Synthesis and pairing properties of oligodeoxynucleotides containing bicyclo-RNA and bicyclo-ANA modifications

Article abstract of DOI:10.1021/jo300554w

The synthesis of the ribo(bc-rT)- and arabino(bc-araT)-version of bicyclothymidine (bc-dT) has been achieved. A conformational analysis by X-ray and/or ¹H NMR spectroscopy on the corresponding 3′,5′- benzyl-protected nucleosides featured a rigid C(2′)-endo conformation for the furanose ring, irrespective of the configuration of the OH group at C(2′). The conformation of the carbocyclic ring in these nucleosides was found to be less defined and thus more flexible. Both nucleosides were converted into the corresponding phosphoramidites and incorporated into oligodeoxynucleotides by standard DNA chemistry. T_m-data of duplexes with cDNA and RNA revealed that a bc-rT unit strongly destabilized duplexes with cDNA and RNA by 6-8 °C/mod, while bc-araT was almost T_m neutral. A rationale based on a previous structure of a bc-DNA mini duplex suggests that the strong destabilization caused by a bc-rT unit arises from unfavorable steric interactions of the equatorial 2′-OH group with the sugar residue of the 3′-neighboring nucleotide unit.

Full text of DOI:10.1021/jo300554w

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pubs.acs.org/joc
Synthesis and Pairing Properties of Oligodeoxynucleotides
Containing Bicyclo-RNA and Bicyclo-ANA Modifications
Arben I. Haziri and Christian J. Leumann*
Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern, Switzerland
S
* Supporting Information
ABSTRACT: The synthesis of the ribo(bc-rT)- and arabino(bc-
araT)-version of bicyclothymidine (bc-dT) has been achieved. A
1
conformational analysis by X-ray and/or H NMR spectroscopy
on the corresponding 3′,5′-benzyl-protected nucleosides featured
a rigid C(2′)-endo conformation for the furanose ring,
irrespective of the configuration of the OH group at C(2′).
The conformation of the carbocyclic ring in these nucleosides
was found to be less defined and thus more flexible. Both
nucleosides were converted into the corresponding phosphor-
amidites and incorporated into oligodeoxynucleotides by stand-
ard DNA chemistry. Tm-data of duplexes with cDNA and RNA revealed that a bc-rT unit strongly destabilized duplexes with
cDNA and RNA by 6−8 °C/mod, while bc-araT was almost Tm neutral. A rationale based on a previous structure of a bc-DNA
mini duplex suggests that the strong destabilization caused by a bc-rT unit arises from unfavorable steric interactions of the
equatorial 2′-OH group with the sugar residue of the 3′-neighboring nucleotide unit.
modulator in cellular assays,23 or as a dowregulator for
INTRODUCTION
■
scavenger receptor B1 mRNA in vivo in a gapmer format.24
In order to address yet unsolved problems in oligonucleotide-
based therapeutics, such as improving cellular uptake, we
recently set out to extend the bc-DNA platform primarily by
substituting position C-6′ with varying functional elements.25,26
As part of this endeavor, we became interested in the ribo- (bc-
RNA) and arabino- (bc-ANA) versions of bc-DNA, the
rationale being to identify the influence of the 2′-OH group
on nucleoside conformation and on nucleic acid affinity (Figure
1). While there exists a previous synthesis of an unprotected bc-
The search for oligonucleotide-based therapeutics is an active
and innovative field that has greatly evolved over the recent
three decades.1 In the early 1990s, the antisense approach was
at the center of attention, in which a mRNA is targeted with an
oligonucleotide to repress translation by either a steric block
mechanism or an RNaseH-mediated ablation of the mRNA.
More recent years have focused on the exploitation of further
oligonucleotide-mediated RNA-silencing mechanisms such as
RNA interference induced by small RNA duplexes (siRNAs)2−5
or by targeting noncoding RNAs that are important in
translational regulation, such as micro-RNAs, with single-
stranded oligonucleotides (antimirs, antagomirs).6−9
Depending on the biological mechanism of action, it is
believed that chemically modified oligonucleotides can outper-
form natural oligonucleotides because of increased biostability
and increased target-RNA affinity via Watson−Crick pairing. In
this context, numerous oligonucleotide analogues have been
evaluated in the past, the biologically most successful ones
being those where the ribonucleoside structure has been 2′-O-
modified, such as 2′-MOE-RNA,10 2′F-RNA,11 or 2′F-ANA,12,13
those with modification in both the internucleosidic linkage and
the nucleoside structure, such as morpholino-NAs,14 or PNA,15
or those with conformationally constrained nucleosides such as
the LNA16,17 or the HNA family.18,19
Our contribution to the field of conformationally constrained
oligonucleotide analogues20 in the past has been the develop-
ment of the bicyclo(bc) and tricyclo(tc)-DNA molecular
platforms.21 In particular, tricyclo(tc)-DNA has shown very
promising antisense results either as steric block inhibitor of
HIV type 1 tat-dependent trans-activation,22 as a splice
Figure 1. Chemical structures of the ribo- and arabino-version of bc-
DNA.
rT nucleoside,27,28 there is no data available on the synthesis of
the bc-araT nucleosides or on oligonucleotides containing both
nucleosides. Here we report on the synthesis of bicyclo
ribothymidine (bc-rT) and bicyclo arabinothymidine (bc-araT),
their structural and conformational preferences as determined
by X-ray and/or 1H NMR, their incorporation into
Received: March 16, 2012
Published: April 9, 2012
© 2012 American Chemical Society
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oligodeoxynucleotides, and finally on their pairing properties
with cDNA and RNA.
conditions).30,31 As expected, only the β-anomers could be
isolated. The β-configuration at C(1′) in both nucleosides 3 and
4 was confirmed by 1H NMR difference NOE experiments (see
the Experimental Section). The 2′-OAc groups in 3 and 4 were
cleaved with NaOMe in the case of 3 and with 0.2 M NaOH in
a mixture of THF/MeOH/H2O in the case of the more delicate
4 to yield nucleosides 5 and 6 that were ready for the
installation of a 2′-O-protecting group suitable for oligonucleo-
tide synthesis. In preliminary experiments, we protected the 2′-
OH with the triisopropylsilyloxy methyl (TOM) group.32 We
later found that this group was not stable during hydrogenolysis
of the benzyl groups. We therefore switched to the alternative
TBS group that was introduced using standard silylation
conditions leading to 7 and 8 in excellent yields. Deprotection
of the benzyl groups in 7 and 8, however, proved to be rather
tricky. Under standard conditions of hydrogenolysis (H2, Pd/
C) the reproducibility was limited, and partial or even complete
decomposition was observed. Hydrogenation of the bases was
another side reaction. After screening various palladium
catalysts and different hydrogen sources and solvents, we
found that H2 and 1,3-cyclohexadiene as an additive with
Pd(OH)2/C as catalyst in EtOAc worked best and gave the 2′-
protected bc-rT nucleoside 9 reproducibly in yields >90%.
Unfortunately, these and related conditions were unsuccessful
for the deprotection of N4-benzoyl bicycloribocytidine 8
without concomitant reduction of the base or loss of the
benzoyl protecting group.
RESULTS AND DISCUSSION
■
Synthesis of Nucleoside Building Blocks. The synthesis
of the bicycloribo- and arabinonucleosides started with the
sugar derivative 1, which was prepared as reported previously
(Scheme 1).29 Acetonide 1was hydrolyzed in boiling acetic acid
a
Scheme 1
At this point, we decided not to optimize the conditions for
benzyl deprotection of 8 but to continue with the synthesis of
the phosphoramidite of bc-rT. Thus, selective tritylation of the
5′OH group with DMTr-Cl in 9 (→10), followed by standard
phosphitylation with (2-cyanoethoxy)(diisopropylamino)-
chlorophosphine, yielded the desired building block 11 in
good yields.
The synthesis of the bicyclo arabinothymidine building block
18 was effected by inverting the configuration at C(2′) in
nucleoside 5 using the “anhydro approach” (Scheme 2).33 In
initial experiments, 4 was treated with diphenyl carbonate to
give the anhydro nucleoside 13 that was isolated and
subsequently reacted with KOH to give the C(2′)-inverted
arabino nucleoside 14. However, the yields were low (20−30%
for each step) and called for improvement of the procedure by
converting the 2′-OH into a better leaving group. Therefore, 4
a
Reagents and conditions: (a) 80% aq AcOH, 90 °C, 16 h; (b) Ac2O,
pyridine, rt, 16 h, 74%; (c) thymine (2 equiv), BSA (5.3 equiv), TMS-
OTf (2 equiv), MeCN, 50 °C, 16 h, 73%; (d) N4Bz-Cytosine (2
equiv), BSA (5 equiv), SnCl4 (3 equiv), MeCN, rt, 12 h, 98%; (e)
NaOMe (2 equiv), MeOH, rt, 4 h, 91%; (f) 0.2 M NaOH/THF/
MeOH, H2O, 0 °C, 45 min, 48%; (g) TBS-Cl (1.3 equiv), imidazole
(1.5 equiv), DMF, 24 h, 7 (66%), 8 (71%); (h) 1,3-cyclohexadiene (10
equiv), H2, Pd(OH)2/C, EtOAc, 6 h, 97%; (i) DMTr-Cl (6 equiv),
pyridine, rt, 16 h, 90%; (j) (iPr)2NP(Cl)OCH2CH2CN (4 equiv),
EtN(iPr2)2 (6 equiv), MeCN, rt, 4 h, 76%.
followed by acetylation to give 2 that was subsequently
converted into nucleosides 3 and 4 in excellent yield with in
situ persilylated thymine or N-4-benzoylated cytosine and
TMSOTf or SnCl4 as Lewis acid promoter (Vorbruggen
̈
a
Scheme 2
a
Reagents and conditions: (a) Ms-Cl (3 equiv), pyridine, rt, 1 h, 89%; (b) 1 M NaOH (4 equiv), EtOH, H2O, reflux, 16 h, 85%; (c) TBS-OTf (1.5
equiv), imidazole (2.5 equiv), DMF, rt, 20 h, 71%; (d) 1,3-cyclohexadiene (10 equiv), H2, Pd(OH)2/C, EtOAc, 2 h, 58%; (e) DMTr-Cl (3 equiv),
pyridine, rt, 5 h, 90%; (f) (iPr)2NP(Cl)OCH2CH2CN (3 equiv), EtN(iPr2)2 (5 equiv), MeCN, rt, 1 h, 97%.
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Figure 2. ORTEP (50% probability ellipsoids) representation of the two symmetry-independent molecules 5A (left) and 5B (right). H-atoms are
given arbitrary displacement parameters for clarity.
was converted to the mesylate 12 that was subsequently treated
with 1 M aq NaOH to give nucleoside 14 (via the anhydro
intermediate 13) in 85% yield. The arabino configuration in 14
conformation with a P of 128°. This is somewhat unusual, as
ribonucleosides typically prefer a 3′-endo (N) conformation.
The orientation of the base (torsion angle χ) is, as expected,
anti. Unfortunately, we were not able to get suitable crystals for
X-ray analysis of 14 in order to compare the solid-state
structures of the two diastereoisomers.
1
was verified by H NMR NOE experiments. As before, the 2′-
OH group was TBS-protected to give 15. Debenzylation as
described before gave 16 in 58% that was subsequently
tritylated (→ 17) and phosphitylated (→ 18) using standard
conditions.
Conformation of 5, 6 and 14 in Solution. Analysis of the
1
vicinal coupling constants in the H NMR spectra of the two
ribo-configured nucleosides 5 and 6 and the arabino-configured
14 using the optimized Karplus relation for nucleosides36 shed
light on the conformation of the C(1′)−C(2′) and the C(5′)−
C(6′) bonds (Table 2). A complete analysis of the sugar pucker
Conformational Properties of Nucleosides. X-ray
Structure of 5. Crystals of benzyl-protected nucleoside 5
were subjected to X-ray analysis to map the conformational
preferences of the bicyclic core structure. Two symmetry-
independent molecules (5A and B) were found in the unit cell,
which essentially differ only in the conformation of the
carbocyclic ring. In molecule 5A (Figure 2, left), the 5′-OBn
group is in a pseudoaxial arrangement while in molecule 5B
(Figure 2, right) it is pseudoequatorially oriented. Thus, the
torsion angle γ for 5A is in a gauche (+sc) orientation as
observed in A- and B-DNA, whereas for 5B it is in an anticlinal
(+ac) orientation, as observed for other bicyclonucleosides.34,35
In the unit cell the 5′-OBn group of molecule 5A almost
perfectly stacks onto the nucleobase of molecule 5B, suggesting
that the orientation of O-5′ in 5A may be biased by stacking
interactions. Most interestingly, in both molecules 5A and 5B,
the ribofuranose conformation is almost perfectly 2′-endo
(South) giving rise to pseudorotation phase angles P of 165°
for 5A and 162° for 5B (Table 1). Thus, the furanose
conformation of bc-rT belongs to the same class as that of the
2′-deoxygenated derivative bc-dT, which shows a 1′-exo
Table 2. Selected Vicinal Coupling Constants and Calculated
Torsion Angles from 1H NMR spectra (400 MHz) in CDCl3
calcd
dihedral
angles
(deg)
derived
nucleoside
torsion angles
(deg)
coupling
compd constants (Hz)
3J
% S
conformation
5
3JH1′H2′
3JH5′H6′
3JH5′H6″
3JH1′H2′
3JH5′H6′
3JH5′H6″
3JH1′H2′
3JH5′H6′
8.1
5.1
4.9
6.6
5.1
5.5
2.4
8.9
145
35
ν1 = 35
80
γ = gauche
37
6
137
35
ν1 = 43
65
γ = gauche
33
14
53
ν1 = 37
149
γ = anticlinal
of the bicyclo[3.3.0] skeleton was unfortunately not possible
due to the coupling barrier at C(3′) and to substantial signal
overlap of C(6′) and C(7′) in the carbocyclic subunit. However,
an estimate for the population of the S-conformation in the
ribo-configured nucleosides (Table 2) can be obtained from the
following relationship
Table 1. Pseudorotational Phase Angles P and Selected
Torsion Angles of bc-rT in Comparison to bc-dT34
nucleoside
furanose pucker P (deg)
δ (deg)
γ (deg)
χ (deg)
5A
2′- endo (2E)
2′- endo (2E)
1′- exo (1E)
165
162
128
150
150
126
75
126
149
−131
−145
−113
5B
%S = 3JH1′H2′ × 100/10.1
bc-dT
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assuming that the N- and S-conformation are the only
populated conformations.37
nucleosides as determined by trityl assay. Crude oligonucleo-
tides were deprotected and detached from the solid support by
standard ammonia treatment followed by TBS removal with
TBAF. All oligonucleotides were purified by HPLC, and their
structural compositions were verified by ESI-MS (Supporting
Information).
Tm Measurements. The UV melting curve analysis was
performed at 260 nm with a cooling−heating−cooling cycle at
a rate of 0.5 °C min−1 in standard saline buffer at pH 7.0. All
curves within a cycle were superimposable, thus ruling out
nonequilibrium association states. The measured Tm data are
summarized in Table 3.
The ν1values (O(4′)−C(1′)−C(2′)−C(3′)) calculated from
the 3JH1′,H2′ data are in all three cases 5, 6, and 14 in agreement
with the furanose unit adopting a C(2′)-endo conformation in
solution (CDCl3), indicating no large deviations from the solid
state structure in this part of the molecule. The calculated
population of the S conformation for the ribo-configured
nucleosides 5 and 6 are 80% and 65%, respectively.
Within the carbocyclic ring we assume envelope conforma-
tions with two major conformers displaying the O(5′)
substituent either in a pseudoaxial or pseudoequatorial position
(Figure 3). In the former case, H-C(5′) is gauche to both H-
As seen here,there are considerable differences in nucleic acid
affinities between the two 2′-epimeric bicyclonucleosides.
Compared to a natural dT unit, the bc-araT modification
shows only a modest destabilization by 1−2 °C per
modification against both DNA and RNA as complements in
a slightly sequence-dependent context. The bc-rT modification
in contrast leads to a dramatic depression of Tm by 6−8 °C per
modification against DNA and 4−7 °C against RNA. No 2′-OH
group, as in the case of bc-dT, leads to subtle increases in Tm
against DNA and modest decreases in Tm against RNA. As
observed on other occasions, contiguous modifications lead to
less Tm depression as noncontiguous modifications.
Some aspects of the presented results deserve further
reflection. An important one concerns the conformation of
the nucleosides as revealed by X-ray and/or NMR. Both the
solid- and liquid-state structures of the two bc-RNA (5, 6)and
the one bc-ANA (14) nucleosides show a 2′-endo furanose
conformation which is very similar to the 1′-exo conformation
of bc-dT. These results confirm previous findings on the
debenzylated derivative of 5, where based on NMR analysis also
a rigid 2′-endo conformation had been proposed.27 Taken
together, this strongly suggests that the specific structural
features of the bicyclo[3.3.0] skeleton drives the furanose unit
into a rigid south (S) type conformation, irrespective of the
presence or the configuration of the OH group at C(2′). It thus
overrules classical stereoelectronic factors (gauche effect) that
are believed to be responsible for driving ribonucleosides
preferentially into the 3′-endo conformation.39 The fact that
ribonucleosides prefer the 3′-endo conformation while that for
arabinonucleosides is a more evenly distributed between S- and
N-type is well documented on many examples.40,41 While there
seems to be a rigid furanose ring conformation, that of the
carbocyclic ring in general seems to be structurally less defined
in the bicyclonucleosides.
Figure 3. Two possible carbocyclic conformations with O(5′) in axial
(left) and equatorial (right) arrangements.
C(6′). Hence, the coupling constants in such cases are expected
to be between 3 and 4 Hz. If, however, O(5′) arranges
pseudoequatorially, a trans-diaxial relationship with a large
coupling constant (∼10 Hz) between H-C(5′) and the β-H-
C(6′) should be expected.
3
3
The coupling constants JH5′,H6′ and JH5′,H6″, both around 5
Hz for 5 and 6, are in agreement with a preference of the O(5′)
for a pseudoaxial arrangement (γ = gauche). In the case of the
arabinonucleoside 14 the situation is less clear since only one of
3
the two JH5′H6′ coupling constants could be resolved. Its
magnitude around 9 Hz might be an indication for a higher
preference of the pseudoequatorial (γ = anticlinal) arrangement
of O(5′).
Oligonucleotide Synthesis. A series of oligonucleotide
dodecamers containing single or double bc-rT or bc-araT
substitutions (Table 3) were synthesized on a 1.3 μmol scale by
standard phosphoramidite chemistry. For incorporation of the
modified building blocks, the coupling step was extended to 12
min and the phosphoramidite concentration was increased to
0.2 M. Coupling efficiencies for building blocks 11 and 18 were
generally lower (∼90%) compared to that of unmodified
Table 3. Tm Data from UV-Melting Curves (260 nm)
(Concentration (Total Single Strand) = 2 μM in 10 mM
NaH2PO4, 150 mM NaCl, pH 7.0)
Perhaps the most striking result of this work is the strong
destabilizing effect on duplex formation of a bc-RNA residue
which is essentially absent in the case of a bc-ANA residue. This
is in contrast to the natural system where RNA forms more
stable duplexes with complementary RNA and DNA as
compared to ANA, a fact that has been explained with steric
DNA
complement
RNA
complement
a
a
oligonucleotides
t =
5′-GGATGTTCTCGA-3′
47.5
49.5
5′-GGATGTTCtCGA-3′
bc-rT
39.3 (−8.1)
45.9 (−1.5)
49.0 (+1.5)
34.6 (−6.4)
42.6 (−2.4)
48.7 (+0.6)
34.0 (−6.7)
42.4 (−2.5)
47.9 (+0.2)
43.0 (−6.7)
48.4 (−1.2)
49.0 (−0.5)
41.0 (−4.3)
46.4 (−1.6)
48.2 (−0.6)
39.3 (−5.1)
44.6 (−2.5)
48.0 (−0.7)
obstruction of base-stacking caused by the β-OH group.42
A
bc-araT
b
possible structural rationale for this behavior may lie in the
specific backbone conformation induced by the bicyclo scaffold.
The only available high-resolution structure of bc-DNA is that
of a bc-C dimer which was shown to form a parallel stranded
C−C+ minihelix.35 Surmising that the backbone structure
would be similar in a classical antiparallel Watson−Crick
duplex, modeling of a 2′-OH group into this structure in the
ribo-configuration (Figure 4, left) clearly shows conflicting
steric interactions between this hydroxyl group and either
O(4′), O(5′), or C(6) of the neighboring 3′-nucleotide. We
bc-dT
5′-GGATGttCTCGA-3′
5′-GGAtGTTCtCGA-3′
bc-rT
bc-araT
b
bc-dT
bc-rT
bc-araT
b
bc-dT
a
b
ΔTm per modification relative to dT in parentheses. Data taken
from ref 38.
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EXPERIMENTAL SECTION
■
General Methods. All reactions were performed under an
atmosphere of argon in oven-dried glassware. Anhydrous solvents
for reactions were obtained by filtration through activated alumina or
by storage over molecular sieves (4 Å). Column chromatography
(CC) was performed on silica gel 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. Visualization was achieved either under UV light or by staining
in dip solutions (vanilline (15 g), ethanol (250 mL), concd H2SO4
(2.5 mL), p-anisaldehyde (10 mL), concentrated H2SO4 (10 mL),
concentrated acetic acid (2 mL), or ethanol (180 mL)) followed by
heating with a heat gun. NMR spectra were recorded at 300 or 400
MHz (1H), at 75 MHz (13C), and at 162 MHz (31P). 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 on DEPT or
Figure 4. (Left) Structure of a bc-dinucleotide unit into which a 2′-OH
group was modeled. The structure is based on an X-ray structure of
the bc-C2 dimer.35 (Right) Exerpt of the X-ray structure of a standard
RNA duplex (PDB ID: 3R1C).
1
13
1
1
APT experiments, and on H, H- and H, C-correlation experiments
(COSY/HMSC). 1H NMR difference-NOE experiments were
recorded at 400 MHz. Chemical shifts for 31P NMR are reported
relative to 85% H3PO4 as external standard. Electrospray ionization in
the positive mode (ESI+) was used for high-resolution mass detection.
(2RS,3R,3aR,6R,6aR)-3a,6-Bis(benzyloxy)-2-methoxyhexahy-
dro-2H-cyclopenta[b]furan-3-yl Acetate (2). Compound 1 (1.0 g,
2.51 mmol) was dissolved in 80% aqueous AcOH (40 mL) and stirred
at 90 °C for 16 h. AcOH was removed by coevaporation with EtOH (3
× 10 mL), toluene (3 × 10 mL), and anhydrous pyridine (1 × 10 mL).
The residue was dissolved in anhydrous pyridine (10 mL), and Ac2O
(10 mL) was added dropwise. After the mixture was stirred at rt for 16
h, the reaction was quenched by the addition of water (20 mL) at 0
°C. The aqueous solution was extracted with CH2Cl2 (3 × 30 mL).
The combined organic phases were washed with satd aq NaHCO3 (2
× 50 mL), dried over MgSO4, filtered, and evaporated. CC (hexane/
EtOAc 2:1) yielded compound 2 (0.820 g, 74%) as a 3:1 mixture of α-
and β-anomers in the form of a colorless oil. TLC (hexane/EtOAc
2:1): Rf 0.31 (β-anomer), 0.25 (α-anomer). 1H NMR (300 MHz,
CDCl3), (data of β-anomer): δ 2.00−2.30 (m, 4H, 2H-4, 2H-5), 2.06,
2.15 (2s, 6H, 2CH3), 3.94−4.02 (m, 1H, H-6), 4.28 (d, J = 11.5 Hz,
1H, 1CH2Ph), 4.64 (d, J = 4.0 Hz, 1H, H-6), 4.68 (d, J = 11.8 Hz, 1H,
CH2Ph), 5.50 (d, J = 11.5 Hz, 1H, 1 CH2Ph), 5.51 (d, J = 11.8 Hz, 1H,
1 CH2Ph), 5.28 (d, J = 0.75 Hz, 1H, H-3), 6.25 (s, 1H, H-2), 7.28−
7.33 (m, 10H, Ph). 1H NMR (300 MHz, CDCl3); data of α-anomer: δ
1.85, 2.15 (2s, 6H, 2 CH3), 1.75−2.00 (m, 4H, 2H-4, 2H-5), 3.90−
4.05 (m, 1H, H-6), 4.13 (dd, J = 7.1 Hz, J = 14.3 Hz, 1H, H-6), 4.52−
4.70 (m, 4H, 2CH2Ph), 5.6 (d, J = 4.5 Hz, 1H, H-3), 6.52 (d, J = 4.5
Hz, 1H, H-2), 7.28−7.33 (m, 10H, Ph). 13C NMR (75 MHz, CDCl3,
anomeric mixture): δ 21.1, 21.4, 21.7, 21.8, 28.9, 29.1, 30.6, 32.4, 68.0,
68.2, 72.6, 78.8, 79.1, 79.7, 79.8, 85.4, 88.0, 88.1, 90.1, 96.4, 101.7,
127.2, 127.6, 128.1, 128.3, 128.5, 128.5, 128.5, 129.0, 129.05, 129.11,
138.58, 138.62, 138.9, 139.5, 169.8, 170.0, 170.1. HRMS (ESI): m/z
[M + Na]+ calcd for C25H28O7Na 463.1733, found 463.1728.
(3′S,5′R)-1-(2′-Acetoxy-3′,5′-O-dibenzyl-3′,5′-ethano-β-D-
ribofuranosyl)thymidine (3). Compound 2 (0.80 g, 1.8 mmol) and
dry thymine (0.45 g, 3.6 mmol) were suspended in anhydrous CH3CN
(35 mL). N,O-Bis(trimethylsilyl)acetamide (BSA) (1.9 g, 9.6 mmol)
was added dropwise, and the solution was stirred at 80 °C for 1 h. The
mixture was then cooled to 0 °C, and TMS-triflate (0.8 g, 3.6 mmol)
was added. The clear solution was stirred at 50 °C for 16 h. The
solution was then cooled to 0 °C and quenched by the addition of sat
aq NaHCO3 (30 mL). The aqueous phase was extracted with CH2Cl2
(3 × 50 mL). The combined organic phases were dried with MgSO4,
filtered, and evaporated. CC (EtOAc/hexane 1:1) yielded nucleoside 3
(0.68 g, 73%, β anomer only) as a colorless foam. TLC (hexane/
EtOAc 1:1): Rf 0.23. 1H NMR (300 MHz, CDCl3): δ 1.51 (d, J = 1.2
Hz, 3H, (CH3)C-5, 2.07 (s, 3H, CH3), 2.00−2.34 (m, 4H, 2H-6′, 2H-
7′), 4.00−4.06 (m, 1H, H-5′), 4.57−4.73 (m, 5H, H-4′, 2CH2Ph), 5.15
(d, J = 8.1 Hz, 1H, H-2′), 6.45 (d, J = 8.1 Hz, 1H, H-1′), 7.28−7.39 (m,
10H, Ph), 7.54 (d, J = 1.3 Hz, 1H, H-6), 8.07 (s, 1H, NH). 1H NMR-
difference NOE (400 MHz, CDCl3): 6.45 (H-1′) → 5.15 (H-2′, 2.1%),
4.63 (H-4′, 3.7%); 5.15 (H-2) → 7.54 (H-6, 12.3%), 6.45 (H-1′, 3.6%);
therefore postulate that internucleotide steric repulsion of this
equatorial hydroxy group is responsible for the strong
destabilization. Such interactions are expected to be absent in
the case of bc-DNA or bc-ANA residues which is well reflected
in the corresponding Tm data. Interestingly, from a X-ray
structure of a standard RNA-duplex in A-conformation (Figure
4, right, PDB ID: 3R1C) it appears that the axial 2′-OH group
shows a distance of ∼3.4 Å to O(4′) of the adjacent 3′-
nucleotide. A hypothetic flipping of the 5′-ribonucleoside into a
2′-endo conformation would lead to steric conflicts between
these two centers also in this case, suggesting that an equatorial
2′-OH substituent would negatively influence the pairing
conformation also in a pure RNA backbone.
By comparing the pairing properties of ANA/RNA with
DNA/RNA duplexes in a mixed sequence context it becomes
evident that ANA/RNA duplexes are typically weaker by ca. 2
°C/mod.42 These data compare well with the differential
stability of bc-ANA/RNA and bc-DNA/RNA duplexes where
we also find bc-ANA units to decrease duplex stability in the
same range, suggesting that the influence of the β-2′-OH group
on hybrid duplex stability (particularly on base-stacking) are
similar in both backbone systems.
CONCLUSIONS
■
We present here the synthesis of two novel members of the
bicyclo[3.3.0] family, namely the ribo and arabino versions of
bicyclothymidine. Incorporation into oligodeoxynucleotides
followed by thermal melting experiments revealed a strong
destabilizing effect of a bicyclo-RNA unit and an almost Tm-
neutral effect of a bicyclo-ANA unit in base-pairing with DNA
and RNA complements. The destabilizing effect of a bc-RNA
unit was rationalized by its preferred 2′-endo furanose
conformation which places the 2′-OH group into an equatorial
position, causing intrastrand steric conflicts with the 3′-
neighboring nucleoside unit. Together with the structural
analysis of the monomeric nucleosides this highlights the
propensity of the bicyclo[3.3.0] scaffold to maintain the south
conformation of the furanose unit irrespective of the
substitution pattern at C(2′).
An interesting extension of this work is to study the effect of
bicyclo-F-RNA or -F-ANA nucleosides in duplex formation.
Recent work on F-RNA43 and F-ANA13,44 as well as on F-
HNA45 has shown a dramatic stabilization exerted by the
fluorine substituent, most likely due to electrostatic effects on
base-pairing. Work into this direction is currently in progress.
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4.63 (H-4′) → 6.45 (H-1′, 2.8%), 4.06 (H-5′, 5.4%); 4.06 (H-5′) →
4.63 (H-4′, 15.6%). 13C NMR (75 MHz, CDCl3): δ 12.0, 20.7, 29.9,
30.2, 67.5, 71.5, 76.6, 78.1, 79.3, 86.7, 87.1, 88.8, 111.7, 126.8, 127.7,
128.1, 128.5, 128.7, 135.3, 137.6, 138.3, 150.4, 163.2, 170.5. HRMS
(ESI): m/z calcd for C28H30O7N2Na (M + Na)+ 529.1951, found
529.1948.
(3′S,5′R)-1-(2′-O-tert-Butydimethylsilyl-3′,5′-O-dibenzyl-
3′,5′-ethano-β-d-ribofuranosyl)thymidine (7). To a solution of 5
(200 mg, 0.43 mmol) in DMF (1 mL) were added imidazole (43 mg,
0.64 mmol) and TBS-Cl (84 mg, 0.56 mmol) at 0 °C. After being
stirred at rt for 24 h, the mixture was diluted with CHCl3 (25 mL) and
washed with water (3 × 25 mL) and brine (25 mL). The combined
organic phases were dried over MgSO4 and evaporated, and the
residue was purified by CC (hexane/EtOAc 2:3 + 1% Et3N) to give 7
(0.166 g, 66%) as a yellow oil. TLC (hexane/EtOAc 2:3): Rf 0.66. 1H
NMR (300 MHz, CDCl3): δ −0.08 (s, 3H, Si-CH3), 0.09 (s, 3H, Si-
(3′S,5′R)-N4-Benzoyl-1-(2′-acetoxy-3′,5′-O-dibenzyl-3′,5′-
ethano-β-D-ribofuranosyl)cytidine (4). Dry N4-Bz-cytosine (0.19
g, 0.9 mmol) and acetylated sugar 2 (0.2 g, 0.45 mmol) were
suspended in anhydrous CH3CN (6 mL) and treated with BSA (0.45
g, 2.2 mmol). The suspension was stirred for 1 h at rt. Then, SnCl4
(0.23 g, 0.9 mmol) was added dropwise, and the mixture was stirred
for another 3 h at rt. Another 1 equiv of SnCl4 (0.11 g, 0.45 mmol)
was added and the mixture stirred overnight. The reaction was
quenched with satd aq NaHCO3 (10 mL) and the mixture extracted
with CH2Cl2 (3 × 20 mL). The organic phases were washed with satd
aq NaHCO3 (3 × 15 mL) and brine (1 × 15 mL). The aqueous phase
was extracted with CH2Cl2 (2 × 50 mL). The combined organic
phases were dried over MgSO4, evaporated, and purified by CC
(hexane/EtOAc 1:2) to give 4 (0.267 g, 98%) as a yellow foam. TLC
t
CH3), 0.85 (s, 9H, Bu), 1.43 (s, 3H, CH3(C-5)), 2.00−2.16 (m, 4H,
2H-6′, 2H-7′), 4.01−4.03 (m, 1H, H-5′), 4.13 (d, J = 8.1 Hz, 1H, H-2′),
4.53 (d, J = 5.3, 1H, H-4′), 4.55−4.97 (m, 4H, CH2Ph), 6.38 (d, J =
8.3 Hz, 1H, H-1′), 7.28−7.40 (m, 10H, Ph), 7.52 (d, J = 1.3 Hz, 1H,
H-6), 8.00 (s, 1H, NH). 13C NMR (75 MHz, CDCl3): δ −4.8, −4.0,
12.0, 14.5, 18.1, 21.3, 25.8, 25.9, 30.0, 30.1, 30.4, 60.6, 68.6, 71.6, 78.5,
81.2, 87.4, 88.7, 88.8, 116.8, 127.2, 127.7, 127.9, 128.3, 128.6, 128.9,
136.0, 138.1, 139.4, 150.6, 163.5. HRMS (ESI): m/z [M + H]+ calcd
for C32H43O6N2Si 579.2890, found 579.2868.
(3′S,5′R)-N4-Benzoyl-1-(2′-O-tert-butydimethylsilyl-3′,5′-O-
dibenzyl-3′,5′-ethano-β-D-ribofuranosyl)cytidine (8). To a sol-
ution of 6 (90 mg, 0.16 mmol) in DMF (1 mL) were added imidazole
(16 mg, 0.24 mmol) and TBS-Cl (31 mg, 0.21 mmol) at 0 °C. After
being stirred for 24 h at rt, the mixture was diluted with CHCl3 (25
mL) and washed with water (3 × 20 mL) and with brine (1 × 20 mL).
The organic phase was dried over MgSO4 and evaporated and the
residue purified by CC (hexane/EtOAc 1:2 + 1% Et3N) to give 8
(0.076 g, 71%) as a white solid. TLC (hexane/EtOAc 1:3): Rf 0.65. 1H
NMR (300 MHz, CDCl3): δ −0.09 (s, 3H, Si-CH3), 0.06 (s, 3H, Si-
1
(hexane/EtOAc 1:2): Rf 0.42. H NMR (300 MHz, CDCl3): δ 2.04−
2.25 (m, 7H, CH3, 2H-6′, 2H-7′), 4.08 (m, 1H, H-5′), 4.58−4.70 (m,
5H, 2CH2Ph, H-4′), 5.17 (d, J = 7.3 Hz, 1H, H-2′), 6.63 (d, J = 7.3 Hz,
1H, H-1′), 7.28−7.70 (m, 14H, 13Ph, H-5), 7.98 (d, J = 7.4 Hz, 2H,
1
2Bz), 8.28 (d, J = 7.4 Hz, 1H, H-6), 8.62 (sb, 1H, NH). H NMR-
NOE (400 MHz, CDCl3): 6.63 (H-1′) → 5.17 (H-2′, 2.6%), 4.71 (H-
4′, 5.5%), 4.08 (H-5′, 0.2%); 4.08 (H-5′) → 4.70 (H-4′, 11.1%). 13C
NMR (75 MHz, CDCl3): δ 20.7, 22.7, 30.5, 67.6, 71.7, 78.4, 81.0, 87.3,
89.0, 89.5, 126.9, 127.6, 128.2, 128.4, 128.5, 128.8, 129.0, 133.1, 137.3,
138.3, 170.4. HRMS (ESI): m/z [M + H]+ calcd for C34H34O7N3
596.2397, found 596.2393.
t
CH3), 0.85 (s, 9H, Bu), 1.52−2.22 (m, 4H, 2H-6′, 2H-7′), 4.10 (m,
2H, H-2′, H-5′), 4.50−4.95 (m, 5H, 2CH2Ph, H-4′), 6.60 (d, J = 7.9
Hz, 1H, H-1′), 7.28−7.60 (m, 14H, 13H-Ph, H-5), 7.92 (sb, 2H-Bz),
8.18 (d, J = 7.5 Hz, 1H, H-6), 8.70 (sb, 1H, NH). 13C NMR (75 MHz,
CDCl3): δ −5.0, −4.4, 1.0, 14.2, 17.8, 21.0, 25.6, 29.7, 30.6, 60.4, 68.3,
71.6, 78.6, 83.0, 87.8, 89.0, 90.1, 127.0, 127.4, 128.3, 128.4, 128.5,
128.9, 129.0, 133.3, 137.4, 139.1. HRMS (ESI): m/z [M + H]+ calcd
for C38H46O6N3Si 668.3156, found 668.3157.
(3′S,5′R)-1-(2′-Hydroxy-3′,5′-O-dibenzyl-3′,5′-ethano-β-d-
ribofuranosyl)thymidine (5). To a solution of nucleoside 3 (0.67 g,
1.3 mmol) in anhydrous MeOH (30 mL) was added NaOMe (0.14 g,
2.6 mmol). The clear solution was stirred at rt for 4 h. After being
cooled to 0 °C, the solution was neutralized with aq HCl and then
extracted with CH2Cl2 (3 × 50 mL). The combined organic phases
were washed with satd aq NaHCO3, dried over MgSO4, filtered, and
evaporated. CC (hexane/EtOAc 2:3) yielded compound 5 (0.560 g,
(3′S,5′R)-1-(3′,5′-Dihydroxy-2′-O-(tert-butyldimethylsilyl)-
3′,5′-ethano-β-D-ribofuranosyl)thymidine (9). To a solution of 7
(0.18 g, 0.3 mmol) in EtOAc (4.5 mL) were added 20% Pd(OH)2/C
(0.1 g) and 1,3-cyclohexadiene (0.24 g, 0.3 mmol). The mixture was
flushed with Ar for 15 min and then set under an atmosphere of H2.
After being stirred for 6 h at rt, the mixture was filtered through a pad
of Celite and the filtrate evaporated. The residue was purified by CC
(hexane/EtOAc 1:3) to yield 9 (0.12 g, 97%) as a white foam. TLC
(hexane/EtOAc 1:3): Rf 0.26. 1H NMR (300 MHz, MeOD): δ −0.11
1
91%) as a white foam. TLC (EtOAc/hexane 3:2): Rf 0.18. H NMR
(300 MHz, CDCl3): δ 1.50 (s, 3H, CH3), 2.04−2.13 (m, 4H, 2H6′,
2H7′), 3.07 (d, J = 10.3 Hz, 1H, 2′−OH), 3.98−4.10 (m, 2H, H-5′, H-
2′), 4.56−4.67 (m, 5H, 2CH2Ph, H-4′), 6.15 (d, J = 8.1 Hz, 1H, H-1′),
1
7.28−7.45 (m, 10H, Ph), 7.49 (s, 1H, H-6), 8.20 (s, 1H, NH). H
NMR-NOE (400 MHz, CDCl3): 6.15 (H-1′) → 4.64 (H-4′, 2.3%),
4.06 (H-2′, 1.3%); 4.65 (H-4′) → 4.04 (H-5′, 5.3%); 4.06 (H-2′) →
8.20 (N−H, 1.3%), 7.49 (C−H, 8.5%), 6.15 (H-1′, 1.7%). 13C NMR
(75 MHz, CDCl3): δ 11.6, 26.8, 29.7, 65.8, 71.3, 77.7, 79.3, 83.2, 88.2,
88.6, 111.2, 127.1, 127.3, 127.8, 128.4, 128.4, 135.1, 137.0, 137.2,
150.5, 162.7. HRMS (ESI): m/z [M + Na]+ calcd for C26H28O6N2Na
487.1845, found 487.1843.
t
(s, 3H, Si-CH3), −0.17 (s, 3H, Si-CH3), 0.92 (s, 9H, Bu), 1.39−1.52
(m, 1H, 1H-7′), 1.70−1.85 (m, 1H, 1H-6′), 1.90 (s, 3H, (CH3)C-5),
2.05−2.10 (m, 1H,1H-6′), 2.25−2.39 (m, 1H, 1H-7′), 3.80 (d, J = 5.1
Hz, 1H, H-4′), 3.97 (d, J = 3.6 Hz, 1H, H-2′), 4.05 (m, 1H, H-5′), 5.91
(d, J = 3.6 Hz, 1H, H-1′), 7.57 (s, 1H, H-6). 13C NMR (75 MHz,
CDCl3): δ −4.9, −4.7, 12.7, 19.1, 26.7, 32.2, 33.7, 58.6, 73.5, 81.6,
87.4, 89.0, 89.6, 109.9, 141.3, 152.3, 166.7. HRMS (ESI): m/z [M +
H]+ calcd for C18H31O6N2Si 399.1951, found 399.1960.
(3′S,5′R)-N4-Benzoyl-1-(2′-hydroxy-3′,5′-O-dibenzyl-3′,5′-
ethano-β-D-ribofuranosyl)cytidine (6). Nucleoside 4 (0.26 g, 0.44
mmol) was dissolved in 0.2 M NaOH in THF/MeOH/H2O 5:4:1 (25
mL) at 0 °C. After 45 min, the reaction was quenched by addition of
NH4Cl (0.347 g, 1.5 equiv relative to NaOH). The solution was stirred
for another 10 min at rt before evaporation. The residue was adsorbed
on silica gel (MeOH) and purified by CC (hexane/EtOAc 1:3) to give
compound 6 (0.120 g, 52%) as a white foam. TLC (hexane/EtOAc
1:3): Rf 0.22. 1H NMR (300 MHz, CDCl3): δ 2.00−2.12 (m, 4H, 2H-
6′, 2H-7′), 4.06 (m, 2H, H-2′, H-5′), 4.16 (sb, 1H, 2′−OH), 4.52−4.82
(m, 5H, 2CH2Ph, H-4′), 6.19 (d, J = 6.6 Hz, 1H, H-1′), 7.28−7.61 (m,
14H, 13Ph, 1H−C5), 7.89 (d, J = 7.4 Hz, 2H, H-Bz), 8.27 (d, J = 7.5
Hz, 1H, H-6), 8.68 (sb, 1H, NH). 1H NMR-NOE (400 MHz, CDCl3):
6.20 (H-1′) → 4.12 (H-2′, 3.3%); 4.78 (H-4′) → 6.23 (H-1′, 2.5%),
4.09 (H-5′, 9.9%). 13C NMR (75 MHz, CDCl3): δ 1.0, 14.1, 22.7, 28.7,
29.3, 29.5, 29.63, 29.67, 30.2, 31.4, 31.9, 67.2, 71.7, 78.2, 82.9, 86.5,
89.9, 93.0, 96.7, 113.6, 127.45, 127.52, 127.8, 128.1, 128.3, 128.5,
128.7, 129.1, 133.2, 137.4, 138.1, 144.9, 162.1. HRMS (ESI): m/z [M
+ H]+ calcd for C32H32O6N3 554.2291, found 554.2291.
(3′S,5′R)-1-[3′-Hydroxy-5′-O-[(4,4′-dimethoxytriphenyl)-
methyl]-2′-O-(tert-butyldimethyllsilyl)-3′,5′-ethano-β-d-
ribofuranosyl]thymidine (10). To a stirred solution of nucleoside 9
(0.29 g, 0.74 mmol) in pyridine (3 mL) was added DMTr-Cl (0.75 g,
2.2 mmol) at rt. After 3 h, another portion of DMTr-Cl (0.75 g, 2.2
mmol) was added and the mixture stirred for 16 h. The reaction was
then quenched with satd aq NaHCO3 (5 mL) and the mixture
extracted with CH2Cl2 (3 × 10 mL). The combined organic phases
were dried over MgSO4, filtered, and evaporated, and the residue was
purified by CC (hexane/EtOAc 2:1 + 1% Et3N) to give 10 (0.47 g,
1
90%) as a yellow foam. TLC (hexane/EtOAc 2:1): Rf 0.30. H NMR
(300 MHz, CDCl3): δ −0.02 (s, 3H, Si-CH3), 0.07 (s, 3H, Si-CH3),
0.87 (s, 9H, tBu), 1.06- 2.23 (m, 7H, (CH3)C-5, 2H-6′, 2H-7), 3.11 (s,
1H, 3′-OH), 3.78 (s, 6H, 2MeO), 3.94 - 3.96 (m, 2H, H-4′, H-2′),
4.00−4.10 (m, 1H, H-5′), 6.03 (d, J = 7.4 Hz, 1H, H-1′), 6.84 (dd, J =
9.0, 2.2 Hz, 4H, H-Ph), 7.28−7.49 (m, 10H, H-Ph, H-6), 8.12 (sb, 1H,
5866
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Featured Article
NH). 13C NMR (75 MHz, CDCl3): δ −4.7, −4.6, −4.5, −4.4, 12.0,
12.5, 14.3, 18.0, 22.9, 25.4, 29.5, 29.8, 34.7, 55.3, 71.5, 73.7, 79.8, 82.8,
87.5, 87.9, 88.2, 89.2, 94.3, 112.8, 113.4, 123.6, 127.3, 128.0, 128.6,
129.3, 130.5, 134.2, 136.5, 138.1, 139.7, 150.4, 158.3, 159.0, 163.9.
HRMS (ESI): m/z [M + Na]+ calcd for C39H48O8N2SiNa 723.3078,
found 723.3087.
5 (0.3 g, 0.64 mmol) in pyridine (3.5 mL) was added dropwise MsCl
(0.22 g, 1.9 mmol) at 0 °C. After 1 h at rt, the reaction was quenched
with water (10 mL) and the mixture extracted with CH2Cl2 (3 × 15
mL). The combined organic phases were washed with satd aq
NaHCO3 (3 × 30 mL), dried over MgSO4, and evaporated, and the
residue was purified by CC (hexane/EtOAc 1:2) to give the product
12 (0.31 g, 89%) as a white solid. TLC (hexane−EtOAc 1:2): Rf 0.48.
1H NMR (300 MHz, CDCl3): δ 1.48 (d, J = 1.1 Hz, 3H, CH3), 2.20−
2.26 (m, 4H, 2H- 6′, 2H- 7′), 3.01 (s, 3H, CH3), 4.06 (m, 1H, H-5′),
4.55−4.84 (m, 5H, 2CH2Ph, H-4′), 5.02 (d, J = 7.9 Hz, 1H, H-2′), 6.47
(d, J = 7.9 Hz, 1H, H-1′), 7.28−7.37 (m, 10H, Ph), 7.54 (d, J = 1.3 Hz,
1H, H-6), 8.48 (s, 1H, NH). 13C NMR (75 MHz, CDCl3): δ 12.0,
28.5, 29.6, 38.5, 67.8, 71.4, 76.6, 77.8, 82.1, 86.7, 87.4, 88.3, 112.1,
126.9, 127.65, 127.67, 128.1, 128.5, 128.7, 135.0, 137.4, 138.3, 150.5,
163.2. HRMS (ESI): m/z [M + H]+ calcd for C27H31O8N2S 543.1801,
found 543.1796.
(3′S,5′R)-1-(2′-Hydroxy-3′,5′-O-dibenzyl-3′,5′-ethano-β-D-
arabinofuranosyl)thymidine (14) (from Compound 12). To a
stirred solution of 12 (0.30 g, 0.55 mmol), in EtOH (10 mL) and H2O
(10 mL) was added 1 M aq NaOH (2.2 mL), and the reaction mixture
was stirred under reflux for 16 h. After being cooled to rt, the mixture
was neutralized with 1 M aq HCl and concentrated in vacuo. The
resulting aqueous solution was saturated with NaCl and then extracted
with CH2Cl2 (3 × 30 mL). The combined organic extracts were
washed with sat aq NaHCO3 (2 × 30 mL), dried over MgSO4, and
evaporated, and the residue was purified by CC (hexane/EtOAc, 1:2)
to give the product 14 (0.22 g, 85%) as a white foam. Analytical data
identical as described above.
(3′S,5′R)-1-{3′-O-[(Cyanoethoxy)(diisopropylamino)-
phosphino]-5′-O-[(4,4′-dimethoxytriphenyl)methyl]-2′-O-(tert-
butyldimethyllsilyl)-3′,5′-ethano-β-D-ribofuranosyl)]thymidine
(11). To a stirred solution of 10 (0.47 g, 0.67 mmol) in MeCN (3 mL)
i
i
was added at rt Pr2NEt (0.5 g, 0.4 mmol), followed by Pr2NP(Cl)-
OCH2CH2CN (0.58 g, 0.26 mmol). After 4 h, the mixture was diluted
with EtOAc (10 mL) and washed with sat aq NaHCO3 (10 mL). The
aqueous phase was extracted with EtOAc (3 × 10 mL) and the
combined organic phase dried over MgSO4, evaporated and the
residue purified by CC (hexane/EtOAc 2:1 + 1% Et3N) to obtain 11
(0.46 g, 76%) as a white foam. TLC (hexane/EtOAc 2:1): Rf 0.34. 1H
NMR (300 MHz, CDCl3): δ 0.00, 0.01, 0.08, 0.13 (4s, 6H, Si-CH3),
t
0.87, 0.88 (2s, 9H, Bu), 0.75−0.95 (m, 2H, 2H-6′), 1.20−1.35 (m,
12H, 4N−CH3), 1.85−1.95 (m, 2H, 2H-7′),2.53−2.65 (m, 2H CH2−
CN), 3.40−3.98 (m, 10H, 2OMe, 2CH2−O, 2 x CH-N), 4.10−4.21
(m, 2H, H-2′, H-5′), 4.34−4.37, 4.55−4.58 (2 m, 1H, H-4′), 6.22−6.26
(m, 1H, H-1′), 6.80−6.85 (m, 4H, Ph), 7.28−7.47 (m, 9H, Ph), 7.54
(s, 1H, H-6), 8.23 (bs, 1H, NH). 13C NMR (75 MHz, CDCl3): δ −4.7,
−4.6, −3.8, 11.4, 14.4, 18.1, 18.2, 20.4, 20.6, 20.7, 24.4, 24.48, 24.51,
24.6, 24.7, 24.77, 24.82, 25.7, 25.8, 43.3, 43.4, 43.5, 43.6, 58.4, 72.8,
73.2, 87.6, 87.77, 87.84, 111.5, 111.6, 113.4, 117.9, 118.1, 127.46,
127.50, 128.1, 128.8, 130.6, 135.9, 136.0, 136.1, 136.2, 136.3, 136.4,
145.0, 145.1, 150.5, 150.6, 159.1, 163.5. 31P NMR (162 MHz, CDCl3):
δ 141.2, 142.1. HRMS (ESI): m/z [M + H]+ calcd for C48H66O9N4 PSi
901.4337, found 901.4352.
(3′S,5′R)-1-(2′-O-tert-Butydimethylsilyl-3′,5′-O-dibenzyl-
3′,5′-ethano-β-D-arabinofuranosyl)thymidine (15). To a solution
of nucleoside 14 (150 mg, 0.32 mmol) in 1 mL of DMF were added
imidazole (55 mg, 0.8 mmol) and TBS-OTf (126 mg, 0.48 mmol).
After being stirred at rt for 20 h, the mixture was diluted with CH2Cl2
(20 mL) and washed with water (15 mL) and brine (15 mL). The
organic phase was dried over MgSO4 and evaporated and the residue
purified by CC (hexane/EtOAc 3:2) to give 15 (0.13 g, 71%) as a
(3′S,5′R)-2,2′-Anhydro-1-(3′,5′-O-dibenzyl-3′,5′-ethano-β-D-
arabinofuranosyl)thymidine (13). A solution of 5 (50 mg, 0.1
mmol) in DMF (2 mL) was treated with diphenyl carbonate (27 mg,
0.13 mmol) and NaHCO3 (2 mg). The mixture was heated to 150 °C
for 1 h and, after cooling to rt, poured into Et2O (5 mL). The organic
phase was evaporated and the residue purified by CC (hexane/EtOAc
1:1) to give the compound 13 (11 mg, 23%) as a white solid. TLC
1
white foam. TLC (hexane/EtOAc 1:2): Rf 0.41 H NMR (300 MHz,
CDCl3): δ −0.14 (s, 3H, Si-CH3), 0.15 (s, 3H, Si-CH3), 0.87 (s, 9H, t-
Bu), 1.57−1.65 (m, 1H, 1H-7′), 1.83 (s, 3H, (CH3) C-5), 1.90−2.08
(m, 2H, 1H-6′, 1H-7′), 2.26−2.30 (m, 1H, H-6′), 3.97−4.01 (m, 1H,
H-5′), 4.31 (d, J = 5.3 Hz, 1H, H-4′), 4.37 (d, J = 3.6 Hz, 1H, H-2′),
4.43 (d, J = 11.1 Hz, 1H, CH2Ph), 4.48 (d, J = 11.1 Hz, 1H, CH2Ph),
4.54 (d, J = 11.5 Hz, 1H, CH2Ph), 4.69 (d, J = 11.5 Hz, 1H, CH2Ph),
4.53 (d, J = 11.5 Hz, 1H, CH2Ph), 4.67 (d, J = 11.5 Hz, 1H, CH2Ph),
6.15 (d, J = 3.6 Hz, 1H, H-1′), 7.28−7.48 (m, 10H, Ph), 7.48 (d, J =
1.32 Hz, 1H, H-6), 8.26 (s, 1H, NH). 13C NMR (75 MHz, CDCl3): δ
−5.2, −4.9, −3.6, −3.0, 12.4, 17.9, 25.6, 25.7, 25.8, 26.5, 30.8, 66.7,
71.7, 75.4, 77.8, 84.3, 86.1, 95.0, 109.0, 127.2, 127.9, 128.46, 128.54,
137.6, 138.0, 139.0, 149.9, 163.3. HRMS (ESI): m/z [M + Na]+ calcd
for C32H42O6N2NaSi 601.2710, found 601.2704.
1
(hexane/EtOAc 1:2): Rf 0.05. H NMR (300 MHz, CDCl3): δ 1.74−
2.03 (m, 6H, 1H-6′, 2H-7′, (CH3)C-5), 2.63−2.65 (m, 1H, 1H-6′),
3.95 (dd, J = 10.5 Hz, 6.3 Hz 1H, H-5′), 4.49 (s, 2H, CH2Ph), 4.62 (d,
J = 11.4 Hz,1H, 1CH2Ph), 4.69 (d, J = 11.4 Hz, 1H, 1CH2Ph), 4.82 (d,
J = 5.3 Hz, 1H, H-4′), 5.33 (d, J = 6.0 Hz, 1H, H-2′), 6.38 (d, J = 6.0
Hz, 1H, H-1′), 7.25−7.44 (m, 11H, 2Ph, H-6). 13C NMR (75 MHz,
CDCl3): δ 13.9, 25.3, 29.7, 67.2, 71.3, 85.5, 92.3, 93.0, 95.4, 119.0,
127.2, 127.6, 127.7, 128.1, 128.4, 128.7, 130.2, 137.1, 137.4, 159.7,
172.3. HRMS (ESI): m/z [M + H]+ calcd for C26H27O5N2: 447.1920;
found: 447.1914.
(3′S, 5′R)-1-(2′-hydroxy-3′,5′-O-dibenzyl-3′,5′-ethano-β-D-
arabinofuranosyl)thymidine (14). A stirred solution of 13 (50
mg, 0.11 mmol) in 0.1 M KOH in EtOH (10 mL) was refluxed for 3 h.
After cooling to rt, the mixture was neutralized with 1 M aq HCl and
concentrated in vacuo. The residual aqueous solution was saturated
with NaCl and extracted with CH2Cl2 (3 × 20 mL). The combined
organic extracts were washed with sat aq NaHCO3 (2 × 30 mL), dried
over MgSO4, and evaporated, and the residue waspurified by CC
(hexane/EtOAc 1:2) to give the product 14 (17 mg, 32%) as a white
foam. TLC (hexane/EtOAc 1:2): Rf 0.24. 1H NMR (300 MHz,
CDCl3): δ 1.60 (sb, 4H, 2′−OH, (CH3)C-5), 1.82 (m, 1H, H-7′), 2.10
(m, 1H, H-7′), 2.23 (m, 1H, H-6′), 2.46 (m, 1H, H-6′), 4.10−4.13 (m,
1H, H-5′), 4.38 (sb, 1H, H-2′), 4.43 (d, J = 4.7 Hz, 1H, H-4′), 4.46−
4.72 (m, 4H, 2CH2Ph), 6.30 (d, J = 2.5, 1H, H-1′), 7.28−7.34 (m,
10H, Ph), 7.46 (d, J = 0.8 Hz, 1H, H-6), 9.06 (sb, 1H, NH). 1H NMR-
NOE (400 MHz, CDCl3): 6.30 (H-1′) → 4.38 (H-2′, 3.99%), 4.31 (H-
4′, 1.41%). 13C NMR (75 MHz, CDCl3): δ 12.2, 14.2, 21.0, 23.8, 30.0,
60.4, 67.3, 71.6, 72.2, 78.9, 88.3, 89.7, 95.5, 108.7, 127.4, 127.8, 127.9,
128.4, 128.5, 128.9, 136.8, 137.9, 138.0, 150.0, 164.1. HRMS (ESI):
m/z [M + H]+ calcd for C26H29O6N2 465.2026, found 465.2020.
(3′S,5′R)-1-(2′-O-Methylsulfonyl-3′,5′-O-dibenzyl-3′,5′-etha-
no-β-D-arabinofuranosyl)thymidine (12). To a stirred solution of
(3′S,5′R)-1-[3′,5′-Dihydroxy-2′-O-(tert-butyldimethylsilyl)-
3′,5′-ethano-β-D-arabinofuranosyl]thymidine (16). To a solution
of 15 (50 mg, 0.086 mmol) in EtOAc (2 mL) were added 20%
Pd(OH)2/C (50 mg) and 1,3-cyclohexadiene (69 mg, 0.86 mmol).
The mixture was flushed with Ar for 15 min and set under an
atmosphere of H2. After being stirred for 2 h, the mixture was filtered
through a pad of Celite, and the solvents were removed under reduced
pressure. The residue was purified by CC (hexane/EtOAc 1:3) to give
the title compound 16 (20 mg, 58%) as a white solid. TLC (hexane/
1
EtOAc 1:3): Rf 0.16. H NMR (300 MHz, MeOD): δ −0.28 (s, 3H,
Si-CH3), 0.01 (s, 3H, Si-CH3), 0.75 (s, 9H, t-Bu), 1.15−1.27 (m, 1H,
1H-7′), 1.60−1.69 (m, 1H, H-6′), 1.72 (s, 3H, (CH3)C-5), 1.79−1.90
(m, 1H, 1H-6′), 2.13−2.21 (m, 1H, 1H-7′), 3.78 (d, J = 5.1 Hz, 1H, H-
4′), 3.96 (d, J = 3.6 Hz, 1H, H-2′), 3.97−4.05 (m, 1H, H-5′), 5.90 (d, J
= 3.6 Hz, 1H, H-1′), 7.57 (d, J = 1.1 Hz, 1H, H-6). 13C NMR (75
MHz, MeOD): δ −4.9, −4.7, 12.7, 19.1, 26.7, 32.2, 33.7, 73.5, 81.6,
87.4, 89.0, 89.6, 109.9, 141.3, 152.3, 166.7. HRMS (ESI): m/z [M +
H]+ calcd for C18H31O6N2Si 399.1951, found 399.1946.
(3′S,5′R)-1-[3′-Hydroxy-5′-O-(4,4′-dimethoxytriphenylmeth-
yl)-2′-O-(tert-butyldimethylsilyl)-3′,5′-ethano-β-D -
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dx.doi.org/10.1021/jo300554w | J. Org. Chem. 2012, 77, 5861−5869

The Journal of Organic Chemistry
Featured Article
arabinofuranosyl]thymidine (17). To a stirred solution of
nucleoside 16 (0.24 g, 0.6 mmol) in pyridine (3 mL) was added
DMTr-Cl (0.60 g, 1.8 mmol) at rt. After 5 h, the reaction was
quenched with satd aq NaHCO3 (5 mL), and the mixture was
extracted with CH2Cl2 (3 × 10 mL). The combined organic phases
were dried over MgSO4, filtered, and evaporated, and the residue was
purified by CC (hexane/EtOAc 2:1 + 1% Et3N) to give the title
compound 17 (0.38 g, 90%) as a white solid. TLC (hexane/EtOAc 1:2
buffers were used for HPLC: A: 25 mM Trizma base in H2O, pH 8.0;
B: 25 mM Trizma Base, 1.25 M NaCl, in H2O, pH 8.0. Linear
gradients of B in A were used. The integrity of all oligonucleotides was
confirmed with ESI−-MS (see the Supporting Information).
Melting Curves. Thermal denaturation experiments were carried
out 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−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 standard saline
buffer (150 mM NaCl, 10 mM Na2HPO4, pH 7.0) at a total
oligonucleotide concentration of 2 μM.
1
+1%Et3N): Rf 0.47. H NMR (300 MHz, CDCl3): δ = −0.13 (s, 3H,
Si-CH3), 0.15 (s, 3H, Si-CH3), 0.89 (s, 9H, t-Bu), 1.07−1.10 (m, 1H,
1H-7′), 1.47−1.51(m, 1H, 1H-6′), 1.61−1.78 (m, 1H, 1H-6′), 1.94 (s,
3H, (CH3)C-5, 2.04−2.19 (m, 1H, 1H-7′), 2.23 (bs, 1H, OH), 3.19 (d,
J = 5.3 Hz, 1H, H-4′), 3.77 (s, 6H, 2OMe), 3.95−3.98 (m, 1H, H-5′),
4.08 (d, J = 3.9 Hz, 1H, H-2′), 6.00 (d, J = 3.9 Hz, 1H, H-1′ (sb, 1H,
NH). 13C NMR (75 MHz, CDCl3): δ −5.0, −4.8, 12.9, 18.2, 26.1,
30.0, 31.2, 31.9, 55.5, 74.1, 80.7, 85.6, 86.9, 87.0, 88.6, 109.3, 113.4,
127.2, 128.1, 128.5, 130.51, 130.54, 137.11, 137.13, 139.3, 146.0,
150.4, 158.9, 163.7. HRMS (ESI): m/z [M + Na]+ calcd for
C39H48O8N2NaSi 723.3078, found 723.3072.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H, 13C, and 31P NMR spectra of compounds 2−18, analytical
data of oligonucleotides, and X-ray structural data and packing
diagram of compound 5. This material is available free of charge
via the Internet at http://pubs.acs.org/.
(3′S,5′R)-1-[3′-O-(Cyanoethoxydiisopropylaminophosphi-
no)-5′-O-[(4,4′-dimethoxytriphenyl)methyl]-2′-O-(tert-butyldi-
methylsilyl)-3′,5′-ethano-β-D-arabinofuranosyl]thymidine (18).
To a stirred solution of 17 (0.34 g, 0.49 mmol) in MeCN (3 mL) were
added iPr2NEt (0.3 g, 2.4 mmol) and iPr2NP(Cl)OCH2CH2CN (0.32
g, 1.4 mmol) at rt. After 1 h, the mixture was diluted with EtOAc (10
mL) and washed with satd aq NaHCO3 (2 × 10 mL). The aqueous
phases were extracted with EtOAc (3 × 10 mL) and the combined
organic phases dried over MgSO4 and evaporated, and the residue was
purified by CC (hexane/EtOAc 1:2 + 1% Et3N) to give 18 (0.43 g,
97%) as a white foam. TLC (hexan/EtOAc 2:1 + 1% Et3N): Rf 0.63.
1H NMR (300 MHz, CDCl3): δ −0.162, −0.168, 0.09, 0.10 (4s, 6H,
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +41-31-631-4355. Fax: +41-31-631-3422. Email:
leumann@ioc.unibe.ch.
Si-CH3), 0.89 (s, 9H, tBu), 1.06−1.15 (m, 12H, 4N−CH3), 1.15- 1.78,
(m, 3H, 2H-6′, 1H-7′), 1.95 (s, 3H, (CH3)C-5, 2.04−2.12 (m, 1H, 1H-
7), 2.54−2.56 (m, 2H, CH2CN), 3.45−3.65 (m, 4H, CH2−O, 2CH-
N), 3.80 (s, 6H, 2OMe), 3.90 (m, 1H, H-5′), 4.29−4.32 (m, 1H, H-4′),
5.91, 5.95 (2d, J = 3.4 Hz, 1H, H-1′), 6.79−6.84 (m, 4H, Ph), 7.15−
7.61 (m, 10H, Ph, H-6), 8.30−8.50 (m, 1H, NH). 13C NMR (75 MHz,
CDCl3): δ −5.3, −5.2, −5.1, −4.99, −4.95, −4.90, 12.6, 14.2, 17.9,
20.08, 20.14, 22.7, 24.1, 24.18, 24.24, 24.3, 24.4, 24.5, 25.8, 29.5, 29.6,
31.8, 32.0, 43.2, 43.3, 43.4, 43.5, 55.2, 55.3, 57.7, 57.9, 73.2, 73.3, 79.0,
79.1, 79.3, 85.4, 85.5, 85.6, 85.9, 86.0, 86.63, 86.65, 92.4, 92.45, 92.48,
92.6, 108.7, 108.8, 113.1, 117.5, 126.8, 127.8, 128.2, 130.2, 130.3,
136.8, 136.9, 138.98, 139.03, 145.6, 149.96, 149.99, 156.6, 163.5. 31P
NMR (162 MHz, CDCl3): δ 140.96, 142.13. HRMS (ESI): m/z [M +
H]+ calcd for C48H66O9N4 PSi 901.4337, found 901.4331.
Oligonucleotide Synthesis and Purification. The 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 dichloroethane was
used for detritylation. The concentrations for the natural phosphor-
amidite solutions were 0.1 M and for the modified phosphoramidites
0.15 or 0.2 M. The coupling times for natural phosphoramidites were
1.5 min and for the modified phosphoramidite 12−14 min. The
coupling efficiencies for 11 and 18 were generally low (∼90%) as
judged from the trityl assay. Deprotection and detachment from solid
support were performed in concentrated NH3/EtOH (3:1, 0.5 mL, 55
°C, 30 h). Removal of the silyl groups was performed by treatment of
the crude oligonucleotides with 1 M TBAF in THF (0.5 mL) at rt for
24 h. After evaporation, the brown residue was taken up in H2O and
filtered through a Sep-Pak C-18 cartridge (Waters). The crude samples
were purified by ion exchange HPLC (Dionex, DNAPac-200, 4.6 ×
250 mm column with precolumn). The product containing fractions
were concentrated and again desalted over Sep-Pak C-18 cartridge
(Waters) according to the protocol of the manufacturer. The following
ACKNOWLEDGMENTS
■
Financial support of this work by the Swiss National Science
Foundation (Grant No. 200020-130373) is gratefully acknowl-
edged. We thank the group of Chemical Crystallography of the
University of Bern (PD Dr. P. Macchi and Dr. J. Hauser) for
the X-ray structure and the Swiss National Science Foundation
(Requip project 206021_128724) for cofunding of the single-
crystal X-ray diffractometer at the Department of Chemistry
and Biochemistry of the University of Bern.
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