Synthesis and properties of isobicyclo-DNA

Article abstract of DOI:10.1002/chem.201300487

We present the synthesis of the isobicyclo-DNA building blocks with the nucleobases A, C, G and T, as well as biophysical and biological properties of oligonucleotides derived thereof. The synthesis of the sugar part was achieved in 5 steps starting from a known intermediate of the tricyclo-DNA synthesis. Dodecamers containing single isobicyclo-thymidine incorporations, fully modified A- and T-containing sequences, and fully modified oligonucleotides containing all four bases were synthesized and characterized. Isobicyclo-DNA forms stable duplexes with natural nucleic acids with a pronounced preference for DNA over RNA as complements. The most stable duplexes, however, arise by self-pairing. Isobicyclo-DNA forms preferentially B-DNA-like duplexes with DNA and A-like duplexes with complementary RNA as determined by circular dichroism (CD) spectroscopy. Self-paired duplexes show a yet unknown structure, as judged from CD spectroscopy. Biochemical tests revealed that isobicyclo-DNA is stable in fetal bovine serum and does not elicit RNaseH activity. Copyright

Full text of DOI:10.1002/chem.201300487

FULL PAPER
DOI: 10.1002/chem.201300487
Synthesis and Properties of Isobicyclo-DNA
Anna-Barbara Gerber and Christian J. Leumann*[a]
Chem. Eur. J. 2013, 00, 0 – 0
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Abstract: We present the synthesis of
the isobicyclo-DNA building blocks
with the nucleobases A, C, G and T, as
well as biophysical and biological prop-
erties of oligonucleotides derived
thereof. The synthesis of the sugar part
was achieved in 5 steps starting from a
known intermediate of the tricyclo-
DNA synthesis. Dodecamers contain-
ing single isobicyclo-thymidine incor-
porations, fully modified A- and T-con-
taining sequences, and fully modified
oligonucleotides containing all four
bases were synthesized and character-
ized. Isobicyclo-DNA forms stable du-
plexes with natural nucleic acids with a
pronounced preference for DNA over
RNA as complements. The most stable
duplexes, however, arise by self-pair-
ing. Isobicyclo-DNA forms preferen-
tially B-DNA-like duplexes with DNA
and A-like duplexes with complemen-
tary RNA as determined by circular di-
chroism (CD) spectroscopy. Self-paired
duplexes show a yet unknown struc-
ture, as judged from CD spectroscopy.
Biochemical tests revealed that isobicy-
clo-DNA is stable in fetal bovine
serum and does not elicit RNaseH
activity.
Keywords: DNA
· nucleosides ·
oligonucleotides · RNA · serum
stability
Introduction
Oligonucleotides are a class of compounds with considerable
therapeutic potential. Unlike small molecule drugs that typi-
cally target proteins, therapeutic oligonucleotides act
through Watson–Crick hybridization to a matched sequence
tract on an RNA of interest, modulating its biological func-
tion.[1] Originally, mRNAs of disease-related proteins have
been the target of classical antisense oligonucleotides that
either sterically block or induce RNaseH degradation of the
mRNA in vivo.[2] The project ENCODE[3] recently revealed
that 80% of the human genome is transcribed into RNA.
Of these RNAs only 2% encode for proteins whereas a con-
siderable subset, such as micro-RNAs (miRNAs) and gener-
ally non-coding RNAs, are involved in gene regulation.
Thus, they play a pivotal role in the onset of disease, which
greatly expands the palette of RNA sequences as therapeu-
tic targets in the future.
Figure 1. A) bicyclo-DNA, B) isobicyclo-DNA, C) tricyclo-DNA.
clic 5-membered ring connecting C(5’) and C(3’) and there-
by rigidifying the rotational freedom around the C(3’)-C(4’)
and the C(4’)-C(5’) bonds (Figure 1, A and C).
Besides conformational restriction, this carbocyclic ring
offers the unique advantage to change the geometry of the
repeating backbone unit in a single strand by moving the hy-
droxyl groups involved in the internucleotidic linkages to
other carbon atoms within the cycle. In this context we
became interested in exploring the properties of isobicyclo-
DNA (Figure 1B) in which the C(5’) hydroxyl group has
been moved to C(6’). Interestingly, this isobicyclo-DNA
shows the same number of bonds within the repeating back-
bone unit as DNA. In this article we report on the synthesis
of the corresponding isobicyclo-DNA building blocks with
all four natural bases, on their incorporation into oligonu-
cleotides, their RNA and DNA recognition properties, as
well as on their structure as determined by circular dichro-
ism (CD) spectroscopy and their biological properties
(serum stability and RNaseH activation).
The basic requirements for therapeutic oligonucleotides
are 1) high affinity towards a target RNA, 2) biostability,
and 3) cellular availability. A large number of chemical
modifications on the DNA bases, sugar, and internucleoside
linkage have been reported over the last two decades, but
only a handful of those have been used in extensive pre-clin-
ical or clinical tests. Amongst those are the class of 2’O-al-
kylated RNA,[4] the morpholino phosphorodiamidates,[5] the
peptide nucleic acids (PNA),[6] and the locked nucleic acids
(LNA)[7]
.
In our laboratory, we have been involved in the design
and synthesis of conformationally constrained oligonucleo-
Results
ACHTUNGTRENNUNGtides over the last two decades. Offsprings of these efforts
were the bicyclo-[8] and tricyclo-DNA (tc-DNA)[9] molecular
platform (Figure 1). Unique to both families is the carbocy-
Molecular modeling of monomers: To investigate the influ-
ence of the C(5’)!C(6’) hydroxyl shift on the structure of
the nucleosides, a conformational search was performed by
using the MM+ force field as implemented in the software
package HyperChem. The C(3’), C(5’) dimethylated isobicy-
lo thymidine was initially built in a 2’-endo conformation
and then energy minimized. In a conformational search ex-
periment all endocyclic torsion angles were then varied, re-
sulting in two different low-energy conformational families
[a] Dr. A.-B. Gerber, Prof. C. J. Leumann
Department of Chemistry & Biochemistry
University of Bern, Freiestrasse 3
3012 Bern (Switzerland)
Fax : (+41)31-631-3422
E-mail: leumann@ioc.unibe.ch
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201300487.
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Isobicyclo-DNA
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defunctionalize C(5’), the alcohols 6a, 6b, and 7a were con-
verted into the thiocarbamates 8a, 8b, and 9a in good
yields. The subsequent Barton–McCombie reduction was
performed with azobisisobutyronitrile (AIBN) as the radical
initiator and tris(trimethylsilyl)silane (TTMSS) as the H-
donor to yield 10a, 10b, and 11a, again in good yields
(Scheme 1).
Synthesis of the phosphoramidite building blocks: The nu-
cleosidation of building block 10b (for nucleosidation of
10a see the Supporting Information) with the bases T, ABz,
and CBz was performed under Vorbrꢁggen conditions lead-
ing to almost 1:1 mixtures of anomers 12a/b, 13a/b, and
14a/b in 75–85% yields that were not separable by standard
chromatography techniques (Scheme 2). Subsequent deace-
tylation under mild conditions yielded 15a/b, 16a/b, and
17a/b. We decided to invert the configuration at C(6’) at
this stage. Therefore, the free nucleosides 15a/b, 16a/b, and
17a/b were converted into the corresponding C(6’) mesy-
lates 21a/b, 22a, 22b, and 23a/b. At this point it was possi-
ble to separate the a- and b-anomers of 22 by using column
chromatography, whereas 21 and 23 had unfortunately still
to be taken forward as mixtures of anomers. Subsequent
acetylation of O(3’) followed by displacement of the 6’-
mesyl function by CsOAc in DMSO yielded 24a/b, 25b, and
26a/b. Saponification (!27a/b, 28b, 29a/b) and subsequent
tritylation gave compounds 30a, 30b, 31b, 32a, and 32b. As
already observed in other cases, the tritylated nucleosides 30
and 32 were now separable by chromatography and isolated
as pure isomers. The relative configuration at the centers
C(1’) in 30b, 31b, and 32b was ascertained by using
1H NMR-ROESY (the Supporting Information). The treat-
ment of the b-nucleosides 30b, 31b, and 32b with 2’-cya-
noethyl N,N-diisopropylchlorophosphoramidite (CEP-Cl)
under standard conditions finally yielded the desired phos-
phoramidites 33b, 34b, and 35b.
Figure 2. Lowest energy conformers of 3’,5’-dimethylated isobicyclo-thy-
midine: a) O(6’) in axial position, furanose in C(1’)-exo conformation;
b) O(6’) in equatorial position, furanose in C(4’)-exo conformation.
(Figure 2). Table 1 summarizes the endocyclic torsion angles
of the furanose unit and the corresponding energies of two
representative structures of each family. The lowest energy
conformer turned out to be that with the 6’ substituent in an
axial position (Figure 2a). This structure fits well into a B-
DNA double helix as judged from simple model building.
Table 1. Results of the conformational search experiment for an isobicy-
clic thymidine. w is the angle between the phosphate oxygen and the C6’
atom.
n0
n1
n2
[8]
n3
n4
w
Furanose
pucker
Energy
[kcalmolꢀ1
]
a)
b)
ꢀ41
ꢀ36
35
15
ꢀ17
ꢀ6
30
41
82
170
C(1’)-exo
C(4’)-exo
29.22
30.26
8
ꢀ30
Synthesis of the isobicyclo sugar: The bicyclic silyl enol
ethers 1a (C(1’)-a), 1b (C(1’)-b) used in the synthesis of the
tricyclo sugar[10] were considered to be the starting materials
of choice for the synthesis of the isobicyclo-DNA scaffold.
Hydroboration of 1a or 1b
yielded the compounds 2a, 2b
and 3a, 3b (Scheme 1) in dia-
stereomeric ratios of 4:1 in the
a-series and 10:1 in the b-
series.[11] The lowest abundant
isomer 3b could not be isolated
as a pure compound and was
not used in further transforma-
tions. All other isomers 2a, 2b,
and 3a were taken forward in
the following nucleoside syn-
thesis. Acetylation of 2a, 2b,
and 3a was performed under
standard conditions by using
Ac2O/ 4-dimethylaminopyridine
(DMAP) in pyridine at room
temperature yielding 4a, 4b,
and 5a. Desilylation with HF/
Et3N (37% HF) in THF yielded
Scheme 1. a) 1) BH3·THF, THF, ꢀ788C!RT, 22 h, 2) Oxone in sat. NaHCO3, RT, 2 h, 80%; b) Ac2O, DMAP,
pyridine, RT, 95%; c) HF/Et3N, THF, RT, 85%; d) TCDI, THF, heat at reflux, 82%; e) AIBN, TTMSS,
compounds 6a, 6b, and 7a. To toluene, heat at reflux, 82%.
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C. J. Leumann and A.-B. Gerber
Scheme 2. a) Thymine (2 equiv), BSA (5 equiv), TMSOTf (3 equiv), MeCN, 08C to RT, 85%/benzoyl-adenine (2 equiv), BSA (4 equiv), TMSOTf
(0.3 equiv), MeCN, 858C, 75%/benzoyl-cytosine (2 equiv), BSA (5 equiv), TMSOTf (3 equiv), 08C to RT, 75%; b) 0.2m NaOH in 5:4:1 THF/MeOH
H2O, 0 8C, 75–90%; c) MsCl (1 equiv), pyridine, 08C!RT, 60–70%; d) Ac2O, DMAP, pyridine, RT, 80–91%; e) CsOAc, DMSO, 908C, 81–93%; f) 0.2m
NaOH in 5:4:1 THF/MeOH, H2O, 0 8C, 60-84%; g) DMT-Cl, pyridine, RT, 48% (30a), 40% (30b), 90% (31b), 62% (32a), 30% (32b); h) Hꢁnigꢂs
base, CEP-Cl, MeCN, RT, 72–87%.
For the synthesis of the isobicyclo guanosine, a slightly
different protecting-group strategy had to be employed due
to the notoriously high degree of insolubility imparted by
the guanine base. Thus the O(3’) and O(5’) acetyl groups in
10b were replaced by TBS groups, leading to 37b, which
was subjected to nucleosidation with 2-amino-6-chloropur-
ine under classical Vorbrꢁggen conditions giving nucleosides
38a/b in an anomeric ratio of 1:1 in acceptable yields
(Scheme 3). Treatment of 38a/b with sodium hydride and 3-
hydroxypropionitrile exchanged the C(6’)-chloro substituent
by oxygen (!39a/b). Further protection of the exo amino
function of the guanine base yielded amidine 40a/b. To
invert the configuration at C(6’), selective desilylation at
this position was desirable. This goal was achieved by the
use of HF/Et3N (!41a/b). Although desilylation with fluo-
ride ions is known to be unspecific,[12] we observed high se-
lectivity towards deprotection of the sterically less-hindered
O(6’). The free hydroxyl group in 41a/b was then mesylated
(!42a/b), inverted (!43a/b), and the acetyl function re-
moved with concomitant removal of the amidine protecting
group, leading to the chromatographically separable anom-
ers 44a and 44b. Re-protection of the N(2) amino function
in the b-anomer (!45b) and dimethoxytrityl (DMT) pro-
tection by using standard conditions yielded 46b in good
yields. Subsequent deprotection with TBAF (!47b) fol-
lowed by phosphitylation finally concluded the synthesis of
the isobicyclo-G building block 48b. Again, the relative con-
figuration at C(1’) was confirmed by using 1H NMR-
ROESY on compound 47b (the Supporting Information).
on a DNA synthesizer on the 1.3 mmol scale. Either natural
deoxynucleoside-derived controlled pore glass (CPG) solid
support was used or, for the fully modified strands, a univer-
sal solid support was used. For the incorporation of the iso-
bicyclo-nucleoside building blocks the coupling time was in-
creased to 9 min. All other steps remained unchanged. After
the chain-assembly the oligonucleotides were cleaved from
the solid support and deprotected by aminolysis at 708C
overnight, then purified by using reverse phase or ion ex-
change HPLC. The expected masses were confirmed by
ESIꢀ mass spectrometry.
Biophysical properties of oligonucleotides containing isobi-
cyclo-thymidines: Oligonucleotides S1–S4 containing single
and consecutive isobicyclo-thymidine modifications were
synthesized and their pairing properties with complementary
DNA and RNA investigated by UV melting curves at
260 nm. (Table 2 and Figures S1 and S2, the Supporting In-
formation). Analysis of the Tm data revealed a stabilization
of 18C per modification with complementary DNA. This
occurs in all four duplexes independently of the position or
the number of modifications in the strand. In the case of
RNA, the isobc-T modification seems to be stabilizing for
single incorporations but destabilizing for consecutive incor-
porations. As expected, the CD-spectra of S1–S4 with com-
plementary DNA and RNA do not deviate significantly in
shape from that of S5, indicating minor structural perturba-
tions imparted by the isobc-T residues (Figures S5 and S6,
the Supporting Information).
Oligonucleotide synthesis: Modified oligonucleotides were
Synthesis and properties of fully modified oligonucleotides
synthesized by using standard phosphoramidite chemistry
containing isobc-T and isobc-A: As for the synthesis with
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Scheme 3. a) 2-Amino-6-chloropurine, BSA, TMSOTf, MeCN, 608C, 5 h, 64%; b) NaH, 3-hydroxypropionitrile, THF, 08C to RT, 3 h; c) N,N-DMF-di-
methylacetal, DMF, 558C, 3 h, 71% over two steps; d) HF/Et3N, THF, RT, 55%; e) MsCl, pyridine, RT, 92%; c) CsOAc, DMSO, 858C, 16 h, 85%; f) 1m
ACHTUNGTRENNUNG
KOH in 5:3 MeOH/H2O, 60 8C, 42 % (53b), 20% (53a); g) N,N-DMF-dimethylacetal, DMF, 558C, 3 h, 64%; h) DMT-Cl, pyridine, RT, 16 h, 94%;
i) TBAF, THF, RT, 5 h, 70%; j) Hꢁnigꢂs base, CEP-Cl, MeCN, RT, 1 h, 88%.
Table 2. Tm values derived from UV-melting-temperature experiments at
260 nm. Conditions: NaH2PO4 (10 mm), NaCl (150 mm), pH 7.0, c=
1.2 mm strand concentration.
Table 3. Tm values derived from UV-melting-temperature experiments at
260 nm. Conditions: NaH2PO4 (10 mm), NaCl (150 mm), pH 7.0, c=
1.2 mm strand concentration.
Sequence[a]
Complementary
DNA
Complementary
RNA
Sequence
Tm vs. DNA
antiparallel
Tm vs. RNA
antiparallel
Tm vs. DNA
parallel
S1
S2
S3
S4
S5
5’-d(GGAtGTTCTCGA)-3’
5’-d(GGATGttCTCGA)-3’
5’-d(GGATGTTCtCGA)-3’
5’-d(GGAtGttCtCGA)-3’
5’-d(GGATGTTCTCGA)-3’
48.0
49.0
48.0
51.0
47.0
49.0
47.5
50.0
49.5
49.0
S6 6’-d(ataatttaataa)-3’
S7 6’-d(ttattaaattat)-3
S8 5’-d(ATAATTTAA-
TAA)-3’
S9 5’-d(TTATTAAAT-
TAT)-3’
25.1
25.9
23.2
<10
21.9
28.1
19.8
<10
n.m.[a]
23.2
11.2
n.m.[a]
[a] Upper case letters: natural nucleotides; lower case letters: isobc-
nucleotides.
[a] Not measured.
single isobc-T incorporations, a non-self-complementary se-
quence motif was chosen. S6 and S7 (Table 3) are comple-
mentary and can therefore give insight into the self-pairing
of isobicyclo-DNA. The melting curves of S6 and S7 with
complementary DNA and RNA partially confirm the results
obtained with the thymidine-modified oligonucleotides. All
duplex melting curves reflect cooperative and reversible
melting (Figure 3). The isobc modifications stabilize duplex-
es with DNA, but not as predicted from single incorpora-
tions with 18C per modification. The overall stabilization is
+2 (S6) and +38C (S7) per duplex, respectively (Table 3).
In the case of RNA as a complement, duplex destabiliza-
tion with a Tm value below 108C is observed. This was pre-
dicted from the earlier experiments in which we have shown
that single incorporations stabilize the duplex and consecu-
tive modifications destabilize it. Thermal denaturation ex-
periments were also performed for the fully modified
duplex S6/S7. The Tm in this case is remarkably high with a
value of 41.98C, which is almost twice as high as that for the
natural DNA duplex. Furthermore, to check the specificity
for the antiparallel orientation in the duplex, melting tem-
peratures for the parallel duplexes with DNA were meas-
ured (Figure S3, the Supporting Information). Both S6 and
S7 showed Tm values that are comparable to the ones meas-
ured for the antiparallel duplexes (ꢀ3 for S6 and +28C for
S7, Table 3). The same is true for the natural duplex, which
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C. J. Leumann and A.-B. Gerber
Figure 3. UV-melting curves (260 nm) of the AT-duplexes. Conditions:
NaH2PO4 (10 mm), NaCl (150 mm), pH 7.0, c=1.2 mm strand concen-
Figure 4. CD spectrum of the duplex S6:S7 at different temperatures.
Conditions: 10 mm NaH2PO4, 150 mm NaCl, pH 7, c=1.2 mm strand con-
centration.
~
!
&
*
DNA:RNA, + DNA:DNA,
G
S7:RNA,
S6:S7,
^
* S6:DNA, S7:DNA.
was slightly destabilized by ꢀ38C compared with the anti-
parallel duplex.
close to the one obtained in our case.[13] However, the nega-
tive band between 260 and 290 nm in the S6:S7 duplex is
most likely not the result of a partial parallel isobc-AT
duplex structure because achieving a melting temperature of
41.98C with a minimum of 6 mismatches in a dodecamer
seems very unlikely. Hence we hypothesize that there exists
a different, yet undetermined duplex conformation for pure
isobc-AT duplexes.
It thus appears that, much like natural DNA,[13] also
isobc-AT sequences have the potential to form parallel re-
verse Watson–Crick duplexes. It also becomes clear that
isobc-AT-oligonucleotides do not have a considerable stabi-
lizing effect on duplexes with DNA but have a significant
destabilizing effect on duplexes with RNA as complement.
In contrary, the fully unnatural duplex is significantly more
stable compared with pure DNA or hybrid (isobicyclo-
DNA/DNA) duplexes. This is not uncommon for sugar-
modified oligonucleotide analogues, such as hexitol nucleic
acids (HNA)[14] or tricycloDNA, and reflects most likely a
more perfect geometric match of the repeating backbone
units in homo-backbone duplexes as compared with hetero-
backbone duplexes.
Thermodynamics of duplex formation: By using standard
curve-fitting procedures to experimental melting curves[16]
we calculated the thermodynamic data of duplex formation
of the fully modified duplex S6:S7 as well as the hybrid
Table 4. Thermodynamic data of duplex formation from curve fitting to
the experimental melting curve. Conditions: NaH2PO4 (10 mm), NaCl
(150 mm), pH 7.0, c=1.2 mm strand concentration.
To gain an insight into the structural properties of these
AT duplexes, CD spectra were measured. The CD spectra
for the natural duplex S8:S9 as well as for the hybrid duplex
S6:S9 (Figure S7 and S8, the Supporting Information)
showed a B-like conformation and, in the case of the fully
modified duplex S6:S7, the CD spectrum showed a signature
that is significantly different from A-, B-, or Z-DNA
(Figure 4). The CD traces of the single strands (at high tem-
perature) are similar to the unmodified ones and the hybrid
duplexes. In the paired state, however, the CD shows an in-
tense negative band between 290 and 260 nm, which mirrors
the band of the unmodified duplex in this part of the spec-
trum (Figure 4). For the parallel duplexes in all three cases a
B-type conformation is observed (Figure S9, the Supporting
Information).
Duplex
DH
DS
DG at 258C
[kcalmolꢀ1
]
[calmolꢀ1 Kꢀ1
]
[kcalmolꢀ1
]
S6:S7
S6:S9
S8:S9
ꢀ73.0
ꢀ84.7
ꢀ76.6
ꢀ202.8
ꢀ254.0
ꢀ228.9
ꢀ12.6
ꢀ8.9
ꢀ8.3
duplex S6:S9 and compared the data to that of the natural
duplex S8:S9 (Table 4). From there it appears that the natu-
ral duplex S8:S9 is enthalpically favored over the fully modi-
fied duplex. The free energy DG at 258C is, as expected
from the Tm data, in favor of the fully modified duplex
S6:S7. Thus, the fully modified duplex is entropically fa-
vored, which is in agreement with its reduced conformation-
al flexibility. The hybrid duplex shows a somewhat unusually
high enthalpy DH, which is counterbalanced by the entropy
term, indicating enthalpy/entropy compensation to be opera-
tive.
Reported CD spectra of natural, parallel AT-duplexes
were found to be sequence specific: A parallel duplex with
alternating AT base pairs shows
a negative band at
290 nm[15] whereas a d(A)10·d(T)10 hairpin shows a spectrum
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Table 6. Tm data for fully modified isobc-oligonucleotides containing all
four bases. Conditions: NaH2PO4 (10 mm), NaCl (150 mm), pH 7.0, strand
concentration for all dodecamers: c=1.2 mm. Total strand concentration
for the natural hexamers: 2.4 mm and for S11: 4.8 mm.
We also investigated the influence of salt concentration
on duplex stability by varying the NaCl concentration of the
buffer in a range from 0.05–1.5m. The uptake of sodium
ions upon duplex formation helps to stabilize the duplex by
reducing the repulsion between the phosphate groups. Since
the negative charge density is higher in a duplex than in
single strands, counterions are screened upon duplex forma-
tion. As expected, an increase of the Tm values with increas-
ing NaCl concentration was observed (Figure 5). The du-
Sequence
DNA
RNA
isobc
antiparallel
antiparallel
duplex
[a]
S10
S11
6’-d(cctactagagct)-3’
51.0 (+4)
<10
30.0 (ꢀ11.5)
–
6’-d
G
34.5
40.7
[a] Not detected. [b] Self-complementary duplex: Tm data refer to the re-
spective backbone type.
Synthesis and properties of oligonucleotides containing all
four isobc-nucleosides: Two fully modified sequences con-
taining isobc-A, -T, -G, and -C residues were synthesized: a
dodecamer (S10) for the investigation of the pairing proper-
ties with complementary DNA and RNA and a self-comple-
mentary hexamer (S11) to get further insight into the struc-
ture of fully modified duplexes (Table 6). Thermal melting
of S10 with complementary DNA shows, as all modified
DNA sequences, a cooperative and reversible melting be-
havior (Figure 6). Again, a stabilization of the duplex was
Figure 5. Plot of the Tm versus lnACHTNUGRTNEUNG[NaCl]; 10 mm NaH2PO4, 50 mm–1.5m
!
&
NaCl, pH=7.0, c=1.2 mm strand concentration.
DNA:DNA, S6:S7,
*
S6:DNA.
plexes S8:S9 and S6:S9 both doubled their Tm values within
the 30-fold increase of salt concentration. The fully modified
duplex S6:S7 increased its Tm by 358C, which is slightly
more than twice the starting value at 0.05m NaCl. We fur-
ther determined the relative counterion uptake Dn (Table 5)
according to classical polyelectrolyte theory,[17] by using the
transition enthalpy (DH) data from Table 4.
Table 5. Calculated counterion uptake. Conditions: NaH2PO4 (10 mm),
NaCl (200 mm), pH 7.
Figure 6. UV melting curves (260 nm) for the modified sequence S7 with
DNA (left) and RNA (right). Conditions: NaH2PO4 (10 mm), NaCl
&
*
(150 mm), pH 7.0, c=1.2 mm strand concentration. S11,
RNA hexa-
S8:S9
S6:S9
S6:S7
~
!
AHCTUNGTRENNGmUN er, S10:DNA, DNA:DNA, * S10:RNA, + DNA:RNA.
Dn
dTm/d(ln
1.1
5.3
1.6
7.2
2.1
11.8
N
observed but, even including the more stable CG base pairs,
the difference in melting temperature is small (+48C/
duplex) and comparable to the one observed in the AT-
series (Table 6). In addition, the melting behavior of S10
with a parallel DNA complement was investigated. No co-
operative transition was observed revealing that isobc-DNA,
as DNA, strongly prefers antiparallel Watson–Crick pairing
in sequences containing all four base-pairs. For the hexamer
S11 a very stable duplex was found with for GC base-pairs
usually low hyperchromicities at 260 nm (Figure 6). This
again confirms that fully modified isobc-duplexes are very
stable, irrespective of the base composition.
2
Dn ¼ ꢀ2DH=RTm ꢁ dTm=dðln½NaClꢂÞ
The fully modified isobc system S6:S7 screens about twice
as much counterions upon duplex formation than the corre-
sponding unmodified duplex and about a third more than
the hybrid duplex. This is in agreement with a higher degree
of spatial compression of the phosphate groups upon transi-
tion from single strands to the duplex. Whether this is due
to a more compressed duplex structure or a more relaxed
single strand structure remains unknown at this point.
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C. J. Leumann and A.-B. Gerber
Table 7. Mismatch discrimination. The DTm values relative to the match-
ed duplex are given in parentheses. Conditions: NaH2PO4 (10 mm), NaCl
(150 mm), pH 7.0, c=1.2 mm strand concentration.
Duplex
X=A X=G X=C X=T
(match)
6’-d(cctactagagct)-3’ (S10)
5’-d(ACGXCTTGTAGG)-3’
5’-d(CCTACAAGAGCT)-3’
5’-d(ACGXCTTGTAGG)-3’
37
(ꢀ14) (ꢀ8)
34 39
(ꢀ13) (ꢀ8)
43
33
51
(ꢀ18)
30
47
(ꢀ17)
complement was also tested. After incubation at 378C in
medium containing 10% FBS, aliquots were taken in regular
intervals and after ethanol precipitation analyzed by PAGE.
The gel was visualized with stains-all solution (Figure 8).
Figure 7. CD spectra of S11:S11, S10:DNA, and S10:RNA. Conditions:
NaH2PO4 (10 mm), NaCl (150 mm), pH 7, c=1.2 mm strand concentra-
tion.
The CD spectra of the hybrid duplex (S10:DNA,
Figure 7) revealed a completely different curve shape com-
pared with the unmodified duplex (Figure S13, the Support-
ing Information), indicating substantial deviation from clas-
sical A- or B-type structures. In contrast, in the case of the
RNA complement, a typical A-form is observed with com-
parable spectra for the unmodified and the modified duplex.
The CD signature of the S11:S11 duplex, however, shows an
intense negative cotton effect at 290 nm and a positive
cotton effect at 260 nm (Figure 7). It thus deviates signifi-
cantly from that of the self-complementary RNA hexamer
(A-type conformation, data not shown) and also to some
extent from the fully modified isobc-AT duplex S6:S7
(Figure 3). It thus shows some similarities to that of Z-
DNA[18] which, given the high GC content, could be a possi-
ble structure for S11. However, more high resolution struc-
tural work is needed to get a reliable picture of the 3D
structure.
Figure 8. PAGE analysis of the serum stability of DNA (left) and isobi-
AHCTUNGTREGcNNUN yclo-DNA (right). 1: 0 h, 2: 0.5 h, 3: 2 h, 4: 6.5 h, 5: 21 h.
The resulting gel shows that S10 is stable over a period of
at least 21 h without signs of degradation. In contrast, the
DNA complement is substantially hydrolyzed after this
period of time, indicating a significantly higher degree of bi-
ostability of isobc-DNA.
RNaseH activity: Due to its ability to cleave DNA/RNA
heteroduplexes, it is desirable for chemically modified oligo-
nucleotides designed to ablate an RNA of interest, to be
able to activate the endogenous enzyme RNaseH. This is,
however, difficult to achieve since only a small number of
DNA analogues are known to be compatible with RNaseH
activity.[20] To investigate this, the RNA complement of S10
was 5’-labeled with [g-32P]-ATP and T4 polynucleotide
kinase (T4 PNK) and annealed to S10. The natural RNA/
RNA and the hybrid DNA/RNA duplexes served as nega-
tive and positive control. As expected, the RNA/RNA
duplex did not show any RNaseH-mediated degradation,
whereas the DNA/RNA hybrid turned out to be a good sub-
strate. The S10/RNA duplex showed no signs of RNA cleav-
age after treatment with RNaseH, clearly showing that
isobc-DNA does not elicit RNaseH activity (Figure 9).
Mismatch discrimination: To test the base-pairing selectivity
of the isobc-DNA system, the melting temperatures of dif-
ferent mismatched duplexes complementary to S10 were
measured. A thymidine residue in the complementary DNA
sequence was replaced by an A, C or G, and the correspond-
ing Tm values were determined (Table 7).
In all cases thermal destabilizations in the mismatched du-
plexes were observed. The A–C mismatch was the strongest
discriminating followed by the A–A and the A–G mismatch-
es. The decrease is generally of about the same extent as in
DNA, indicating an equal selectivity of the two Watson–
Crick pairing systems.
Serum stability: The serum stability of S10 was measured in
heat deactivated fetal bovine serum (FBS) in which the pre-
dominant nucleases are the 3’-exophosphodiesterases.[19] As
a control the stability of the corresponding natural DNA
Discussion
We synthesized all four isobicyclo nucleosides and incorpo-
rated them successfully into DNA. Single isobicyclo-T incor-
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Isobicyclo-DNA
FULL PAPER
to low electrolyte concentration, which supports the prefer-
ence of an X-type conformation of isobc-DNA duplexes at
physiological salt concentration.
The fully modified isobc-AT oligonucleotides form also
stable parallel duplexes. Parallel duplex formation in poly-
AHCTUNGTRENNUNG
(dAT) sequences is well known and investigated.[15,24] They
require reversed Watson–Crick base-pairs and their thermal
stability is highly sequence dependent. This explains the var-
iability in the Tm values for the two duplexes S6 and S7 with
parallel complements. However, no melting transition was
found for the fully modified S10, containing all four bases,
with a parallel DNA complement, revealing that isobc-DNA
prefers strictly antiparallel pairing in sequences containing
also G–C base-pairs. These preferences are identical to that
of natural DNA where parallel reversed Watson Crick is
also restricted to AT sequences.
Figure 9. PAGE analysis of the RNaseH acitvity of the duplexes:
RNA:32P-RNA (C12:32P-C9), isobicyclo-DNA:32P-RNA (S7:32P-C9),
DNA:32P-RNA (C8:32P-C9). 1: 5 min, 2: 45 min, 3: 120 min, 4: 300 min.
Isobc-DNA was found to be unable to activate RNaseH
in isobc-DNA/RNA hybrids. RNaseH is known to prefera-
bly bind to A-form rather than B-form duplexes. Hence, as
a consequence the enzyme binds RNA/RNA duplexes and
porations in natural DNA showed stable duplexes with com-
plementary DNA and RNA. Consecutive incorporations de-
stabilize the RNA hybrid. The same result was obtained for
the fully modified AT duplexes where a stabilized duplex
with DNA and a significantly destabilized duplex with com-
plementary RNA was found. An interesting finding in the
AT series was the very stable self-duplex with a Tm of
41.98C. The CD spectrum of this fully modified isobicyclo
duplex (Figure 3) revealed a structure different from A- or
B-form duplex.
DNA/RNA hybrids much tighter than
a DNA/DNA
duplex.[25] The RNA in dsRNA duplexes is not cleaved since
the 2’-OH groups affect the hydration pattern in the minor
groove.[26] Since the CD spectrum of the isobicyclo-DNA/
RNA duplex shows an A-like structure and is very similar
to that of the DNA/RNA duplex, an activation of RNaseH
could have been expected. However, it has also been shown
that the enzyme is very sensitive to structural changes
within the minor groove. This is the reason why all 2’ modi-
fied oligonucleotides (e.g., 2’-OMe[27]) as well as the sterical-
ly constrained (e.g., LNA[28]; tcDNA[9a]) do not activate
RNaseH.[26] From this point of view it is not surprising that
isobc-DNA is unable to do so.
It is known that the duplex conformation as revealed by
CD-spectroscopy is dependent on the base sequence and on
the salt concentration of the buffer. Increasing the salt con-
centration typically leads to a decrease of the long wave-
length band between 290 and 260 nm corresponding to var-
iants of B-DNA structures differing in the number of base
pairs per helix turn.[21] Vorlꢃckovꢄ and Kypr investigated the
ˇ
conformational changes of polyACTHNUGRTNEUNG(A–T) sequences at different
CsF concentrations and found that the long wavelength part
of the CD spectra falls off, inverts and becomes negative at
high salt concentrations.[22] They called that structure X-
DNA and suggested that this X-DNA is a right handed anti-
parallel double helix with Hoogsteen base-pairs that were
found by Abrescia et al. some years ago.[21,23] In this Hoog-
Conclusion
We have achieved the synthesis of all four isobc-DNA build-
ing blocks starting from the silyl enol ethers 1a or 1b. These
phosphoramidites were successfully incorporated into natu-
ral deoxyoligonucleotides or were used for the synthesis of
fully modified isobc-DNA by using standard solid-phase
DNA synthesis. Oligodeoxynucleotides containing single in-
corporations as well as fully modified isobc-DNA show
stable duplexes with DNA complements with a slightly en-
hanced stability (ca., 0.38C per mod). RNA is discriminated
as a complement in all the investigated duplexes by approxi-
mately 18C per modification. All hybrid duplexes show B-
conformation with DNA and A-conformation with RNA as
judged from CD-experiments. Base mismatches are discrimi-
nated in the same way as in natural DNA. Fully modified
isobicyclo oligonucleotides form very stable duplexes within
their own backbone series that are of higher thermodynamic
stability than the corresponding natural duplexes. Fully
modified duplexes containing only AT base-pairs show a
CD-signature that is significantly different from that of can-
onical DNA conformations. Based on the available biophysi-
ACHTUNGTRENNUNG
steen DNA[23] the overall helical parameters are similar to
that of B-DNA with the adenines in a syn-conformation.
Therefore one hypothesis is that the fully modified isobicy-
clo-AT duplex S6:S7 forms such an X-DNA structure.
Further evidence for such a X-DNA structure comes from
CD-experiments with S6:S7 as well as the hybrid DNA du-
plexes of S6 at different salt concentrations. While no major
changes occur in the case of the unmodified duplex S8:S9
and the hybrid duplex S6:S9 (Figures S7 and S8, the Sup-
porting Information), the CD of the fully modified duplex
S6:S7 shows a high dependence on the sodium chloride con-
centration (Figure S10, the Supporting Information). The
negative band at 275 nm increases in intensity upon increas-
ing the NaCl concentration. This could indeed be an indica-
tion of a partial structural transition from the more compact
X- towards a B-type conformation when changing from high
Chem. Eur. J. 2013, 00, 0 – 0
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C. J. Leumann and A.-B. Gerber
(3R,5S,8R,7S)-8-[(1H-Imidazole-1-carbonthioyl)oxy]-3-methyoxyhexahy-
dro-2H-cyclopenta[b]furan-3a,6-diyl diacetate (8b): TCDI (4.2 g,
23.3 mmol) was added to a solution of 6b (3.2 g, 11.7 mmol) in THF
(60 mL). The yellow solution was heated at reflux at 758C for 3 h. The
THF was then evaporated and the crude mixture was purified by FC
(ethyl acetate/hexane 1:1) to yield 8b (4.2 g, 10.9 mmol, 93%) as a
yellow foam. Rf =0.40 (ethyl acetate/hexane 1:1); 1H NMR (400 MHz,
CDCl3): d=8.39 (m, 1H) 7.67 (m, 1H), 7.04 (m, 1H) 5.92–5.85 (m, 1H)
5.70–5.67 (m, 1H), 5.08, 5.06 (2m, 2H) 3.34 (s, 3H), 2.94 (dd, 1H, J=
13.9, 8.1), 2.58 (d, 1H, J=14.0), 2.49 (ddd, 1H, J=14.1, 5.7, 1.8), 2.14–
2.10 (m, 1H) 2.08, 2.05 ppm (2s, 6H); 13C NMR (101 MHz, CDCl3): d=
183.54, 170.40, 170.29, 137.40, 131.24, 118.26, 106.14, 89.25, 84.34, 82.33,
73.85, 55.33, 47.82, 39.38, 21.55, 21.15 ppm; HRMS (NSI+, EtOAc): m/z
calcd for C16H20N2O7S: 385.1064; found: 385.1051 [M+H+].
cal data we propose a double helical structure with Hoog-
ACHTUNGTRENNUNGsteen base-pairs. Furthermore, isobc-DNA is stable in
bovine serum and does not activate RNaseH.
Thus it appears that the phosphate shift from C(5’) to
C(6’) in the bicyclic system is well tolerated in duplexes with
DNA and less well in duplexes with RNA. Pure isobc-DNA
duplexes, however, display considerable structural differen-
ces of a yet unknown type. The feature of isobc-DNA to
stablize DNA duplexes and discriminate RNA is interesting
and could find possible applications in diagnostics, as for ex-
ample, in primers for the polymerase chain reaction (PCR).
(3R,5S,7S)-3-Methoxyhexahydro-2H-cyclopenta[b]furan-3a,6-diyl diace-
tate (10b): The sugar 8b (4.2 g, 10.9 mmol), AIBN (270 mg, 2.2 mmol),
and TTMSS (6.7 mL, 22 mmol) were dissolved in toluene (87 mL) and
stirred for 2 h at 858C. The toluene was then evaporated and the crude
mixture purified by FC (ethyl acetate/hexane 1:3). Compound 10b (2.6 g,
10.1 mmol, 92%) was obtained as a colorless oil. Rf =0.6 (ethyl acetate/
hexane 1:1); 1H NMR (300 MHz, CDCl3): d=5.39–5.31 (p, 1H, J=6.3),
5.04 (dd, 1H, J=5.6, 0.7), 4.71 (dd, 1H, J=7.0, 3.5) 3.33 (s, 3H), 2.78–
2.71 (dd, 1H, J=14.6, 6.2), 2.60 (d, 1H, J=14.0), 2.33 (ddd, 1H, J=14.2,
5.7, 0.8), 2.29–2.07 (m, 3H) 2.02, 2.00 ppm (2s, 6H); 13C NMR (75 MHz,
CDCl3): d=170.58, 170.37, 106.61, 92.63, 87.86, 75.03, 54.99, 46.55, 44.15,
39.02, 21.65, 21.28 ppm; HRMS (NSI+, MeOH): m/z calcd for C12H18O6:
281.0996, found: 281.1002 [M+Na+].
Experimental Section
General: All reactions were performed under Ar and in dried glassware.
Anhydrous solvents for reactions were obtained by filtration through ac-
tivated alumina or by storage over molecular sieves (4 ꢅ). Column chro-
matography (CC) was performed on silica gel with an average particle
size of 40 mm. All solvents for column chromatography were of technical
grade and distilled prior to use. Thin-layer chromatography was per-
formed on silica gel plates. Visualization was achieved either under UV
light or by dipping in staining solution (CerIV-sulfate (10.5 g), phosphor-
molybdenic acid (21 g), conc. H2SO4 (60 mL), H2O (900 mL) or p-anisal-
dehyde (10 mL), conc. H2SO4 (10 mL), glacial acetic acid (2 mL), ethanol
(180 mL)) followed by heating with a heat gun. NMR spectra were re-
corded at 300 or 400 MHz field width (1H) in either CDCl3 or CD3OD.
Chemical shifts (d) are reported in ppm relative to residual undeuterated
solvent CHCl3: 7.26 ppm (1H) and 77.16 ppm (13C); CHD2OD: 3.31 ppm
(1H) and 49.0 ppm (13C), J in Hz. Signal assignments are based on DEPT
(3’S,6’S(6’R))-1-[3’,6’-Di-O-acetyl-2’-deoxy-3’,5’-ethano-a- and -b-d-ribo-
furanosyl] thymine (12a/b): Thymine (293 mg, 2.3 mmol) was suspended
in MeCN (12 mL) and BSA (1.4 mL, 5.8 mmol) was added. The suspen-
sion was stirred until the solution became clear. The solution was then
cooled to 08C and 10b (300 mg, 1.2 mmol, dissolved in 6 mL MeCN) and
TMSOTf (0.63 mL, 3.5 mmol) were added. After 1 h at 08C the mixture
was allowed to warm to r.t. and the stirring was continued for another
16 h. It was then diluted with ethyl acetate and washed with sat.
NaHCO3. Purification by using FC (ethyl acetate/hexane 4:1) yielded the
desired nucleoside 12a/b (355 mg, 1.0 mmol, 87%) in a anomeric ratio
(a/b) of approximately 1:1 as a white foam. Rf =0.49 (ethyl acetate/
and on H-1H and H-13C correlation experiments (COSY/HMSC). High-
resolution mass spectra were recorded on an Applied Biosystems Sciex
QSTAR Pulsar.
1
1
(3R,5S,8R,7S)-8-[(tert-Butyldimethylsilyl)oxy]-3-methoxyhexahydro-2H-
cyACHTUNGTRENNUNGclopenta[b]furan-3a,6-diyl diacetate (4b): A solution of 2b (4.3 g,
1
hexane 4:1); H NMR (400 MHz, CDCl3): d=8.29 (m, 2H), 7.27 (s, 1H),
7.11 (s, 1H), 6.12 (m, 2H), 5.23 (m, 2H), 4.93 (dd, 1H, J=6.4, 3.1), 4.56
(d, 1H, J=5.6), 2.86 (m, 2H), 2.79 (ddd, 1H, J=13.8, 6.3, 1.3), 2.66 (m,
1H), 2.54 (m, 1H), 2.36 (m, 1H), 2.25 (m, 2H), 2.16 (m, 1H), 2.10 (m,
2H), 2.08 (s, 3H), 2.06 (s, 3H), 2.04 (2s, 6H), 2.00 (m, 1H)1.96 (m, 3H),
1.95 ppm (m, 3H); 13C NMR (75 MHz, CDCl3): d=170.69, 170.49,
170.35, 170.15, 163.73, 163.44, 150.30, 150.28, 135.31, 134.44, 112.04,
111.11, 91.21, 90.17, 87.69, 86.78, 85.33, 83.74, 73.65, 73.23, 44.46, 44.35,
43.64, 43.37, 37.84, 36.47, 21.73, 21.63, 21.19, 21.16, 12.86, ꢀ1.73 ppm;
HRMS (NSI+, MeCN): m/z calcd for C16H20N2O7: 375.1163; found:
375.1145 [M+Na+].
14.2 mmol) in pyridine (35 mL) was cooled to 08C and DMAP (171 mg,
1.4 mmol) and Ac2O (4.0 mL, 42.7 mmol) were added. The mixture was
stirred for 16 h at RT and, after cooling to 08C, was carefully quenched
and washed with sat. NaHCO3. The aqueous layers were extracted with
CH2Cl2. The combined organic layers were dried over MgSO4 and evapo-
rated. The product was separated by flash chromatography (FC) (ethyl
acetate/hexane, 1:3) yielded 4b (4.9 g, 12.6 mmol, 90%) as a white solid.
Rf =0.9 (hexane/ethyl acetate 1:1); 1H NMR (400 MHz, CDCl3): d=5.24
(m, 1H), 5.06 (d, 1H, J=5.3), 4.36 (d, 1H, J=6.4), 4.20 (dd, 1H, J=8.6,
6.4), 3.36 (s, 3H), 2.86 (dd, 1H, J=13.9, 7.6), 2.55 (d, 1H, J=13.8), 2.36
(ddd, 1H, J=13.8, 5.7, 1.8), 2.02 (s, 6H), 1.76 (ddd, 1H, J=13.8, 9.7, 1.6),
0.90 (s, 9H), 0.11 (s, 3H), 0.08 ppm (s, 3H); 13C NMR (101 MHz, CDCl3):
d=170.55, 170.17, 105.42, 89.29, 85.33, 77.97, 75.80, 54.62, 47.69, 40.15,
25.89, 21.62, 21.16, ꢀ4.60, ꢀ4.71 ppm; HRMS (NSI+, MeCN): m/z calcd
for C18H32O7Si: 411.1813; found: 411.1810 [M+Na+].
(3’S,6’S)-N6-Benzoyl-9-[3’,6’-di-O-acetyl-2’-deoxy-3’,5’-ethano-a- and -b-d-
ribofuranosyl] adenine (13a/b): BSA (1.9 mL, 7.6 mmol) was added to a
suspension of N6-benzoyl-adenine (926 mg, 3.9 mmol) in MeCN (15 mL).
The mixture was stirred at RT until the solution was clear. Compound
10b (500 mg, 1.9 mmol, solved in 4 mL MeCN) and TMSOTf (0.1 mL,
0.6 mmol) were added and the solution was stirred for 2 h at 858C. After
cooling to RT the mixture was quenched with sat. NaHCO3 and extracted
with ethyl acetate. The combined organic layers were dried over MgSO4
and evaporated. FC (3% methanol in CH2Cl2) yielded 13a/b (654 mg,
1.4 mmol, 74%) in an anomeric ration of ꢃ1:1 as a white foam. Rf =0.65
(10% methanol in CH2Cl2); 1H NMR (300 MHz, CDCl3): d=8.81, 8.79
(2s, 2H) 8.24, 8.15, (2s, 2H) 8.03 (m, 4H), 7.64–7.59 (m, 2H), 7.55–7.50
(m, 4H), 6.45 (dd, 1H, J=6.9, 2.7), 6.34 (dd, 1H, J=8.4, 6.4), 5.55–5.46
(m, 1H, H-C(6’), b), 5.31–5.21 (m, 1H), 4.89 (dd, 1H J=6.1, 1.0), 4.73
(dd, 1H, J=6.5, 1.7), 3.44 (dd, 1H, J=15.4, 2.7), 3.03–2.90 (m, 4H),
2.88–2.81 (m, 1H) 2.45–2.36 (m, 2H), 2.22–2.12 (m, 4H), 2.12 (s, 3H),
2.06, 2.05 (2s, 6H), 1.95 ppm (s, 3H); 13C NMR (101 MHz, CDCl3): d=
170.60, 170.55, 170.47, 170.21, 164.72, 153.09, 152.97, 149.89, 149.72,
141.43, 141.25, 133.85, 133.07, 133.03, 129.13, 128.07, 123.67, 91.03, 90.99,
(3R,5S,8R,7S)-8-Hydroxy-3-methoxyhexahydro-2H-cyclopenta[b]furan-
3a,6-diyl diacetate (6b): A solution of 4b (10.4 g, 26.8 mmol) in THF
(80 mL) was cooled to 08C. HF/Et3N (37% HF, 23 mL, 53.6 mmol) was
added and the solution was allowed to warm to RT. The reaction mixture
was stirred for 60 h at RT and subsequently quenched with silica. The sol-
vent was evaporated and the crude mixture purified by using FC (ethyl
acetate/hexane 1:1). Compound 6b (5.9 g, 21.5 mmol, 80%) was obtained
as a colorless oil. Rf =0.4 (ethyl acetate/hexane 1:1), 1H NMR (300 MHz,
CDCl3): d=5.10–5.13 (m, 2H), 4.53 (d, J=6.4, 1H), 4.10 (m, 1H), 3.39
(s, 3H); 2.78–2.80 (m, 2H), 2.48 (m, 2H), 2.03 (s, 3H); 2.00 (s, 3H), 1.95–
1.86 ppm (m, 1H); 13C NMR (75 MHz, CDCl3): d=171.06, 170.09,
106.93, 89.23, 85.93, 78.71, 74.79, 55.79, 47.14, 39.93, 21.51, 21.14 ppm;
HRMS (NSI+, MeCN/H2O, 1% HF): m/z calcd for C12H18O7: 279.09447;
found: 297.09538 [M+Na+].
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Isobicyclo-DNA
FULL PAPER
87.65, 86.25, 85.85, 84.84, 77.55, 77.23, 76.91, 73.46, 73.26, 44.25, 44.17,
43.77, 37.02, 36.88, 21.81, 21.52, 21.23, 21.18 ppm; HRMS (NSI+, MeOH):
m/z calcd for C23H23N5O6: 466.1721; found: 466.1724 [M+H+].
7.5), 7.99 (m, 2H), 7.96 (m, 2H), 7.63 (m, 4H), 7.55 (m, 4H), 6.10 (m,
2H), 4.72 (dd, 1H, J=7.2, 2.1), 4.40–4.28 (m, 3H), 2.69 (dd, 1H, J=13.7,
5.2), 2.59 (dd, 1H, J=14.8, 6.7), 2.45 (dd, 1H, J=14.8, 2.3), 2.38–2.29 (m,
2H), 2.23 (m, 1H), 2.14 (m, 1H), 1.96–1.85 (m, 3H), 1.81–1.69 ppm (m,
2H); 13C NMR (101 MHz, MeOD): d=164.81, 146.56, 146.10, 145.61,
134.74, 134.69, 134.10, 134.04, 129.83, 129.82, 129.16, 129.11, 98.80, 97.96,
92.69, 91.59, 90.35, 88.28, 87.99, 87.32, 85.96, 85.89, 82.54, 80.66, 79.66,
72.15, 71.89, 71.53, 58.32, 57.06, 57.01, 48.88, 48.31, 46.79, 45.89, 43.79,
42.06, 41.63, 41.24, 18.36 ppm; HRMS (NSI+, MeCN) m/z calcd for
C18H19N3O7: 358.1397; found: 358.1387 [M+H+].
(3’S,6’S)-N6-Benzoyl-1-[3’,6’-di-O-acetyl-2’-deoxy-3’,5’-ethano-a- and -b-d-
ribofuranosyl] cytosine (14a/b): N-Benzoylcytosine (833 mg, 3.9 mmol)
was suspended in MeCN (20 mL), BSA (2.3 mL, 9.5 mmol) was added
and the suspension was stirred until the solution became clear. The solu-
tion was then cooled to 08C and 10b (500 mg, 1.9 mmol, dissolved in
20 mL MeCN) and TMSOTf (1 mL, 3.9 mmol) were added. After 1 h at
08C the mixture was allowed to warm to RT and the stirring was contin-
ued for another 16 h. It was diluted with ethyl acetate and washed with
sat. NaHCO3. Purification by FC (ethyl acetate/hexane 4:1) yielded the
desired nucleoside 14a/b (638 mg, 1.5 mmol, 76%) in an anomeric ratio
(a/b) of 1.5:1 as a white foam. Rf =0.26 (ethyl acetate/hexane 4:1);
1H NMR (300 MHz, CDCl3): d=8.87 (br, 2H), 7.92 (m, 4H), 7.63–7.47
(m, 8H), 6.10 (m, 2H), 5.20 (m, 2H), 5.03 (dd, 1H, J=6.8, 3.2), 4.66 (d,
1H, J=5.7), 3.19 (dd, 1H, J=14.8, 5.4), 2.91 (dd, 1H, J=15.4, 6.5), 2.70
(m, 3H), 2.30 (m, 1H), 2.25–2.11 (m, 5H), 2.07 (s, 3H), 2.04, 2.03 (2s,
6H), 1.99 (m, 1H), 1.93 ppm (s, 3H); 13C NMR (101 MHz, MeOD): d=
172.40, 172.25, 172.03, 171.74, 164.98, 146.02, 145.77, 134.66, 134.14,
134.12, 130.47, 129.84, 129.57, 129.17, 129.14, 123.36, 120.19, 98.89, 98.11,
92.31, 92.07, 91.40, 90.17, 89.95, 89.44, 88.06, 87.65, 75.13, 61.55, 45.90,
45.44, 44.12, 43.44, 38.32, 37.57, 21.47, 21.30, 20.90, 20.88, 20.86,
14.45 ppm; HRMS (NSI+, MeCN): m/z calcd for C22H23N3O7: 442.1609;
found: 442.1617 [M+H+].
(3’S,6’S)-1-[2’-Deoxy-6’-O-(methylsulfonyl)-3’,5’-ethano-a- and -b-d-ribo-
furanosyl] thymine (18a/b): A solution of 15a/b (294 mg, 1.09 mmol) was
dissolved in pyridine (15 mL) and cooled to 08C. MsCl (85 mL,
1.09 mmol) was added and the mixture was stirred at 08C and overnight
at 48C. After quenching by the addition of sat. NaHCO3 and washing
with ethyl acetate the combined organic layers were dried over MgSO4
and purified by FC (5% methanol in CH2Cl2) to yield 18a/b (250 mg,
0.72 mmol, 66%) in an anomeric ratio of a/bꢃ1.25:1 as a white solid.
Rf =0.45 (10% methanol in CH2Cl2); 1H NMR (400 MHz, DMSO): d=
11.35 (s, 1H), 11.27 (s, 1H), 7.68ACTHNUGTRENNUG(s, 1H), 7.55AHCTUNGTRENN(NGU s, 1H), 6.08 (m, 2H), 5.18
(m, 1H), 5.07 (m, 1H), 4.50 (dd, 1H, J=6.2, 2.0), 4.10 (dd, 1H, J=7.1,
2.2), 3.22 (s, 3H), 3.20 (s, 3H), 2.52 (m, 3H), 2.20 (m, 5H), 2.08 (m, 2H),
1.99 (m, 1H), 1.90 (m, 1H), 1.81 (m, 3H), 1.79 ppm (m, 3H); 13C NMR
(101 MHz, DMSO): d=163.80, 163.61, 150.45, 150.43, 136.44, 135.96,
109.70, 109.11, 88.09 (C4’), 86.61 (C4’), 85.51 (C1’), 84.10 (C1’), 83.45,
83.11, 80.49, 80.23, 45.40, 45.21, 45.05, 44.06, 38.32, 37.70, 37.55, 37.42,
12.2, 12.11 ppm; HRMS (NSI+, MeOH): m/z calcd for C13H18N2O7S:
347.0907; found 347.0902 [M+H+].
(3’S,6’S)-1-[2’-Deoxy-3’,5’-ethano-a- and -b-d-ribofuranosyl] thymine
(15a/b): The nucleoside 12a/b (480 mg, 1.36 mmol) was dissolved in 0.2m
NaOH in 5:4:1 THF/methanol/H2O (65 mL) at 08C: The solution was
stirred for one hour at 08C and quenched by the addition of ammonium
chloride. The solvents were then evaporated and the remains absorbed
on silica gel. Purification by FC (10% methanol in CH2Cl2) yielded com-
pound 15a/b (294 mg, 1.1 mmol, 81%) in an anomeric ratio of a/b
ꢃ1.25:1 as a white foam. Rf =0.19 (10% methanol in CH2Cl2), 1H NMR
(400 MHz, MeOD): d=7.74 (s, 1H), 7.40 (s, 1H), 6.10 (m, 2H), 4.55 (dd,
1H, J=6.6, 1.9), 4.38 (m, 1H,), 4.28 (m, 1H) 4.18 (dd, 1H, J=6.7, 1.0),
2.52 (dd, 1H, J=14.7, 7.1), 2.34 (m, 4H), 2.14 (m, 1H), 2.05 (m, 2H), H-
C(7’)), 1.90 (m, 3H), 1.89 (m, 3H), 1.88 (m, 2H), 1.80 (m, 1H), 1.71 ppm
(m, 1H); 13C NMR (101 MHz, MeOD): d=166.55, 166.22, 152.52, 152.23,
138.60 (C-6), 137.07 (C-6), 111.98, 111.06, 91.65, 89.49 88.58, 86.24, 85.50,
84.89, 71.98, 71.69, 49.33, 47.57, 46.94, 42.08, 41.15, 20.84, 12.51,
12.41 ppm; HRMS (NSI+, MeCN): m/z calcd for C12H16N2O5: 269.1132;
found: 268.1141 [M+H+].
(3’S,6’S)-N6-Benzoyl-9-[2’-deoxy-6’-O-(methylsulfonyl)-3’,5’-ethano-a-
and -b-d-ribofuranosyl] adenine (19a/b): A solution of 16a/b (153 mg,
0.4 mmol) was dissolved in pyridine (6 mL) and cooled to 08C. MsCl
(0.03 mL, 0.4 mmol) was added and the mixture was stirred for 2 h at RT.
Silica was then added and the pyridine was evaporated and shortly high
vac dried. FC (7% methanol in CH2Cl2) yielded the corresponding
anomers 19a (105 mg. 0.23 mmol, 57%) and 19b (73 mg, 0.16 mmol,
39%) as white foams. Compound 19a: Rf =0.5 (10% methanol in
CH2Cl2); 1H NMR (400 MHz, MeOD): d=8.74 (2 s, 2H), 8.09 (m, 2H),
7.65 (m, 1H), 7.57 (m, 2H), 6.56 (dd, 1H, J=7.1, 2.6), 5.21–5.14 (m, 1H),
4.59 (dd, 1H, J=6.3, 1.5), 3.12 (s, 3H), 2.92 (dd, 1H, J=14.9, 2.6), 2.84
(dd, 1H, J=14.8, 7.2), 2.67 (ddd, 1H, J=13.4, 6.5, 1.3), 2.40–2.35 (m,
1H), 2.26 –2.14 ppm (m, 2H); 13C NMR (101 MHz, MeOD): d=168.16,
153.11, 151.22, 144.78, 138.41, 134.99, 133.92, 129.78, 129.43, 125.29, 90.39,
86.96, 85.87, 81.13, 47.38, 47.26, 39.44, 38.11 ppm; HRMS (NSI+,
MeOH): m/z calcd for C20H21N5O6S: 460.1285; found: 460.1272 [M+H+];
Compound 19b: Rf =0.4 (10% methanol in CH2Cl2); 1H NMR
(400 MHz, MeOD): d=8.73 (s, 1H), 8.54 (s, 1H), 8.08 (m, 2H), 7.63 (m,
1H), 7.55 (m, 2H), 6.37 (dd, 1H, J=9.3, 5.9), 5.66–5.58 (m, 1H), 4.38
(dd, 1H, J=6.9, 1.8), 3.13 (s, 3H), 3.05 (m, 1H), 2.75 (ddd, 1H, J=13.0,
6.2, 1.3), 2.63 (dd, 1H, J=13.8, 5.9), 2.43 (m, 1H), 2.20 (ddd, 1H, J=
14.1, 8.5, 7.0), 2.10 ppm (dd, 1H, J=13.0, 8.9); 13C NMR (101 MHz,
MeOD): d=168.15, 153.20, 151.22, 144.75, 138.43, 134.96, 133.89, 129.79,
129.75, 129.41, 125.56, 89.26, 86.34, 85.54, 81.11, 46.85, 46.41, 39.32, 38.16,
18.39 ppm; HRMS (NSI+, MeOH): m/z calcd for C20H21N5O6S:
460.1285; found: 460.1273 [M+H+].
(3’S,6’S)-N6-Benzoyl-9-[2’-deoxy-3’,5’-ethano-a- and -b-d-ribofuranosyl]
adenine (16a/b): The nucleosides 13a/b (606 mg, 1.3 mmol) were dis-
solved in 0.2m NaOH in 5:4:1 THF/methanol/H2O (90 mL) at 08C: The
solution was stirred for one hour at 08C and quenched by the addition of
ammonium chloride. The solvents were then evaporated and the remains
absorbed on silica gel. Purification by FC (10% methanol in CH2Cl2)
yielded compound 16a/b (437 mg, 1.14 mmol, 88%) in anomeric ratio of
a/bꢃ1:1 as a white foam. Rf =0.2 (10% methanol in CH2Cl2); 1H NMR
(300 MHz, MeOD): d=8.76 (s, 1H), 8.70 (2s, 2H), 8.54 (s, 1H), 8.07 (m,
4H), 7.67–7.61 (m, 2H), 7.57–7.52 (m, 4H), 6.51 (dd, 1H J=7.2, 1.8),
6.33 (dd, 1H, J=9.7, 5.3), 4.64–4.58 (m, 1H), 4.49 (d, J=5.9), 4.37–4.26
(m, 2H), 2.91–2.83 (m, 2H), 2.72 (dd, 1H J=14.9, 7.3), 2.59 (dd, 1H, J=
13.5, 5.3), 2.48–2.37 (m, 2H), 2.21–2.07 (m, 2H), 1.92–1.74 ppm (m, 4H);
13C NMR (75 MHz, MeOD): d=153.36, 153.18, 152.95, 151.12, 151.08,
144.82, 144.29, 134.95, 133.88, 129.74, 129.41, 91.51, 90.22, 86.86, 86.06,
85.82, 85.41, 71.74, 71.54, 47.85, 47.03, 41.50, 41.32 ppm; HRMS (NSI+,
MeOH): m/z calcd for C19H19N5O4: 382.1510; found: 382.1512 [M+H+].
(3’S,6’S)-N6-Benzoyl-1-[2’-deoxy-6’-O-(methylsulfonyl)-3’,5’-ethano-a-
and -b-d-ribofuranosyl] cytosine (20a/b): A solution of 17a/b (415 mg,
1.16 mmol) in pyridine (16 mL) was cooled to 08C and MsCl (90 mL,
1.16 mmol) was added. The mixture was stirred overnight at 48C. Sat.
NaHCO3 was added and the aqueous phase was extracted with ethyl ace-
tate. The crude product 20a/b precipitated in the organic phase and was
used in the next experiment without further purification. Rf =0.49 (10%
methanol in CH2Cl2); 1H NMR (400 MHz, DMSO): d=11.21 (br, 2H),
8.23 (m, 2H), 8.00 (m, 4H), 7.62 (m, 2H), 7.51 (m, 4H), 7.37 (m, 2H),
6.11 (dd, 1H, J=9.3, 5.1), 6.04 (dd, 1H, J=6.8, 2.7), 5.68 (s, 1H), 5.53 (s,
1H), 5.20 (m, 1H), 5.10 (m, 1H), 4.67 (dd, 1H, J=6.9, 2.3), 4.23 (dd, 1H,
J=7.1, 1.9), 3.23 (s, 3H), 3.21 (s, 3H), 2.63 (dd, 1H, J=14.5, 6.9), 2.47
(m, 2H), 2.31 (m, 2H), 2.24 (m, 2H), 2.14 (m, 2H), 2.06 (m, 1H)
1.96 ppm (m, 2H); 13C NMR (101 MHz, DMSO): d=162.99, 145.29,
(3’S,6’S)-N6-Benzoyl-1-[2’-deoxy-3’,5’-ethano-a- and -b-d-ribofuranosyl]
cytosine (17a/b): The nucleoside 14a/b (316 mg, 0.72 mmol) was dis-
solved in 0.2m NaOH in 5:4:1 THF/methanol/H2O (50 mL) at 08C. The
solution was stirred for one hour at 08C and quenched by the addition of
ammonium chloride. The solvents were then evaporated and the remains
absorbed on silica gel. Purification by FC (10% methanol in CH2Cl2)
yielded compound 17a/b (191 mg, 0.53 mmol, 74%) in an anomeric ratio
of a/bꢃ1:1 as a white foam. Rf =0.36 (10% methanol in CH2Cl2);
1H NMR (300 MHz, MeOD): d=8.26 (d, 1H, J=7.5), 8.18 (d, 1H, J=
Chem. Eur. J. 2013, 00, 0 – 0
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
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These are not the final page numbers!

C. J. Leumann and A.-B. Gerber
133.20, 132.74, 132.65, 128.45, 128.40, 95.63, 89.25, 88.90, 87.29, 85.21,
83.76, 80.22, 79.86, 46.10, 45.99, 45.16, 45.10, 38.38, 37.71, 37.40,
21.01 ppm; HRMS (NSI+, THF): m/z calcd for C19H21N3O7S: 436.1173;
found: 436.1172 [M+H+].
ꢃ1.25:1 was obtained as a white foam. Rf =0.88 (10% methanol in
CH2Cl2); 1H NMR (400 MHz, CDCl3): d=7.36 (m, 1H), 7.30 (m, 1H,),
6.41 (t, 1H, J=6.4), 6.20 (dd, 1H, J=9.7, 5.2), 5.43 (m, 1H), 5.27 (m,
1H), 4.94 (dd, 1H, J=6.8, 1.4), 4.62 (d, 1H, J=6.8), 2.98 (m, 1H,), 2.84
(dd, 1H, J=14.3, 5.2), 2.55 (m, 2H), 2.43 (m, 1H), 2.38 (m, 3H), 2.30 (m,
1H), 2.15 (m, 2H), 2.10, 2.09 (2s, 6H), 2.06–2.01 (m, 1H), 2.04, 2.03 (2s,
6H), 1.95 (m, 3H), 1.93 ppm (m, 3H); 13C NMR (101 MHz, CDCl3): d=
170.51, 170.27, 169.74, 163.97, 163.70, 150.64, 150.52, 135.17, 134.65,
111.64, 111.37, 93.65, 93.53, 88.38, 87.53, 86.04, 83.88, 77.43, 77.28, 75.10,
45.15, 45.04, 44.65, 44.36, 42.85, 38.75, 37.50, 21.76, 21.64, 21.40, 12.86,
12.81 ppm; HRMS (NSI+, MeOH): m/z calcd for C16H20N2O7: 353.1343;
found: 353.1341 [M+H+].
(3’S,6’S)-1-[3’-O-Acetyl-2’-deoxy-6’-O-(methylsulfonyl)-3’,5’-ethano-a- and
-b-d-ribofuranosyl] thymine (21a/b):
A solution of 18a/b (250 mg,
0.72 mmol) in pyridine (7 mL) was cooled to 08C and DMAP (9 mg,
0.07 mmol) and Ac2O (0.08 mL, 0.8 mmol) were added. The mixture was
stirred over night at RT and, after cooling to 08C, carefully quenched
and washed with sat. NaHCO3. The aqueous layers were extracted with
CH2Cl2. The combined organic layers were dried over MgSO4 and evapo-
rated. FC (3% methanol in CH2Cl2) yielded 21a/b (218 mg, 0.56 mmol,
78%) an anomeric ratio of a/bꢃ1.25:1 as a white foam. Rf =0.49 (10%
(3’S,6’R)-N6-Benzoyl-9-[3’,6’-di-O-acetyl-2’-deoxy-3’,5’-ethano-b-d-ribofu-
ACHUTNGRENUrNG aACTHUNGTRENNUnG osyl] adenine (25b): A solution containing 22b (219 mg, 0.41 mmol)
1
methanol in CH2Cl2); H NMR (400 MHz, CDCl3): d=7.24 (m, 1H), 7.06
(m, 1H), 6.11 (t, 1H, J=6.2), 6.03 (dd, 1H, J=9.2, 5.6), 5.24–5.15 (m,
2H), 4.95 (dd, 1H, J=6.2, 3.3), 4.58 (dd, 1H, J=6.4, 1.4), 3.05, 3.03 (2s,
6H), 2.92 (dd, 1H, J=14.8, 6.6), 2.84 (m, 2H), 2.68 (dd, 1H, J=15.3,
6.0), 2.54 (m, 2H), 2.48–2.29 (m, 5H), 2.14 (m, 1H), 2.09, 2.07 (2s, 6H),
1.97 (m, 3H), 1.96 ppm (m, 3H); 13C NMR (101 MHz, CDCl3): d=
170.02, 163.14, 162.93, 149.91, 149.81, 134.92, 134.66, 111.82, 111.20, 90.48,
89.56, 86.93, 86.34, 84.73, 84.62, 79.91, 78.92, 77.33, 77.01, 76.69, 44.27,
43.96, 43.89, 43.79, 38.55, 38.46, 37.24, 21.05, 12.68, 12.63 ppm; HRMS
(NSI+, MeCN): m/z calcd for C15H20N2O8S: 389.1013; found 389.1019
[M+H+].
and CsOAc (1.1 g, 6 mmol) in DMSO (8 mL) was heated to 858C and
stirred for 16 h. After cooling to RT the mixture was diluted with ethyl
acetate and washed with sat. NaHCO3. The combined organic layers
were washed with H2O and dried over MgSO4. FC purification (5%
methanol in CH2Cl2) yielded 25b (155 mg, 0.33 mmol, 81%) as a white
foam. Rf =0.75 (10% methanol in CH2Cl2); 1H NMR (400 MHz, CDCl3):
d=9.11 (br, 1H), 8.77 (s, 1H), 8.28 (s, 1H), 8.03 (m, 2H), 7.60 (m, 1H),
7.52 (m, 2H), 6.61 (dd, 1H, J=6.8, 3.1), 5.35 (m, 1H), 4.96 (d, 1H), 3.33
(m, 1H), 3.14 (m, 1H), 2.40 (m, 4H), 2.11 (s, 3H), 1.96 ppm (s, 3H);
13C NMR (101 MHz, CDCl3): d=170.38, 170.30, 152.85, 141.23, 141.15,
136.16, 133.04, 130.49, 129.06, 128.15, 94.17, 89.59, 86.63, 86.10, 85.24,
76.29, 45.24, 44.58, 38.61, 37.99, 29.90, 21.58, 21.48 ppm; HRMS (NSI+,
MeCN): m/z calcd for C23H23N5O6: 466.1721; found: 466.1727 [M+H+].
(3’S,6’S)-N6-Acetyl-9-[3’-O-acetyl-2’-deoxy-6’-O-(methylsulfonyl)-3’,5’-
ethano-b-d-ribofuranosyl]-N6-benzoyladenine (22b): A solution of 19b
(195 mg, 0.42 mmol) in pyridine (12 mL) was cooled to 08C and DMAP
(5 mg, 42 mmol) and Ac2O (0.12 mL, 1.3 mmol) were added. The mixture
was stirred for 16 h at RT and, after cooling to 08C, carefully quenched
and washed with sat. NaHCO3. The aqueous layers were extracted with
CH2Cl2. The combined organic layers were dried over MgSO4 and evapo-
rated. FC (5% methanol in CH2Cl2) yielded 22b (192 mg, 0.38 mmol,
91%) as a white foam. Rf =0.8 (10% methanol in CH2Cl2); 1H NMR
(400 MHz, CDCl3): d=8.69 (s, 1H), 8.31 (s, 1H), 7.71 (m, 2H), 7.42 (m,
1H), 7.32 (m, 2H), 6.42 (dd, 1H, J=6.9, 2.9), 5.18 (m, 1H) 4.90 (dd, 1H,
J=6.1, 1.7), 3.41 (m, 1H), 3.03 (s, 3H), 2.89 (m, 2H), 2.44 (m, 2H), 2.34
(m, 1H), 2.09 ppm (s, 3H); 13C NMR (101 MHz, CDCl3): d=175.38,
172.86, 172.05, 170.24, 152.94, 152.53, 152.43, 151.16, 134.41, 133.07,
129.44, 129.01, 128.78, 90.34, 87.23, 79.21, 44.41, 43.96, 38.65, 37.68, 25.61,
21.37 ppm; HRMS (NSI+, EtOAc): m/z calcd for C22H23N5O4S:
502.1391; found: 502.11385 [M+H+].
(3’S,6’R)-N6-Benzoyl-1-[3’,6’-Di-O-acetyl-2’-deoxy-3’,5’-ethano-a- and -b-
d-ribofuranosyl] cytosine (26a/b): A solution containing 23a/b (523 mg,
1.09 mmol) and CsOAc (2.9 g, 15.1 mmol) in DMSO (20 mL) was heated
to 858C and stirred for 16 h. After cooling to RT the mixture was diluted
with ethyl acetate and washed with sat. NaHCO3. The combined organic
layers were washed with H2O and dried over MgSO4. The crude product
26a/b was used for the following step without purification. Rf =0.82
(10% methanol in CH2Cl2); HRMS (NSI+, EtOAc): m/z calcd for
C22H23N3O7: 442.1609; found: 442.1601 [M+H+].
(3’S,6’R)-1-[2’-Deoxy-3ꢀ,5ꢀ-ethano-a- and -b-d-ribofuranosyl] thymine
(27a/b): The nucleoside 24a/b (254 mg, 0.72 mmol) was dissolved in 0.2m
NaOH in a solution of THF/methanol/H2O (5:4:1, 40 mL) at 08C. The
solution was stirred for one hour at 08C and quenched by the addition of
ammonium chloride. The solvents were then evaporated and the remains
absorbed on silica gel. Purification by FC (10% methanol in CH2Cl2)
yielded compound 27a/b (123 mg, 0.45 mmol, 64%) in anomeric ratio of
(3’S,6’S)-N6-Acetyl-1-[3’-O-acetyl-2’-deoxy-6’-O-(methylsulfonyl)-3’,5’-
ethano-a- and b-d-ribofuranosyl]-N6-benzoylcytosine (23a/b): A solution
of 20a/b (769 mg, 1.76 mmol) in pyridine (35 mL) was cooled to 08C and
DMAP (22 mg, 0.18 mmol) and Ac2O (0.5 mL, 5.28 mmol) were added.
The mixture was stirred for 20 h at RT and, after cooling to 08C, carefully
quenched and washed with sat. NaHCO3. The aqueous layers were ex-
tracted with CH2Cl2. The combined organic layers were dried over
MgSO4 and evaporated. FC (5% methanol in CH2Cl2) yielded 23a/b
(628 mg, 1.3 mmol, 75% over two steps) in an anomeric ratio of a/bꢃ2:1
a/bꢃ1.25:1 as
a white foam. Rf =0.2 (10% methanol in CH2Cl2);
1H NMR (400 MHz, DMSO): d=11.25 (br, 2H), 7.76 (s, 1H), 7.66 (s,
1H), 6.20 (m, 2H), 5.31 (s, 1H), 5.26 (s, 1H), 4.94 (d, 1H, J=2.7), 4.71
(d, 1H, J=3.8), 4.39 (dd, 1H, J=7.1, 3.0), 4.31 (m, 1H), 4.23 (m, 1H),
4.08 (m, 1H), 2.62 (dd, 1H, J=13.7, 6.9), 2.31 (m, 1H), 2.22 (m, 1H),
2.15 (m, 1H), 2.09 (m, 1H), 2.02 (m, 2H), 1.95 (m, 1H), 1.85 (m, 1H),
1.80 (m, 3H), 1.76 (m, 3H), 1.77 (m, 2H), 1.55 ppm (m, 1H); 13C NMR
(101 MHz, DMSO): d=163.79, 163.63, 150.40, 150.36, 136.50, 109.57,
109.13, 90.11, 89.98, 86.81, 86.10, 85.59, 83.66, 72.36, 70.90, 48.33, 47.92,
46.80, 46.02, 41.32, 40.66, 12.34, 12.25 ppm; HRMS (NSI+, MeOH) for
C12H16N2O5: m/z calcd for 268.0951; found: 268.0955 [M+].
as
a
white foam. Rf =0.9 (10% methanol in CH2Cl2); 1H NMR
(400 MHz, CDCl3): d=7.93 (m, 6H), 7.61 (m, 4H), 7.55 (m, 4H), 6.10
(dd, 1H, J=6.4, 4.1), 6.04 (dd, 1H, J=8.5, 5.6), 5.19 (m, 2H), 5.04 (dd,
1H, J=5.9, 3.7), 4.69 (d, 1H), 3.15 (m, 1H), 3.04 (s, 3H), 3.03 (s, 3H),
2.99 (m, 1H), 2.83–2.67 (m, 3H), 2.50 (m, 2H), 2.35 (m, 4H), 2.08 (m,
6H), 2.04 ppm (m, 1H); 13C NMR (101 MHz, CDCl3): d=176.17, 170.32,
170.09, 163.25, 163.18, 154.73, 149.28, 144.01, 136.97, 133.47, 132.99,
129.09, 128.19, 97.45, 96.72, 90.54, 90.08, 89.53 (C(1’)), 88.09, 86.89, 85.77,
79.77, 79.31, 45.55, 45.11, 44.25, 43.74, 38.64, 38.61, 38.58, 37.66, 29.87,
29.53, 21.66, 21.53, 21.15 ppm; HRMS (NSI+, EtOAc): m/z calcd for
C21H23N3O8S: 478.1279; found: 478.1268 [M+H+].
(3’S,6’R)-N6-Benzoyl-9-[2’-deoxy-3’,5’-ethano-b-d-ribofuranosyl] adenine
(28b): The nucleoside 25b (115 mg, 0.25 mmol) was dissolved in 0.2m
NaOH in THF/methanol/H2O (5:4:1, 17 mL) at 08C. The solution was
stirred for one hour at 08C and quenched by the addition of ammonium
chloride. The solvents were then evaporated and the remains absorbed
on silica gel. Purification by FC (10% methanol in CH2Cl2) yielded com-
pound 28b (80 mg, 0.21 mmol, 84%) as a white foam. Rf =0.3 (10%
(3’S,6’R)-1-[3’,6’-Di-O-acetyl-2’-deoxy-3’,5’-ethano-a- and -b-d-ribofurano-
syl] thymine 24a/b: A solution containing 21a/b (301 mg, 0.77 mmol) and
CsOAc (2.1 g, 10.7 mmol) in DMSO (15 mL) was heated to 858C and
stirred for 16 h. After cooling to RT the mixture was diluted with ethyl
acetate and washed with sat. NaHCO3. The combined organic layers
were washed with H2O and dried over MgSO4. By using FC (5% metha-
nol in CH2Cl2) 24a/b (254 mg, 0.72 mmol, 93%) an anomeric ratio of a/b
1
methanol in CH2Cl2); H NMR (300 MHz, MeOD): d=8.71 (s, 1H), 8.65
(s, 1H), 8.09 (m, 2H), 7.65 (m, 1H), 7.55 (m, 1H), 6.55 (dd, 1H, J=9.3,
5.9), 4.50 (m, 1H), 4.38 (dd, J=6.9, 1.4, 1H), 3.01 (dd, 1H, J=13.5, 9.4),
2.70 (dd, 1H, J=13.6, 5.9), 2.33–2.24 (m, 2H), 2.03 ppm (m, 2H);
13C NMR (75 MHz, MeOD) d=153.22, 153.09, 151.14, 144.27, 134.98,
133.90, 129.76, 129.42, 125.05, 92.42, 88.68, 85.90, 74.93, 54.79, 49.46,
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Isobicyclo-DNA
FULL PAPER
41.83 ppm; HRMS (NSI+, MeOH): m/z calcd for C19H19N5O4: 382.1510;
found: 382.1505 [M+H+].
3.3 mmol) was added in three portions to a solution of nucleoside 29a/b
(394 mg, 1.1 mmol) in pyridine (6 mL) and it was stirred for 24 h at RT.
The solution was then diluted with ethyl acetate, washed with sat.
NaHCO3 and dried over MgSO4. Purification by FC (ethyl acetate/
hexane, 4:1 to 5% methanol in CH2Cl2) yielded the corresponding anom-
ers a and b, 32a (450 mg, 0.68 mmol, 62%) and 32b (217 mg, 0.33 mmol,
30%) as white foams. Compound 32a: Rf =0.22 (ethyl acetate/hexane,
4:1); 1H NMR (400 MHz, CDCl3): d=9.00 (br, 1H), 7.87 (m, 3H), 7.55
(m, 1H), 7.46 (m, 5H), 7.36 (m, 4H), 7.26 (m, 2H), 7.21 (m, 1H), 6.83
(m, 4H), 6.05 (dd, 1H, J=7.1, 2.1), 4.48 (dd, 1H, J=7.3, 3.8), 4.25 (m,
1H), 3.79 (s, 6H), 3.03 (dd, 1H), 2.64 (dd, 1H, J=14.8, 2.0), 1.64 ppm
(m, 3H); 13C NMR (101 MHz, CDCl3): d=162.60, 158.72, 155.27, 146.01,
145.82, 137.07, 137.03, 133.27, 133.14, 130.26, 129.08, 128.32, 128.10,
127.87, 126.98, 113.43, 96.20, 93.05, 92.81, 87.17, 86.83, 75.04, 60.59, 55.40,
47.40, 46.27, 40.51, 21.22, 14.37 ppm; HRMS (NSI+, MeCN): m/z calcd
for C39H37N3O7: 660.2704, found: 660.2724 [M+H+]; Compound 43b:
Rf =0.05 (ethyl acetate/hexane, 4:1); 1H NMR (300 MHz, CDCl3): d=
8.77 (br, 1H), 8.23 (m, 1H), 7.89 (m, 2H), 7.60 (m, 1H), 7.51 (m, 4H),
7.37–7.19 (m, 8H), 6.84 (m, 4H), 6.45 (dd, 1H, J=8.6, 5.5), 4.37 (m, 1H),
4.23 (d, 1H, J=5.8), 3.79, 3.77 (2 s, 6H), 2.84 (dd, 1H, J=13.5, 5.4), 2.36
(dd, 1H, J=13.5, 8.7), 1.88- 1.73 (m, 3H), 1.42 ppm (m, 1H); 13C NMR
(126 MHz, CDCl3): d=158.87, 158.37, 145.45, 136.83, 136.71, 133.38,
130.28, 130.20, 129.26, 128.35, 128.31, 127.76, 127.17, 113.62, 113.51, 90.84,
89.31, 88.06, 87.68, 87.12, 76.79, 55.43, 55.42, 47.10, 39.46 ppm; HRMS
(NSI+, MeCN): m/z calcd for C39H37N3O7: 660.2704; found: 660.2704
[M+H+].
(3’S,6’R)-N6-Benzoyl-1-[2’-deoxy-3’,5’-ethano-a- and -b-d-ribofuranosyl]
cytosine (29a/b): The nucleoside 26a/b (386 mg, 0.87 mmol) was dis-
solved in 0.2m NaOH in THF/methanol/H2O (5:4:1, 50 mL) at 08C. The
solution was stirred for one hour at 08C and quenched by the addition of
ammonium chloride. The solvents were then evaporated and the remains
absorbed on silica gel. Purification by FC (5% methanol in CH2Cl2)
yielded compound 29a/b (183 mg, 0.51 mmol, 59% over two steps) in an
anomeric ratio of a/bꢃ2:1as a white foam. Rf =0.43 (10% methanol in
CH2Cl2); 1H NMR (400 MHz, MeOD): d=8.46 (d, 1H, J=7.5), 8.28 (d,
1H, J=7.5), 7.98 (m, 4H), 7.63 (m, 4H), 7.54 (m, 4H), 6.33 (dd, 1H, J=
8.9, 5.8), 6.21 (dd, 1H, J=6.8, 3.2), 4.68 (dd, 1H, J=7.2, 3.1), 4.49 (m,
1H), 4.43 (m, 1H), 4.35 (d, 1H), 3.03 (dd, 1H, J=14.4, 6.8), 2.67 (dd,
1H, J=13.6, 5.8), 2.37 (m, 3H), 2.24 (m, 1H), 2.15 (m, 1H), 2.09 (m,
2H), 2.04 (m, 1H), 1.94 (m, 1H), 1.85 ppm (m, 1H); 13C NMR
(101 MHz, MeOD): d=169.13, 164.79, 157.92, 146.88, 146.56, 134.76,
134.72, 134.10, 134.06, 129.83, 129.17, 129.13, 98.76, 98.06, 93.64, 92.52,
92.07, 88.92, 88.10, 87.97, 74.88, 73.43, 50.01, 48.88, 42.27, 41.63 ppm;
HRMS (NSI+, MeOH): m/z calcd for C18H19N3O5: 380.1217; found:
380.1220 [M+Na+].
(3’S,6’R)-1-{2’-Deoxy-6’-O-[(4,4’-dimethoxytriphenyl)methyl]-3’,5’-ethano-
a- and -b-d-ribofuranosyl} thymine (30a/b): To a solution of nucleoside
27a/b (456 mg, 1.7 mmol) in pyridine (8 mL) was added DMT-Cl (1.7 g
5.1 mmol) in three portions and it was stirred for 24 h at RT. The solution
was then diluted with ethyl acetate, washed with sat. NaHCO3 and dried
over MgSO4. Purification by FC (2.5% methanol in CH2Cl2) yielded the
corresponding anomers a and b, compound 30a (470 mg, 0.82 mmol,
48%) and 30b (384 mg, 0.67 mmol, 40%), respectively, as pale-yellow
foams. Compound 30a: Rf =0.23 (3% methanol in CH2Cl2); 1H NMR
(400 MHz, CDCl3): d=7.47 (m, 2H), 7.35 (m, 4H), 7.29 (m, 2H), 7.22
(m, 2H), 6.83 (m, 4H), 5.95 (dd, 1H, J=7.8, 3.7), 4.40 (dd, 1H, J=7.0,
3.1), 4.24 (m, 1H), 3.79 (s, 6H), 2.89 (dd, 1H, J=14.5, 7.8), 2.30 (dd, 1H,
J=14.5, 3.7), 1.95 (m, 1H), 1.91 (m, 3H), 1.67 (m, 1H), 1.53 ppm (m,
2H); 13C NMR (101 MHz, CDCl3): d=163.87, 158.81, 150.59, 145.86,
138.45, 137.10, 136.98, 130.28, 130.25, 128.28, 128.16, 127.04, 113.49,
110.80, 91.87, 91.50, 87.46, 87.22, 75.18, 55.45, 47.22, 46.53, 46.39, 40.46,
12.63, 11.67 ppm; HRMS (NSI+, MeCN): m/z calcd for C33H34N2O7:
593.2258; found: 593.2250 [M+Na+]; Compound 30b: Rf =0.17 (3%
methanol in CH2Cl2), 1H NMR (400 MHz, CDCl3): d=8.69 (br, 1H), 7.45
(m, 2H), 7.35 (m, 4H), 7.32 (m, 1H), 7.27 (m, 2H), 7.21 (m, 1H), 6.82
(m, 4H), 6.27 (dd, 1H, J=8.8, 5.9), 4.33 (m, 1H), 4.05 (dd, 1H, J=7.5,
2.1), 3.78 (s, 6H), 2.49 (m, 2H), 2.10 (m, 1H), 1.77 (m, 2H), 1.50 (m,
3H), 1.18 ppm (m, 1H); 13C NMR (101 MHz, CDCl3): d=163.72, 158.91,
158.89, 150.54, 145.65, 136.86, 136.66, 135.61, 130.22, 130.15, 128.34,
128.18, 127.18, 113.67, 111.57, 89.85, 88.10, 87.68, 85.01, 76.40, 55.47,
46.99, 46.62, 46.08, 39.32, 12.31 ppm; HRMS (NSI+, MeCN): m/z calcd
for C33H34N2O7: 593.2258; found: 593.2251 [M+Na+].
(3’S,6’R)-1-{6’-O-[(2-Cyanoethoxy)(diisopropylamino)phosphino]-2’-de-
AHCTUNGTERGoNNUN xy-6’-O-[(4,4’-dimethoxytriphenyl)methyl]-3’,5’-ethano-b-d-ribofurano-
syl} thymine (33b): Compound 30b (163 mg, 0.29 mmol) was dissolved in
MeCN (2.5 mL) and Hꢁnigꢂs base (0.24 mL, 1.43 mmol) and CEP-Cl
(0.19 mL, 0.87 mmol) were added. The solution was stirred at RT for 1 h,
diluted with ethyl acetate and washed with sat. NaHCO3. The aqueous
layer is extracted with ethyl acetate and dried over MgSO4. Purification
by using FC (ethyl acetate/hexane 1:1) yielded phosphoramidite 33b
(214 mg, 0.24 mmol, 72%) as a white foam. 31P NMR showed no phos-
phonate peak, therefore no further purification was necessary. Rf =0.63
(ethyl acetate/hexane 2:1); 1H NMR (400 MHz, CDCl3): d=8.11 (br,
1H), 7.45 (m, 2H), 7.35 (m, 4H), 7.27 (m, 4H), 7.20 (m, 1H), 6.82 (m,
4H), 6.23 (dd, 1H, J=9.6, 4.9), 4.33 (m, 1H), 4.27 (dd, 1H, J=14.8, 7.5),
3.79 (m, 6H), 3.80–3.55 (m, 4H), 2.85 (m, 1H), 2.64 (m, 1H), 2.58 (t, 1H,
J=6.3), 2.52 (m, 1H), 2.19 (m, 2H), 1.70 (m, 1H), 1.50 (m, 3H), 1.16 (m,
9H), 1.10 (m, 3H), 1.05 ppm (m, 1H); 13C NMR (101 MHz, CDCl3): d=
163.59, 158.89, 150.19, 145.61, 136.87, 136.59, 135.70, 130.24, 130.14,
128.35, 128.19, 127.18, 113.69, 111.30, 91.69, 89.46, 88.19, 85.18, 77.44,
76.32, 76.20, 60.62, 58.17, 57.99, 55.47, 43.66, 43.51, 38.94, 38.85, 24.76,
24.53, 24.45, 20.56, 14.41, 12.30, 12.26 ppm; 31P NMR (122 MHz, CDCl3):
d=142.13, 141.81; HRMS (NSI+, MeCN): m/z calcd for C42H51N4O8P:
771.3517; found: 771.3531 [M+H+]
(3’S,6’R)-N6-Benzoyl-9-{3’-O-[(2-cyanoethoxy)(diisopropylamino)phos-
phino]-2’-deoxy-6’-O-[4,4’-dimethoxytriphenyl)methyl]-3’,5’-ethano-b-d-ri-
bofuranosyl} adenine (34b): Compound 31b (290 ng, 0.42 mmol) was dis-
solved in MeCN (4 mL) and Hꢁnigꢂs base (0.36 mL, 2.1 mmol) and CEP-
Cl (0.28 mL, 1.3 mmol) were added. The solution was stirred at RT for
1 h, diluted with ethyl acetate and washed with sat. NaHCO3. The aque-
ous layer was extracted with ethyl acetate and the organic layers dried
over MgSO4. Purification by FC (ethyl acetate/hexane 1:1) yielded phos-
phoramidite 34b (310 mg, 0.35 mmol, 83%) as a white foam. 31P NMR
showed a H-phosphonate peak. Compound 34b had therefore to be fur-
ther purified and was dissolved in CH2Cl2 (1 mL) and added dropwise to
ice-cold hexane. The filter cake was collected by washing the filter with
CH2Cl2 and evaporating the solvent. 31P NMR showed no more phospho-
nate so the pure phosphoramidite 34b (325 mg, 0.37 mmol, 87%) could
be collected. Rf =0.48 (ethyl acetate/hexane 2:1); 1H NMR (300 MHz,
CDCl3): d=9.07 (s, 1H), 8.83 (d, 1H, J=3.2), 8.26 (s, 1H), 8.04 (m, 2H),
7.61 (m, 1H), 7.55 (m, 2H)), 7.46 (m, 2H), 7.36 (m, 4H), 7.32 (m, 2H),
6.82 (m, 4H), 6.45 (m, 1H), 4.36 (m, 2H), 3.78 (s, 6H), 3.74–3.54 (m,
4H), 3.05 (m, 2H), 2.63 (t, 1H, J=6.3), 2.55 (t, 1H, J=6.3), 2.08 (m,
1H), 1.90 (m, 2H), 1.50 (m, 1H), 1.16 (m, 9H,), 1.10 ppm (d, J=6.8,
3H); 13C NMR (101 MHz, CDCl3): d=164.71, 158.73, 153.02, 151.71,
(3’S,6’R)-N6-Benzoyl-9-{2’-Deoxy-6’-O-[4,4’-(dimethoxytriphenyl)methyl]-
3’,5’-ethano-b-d-ribofuranosyl} adenine (31b): To a solution of nucleoside
28b (80 mg, 0.21 mmol) in pyridine (2 mL) was added DMT-Cl (213 mg
0.63 mmol) in three portions and the mixture was stirred for 24 h at RT.
The solution was then diluted with ethyl acetate, washed with sat.
NaHCO3 and dried over MgSO4. Purification by FC (5% methanol in
CH2Cl2) yielded compound 31b (129 mg, 0.19 mmol, 90%) as a white
foam. Rf =0.42 (10% methanol in CH2Cl2); 1H NMR (400 MHz, CDCl3):
d=8.81 (s, 1H), 8.27 (s, 1H), 8.04 (m, 2H), 7.61 (m, 1H,), 7.53 (m, 2H),
7.45 (m, 2H), 7.35 (m, 4H), 7.28 (m, 2H), 7.22 (m, 1H), 6.81 (m, 4H),
6.46 (dd, 1H, J=9.1, 5.4), 4.35 (m, 1H), 4.21 (dd, 1H, J=7.4, 3.5), 3.78
(s, 6H), 2.92 (dd, 1H, J=13.2, 9.2), 2.64 (dd, 1H, J=13.2, 5.5), 2.02 (m,
1H), 1.83–1.58 ppm (m, 3H); 13C NMR (101 MHz, CDCl3): d=171.37,
171.24, 158.84, 153.04, 150.05, 149.74, 145.65, 141.30, 136.90, 136.87,
136.18, 132.97, 130.25, 129.09, 128.28, 128.23, 128.11, 127.14, 123.95,
113.57, 90.02, 87.58, 87.17, 85.18, 77.43, 75.85, 55.46, 47.26, 46.60, 46.08,
39.91 ppm; HRMS (NSI+, MeOH): m/z calcd for C40H37N5O6: 684.2817;
found: 684.2830 [M+H+].
(3’S,6’R)-N6-Benzoyl-1-{2’-Deoxy-6’-O-[4,4’-(dimethoxytriphenyl)methyl]-
3’,5’-ethano-a and -b-d-ribofuranosyl] cytosine (32a/b): DMT-Cl (1.1 g
Chem. Eur. J. 2013, 00, 0 – 0
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C. J. Leumann and A.-B. Gerber
145.59, 141.41, 141.28, 136.85, 136.80, 134.02, 132.86, 130.21, 130.16,
129.02, 128.22, 128.15, 128.00, 127.02, 123.21, 117.78, 117.72, 113.49, 91.16,
91.07, 91.04, 90.95, 89.89, 89.71, 89.68, 89.64, 87.60, 87.55, 85.26, 85.11,
77.36, 75.67, 75.53, 58.11, 58.03, 57.93, 57.85, 55.38, 45.48, 45.40, 45.12,
43.60, 43.56, 43.48, 43.43, 39.35, 39.32, 24.73, 24.67, 24.50, 24.47, 24.39,
20.56, 20.48, 20.40 ppm; 31P NMR (122 MHz, CDCl3): d=142.13, 142.07;
HRMS (NSI+, MeCN): m/z calcd for C49H54N7O7P: 884.3901; found:
884.3902 [M+H+].
was added BSA (2.3 mL. 9.6 mmol). The mixture was stirred at r.t. until
a clear solution appeared. Compound 46 (858 mg, 2.1 mmol, in 5 mL
MeCN) and TMSOTf (0.13 mL, 0.72 mmol) was then added and the mix-
ture was stirred for 6 h at 558C. The brownish solution was diluted by the
addition of ethyl acetate, washed with sat. NaHCO3 and dried over
MgSO4. Purification by FC (ethyl acetate/hexane, 1:3) yielded 38b
(411 mg, 0.76 mmol, 36%) and 38a (316 mg, 0.58 mmol, 28%) as white
foams. Compound 38b: Rf =0.4 (hexane/ethyl acetate, 3:1); 1H NMR
(400 MHz, CDCl3): d=7.87 (s, 1H), 6.00 (dd, 1H, J=9.1, 5.6), 5.13 (br,
2H), 4.40 (m, 1H), 4.31 (d, 1H, J=6.3), 2.54 (m, 2H), 2.34 (m, 1H), 2.12
(m, 1H), 1.84 (m, 2H), 0.90 (m, 18H), 0.16 (m, 6H), 0.08 ppm (s, 6H);
13C NMR (101 MHz, CDCl3): d=159.18, 153.72, 151.77, 140.60, 126.19,
89.88, 86.42, 83.86, 71.80, 48.95, 47.24, 40.71, 26.05, 25.87, 18.33, 18.03,
ꢀ2.37, ꢀ2.45, ꢀ4.42, ꢀ4.48 ppm; HR-MS (NSI+, EtOAc): m/z calcd for
C24H43N5O3ClSi2: 540.2587; found: 540.2608 [M+H+]; Compound 38a:
Rf =0.26 (hexane/ethyl acetate 3:1); 1H NMR (400 MHz, CDCl3): d=
8.19 (s, 1H), 6.24 (dd, 1H, J=7.1, 1.6), 5.11 (br, 2H), 4.45 (d, 1H, J=
6.1), 4.25 (m, 1H), 2.85 (dd, 1H, J=14.6, 1.5), 2.48 (dd, 1H,=14.6, 1.5),
2.30 (m, 1H), 2.12 (m, 1H), 1.90 (m, 1H), 1.81 (m, 1H), 0.89, 0.80 (2 s,
18H3), 0.12 (s, 3H), 0.07 (s, 6H), 0.02 ppm (s, 3H); 13C NMR (101 MHz,
CDCl3): d=159.15, 153.63, 151.52, 141.11, 125.92, 91.16, 86.93, 84.80,
71.49, 50.35, 48.48, 40.79, 26.04, 25.79, 18.31, 17.97, ꢀ2.26, ꢀ2.55, ꢀ4.47,
ꢀ4.56 ppm; HRMS (NSI+, EtOAc): m/z calcd for C24H43N5O3ClSi2:
540.2587; found: 540.2607 [M+H+].
(3’S,6’R)-N6-Benzoyl-1-{3’-O-[(2-cyanoethoxy)(diisopropylamino)phos-
phino]-2’-deoxy-6’-O-[4,4’-dimethoxytriphenyl)methyl]-3’,5’-ethano-b-d-ri-
bofuranosyl} cytosine (35b): Compound 32b (216 mg, 0.33 mmol) was
dissolved in MeCN (3 mL) and Hꢁnigꢂs base (0.28 mL, 1.6 mmol) and
CEP-Cl (0.22 mL, 0.99 mmol) were added. The solution was stirred at
RT for 2 h, diluted with ethyl acetate and washed with sat. NaHCO3. The
aqueous layer was extracted with ethyl acetate and dried over MgSO4.
Purification using by FC (ethyl acetate/hexane 1:1) yielded phosphorami-
dite 35b (214 mg, 0.24 mmol, 72%) as a white foam. 31P NMR showed a
phosphonate peak. Compound 35b had therefore to be further purified;
it was dissolved in of CH2Cl2 (1 mL) and added dropwise to ice-cold
hexane. The filter cake was collected by washing the filter with CH2Cl2
and evaporating the CH2Cl2. After three cycles and another FC the
31P NMR spectra showed no more phosphonate so the pure phosphora-
midite 35b could be collected. Rf =0.4 (ethyl acetate/hexane, 2:1);
1H NMR (400 MHz, CDCl3): d=8.70 (br, 1H), 8.20 (m, 1H), 7.90 (m,
2H), 7.62 (m, 1H), 7.52 (m, 2H), 7.44 (m, 2H), 7.33 (m, 5H), 7.25 (m,
3H), 6.84 (m, 4H), 6.24 (m, 1H), 4.40 (m, 2H), 3.78 (2 s, 6H), 3.77–3.53
(m, 4H), 3.11 (dd, 1H, 13.9, 4.7), 2.65 (t, 1H, J=6.3), 2.57 (t, 1H, J=
6.4), 2.38 (m, 1H), 2.09 (m, 1H), 1.85 (m, 1H), 1.81 (m, 1H), 1.44 (m,
1H) 1.15 (m, 9H), 1.08 ppm (m, 3H); 13C NMR (101 MHz, CDCl3): d=
162.21, 158.87, 158.84, 145.37, 136.79, 136.63, 133.33, 130.26, 130.16,
129.25, 128.32, 127.75, 127.17, 113.63, 91.68, 91.59, 91.52, 91.43, 90.40,
90.34, 90.17, 90.10, 88.12, 88.09, 87.38, 87.34, 77.43, 76.44, 76.43, 58.25,
58.08, 58.00, 55.48, 55.42, 46.98, 46.96, 46.74, 46.64, 46.56, 46.33, 46.25,
45.79, 45.68, 43.64, 43.51, 39.09, 24.79, 24.72, 24.53, 24.47, 24.40, 24.21,
24.13, 24.10, 22.54, 20.54, 20.50, 20.46, 20.42, 14.41, 14.33 ppm; 31P NMR
(122 MHz, CDCl3): d=149.96, 149.68 ppm; HRMS (NSI+, THF): m/z
calcd for C48H54N5O8P: 860.3788; found: 860.3789 [M+H+].
(3’S,6’S)-)-9-{3’,6’-Di-O-[(tert-butyl)-dimethylsilyl]-2’-deoxy-3’,5’-ethano-a-
and -b-d-ribofuranosyl} guanine (39a/b): NaH (60%, 285 mg, 7.1 mmol)
was carefully added to a solution of 3-hydroxypropionitrile (0.4 mL,
5.9 mmol) in THF (8 mL) at 08C and the suspension was stirred for 1 h
at RT. The anomeric compound 38a/b (700 mg, 1.3 mmol in 8 mL THF)
was added and the brown mixture was stirred for another 3 h at RT. Ad-
dition of silica, evaporation of the solvents and filtration (10% methanol
in CH2Cl2) over a patch of silica yielded the crude product 39a/b. The
brown foam was used without further purification in the next step. Rf =
0.5 (10% methanol in CH2Cl2); HRMS (NSI+, MeCN) m/z calcd for
C24H43N5O4Si2: 521.81; found 522.29 [M+H+].
(3’S,6’S)-N2-(N,N-Dimethylformamidino)-9-{3’,6’-di-O-[(tert-butyl)-di-
ACHUTNGRENUNmG ethACHTUGNTRENyNUGN lsilyl]-2’-deoxy-3’,5’-ethano-a- and -b-d-ribofuranosyl} guanine
(3R,5S,7S)-3-Methoxyoctahydropentalene-7,5-diol (36b): The sugar 10b
(1.0 g, 3.6 mmol) was dissolved in 0.2m NaOH in THF/methanol/H2O
(5:4:1, 150 mL) at 08C. The solution was stirred for one hour at 08C and
quenched by the addition of ammonium chloride. The solvents were then
evaporated and the remains absorbed on silica gel. Purification by using
FC (5% methanol in CH2Cl2) yielded compound 36b (618 mg, 3.5 mmol,
97%) as a colorless oil. Rf =0.14 (ethyl acetate/hexane 1:1); 1H NMR
(300 MHz, MeOD): d=4.98 (dd, 1H, J=5.8, 2.0), 4.40 (m, 1H), 4.23 (dd,
1H,=6.7, 2.3), 3.27 (s, 3H), 2.27 (m, 2H), 2.00 (m, 1H), 1.94 (m, 1H),
1.85 (m, 1H), 1.74 ppm (dd, 1H, J=13.0, 7.9); 13C NMR (75 MHz,
MeOD): d=107.38, 90.61, 86.83, 72.44, 55.27, 49.70, 48.90, 42.88 ppm;
HRMS (NSI+, MeOH): m/z calcd for C8H14O4: 197.0784; found:
197.0788 [M+Na+].
(40a/b): The crude compound 39a/b (750 mg, ꢃ1.4 mmol) was dissolved
in DMF (12 mL) and N,N-dimethylformamide dimethyl acetate (0.37 mL,
2.8 mmol) was added. The clear yellow solution was stirred for 2 h at
558C. The solvent was evaporated through Kugelrohr distillation and pu-
rified by using FC (5% methanol in CH2Cl2) to obtain nucleoside 40a/b
(715 mg, 1.2 mmol, 71% over 2 steps) in an anomeric ratio of a/bꢃ2:1as
yellow oil. Rf =0.5 (10% methanol in CH2Cl2); 1H NMR (300 MHz,
MeOD): d=8.66 (s, 1H), 8.62 (s, 1H), 8.01 (2 s, 2H), 6.25 (dd, 1H, J=
6.8, 1.7), 6.14 (dd, 1H, J=9.6, 5.1), 4.57 (d, 1H, J=6.1), 4.45 (m, 1H),
4.34 (m, 2H), 3.20 (m, 6H), 3.12 (m, 6H), 2.99 (s, 1H), 2.86 (m, 2H),
2.64–2.36 (m, 5H), 2.12 (m, 2H), 1.85 (m, 4H), 0.96 (s, 18H), 0.92 (2 s,
18H), 0.77 (m, 12H), 0.21 ppm (m, 12H); 13C NMR (75 MHz, CDCl3):
d=158.38, 158.28, 158.22, 157.10, 156.90, 150.37, 135.72, 118.34, 120.54,
90.85, 89.89, 87.00, 86.54, 84.23, 82.88, 77.43, 71.95, 71.65, 58.07, 48.82,
48.72, 41.56, 40.80, 35.43, 26.02, 25.90, 25.83, 21.70, 18.27, 18.06, 18.00,
ꢀ2.29, ꢀ2.46, ꢀ2.52, ꢀ4.47, ꢀ4.54 ppm; HRMS (NSI+, EtOAc): m/z
calcd for C27H49N6O4Si2: 577.3348; found: 577.3339 [M+H+].
tert-Butyl-(((3R,5S,7S)-3-methoxy-7-(((2,3,3-trimethylbutan-2-yl)silyl)oxy)-
ACHTUNGTRENNUNGoctahydropentalen-3a-yl)oxy)dimethylsilane (37b): Compound 36b (1.7 g,
10.0 mmol) was dissolved in DMF (40 mL). TBSCl (3.8 g, 25.0 mmol)
and imidazole (2.1 g, 30.0 mmol) were added and the mixture was stirred
at 858C for 2 days. The solution was then diluted with ethyl acetate,
washed with brine and H2O and dried over MgSO4. Purification by using
FC (ethyl acetate/hexane, 3:1) yielded 46 (3.3 g, 8.2 mmol, 82%) as a col-
orless oil. Rf =0.9 (ethyl acetate/hexane 1:1); 1H NMR (300 MHz,
CDCl3): d=4.99 (dd, 1H, J=5.7, 2.2), 4.49 (m, 1H), 4.25 (d, 1H, J=6.4),
3.33 (s, 3H), 2.33 (m, 2H), 2.00 (m, 2H), 1.77 (m, 2H), 0.88 (m, 18H),
0.08 ppm (m, 12H); 13C NMR (101 MHz, CDCl3): d=105.87 (C(3)),
89.93 ((C1)), 71.85 (C(7)), 55.13 (OMe), 50.60 (C(4)), 49.47 (C(6)), 41.98
(C(8)), 26.08 (TBS), 25.89 (TBS), 18.42 (TBS), 17.99 (TBS), ꢀ2.50
(TBS), ꢀ2.55 ppm (TBS); HRMS (NSI+, THF): m/z calcd for
C20H42O4Si2: 403.2694; found: 403.2693 [M+H+].
(3’S,6’S)-N2-(N,N-Dimethylformamidino)-9-{3’-O-[(tert-butyl)-dimethylsil-
yl]-2’-deoxy-3’,5’-ethano-a- and -b-d-ribofuranosyl} guanine (41a/b): A
solution of 40a/b (1.5 g, 2.6 mmol) in THF (20 mL) was cooled to 08C.
HF/Et3N (37% HF, 1.7 mL, 3.9 mmol) was added and allowed to warm
to RT. The reaction mixture was stirred at RT overnight and then
quenched with silica. The solvent was evaporated and the crude mixture
purified by using FC (5% methanol in CH2Cl2). 41a/b (658 mg,
1.4 mmol, 55%) in an anomeric ratio of a/bꢃ2:1was obtained as a white
foam. Rf =0.5 (10% methanol in CH2Cl2), 1H NMR (400 MHz, MeOD):
d=8.66 (s, 1H), 8.62 (s, 1H), 8.01 (s, 1H), 7.97 (s, 1H), 6.24 (dd, 1H, J=
6.7, 1.6), 6.09 (dd, 1H, J=10.0, 5.1), 4.59 (m, 1H), 4.53 (m, 1H), 4.30 (m,
1H), 4.25 (m, 1H), 3.21 (m, 6H), 3.12 (m, 6H), 2.92 (dd, 1H, J=14.6,
1.6), 2.71 (m, 1H), 2.60 (m, 2H), 2.49 (m, 1H), 2.41 (m, 1H), 2.15 (m,
2H), 1.89–1.79 (m, 4H), 0.95 (s, 9H), 0.76 (s, 9H), 0.22 (m, 6H), 0.12 (s,
(3’S,6’S)-2-Amino-6-chloro-9-{3’,6’-di-O-[(tert-butyl)-dimethylsilyl]-2’-de-
ACHTUNGTRENNUNG
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Isobicyclo-DNA
FULL PAPER
3H), ꢀ0.02 ppm (s, 3H); 13C NMR (101 MHz, MeOD): d=160.16,
159.69, 159.62, 159.00, 158.85, 151.96, 151.43, 138.82, 138.31, 121.07,
120.95, 92.76, 91.03, 88.52, 88.12, 87.23, 84.98, 71.63, 71.39, 50.21, 47.74,
41.62, 41.45, 41.18, 40.91, 35.34, 26.24, 26.07, 18.73, 18.56, ꢀ2.41, ꢀ2.45,
ꢀ2.48, ꢀ2.75 ppm; HRMS (NSI+, MeCN): m/z calcd for C21H34N6O4Si:
463.2484; found: 463.2472 [M+H+].
41.35, 26.06, 18.57, ꢀ2.56, ꢀ2.89 ppm; HRMS (NSI+, MeOH): m/z calcd
for C18H29N5O4Si: 408.2062; found: 408.2057 [M+H+].
(3’S,6’R)-N2-(N,N-Dimethylformamidino)-9-{3’-O-[(tert-butyl)-dimethyl-
silyl]-2’-deoxy-3’,5’-ethano-b-d-ribofuranosyl} guanine (45b): The nucleo-
side 44b (170 mg, 0.41 mmol) was dissolved in DMF (4.5 mL) and N,N-
dimethylformamide dimethyl acetate (0.11 mL, 0.82 mmol) was added.
The solution was stirred for 3 h at 558C. The DMF was then evaporated
by Kugelrohr distillation and the crude product purified by FC (10%
methanol in CH2Cl2). Compound 45b (122 mg, 0.26 mmol, 64%) was ob-
tained as a white foam. Rf =0.3 (10% methanol in CH2Cl2); 1H NMR
(400 MHz, CDCl3): d=9.13 (s, 1H), 8.68 (s, 1H), 7.87 (s, 1H), 6.04 (dd,
1H, J=10.0, 5.5), 4.53 (m, 1H), 4.35 (d, 1H, J=6.3), 3.22 (dd, 1H, J=
13.4, 10.1), 3.17 (s, 3H), 3.07 (s, 3H), 2.53 (dd, 1H, J=13.4, 5.5), 2.40 (m,
1H), 2.18 (m, 1H) 2.10 (m, 1H), 2.01 (m, 1H), 0.90 (m, 9H), 0.15 (s,
3H), 0.13 ppm (m, 3H); 13C NMR (75 MHz, CDCl3): d=81.93, 80.54,
79.99, 72.95, 60.80, 44.42, 15.14, 12.56, 7.82, 0.20, ꢀ1.84, ꢀ27.06, ꢀ29.22,
ꢀ35.90, ꢀ36.81, ꢀ42.05, ꢀ51.34, ꢀ59.19, ꢀ79.57, ꢀ79.66; HRMS (NSI+,
MeCN): m/z calcd for C21H34N6O4Si: 463.2484; found: 463.2475
[M+H+].
(3’S,6’S)-N2-(N,N-Dimethylformamidino)-9-{6’-O-(methylsulfonyl)-3’-O-
[(tert-butyl)-dimethylsilyl]-2’-deoxy-3’,5’-ethano-a- and -b-d-ribofuranosyl}
guanine (42a/b): A solution of 41a/b (243 mg, 0.53 mmol) in pyridine
(5.5 mL) was cooled to 08C and MsCl (40 mL, 0.58 mmol) was added.
The mixture was stirred for 4 h at RT. After the addition of silica the pyr-
idine was evaporated and the crude product purified by using FC (5%
methanol in CH2Cl2). Compound 42a/b (263 mg, 0.49 mmol, 92%) in an
anomeric ratio of a/bꢃ2:1was obtained as a white solid. Rf =0.4 (10%
methanol in CH2Cl2), 1H NMR (400 MHz, CDCl3): d=8.62 (s, 1H, N=
CHNACHTUNGTRENNUNG(CH3)2), 8.53 (s, 1H), 7.93 (s, 1H), 7.79 (s, 1H), 6.24 (dd, 1H, J=
7.1, 3.7), 6.04 (dd, 1H, J=9.4, 5.4), 5.24–5.13 (m, 2H), 4.54 (d, 1H, J=
5.4), 4.35 (dd, 1H, J=6.9, 1.6), 3.20 (s, 3H), 3.18 (s, 3H), 3.11 (s, 6H),
3.05 (s, 3H), 3.04 (s, 3H), 2.72 (m, 2H), 2.61 (m, 4H), 2.42 (m, 2H),
2.26–2.12 (m, 4H), 0.92 (s, 9H), 0.87 (s, 9H), 0.19 (s, 3H), 0.18 (s, 3H),
0.15 (s, 3H), 0.08 ppm (s, 3H); 13C NMR (101 MHz, CDCl3): d=158.06,
157.99, 156.93, 150.34, 136.75, 136.43, 121.20, 89.19, 88.54, 86.97, 86.46,
83.87, 79.69, 79.22, 47.39, 47.30, 46.53, 46.39, 41.70, 38.59, 38.00, 37.84,
35.50, 25.83, 25.80, 18.04, ꢀ2.46, ꢀ2.48 ppm; HRMS (NSI+, EtOAc): m/z
calcd for C22H36N6O6SSi: 540.2186; found: 541.2183 [M+].
(3’S,6’R)-N2-(N,N-Dimethylformamidino)-9-{5’-O-[4,4’-(dimethoxytriphe-
nyl)methyl]-3’-O-[(tert-butyl)-dimethylsilyl]-2’-deoxy-3’,5’-ethano-b-d-ribo-
furanosyl} guanine (46b): DMT-Cl (154 mg 0.45 mmol) was added in
three portions to a solution of nucleoside 45b (70 mg, 0.15 mmol) in pyri-
dine (0.7 mL) and it was stirred for 24 h at RT. The solution was then di-
luted with ethyl acetate, washed with sat. NaHCO3 and dried over
MgS04. Purification by FC (5% methanol in CH2Cl2) yielded the nucleo-
side 46b (108 mg, 0.14, mmol, 94%) as a white foam. Rf =0.66 (10%
methanol in CH2Cl2), 1H NMR (300 MHz, CDCl3): d=8.94 (br, 1H), 8.61
(s, 1H), 7.88 (m, 1H), 7.47 (m, 2H), 7.36 (m, 4H), 7.30 (m, 1H), 7.20 (m,
1H), 6.83 (m, 4H), 6.19 (dd, 1H, J=9.6, 5.1), 4.27 (m, 1H), 4.12 (dd, 1H,
J=7.5, 3.9), 3.79 (s, 6H), 3.13 (s, 3H), 3.10 (s, 3H), 2.73 (dd, 1H, J=13.0,
9.6), 2.42 (dd, 1H, J=13.0, 5.1), 1.98 (m, 1H), 1.68 (m, 1H), 1.60 (m,
2H), 0.83 (s, 9H,), 0.04 (s, 3H), ꢀ0.04 ppm (s, 3H); 13C NMR (75 MHz,
CDCl3): d=158.86, 158.29, 157.96, 156.81, 150.17, 145.69, 136.96, 136.21,
130.28, 128.28, 128.22, 127.11, 113.55, 90.76, 88.62, 87.44, 84.73, 75.66,
55.47, 46.96, 45.56, 41.51, 39.70, 35.37, 25.88, 18.02, ꢀ2.63 ppm; HRMS
(NSI+, MeCN): m/z calcd for C42H52N6O6Si: 765.3790; found: 765.3779
[M+H+].
(3’S,6’R)-N2-{N,N-Dimethylformamidino}-9-{6’-O-acetyl-3’-O-[(tert-bu-
ACHTUNGTRENNUNGtyl)-dimethylsilyl]-2’-deoxy-3’,5’-ethano-a- and -b-d-ribofuranosyl} gua-
nine (43a/b): A solution containing 42a/b (620 mg, 1.15 mmol) and
CsOAc (3.0 g, 15.9 mmol) in DMSO (16 mL) was heated to 858C and
stirred for 16 h. After cooling to RT the mixture was diluted with ethyl
acetate and washed with sat. NaHCO3. The combined organic layers
were washed with H2O and dried over MgSO4. By using FC purification
(5% methanol in CH2Cl2) 43a/b (491 mg, 0.97 mmol, 85%) was obtained
in an anomeric ratio of a/bꢃ2:1 as a white foam. The 1H NMR spectra
still showed a considerable amount of DMSO. Rf =0.5 (10% methanol in
CH2Cl2); 1H NMR (400 MHz, CDCl3): d=9.72 (br, 2H), 8.60 (s, 1H),
8.58 (s, 1H), 7.95 (s, 1H), 7.89 (s, 1H), 6.32 (m, 1H), 6.22 (m, 1H), 5.34
(m, 2H), 4.59 (m, 1H), 4.36 (dd, 1H, J=6.9, 1.3), 3.17 (m, 6H), 3.10 (m,
6H), 2.70–2.53 (m, 4H), 2.41- 2.28 (m, 6H) 2.16 (m, 2H), 2.06 (s, 3H),
2.05 (s, 3H), 0.90 (s, 9H), 0.87 ppm (s, 9H,), 0.14 (m, 12H); 13C NMR
(101 MHz, CDCl3): d=170.49, 158.39, 158.13, 157.17, 156.95, 150.16,
136.83, 135.83, 132.06, 120.35, 95.82, 91.14, 91.03, 89.43, 89.36, 84.92,
83.83, 76.72, 76.30, 48.04, 46.85, 46.22, 45.88, 41.57, 37.76, 37.59, 35.40,
25.85, 25.80, 25.74, 21.52, 21.40, 17.99, ꢀ2.56, ꢀ2.63, ꢀ2.73 ppm; HRMS
(NSI+, EtOAc) calcd for C23H36N6O5Si: 505.2589; found: 505.2582 [M+
H+].
(3’S,6’R)-N2-(N,N-Dimethylformamidino)-9-{6’-O-[4,4’-(dimethoxytriphe-
nyl)methyl]-2’-deoxy-3’,5’-ethano-b-d-ribofuranosyl} guanine (47b): Nu-
cleoside 46b (74 mg, 0.1 mmol) was dissolved in THF (2.2 mL) and
TBAF (1m in THF, 0.1 mL, 0.1 mmol) was added. The clear solution was
stirred for 5 h at RT and then quenched by the addition of silica. Purifica-
tion by FC (5% methanol in CH2Cl2) yielded 47b (44 mg, 70%) as a
white foam. Rf =0.4 (10% methanol in CH2Cl2); 1H NMR (400 MHz,
MeOD): d=8.68 (s, 1H), 7.97 (s, 1H), 7.46 (m, 2H), 7.34 (m, 4H), 7.27
(m, 2H), 7.21 (m, 1H), 6.84 (m, 4H), 6.24 (dd, 1H, J=9.4, 5.4), 4.33 (m,
1H), 4.08 (dd, 1H, J=7.4, 3.4), 3.78 (s, 6H), 3.16 (s, 3H), 3.12 (s, 3H),
2.91 (dd, 1H, J=13.1, 9.5), 2.49 (dd, 1H, J=13.2, 5.4), 1.95 (m, 1H), 1.82
(m, 1H), 1.67 (dd, 1H, J=14.0, 6.1), 1.57 ppm (m, 1H); 13C NMR
(101 MHz, MeOD): d=160.25, 159.82, 159.17, 151.84, 147.10, 138.25,
138.09, 138.06, 131.32, 129.33, 128.92, 127.86, 120.65, 114.27, 90.89, 88.62,
87.54, 85.98, 77.02, 55.73, 47.44, 46.93, 41.52, 40.74, 35.33 ppm; HRMS
(NSI+, MeCN, 0.1% HCOOH): m/z calcd for C36H38N6O6: 651.2926;
found: 651.2925 [M+H+].
(3’S,6’R)-9-{3’-O-[(tert-Butyl)-dimethylsilyl]-2’-deoxy-3’,5’-ethano-a- and
-b-d-ribofuranosyl} guanine (44a/b): The anomeric mixture 43a/b
(512 mg, 1.0 mmol) was dissolved in 1m KOH in methanol/H2O (5:3,
12 mL). The solution was stirred for 6 h at 608C and quenched by the ad-
dition of ammonium chloride. The solvents were then evaporated and
the remains absorbed on silica gel. FC (5% methanol in CH2Cl2) yielded
the anomers 44b (170 mg, 0.42 mmol, 42%) and 44a (78 mg, 0.2 mmol,
20%) as white solids. Compound 44a: Rf =0.27 (10% methanol in
1
CH2Cl2); H NMR (400 MHz, MeOD): d=7.96 (s, 1H), 6.19 (dd, 1H, J=
(3’S,6’R)-N2-(N,N-Dimethylformamidino)-9-{3’-O-[(2-cyanoethoxy)(diiso-
propylamino)phosphino]-6’-O-[4,4’-(dimethoxytriphenyl)methyl]-3’-O-
9.5, 5.7), 4.48 (m, 1H), 4.34 (dd, 1H, J=7.0, 1.7), 2.93 (dd, 1H, J=13.4,
9.6), 2.59 (dd, 1H, J=13.4, 5.7), 2.26 (m, 2H), 2.08 (dd, 1H, J=13.7, 5.4),
1.95 (m, 1H), 0.93 (s, 9H), 0.19 (s, 3H), 0.17 ppm (s, 3H); 13C NMR
(101 MHz, MeOD): d=159.29, 155.44, 152.66 137.90, 117.66, 93.15, 91.18,
85.41, 74.81, 49.30, 41.34, 26.18, 18.69, ꢀ2.53, ꢀ2.59 ppm; HRMS (NSI+,
MeOH): m/z calcd for C18H29N5O4Si: 408.2062; found: 408.2056 [M+H+
]; Compound 44b: TLC Rf =0.27 (10% methanol in CH2Cl2); 1H NMR
(400 MHz, MeOD): d=7.89 (s, 1H), 6.26 (dd, 1H, J=6.7, 3.0), 4.66 (dd,
1H, J=7.0, 2.0), 4.45 (m, 1H), 2.95 (dd, 1H, J=13.9, 6.7), 2.75 (dd, 1H,
J=13.9, 3.0), 2.31 (m, 1H), 2.14 (m, 2H), 1.84 (m, 1H), 0.78 (s, 9H), 0.11
(s, 3H), ꢀ0.02 ppm (s, 3H); 13C NMR (101 MHz, MeOD): d=159.33,
155.17, 152.30, 137.30, 118.09, 94.21, 91.23, 88.09, 74.20, 49.66, 48.90,
[(tert-butyl)-dimethylsilyl]-2’-deoxy-3’,5’-ethano-b-d-ribofuranosyl}
gua-
nine (48b): Hꢁnigꢂs base (0.06 mL, 0.34 mmol) and CEP-Cl (0.04 mL,
0.2 mmol) was added to a solution of 47b (44 mg, 0.07 mmol) in MeCN
(1 mL). The mixture was stirred at RT for 1 h then diluted with ethyl ace-
tate and washed with sat. NaHCO3. Purification by FC (3% methanol in
CH2Cl2) yielded the desired phosphoramidite 48b (50 mg, 0.06 mmol,
88%) as a white foam. Rf =0.7 (10% methanol in CH2Cl2), 1H NMR
(400 MHz, CDCl3): d=8.95 (br, 1H), 8.60 (m, 1H), 7.85 (m, 1H), 7.45
(m, 2H), 7.35 (m, 4H), 7.28 (m, 2H), 7.21 (m, 1H), 6.83 (m, 4H), 6.22
(m, 1H), 4.32 (m, 2H), 3.79 (m, 6H), 3.77–3.54 (m, 4H), 3.13 (s, 3H),
3.09 (m, 3H), 2.84 (m, 2H), 2.57 (t, 1H, J=6.3), 2.51 (t, 1H, J=6.3), 2.09
Chem. Eur. J. 2013, 00, 0 – 0
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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C. J. Leumann and A.-B. Gerber
(m, 1H), 1.86 (m, 2H), 1.60 (m, 1H), 1.15 (m, 9H), 1.09 ppm (d, 3H, J=
6.8); 13C NMR (101 MHz, CDCl3): d=158.80, 158.33, 158.00, 156.94,
156.88, 150.38, 150.30, 145.66, 136.87, 136.83, 136.38, 130.30, 130.25,
128.24, 127.08, 120.90, 117.75, 113.53, 90.91, 90.82, 90.76, 89.12, 87.55,
87.52, 84.33, 75.62, 75.51, 58.20, 58.02, 57.92, 55.46, 44.92, 44.60, 43.59,
43.47, 41.50, 39.41, 35.36, 29.89, 24.75, 24.50, 20.53 ppm; 31P NMR
(122 MHz, CDCl3): d=141.88 ppm; HRMS (NSI+, MeCN) for
C45H55N8O7P: m/z calcd for: 851.4010; found: 851.4009 [M+H+].
PFO-350S temperature controller. The temperature was measured from
the heating block. The graphs were subsequently smoothed with a noise
filter. The phosphate buffer was used as blank. Spectra are given in mdeg
from 210 to 320 nm.
Molecular modeling: Molecular modeling was carried out with the
Amber force field as incorporated in the software package HyperChem
Release 8.0.4 for Windows of Hypercube, Inc. Only original Amber pa-
rameters were used and no explicit water or counterions were included.
RNaseH activity: A solution (0.2 mm) of the oligonucleotide S10 and of
the corresponding DNA and RNA strands was solved in a buffer system
(containing 75 mm KCl, 50 mm Tris-HCl, 3 mm MgCl2 and10 mm dithio-
threitol) and was then hybridized with 0.1 mm of the 32P labeled comple-
mentary RNA strand to give a total volume of 60 mL. After heating to
808C the solution was incubated for 15 min at 378C. The enzyme (2.5 U)
was then added and the mixture was kept at 258C. Aliquots were taken
at 5, 45, 120, and 300 min and were quenched by the addition of a load-
ing buffer (100% formamide, bromphenol blue, and xylencyanol). The
samples were stored at 48C overnight, analyzed on a 20% denaturing
polyacrylamide gel and the bands visualized by autoradiography.
Acknowledgements
We thank the Swiss National Science Foundation and the Association
Monꢇgasque contre les Myopathies (AMM) for financial support of this
project.
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taken up in Dulbeccoꢂs Modified Eagle Medium DMEM/F12 (386 mL),
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Gene Assembler Special DNA-synthesizer by using standard phosphor-
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was used for the detritylation step. The concentrations of the phosphor-
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
The oligonucleotides S1–S4 were cleaved from the resin and deprotected
in 33% NH3 solution at 558C overnight. Also the fully modified sequen-
ces S6, S7, S10, and S11 were cleaved and deprotected in 33% NH3 but
at 708C overnight. After evaporation the crude samples were filtered,
taken up in water and purified by reversed phase (Source 15 RPC ST
100/4.6 Polystyrene-15 column from Pharmacia Biotech) or DEAE-
HPLC (DNAPac-200 4ꢆ250 mm column with pre-column both from
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Isobicyclo-DNA
FULL PAPER
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Received: && &&
Published online: && &&
,
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C. J. Leumann and A.-B. Gerber
DNA
A.-B. Gerber,
C. J. Leumann* ............... &&&& — &&&&
Synthesis and Properties of Isobicyclo-
DNA
A twist to DNA structure: Moving the
phosphodiester backbone from the
natural C(5’) position into the C(6’)
position, which is only available on the
bicyclo-DNA skeleton, leads to isobi-
cyclo-DNA (Isobc-DNA; see scheme).
Isobc-DNA forms stable duplexes with
DNA and discriminates RNA as a
complement. In addition, it forms very
stable antiparallel self-duplexes with a
structure that is different from A-, B-,
or Z-DNA.
DNA
The synthesis of isobicyclo-DNA (iso-bc-DNA) building
blocks with all four natural bases is presented, as well as
their incorporation into oligonucleotides and their RNA
and DNA recognition properties. Their structure was
determined by circular dichroism (CD) spectroscopy and
their biological properties through serum stability and
RNaseH activation. Slight changes in the DNA backbone
geometry lead to remarkable consequences on the duplex
structure, as can be seen from the properties of isobicyclo-
DNA. For more details see the Full Paper by C. J.
Leumann and A.-B. Gerber on page &&ff.
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ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 0000, 00, 0 – 0
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These are not the final page numbers!