Screening the structural space of bicyclo-DNA: Synthesis and thermal melting properties of bc4,3-DNA

Article abstract of DOI:10.1002/ejoc.200801034

In attempts to screen the structural and functional properties of bicyclo-DNA, in which the ribose C(3′) and C(5′) centers are integrated into an additional five-membered carbocyclic ring ([3.3.0]-series) we have now synthesized and investigated a ring en

Full text of DOI:10.1002/ejoc.200801034

FULL PAPER
DOI: 10.1002/ejoc.200801034
Screening the Structural Space of Bicyclo-DNA: Synthesis and Thermal
Melting Properties of bc4,3-DNA
Andrea Stauffiger[a] and Christian J. Leumann*[a]
Keywords: Oligonucleotides / Nucleosides / RNA recognition / DNA structures / Nucleosides
In attempts to screen the structural and functional properties
of bicyclo-DNA, in which the ribose C(3Ј) and C(5Ј) centers
are integrated into an additional five-membered carbocyclic
ring ([3.3.0]-series) we have now synthesized and investi-
gated a ring enlarged analogue in which C(5Ј) and C(3Ј) are
spanned by a six-membered carbocyclic ring ([4.3.0]-series).
The synthesis of the corresponding bc4,3-T nucleoside 13 was
performed in 12 steps by starting from known allyl furanose
1. X-ray analysis of its benzyl protected precursor 12 showed
the cyclohexane ring to adopt a chair conformation with the
O(5Ј) substituent in an axial position. The furanose part
block 15 and incorporated into oligodeoxynucleotides by
standard phosphoramidite chemistry. The thermal stabilities
of oligonucleotides with single or double incorporations of
bc4,3-T residues, paired to complementary DNA or RNA,
were found to be similar to those of unmodified oligonucleo-
tides (–2.3 to +0.7 °C per modification) and to those with the
known bc-T modifications. We also found that mismatch dis-
crimination in the bc4,3-T series was similar to that of the nat-
ural series but less discriminative in comparison to the
known bc-T series.
shows clearly S-type sugar pucker. This nucleoside was con- (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
verted into the corresponding phosphoramidite building
Germany, 2009)
angle γ [C(4Ј)–C(5Ј), Figure 1], which as a consequence of
the carbocyclic ring prefers an ac [pseudoequatorial O(5Ј)-
substituent] conformation in nucleoside monomers and di-
mers.[8c,8d]
Introduction
In vitro and in vivo attenuation of gene expression can
be achieved by complementary Watson–Crick base-pairing
of antisense oligonucleotides (ASOs) with RNA targets. A
wide variety of strategies in the design and synthesis of
modified ASOs have been applied in the last 30 years to
increase target specificity and affinity and to decrease in
vivo toxicity.[1] The concept of conformational restriction[2]
has been successfully applied in nucleic acid chemistry and
has brought forward a large variety of analogues, such as
the bridged nucleic acids (LNA/BNA),[3] the hexitol nucleic
acids (HNA),[4] and tricyclo-DNA (tc-DNA),[5] all of which
show increased affinity towards complementary RNA with-
out base-pairing properties being compromised. Not only
are these advanced analogues expected to replace the phos-
phorothioates and 2Ј-modified analogues in therapy,[6] but
in some cases they have also proven to increase siRNA effi-
cacy.[7]
Our first-generation, conformationally restricted oligo-
nucleotide analogue [3.3.0]bicyclo-DNA (bc-DNA)[8a,8b]
shows no significant improvement in Watson–Crick pairing
to RNA relative to that of DNA, probably due to a struc-
tural mismatch in the repetitive backbone unit at torsion
[a] Department of Chemistry and Biochemistry, University of Bern
Freiestrasse 3, 3012 Bern, Switzerland
Figure 1. Chemical structures of [3.3.0]bicyclo-DNA (bc-DNA),
bcox-DNA, [4.3.0]bicyclo-DNA, (bc4,3-DNA), and tricyclo-DNA
(tc-DNA), as well as conformational features of the carbocyclic
rings in the bc[4.3.0]- and [3.3.0]-series (box).
Fax: +41-31-631-34-22
E-mail: leumann@ioc.unibe.ch
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
Eur. J. Org. Chem. 2009, 1153–1162
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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A. Stauffiger, C. J. Leumann
FULL PAPER
With the background of establishing a structure–affinity
In an energy-minimized structure of a fully modified
relationship in the bicyclo-DNA family, several analogues, [4.3.0]bicyclo-DNA oligonucleotide paired to its comple-
such as bcox-DNA,[9] are currently under investigation in mentary DNA, the six-membered ring adopts a twist-boat
our laboratory. An interesting candidate in this context is conformation with the O(5Ј) atom in an pseudoaxial orien-
[4.3.0]bicyclo-DNA (bc4,3-DNA, Figure 1). We reasoned tation. The furanose puckering is O(4Ј)-endo (Figure 3).
that annealing a six-membered instead of a five-membered
ring to the furanose unit will have an additional rigidifying
effect on the carbocyclic structure, because of the reduced
number of different conformational states of a cyclohexane
ring relative to those of a cyclopentane ring. Furthermore,
the transition from a five- to a six-membered ring is ex-
pected to shift specifically the conformational preferences
of the backbone torsion angles γ and δ. (Figure 1). A bc4,3
-
nucleoside precursor with the base thymine was previously
synthesized by Nielsen and coworkers.[10a] An alternative
synthesis for bc4,3-T was disclosed recently by our labora-
tory.[11] However, this nucleoside has never been structurally
investigated nor inserted into oligonucleotides. Here we re-
port on an alternative synthesis of [4.3.0]bicyclothymidine,
its conformational preferences as determined by computer
modeling and X-ray crystallography, as well as on the incor-
poration into oligodeoxyribonucleotides by solid-phase
phosphoramidite chemistry and on the base-pairing proper-
ties with complementary DNA and RNA.
Results and Discussion
Molecular Modeling
Figure 3. Top: Energy-minimized molecular model of a DNA du-
plex containing a fully modified [4.3.0]bicyclo-DNA oligonucleo-
A conformational search of the 5ЈO- and 3ЈO-methylated
T-monomer as a model for the nucleoside [no H-bond do- tide. Bottom: Structure of an average monomeric unit in the du-
plex.
nor at O(3Ј,5Ј)] by molecular modeling with the use of Hy-
perchem software[12] revealed the lowest-energy conformer
with a six-membered ring chair conformation to be that
with the O(5Ј) atom in an equatorial position
(28.08 kcalmol–1). This compares to the energy of the other Synthesis of the Nucleoside Building Block
possible chair conformation with the O(5Ј) atom in the ax-
The synthesis of phosphoramidite building block 15
started from C(3Ј)-allylfuranose 1, which was obtained in
three steps from diacetone--glucose (Scheme 1)[10e,10f] by
following a strategy similar to that described previously by
Nielsen et al.[10a–10d] Oxidative and selective cleavage of the
exposed glycol was efficiently performed with periodic acid
in ethyl acetate to give the corresponding 5Ј-carboxal-
dehyde.[13] Subsequent Grignard reaction with vinylmagne-
sium bromide yielded 2 as a mixture of stereoisomers. Both
isomers were smoothly converted into [4.3.0]bicyclo deriva-
tives 3 and 4 (S/R, 8.5:1) by ring-closing metathesis with
the use of Grubb’s 2nd generation catalyst (2 mol-%). Un-
desired 5ЈS isomer 3, the relative configuration of which
was unambiguously assigned by NMR-ROESY spec-
troscopy, was subsequently oxidized with Dess–Martin
periodinane to enone 5, followed by Luche reduction to
give again a mixture of stereoisomers 3 and 4, however, now
in a ratio of 2:1 in favor of desired R isomer 4. After cata-
lytic hydrogenation (Ǟ6) and benzylation of the OH func-
ial position (31.50 kcalmol–1), which is higher in energy by
3.42 kcalmol–1 (Figure 2).
Figure 2. Conformational search. The O(5Ј) atom in the equatorial
(left) and axial (right) orientations. The two structures differ by
3.42 kcalmol–1.
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Eur. J. Org. Chem. 2009, 1153–1162

Screening the Structural Space of Bicyclo-DNA
tion (Ǟ7), the dioxolane ring was hydrolyzed in boiling ace-
tic acid, followed by acetylation to give sugar building block
8 in excellent yield.
Scheme 1. Synthesis of [4.3.0]bicyclosugar. Reagents and condi-
tions: (a) H5IO6 (1.1 equiv.), EtOAc, room temp., 90 min; (b) vi-
nylMgBr (1.8 equiv.), THF, room temp., 16 h; (c) Grubbs’ catalyst
2nd generation (2 mol-%), CH2Cl2, 40 °C, 3 h (ratio of 3/4, 8.5:1);
(d) Dess–Martin periodinane (1.5 equiv.), CH2Cl2, room temp., 4 h;
(e) CeCl3·7H2O (2 equiv.), NaBH4 (2 equiv.), MeOH, 0 °C, 30 min
(ratio of 3/4, 1:2); (f) 20% Pd(OH)2/C, H2, MeOH, room temp.,
3 h; (g) NaH (2 equiv.), DMF, 50 °C, 1 h, then BnBr (2 equiv.),
DMF, room temp., 16 h; (h) 80% aq. AcOH, 90 °C, 16 h; (i) Ac2O,
pyridine, room temp., 16 h.
Scheme 2. Synthesis of [4.3.0]bicyclothymidine phosphoramidite.
Reagents and conditions: (a) thymine (2 equiv.), BSA (5 equiv.),
TMSOTf (1.4 equiv.), CH3CN, 40 °C, 16 h; (b) NaOMe (2 equiv.),
MeOH, room temp., 16 h; (c) 1,1Ј-thiocarbonyldiimidazol
(1.5 equiv.), room temp., DMF, 6 h; (d) AIBN (1 equiv.), Bu3SnH
(3 equiv.), toluene, 80 °C, 4 h; (e) 20% Pd(OH)2/C, cyclohexa-1,4-
diene (10 equiv.), H2, MeOH, room temp., 6 h; (f) DMTOTf
(1.5 equiv.), pyridine, room temp., 24 h; (g) CEPCl (3 equiv.),
Hünig’s base (5 equiv.), CH3CN, room temp., 2 h.
Nucleosidation of 8 with persilylated thymine under
Vorbrüggen conditions[14] (Scheme 2) led to nucleoside 9
with high selectivity for the β-anomer (ratio α/β, 1:10) and
in good yield. 2Ј-O-Deprotected analogue 10 was obtained
by treating 9 with sodium methoxide in methanol.
In preparing for removal of the C(2Ј)–OH group, the Table 1. Reagents and conditions for the reduction of 11.
thiocarbonylimidazole substituent was introduced to yield
Entry Conditions
Products (yield, %)
11 in excellent yield. Deoxygenation under Barton–
McCombie conditions[15] gave deoxy analogue 12, however,
only in poor yield (37%). Various conditions were tested to
improve the outcome of this reaction (summarized in
Table 1). Best yields were found with AIBN as a radical
starter and Bu3SnH (3 equiv.) in toluene (Table 1, Entry 1).
Very high concentrations of Bu3SnH led to complete turn-
over of the starting material; however, there was a 3:1 pref-
erence for starting alcohol 10 over deoxygenated product 12
(Table 1, Entry 2). A change in the solvent to tBuOH did
not improve the reaction either (Table 1, Entry 3). As an
AIBN (1 equiv.), Bu3SnH (3 equiv.),
toluene, 90 °C, 4 h
1
2
3
4
12/11/10, 2:1:1 (79)
12/10, 1:3 (n.d.)[a]
AIBN (0.8 equiv.), Bu3SnH
(32 equiv.), toluene, 75 °C, 2 h
AIBN (0.8 equiv.), Bu3SnH
(3 equiv.), tBuOH, 75 °C, 16 h
AIBN (0.1 equiv.), TTMSS[b]
(2 equiv.), toluene, 80 °C, 48 h
12/11/10, 2:1:1
(n.d.)[a]
11a (Ͻ20)
[a] n.d. = yield not determined. [b] TTMSS = tris(trimethylsilyl)-
silane.
alternative to tin hydride as a hydrogen donor, tris(trime- 13 in 55% yield. As a side reaction, reduction of the base
thylsilyl)silane (TTMSS), introduced by Chatgilialoglu[16] was observed Although different conditions (e.g., different
was used (Table 1, Entry 4). However, the only product iso- palladium catalysts, high or normal pressure of H2, ad-
lated was side product 11a (Scheme 2), which is known to dition of cyclohexa-1,4-diene as hydrogen source) were
occur in deoxygenations as a consequence of an alternative tested, no optimal deprotection scheme was found.
radical pathway.[15a,15b,16a,17]
Attempts to reoxidize 5,6-dihydrothymine to 13 according
Despite the described problems with the defunctionaliz- to literature procedures[18] were unsuccessful. Despite these
ation at the C(2Ј) position, deoxy derivative 12 was ob- difficulties, free monomer 13 was further converted into
tained in sufficient quantities to continue the synthesis. De- phosphoramidite building block 15 by dimethoxytritylation
protection by catalytic hydrogenation gave free monomer (Ǟ14) and phosphitylation.
Eur. J. Org. Chem. 2009, 1153–1162
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A. Stauffiger, C. J. Leumann
FULL PAPER
X-ray Structure of Nucleoside 12
are comparable to the recently published structure of ben-
zyloximobicyclothymidine (bcox-T).[9]
Crystals of benzyl-protected nucleoside 12 were sub-
jected to X-ray analysis to map the conformational prefer-
ences of the bicyclic core structure (Figure 4, see also Sup-
porting Information). Two individual molecules (12a,b) co-
exist in the unit cell, which essentially differ in the orienta-
tion of the O(3Ј) benzyl group. There are no intramolecular
hydrogen bonds detectable that could influence the local
conformation of the sugar ring.
There is clearly a discrepancy between the X-ray and en-
ergetically preferred modeled structure of 12. In the X-ray
structure, the cyclohexane ring exists in the chair conforma-
tion with the O(5Ј) substituent in an axial position, which
by modeling was found to be higher in energy by
3.42 kcalmol–1 relative to the alternative chair conforma-
tion with the O(5Ј) substituent in an equatorial position.
Interestingly, in the X-ray structure C(5)–H and the methyl
group of thymine make close contacts to the π system of
the O(5Ј) benzyl group, reminiscent of π–σ* interactions.
Although 12 adopts a conformation in the X-ray structure
that is much closer to that of a natural nucleotide in the
DNA backbone, it cannot be ruled out that intramolecular
or packing forces in 12 are enforcing a higher energy con-
former.
Oligonucleotide Synthesis
A series of dodecamers (ON1, ON2, and ON4; Table 3)
and a decamer (ON6; Table 5) of mixed-base oligonucleo-
tides containing single or double [4.3.0]bc-T mutations were
synthesized on a 1 µ or 1.3 µ scale by standard phos-
phoramidite chemistry. For incorporation of the modified
building blocks, the standard coupling step was extended
to 12–14 min and the phosphoramidite concentration was
increased up to 0.2 . Coupling efficiencies were generally
lower (≈90%) relative to those of [3.3.0]bc-amidites. Crude
oligonucleotides were deprotected and detached from the
solid support by using standard conditions (conc. NH3,
55 °C, 16 h). All oligonucleotides were purified by HPLC
and their structural composition was analyzed by ESI-MS
(for details see the Supporting Information).
Figure 4. X-ray structure of nucleoside 12a (left) and 12b (right).
The two independent molecules per asymmetric unit differ mostly
in the orientation of the O(3Ј) benzyl groups.
The furanose ring adopts a C(2Ј)-endo conformation (2E,
south-type) and thus gives rise to a pseudorotational phase
angle P of 174° for 12a and 166° for 12b (Table 2).[19] For
torsion angle ν4 [C(3Ј)–C(4Ј)–O(4Ј)–C(1Ј)][20] the values are
–9.1 (for 12a) and –3.7 (for 12b), hence showing a qua-
siplanar alignment of the four atoms. The orientation of the
base (torsion angle χ) is, as expected, anti.
Table 2. Pseudorotation phase angles and selected nucleoside tor-
sion angles of [4.3.0]bicyclo-DNA nucleoside 12 in relation to bc-
nucleosides and natural deoxyribonucleosides in the B conforma-
tion.
Tm Measurements
UV melting curve analysis was performed at 260 nm with
a cooling–heating–cooling cycle at a rate of 0.5 °Cmin–1 in
standard saline buffer at pH 7.0. All curves within a cycle
were superimposable, thus ruling out nonequilibrium states.
The experimental Tm data are summarized in Tables 3 and 5.
Nucleoside
P[a] [°]
γ [°]
δ [°]
χ [°]
12a
174
166
128
163
144
70
71
149
88
162
154
126
148
122
–105
–120
–113
–118
–119
12b
bc-T[b]
bcox-T[c]
dN[d]
57
Table 3. Tm data from UV melting curves (260 nm) of modified
dodecamer duplexes with complementary DNA and RNA.
[a] Pseudorotation phase angle. [b] Taken from ref.[8c] [c] Taken
from ref.[9] [d] Average deoxynucleotide conformation in B-DNA
(ref.[20]).
X
Tm vs.
DNA
[°C][b]
Tm vs.
RNA
[°C][b]
Oligonucleotide[a]
The carbocyclic ring shows a chair-like conformation
with the hydroxy group at C(5Ј) in an axial position. As a
consequence, torsion angle γ is in a gauche (+sc) orientation
as observed in A- and B-DNA. Comparison with the X-ray
structure of the parent bc-T shows a shift in the conforma-
tion of the furanose ring from the C(1Ј)-exo to the C(2Ј)-
endo form and a change in the torsion angle γ from the
ON1 d(GGAXGTTCTCGA)bc4,3-T
ON2 d(GGATGTTCXCGA)bc4,3-T
ON3 d(GGATGTTCXCGA) bc-T
ON4 d(GGATGXXCTCGA)bc4,3-T
ON5 d(GGATGXXCTCGA) bc-T
46.1 (–1.4) 47.4 (–2.3)
47.3 (–0.2) 48.9 (–0.8)
49.0 (+1.5) 49.0 (–0.7)
47.0 (–0.3) 51.0 (+0.7)
48.7 (+0.6) 48.2 (–0.7)
[a] Tm of unmodified oligodeoxynucleotide: 47.5 °C (vs. DNA);
49.7 °C (vs. RNA). [b] ∆Tm per modification in parenthesis; c =
anticlinal to the gauche range. These structural properties 2 µ in 10 m NaH2PO4, 150 m NaCl, pH 7.0.
1156 Eur. J. Org. Chem. 2009, 1153–1162
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Screening the Structural Space of Bicyclo-DNA
Analysis of the Tm data revealed a slight reduction in the stabilization (–3.0 °C against DNA and –3.7 °C against
thermal stability of duplexes containing one or two [4.3.0]- RNA) of the duplex, both relative to the natural system
bicyclothymidines against DNA and RNA. Duplexes in- and the tc-system (ON7). In contrast, the tc-T showed a
volving ON1 with the modification in between two purine stabilizing effect against its natural DNA complement. The
bases and closer to the 5Ј-end show the highest degree of alteration in the backbone of the [4.3.0]bicyclo system thus
destabilization. Duplexes with ON2 and ON4 show about has an effect on the stability of the duplexes in comparison
the same stability as natural duplexes. A slight stabilizing with the sequence and length of the oligonucleotide chosen.
effect can be seen for the ON4/RNA duplex (+0.7 °C/modi-
Table 5. Tm data from UV melting curves (260 nm) of a bc4,3-T and
fication), an observation which might indicate that consecu-
tc-T modified decamer with complementary DNA and RNA.
tive modifications lead to stabilization of the duplex in
Code
Oligonucleotide[a]
Tm vs. DNA
[°C][b]
Tm vs. RNA
[°C][b]
comparison to the natural system. Relative to the parent
[3.3.0]bicyclo series (ON3 and ON5) there exist only minor
variations in the values of Tm. There is a weak tendency for
more stable duplexes of bc-T with complementary DNA
and for bc4,3-T with complementary RNA.
To determine the relative effect of the modifications on
pairing selectivity we measured the Tm data for the singly
modified oligonucleotide ON2 with complementary DNA
carrying a mismatched base opposite the modification. For
comparison, the sense strands carrying bc-T, bcox-T, and a
natural thymidine were also measured (data taken from
ref.[9]; Table 4).
ON6
d(AACTGtCACG) 40.3 (–3.0)
41.3 (–3.7)
ON7[c] d(pAACTGtctCACG) 45.3 (+0.9)[c]
43.5 (+0.5)[c]
[a] Tm of unmodified duplex: 43.3 °C (vs. DNA); 45.0 °C (vs.
RNA). [b] ∆Tm per modification in parenthesis. [c] Taken from
ref.[5c]; Tm is compared to natural oligonucleotides also bearing a
5Ј-phosphate. The values of Tm are 44.4 °C against DNA and
43.0 °C against RNA. Conditions as for Table 4.
Conclusions
We prepared the novel nucleoside [4.3.0]bicyclothymidine
and incorporated this analogue into oligonucleotides
through standard solid-phase synthesis. Whereas the syn-
thesis of the analogous bc4,3-ribothymidine proceeded
smoothly and with high selectivity to the β-anomeric nucle-
oside, removal of the 2ЈOH function and debenzylation of
12 were the bottleneck of the synthesis, as the maximum
yield of the former step remained below 40%. We believe
that the difficulties of both steps are due to the high steric
congestion around the C(2Ј,3Ј) centers. Because the synthe-
sis of the parent bc-nucleosides follows a different route, we
cannot compare directly the influence of the six- versus five-
membered carbocyclic ring on the reactivity at these cen-
Table 4. Tm data from UV melting curves (260 nm) of duplexes of
d(GGATGTTCXCGA) with DNA complements carrying a mis-
matched base opposite X.
Mis-
Tm [°C]
X = bcox-T[c]
match[a]
X = dT[b]
X = bc-T[c]
X = bc4,3-T[b]
G–X
C–X
T–X
39.7 (–7.8)
36.0 (–11.5)
38.0 (–9.5)
37.0 (–12.0)
35.0 (–14.0)
32.0 (–17.0)
31.9 (–11.1)
32.6 (–10.4)
34.9 (–8.1)
40.4 (–6.9)
36.3 (–11.1)
38.6 (–8.7)
[a] Values in parentheses are ∆Tm values relative to the matched
duplex. [b] Tm value for matched duplex, see Table 3. [c] Tm values
for matched duplexes: bc-T: 49.0 °C, bcox-T: 43.0 °C.
As expected, a thermal destabilization of the mismatched ters. It is also noteworthy that oligonucleotide synthesis,
duplexes was observed. The high mismatch discrimination probably for the very same reason, was significantly less
observed for [3.3.0]bc-DNA is reduced in [4.3.0]bicyclo- efficient relative to that of the parent bc-DNA. With coup-
DNA. The destabilization is generally of about the same ling efficiencies of only around 90% the synthesis of fully
extent as that observed with the natural mismatched du- modified bc4,3-oligonucleotides will be extremely difficult if
plexes. In the special case of the G–T mismatch (wobble not impossible without improvements in the coupling step.
pair), the [4.3.0]bc-T is far less destabilizing than the bc-T
In order to investigate the effect of the altered backbone
and the bcox-modification. From the results obtained, it can on the oligonucleotide level, three dodecamers bearing
be concluded that the replacement of the “ethano” with a either one or two modified thymidine residues were synthe-
“propano” bridge between C(3Ј) and C(5Ј) leads to a sized and analyzed. These oligonucleotides were able to
slightly less specific pairing system with no major effect on form stable duplexes both with complementary DNA and
overall affinity.
RNA and proved to be of more or less the same thermal
The effect of the [4.3.0]bicyclo modification on the du- stability as their natural counterparts. It is noteworthy that
plex structure was studied by CD spectroscopy in the cases the mismatch-discrimination was lower than that in the cor-
of ON4 in complexation with RNA and DNA (Supporting responding bc-series with the five-membered carbocycle
Information). As anticipated, no major deviations in the and similar to that of the natural system.
CD spectra from those of the unmodified DNA/DNA and
One of the aims of this work was to contribute to estab-
DNA/RNA duplexes were found, indicating no major lishing a structure–affinity relationship of the bicyclo-DNA
structural changes induced by the two modified thymidine family and ultimately to understand the effect of conforma-
moieties in the core of the sense strand.
tional restriction on nucleic acid affinity. Whereas a confor-
For comparison with tricyclo-DNA[5c] a decamer oligo- mational search on the bc4,3-T monomer by computer sim-
nucleotide bearing one [4.3.0]bc-T modification (ON6) was ulation suggested the O(5Ј) substituent to adopt preferen-
synthesized (Table 5). This modification led to a strong de- tially an equatorial orientation (with torsion angle γ being
Eur. J. Org. Chem. 2009, 1153–1162
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A. Stauffiger, C. J. Leumann
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nylmagnesium bromide (1  in THF, 37.7 mL, 37.69 mmol,
1.8 equiv.) was added dropwise. The solution was stirred under an
atmosphere of argon for 16 h and then quenched at 0 °C by the
addition of ice and water. The solution was neutralized by addition
of 4  AcOH and then concentrated. The aqueous phase was ex-
tracted with CH2Cl2 (3ϫ150 mL), and the combined organic phase
was dried with MgSO4, filtered, and concentrated. CC (hexane/
EtOAc, 4:1) yielded diene 2 (isomeric mixture, 6.06 g, 83.6%) as a
colorless oil. Rf (hexane/EtOAc, 9:1) = 0.65 (for 2R), 0.37 (for 2S).
in the anticlinal range), X-ray analysis of benzyl-protected
precursor 12 revealed a pseudoaxial arrangement with tor-
sion angles γ and δ that are much closer to that of the DNA
backbone. Given the uncertainty that the structure of 12
in the crystal does not necessarily reflect the ground state
conformation of a bc4,3-nucleotide in solution, the in-
terpretation of its effect in terms of conformational restric-
tion remains speculative unless a high-resolution structure
of a modified oligonucleotide duplex becomes available. If
1
Data for 2R: H NMR (300 MHz, CDCl3): δ = 7.35 (m, 5 H, Ph),
the ground-state conformation of the cyclohexyl ring is in- 6.01 [m, 2 H, H-C(2), H-C(6Ј)], 5.68 [d, J = 3.78 Hz, 1 H, H-C(1Ј)],
5.45–5.21 [m, 4 H, H-C(7Ј), H-C(1)], 4.74 (m, 2 H, CH2Ph), 4.52
[d, J = 3.75 Hz, 1 H, H-C(2Ј)], 4.36 [m, 1 H, H-C(5Ј)], 3.96 [d, J =
9.21 Hz, 1 H, H-C(4Ј)], 2.74 (d, J = 2.64 Hz, 1 H, OH), 2.67 [m, 2
H, H-C(3)], 1.61 (s, 3 H, CH3), 1.36 (s, 3 H, CH3) ppm. 13C NMR
(75 MHz, CDCl3): δ = 138.0 (Ph1), 132.1 [C(2)], 131.3 [C(6Ј)], 128.3
(Phortho), 127.6 (Phpara), 127.4 (Phmeta), 119.3 [C(1)], 116.2 [C(7Ј)],
112.9 [C(Me2)], 104.1 [C(1Ј)], 84.8 [C(3Ј)], 81.7 [C(4Ј)], 81.6 [C(2Ј)],
70.2 [C(5Ј)], 67.8 (CH2Ph), 36.0 [C(3)], 26.8 (CH3), 26.6 (CH3).
deed that with the 5Ј-substituent in the axial position
(DNA-duplex-like structure), then the change in the geome-
try of torsion angle γ from anticlinal to gauche had to be
considered as irrelevant on duplex stability. If, however, the
ground state conformation is that with the O5Ј-substituent
in the equatorial position (DNA-duplex-unlike), then the
same structural situation as in the case of bc-DNA is en-
countered and the potential energetic benefit for changing
γ into a DNA-duplex-like geometry remains elusive. A clear
1
Data for 2S: H NMR (300 MHz, CDCl3): δ = 7.33 (m, 5 H, Ph),
6.02 [m, 2 H, H-C(2), H-C(6Ј)], 5.71 [d, J = 3.78 Hz, 1 H, H-C(1Ј)],
conclusion, however, that can be drawn is that the shift 5.45–5.14 [m, 4 H, H-C(7Ј), H-C(1)], 4.72 (m, 2 H, CH2Ph), 4.51
[d, J = 3.75 Hz, 1 H, H-C(2Ј)], 4.42 [m, 1 H, H-C(5Ј)], 4.12 [d, J =
6.00 Hz, 1 H, H-C(4Ј)], 2.67 [dd, J = 14.69, 7.61 Hz, 1 H, H-C(3)],
2.52 [dtd, J = 14.70, 6.75, 1.32, 1.32 Hz, 1 H, H-C(3)], 2.29 (d, J
= 5.10 Hz, 1 H, OH), 1.60 (s, 3 H, CH3), 1.36 (s, 3 H, CH3) ppm.
13C NMR (75 MHz, CDCl3): δ = 138.8 (Ph1), 136.7 [C(2)], 132.7
[C(6Ј)], 128.2 (Phortho), 127.3 (Phpara), 127.2 (Phmeta), 118.8 [C(1)],
116.4 [C(7Ј)], 112.8 [C(Me2)], 103.7 [C(1Ј)], 83.4 [C(3Ј)], 83.1
[C(4Ј)], 82.7 [C(2Ј)], 70.1 [C(5Ј)], 66.9 (CH2Ph), 36.2 [C(3)], 26.9
(CH3), 26.6 (CH3) ppm. HRMS (ESI+): calcd. for C20H26O5Na [M
+ Na]+ 369.1677; found 369.1676.
from the bc[3.3.0]- to the bc[4.3.0]-scaffold does not dra-
matically alter nucleic acid affinity.
Experimental Section
General: All reactions were performed under an atmosphere of ar-
gon in dried glassware. Anhydrous solvents for reactions were ob-
tained by filtration through activated alumina or by storage over
molecular sieves (4 Å). Column chromatography (CC) was per-
formed on silica gel (Fluka) with an average particle size of 40 µm.
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
achieved either under UV light or by staining in dip solution [vanil-
lin (15 g), absolute ethanol (250 mL), concentrated H2SO4 (2.5 mL)
or p-anisaldehyde (10 mL) concentrated H2SO4 (10 mL), concen-
trated acetic acid (2 mL), ethanol (180 mL)] followed by heating
with a heat gun. NMR spectra were recorded at room temperature
with a Bruker AC-300 or Bruker DRX-400 instrument. Chemical
shifts (δ) are reported relative to the undeuterated residual solvent
peak [CHCl3: 7.27 ppm (1H) and 77.0 ppm (13C); CHD2OD:
3.35 ppm (1H) and 49.3 ppm (13C)]. Signal assignments are based
3 and 4: To the isomeric mixture of 2 (4.45 g, 12.85 mmol) dissolved
in anhydrous CH2Cl2 (300 mL) was added the 2nd generation
Grubbs catalyst (218.0 mg, 0.26 mmol, 2 mol-%). The slightly pur-
ple solution was stirred at 40 °C under an atmosphere of argon for
3 h. CH2Cl2 was then removed in vacuo, and the crude product
was purified by CC (hexane/EtOAc, 7:3) to yield isomers 3 (3.13 g,
76.3%) and 4 (338.4 mg, 8.5%) as yellow oils. Rf (hexane/EtOAc,
1
7:3) = 0.38 (for 3), 0.21 (for 4). Data for 3: H NMR (400 MHz,
CDCl3): δ = 7.28 (m, 5 H, Ph), 5.92 [m, 1 H, H-C(3)], 5.68 [d, J =
3.68 Hz, 1 H, H-C(8)], 5.65 [dddd, J = 11.00, 5.43, 2.18, 1.06 Hz,
1 H, H-C(4)], 4.64 (d, J = 11.12 Hz, 1 H, CH2Ph), 4.51 (d, J =
11.16 Hz, 1 H, CH2Ph), 4.34 [d, J = 3.68 Hz, 1 H, H-C(7)], 4.30 [s,
1 H, H-C(1)], 4.10 [dd, J = 10.80, 3.66 Hz, 1 H, H-C(2)], 2.50 [dd,
J = 18.71, 5.47 Hz, 1 H, H-C(5)], 1.89 (d, J = 10.97 Hz, 1 H, OH),
1.68 [ddd, J = 18.74, 4.83, 2.27 Hz, 1 H, H-C(5)], 1.64 (s, 3 H,
CH3), 1.31 (s, 3 H, CH3). 13C NMR (101 MHz, CDCl3): δ = 138.1
(Ph1), 131.2 [C(3)], 128.2 (Phortho), 127.7 (Phpara), 127.6 (Phmeta),
123.8 [C(4)], 113.4 [C(Me2)], 104.7 [C(8)], 81.5 [C(7)], 79.4 [C(6)],
78.1 [C(1)], 67.2 (CH2Ph), 63.4 [C(2)], 26.8 [C(5)], 26.5 (2ϫCH3)
1
1
on DEPT or APT experiments, and on H–1H and H–13C corre-
lation experiments (COSY/HMSC). Difference [1H]1H-NOE ex-
periments were recorded at 400 MHz. Chemical shifts for 31P NMR
are reported relative to 85% H3PO4 as external standard. Electron
impact (EI) spectra were recorded with a Micromass AutoSpeq Q
VG with an ionization energy of 70 eV. Electrospray ionization
mass spectra (ESI) were recorded with either a Fisons Instrument
VG Platform (low resolution) or an Applied Biosystems, Sciex
QSTAR Pulsar (high resolution). UV spectra were measured with
a Varian Cary 3E UV/Vis spectrophotometer.
1
ppm. Data for 4: H NMR (300 MHz, CDCl3): δ = 7.35 (m, 5 H,
Ph), 5.81 [d, J = 3.57 Hz, 1 H, H-C(8)], 5.76 [m, 1 H, H-C(3)], 5.63
[m, 1 H, H-C(4)], 4.69 (d, J = 11.31 Hz, 1 H, CH2Ph), 4.61 (d, J
= 11.31 Hz, 1 H, CH2Ph), 4.51 [d, J = 3.03 Hz, 1 H, H-C(7)], 4.48
[d, J = 4.53 Hz, 1 H, H-C(1)], 4.41 [m, 1 H, H-C(2)], 2.49 [m, 1 H,
H-C(5)], 2.18 (s, 1 H, OH), 1.79 [qd, J = 19.09, 2.77, 2.77, 2.74 Hz,
1 H, H-C(5)], 1.65 (s, 3 H, CH3), 1.39 (s, 3 H, CH3) ppm. 13C
NMR (101 MHz, CDCl3): δ = 138.3 (Ph1), 128.4 (Phortho), 128.2
[C(3)], 127.6 (Phpara), 127.5 (Phmeta), 123.5 [C(4)], 113.6 [C(Me2)],
104.2 [C(8)], 82.9 [C(1)], 81.9 [C(6)], 75.8 [C(7)], 66.7 (CH2Ph), 65.1
[C(2)], 28.0 [C(5)], 26.6 (CH3), 26.5 (CH3) ppm. HRMS (ESI+):
calcd. for C18H22O5Na [M + Na]+ 341.1364; found 341.1362.
2: To a solution of furanose derivative 1[12] (8.17 g, 20.94 mmol)
dissolved in EtOAc (250 mL) was added H5IO6 (5.25 g,
23.03 mmol, 1.1 equiv.). The white suspension was stirred at room
temperature under an atmosphere of argon for 90 min. Solids
where then filtered off, and EtOAc was removed in vacuo. The
intermediate aldehyde was dried under high vacuum for 6 h before
further use. The slightly red-colored solid was taken up in anhy-
drous THF (150 mL) and cooled to 0 °C. Slowly, a solution of vi-
1158
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© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 1153–1162

Screening the Structural Space of Bicyclo-DNA
5: To a solution of allyl alcohol 3 (7.84 g, 24.63 mmol) dissolved in
anhydrous CH2Cl2 (200 mL) was added Dess–Martin periodinane
(15.74 g, 36.94 mmol, 1.5 equiv.). The mixture was stirred under an = 0.58. H NMR (300 MHz, CDCl3): δ = 7.35 (m, 10 H, Ph), 5.83
centrated. CC (hexane/EtOAc, 4:1) yielded title compound 7
(654.8 mg, 1.59 mmol, 95%) as a yellow oil. Rf (hexane/EtOAc, 7:3)
1
atmosphere of argon at room temperature for 4 h. The solvent was
removed in vacuo, and the remaining solids were taken up in
EtOAc. After filtration through a bed of Celite/silica (1:1), the sol-
vent was removed to yield the crude product. Remaining Dess–
Martin reagent was removed by taking the product up in hexane/
[d, J = 3.78 Hz, 1 H, H-C(8)], 4.61 (d, J = 10.20 Hz, 2 H, CH2Ph),
4.55 (d, J = 9.6 Hz, 2 H, CH2Ph), 4.39 [d, J = 3.21 Hz, 1 H, H-
C(1)], 4.33 [d, J = 3.75 Hz, 1 H, H-C(7)], 3.64 [ddd, J = 11.50,
4.88, 3.34 Hz, 1 H, H-C(2)], 1.94 [d, J = 14.5 Hz, 1 H, H-C(3)],
1.83 [m, 1 H, H-C(6)], 1.65 (m, 1 H), 1.62 (s, 3 H, CH3), 1.58 (m,
EtOAc (2:1) followed by filtration. Evaporation yielded enone 5 1 H), 1.51 (m, 1 H), 1.38 (s, 3 H, CH3), 1.09 (m, 1 H) ppm. 13C
(7.8 g, quantitative) as a clear oil. Rf (hexane/EtOAc, 7:3) = 0.25.
1H NMR (300 MHz, CDCl3): δ = 7.31 (m, 5 H, Ph), 6.84 [ddd, J
= 10.27, 5.97, 2.34 Hz, 1 H, H-C(4)], 6.21 [ddd, J = 10.35, 2.08,
NMR (75 MHz, CDCl3): δ = 138.7 (Ph1), 138.5 (Ph1), 128.2, 127.7,
127.6, 127.5, 127.3 (Ph), 112.6 [C(Me2)], 104.8 [C(8)], 83.6 [C(6)],
81.6 [C(1)], 76.0 [C(7)], 74.9 [C(2)], 66.2 (CH2Ph), 60.3 (CH2Ph),
0.99 Hz, 1 H, H-C(3)], 5.93 [d, J = 3.59 Hz, 1 H, H-C(8)], 4.71 (d, 26.7 (CH3), 26.5 (CH3), 26.2, 25.8, 19.2 [C(3), C(4), C(5)] ppm.
J = 11.10 Hz, 1 H, CH2Ph), 4.58 (d, J = 11.13 Hz, 1 H, CH2Ph),
HRMS (ESI+): calcd. for C25H30O5Na [M + Na]+ 433.1990; found
4.52 [d, J = 3.57 Hz, 1 H, H-C(7)], 4.32 [s, 1 H, H-C(1)], 2.80 [dd, 433.1995.
J = 19.18, 5.98 Hz, 1 H, H-C(5)], 2.20 [td, J = 19.22, 2.89, 2.89 Hz,
8: Compound 7 (547.5 mg, 1.33 mmol) was dissolved in 80% aque-
1 H, H-C(5)], 2.13 [m, 1 H, H-C(5)], 1.66 (s, 3 H, CH3), 1.40 (s, 3
H, CH3) ppm. 13C NMR (75 MHz, CDCl3): δ = 191.5 [C(2)], 145.5
[C(3)], 137.8 (Ph1), 128.8 (Phortho), 128.3 (Phortho), 127.7 (Phortho),
127.3 [C(4)], 114.0 [C(Me2)], 105.8 [C(8)], 84.8 [C(6)], 81.8 [C(1)],
81.8 [C(7)], 67.7 (CH2Ph), 28.8 [C(5)], 26.7 (CH3), 26.6 (CH3) ppm.
HRMS (ESI+): calcd. for C18H20O5Na [M + Na]+ 339.1208; found
339.1196.
ous AcOH (25 mL) and stirred at 90 °C for 16 h. AcOH was evapo-
rated by using EtOH (3ϫ), toluene (3ϫ), and anhydrous pyridine
(1ϫ) as cosolvents. The residue was dissolved in anhydrous pyr-
idine (10 mL) and Ac2O (10 mL) was added dropwise. The solution
was stirred at room temperature under an atmosphere of argon for
16 h. It was then cooled to 0 °C and quenched by the addition of
ice-cold water. The aqueous solution was extracted with CH2Cl2
(3ϫ). The organic phase was washed with a solution of aqueous
saturated NaHCO3 (2ϫ), dried with MgSO4, and filtered, and the
solvents were then evaporated. CC (hexane/EtOAc, 7:3) yielded
sugar 8 (mixture of anomers, 565.4 mg, 92%) as a colorless oil. Rf
(hexane/EtOAc, 7:3) = 0.53, 0.46. Data for the mixture of anomers:
1H NMR (300 MHz, CDCl3): δ = 7.35 (m, 20 H, Ph), 6.46 [d, J =
4.50 Hz, 1 H, H-C(8β)], 6.19 [s, 1 H, H-C(8α)], 5.26 [d, J = 4.50 Hz,
1 H, H-C(7β)], 5.12 [s, 1 H, H-C(7α)], 4.47 [m, 10 H, 4ϫCH2Ph,
H-C(1α), H-C(1β)], 3.67 [m, 2 H, H-C(2α), H-C(2β)], 2.09 (s, 3 H,
CH3), 2.07 (s, 3 H, CH3), 2.03 (s, 3 H, CH3), 2.02 (s, 3 H, CH3),
1.82–1.70 (m, 8 H), 1.53–1.37 [m, 4 H, 2ϫ2 H-C(3), 2ϫ2 H-C(4),
2ϫ2 H-C(5)] ppm. 13C NMR (75 MHz, CDCl3): δ = 169.8, 169.1,
168.8, 138.5, 138.4, 128.3, 127.7, 127.6, 127.5, 127.1 (Ph), 99.9 (C-
8), 95.3, 82.2, 81.1, 79.5, 78.9, 77.6, 77.4, 75.1, 74.7, 73.2, 70.7,
70.6, 66.4, 66.2, 60.3, 26.7, 25.4, 25.2, 25.1, 21.1, 20.9, 20.8, 20.5,
19.2, 19.0, 14.2 ppm. HRMS (ESI+): calcd. for C26H30O7Na [M +
Na]+ 477.1889; found 477.1882.
Luche Reduction to Enols 3 and 4: To a solution of enone 5 (7.79 g,
24.63 mmol) dissolved in anhydrous MeOH (250 mL) was added
CeCl3·7H2O (18.35 g, 49.26 mmol, 2 equiv.). The solution was co-
oled to 0 °C and NaBH4 (1.86 g, 49.26 mmol, 2 equiv.) was added
portionwise. The clear solution was stirred at 0 °C for 30 min, and
then quenched by the addition of water and neutralized with 4 
AcOH. The aqueous phase was extracted with CH2Cl2 (3ϫ), and
the organic phase was dried with MgSO4, filtered, and evaporated
to yield products 3 (2.21 g, 28.2%) and 4 (4.33 g, 55.2%).
6: To a solution of allyl alcohol 4 (4.33 g, 13.61 mmol) dissolved
in anhydrous MeOH (200 mL) was added Pd(OH)2/C (20 wt.-%,
866 mg) was added. The black solution was degassed with argon
and then flooded with H2 for 5 min. The solution was stirred at
room temperature under an atmosphere of H2 for 3 h. Palladium
was then filtered off over Celite, and the solvent was evaporated in
vacuo to yield product 6 (4.24 g, 97%) as a colorless oil. Rf (hexane/
1
EtOAc, 7:3) = 0.44. H NMR (300 MHz, CDCl3): δ = 7.35 (m, 5
9: Sugar 8 (3.98 g, 8.76 mmol) and thymine (previously dried under
high vacuum, 2.20 g, 17.52 mmol, 2 equiv.) were dissolved in anhy-
drous CH3CN (95 mL). N,O-Bis(trimethylsilyl)acetamide (BSA)
(10.7 mL, 43.8 mmol, 5 equiv.) was added dropwise, and the solu-
tion was stirred at 80 °C under an atmosphere of argon for 1 h.
the mixture was then cooled to 0 °C and TMS-triflate (2.1 mL,
11.6 mmol, 1.4 equiv.) was added. The clear solution was stirred
under an atmosphere of argon at 40 °C for 16 h. The solution was
then cooled to 0 °C and quenched by the addition of saturated
aqueous NaHCO3. The aqueous phase was extracted with CH2Cl2
(3ϫ). The combined organic phase was dried with MgSO4, filtered,
and concentrated. CC (EtOAc/hexane, 3:2) yielded nucleoside 9
(3.01 g, 66%, 10:1 β:α) as a white foam. Rf (EtOAc/hexane, 3:2) =
H, Ph), 5.70 [d, J = 3.60 Hz, 1 H, H-C(8)], 4.61 (d, J = 10.74 Hz,
1 H, CH2Ph), 4.54 (d, J = 10.92 Hz, 1 H, CH2Ph), 4.37 [d, J =
3.57 Hz, 1 H, H-C(7)], 4.29 [d, J = 3.21 Hz, 1 H, H-C(1)], 3.80 [br.
s, 1 H, H-C(2)], 1.95 (d, J = 14.31 Hz, 2 H), 1.82 (d, J = 6.96 Hz,
1 H), 1.62 (s, 3 H, CH3), 1.55 (m, 2 H), 1.37 (s, 3 H, CH3), 1.02
(m, 1 H) ppm. 13C NMR (75 MHz, CDCl3): δ = 138.5 (Ph1), 128.3
(Phortho), 127.6 (Phortho), 127.5 (Phortho), 113.1 [C(Me2)], 104.6
[C(8)], 83.5 [C(6)], 82.2 [C(7)], 78.6 [C(1)], 68.2 [C(2)], 66.4
(CH2Ph), 29.1, 26.1, 19.2 [C(3), C(4), C(5)], 26.7 (CH3), 26.6 (CH3)
ppm. HRMS (ESI+): calcd. for C18H24O5Na [M + Na]+ 343.1521;
found 343.1510.
7: NaH (55–65% in oil, 160.7 mg, 3.35 mmol, 2 equiv.) was dis-
solved in anhydrous DMF (6 mL) and cooled to 0 °C. Compound
6 (536.6 mg, 1.67 mmol) in anhydrous DMF (3 mL) was added
dropwise, and the suspension was stirred at 50 °C under an atmo-
sphere of argon for 1 h. The brown solution was then cooled to
0 °C and benzyl bromide (0.4 mL, 3.35 mmol, 2 equiv.) was added.
The mixture was stirred at room temperature under an atmosphere
of argon for 16 h. DMF was then removed by distillation under
reduced pressure, and the yellow residue was taken up in CH2Cl2.
The organic phase was washed with a solution of saturated aque-
ous NaHCO3 (2ϫ) and then dried with MgSO4, filtered and con-
1
0.64. H NMR (400 MHz, CDCl3): δ = 7.94 (br. s, 1 H, NH), 7.61
[d, J = 1.32 Hz, 1 H, H-C(6)], 7.35 (m, 10 H, Ph), 6.02 [d, J =
4.52 Hz, 1 H, H-C(1Ј)], 5.40 [d, J = 4.52 Hz, 1 H, H-C(2Ј)], 4.72
(d, J = 12.12 Hz, 1 H, CH2Ph), 4.64 (d, J = 11.96 Hz, 1 H, CH2Ph),
4.56 (d, J = 11.36 Hz, 1 H, CH2Ph), 4.48 (d, J = 11.48 Hz, 1 H,
CH2Ph), 4.29 [d, J = 3.80 Hz, 1 H, H-C(4Ј)], 3.86 [m, 1 H, H-
C(5Ј)], 2.06 (s, 3 H, OAc), 1.84 [m, 4 H, H-C(6Ј), H-C(8Ј)], 1.70 [d,
J = 1.12 Hz, 3 H, C(5)-CH3], 1.65 [m, 1 H, H-C(7Ј)], 0.88 [m, 1
H, H-C(7Ј)] ppm. 13C NMR (101 MHz, CDCl3): δ = 165.9, 143.1,
(thymine and acetoxy), 138.7 (Ph1), 138.5 (Ph1), 135.8 [C(6)], 128.6,
Eur. J. Org. Chem. 2009, 1153–1162
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1159

A. Stauffiger, C. J. Leumann
FULL PAPER
128.4, 128.1, 127.9, 127.7, 127.5, 126.9 (phenyl), 121.3, 110.7
[C(1Ј)], 103.2, 80.3, 76.3, 74.5, 70.7, 66.8, 66.3, 60.8, 27.5, 25.6,
21.4, 20.8, 12.5, 12.2 ppm. HRMS (ESI+): calcd. for
C29H32O7N2Na [M + Na]+ 543.2107; found 543.2116.
white solid and 11 (920.8 mg, 23.5%), and 10 (593.4 mg, 18.7%) as
white foams. Data for 12: Rf (EtOAc/hexane, 1:1) = 0.38. 1H NMR
(300 MHz, CDCl3): δ = 8.20 (br. s, 1 H, NH), 7.72 [d, J = 1.20 Hz,
1 H, H-C(6)], 7.35 (m, 10 H, Ph), 6.21 [dd, J = 7.60, 6.01 Hz, 1 H,
H-C(1Ј)], 4.59 (2ϫdd, 4 H, 2ϫCH2Ph), 4.12 [d, J = 4.44 Hz, 1 H,
H-C(4Ј)], 3.93 [m, 1 H, H-C(5Ј)], 2.70 [dd, J = 13.08, 5.92 Hz, 1 H,
H-C(2Јeq)], 2.14 [dd, J = 13.08, 7.60 Hz, 1 H, H-C(2Јax)], 1.95–1.31
[m, 6 H, H-C(6Ј), H-C(7Ј), H-C(8Ј)], 1.56 [d, J = 0.78 Hz, 3 H,
C(5)-CH3] ppm. 13C NMR (75 MHz, CDCl3): δ = 163.3, 151.1
(quart. thymine), 137.9 (Phquart), 136.2 [C(6)], 128.7–126.1 (phenyl),
110.5 [C(5)], 84.8 [C(1Ј)], 83.3, 75.0 [C(4Ј), C(5Ј)], 70.6 (CH2Ph),
64.8 (CH2Ph), 40.4 [C(2Ј)], 30.1, 25.2, 17.1 [C(6Ј), C(7Ј), C(8Ј)],
12.1 [C(5)-CH3] ppm. HRMS (ESI+): calcd. for C27H30N2O5Na
[M + Na]+ 485.2025; found 485.2059.
10: To a solution of nucleoside 9 (171.2 mg, 0.33 mmol) dissolved
in anhydrous MeOH (4 mL) was added sodium methoxide
(35.5 mg, 0.66 mmol, 2 equiv.). The clear solution was stirred under
an atmosphere of argon at room temperature for 16 h. After cool-
ing to 0 °C, the solution was quenched by the addition of 1  aque-
ous HCl and then extracted with CH2Cl2 (3ϫ). The organic phase
was washed with a solution of saturated aqueous NaHCO3 and
then dried with MgSO4, filtered, and concentrated. CC (EtOAc/
hexane,3:2) yielded compound 10 (128.4 mg, 0.27 mmol, 82%) as
a white foam. Rf (EtOAc/hexane, 3:2) = 0.42. 1H NMR (400 MHz,
CDCl3): δ = 8.45 (br. s, 1 H, NH), 7.68 [d, J = 1.24 Hz, 1 H, H-
C(6)], 7.34 (m, 10 H, Ph), 5.93 [d, J = 5.24 Hz, 1 H, H-C(1Ј)], 4.61
(m, 4 H, 2ϫCH2Ph), 4.33 [d, J = 4.40 Hz, 1 H, H-C(4Ј)], 4.24 [dd,
J = 7.36, 5.52 Hz, 1 H, H-C(2Ј)], 3.92 [ddd, J = 7.20, 4.30, 2.94 Hz,
1 H, H-C(5Ј)], 3.19 (d, J = 7.32 Hz, 1 H, OH), 1.95 [m, 2 H, H-
C(6Ј)], 1.77 [m, 2 H, H-C(8Ј)], 1.57 [d, J = 1.12 Hz, 3 H, C(5)-
CH3], 1.33 [d, J = 1.08 Hz, 1 H, H-C(7Ј)], 0.88 [m, 1 H, H-C(7Ј)]
ppm. 13C NMR (101 MHz, CDCl3): δ = 163.3, 151.1 (quart. thy-
mine), 137.9 (Phquart), 135.8 [C(6)], 128.7–126.1 (phenyl), 110.5
[C(5)], 90.7 [C(1Ј)], 82.1 [C(3Ј)], 79.1 [C(4Ј)], 77.0 [C(2Ј)], 74.7
[C(5Ј)], 70.1 (CH2Ph), 65.6 (CH2Ph), 26.8 [C(8Ј)], 25.7 [C(6Ј)], 16.9
[C(7Ј)], 12.0 [C(5)-CH3] ppm. 1H NMR difference-NOE (400 MHz,
CDCl3): δ (%) = 5.93 Ǟ 4.33 (2.2), 4.24 (2.1); 4.24 Ǟ 7.68 (6.7),
5.93 (3.0), 1.95 (1.1), 1.33 (4.0); 3.92 Ǟ 7.24 (3.1), 4.61 (6.6), 4.33
(9.3), 1.95 (1.6), 1.33 (1.1); 3.19 (OH) Ǟ 5.93 (9.1), 4.24 (5.6) ppm.
HRMS (ESI+): calcd. for C27H30N2O6Na [M + Na]+ 501.2001;
found 501.1992.
13: Compound 12 (85 mg, 0.18 mmol) was dissolved in anhydrous
MeOH (5 mL) and degassed with argon. Pd(OH)2/C (20%, 55 mg)
and cyclohexa-1,4-diene (0.17 mL, 1.84 mmol, 10 equiv.) were then
added. The black solution was once again degassed with argon and
then flushed with H2. The solution was stirred under an atmo-
sphere of H2 at room temperature for 6 h. Palladium residues were
filtered and washed with MeOH. The solvent was evaporated, and
the crude product was purified by CC (CH2Cl2/MeOH, 95:5) to
yield monomer 13 (28.3 mg, 55%) as a white solid. Rf (CH2Cl2/
1
MeOH, 95:5) = 0.15. H NMR (400 MHz, MeOD): δ = 8.20 [d, J
= 1.17 Hz, 1 H, H(6)], 6.22 [dd, J = 7.72, 6.24 Hz, 1 H, H-C(1Ј)],
4.03 [dtd, J = 4.37, 4.37, 6.30, 6.45 Hz, 1 H, H-C(5Ј)], 3.80 [d, J =
4.43 Hz, 1 H, H-C(4Ј)], 2.19 [ddd, J = 18.95, 12.73, 6.98 Hz, 2 H,
H-C(2Ј)], 1.91–1.86 (m, 1 H), 1.89 [d, J = 1.20 Hz, 3 H, C(5)-CH3],
1.78–1.49 [m, 5 H, H-C(6Ј), H-C(7Ј), H-C(8Ј)] ppm. 13C NMR
(101 MHz, MeOD): δ = 163.3, 151.1 (quart. thymine), 139.0 [C(6)],
110.5 [C(5)], 86.3 [C(4Ј)], 86.0 [C(1Ј)], 78.5 [C(3Ј)], 68.4 [C(5Ј)], 44.8
[C(2Ј)], 36.0, 30.8, 18.3 [C(6Ј), C(7Ј), C(8Ј)], 12.5 [C(5)-CH3] ppm.
1H NMR difference-NOE (400 MHz, MeOD): δ (%) = 8.20 Ǟ 6.22
(2.0), 4.03 (0.7), 3.80 (0.4), 2.21 (4.1), 1.89 (7.6); 6.22 Ǟ 8.20 (1.7),
3.80 (2.8), 2.16 (5.6); 4.03 Ǟ 8.20 (0.9), 3.80 (10.3), 1.60 (1.6); 3.80
Ǟ 6.22 (3.3), 4.03 (8.9); 2.21 Ǟ 8.20 (9.3), 6.22 (4.1), 2.16 (8.5);
2.16 Ǟ 6.22 (12.7), 3.80 (1.3), 2.21 (7.8) ppm. HRMS (ESI+):
calcd. for C13H18N2O5Na [M + Na]+ 305.1113; found 305.1121.
11: Nucleoside 10 (1.74 g, 3.63 mmol) was dissolved in anhydrous
DMF (35 mL) and treated with 1,1Ј-thiocarbonyldiimidazole
(971.5 mg, 5.45 mmol, 1.5 equiv.). The yellow solution was stirred
at room temperature under an atmosphere of argon for 6 h. DMF
was evaporated, and the yellow oil was taken up in water. Extrac-
tion with CH2Cl2 (3ϫ) was followed by drying over MgSO4 and
filtration. After concentration, CC (1:1 EtOAc/hexane) yielded
compound 11 (2.01 g, 94%) as a white foam. Rf (EtOAc/hexane,
1:1) = 0.11. 1H NMR (300 MHz, CDCl3): δ = 8.31 [s, 1 H, H-
C(imidazole)], 7.88 (br. s, 1 H, NH), 7.71 [d, J = 1.32 Hz, 1 H, H-
C(6)], 7.58 [t, J = 3.00, 1.68 Hz, 1 H, H-C(imidazole)], 7.37 (m, 10
H, Ph), 7.02 [dd, J = 1.71, 0.96 Hz, 1 H, H-C(imidazole)], 6.41 [s,
2 H, H-C(2Ј), H-C(1Ј)], 4.82 (d, J = 11.85 Hz, 1 H, CH2Ph), 4.62
(m, 3 H, CH2Ph), 4.33 [d, J = 4.35 Hz, 1 H, H-C(4Ј)], 4.03 [m, 1
H, H-C(5Ј)], 2.07–1.97 [m, 4 H, 2ϫCH2, H-C(6Ј), H-C(8Ј)], 1.62
[m, 2 H, CH2, H-C(7Ј)], 1.48 [d, J = 0.93 Hz, 3 H, C(5)-CH3] ppm.
13C NMR (101 MHz, CDCl3): δ = 222.0 (C=S), 154.8, 137.9
(Phquart), 137.4, 135.7, 128.7, 128.7, 128.5, 128.1, 127.8, 127.2,
126.8, 111.1, 82.3, 80.8, 74.6, 707, 66.3, 31.4, 27.3, 11.8 ppm.
HRMS (ESI+): calcd. for C31H32N4O6S [M]+ 589.6924; found
589.1404.
14: Nucleoside 13 (210 mg, 0.74 mmol) was coevaporated with an-
hydrous benzene (3 mL) and pyridine (3 mL). It was then taken up
again in anhydrous pyridine (3 mL) and (4,4Ј-dimethoxytriphenyl)
methyl triflate (DMTOTf, 510 mg, 1.13 mmol, 1.5 equiv.) was
added. The red-brown solution was stirred at room temperature
under an atmosphere of argon for 3 h and then another portion of
DMTOTf (170 mg, 0.38 mmol, 0.5 equiv.) was added. After 7 h, a
third portion of DMTOTf (170 mg, 0.38 mmol, 0.5 equiv.) was
added, and the solution was stirred for another 12 h. The reaction
was then quenched by the addition of a solution of saturated aque-
ous NaHCO3. The aqueous phase was extracted with CH2Cl2 (3ϫ),
and the organic phase was dried with MgSO4, filtered, and concen-
trated. CC (EtOAc/hexane, 9:1 + 1% Et3N) yielded nucleoside 14
(282.6 mg, 65%) as a yellow foam. Rf (EtOAc/hexane, 9:1) = 0.22.
1H NMR (400 MHz, CDCl3): δ = 8.09 (s, 1 H, NH), 7.57 [d, J =
1.22 Hz, 1 H, H-C(6)], 7.55 (m, 2 H, arom.), 7.46–7.43 (m, 4 H,
arom.), 7.32–7.23 (m, 3 H, arom.), 6.85 (s, 2 H, arom.), 6.83 (s, 2
H, arom.), 5.87 [dd, J = 7.81, 4.70 Hz, 1 H, H-C(1Ј)], 3.83 [m, 1
H, H-C(5Ј)], 3.81 (s, 6 H, PhMeO), 3.15 [d, J = 3.51 Hz, 1 H, H-
C(4Ј)], 2.31 [dd, J = 13.75, 7.86 Hz, 1 H, H-C(2Јα)], 1.96 [dd, J =
14.70, 9.98 Hz, 1 H, H-C(2Јβ)], 1.91 [d, J = 1.20 Hz, 3 H, C(5)-
CH3], 1.51–1.31 and 0.91–0.84 [m, 6 H, H-C(6Ј), H-C(7Ј), H-C(8Ј)]
ppm. 13C NMR (101 MHz, MeOD): δ = 163.2 (C=O), 158.7 (C-
O-CH3), 149.8, 145.7, 136.8 (Car), 135.2 [C(6)], 130.4, 129.3, 128.5,
128.3, 127.8, 126.9 (Car), 113.1 (Car), 110.2 [C(5)], 86.9 [C(3Ј)], 83.5
12: Thioester 11 (3.9 g, 6.63 mmol) was dissolved in toluene
(100 mL) and flooded with argon for 15 min. AIBN (544 mg,
3.31 mmol, 0.5 equiv.) was then added, and the solution was again
flushed with argon. Bu3SnH (3.50 mL, 13.25 mmol, 2 equiv.) was
then added dropwise, and the clear solution was stirred at 80 °C
under an atmosphere of argon for 4 h. Another portion of AIBN
(544 mg, 3.31 mmol, 0.5 equiv.) and of Bu3SnH (1.75 mL,
6.63 mmol, 1 equiv.) was added, and the solution was stirred for
another 12 h. Toluene was evaporated in vacuo followed by CC
(1:1 EtOAc/hexane) to give compounds 12 (1.14 g, 37.2%) as a
1160
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© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 1153–1162

Screening the Structural Space of Bicyclo-DNA
[C(1Ј)], 83.0 [C(4Ј)], 70.1 [C(5Ј)], 55.2 (O-CH3), 47.4 [C(2Ј)], 35.1 thane was used for detritylation. The concentrations for the natural
[C(8Ј)], 27.3 [C(7Ј)], 19.8 [C(6Ј)], 12.7 [C(5)-CH3] ppm. HRMS
(ESI+): calcd. for C43H36N2O7Na [M + Na]+ 607.2420; found
607.2439.
phosphoramidite solutions were 0.1  and for the modified phos-
phoramidites 0.15  or 0.2 . The coupling times for natural phos-
phoramidites were 1.5 min and for the modified phosphoramidite
12–14 min. The coupling efficiencies for 15 were generally low
(≈90%) as judged from the trityl assay. Deprotection and detach-
ment of the oligonucleotides were performed in concentrated NH3
(0.5 mL, 55 °C, 16 h). RNA oligonucleotides were instead treated
with concentrated NH3/EtOH (3:1, 0.5 mL, 55 °C, 30 h) then evap-
orated before TBAF (0.5 mL) was added (room temp., 24 h). After
evaporation, the brown deposit was taken up in H2O then filtered
through a SEP-PACK® C-18 cartridge (Waters). The crude samples
were purified by either RP-HPLC (Source 15 RPC ST 100/4.6 Poly-
styrene-15 column from Pharmacia Biotech or VA 15/4.6 Nucleogel
RP 300–5 column from Macherey–Nagel) or ion exchange–HPLC
(DNAPac-200 4ϫ250 mm column with precolumn both from Di-
onex). Samples from ion-exchange HPLC were desalted over SEP-
PACK® C-18 cartridge (Waters) according to the protocol of the
manufacturer. The following buffers were used for HPLC: RP-
HPLC: A: 0.1  triethylammonium acetate in H2O, pH 7.0; B:
0.1  triethylammonium acetate in H2O/CH3CN (1:4), pH 7.
DEAE–HPLC: A: 20 m KH2PO4 in H2O/CH3CN (4:1), pH 6.0;
B: 20 m KH2PO4, 1  KCl in H2O/CH3CN (4:1), pH 6.0. Linear
gradients of B in A were used. The integrity of all oligonucleotides
was confirmed with MS (ESI–; see Supporting Information). Con-
centrations of the oligonucleotide solutions were determined by
UV absorption at 260 nm.
15: DMT-protected nucleoside 14 (275.0 mg, 0.47 mmol) was dis-
solved in anhydrous benzene (3 mL) and the solvent was evapo-
rated. It was then taken up in anhydrous CH3CN (3 mL) and
Hünig’s base (0.40 mL, 2.36 mmol, 5 equiv.) and freshly distilled 2-
cyanoethoxydiisopropylaminochlorophosphane (CEPCl, 0.32 mL,
1.43 mmol, 3 equiv.) were added. The yellow solution was stirred
at room temperature under an atmosphere of argon for 2 h and
then diluted with EtOAc. The organic phase was washed with satu-
rated aqueous NaHCO3 (1ϫ) and the aqueous phase was extracted
with EtOAc (2ϫ). The combined organic phase was dried with
NaSO4, filtered, and concentrated. CC (EtOAc/hexane, 2:1 + 1%
Et3N) yielded phosphoramidite 15 (260 mg, 70%) as a white foam.
1
Rf (EtOAc/hexane, 4:1) = 0.47. H NMR (400 MHz, CDCl3): δ =
8.06 (br. s, 1 H, NH), 7.57 [dd, J = 2.21, 1.23 Hz, 1 H, H-C(6)],
7.53 (m, 3 H, arom. DMT), 7.44–7.40 (m, 4 H, arom. DMT), 7.30–
7.21 (m, 2 H, arom. DMT), 6.82 (s, 2 H, arom.), 6.82 (s, 2 H,
arom.), 5.94 [ddd, J = 18.91, 8.01, 4.32 Hz, 1 H, H-C(1Ј)], 3.83 [m,
1 H, H-C(5Ј)], 3.80 (s, 6 H, PhMeO), 3.68 [m, 1 H, H-C(4Ј)], 3.52
(m, 4 H, 2ϫ H-CiPr, OCH2), 2.73 [dd, J = 13.81, 8.24 Hz, 1 H, H-
C(2Јβ)], 2.53 (m, 2 H, CH2CN), 1.96 [m, 1 H, H-C(2Јα)], 1.91 [dd,
J = 7.41, 1.00 Hz, 3 H, C(5)-CH3], 1.43–1.20 and 0.88–0.84 [m, 6
H, H-C(6Ј), H-C(7Ј), H-C(8Ј)], 1.12 [dd, J = 6.77, 2.06 Hz, 6 H,
(CH3)2CH], 1.03 [dd, J = 12.06, 6.77, (CH Hz, 6 H3)2CH] ppm.
13C NMR (101 MHz, CDCl3): δ = 163.3 (C=O), 158.7 (C-O-CH3),
149.9, 145.8, 136.9, 136.8 (Car), 135.3, 135.2 [C(6)], 130.4, 130.3,
128.4, 127.7, 126.8 (Car), 117.7, 117.6 (CN), 113.0 (Car), 110.2
[C(5)], 86.8, 86.7 [C(3Ј)], 84.1, 83.9 [C(1Ј)], 83.3, 83.2 [C(4Ј)], 77.3,
77.3 [C(Ph3)], 70.2 [C(5Ј)], 57.7, 57.6, 57.5, 57.4 (OCH2), 55.2
(OCH3), 45.0, 44.9 [C(2Ј)], 43.3, 43.2, 43.1, 43.0 (CiPr), 34.7 [C(8Ј)],
27.4, 27.3 [C(7Ј)], 24.5, 24.4, 24.3, 24.2 (H3CiPr), 20.4, 20.3, 20.3,
20.2 (CH2-CN), 19.8, 19.7 [C(6Ј)], 12.7 [C(5)-CH3] ppm. 31P NMR
(161.9 MHz, CDCl3): δ = 142.90, 140.87 ppm. HRMS (ESI+):
calcd. for C43H53N4O8NaP [M + Na]+ 807.3498; found 807.3494.
Melting Curves: Thermal denaturation experiments were carried
out with a Varian Cary 3E UV/Vis spectrophotometer. Ab-
sorbances were monitored at 260 nm and the heating rate was set
to 0.5 °Cmin–1. A cooling–heating–cooling cycle in the temperature
range of 80–15 °C was applied. The first derivative of the melting
curves were calculated with the Varian WinUV software. To avoid
evaporation of solvents, a layer of dimethylpolysiloxane was added
over the samples within the cell. All measurements were carried out
in NaCl (150 m)/Na2HPO4 (10 m) at pH 7.0 at a total oligonu-
cleotide concentration of 2 µ.
Molecular Modeling: The conformational search was performed in
an MM+ force field with the conformational search software (Hy-
perChem Conformational Search, Version 8.0) as included in Hy-
perChemTM Professional 8.0.4. Torsion angles of the six-membered
ring as well as γ and δ were varied. The standard parameter set up
was used. The structures were energy minimized to a RMS gradient
of 0.01 kcalÅ–1 mol–1. Wrong chirality structures were discarded.
CCDC-705765 (for 12) contains the supplementary crystallo-
graphic data for this paper. These data can be obtained free of
charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.
Supporting Information (see footnote on the first page of this arti-
cle): Structure refinement details; UV melting curves; CD spectra.
Energy minimizations for duplexes, were carried out with the Am-
ber force field as incorporated in the software package Hyper-
ChemTM Professional 8.0.4. Explicit H2O molecules and counteri-
ons were not included. A distance-dependent permittivity of ε = 4r
was used instead as a screening function. The double helical struc-
ture was built on the basis of the parameters of a B-DNA. It was
minimized by using a Polak–Ribiere algorithm with an RMS gradi-
ent of 0.01 kcalÅ–1 mol–1.
Acknowledgments
We thank the BENEFRI small molecule crystallography service
(Prof. H. Stöckli-Evans) for solving the X-ray structure of 12. Fin-
ancial support from the Swiss National Science Foundation (grant
200020-115913) is gratefully acknowledged.
Oligonucleotide Synthesis and Purification: The chemical synthesis
of oligonucleotides was performed either on a 1.3 µmol scale with
a Pharmacia LKB Gene Assembler Special DNA-synthesizer or on
a 1 µmol scale with a Polygen DNA synthesizer by using standard
phosphoramidite chemistry. The phosphoramidite building blocks
of the natural nucleosides and the nucleosides bound to CPG-solid
support were purchased from Glen Research or Vivotide. Solvents
and reagents used for the synthesis were prepared according to the
indications of the manufacturer. 5-(Ethylthio)-1H-tetrazole (ETT)
was used as an activator and 3% dichloroacetic acid in dichloroe-
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Received: October 22, 2008
Published Online: December 18, 2008
1162
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