Parallel synthesis and nucleic acid binding properties of C(6′)-α-functionalized bicyclo-DNA

Article abstract of DOI:10.1016/j.bmc.2010.09.064

Two novel bicyclo-T nucleosides carrying a hydroxyl or a carboxymethyl substituent in C(6′)-α-position were prepared and incorporated into oligodeoxynucleotides. During oligonucleotide deprotection the carboxymethyl substituent was converted into different amide substituents in a parallel way. T_m-measurements showed no dramatic differences in both, thermal affinity and mismatch discrimination, compared to unmodified oligonucleotides. The post-synthetic modification of the carboxymethyl substituent allows in principle for a parallel preparation of a library of oligonucleotides carrying diverse substituents at C(6′). In addition, functional groups can be placed into unique positions in a DNA double helix.

Full text of DOI:10.1016/j.bmc.2010.09.064

Bioorganic & Medicinal Chemistry 18 (2010) 7786–7793
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Parallel synthesis and nucleic acid binding properties of C(60)-
bicyclo-DNA
a-functionalized
⇑
Peter Šilhár, Christian J. Leumann
Department of Chemistry and 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
Two novel bicyclo-T nucleosides carrying a hydroxyl or a carboxymethyl substituent in C(60)-
a-position
Article history:
Received 23 August 2010
Revised 20 September 2010
Accepted 24 September 2010
Available online 1 October 2010
were prepared and incorporated into oligodeoxynucleotides. During oligonucleotide deprotection the
carboxymethyl substituent was converted into different amide substituents in a parallel way. Tm-mea-
surements showed no dramatic differences in both, thermal affinity and mismatch discrimination, com-
pared to unmodified oligonucleotides. The post-synthetic modification of the carboxymethyl substituent
allows in principle for a parallel preparation of a library of oligonucleotides carrying diverse substituents
at C(60). In addition, functional groups can be placed into unique positions in a DNA double helix.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Oligonucleotides
Nucleosides
Antisense
DNA diagnostics
Bicyclo-DNA
1. Introduction
Some years ago we developed bicyclo-DNA (bc-DNA) as a first
generation conformationally constrained oligonucleotide analog.13
Oligonucleotide based gene silencing was discovered in the late
seventies of the last century,1,2 and has evolved over the years into
oligonucleotide based therapeutic strategies, the two most impor-
tant ones being antisense and RNA interference.3,4 In the therapeu-
tic 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.
Over the years a large number of chemically modified oligonucleo-
tide analogs have been synthesized and their biological properties
evaluated. The field has been reviewed on several occasions before,
and so also very recently.5 One successful concept applied to the de-
sign of oligonucleotide analogs is that of conformational restriction,
resulting in structures such as locked-nucleic acids (LNA),6,7 hexitol
nucleic acids (HNA),8 and tricyclo-DNA (tc-DNA, Fig. 1).9,10 These
analogs typically exhibit increased affinity to RNA and feature high-
er nuclease resistance. Due to their properties, some of these mod-
ified nucleic acids are now considered to become next generation
oligonucleotide drugs.4,11 While the challenges linked to target
affinity and nuclease resistance have largely been met in the past,
several drawbacks, the most prominent ones being cellular uptake
and distribution, largely remained unsolved. A promising strategy
for the latter problem may be bioconjugation of oligonucleotides
to specific or unspecific cell targeting molecular entities.12
It turned out that bc-DNA base-pairs to natural nucleic acids with
about equal stability as DNA itself.14 One of the unique structural
features of bc-DNA is the ethylene bridge between the centers
C(30) and C(50) of the ribose unit which lends itself for further
chemical substitution. We reasoned that additional substituents
O
P
O-
O
6'
P
O-
4'
O
P
O-
O
O
5'
O
O
O
O
RO
Base
Base
Base
1'
3'
O
7'
2'
O
bcOalk-DNA
O
bc-DNA
tc-DNA
R = H
O-
O
P
O-
O
P
+
R = CH2CONH(CH2)2NH3
R = CH2CONH(CH2)3NH3
+
O
O
O
O
N
Base
H2N
Base
BnO
O
O
6'-amino-bc-DNA
bcox-DNA
Figure 1. Conformationally constrained oligonucleotide analogs of the bicyclo-DNA
type: left 60- -O-derivatized bicyclo-DNA (bcOalk-DNA), center bicyclo-DNA
(bc-DNA), right: tricyclo-DNA (tc-DNA).
⇑
Corresponding author. Tel.: +41 31 631 4355; fax: +41 31 631 3422.
E-mail address: leumann@ioc.unibe.ch (C.J. Leumann).
a
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.09.064

P. Šilhár, C. J. Leumann / Bioorg. Med. Chem. 18 (2010) 7786–7793
7787
at position C(60) could be interesting, for example, for controlling
the conformation of the carbocyclic ring, thus contributing to the
structure activity profile of the bc-DNA molecular platform. In
addition, incorporation of a post-synthetically transformable group
could be used either to attach cell targeting or reporter groups, or
to improve the lipophilic character of bc-DNA, thus improving its
cellular uptake properties. Earlier work on C(60)-oxime modified
bc-DNA (bcox-DNA) turned out to be encouraging in this context.15
Another candidate of interest, particularly in the latter context, is
bcJalk-DNA ( Fig. 1). Here we report on the synthesis of the
bcOalk-T nucleoside carrying an alkoxy substituent in
a-position
at C(60), on its conformational preference as determined by X-ray
crystallography, on its incorporation into oligonucleotides and on
the base-pairing properties with DNA and RNA.
Figure 2. X-ray structure of nucleoside 6.
2. Results and discussion
Table 1
Pseudorotation phase angles (P) and selected nucleoside torsion angles
2.1. Synthesis of nucleosides and building blocks
P (°)
c
(°)
d (°)
v (°)
6
108
128
144
158.2
149.3
57
112.6
126.5
122
ꢀ133.5
ꢀ112.8
ꢀ119
The synthesis of the building block 8, leading to bc-DNA con-
taining a hydroxyl group at C(60), started from the already known
diol 1 (Scheme 1) that was used previously in the synthesis of
60-amino-bc-DNA.16 The secondary hydroxyl group of diol 1 was
selectively acetylated with Ac2O to give compound 2. Looking for
a b-selective nucleosidation reaction we focused on a two step pro-
cedure involving NIS mediated regio- and stereoselective addition
of persilylated thymine to glycals,17,18 followed by radical removal
of the iodo-substituent. This reaction sequence proved to be very
useful for the b-selective synthesis of tricyclo-pyrimidine nucleo-
sides.19,20 Therefore, intermediate 2 was transformed to the fura-
nose glycal 3 with TMS-OTf in the presence of 2,6-lutidine. Under
these conditions the free 30-OH group was simultaneously silylat-
ed. NIS mediated addition of persilylated thymine to glycal 3 then
bc-Ta
dNb
a
Taken from (Ref. 21).
Average deoxynucleotide conformation in B-DNA (Ref. 22).
b
appears in an 60-endo conformation with the 50- and 60-subsituents
pseudoequatorially aligned. A comparison with the X-ray struc-
tures of the bc-T nucleoside shows a high degree of similarity of
the torsion angles c, d and v a-substituent
, indicating that a 60-
does not cause any significant changes to the bicyclic core
conformation.
The building block 8 for oligonucleotide synthesis was then pre-
pared by standard protocols in DNA chemistry. The secondary OH
function in 6 was selectively tritylated with 4,40-dimethoxytrityl
chloride (?7) followed by phosphitylation of the 30-OH function
leading to phosphoramidite 8.
The synthesis of the alkoxy substituted building block 17 was
first tried by alkylation of 1 using ethyl bromoacetate and a variety
of bases such as Pr2EtN, DBU, pyridine, K2CO3. None of these at-
tempts proved to be successful and typically ended with uncon-
verted starting material 1 or decomposition. In situ addition of
NaI or AgOTf did not solve the problem either, as it lead to only
low yields of the desired alkylation product, or to double (O(5),
O(7)) alkylation of the oxabicyclic ring system. The finally
proceeded smoothly and yielded 4 in an anomeric ratio b/
a of
85:15 (1H NMR). The inseparable mixture of nucleosides 4 was
deiodinated via radical reduction and the anomeric mixture re-
solved by chromatography (?5). Nucleoside 6 was then obtained
by desilylation with HF in pyridine.
Suitable crystals of nucleoside 6 were subjected to X-ray analy-
sis in order to prove the anomeric configuration as well as to map
the conformational preferences of the bicyclic core structure
(Fig. 2). In the crystal the furanose ring shows a perfect 01T confor-
i
mation (twisted O(40)-endo/C(10)-exo) with
a pseudorotation
phase angle P of 108° and thus adopts S-type conformation as also
the natural 20-deoxynucleosides (Table 1). The carbocyclic ring
O
TBSO
TBSO
HO
TBSO
AcO
TBSO
O
O
O
O
a
b
c
d
OMe
N
OMe
NH
86%
92%
81%
80%
AcO
AcO
O
I
OH
OH
OTMS
TMSO
1
2
3
4
β:α = 85 :15
O
O
O
g
O
TBSO
AcO
HO
DMTrO
DMTrO
O
O
O
O
N
O
N
N
N
e
f
NH
NH
NH
NH
93%
93%
90%
AcO
AcO
AcO
O
O
O
OTMS
OH
OH
O
(iPr)2N
P
5
6
7
OCH2CH2CN
8
+ 14% α-nucleoside
Scheme 1. Synthesis of nucleoside building block 8 (a) Ac2O (1.5 equiv), pyridine (2 equiv), ClCH2CH2Cl, 55 °C, 20 h. (b) TMS-OTf (2.5 equiv), 2,6-lutidine (5 equiv), CH2Cl2, rt,
3 h. (c) thymine (1.5 equiv), BSA (1.6 equiv), CH2Cl2, rt, 1 h, NIS (1.2 equiv), rt, 8 h. (d) Bu3SnH (1.2 equiv), AIBN (0.3 equiv), toluene, 110 °C, 2 h. (e) HF.py, pyridine/DCM, rt,
16 h. (f) DMTr-Cl (1.2 equiv), pyridine (2 equiv), CH2Cl2, rt, 12 h. (g) iPr2NP(Cl)CH2CH2CN (2.5 equiv), iPr2NEt (4 equiv), MeCN, rt, 2 h.

7788
P. Šilhár, C. J. Leumann / Bioorg. Med. Chem. 18 (2010) 7786–7793
TBSO
TBSO
HO
TBSO
TBSO
O
O
O
O
O
OMe
OMe
OMe
a, b
d
e
c
80%
92%
77%
AcO
77%
O
OH
OTBS
OTBS
OTBS
EtOOC
EtOOC
2
9
10
11
g
94%
O
O
O
TBSO
TBSO
O
O
O
TBSO
N
O
O
N
N
NH
NH
f
NH
+
O
O
99%
O
O
OTBS
OTBS
I
TBSO
12
EtOOC
EtOOC
14
EtOOC
13
h
β:α = 5 :6
80%
O
O
O
HO
DMTrO
DMTrO
O
O
O
O
N
N
N
O
i
j
NH
NH
NH
92%
82%
O
O
O
O
OH
OH
16
O
EtOOC
EtOOC
EtOOC
P
(iPr)2N
15
OCH2CH2CN
17
Scheme 2. Synthesis of bc-DNA building block 17 (a) TBDMS-Cl (2 equiv), imidazole (3 equiv), DMF, 75 °C, 2 days. (b) K2CO3 (2 equiv), EtOH, rt, 8 h. (c) N2CHCOOEt (2 equiv),
Cu(acac)2 (5 mol %), CH2Cl2, 70 °C, 5 h. (d) TMS-OTf (2 equiv), 2,6-lutidine (6 equiv), CH2Cl2, rt, 3 h. (e) thymine (3 equiv), BSA (2.5 equiv), CH2Cl2, 40 °C, 2 h, N-Iodosuccinimide
(1.3 equiv), rt, 7 h. (f) Bu3SnH (1.5 equiv), AIBN (0.4 equiv), toluene, 110 °C, 2 h. (g) TMS-OTf (2.5 equiv), 2,6-lutidine (7 equiv), ClCH2CH2Cl, 85 °C, 3 h. (h) HF.py, pyridine/THF,
50 °C, 2 days. (i) DMTr-Cl (1.5 equiv), pyridine (3 equiv), CH2Cl2, rt, 12 h. (J=) (iPr)2NP(Cl)OCH2CH2CN (2.5 equiv), iPr2NEt (4 equiv), MeCN, rt, 2 h.
successful synthesis started with intermediate 2 and is depicted in
Scheme 2. To prevent alkylation of the O(5) function, it was TBS-
protected to give after deacetylation compound 9. O(7)-alkylation
was then accomplished by Cu(II) catalyzed carbene insertion of
ethyl diazoacetate yielding 10. Applying the same strategy as
before, glycal 11, obtained from 10 by elimination, was subjected
to NIS mediated nucleosidation and produced the iodonucleosides
converted to the phosphoramidite building block 17 following
analogous procedures as for amidite 8.
2.2. Oligonucleotide synthesis and deprotection
A series of oligodeoxyribonucleotides (Table 2) containing sin-
gle or double substitutions were synthesized on a 1.3 lmol scale
12, however in a disappointing anomeric ratio b/
a
= 5:6 (1H NMR).
by standard automated phosphoramidite chemistry. For incorpora-
tion of the modified building blocks the coupling time was ex-
tended to 6 min for 8 and 12 min for 17. No further changes to
the synthesis cycle were necessary. Coupling yields, as monitored
by the trityl assay, were in all cases in the range of 97–99 %. After
completion of chain assembly, the detachment from the solid sup-
port and deprotection was carried out by standard treatment with
conc. NH3 (55 °C, 16 h 70 °C) for oligonucleotides that included
building block 8. Solid supported oligonucleotides constructed
We believe that the increase in steric demand of the TBS group
compared to the TMS group (as in 3) is primarily responsible for
the loss of selectivity. The anomeric mixture of nucleosides 12
were deiodinated as before and the nucleosides 13 and 14 could
be isolated in pure form after chromatographic separation. The
undesired
a-nucleoside 14 could be recycled to glycal 11 while
the b-nucleoside 13, the configuration at the anomeric center of
which was confirmed by 1H NMR-NOE spectroscopy, was further
Table 2
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)
t =
R
N
O
O
Sequence
ESIꢀ-MS m/z calc
ESIꢀ-MS m/z found
(
D
(D
NH
ODNA
ON1
ON2
ON3
ON4
ON5
ON6
ON7
ON8
ON9
ON10
ON11
ON12
d(GGA TGT TCt CGA)
R = H
R = OH
R = OCH2CONH(CH2)2NH3
R = OCH2CONH(CH2)3NH3
R = H
3701.2
3718.4
3818.6
3832.6
3727.1
3760.5
3960.7
3988.8
3727.1
3760.5
3960.7
3988.8
3701.0
3718.8
3817.9
3832.6
3728.4
3761.2
3960.5
3988.4
3728.6
3760.9
3960.9
3988.5
49.1 (+1.5)
45.3 (ꢀ2.3)
48.0 (+0.4)
47.0 (ꢀ0.6)
48.0 (+0.2)
45.6 (ꢀ1.0)
47.3 (ꢀ0.2)
47.3 (ꢀ0.2)
48.8 (+0.6)
47.0 (ꢀ0.3)
47.0 (ꢀ0.3)
46.7 (ꢀ0.5)
49.2 (ꢀ0.5)
48.3 (ꢀ1.4)
47.6 (ꢀ2.1)
47.2 (ꢀ2.5)
51.1 (+0.7)
46.9 (ꢀ1.4)
46.2 (ꢀ1.7)
46.2 (ꢀ1.7)
48.5 (ꢀ0.6)
47.9 (ꢀ0.9)
46.6 (ꢀ1.5)
46.6 (ꢀ1.5)
+
+
d(GGA tGT TCt CGA)
d(GGA TGt tCT CGA)
R = OH
+
+
R = OCH2CONH(CH2)2NH3
R = OCH2CONH(CH2)3NH3
R = H
R = OH
R = OCH2CONH(CH2)2NH3
R = OCH2CONH(CH2)3NH3
+
+
a
b
c
Total strand conc 2 lM in 10 mM NaH2PO4, 150 mM NaCl, pH 7.0. Estimated error in Tm = 0.5 °C.
Tm of unmodified duplex: 47.6 °C.
Tm of unmodified duplex: 49.7 °C.

P. Šilhár, C. J. Leumann / Bioorg. Med. Chem. 18 (2010) 7786–7793
Table 3
7789
ODNA
ODNA
Tm data (°C) from UV-melting curves (260 nm) of ON1 and ON2 with DNA
complements carrying a mismatched base opposite the modification
H2N(CH2)nNH2 / H2O
O
O
O
Thy
O
Thy
60°C, 20 h
Mismatchb
ON2
ON1
Unmodified DNAa
ODNA
ODNA
COOEt
+H3N(H2C)nHNOC
G–T
C–T
T–T
37.6 (ꢀ7.7)
36.7 (ꢀ8.6)
32.3 (ꢀ13.0)
37.0 (ꢀ12.0)
35.0 (ꢀ14.0)
32.0 (ꢀ17.0)
39.7 (ꢀ7.8)
36.0 (ꢀ11.5)
38.0 (ꢀ9.5)
Scheme 3. Conditions for detachment/deprotection with concomitant transforma-
tion of ester into amide functions of oligonucleotides built from phosphoramidite
a
2
l
M concn of strands, 150 mM NaCl, 10 mM phosphate buffer (pH 7.0).
17.
b
Tm of matched duplexes see Table 2. Values in parenthesis are Tm relative to
D
the matched duplex.
from building block 17 were split into two parts and were treated
in a parallel fashion either with 40% aqueous 1,2-diaminoethane or
1,3-diaminopropane at 60 °C for 20 h. Under these conditions, not
only deprotection and detachment from solid support, but also
aminolysis of the unique ester functions occurred (Scheme 3). With
this procedure an aminoalkyl chain is covalently introduced which
is expected to stabilize duplexes by electrostatic means and which
could also beneficially influence cellular uptake of oligonucleo-
tides.23 The crude oligonucleotides were then purified by ion-ex-
difficult as the studies on 60-amino-bc-DNA have been conducted
on oligothymidylate sequences exclusively which may behave dif-
ferently as the more general sequences chosen for this study.
2.4. Mismatch discrimination
For the case of ON2 as an example we determined Tm data with
complementary DNA carrying a mismatched base opposite the
modification to determine the relative effect of the modification
on pairing selectivity (Table 3). Generally, the OH function on the
bc-T unit leads to less discrimination of a mismatch situation com-
pared with an unmodified bc-T unit. On the other hand, an unmod-
ified bc-T unit shows higher discriminative power compared with a
natural dT unit. Differences are particularly manifest in the case of
the wobble (G–T) mismatch. The molecular origin of these thermo-
chemical variations remains elusive at this point.
change HPLC. Table
2 gives an overview on the sequences
ON1–12 obtained in this way as well as a confirmation of their
structure by mass spectrometry. In addition the thermal melting
data (Tm) with complementary DNA and RNA are summarized.
2.3. Tm measurements
UV-melting curves were measured at 260 nm with a cooling–
heating–cooling cycle at a rate of 0.5 °C/min in standard saline buf-
fer (10 mM Na-phosphate, 150 mM NaCl, pH 7.0). Analysis of the
Tm data (Table 2) revealed that oligonucleotides containing single
(ON2–4) or double substitutions in a discontinuous (ON6–8) or
continuous (ON10–12) fashion either slightly destabilized du-
3. Conclusions
We successfully prepared the two phosphoramidite building
blocks 8 and 17 containing 60-alkoxy bc-T modifications and incor-
porated them into oligodeoxynucleotides. While building block 8
led to 60-hydroxylated bc-T-units, 17 is versatile and can post-syn-
thetically be transformed by ester aminolysis into a variety of
structurally diverse oligonucleotides, depending on the deprotec-
tion conditions. We found that neither of the investigated 60-mod-
ifications had a dramatic effect on Tm with complementary DNA
and RNA. Thus, the synthetic strategy with building block 17 opens
the door for rapid, parallel synthesis of oligonucleotides with vary-
ing functional groups that can be beneficial for bioanalytical or
therapeutic purposes. In addition, the bc-DNA scaffold offers possi-
bilities for introducing unique functional entities in positions of the
DNA double helix without significantly compromising with DNA or
RNA affinity that are otherwise not accessible. This may be
exploited in the future for post-synthetic conjugation to improve
the performance of diagnostic or therapeutic oligonucleotides.
plexes with complementary DNA or were Tm neutral.
DTm per
modification range from ꢀ2.3 to +0.4 °C. The 60-OH-bc-T modifica-
tion tends to destabilize more than the diaminoethyl- or diamino-
propyl-bc-T units. This may be a consequence of the additional
positive charge in the latter modifications, counterbalancing the
repulsive effect of the two phosphodiester backbones in the duplex
to some extent. Interestingly, the difference in the linker length
had no significant effect on Tm. In the case of two consecutive mod-
ifications (ON10–12) almost no destabilization was observed
against complementary DNA. This indicates that reducing the
number of junctions between natural and bc-units in the backbone
has a positive effect on Tm. The same trend, but somewhat more
pronounced, also occurs with RNA as complement. All modifica-
tions show also slightly reduced affinity compared to duplexes
containing unmodified bc-T units (ON1, 5, 9). The substitution of
a 60-
sumably less due to conformational changes but more due to the
change in hydrophobicity. Structural models of 60- -bcOalk-DNA
a-hydrogen atom by a hydroxyl function thus destabilizes pre-
a
shows that the substituents point away in a perpendicular way
from the helix axis, directly into the solvent. We thus exclude that
interference of these substituents with base-pairing or with the
phosphodiester backbone cause the slight drop in Tm.
4. Experimental
4.1. General
Compared with 60-amino-bicyclo-DNA (Fig. 1) which carries an
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 4 Å molecu-
lar sieves. Column chromatography (CC) was performed on silica
amino function in b-position,16 the C(60)-
a-OR modifications are
slightly more destabilizing. This could be a consequence of the
missing or differentially aligned positive charges in the latter mod-
ifications which might shield the negative charges of the phospho-
diester backbone in a less efficient way. Alternatively, it could also
be a conformational effect of the carbocyclic ring. In the case of
C(50,60)-cis-substitution, as in 60-amino-bc-DNA, the 50-OH group
can be expected to have a higher tendency to occur in a pseudoax-
ial position matching more closely the natural +sc conformation of
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
(Macherey-Nagel, 0.25 mm, UV254). Visualization was performed
either by UV or by staining in dip solution (10.5 g Cer(IV)-sulfate,
21 g phophormolybdenic acid, 60 ml conc. sulfuric acid, 900 ml
H2O) followed by heating with a heat gun. NMR spectra were re-
corded on a Bruker DRX-400 or a Bruker AC-300 spectrometer at
400 or 300 MHz (1H NMR) or 100 MHz (13C NMR) in either CDCl3
torsion angle
bcOalk-DNA, where
needs, however, to be pointed out that direct comparison is
c
compared to C(50,60)-trans-substitution, as in 60-
is more constrained to the ap-conformation. It
a-
c

7790
P. Šilhár, C. J. Leumann / Bioorg. Med. Chem. 18 (2010) 7786–7793
or CD3OD. d in ppm relative to residual undeuterated solvent
(CHCl3: 7.26 ppm (1H) and 77.0 ppm (13C). CHD2OD: 3.35 ppm
(1H) and 49.3 ppm (13C)), J in Hz. 13C-multiplicities were deter-
mined from DEPT-spectra and signal assignments are based on
13C/1H-HMBC spectra. Proton signal assignments were based on
COSY and HMBC. 1H NMR difference-NOE spectra were recorded
on a Bruker DRX-500 instrument at 500 MHz. High resolution elec-
trospray ionization (ESI) mass spectra (MS, m/z) were recorded on
an Applied Biosystems Sciex QSTAR Pulsar instrument.
1H NMR (300 MHz, CDCl3) d: 0.09, 0.12 (6H, 2 ꢁ s, Me2Si), 0.25
(9H, s, Me3Si), 0.89 (9H, s, Me3CSi), 1.64 (1H, dd, J = 13.8, 8.7 Hz,
H–C(70)), 1.94 (3H, d, J = 1.3 Hz, Me–C(5)), 2.10 (3H, s, Ac), 2.38
(1H, dd, J = 13.8, 6.4 Hz, H–C(70)), 3.84 (1H, d, J = 9.8 Hz, H–C(20)),
4.14 (1H, t, J = 6.8 Hz, H–C(50)), 4.27 (1H, d, J = 7.2, H–C(40)), 4.94
(1H dt, J = 8.7, 6.4 Hz, H–C(60)), 6.34 (1H, d, J = 9.8 Hz, 1H–C(10)),
7.22 (1H, d, J = 1.3, 1H–C(6)), 8.17 (1H, s, br, H–N(3)). 13C NMR
(100 MHz, CDCl3) d: ꢀ5.12, ꢀ4.74 (Me2Si), 2.02 (Me3Si), 12.54
(Me–C(5)), 18.29 (Me3CSi), 20.87 (Ac), 25.66 (Me3CSi), 36.59
(C20), 36.72 (C70), 74.38 (C50), 78.79 (C60), 84.36 (C40), 84.51 (C30),
88.70 (C10), 111.92 (C5), 134.19 (C6), 150.52 (C2), 163.46 (C4),
170.14 (Ac). ESI+-HRMS: calcd for C23H39N2O7NaSi2I ([M+Na]+)
661.1238. Found: 661.1226.
4.1.1. (1R,3R,5S,7S,8R)-7-[(Acetyl)oxy]-8-[(tert-butyldimethyl
silyl)oxy]-3-methoxy-2-oxabicyclo[3.3.0]octan-5-ol (2)
Acetic anhydride (0.25 ml, 2.56 mmol) was added to a solution
of 1 (520 mg, 1.708 mmol) and pyridine (0.28 ml, 3.42 mmol) in
1,2-dichloroethane (10 ml) at rt. The reaction mixture was stirred
and heated to 55 °C for 20 h. After quenching with satd NaHCO3
(30 ml) and extraction with CH2Cl2 (3 ꢁ 30 ml) the organic phases
were combined, dried (MgSO4) and evaporated. Column chroma-
tography (hexane/EtOAc 3:1?1/1) afforded the monoacetylated
product 2 (510 mg, 86 %) as a colorless oil. 1H NMR (300 MHz,
CDCl3) d: 0.09, 0.11 (6H, 2 ꢁ s, Me2Si), 0.91 (9H, s, Me3CSi), 1.65
(1H, ddd J = 13.6, 9.0, 1.3 Hz, H–C(6)), 2.04 (3H, s, Ac), 2.21 (1H,
dd, J = 13.4, 1.5 Hz, H–C(4)), 2.31 (1H, ddd, J = 13.4, 5.5, 1.5 Hz,
H–C(4)), 2.67 (1H, dd, J = 13.6, 7.3 Hz, H–C(6)), 3.37 (3H, s, OMe),
4.06 (1H, d, J = 6.0 Hz, H–C(1)), 4.11 (1H, dd, J = 7.9, 6.0 Hz,
H–C(8)), 5.09 (1H, dd, J = 5.5, 1.5 Hz, H–C(3)), 5.20 (1H, dt, J = 9.0,
7.7 Hz, H–C(7)). 13C NMR (100 MHz, CDCl3) d: ꢀ4.92, ꢀ4.78
(Me2Si), 18.19 (Me3CSi), 21.03 (Ac), 25.69 (Me3CSi), 42.20 (C6),
49.43 (C4), 54.49 (MeO), 76.16 (C8), 78.27 (C-7), 83.52 (C5),
87.44 (C1), 105.56 (C3), 170.50 (Ac). ESI+-HRMS: calcd for
4.1.4. 1-[(30S,50R,60S)-60-O-Acetyl-50-O-(tert-butyldimethylsilyl)-
30-O-(trimethylsilyl)-20-deoxy-30,50-ethano-b-
D-
ribofuranosyl]thymine (5)
Azoisobutyronitril (AIBN, 40 mg, 0.24 mmol) was added at rt to
a solution of the mixture of anomers 4 (500 mg, 0.78 mmol) and
Bu3SnH (0.25 ml, 0.93 mmol) in toluene (10 ml). After heating at
reflux for 1 h the solvent was evaporated and the residue purified
by column chromatography (hexane/EtOAc 5:1?3:1) to give
a
-anomer of 5 (58 mg, 14 %) and b-anomer of 5 (320 mg, 80 %) both
as a white solid. Data for b-anomer: 1H NMR (300 MHz, CDCl3) d:
0.10, 0.12 (6H, 2 ꢁ s, Me2Si), 0.17 (9H, s, Me3Si), 0.90 (9H, s, Me3C-
Si), 1.67 (1H, dd, J = 12.8, 10.2 Hz, H–C(70)), 1.72 (1H, dd, J = 13.8,
9.2 Hz, H–C(20)), 1.92 (3H, d, J = 1.1 Hz, Me–C(5)), 2.08 (3H, s, Ac),
2.47 (1H, dd, J = 12.8, 6.4 Hz, H–C(70)), 2.65 (1H, dd, J = 13.8,
5.1 Hz, H–C(20)) 4.09–4.14 (2H, m, H–C(40), H–C(50)), 4.86 (1H, m,
H–C(60)), 6.23 (1H, dd, J = 9.2, 5.1 Hz, H–C(10)), 7.53 (1H, d,
J = 1.1 Hz, H–C(6)), 8.23 (1H, s, br, H–N(3)). 13C NMR (100 MHz,
CDCl3) d: ꢀ5.09, ꢀ4.81 (Me2Si), 1.87 (Me3Si), 12.56 (Me–C(5)),
18.27 (Me3CSi), 20.91 (Ac), 25.65 (Me3CSi), 39.14, 47.64 (C20, C70),
74.41 (C50), 78.41 (C60), 85.10 (C30), 85.33 (C40), 86.93 (C10),
111.00 (C5), 135.15 (C6), 149.97 (C2), 163.48 (C4, Ac). ESI+-HRMS:
calcd for C23H40N2O7NaSi2 ([M+Na]+) 535.2271. Found: 535.2266.
C
16H30O6NaSi ([M+Na]+) 369.1709. Found: 369.1708.
4.1.2. (1R,5S,7S,8R)-7-[(Acetyl)oxy]-8-[(tert-butyldimethylsilyl)
oxy]-5-[(trimethylsilyl)oxy]-2-oxabicyclo[3.3.0]oct-3-ene (3)
To a solution of 2 (1.92 g, 5.54 mmol) and 2,6-lutidine (3.3 ml,
28.3 mmol) in dry CH2Cl2 (10 ml) was added dropwise at rt
TMSOTf (2.5 ml, 13.85 mmol). After stirring for 4 h at rt the reac-
tion mixture was diluted with AcOEt (50 ml), washed with satd
NaHCO3 (2 ꢁ 25 ml) and the aqueous phases extracted with AcOEt
(2 ꢁ 25 ml). The combined organic phases were dried over MgSO4
evaporated and the crude product purified by flash chromatogra-
phy (hexane/tBuOMe 10:1?5:1) to give 3 (1.97 g, 92%) as a color-
less oil. 1H NMR (300 MHz, CDCl3) d: 0.10, 0.11 (15H, 2 ꢁ s, Me3Si,
Me2Si), 0.90 (9H, s, Me3CSi), 1.79 (1H, dd, J = 12.6, 9.8 Hz, H–C(6)),
2.04 (3H, s, Ac), 2.42 (1H, dd, J = 12.6, 6.2 Hz, H–C(6)), 4.22 (1H, dd,
J = 8.1, 6.8 Hz, H–C(8)), 4.36 (1H, d, J = 6.8, H–C(1)), 4.75 (1H, ddd,
J = 9.8, 8.1, 6.2 Hz, H–C(7)), 4.99 (1H, d, J = 2.6 Hz, H–C(4)), 6.46
(1H, d, J = 2.6 Hz, H–C(3)). 13C NMR (100 MHz, CDCl3) d: ꢀ4.98,
ꢀ4.82 (Me2Si), 1.83 (Me3Si), 18.24 (Me3CSi), 20.94 (Ac), 25.66
(Me3CSi), 41.31 (C6), 76.28 (C8), 77.04 (C7), 87.20 (C5), 88.00
(C1), 106.99 (C4), 149.31 (C3), 170.34 (Ac). ESI+-HRMS: calcd for
4.1.5. 1-[(30S,50R,60S)-60-O-Acetyl-20-deoxy-30,50-ethano-b-
D-
ribofuranosyl]thymine (6)
To a solution of nucleoside 5 (260 mg, 0.51 mmol) and pyridine
(1 ml) in CH2Cl2 (5 ml) was added HF-pyridine (0.25 ml,
10.1 mmol) at 0 °C. After stirring for 15 h at rt, silica gel (ca. 1 g)
was added and the mixture stirred for another 15 min. After evap-
oration the adsorbed product was purified by column chromatog-
raphy (EtOAc/EtOH 10:1) to yield the title compound 6 (148 mg,
90%) as white crystals. Mp 210–211 °C. 1H NMR (300 MHz,
DMSO-d6) d: 1.58 (1H, dd, J = 13.6, 7.1 Hz, H–C(70)), 1.78 (3H, d,
J = 1.1 Hz, Me–C(5)), 1.95 (1H, dd, J = 13.2, 9.9 Hz, H–C(20)), 2.03
(3H, s, Ac), 2.25 (1H, dd, J = 13.2, 5.3 Hz, H–C(20)), 2.40 (1H, dd,
J = 13.6, 6.4 Hz, H–C(70)), 3.91–3.99 (2H, m, H–C(40), H–C(50)),
4.90 (1H, m, H–C(60)), 5.43 (1H, d, J = 5.8 Hz, H–O(50)), 5.54 (1H,
s, H–O(30)), 6.16 (1H, dd, J = 9.9, 5.3 Hz, H–C(10)), 7.78 (1H, d,
J = 1.1, H–C(6)), 11.34 (1H, s, H–N(3)). 13C NMR (100 MHz,
DMSO-d6) d: 12.19 (Me–C(5)), 20.86 (Ac), 40.68, 46.11 (C20, C70),
73.09 (C50), 78.46 (C60), 82.33 (C30), 84.75 (C40), 87.18 (C10),
109.33 (C5), 136.11 (C6), 150.31 (C2), 163.64 (C4), 169.91 (Ac).
ESI+-HRMS: calcd for C14H18N2O7Na ([M+Na]+) 349.1011. Found:
349.1012.
C
18H34O5NaSi2 ([M+Na]+) 409.1842. Found: 409.1827.
4.1.3. 1-[(20R,30S,50R,60S)-60-O-Acetyl-50-O-(tert-butyldimethyl
silyl)-30-O-(trimethylsilyl)-20-deoxy-20-iodo-30,50-ethano-b-
D-
ribofuranosyl]thymine (4)
To a suspension of thymine (190 mg, 1.5 mmol) and glycal 3
(390 mg, 1.0 mmol) in CH2Cl2 (5 ml) was added BSA (0.37 ml,
1.5 mmol) and the mixture was stirred at rt for 1 h. Then solid
N-iodsuccinimide (270 mg, 1.2 mmol) was added and the mixture
stirred for 8 h at rt. The reaction was quenched with satd NaHCO3
(15 ml) and Na2S2O3 (10 %, 5 ml). The aqueous phase was extracted
with AcOEt (3 ꢁ 25 ml) and the combined organic phases were
dried over MgSO4 and evaporated. Column chromatography (hex-
4.1.6. 1-[(30S,50R,60S)-60-O-Acetyl-50-O-[(4,40-
dimethoxytriphenyl)methyl)-20-deoxy-30,50-ethano-b-
D-
ribofuranosyl]thymine (7)
To a solution of nucleoside 6 (630 mg, 1.9 mmol) and pyridine
(0.3 ml, 3.8 mmol) in CH2Cl2 (10 ml) was added (4,40-dimethoxytri-
phenyl)methyl chloride (DMT-Cl, 773 mg, 2.28 mmol) and the mix-
ture stirred at rt overnight. The reaction was quenched with satd
ane/EtOAc 5:1?3:1) afforded the unseparable mixture of
a- and
b-anomers 4 (524 mg, 81 %, /b 15:85). Data for the b-anomer:
a

P. Šilhár, C. J. Leumann / Bioorg. Med. Chem. 18 (2010) 7786–7793
7791
NaHCO3 (20 ml) and the aqueous phase was extracted with EtOAc
(3 ꢁ 20 ml). The combined organic phases were dried (MgSO4),
evaporated and the crude product purified by column chromatog-
raphy (hexane/EtOAc 1:3, 1% NEt3) to give the title compound 7
(1.19 g, 98%) as a white foam. 1H NMR (300 MHz, CDCl3) d: 1.57
(3H, d, J = 1.1 Hz, Me–C(5)), 1.59 (1H, dd, J = 13.8, 7.3 Hz,
H–C(70)), 1.92 (1H, dd, J = 13.6, 9.6 Hz, 1H–C(20)), 1.99 (3H, s, Ac),
2.43 (1H, dd, J = 13.8, 5.9 Hz, H–C(70)), 2.55 (1H, dd, J = 13.6,
5.1 Hz, H–C(20)), 3.40 (1H, d, J = 6.0, H–C(40)), 3.78, 3.79 (6H,
2 ꢁ s, MeO), 4.17 (1H, m, H–C(50)), 4.58 (1H, m, H–C(60)), 6.21
(1H, dd, J = 9.6, 5.1, H–C(10)), 6.80–6.82 (4H, m, H-arom), 7.24–
7.30, 7.35–7.39, 7.47–7.50 (3H, 4H, 2H, 3 ꢁ m, H-arom), 7.60
(1H, d, J = 1.1 Hz, 1H–C(6)). 13C NMR (100 MHz, CDCl3) d: 12.09
(Me–C(5)), 21.09 (Ac), 40.59, 47.69 (C20, C70), 55.36 (MeO), 75.88
(C50), 77.81 (C60), 83.65 (C30), 85.46 (C40), 87.10 (C10), 111.63
(C5), 113.20, 127.37, 127.87, 128.69, 130.70, 130.80 (HC-arom),
135.24 (C6), 135.81 135.99, 144.88 (C-arom), 150.41 (C2), 159.03,
159.08 (C-arom), 163.62 (C4), 170.07 (Ac). ESI+-HRMS: calcd for
C35H36N2O9Na ([M+Na]+) 651.2318. Found: 651.2311.
red at rt for 8 h. The reaction mixture was filtered through a pad of
silica gel, washed with EtOAc/EtOH 1:1 and the filtrate adsorbed on
silica gel. Column chromatography (silica gel, hexane/EtOAc
8:1?1:1) afforded product 9 (2.13 g, 80%) as a colorless oil. 1H
NMR (300 MHz, CDCl3) d: 0.11, 0.15 (12H, 2 ꢁ s, 2 ꢁ Me2Si), 0.86,
0.94 (18H, 2 ꢁ s, 2 ꢁ Me3CSi), 1.73 (1H, dd, J = 12.9, 9.5 Hz,
H–C(6)), 1.96 (1H, d, J = 3.8 Hz, OH), 2.03 (1H, dd, J = 13.7, 5.2 Hz,
H–C(4)), 2.18–2.25 (2H, m, H–C(4), H–C(6)), 3.33 (3H, s, OMe),
3.86 (1H, dd, J = 8.0, 5.0 Hz, H–C(8)), 3.97 (1H, m, H–C(7)), 4.06
(1H, d, J = 5.0 Hz, H–C(1)), 5.02 (1H, dd, J = 5.2, 1.6 Hz, H–C(3)).
13C NMR (100 MHz, CDCl3) d: ꢀ4.65 (Me2Si), 18.20 (Me3CSi),
25.61, 25.93 (Me3CSi), 45.25 (C6), 49.71 (C4), 54.51 (MeO), 75.36
(C8), 78.97 (C7), 83.62 (C5), 86.76 (C1), 105.34 (C3). ESI+-HRMS:
calcd for C20H42O5NaSi2 ([M+Na]+) 441.2468. Found: 441.2463.
4.1.9. (1R,5S,7S,8R)-5,8-[Di-(tert-butyldimethylsilyl)oxy]-7-
[(ethoxycarbonyl)methyl]oxy-3-methoxy-2-
oxabicyclo[3.3.0]octane (10)
A
solution of ethyl diazoacetate (1.5 ml, 13.0 mmol) in
1,2-dichloroethane (8 ml) was added dropwise within 4 h at
70 °C to a solution of 9 (2.1 g, 5.01 mmol) and Cu(acac)2 (66 mg,
5 mol %) in 1,2-dichloroethane (10 ml). The reaction mixture was
stirred for 3 h at 70 °C. After the reaction was complete, the mix-
ture was evaporated and the crude product was purified by column
chromatography (hexane/EtOAc 10:1?4:1) to give the title com-
4.1.7. 1-[(30S,50R,60S)-60-O-Acetyl-50-O-[(4,40-
dimethoxytriphenyl)methyl)-30-O-(2-cyanoethoxy
diisopropylaminophosphanyl)-20-deoxy-30,50-ethano-b-
D-
ribofuranosyl]thymine-) (8)
2-Cyanoethoxy diisopropylamino chlorophosphine (CEP-Cl,
1.0 ml, 4.47 mmol) was added at rt to a solution of nuleoside 7
(1.18 g, 1.88 mmol) and diisopropylethylamine (1.3 ml, 7.48
mmol) in MeCN (8 ml). After stirring for 2 h at rt, the mixture
was diluted with EtOAc (50 ml), washed with satd NaHCO3
(2 ꢁ 20 ml) and brine (5 ml). The aqueous phases were extracted
with EtOAc (3 ꢁ 25 ml). The combined organic phases were dried
(MgSO4), evaporated and the crude product purified by column
chromatography (hexane/EtOAc 1:2 + 1% NEt3) to give the title
compound 8 (1.39 g, 90 %) as a white foam. 1H NMR (300 MHz,
CDCl3) d: 1.08–1.14 (12H, m, (Me2CH)2N), 1.45, 1.60 (3H, 2 ꢁ d,
J = 1.1 Hz, Me–C(5)), 1.80–1.95 (2H, m, C(20), C(70)), 1.96, 2.00
(3H, 2 ꢁ s, Ac), 2.40–2.50, 2.55–2.61 (2H, 2 ꢁ m, C(20), C(70)),
2.90–2.98 (1H, m, (Me2CH)2N), 3.50–3.77 (5H, m, C(40),
OCH2CH2CN), 3.79 (6H, s, MeO), 4.12–4.17 (1H, m, C(50)), 4.41,
4.59 (1H, 2 ꢁ m, C(60)), 6.13, 6.24 (1H, 2 ꢁ dd, J = 9.8, 4.8 Hz,
C(10)), 6.81 (4H, m, H-arom), 7.28, 7.38, 7.48 (9H, 3 ꢁ m, H-arom),
7.59 (1H, d, J = 1.1 Hz, C(6)), 8.22 (1H, s, br, H–N(3)). 13C NMR
(100 MHz, CDCl3) d: 11.76, 12.03 (Me–C(5)), 20.15, 20.23 (d,
pound 10 (2.37 g, 77% yield) as
a
yellowish oil. 1H NMR
(300 MHz, CDCl3) d: 0.09, 0.15, 0.16 (12H, 3 ꢁ s, 2 ꢁ Me2Si), 0.85,
0.94 (18H, 2 ꢁ s, 2 ꢁ Me3CSi), 1.27 (3H, t, J = 7.2 Hz, CH3-Et), 1.81
(1H, dd, J = 13.1, 9.9 Hz, H–C(6)), 2.07 (1H, dd, J = 13.8, 5.3 Hz,
H–C(4)), 2.22 (1H, dd, J = 13.8, 1.5 Hz, H–C(4), 2.31 (1H, dd,
J = 13.1, 7.0 Hz, H–C(6)), 3.32 (3H, s, OMe), 3.78 (1H, ddd, J = 9.9,
8.0, 7.0 Hz, H–C(7)), 3.99–4.07 (2H, m, H–C(1), H–C(8)), 4.26 (2H,
q, J = 7.2, CH2-Et), 4.28 (2H, s, OCH2CO), 5.01 (1H, dd, J = 5.3,
1.5 Hz, 1H–C(3)). ESI+-HRMS: calcd for C24H48O7NaSi2 ([M+Na])+
527.2836. Found: 527.2834.
4.1.10. (1R,5S,7S,8R)-5,8-[Di-(tert-butyldimethylsilyl)oxy]-7-
[(ethoxycarbonyl)methyl]oxy-2-oxabicyclo[3.3.0]oct-3-ene (11)
To a solution of 10 (1.13 g, 2.24 mmol) and 2,6-lutidine (1.6 ml,
13.44 mmol) in dry CH2Cl2 (15 ml) was added dropwise TMS-OTf
(0.81 ml, 4.48 mmol) at rt. After stirring for 3 h the reaction mix-
ture was quenched with satd NaHCO3 (25 ml) and extracted with
CH2Cl2 (3 ꢁ 25 ml). The combined organic phases were dried over
MgSO4, evaporated and the crude product purified by column chro-
matography (hexane/EtOAc 10:1?4:1) to give the title compound
11 as a colorless oil (0.98 g, 92 %). 1H NMR (300 MHz, CDCl3) d:
0.06, 0.07, 0.13 (12H, 4 ꢁ s, 2 ꢁ Me2Si), 0.84, 0.92 (18H, 2 ꢁ s,
2 ꢁ Me3CSi), 1.27 (3H, t, J = 7.2 Hz, CH3-Et), 1.90 (1H, dd, J = 12.6,
10.2 Hz, H–C(6)), 2.30 (1H, dd, J = 12.6, 6.1 Hz, H–C(6)), 3.62 (1H,
ddd, J = 10.2, 7.7, 6.1 Hz, H–C(7)), 4.16 (1H, dd, J = 7.7, 6.6 Hz,
H–C(8)), 4.22 (2H, s, OCH2CO), 4.26 (2H, q, J = 7.2 Hz, CH2-Et),
4.30 (1H, d, J = 6.6 Hz, H–C(1)), 4.94 (1H, d, J = 2.6 Hz, H–C(4)),
6.41 (1H, d, J = 2.6, H–C(3)). 13C NMR (100 MHz, CDCl3) d: ꢀ4.93,
ꢀ4.68, ꢀ3.13, ꢀ2.58 (2 ꢁ Me2Si), 14.19 (CH3-Et), 17.76, 18.22
(2 ꢁ Me3CSi), 25.64, 25.79 (2 ꢁ Me3CSi), 41.86 (C6), 60.73 (CH2-
Et), 67.91 (OCH2CO), 78.66 (C8), 83.34 (C7), 86.53 (C5), 88.86
(C1), 107.42 (C4), 148.86 (C3), 170.47 (C@O). ESI+-HRMS: calcd
for C23H44O6NaSi2 ([M+Na])+ 495.2574. Found: 495.2573.
J
C,P = 8.3 Hz, OCH2CH2CN), 20.89, 20.97 (Ac), 24.14, 24.17, 24.27,
24.38, 24.46, 24.53 (Me2CH)2N), 39.20, 39.37 d, (JC,P = 11.5 Hz, C-
20 or C-70), 43.31, 43.48 (d, JC,P = 8.3 Hz, Me2CH)2N), 45.99, 46.27
(d, JC,P = 10.7 Hz, C-20 or C-70), 55.26 (MeO), 57.74, 57.97 (d,
J
C,P = 7.0 Hz, OCH2CH2CN), 75.40, 75.45 (C50), 77.84 (C60), 85.28,
86.16 (2 ꢁ d, JC,P = 9.0, 4.1 Hz, C40), 87.01 (OC(Ar)3), 87.08, 87.16
(C10), 87.63, 87.79 2 ꢁ d, (JC,P = 18.1, 9.9 Hz, C30), 111.30 (C5),
117.55, 117.81 (CN), 113.10, 127.28, 127.39, 127.77, 127.82,
128.61, 128.67, 130.64, 130.70, 130.76 (HC-arom), 135.13, 135.36
(C6), 135.60 135.67, 135.72, 135.88, 144.58, 144.78 (C-arom),
150.00, 150.10 (C2), 158.95, 159.02, 159.06 (C-arom), 163.42,
163.45 (C4), 169.72, 170.07 (Ac). 31P NMR (162 MHz, CDCl3) d:
142.08, 142.53. ESI+-HRMS: calcd for C44H53N4O10NaP ([M+Na]+)
851.3397. Found: 851.3406.
4.1.8. (1R,5S,7S,8R)-5,8-[Di-(tert-butyldimethylsilyl)oxy]-3-
methoxy-2-oxabicyclo[3.3.0]octan-7-ol (9)
4.1.11. 1-[(30S,50R,60S)-30,50-O-[Di-(tert-butyldimethylsilyl)]-60-
To a solution of 2 (2.20 g, 6.35 mmol) and imidazole (1.29 g,
19 mmol) in DMF (25 ml) was added TBDMS-chloride (1.92 g,
12.7 mmol) at rt. The reaction mixture was stirred at 80 °C for
2 days. The reaction mixture was then diluted with EtOH (2 ml)
and evaporated. The residue was dissolved in abs EtOH (30 ml)
and K2CO3 (2.0 g, 14.5 mmol) was added. The suspension was stir-
O-(ethoxycarbonyl)methyl-20-deoxy-20-iodo-30,50-ethano-
a,b-D-
ribofuranosyl]thymine (12)
To a suspension of thymine (670 mg, 5.27 mmol) and glycal 11
(830 mg, 1.755 mmol) in CH2Cl2 (30 ml) was added BSA (0.9 ml,
3.5 mmol) and the mixture was stirred at 40 °C for 2 h. Then solid
N-iodsuccinimide (515 mg, 2.29 mmol) was added at 0 °C and the

7792
P. Šilhár, C. J. Leumann / Bioorg. Med. Chem. 18 (2010) 7786–7793
mixture was stirred for 7 h at rt. After quenching with satd NaHCO3
(15 ml) and aq Na2S2O3 (10%, 5 ml) the phases were separated and
the aqueous phase extracted with AcOEt (3 ꢁ 25 ml). The com-
bined organic phases were dried over MgSO4, evaporated and the
crude product purified by column chromatography (hexane/EtOAc
14.20 (CH3-Et), 17.74, 18.11 (2 ꢁ Me3CSi), 25.57, 25.75
(2 ꢁ Me3CSi), 40.02 (C70), 48.30 (C20), 60.92 (CH2-Et), 67.97 (OCH2-
CO), 76.65 (C50), 84.40 (C30), 84.98 (C60), 85.34 (C10), 87.90 (C40),
110.70 (C5), 135.20 (C6), 149.82 (C2), 163.40 (C4), 170.17 (C@O).
ESI+-MS: 599 ([M+H])+.
8:1?2:1) to give the unseparable mixture of
a
- and b-anomers 12
(980 mg, 77 %, /b = 6/5) as a colorless oil. Data for
a
a
+ b-anomers:
4.1.13. 1-[(30S,50R,60S)-60-O-(Ethoxycarbonyl)methyl-20-deoxy-
1H NMR (300 MHz, CDCl3): 0.10, 0.12, 0.15, 0.20, 0.21, 0.22, 0.24
(24H, 7 ꢁ s, 4 ꢁ Me2Si), 0.89, 0.91, 0.95 (36H, 3 ꢁ s, 4 ꢁ Me3CSi),
1.30 (6H, t, J = 7.2 Hz, 2 ꢁ CH3-Et), 1.74 (1H, dd, J = 13.8, 8.7 Hz,
1H–C(70)), 1.93, 1.96 (6H, 2 ꢁ d, J = 1.1 Hz, 2 ꢁ Me–C(5)), 1.99 1H,
(m, H–C(70)), 2.27 (1H, dd, J = 13.8, 6.1 Hz, 1H–C(70)), 2.61 (1H,
dd, J = 14.9, 7.3 Hz, H–C(70)), 3.78 (1H, d, J = 9.8 Hz, H–C(20)),
3.75–3.88 (2H, m, 2 ꢁ H–C(60)), 4.10–4.16 (2H, m, 2 ꢁ H–C(50)),
4.19, 4.21 (4H, 2 ꢁ s, 2 ꢁ OCH2CO), 4.22, 4.23 (4H, 2 ꢁ q,
J = 7.2 Hz, 2 ꢁ CH2-Et), 4.28 (1H, m, H–C(40)), 4.35 (1H, d, J = 4.7,
H–C(40)), 4.48 (1H, d, J = 9.1, H–C(20)), 5.92 (1H, d, J = 9.1 Hz,
H–C(10a)), 6.31 (1H, d, J = 9.8 Hz, H–C(10b)), 7.02, 7.20 (2H, 2 ꢁ d,
J = 1.1 Hz, 2 ꢁ H–C(6)), 8.05, 8.09 (2H, 2 ꢁ s, br, 2 ꢁ H–N(3)). 13C
NMR (100 MHz, CDCl3): ꢀ5.02, ꢀ4.92, ꢀ4.71, ꢀ4.55, ꢀ2.71,
ꢀ2.48, ꢀ2.41, ꢀ2.10 (4 ꢁ Me2Si), 12.59 (Me–C(5)), 14.20 (CH3-Et),
17.87, 18.24, 18.27, 18.41 (4 ꢁ Me3CSi), 25.55, 25.77, 25.80, 25.89
(4 ꢁ Me3CSi), 36.50, 36.77 (2 ꢁ C20), 37.25, 43.04 (2 ꢁ C70), 60.90,
61.03 (2 ꢁ CH2-Et), 67.86, 67.88 (2 ꢁ OCH2CO), 76.08, 78.88
(2 ꢁ C50), 83.97, 85.69 (2 ꢁ C30), 84.27, 84.59 (2 ꢁ C60), 85.88,
86.63 (2 ꢁ C40), 88.59, 92.36 (2 ꢁ C10), 111.72, 111.90 (2 ꢁ C5),
134.36, 135.37 (2 ꢁ C6), 149.77, 150.19 (2 ꢁ C2), 163.10, 163.17
(2 ꢁ C4), 169.92, 170.16 (2 ꢁ C@O). ESI+-MS: 725.2 ([M+H])+.
30,50-ethano-b-
D-ribofuranosyl]thymine (15)
To a solution of nucleoside 13 (330 mg, 0.55 mmol) and pyri-
dine (2.2 ml) in THF (10 ml) was added HF-pyridine (0.47 ml,
16.5 mmol) at 0 °C and the mixture stirred at 50 °C for 2 days. After
complete conversion of 13, silica gel (ca. 2 g) was added and the
mixture stirred for another 15 min. Then the solvents were evapo-
rated and the residue purified by column chromatography (EtOAc/
EtOH 10:0?10:1) to give nucleoside 15 (160 mg, 80%) as a white
foam. 1H NMR (300 MHz, CDCl3) d: 1.29 (3H, t, J = 7.2 Hz, CH3-Et),
1.91 (3H, d, J = 1.1 Hz, Me–C(5)), 1.99 (1H, dd, J = 14.4, 3.1 Hz, H–
C(70)), 2.32 (1H, dd, J = 13.3, 9.6 Hz, H–C(20)), 2.40 (1H, dd,
J = 14.4, 4.5 Hz, H–C(70)), 2.45 (1H, dd, J = 13.3, 5.7 Hz, H–C(20)),
3.62 (1H, d, J = 3.0 Hz, H–O(50)), 3.87 (1H, s, br, H–O(30)), 4.05
(1H, m, H–C(60)), 4.07, 4.30 (2H, 2 ꢁ d, J = 17.0 Hz, OCH2CO), 4.22
(1H, m, H–C(50)), 4.23 (2H, q, J = 7.2 Hz, CH2-Et), 4.42 (1H, d,
J = 5.5 Hz, 1H–C(40)), 6.17 (1H, dd, J = 9.6, 5.7 Hz, H–C(10)), 7.21
(1H, d, J = 1.1 Hz, 1H–C(6)), 8.64 (1H s, H–N(3)). 13C NMR
(100 MHz, CDCl3) d: 12.43 (Me–C(5)), 14.11 (CH3-Et), 41.63,
44.50 (C70, C20), 61.51 (CH2-Et) 67.14 (OCH2CO), 74.11 (C50),
85.40 (C60), 88.03 (C30), 90.93, 91.34 (C10, C40), 111.40 (C5),
137.34 (C6), 150.27 (C2), 163.24 (C4), 171.55 (C@O). ESI+-HRMS:
calcd for C16H22N2O8Na ([M+Na])+, 393.1274. Found: 393.1257.
4.1.12. 1-[(30S,50R,60S)-30,50-O-[Di-(tert-butyldimethylsilyl)]-60-
O-(ethoxycarbonyl)methyl-20-deoxy-30,50-ethano-
a,b-
D-
4.1.14. 1-[(30S,50R,60S)-50-O-[(4,40-Dimethoxytriphenyl)methyl)-
ribofuranosyl]thymine (14/13)
60-O-(ethoxycarbonyl)methyl-20-deoxy-30,50-ethano-b-
D-ribo-
A solution of azoisobutyronitril (AIBN, 60 mg, 0.366 mmol) in
toluene (2 ml) was added at 100 °C in four portions within 2 h to
a solution of the mixture of anomers 12 (535 mg, 0.738 mmol)
and Bu3SnH (0.35 ml, 1.18 mmol) in dry toluene (15 ml). The reac-
tion was complete after 2 h. The solvents were evaporated and the
anomers separated by column chromatography (hexane/EtOAc
furanosyl]thymine (16)
To a solution of nucleoside 15 (150 mg, 0.405 mmol) and pyri-
dine (0.1 ml, 1.25 mmol) in CH2Cl2 (10 ml) was added (4,40-
dimethoxytriphenyl)methyl chloride (DMT-Cl, 210 mg, 0.62 mmol)
and the mixture was stirred at rt for 12 h. The reaction was diluted
with satd NaHCO3 (20 ml) and the aqueous phase extracted with
CH2Cl2 (3 ꢁ 20 ml). The combined organic phases were dried
(MgSO4), evaporated and the crude product purified by column
chromatography (hexane/EtOAc 1:1?0:1, 1% NEt3) to give the title
compound 16 (250 mg, 92%) as white foam. 1H NMR (300 MHz,
CDCl3) d: 0.99 (3H, d, J = 1.3 Hz, Me–C(5)), 1.23 (3H, t, J = 7.2 Hz,
CH3-Et), 1.85 (1H, d, J = 14.9 Hz, H–C(70)), 2.00 (1H, dd, J = 12.8,
9.9 Hz, H–C(20)), 2.20 (1H, dd, J = 14.9, 3.6 Hz, H–C(70)), 2.43 (1H,
m, H–C(60)), 2.59 (1H, dd, J = 12.8, 4.9 Hz, H–C(20)), 3.41, 3.86
(2H, 2 ꢁ d, J = 16.8 Hz, OCH2CO), 3.61 (1H, s, br, H–O(30)), 3.80,
3.81 (6H, 2 ꢁ s, MeO-DMTr), 4.12 (2H, q, J = 7.2 Hz, CH2-Et), 4.27
(1H, m, H–C(50)), 4.45 (1H, d, J = 5.1, H–C(40)), 6.62 (1H, dd,
J = 9.9, 4.9 Hz, H–C(10)), 6.82–6.87 (4H, m, H-arom), 7.29–7.39,
7.44–7.47 (9H, 2 ꢁ m, H-arom), 7.68 (1H, d, J = 1.3 Hz, H–C(6)).
13C NMR (100 MHz, CDCl3) d: 10.97 (Me–C(5)), 14.05 (CH3-Et),
41.79 (C-70), 45.65 (C-20), 55.30 (MeO-DMTr), 61.40 (CH2-Et),
67.01 (OCH2CO), 75.74 (C50), 86.72 (C30), 87.95 (C60), 88.17 (C10),
91.77 (C40), 111.21 (C5), 113.32, 127.57, 128.06, 128.75, 130.51
(HC-arom), 135.57, 135.72 (C-arom), 135.87 (C6), 149.89 (C2),
159.11, 159.13 (C-arom), 163.41 (C4), 171.20 (C@O). ESI+-MS:
695.2 ([M+Na])+.
7:1?2:1) to give
a-anomer 14 (240 mg, 54%) and b-anomer 13
(200 mg, 45 %), both as a white foam. Data of 14. 1H NMR
(300 MHz, CDCl3) d: ꢀ0.02, 0.11, 0.12 (12H, 3 ꢁ s, 2 ꢁ Me2Si),
0.83, 0.91 (18H, 2 ꢁ s, 2 ꢁ Me3CSi), 1.29 (3H, t, J = 7.1 Hz, CH3-Et),
1.92 (3H, d, J = 1.1 Hz, Me–C(5)), 1.98 (1H, dd, J = 13.9, 4.9 Hz, H–
C(70)), 2.28 (1H, dd, J = 14.3, 2.8 Hz, H–C(20)), 2.38 (1H, dd,
J = 13.9, 5.8 Hz, H–C(70)), 2.47 (1H, dd, J = 14.3, 7.0 Hz, H–C(20)),
3.77 (1H, m, H–C(60)), 4.09 (1H, m, H–C(50)), 4.10, 4.16 (2H, 2 ꢁ d,
J = 16.4 Hz, OCH2CO), 4.22 (2H, q, J = 7.2 Hz, CH2-Et), 4.58 (1H, d,
J = 5.5 Hz, H–C(40)), 6.17 (1H, dd, J = 7.0, 2.8 Hz, H–C(10)), 7.36
(1H, d, J = 1.1, H–C(6)), 8.15 (1H, s, br, H–N(3)). 1H NMR difference
NOE: H(10)?H(40) 0.5 %, H(60) 1.1%. 13C NMR (100 MHz, CDCl3) d:
ꢀ5.12, ꢀ4.71, ꢀ2.98, ꢀ2.82 (2 ꢁ Me2Si), 12.61 (Me–C(5)), 14.20
(CH3-Et), 17.74, 18.11 (2 ꢁ Me3CSi), 25.46, 25.76 (2 ꢁ Me3CSi),
41.64 (C70), 49.17 (C20), 60.96 (CH2-Et), 67.36 (OCH2CO), 75.71
(C50), 85.81 (C60), 86.07 (C30), 89.55 (C10), 93.12 (C40), 109.88
(C5), 136.23 (C6), 149.87 (C2), 163.58 (C4), 170.08 (C@O). ESI+-
MS: 599.3 ([M+H])+. Data for 13: 1H NMR (300 MHz, CDCl3) d:
0.12, 0.15 (12H, 2 ꢁ s, 2 ꢁ Me2Si), 0.88, 0.93 (18H, 2 ꢁ s,
2 ꢁ Me3CSi), 1.29 (3H, t, J = 7.2 Hz, CH3-Et), 1.63 (1H, dd, J = 13.6,
9.2 Hz, H–C(20)), 1.79 (1H, dd, J = 12.7, 10.2 Hz, H–C(70)), 1.92
(3H, d, J = 1.1, Me–C(5)), 2.37 (1H, dd, J = 12.7, 6.2 Hz, H–C(70)),
2.62 (1H, dd, J = 13.6, 5.1 Hz, H–C(20)), 3.73 (1H, m, H–C(60)),
4.02–4.10 (2H, m, H–C(40), H–C(50)), 4.22 (2H, q, J = 7.2, CH2-Et),
4.25 (2H, s, OCH2CO), 6.16 (1H, dd, J = 9.2, 5.1 Hz, H–C(10)), 7.52
(1H, d, J = 1.1 Hz, H–C(6)), 8.19 (1H, s, br, H–N(3)). 1H NMR differ-
ence NOE: H(10)?H(40) 2.1%. H(60)?H(6) 4.3%. 13C NMR (100 MHz,
CDCl3) d: ꢀ5.03, ꢀ4.62, ꢀ2.77, ꢀ2.74 (2 ꢁ Me2Si), 12.54 (Me–C(5)),
4.1.15. 1-[(30S,50R,60S)-50-O-[(4,40-Dimethoxytriphenyl)methyl)-
60-O-(ethoxycarbonyl)methyl]-30-O-(2-cyanoethoxy diisopro-
pylaminophosphanyl-20-deoxy-30,50-ethano-b-
D-ribofuranosyl]
thymine) (17)
To a solution of nucleoside 16 (240 mg, 0.357 mmol) and diiso-
propylethylamine (0.25 ml, 1.44 mmol) in MeCN (8 ml) was added
at rt 2-cyanoethoxy diisopropylamino chlorophosphine (CEP-Cl,

P. Šilhár, C. J. Leumann / Bioorg. Med. Chem. 18 (2010) 7786–7793
7793
0.17 ml, 0.71 mmol). After stirring for 2 h the mixture was diluted
with EtOAc (50 ml) and washed with satd NaHCO3 (2 ꢁ 20 ml) and
brine (5 ml). The aqueous phases were extracted with EtOAc
(3 ꢁ 25 ml). The combined organic phases were dried (MgSO4),
evaporated and the crude product purified by column chromatog-
raphy (hexane/EtOAc 3:1?1:2, 0.5% NEt3) to give the title com-
pound 17 (255 mg, 82%) as a white foam. 1H NMR (300 MHz,
CDCl3) d: 1.11–1.18 (12H, m, (Me2CH)2N), 1.20, 1.25 (3H, 2 ꢁ d,
J = 1.3 Hz, Me–C(5)), 1.25, 1.26 (3H, 2 ꢁ t, J = 7.2 Hz, CH3-Et), 1.91
(1H, m, H–C(20)), 2.12–2.17 (1H, m, H–C(70)), 2.28 (1H, m,
H–C(70)), 2.58–2.68 (2H, m, OCH2CH2CN), 2.90–2.95 (1H, m,
H–C(20)), 3.06–3.13 (1H, m, H–C(60)), 3.54–3.70 (4H, m, (Me2CH)2N,
OCH2CH2CN), 3.70, 3.74, 3.86, 3.88 (2H, 4 ꢁ d, J = 16.0 Hz, OCH2-
CO)), 3.79 (6H, s, MeO), 3.97, 4.06 (1H, 2 ꢁ d, J = 4.8 Hz, H–C(40)),
4.16 (2H, q, J = 7.2 Hz, CH2-Et), 4.16–4.20 (1H, m, H–C(50)), 6.31
(1H, m, H–C(10)), 6.81–6.84 (4H, m, H-arom), 7.27–7.31, 7.37–
7.42, 7.50–7.53 (9H, 3 ꢁ m, H-arom), 7.60, 7.61 (1H, 2 ꢁ d,
J = 1.3 Hz, C(6)), 8.05 (1H, s, br, H–N(3)). 13C NMR (75 MHz, CDCl3)
d: 11.26, 11.35 (Me–C(5)), 14.16 (CH3-Et), 20.15, 20.29 (JC,P = 9 Hz,
OCH2CH2CN), 24.05, 24.15, 24.25, 24.45, 24.53 (Me2CH)2N), 39.00,
39.75 (JC,P = 10 Hz, C70), 43.35, 43.52 (JC,P = 13 Hz, Me2CH)2N),
46.01, 46.12 (C20), 55.23 (MeO), 57.73, 58.02 (JC,P = 16 Hz,
OCH2CH2CN), 60.76, 60.78 (CH2-Et), 67.37, 67.40 (OCH2CO),
75.68, 75.70 (C50), 85.15, 85.38 (C60), 86.45, 86.68 (C10), 87.85,
88.62 (Ar2CPh), 88.75, 88.82 (C40), 88.67, 88.86, (C30), 111.07,
111.10 (C5), 117.83 (CN), 113.14, 113.20, 127.36, 127.89, 127.92,
128.76, 130.69, 130.76, (HC-arom), 135.74 (C6), 135.71, 135.78,
144.55, 144.58 (C-arom), 149.86, 149.94 (C2), 159.00, 159.03 (C-
arom), 163.35, 163.36 (C4), 169.65, 169.76 (C@O). 31P NMR
(121 MHz, CDCl3) d: 141.43, 141.81. ESI+-MS: 895.4 ([M+Na])+.
ethylpolysiloxane. All measurements were carried out in 150 mM
NaCl, 10 mM Na-phosphate, pH 7.0 with duplex concentration of
2 lM.
4.4. X-ray structure of 6
Suitable crystals of 6 were obtained as colourless rods byrecrys-
tallization from EtOH/EtOAc/toluene (2:1:2). The intensity data
were collected at 173 K on a Stoe Image Plate Diffraction System
(Stoe & Cie, 2000, IPDS Software, Stoe & Cie GmbH, Darmstadt,
Germany) using MoK
plate distance 70 mm, / oscillation scans 0–150°, step
exposures of 6 min per image, 2h range 3.27–52.1°, dmin
a
graphite monochromated radiation. Image
D
/ = 1.0°,
ꢀ
dmax = 12.45–0.81 Å. The structure was solved by Direct methods
using the programme SHELXS-97.24 The refinement and all further
calculations were carried out using SHELXL-97.24 The H-atoms could
all be located in Fourier difference maps but were included in cal-
culated positions and treated as riding atoms using SHELXL default
parameters. The non-H atoms were refined anisotropically, using
weighted full-matrix least-squares on F2. Crystallographic data
(excluding structure factors) for compound 6 have been deposited
with the Cambridge Crystallographic Data Centre as supplemen-
tary publication No. CCDC 782500. Copies of the data can be
obtained, free of charge, on application to CCDC, 12 Union Road,
Cambridge CB2 1EZ, UK, (fax: +44 (0)1223 336033 or e-mail:
deposit@ccdc.cam.ac.uk).
Acknowledgments
This work was supported by the Swiss National Science Founda-
tion (Grant No.: 200020-115913). We thank the BENEFRI small
molecule crystallography service (Professor H. Stöckli-Evans) for
solving the X-ray structure of 6.
4.2. Oligonucleotides synthesis and purification
Oligonucleotides were synthesized by standard solid phase
phosphoramidite methodology on the 1.3 lmol scale on a Pharma-
cia LKB Gene Assembler Special DNA Synthesizer. Solvents and
solutions were prepared according to the manufacturer0s protocol.
Ethylthiotetrazole (ETT, 0.25 M in MeCN) was used as activator in
the coupling step. The coupling time was prolonged to 6–12 min
for modified phosphoramidites 8 and 17. Average coupling yields,
as monitored by the on-line trityl assay, were in the range of
97–99%. Deprotection and detachment of the oligonucleotides
built from 17 was effected either in aqueous 1,2-diaminoethane
or 1,3-diaminopropane (1 ml, 40% aq) at 60 °C for 20 h. All other
oligonucleotides were deprotected using standard conditions
(33% aq NH, 55 °C, 16 h). The crude oligomers were purified by
ion-exchange HPLC using a DNAPAC PA200, 4 ꢁ 250 mm analytical
column (Dionex). Mobile phases A: 25 mM TRIZMA in H2O, pH 8.0.
B: 25 mM TRIZMA, 1.25 M NaCl in H2O, pH 8.0, flow 1 ml/min
detection at 260 nm. Purified oligonucleotides were desalted over
Sep-Pak Classic C18 Cartridges (Waters) and were analyzed by ESI-
mass spectrometry (Table 2).
References and notes
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. Bell, N. M.; Micklefield, J. ChemBioChem 2009, 10, 2691.
6. Koshkin, A. A.; Nielsen, P.; Meldgaard, M.; Rajwanshi, V. K.; Singh, S. K.; Wengel,
J. J. Am. Chem. Soc. 1998, 120, 13252.
7. Obika, S.; Nanbu, D.; Hari, Y.; Andoh, J. I.; Morio, K. I.; Doi, T.; Imanishi, T.
Tetrahedron Lett. 1998, 39, 5401.
8. Hendrix, C.; Rosemeyer, H.; Verheggen, I.; Seela, F.; Van Aerschot, A.;
Herdewijn, P. Chem. Eur. J. 1997, 3, 110.
9. Steffens, R.; Leumann, C. J. J. Am. Chem. Soc. 1997, 119, 11548.
10. Renneberg, D.; Leumann, C. J. J. Am. Chem. Soc. 2002, 124, 5993.
11. Tiemann, K.; Rossi, J. J. E. M. B. O. Molecular Medicine 2009, 1, 142.
12. Lönnberg, H. Bioconjugate Chem. 2009, 20, 1065.
13. Tarköy, M.; Bolli, M.; Leumann, C. Helv. Chim. Acta 1994, 77, 716.
14. Bolli, M.; Trafelet, H. U.; Leumann, C. Nucleic Acids Res. 1996, 24, 4660.
15. Luisier, S.; Leumann, C. J. ChemBioChem 2008, 9, 2244.
16. Meier, R.; Grüschow, S.; Leumann, C. J. Helv. Chim. Acta 1999, 82, 1813.
17. Mota, A. J.; Castellanos, E.; de Cienfuegos, L. A.; Robles, R. Tetrahedron:
Asymmetry 2005, 16, 1615.
18. Kim, C. U.; Misco, P. F. Tetrahedron Lett. 1992, 33, 5733.
19. Luisier, S.; Silhar, P.; Leumann, C. J. Nucleic Acids Symp. Ser. 2008, 581.
20. Ittig, D.; Renneberg, D.; Vonlanthen, D.; Luisier, S.; Leumann, C. J.; Coll. Symp.
Series, Hocek, M., Ed., Academy of Sciences of the Czech Republic: Prague, 2005,
pp 21–26.
21. Tarköy, M.; Bolli, M.; Schweizer, B.; Leumann, C. Helv. Chim. Acta 1993, 76, 481.
22. Saenger, W. Principles of Nucleic Acid Structure; Springer: New York, 1984.
23. 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.
4.3. UV-melting curves
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–cooling cycle in the temperature range 20–80 °C was
applied. Tm values were obtained from the derivative curves using
the Varian WinUV software. To avoid evaporation of the solution,
24. Sheldrick, G. M. Acta Crystallogr., Sect. A 1990, 46, 467.
the sample solutions were covered with
a layer of dim-