(C(20)), 43.95 (C(70)), 12.64 (Me-C(5)). ESI+-HRMS: calcd for
C
33H37O7N3 ([M+H]+) 586.2548, found 586.2535.
4.1.2. 1-[(30S,50R,60R)-50-O-[(4,40-dimethoxytriphenyl)methyl]-
60-[300-(N-Fmoc-aminopropyl)]amino-20-deoxy-30,50-ethano-b-
ribofuranosyl]thymine 3
D
-
To a solution of nucleoside 2 (168 mg, 0.286 mmol) in dry
methanol (8 ml) containing molecular sieves (3 Å) was added
FmocNH(CH2)2CHO (169 mg, 0.572 mmol, 2 equiv) at rt. After stir-
ring for 90 min, NaCNBH3 (54 mg, 0.859 mmol, 3 equiv) was added
in three equal portions over 2 h. After 3 h the mixture was diluted
with 25 ml EtOAc and washed with satd aq NaHCO3 (2 ꢁ 25 ml),
and sat aq Na2CO3 solution (2 ꢁ 25 ml). The aqueous phases were
extracted with EtOAc (2 ꢁ 100 ml) and the combined organic
phases dried over MgSO4, filtered, evaporated and the faintly yel-
low residue purified by CC (hexane/EtOAc, 75%?90%) to give the
title compound 3 (210 mg, 85%) as a white foam. TLC (CH2Cl2/EtOH
9:1) R = 0.48. 1H NMR (CDCl3, 300 MHz) d: 9.12 (s, 1H, NH), 8.15 (s,
1H, H–C(6)), 7.74 (d, J = 7.5 Hz, 2H-arom(Fmoc)), 7.54 (t, J = 8.5 Hz,
4H-arom(Fmoc)), 7.44–7.40, 7.40–7.34 (2 m, 6H-arom), 7.30–7.25,
7.25–7.18 (2 m, 3H-arom), 6.82 (dd, J = 9.2, 3.5 Hz, 4Harom(DMTr)),
6.55 (dd, J = 9.8, 5.4 Hz, H–C(10)), 4.83–4.80 (m, 1H, H–N), 4.39 (d,
J = 6.9 Hz, 2H, CH2-Fmoc), 4.21–4.16 (m, 2H, H–C(40), CH-Fmoc),
4.03 (t, J = 5.9 Hz, 1H, H–C(50)), 3.76 (s, 6H, OCH3), 3.13 (m, 2H,
H–C(300)), 2.65 (dd, J = 14.1, 10.4 Hz, 1H, H–C(20)), 2.38 (dd,
J = 13.2, 5.2 Hz, 1H, H–C(20)), 2.32–2.24 (m, 1H, 1H–C(100)), 1.98
(s, 4H, H3C–C(5), 1H–C(70)), 1.86–1.80, 1.78–1.75, (2 m, 2H, H–
C(60), 1H–C(100)), 1.56–1.47 (m, 2H, H–C(200)), 1.25–1.19 (m, 1H,
H–C(70)). 13C NMR (CDCl3, 100 MHz) d: 163.99 (C(4)), 159.00
4.1.4. 1-[(30S,50R,60R)-50-O-[(4,40-dimethoxytriphenyl)methyl]-
60-[300-(N-Fmoc-aminopropyl)]amino- 30-O-(2-cyanoethoxy)
diisopropylaminophosphanyl-20-deoxy-30,50-ethano-b-
D-ribo-
furanosyl]thymine 5
To a mixture of nucleoside 4 (125 mg, 0,13 mmol) and EtNiPr2
(0.089 ml, 0.52 mmol, 4 equiv) in THF (2 ml) was added 2-cyano-
ethoxy diisopropylamino chlorophosphine (0.058 ml, 0.26 mmol,
2 equiv) at rt. After 45 min the mixture was diluted with EtOAc
(25 ml) and washed with a satd aq NaHCO3 (2 ꢁ 25 ml). The aque-
ous phases were extracted with EtOAc (2 ꢁ 50 ml) and the com-
bined organic phases dried over MgSO4, filtered, evaporated and
the crude material purified by CC (hexane/EtOAc 50%?75%) to give
phosphoramidite 5 (106 mg, 70%) as a white foam. TLC (EtOAc/
Hexane 4:1) R = 0.74. 1H NMR (400 MHz, DMSO-d6) d: 7.90–7.87
(m, 2H, H-arom(Fmoc)), 7.69–7.67 (m, 2H, H-arom(Fmoc)), 7.44–
7.38 (m, 3H, H-arom, H–C(6)), 7.32–7.29 (m, 4H, H-arom), 7.25–
7.14 (m, 7H, H-arom), 6.79–6.76 (m, 4H, H-arom(DMTr)), 5.71
(m, 1H, H–C(10)), 4.38–4.28 (m, 4H, CH2-Fmoc, H–C(60), H–C(50)),
4.23–4.20 (m, 1H, CH-Fmoc), 3.81 (br, 1H, H–C(300)), 3.71–3.69
(3s, 6H, MeO-DMTr), 3.64–3.45 (m, 5H, OCH2CH2CN, (Me2CH)2N,
H–C(300)), 3.10–3.02 (m, 3H, H–C(100), H–C(40)), 2.78–2.75 (m, 1H,

5874
J. Lietard et al. / Bioorg. Med. Chem. 19 (2011) 5869–5875
H–C(20)), 2.71–2.70 (m, 2H, OCH2CH2CN), 2.47–2.33 (m, 2.3H, H–
C(20), H–C(70)), 1.86 (sbr 1H, H–C(200)), 1.80, 1.73 (2s, 3H, Me-
C(5)), 1.65–1.57 (m, 1H, H–C(200)), 1.10–1.01 (m, 12H, (Me2CH)2N).
13C NMR (101 MHz, DMSO-d6) d: 163.62 (C(4)), 158.35, 158.26,
156.15 (C-arom), 150.16, 150.10 (C(2)), 144.82, 143.88, 143.81,
140.67 (C-arom), 136.91, 136.53 (C(6)), 135.55, 135.25 (C-arom),
130.49, 130.28, 128.15, 127.55, 127.01, 125.13, 120.07 (CH-arom),
118.86, 118.75 (CN), 112.77 (C-arom(DMTr), 109.58, 109.46 (C(5)),
88.08 (C(30)), 85.44 (C(10)), 85.19, 85.00 (C(40), C(Ph)3), 73.70, 73.53
(C(50)), 65.40 (CH2-Fmoc), 57.86, 57.70 (OCH2CH2CN), 54.95, 54.92
(CH3O-DMTr), 46.70 (CH-Fmoc), 45.05 (C(100)), 42.77, 42.76
((Me2CH)2N), 40.76 (C(70)), 39.85 (C(20)), 38.29 (C(80)), 27.82
(C(90)), 24.09, 23.93, 23.85 ((Me2CH)2N), 19.67, 19.60
(OCH2CH2CN), 11.93 (Me-C(5)). 19F NMR (376 MHz, DMSO-d6) d
ꢀ67.46, ꢀ67.53. 31P NMR (122 MHz, DMSO-d6) d 142.01, 141.32,
141.28, 140.94. ESI+-HRMS: calcd for C62H68O11N6F3NaP ([M+H]+)
1183.4528, found 1183.4561.
EtOAc (2 ꢁ 200 ml) and the combined organic phases dried over
MgSO4, filtered and evaporated. The crude yellow oil was purified
by CC (EtOAc/hexane 3:1?5:1) to yield phosphoramidite
7
(470 mg, 80%) as a white foam. 1H NMR (300 MHz, DMSO-d6) d:
11.49, 11.48 (br, 1H, H–N), 7.89–7.86 (m, 4H, H-arom(Fmoc)),
7.68–7.61 (m, 6H, H–C(6), H-arom), 7.44–7.23 (m, 18H, H-ar-
om(DMTr), H-arom(Fmoc)), 7.16 (t, 2H,), 6.99 (m, 1H,), 6.83–6.77
(m, 4H, H-arom(DMTr)), 5.82–5.76 (m, 1H, H–C(10)), 4.28–4.26 (m,
2.5H, CH2-Fmoc), 4.21–4.19 (m, 2H, CH2-Fmoc, CH-Fmoc), 4.09,
4.04 (2t, 1H, CH-Fmoc), 3.97–3.90 (m, 2H, H–C(a
), H–C(50)), 3.73–
3.67 (m, 0.5H, CH2-Fmoc), 3.65, 3.64 (2s, 6H, MeO-DMTr), 3.61–
3.42 (m, 5H, OCH2CH2CN, (Me2CH)2N, H–C(40)), 3.32–3.24 (m, 1H,
H–C(60)), 2.95 (m, 2H, H–C( ), 2.69–2.62 (m, 3H, H–C(20),
e
OCH2CH2CN), 2.33–2.21 (m, 1.5H, H–C(20)), 2.16–2.11 (m, 1H, H–
C(70)), 1.91–1.85 (m 1H, H–C(70)), 1.82 (br, 3H, Me-C(5)), 1.60 (br,
2H, H–C(b)), 1.37 (br, 2H, H–C(d)), 1.24 (m, 2H, H–C(c)), 1.08–0.99
(m, 12H, (Me2CH)2N). 13C NMR (100 MHz, DMSO-d6) d 171.27
(C(80)), 163.66 (C(4)), 158.21, 158.16 (MeO-C-arom), 156.35,
156.02 (C-arom, HN-COO-Fmoc), 150.27, 150.23 (C(2)), 145.33,
143.90, 143.67, 140.69, 140.62 (C-arom), 135.69, 135.62 (C(6))
129.79, 129.60, 127.68, 127.52, 126.97, 125.09 (CH-arom), 120.04,
119.95 (CH-arom(Fmoc)), 118.88, 118.85 (CN), 113.19, 113.13
(CH-arom(DMTr)), 110.00 (C(5)), 87.21, 87.15 (C(40)), 85.70 (C(10)),
72.35, 72.29 (C(50)), 65.71, 65.15 (CH2-Fmoc), 57.84 (OCH2CH2CN),
4.1.5. 1-[(30S,50R,60R)-50-O-[(4,40-dimethoxytriphenyl)methyl]-
60-[(N ,N -di-Fmoc)- -lysine]amino-20-deoxy-30,50-ethano-b-
L D-
a
e
ribofuranosyl]thymine 6
To a solution of nucleoside 2 (204 mg, 0.35 mmol) in dry THF
a
x
(3.2 ml), was added N ,N -Fmoc-protected lysine (246 mg,
0.42 mmol, 1.2 equiv) and 4-dimethylaminopyridine (21 mg,
0.17 mmol, 0.5 equiv) at rt. The mixture was cooled to 0 °C and a
54.92 (MeO-DMTr), 54.30 (C(
46.73, 46.49 (CH-Fmoc), 42.78, 42.65 ((Me2CH)2N), 40.88 (C(70)),
40.14–38.88 (C( )), 30.23, 30.10 (C(b)), 29.21 (C(d)), 24.23–23.77
a
)), 52.44 (C(60)), 44.96, 44.89 (C(20)),
solution
of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (EDC, 100 mg, 0.52 mmol, 1.5 equiv) in dry CH2Cl2
(2 ml) was slowly added. After 10 min at 0 °C the cooling bath
was removed and after 1 h the mixture was diluted with EtOAc
(100 ml) and washed with sat aq NaHCO3 (2 ꢁ 100 ml) The aq
phases were extracted with EtOAc (2 ꢁ 200 ml), the combined or-
ganic phases dried over MgSO4, filtered, evaporated and the crude
material purified by CC (EtOAc + 1% Et3N?EtOAc/EtOH 98:2) to
give the title compound 6 (373 mg, 93%) as a white foam. 1H
NMR (300 MHz, CD3OD) d: 7.74 (d, J = 7.4 Hz, 4H, H-arom), 7.61–
7.48 (m, 7H, H-arom), 7.41–7.18 (m, 15H, H-arom), 6.80–6.75 (m,
4H, H-arom(DMTr), 5.66 (dd, J = 8.8, 5.2 Hz, 1H, H–C(10)), 4.33–
4.29 (m, 2H, CH2-Fmoc), 4.24–4.17 (m, 4H, CH2-Fmoc, CH-Fmoc,
e
(Me2CH)2N), 22.72 (C(c)), 19.64, 19.57 (OCH2CH2CN), 11.93 (Me-
C(5)). 31P NMR (122 MHz, DMSO-d6) d 141.73, 140.95.
4.1.7. Oligonucleotides synthesis and purification
Oligonucleotide syntheses were performed on the 1.3 lmol
scale on a LKB Gene Assembler Plus (Pharmacia) DNA synthesizer
using standard solid-phase phosphoramidite chemistry. Natural
phosphoramidites were from Vivotide or Applied Biosystems. Solid
support was from Glen Research. Reagents were prepared accord-
ing to the manufacturer’s protocols. As coupling agent, 5-(ethyl-
thio)-1H-tetrazole (Azco PharmChem, 0.25 M in CH3CN) was
used. Fluorescein (FAM)-labeled oligonucleotides were synthe-
sized on FAM-solid support (Roche diagnostics). For the modified
phosphoramidites 5 and 7, an extended coupling time of 9 min
was applied and average coupling efficiencies, as monitored by tri-
tyl assay, were P93% for 7 (except for consecutive couplings,
where the yield dropped to 68%) and 90–95% for 5. Deprotection
of the oligonucleotides and detachment from the solid support
was carried out in concd aq NH3 for 16 h at 55 °C. Crude oligomers
were purified by ion-exchange HPLC on an Äkta basic 10/100 sys-
tem (GE healthcare) with a DNAPAC PA200, 4 ꢁ 250 mm analytical
column (Dionex). Buffer A) 25 mM Trizma (2-amino-2-hydroxy-
methyl-1,3-propanediol) in H2O, pH 8.0; buffer B) 25 mM Trizma,
1.25 M NaCl in H2O, pH 8.0. Linear gradients of B in A were used,
with a 1 ml/min flow rate and detection at 260 nm. Purified oligo-
nucleotides were desalted over Sep-Pak C18 Cartridges (Waters),
quantified on a Nanodrop spectrophotometer (Thermo Scientific),
and analyzed by ESI- mass spectrometry.
H–C(a
)), 4.11–4.04 (m, 1H, H–C(50)), 3.99 (t, J = 6.4 Hz, 1H, CH-
Fmoc), 3.88–3.85 (m, 1H, CH2-Fmoc), 3.67, 3.66 (2s, 6H, MeO-
DMTr), 3.53–3.51 (m, 1H, H–C(40)), 3.44–3.40 (m, 1H, H–C(60)),
3.14–3.06 (m, 2H, H–C(e
)), 2.52–2.45 (m, 1H, H–C(20)), 2.30 (dd,
J = 13.2, 5.2 Hz, 1H, H–C(20)), 2.07 (d, J = 14.6 Hz, 1H, H–C(70)),
1.91, 1.88 (2sbr, 3H, Me-C(5)), 1.75–1.33 (m, 7H, CH2-lysine, H–
C(70)). 13C NMR (101 MHz, CD3OD) d 180.10, 177.40, 166.59,
166.47 (C(4)), 160.43 (MeO-C-arom), 149.23 (C(2)), 147.03,
146.98, 145.53, 145.13, 142.76, 141.57, 139.47 (C-arom), 137.57
(C(6)), 131.53, 130.06, 129.32, 129.02, 128.87, 128.27, 126.35
(CH-arom), 122.19, 121.03 (CH-arom(Fmoc)), 114.42 (CH-ar-
om(DMTr), 89.80 (C(10)), 89.19 (C(40)), 84.80, 74.47 (C(50)), 67.82
(CH2-Fmoc), 55.88 (C(
a
)), 55.81 (CH3O-DMTr), 54.20 (C(60)),
)),
48.10, 48.02 (CH-Fmoc), 47.38 (C(20)), 43.10 (C(70)), 41.44 (C(
e
31.70 (CH2-lysine), 30.20 (CH2-lysine), 24.21 (CH2-lysine), 12.63
(Me-C(5)). ESI+-HRMS: calcd for C69H67O5N12Na ([M+Na]+)
1180.4678, found 1180.4660.
4.1.6. 1-[(30S,50R,60R)-50-O-[(4,40-dimethoxytriphenyl)methyl]-
4.1.8. UV-melting curves
60-[(N ,N -di-Fmoc)-
L
-lysine]amino-30-O-(2-cyanoethoxy)
UV-melting curves were recorded on a Varian Cary 100 Bio UV–
vis spectrophotometer. Absorbances were monitored at 260 nm
and the heating rate was set to 0.5 °C/min. A cooling–heating–cool-
ing cycle in the temperature range 15–80 °C was applied. Tm values
were obtained from the derivative curves using the Varian WinUV
software. To avoid evaporation, the sample solutions were covered
with a layer of dimethylpolysiloxane. All measurements were car-
ried out in 150 mM NaCl, 10 mM Na2HPO4, pH 7.0 with duplex
a
e
diisopropylaminophosphanyl-20-deoxy-30,50-ethano-b-
D-
ribofuranosyl]thymine7
To
a
solution of DMT-protected nucleoside
6
(505 mg,
0.43 mmol) in dry THF (8.5 ml) was added, iPr2NEt (0.3 ml,
1.73 mmol, 4 equiv) and 2-cyanoethoxy diisopropylamino chloro-
phosphine (0.2 ml, 0.87 mmol, 2 equiv) at rt. The solution was stir-
red for 45 min, diluted with EtOAc (100 ml) and washed with satd
aq NaHCO3 (2 ꢁ 100 ml). The aqueous phases were extracted with
concentrations of 2 lM

J. Lietard et al. / Bioorg. Med. Chem. 19 (2011) 5869–5875
5875
4.1.9. Cell cultures and transfection
References and notes
Hela and HEK293T cells were grown at 37 °C in Dulbecco’s Mod-
ified Eagle’s Medium (DMEM, Invitrogen) supplemented with 10%
(v/v) Fetal Calf Serum (Amimed), 100 units/ml penicillin (Invitro-
1. Stephenson, M. L.; Zamecnik, P. C. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 285.
2. Zamecnik, P. C.; Stephenson, M. L. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 280.
3. Sibley, C. R.; Seow, Y.; Wood, M. J. A. Mol. Ther. 2010, 18, 466.
4. Bennett, C. F.; Swayze, E. E. Annu. Rev. Pharmacol. Toxicol. 2010, 50, 259.
5. Koshkin, A. A.; Nielsen, P.; Meldgaard, M.; Rajwanshi, V. K.; Singh, S. K.; Wengel,
J. J. Am. Chem. Soc. 1998, 120, 13252.
6. Obika, S.; Nanbu, D.; Hari, Y.; Andoh, J. I.; Morio, K. I.; Doi, T.; Imanishi, T.
Tetrahedron Lett. 1998, 39, 5401.
7. Hendrix, C.; Rosemeyer, H.; Verheggen, I.; Seela, F.; Van Aerschot, A.;
Herdewijn, P. Chem. Eur. J. 1997, 3, 110.
8. Steffens, R.; Leumann, C. J. J. Am. Chem. Soc. 1997, 119, 11548.
9. Renneberg, D.; Leumann, C. J. J. Am. Chem. Soc. 2002, 124, 5993.
10. Lönnberg, H. Bioconjugate Chem. 2009, 20, 1065.
11. Debart, F.; Abes, S.; Deglane, G.; Moulton, H. M.; Clair, P.; Gait, M. J.; Vasseur, J.
J.; Lebleu, B. Curr Top Med Chem 2007, 7, 727.
12. Chang, C. I.; Kim, H. A.; Dua, P.; Kim, S.; Li, C. J.; Lee, D.-k. Oligonucleotides 2011.
13. Letsinger, R. L.; Singman, C. N.; Histand, G.; Salunkhe, M. J. Am. Chem. Soc. 1988,
110, 4470.
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gen) and 100 lg/ml streptomycin (Invitrogen). For transfection
experiments, 1 ꢁ 105 HeLa and 2 ꢁ 105 HEK293T cells were seeded
in duplicate in six-well plates, half of them containing cover slips,
24 h before transfection. Then, the medium was replaced by a solu-
tion of fluorescein-labeled oligonucleotide ON10 or d(T10)FAM
(10 lM final concentration) in DMEM +/+ (FCS, P/S). The transfec-
tion medium was removed after 48 h at 37 °C and cells washed with
2 ꢁ 1 ml PBS and resuspended in 1 ml fresh DMEM +/+.
4.1.10. Fluorescence measurements and FACS analysis
Fixation of the cells on the cover slips was carried out using a
solution of paraformaldehyde (1 ml, 3.7% in PBS) for 10 min
followed by washing with PBS (2 ꢁ 1 ml), permeabilization of the
cell membrane with Triton x-100 (0.2%, Promega) for 10 min and
washing with PBS (2 ꢁ 1 ml).The cover slips were treated with a
few drops of polyvinylalcohol (Mowiol) and nuclear stain 40,60-
diamidino-2-phenylindole (DAPI). Cells were analyzed by fluores-
cence microscopy (Leica DMI6000 B, Leica Microsystems; software:
Leica Application Suite) 48 h post transfection. Cellular uptake of
fluorescein-labeled oligonucleotides was determined by fluores-
cence-activated cell sorting (FACS, BD FACSCalibur, BD Biosciences).
HeLa and HEK293T cells grown without cover slips were washed in
PBS and trypsinized. After dilution in 1 ml DMEM +/+, the suspen-
sion was centrifuged, the supernatant removed and the pellets
resuspended in 1 ml cold PBS. They were then stored on ice. Fluores-
cence intensity was measured using FACSscan and Cell Quest Pro
software. Viable cells were gated for forward, side scatter and fluo-
rescence activity. 20,000 events were captured per sample.
20. Winkler, J.; Gilbert, M.; Kocourková, A.; Stessl, M.; Noe, C. R. Chem. Med. Chem.
2008, 3, 102.
21. Winkler, J.; Urban, E.; Noe, C. R. Bioconjugate Chem. 2005, 16, 1038.
22. Luisier, S.; Leumann, C. J. Heterocycles 2010, 82, 775.
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24. Luisier, S.; Leumann, C. J. Chem. Bio. Chem. 2008, 9, 2244.
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Acknowledgments
This work was supported by the Swiss National Science Founda-
tion (Grant No.: 200020-115913).