Synthesis and incorporation into α-DNA of a novel conformationally constrained α-nucleoside analogue

Article abstract of DOI:10.1055/s-2002-25755

The synthesis and incorporation into α-DNA of a novel conformationally constrained α-nucleoside analogue is described. The carbohydrate part of this analogue was prepared in 4 steps from the known bicyclic precursor 1 via a stereospecific, intramolecular, Et₃B-mediated radical addition to a keto function as the key step. The thus obtained intermediate 4 was transformed stereoselectively into the corresponding α-nucleoside analogues 7 and 8 containing the bases adenine and thymine, and were further elaborated into the phosphoramidite building blocks 11 and 12. Both building blocks were incorporated into α-oligodeoxynucleotides and their pairing behavior to parallel complementary DNA was studied by UV-melting experiments. Single substitutions of α-deoxyribnucleoside units by the new analogues in the center of duplexes were found to be thermally destabilizing by only -0.8 to -3.1°C.

Full text of DOI:10.1055/s-2002-25755

SPECIAL TOPIC
789
Synthesis and Incorporation into -DNA of a Novel Conformationally
Constrained -Nucleoside Analogue
A
N
ovel Conform
e
ationally
C
r
onstrain
n
ed -Nucleo
h
side
A
nalogu
a
e
rd M. Keller, Christian J. Leumann*
Department of Chemistry & Biochemistry, University of Bern, Freiestrasse 3, 3012 Bern, Switzerland
Fax +41(31)6313422; E-mail: leumann@ioc.unibe.ch
Received 9 February 2002
ture with an overall geometry that is close to the B-confor-
mation of DNA.8 We have investigated earlier the
conformationally constrained -DNA analogue -bicyc-
lo-DNA and found it to pair to complementary DNA and
Abstract: The synthesis and incorporation into -DNA of a novel
conformationally constrained -nucleoside analogue is described.
The carbohydrate part of this analogue was prepared in 4 steps from
the known bicyclic precursor 1 via a stereospecific, intramolecular,
Et3B-mediated radical addition to a keto function as the key step. RNA in a parallel fashion with similar affinity as DNA.9
The thus obtained intermediate 4 was transformed stereoselectively
Given the fact that in -bicyclo-DNA one of the backbone
into the corresponding -nucleoside analogues 7 and 8 containing
torsion angles ( ) deviates intrinsically (anti, pseudoequa-
the bases adenine and thymine, and were further elaborated into the
torial orientation of the oxy-substituent at C-5 ) from that
phosphoramidite building blocks 11 and 12. Both building blocks
observed in DNA and -DNA/DNA duplexes (syn) we
were incorporated into -oligodeoxynucleotides and their pairing
wished to correct for this local structural inconsistency
and further constrict conformational flexibility, and de-
signed the fixed nucleic acid analogue FNA (Figure 1).
behavior to parallel complementary DNA was studied by UV-melt-
ing experiments. Single substitutions of -deoxyribnucleoside units
by the new analogues in the center of duplexes were found to be
thermally destabilizing by only –0.8 to –3.1 °C.
In this communication we report on the synthesis of the
underlying nucleoside analogues 7 and 8, as well as their
building blocks for oligodeoxynucleotide synthesis. Fur-
thermore, we disclose first pairing properties of -oli-
godeoxynucleotides carrying single -FNA nucleotide
residues with complementary DNA, as determined by
UV-melting curve analysis and CD-spectroscopy.
Key words: radical reactions, DNA, nucleosides, antisense agents,
bioorganic chemistry
Introduction
Oligonucleotide analogues continue to be an attractive
goal in medicinal chemistry as specific inhibitors
(antisense agents) for the expression of disease related
genes.1–3 While a first generation phosphorothioate oli-
godeoxynucleotide 21-mer (ISIS 2922, Vitravene®), act-
ing against cytomegalovirus (CMV) induced retinitis, is
available on the market since 1998, a number of first and
second generation (2 - -modified-RNA) oligonucle-
otides are currently in clinical trials. A prerequisite for ef-
ficient inhibition of the expression of the genetic message
is high affinity of an oligonucleotide analogue to its target
RNA, as well as high biological stability in a cellular en-
vironment. Within this context others and we have suc-
cessfully applied the principle of oligonucleotide single
strand preorganization via conformational constriction of
nucleosides as one way to improve the binding properties
of oligonucleotides to their RNA target.4
O
5'
O
O
4' O
2'
O
O
γ
δ
γ
δ
γ
δ
1'
3'
Base
O
Base
Base
O
O
O
α-Bicyclo-DNA
α-DNA
α-FNA
Figure 1 Chemical structures of -bicyclo-DNA, -DNA and
-
FNA. The red part of the structures conformationally constrict the
C3 –C4 and the C4 –C5 bonds (cf. torsion angles and ) of the de-
oxyribose unit. In -FNA, the torsion angle is locked in the syn-con-
formation, as observed in -DNA/DNA duplexes, while it is
preferentially anti in -bicyclo-DNA. The conformation of the fura-
nose units is in the south (S) conformational range in all cases.
Results
The -anomeric isomer of DNA ( -DNA, Figure 1) was
shown earlier to bind to complementary DNA and RNA in
a parallel fashion with similar affinity as DNA.5,6 Further-
more, -oligodeoxynucleotides are more stable against
degradation by nucleases.7 NMR structural analyses on -
DNA/DNA duplexes showed that these are fully Watson–
Crick base-paired and adopt a right-handed helical struc-
Synthesis of Monomers
The synthesis of the nucleosides 7 and 8 started from the
diol 1 for which we had already elaborated a productive
synthetic access.10 In order to obtain the tricyclic sugar
surrogate 4 as the first key intermediate, we planned to in-
troduce a suitably functionalized C2 synthon into ketone
2, which was easily obtained in high yield by Dess–Martin
oxidation. First experiments with the allyl Grignard re-
agent not unexpectedly yielded exclusively the addition
Synthesis 2002, No. 6, 29 04 2002. Article Identifier:
1437-210X,E;2002,0,06,0789,0796,ftx,en;C00302SS.pdf.
© Georg Thieme Verlag Stuttgart · New York
ISSN 0039-7881

790
B. M. Keller, C. J. Leumann
SPECIAL TOPIC
product with the undesired relative configuration at C-6.
In order to control the relative stereochemistry at this cen-
ter, an intramolecular approach using the tertiary hydroxy
function in 2 as an anchor was envisaged. Exploratory ex-
OTBDMS
O
OTBDMS
a
O
74% (5)
58% (6)
Base
O
OEt
O
OH
OR
periments into this direction with the corresponding
-
5 Base = Thy, R = H, (α:β = 9:1)
6 Base = Bz6Ade, R = TMS (α:β > 9:1)
4
bromoacetate ( intramolecular Reformatsky reaction)
failed due to the unreliable and low yielding formation of
the sterically hindered ester. Encouraged by a recent re-
port on intramolecular addition of alkyl radicals to alde-
hydes and ketones by Malacria 11 we decided to explore a
similar approach. To this end the necessary iodoacetal 3
was conveniently prepared as a ca. 1:1 mixture of dia-
stereoisomers by treatment of 2 with ethyl vinyl ether in
the presence of N-iodosuccinimide (NIS).12 The following
intramolecular radical addition with Et3B as the radical
initiator and terminator lead to 4 in an appreciable yield
(55%) for this type of reaction (Scheme 1). The enhanced
electrophilicity of the keto group in 3, due to the neighbor-
ing electronegative substituents, might certainly have
contributed to the efficiency of this reaction. Interestingly,
4 was isolated as a : = 7:1 mixture with respect to the
configuration at the acetal center. This can only be ex-
plained by epimerization during or after the ring closing
reaction, most probably induced by Et3B acting as a Lewis
acid. This observation was important with respect to the
stereochemical outcome of the following nucleosidation
reactions.
O
NHBz
N
NH
N
Bz6Ade =
Thy =
N
O
N
N
Scheme 2 Reagents and conditions: (a) i) thymine or N6-benzoyla-
denine (2–3 equiv), BSA (6–8 equiv), MeCN (for 5), ClCH2CH2Cl
(for 6), r.t., –60 °C, 30 min, ii) Me3SiOTf (1.5–2.0 equiv), 60–80 °C,
2–7 h
Bu4NF in THF, the nucleoside analogues 7 and 8
(Scheme 3) bearing free OH groups became available.
The -(3S)-configuration at the anomeric center in 7 and
8 could unambiguously be assigned by H NMR differ-
ence NOE spectroscopy (see experimental section).
1
OTBDMS
O
OH
O
a
b
84% (7)
67% (8)
80% (9)
79% (10)
O
Base
O
Base
OR
OH
OTBDMS
OH
OTBDMS
OH
5 Base = Thy, R = H
6 Base = Bz6Ade, R = TMS
7 Base = Thy
8 Base = Bz6Ade
b
a
72%
95%
O
O
O
OPix
O
OPix
OH
O
O
1
2
c
90% (11)
85% (12)
O
Base
N(iPr)2
O
Base
OTBDMS
O
OTBDMS
O
O
OH
P
OEt
I
c
9 Base = Thy
10 Base = Bz6Ade
OCH2CH2CN
55%
O
OEt
11 Base = Thy
12 Base = Bz6Ade
O
OH
3
4
O
Scheme 1 Reagents and conditions: (a) Dess–Martin reagent,
CH2Cl2, r.t., 30 min; (b) NIS (2.5 equiv), ethyl vinyl ether (2.5 equiv),
CH2Cl2, –78 °C, 12 h; (c) 15% Et3B in hexane, –20 °C, air, 72 h, 55%
Pix =
Scheme 3 Reagents and conditions: (a) Bu4NF (2.0–2.5 equiv),
THF, 2–4 h, r.t.–40 °C; (b) 9-chloro-9-phenylxanthene (4–8 equiv),
pyridine, r.t., 18 h; (c) (NCCH2CH2O)(i-Pr2N)PCl (1.5 equiv), i-
Pr2NEt (6 equiv), MeCN, r.t.
With 4 in our hands we approached the nucleoside synthe-
sis via the classical Vorbrüggen one-pot procedure
(Scheme 2).13
Me3SiOTf-mediated condensation of 4 with in situ persi-
lylated thymine in MeCN afforded 5 as an
:
Selective introduction of the 9-phenyl-9-xanthenyl (pixyl)
group into the secondary hydroxyl function could be
achieved with the corresponding chloride in absolute py-
ridine, yielding 9 and 10 in very good yields. The pixyl
group has been shown in the past to be a valuable alterna-
tive for the commonly used, sterically more demanding
dimethoxy trityl group (DMT) in DNA or RNA synthesis
for the protection of less accessible, secondary hydroxy
groups.14,15 The phosphoramidite building blocks 11 and
(3S:3R = 9:1) mixture (1H NMR) in 74% yield. Variations
in temperature, solvent and Lewis acid, had no effect on
the ratio of anomers. In the case of the purine base, the
condensation of 4 with persilylated N6-benzoyladenine
was best performed in 1,2-dichloroethane and yielded the
bis-silyl nucleoside 6 in 58%. Only small amounts of the
corresponding -(3R)-isomer (<10% relative to -isomer)
1
could be detected by H NMR. After deprotection with
Synthesis 2002, No. 6, 789–796 ISSN 0039-7881 © Thieme Stuttgart · New York

SPECIAL TOPIC
A Novel Conformationally Constrained -Nucleoside Analogue
791
12 were obtained by reaction of 9 and 10 with the com-
monly used phosphitylation reagent (Scheme 3). As ex-
pected, 11 and 12 appear as diastereoisomeric mixtures at
phosphorous (1H NMR and 31P NMR).
Table 2 Tm-Data (°C) from UV-Melting Curves (260 nm) in 10
mM Na-Cacodylate, pH 7.0 and 1 M NaCl (duplex concd = 2 M)
Entry Duplex
Tm
Tm/mod.
1
-d(TTTTTTTTTT)
40.1
-
-d(AAAAAAAAAA)
Oligonucleotide Synthesis
2
-d(TTTTT-t-TTTT)
-d(AAAAA-A-AAAA)
37.6
37.0
28.5
16.5
15.7
n.d.a
n.d.a
n.d.a
n.d.a
–2.5
The synthesis of oligonucleotides 13–18 (Table 1) was ac-
complished on a DNA synthesizer on the 1.3 mol scale
by standard cyanoethyl phosphoramidite chemistry. The
chain extension cycles were essentially identical to those
for natural oligodeoxynucleotide synthesis with coupling
times increased to 15–20 min for the building blocks 11
and 12 and replacement of the usual activator 1H-tetrazole
by the more powerful (S-alkyl)-1H-tetrazoles. Coupling
yields of >95% per step (trityl assay) were obtained for
DNA or -DNA building blocks. The incorporation of the
-FNA building blocks, however, proceeded only with
yields of 60% even at increased phosphoramidite con-
centration. This inefficiency in the coupling step is most
likely due to steric hindrance around the reacting groups
in 11 and 12 and clearly needs to be addressed in the fu-
ture. This precluded the synthesis of multiply or fully
modified -FNA oligonucleotides. The oligonucleotides
were deprotected and cleaved from solid support in a stan-
dard manner. Crude oligonucleotides were purified to ho-
mogeneity by DEAE ion-exchange HPLC, controlled by
RP-HPLC, and analyzed by ESI-MS. Table 2 gives an
overview of the -oligodeoxynucleotides prepared and
used for the following investigations.
3
-d(TTT-t-TTTTTT)
-d(AAA-A-AAAAAA)
–3.1
4
-d(T-t-TTTTTTTT)
-d(A-A-AAAAAAAA)
–11.5
5
-d(TAAAATATAA)
-d(ATTTTATATT)
-
6
-d(TAAA-a-TATAA)
-d(ATTT-T-ATATT)
–0.8
7
-d(TAAAATATAA)
-d(ATTTCATATT)
-
-
-
-
8
-d(TAAA-a-TATAA)
-d(ATTT-C-ATATT)
9
-d(TAAAATATAA)
(ATTTTATATT)d-
10
-d(TAAA-a-TATAA)
(ATTT-T-ATATT)d-
a n.d.= not detected.
at pH 7.0. The corresponding Tm-data are summarized in
Table 2.
Table 1 Sequence Information and MS Data of -Oligonucleotides
Prepared for the Present Studya
In a first series of experiments, we focused on the pairing
properties of the homobasic -decanucleotides 13–15,
containing one modified -T-FNA unit at various posi-
tions, with their DNA complement. By comparing the
Tm-data (Table 2) it becomes obvious, that incorporation
of a -T-FNA unit in the center of the sequence leads to a
decrease in Tm by ca. 2.5 to 3.1 °C (entries 2 and 3). From
these experiments we conclude that complementary base-
pairing occurs at the site of modification, albeit on an
Product
Oligonucleotides (5 3 )
[M–H]–
calcd
[M–H]–
found
13
14
15
16
17
18
-d(TTTTT-t-TTTT)
-d(TTT-t-TTTTTT)
-d(T-t-TT TTTTTT)
-d(TTTTTTTTTT)
-d(TAAA-a-TATAA)
-d(TAAAATATAA)
3048.2
3048.2
3048.2
2980.0
3111.2
3043.0
3047.8
3047.7
3048.5
2979.9
3110.8
3043.6
overall lower affinity-level compared to unmodified
-
DNA. Surprisingly, incorporation near to the end of the
sequence leads to a decrease in Tm of 11.5 °C (entry 4).
This points to a negative cooperative effect of the -T-
FNA, probably disrupting base-pairing of the 5 -terminal
nucleotide unit in 15.
a Capital letters denote 2 - -deoxyribonucleosides, small letters be-
tween hyphens FNA units.
In order to further explore effects on preferred strand ori-
entation and base-pairing selectivity we investigated the
asymmetric, modified decamer 17 and compared its pair-
ing properties to that of the unmodified -oligonucleotide
18. These experiments also allowed for the detection of
eventual effects arising from the bases, as the -A-FNA
unit was used here. Also in this case, a stable duplex was
formed with the parallel -DNA complement (entry 6)
with a Tm-value of only –0.8 °C. As expected, base-
pairing is selective. No duplex formation could be ob-
served if cytosine was introduced opposite the -A-FNA
Pairing Properties of Modified -Oligonucleotides
In order to assess the influence of the backbone modifica-
tion on duplex stability we recorded UV-melting curves of
the modified -oligonucleotides 13–15 and 17 with their
-DNA complements in parallel, antiparallel and base-
mismatch arrangements and compared their properties
with those of the unmodified -oligonucleotides 16 and
18. All measurements were performed in standard buffer
Synthesis 2002, No. 6, 789–796 ISSN 0039-7881 © Thieme Stuttgart · New York

792
B. M. Keller, C. J. Leumann
SPECIAL TOPIC
residue (entry 8). Again, no antiparallel duplex formation
could be observed, neither in the modified nor in the un-
Discussion and Conclusions
modified duplex (entry 9, 10). From these data we con- Within the growing family of conformationally con-
clude that within the duplex the FNA-residues are fully strained DNA analogues there are two members that are
base-paired, recognize complementary bases with high of particular interest here, namely the -bicyclo-DNA 9,16
selectivity and do not perturb the preferred parallel strand (Figure 1) and -D-LNA.17,18 A direct comparison of
orientation in duplexes of -DNA with DNA.
properties with -bicyclo-DNA is difficult due to the fact
that no data on single substitutions of -DNA with -bi-
cyclo-DNA exists. The comparison of homogeneous
backbone (fully modified) oligonucleotides with that of
non-homogeneous (partially modified) ones is difficult
Structural Investigations by CD-Spectroscopy
In order to evaluate structural differences between modi- and can be misleading as has been demonstrated before in
fied and unmodified duplexes we recorded CD-spectra of the case of the pairing of -D-LNA to RNA.18 Data of -
the duplexes corresponding to entries 1,2 and 4 of Table 2 DNA sequences containing single -D-LNA residues,
(Figure 2). Comparison of the CD Spectra at low temper- however, are available. These data show that -LNA res-
atures reveals a distinct negative maximum around 270 idues destabilize pairing to complementary DNA and
nm in the case of the modified duplexes (Figure 2b and c) RNA thermally by –6.5 to –8.0 °C/mod. This is distinctly
which dissapears upon melting of the duplex (at higher more than observed for -FNA units. The main structural
temperatures). This behavior is essentially absent in the difference between -LNA and -FNA, presented here,
case of the unmodified duplex (Figure 2a). Thus, the CD concerns the conformation of the furanose ring that is N in
spectra indicate subtle structural variations upon introduc- the former and S in the latter case, and the torsion angle
tion of a single -T-FNA unit. This is not unusual and which is unrestricted in -D-LNA. The overall B-structur-
shows local, probably cooperative conformational alter- al features of parallel -/ -DNA duplexes,8 are thus better
ations around the modified unit. These structural varia- accommodated in the FNA units, showing a more appro-
tions could be the reason that substitutions near to the priate geometric preorganization than -D-LNA. In fact,
strand-terminus are more destabilizing than those in the the latter analogue fails to form duplexes with comple-
center. An extrapolation of these observations would mentary DNA but forms stable duplexes with RNA as a
point to a different conformation of a fully modified - complement.
FNA/DNA duplex as compared to a -DNA/DNA duplex.
In conclusion, we have presented a concise synthesis of
This does not preclude, however, that the thermal stability
the adenine and thymine containing building blocks of the
of such a fully modified -FNA/DNA duplex could not be
new conformationally constrained DNA-analogue
-
higher.
FNA. As determined from single incorporations into -
Figure 2 CD-spectra of selected -DNA/DNA duplexes in 10 mM Na-cacodylate, 1 M NaCl, pH 7.0 (c = 2 M).
Synthesis 2002, No. 6, 789–796 ISSN 0039-7881 © Thieme Stuttgart · New York

SPECIAL TOPIC
A Novel Conformationally Constrained -Nucleoside Analogue
793
organic layers were dried (MgSO4) and evaporated. FC (100 g SiO2,
hexane–EtOAc, 9:1) afforded 3 (2.20 g, 72%) as a colorless oil.
TLC (EtOAc–hexane, 2:1): Rf 0.77.
DNA, this analogue diminishes affinity to complementary
DNA only slightly. The limiting factor of a more detailed
study of this new DNA-analogue is the coupling yield
upon oligomerization, which precludes at this point the
study of fully modified -FNA. Experiments to improve
the coupling chemistry, as well as towards the synthesis of
-FNA are currently on our plans.
IR (CHCl3): 3504w(br), 2957s, 2930s, 1755m, 1471m, 1431m,
1256m, 1109s, 973m, 838s cm–1.
1H NMR (300 MHz, CDCl3): = 5.48, 5.2 (2 dd, J = 8.09, 3.31 Hz,
1 H, OCHO), 4.39–4.30 [2 dd, J = 5.89, 2.21 Hz, 1 H, H-C(1)],
4.06, 3.89 [2 s, 1 H, H-C(8)], 3.9–3.1 [m, 6 H, 2 H-C(3)], 2.70, 2.64
[2 dd, J = 5.9, 2.2 Hz, 1 H, H-C(7)], 2.39, 2.32 [2 dd, J = 5.5, 1.8
Hz, 1 H, H-C(7)], 2.28–1.92 [m, 1 H, H-C(4)], 1.86 [dd, J = 12.5,
3.7 Hz, 1 H, H-C(4)], 1.20 (m, 3 H, OCH2CH3), 0.86, 0.85 [2 s, 9 H,
(CH3)3CSi], 0.14, 0.10 [2 s, 6 H, (CH3)2Si].
13C NMR (75 MHz, CDCl3): = 214.2, 214 (2 s, C-6), 97.04, 96.6
(2 d, OCHO), 84.8, 84.3 (2 s, C-5), 78.8 (d, C-8), 74.51, 74.37 (2 d,
C-1), 59.3, 59.24 (2 t, C-3), 59.2, 59.08 (2 t OCH2CH3), 38.55,
38.25 (2 t, C-7), 35.58, 34.36 (2 t, C-4), 25.68, 25.62 [2 q,
(CH3)3CSi], 18.06, 17.92 [2 s, (CH3)3CSi], 15.2, 14.9 (2 q,
OCH2CH3), 6.12, 5.99 (2 t, CH2I), –4.40, –4.52, –4.68, –4.96 [4 q,
CH3)2Si].
Solvents for extraction: technical grade, distilled. Solvents for reac-
tions: reagent grade, distilled over CaH2 (MeCN, CH2Cl2, pyridine),
LiAlH4 (Et2O) or Na (THF). All reactions were performed under an-
hydrous conditions in an argon atmosphere. Reagents were, unless
otherwise stated, from Fluka, highest quality available. Optical ro-
tation: Perkin Elmer-241 polarimeter. IR: Perkin-Elmer FTIR 1600.
NMR: Bruker AC-300, DRX500, in ppm, 13C multiplicities from
DEPT spectra, J in Hz. TLC: Merck SiL G-25 UV254. Flash column
chromatography (FC): silica gel (30–60 micron) from Fluka.
CD-spectra were recorded on a Jasco J-715 spectropolarimeter with
a Jasco PFO-350S temperature controller. The temperature was
measured directly in the cell (path length 10 mm). UV-melting
curves were determined at 260 nm on a Varian Cary 3E spectropho-
tometer that was equipped with a Peltier block using the Varian
WinUV software. Complementary oligodeoxynucleotides were
mixed to 1:1 stoichiometry with a 2 M single strand oligonucle-
otide concentration. A heating-cooling-heating cycle (0 90 °C)
was applied with a temperature gradient of 0.5 °C/min. Tm values
were defined as the maximum of the first derivative of the melting
curve using the software package Origin™v5.0.
HRMS (ESI-TOF, [(M + H2O)– H]–, C17H32O6Si): m/z calcd
487.2892, found: 487.1012.
(1R,3S,5S,7S,11R)-11-{[tert-Butyl(dimethyl)silyl]oxy}-3-eth-
oxy-2,8-dioxatricyclo[5.3.1.01,5]undecan-5-ol (4)
A suspension of 3 (820 mg, 1.74 mmol) in a solution of 15% Et3B
in hexane was treated at –20 °C with air (20 mL). The mixture was
kept at –20 °C for 72 h. The solution was then diluted with EtOAc
(80 mL) and washed with aq sat. NaHCO3 (50 mL). The aqueous
phase was extracted with EtOAc (2 50 mL) and the combined or-
ganic layers were dried (MgSO4) and concentrated. FC (60 g SiO2,
(1S,5R,8R)-8-{[tert-Butyl(dimethyl)silyl]oxy}-5-hydroxy-2-oxa-
bicyclo[3.2.1]octan-6-one (2)
hexane hexane–EtOAc, 5:1) afforded
4 (325 mg, 55%,
3S:3R = 7:1), 15% starting material 3 and 15% of 2. TLC (5% Et2O
in CH2Cl2): Rf 0.34.
To a solution of 110 (1.00 g, 3.6 mmol) in CH2Cl2 (45 mL) was add-
ed 1,1,1-tris(acetyloxy)-1,1-dihydro-1,2-benziodoxol-3(1H)-one
(Dess–Martin reagent,19 1.55 g, 7.2 mmol) and the mixture was
stirred at r.t. After 30 min, the solution was washed with aq 20%
Na2S2O3 (50 mL) and aq sat. NaHCO3 (50 mL) and the aqueous
phase was extracted with CH2Cl2 (2 40 mL). The combined or-
ganic layers were dried (MgSO4) and evaporated. FC (40 g SiO2,
hexane–EtOAc, 1:2) afforded 2 (960 mg, 95%) as a colorless oil.
TLC (EtOAc–hexane, 2:1): Rf 0.67.
IR (CHCl3): 3503w (br), 3007m, 2957s, 2930s, 2858m, 1471w,
1430w, 1375w, 1335w, 1307w, 1258m, 1228w, 1130s, 1109s,
968m, 929m, 854s cm–1.
1H NMR (300 MHz, CDCl3): = 5.48 [dd, J = 5.7, 3.68 Hz, 0.12 H,
H-C(3)], 5.19 [d, J = 4.78 Hz, 0.88 H, H-C(3)], 4.34–4.25 [m,
H-C(9)], 4.06 [dd, J = 5.14, 1.47 Hz, 1 H, H-C(7)], 3.82–3.61 [m, 2
H, H-C(9), OCH2], 3.55 [d, J = 1.84 Hz, 1 H, H-C(11)], 3.46–3.41
(m, OCH2), 3.32 (s, 1 H, OH), 2.59 [dd, J = 13.23, 4.78 Hz, 1 H, H-
C(4)], 2.37 [dd, J = 15.07, 5.15 Hz, 1 H, H-C(6)], 2.1–1.75 [m, 4 H,
H-C(4), 2 H-C(10), H-C(6)], 1.19 [t, J = 6.9 Hz, 3 H, CH3), 0.86
[s, 9 H, (CH3)3CSi], 0.07 [s, 6 H, (CH3)2Si].
13C NMR (75 MHz, CDCl3): 107.52 (d, C-3), 83.98 (s, C-1), 82.18
(d, C-11), 80.99 (d, C-7), 76.58 (s, C-5), 62.7 (t, OCH2), 61.11 (t, C-
9), 47.15 (t, C-6), 38.51 (t, C-4), 34.57 (t, C-10), 25.72, 25.65 [2 q,
(CH3)3CSi], 18.01 [s, SiC(CH3) 3], 15.33 (q, CH3CH2), –4.8, –5.02
(2 q, [CH3)2Si].
IR (CHCl3): 2956s, 2931s, 2859m, 1756s, 1471w, 1379w, 1258m,
1098s, 838s cm–1.
1H NMR (300 MHz, CDCl3): = 4.36 [dd, J = 5.7, 2.2 Hz, 1 H, H-
C(1)], 3.86 [s, 1 H, H-C(8)], 3.83 [dd, J = 12.8, 4.0 Hz, 1 H, H-
C(3)], 3.65 [dd, J = 12.8, 4.0 Hz, 1 H, H-C(3)], 2.80 [br s, 1 H, OH),
2.55 [dd, J = 19.1, 5.7 Hz, 1 H, H-C(7)], 2.40 [dd, J = 19.1, 1.5 Hz,
1 H, H-C(7)], 2.05 [dt, J = 12.9, 7.3 Hz, 1 H, H-C(4)], 1.71 [dd,
J = 12.5, 4.0 Hz, 1 H, H-C(4)], 0.82 [s, 9 H, (CH3)3CSi], 0.08, 0.02
[2 s, 6 H, (CH3)2Si].
HRMS (ESI-TOF, [M – H]–, C17H31O5Si): m/z calcd 343.1938,
found: 343.1940.
13C NMR (75 MHz, CDCl3): = 199.4 (s, C-6), 78.67 (s, C-5),
74.11 (d, C-8), 65.95 (d, C-1), 59.48 (t, C-3), 43.7 (t, C-7), 39.7 (t,
C-4), 25.66 [q, (CH3)3Csi], 18.2 [s, (CH3)3CSi], –4.8, –5.31 [2 q,
CH3)2Si].
HRMS (ESI-TOF, [M – H]–, C13H23O4Si): m/z calcd 271.1354,
found: 271.1365.
(1R,3S,5S,7S,11R)-1-{11-[tert-Butyl(dimethyl)silyl]oxy}-5-hy-
droxy-2,8-dioxatricyclo[5.3.1.01,5]undec-3-yl]-5-methyl-1H-
pyrimidine-2,4-dione (5)
A suspension of thymine (300 mg, 2.37 mmol) and 4 (400 mg, 1.16
mmol) in MeCN (20 mL) was treated with N,O-bis(trimethylsi-
lyl)acetamide (BSA, 2.8 mL, 11.8 mmol) at 60 °C until a clear so-
lution was formed. After addition of TMSOTf (0.4 mL, 1.75 mmol)
the reaction was kept at 60 °C for 2 h. The solution was then diluted
with EtOAc (60 mL) and washed with aq sat. NaHCO3 (40 mL).
The aqueous phase was extracted with EtOAc (2 40 mL), the
combined organic layers dried (MgSO4) and evaporated. FC (40 g
(1S,5R,8R)-8-{[tert-Butyl(dimethyl)silyl]oxy}-5-(1-ethoxy-2-
iodoethoxy)-2-oxabicyclo[3.2.1]octan-6-one (3)
To a solution of 2 (1.75 g, 6.43 mmol) in CH2Cl2 (20 mL) were add-
ed N-iodosuccinimide (3.50 g, 16 mmol) and ethyl vinyl ether (2.1
mL, 16 mmol) at –78 °C. The mixture was then allowed to warm up
to r.t. After the reaction was complete, the solution was diluted with
EtOAc (150 mL) and extracted with aq sat. NaHCO3 (100 mL). The
aqueous phase was washed with EtOAc (2 50mL), the combined
Synthesis 2002, No. 6, 789–796 ISSN 0039-7881 © Thieme Stuttgart · New York

794
B. M. Keller, C. J. Leumann
SPECIAL TOPIC
SiO2, hexane–EtOAc, 1:1) afforded 5 (425mg, 74%) as a mixture of
: (3S/3R = 9:1); white foam.
(1R,3S,5S,7S,11R)-1-(5,11-Dihydroxy-2,8-dioxatricyclo-
[5.3.1.01,5]undec-3-yl)-5-methyl-1H-pyrimidine-2,4-dione (7)
A solution of 5 (200 mg, 0.47 mmol) in THF (4 mL) was treated
with Bu4NF 3H2O (380 mg, 1.2 mmol) at r.t. After 2 h, SiO2 (1 g)
was added, the mixture evaporated and the residue was subjected to
-(3S) Isomer
TLC (hexane–EtOAc, 1:1): Rf 0.60.
FC (8 g SiO2, EtOAc
84%) as a white foam. TLC (EtOAc–hexane, 3:1): Rf 0.10;
[ ]D +51.2 (c = 1.0, MeOH).
EtOAc + 2% MeOH) to give 7 (104 mg,
IR (CHCl3): 3392w, 3027w, 2957w, 1688s, 1471m, 1361w, 1256m,
1109m, 908m, 853m cm–1.
1H NMR (300 MHz, CDCl3): = 8.25 (br s, 1 H, NH), 7.09 [d,
J = 1.21 Hz, 1 H, H-C(6)], 5.84 [dd, J = 9.27, 2.82 Hz, 1 H, H-
C(3 )], 4.40 (s, 1 H, OH), 4.26–4.19 [m, 1 H, H-C(9 )], 4.04 [dd,
J = 5.23, 1.47 Hz, 1 H, H-C(7 )], 3.82–3.77 [m, 1 H, H-C(9 )], 3.67
[s, 1 H, H-C(11 )], 2.9 [dd, J = 14.31, 9.27 Hz, 1 H, H-C(4 )], 2.50–
2.44 [m, 2 H, H-C(4 ), H-C(6 )], 2.10–1.95 [m, 3 H, 2 H-C(10 ), H-
C(6 )], 1.91 (s, 3 H, CH3), 0.92 [s, 9 H, (CH3)3CSi], 0.15, 0.12 [2 s,
6 H, (CH3)2Si].
13C NMR (75 MHz, CDCl3): = 163.76 (s, C-4), 150.0 (s, C-2),
136.7 (d, C-6), 109.7 (s, C-5), 96.0 (s, C-1 ), 88.49 (d, C-3 ), 83.84
(s, C-5 ), 80.7 (d, C-11 ), 74.6 (d, C-7 ), 59.37 (t, C-9 ), 50.54 (t, C-
6 ), 41.2 (t, C-4 ), 32.5 (t, C-10 ), 25.98, 25.81, 25.67 [3 q,
(CH3)3CSi], 18.06 [s, (CH3)3CSi], 12.55 (q, CH3-thymine), –4.9,
–5.1 [2 q, (CH3)2Si].
IR (CHCl3): 3392w, 3027w, 2957w, 1688s, 1471m, 1361w, 1256m,
1109m, 908m, 852m cm–1.
1H NMR (300 MHz, CD3OD): = 7.79 [d, J = 1.47 Hz, 1 H, H-
C(6)], 6.28 [dd, J = 8.83, 2.21 Hz, 1 H, H-C(3 )], 4.13–3.98 [m, 2
H, H-C(9 ), H-C(7 )], 3.7 [dd, J = 11.4, 6.25 Hz, 1 H, H-C(9 )], 3.48
(s, 1 H, OH), 3.21 [t, J = 1.47 Hz, 1 H, H-C(11 )], 2.9 [dd, J = 14.3,
8.83 Hz, 1 H, H-C(4 )], 2.42 [dd, J = 15.4, 5.52 Hz, 1 H, H-C(4 )],
2.14–1.87 [m, 3 H, H-C(6 ), 2 H-C(10 )], 1.77 (s, 3 H, CH3).
13C NMR (75 MHz, CD3OD): = 164.8 (s, C-4), 150.9 (s, C-2),
138.51 (d, C-6), 109.65 (s, C-5), 94.8 (s, C-1 ), 88.98 (d, C-3 ),
81.03 (s, C-5 ), 80.32 (d, C-7 ), 80.07 (d, C-11 ), 57.98 (t, C-9 ),
49.49 (t, C-6 ), 41.21 (t, C-4 ), 31.79 (t, C-10 ), 12.4 (q, CH3).
HRMS (ESI-TOF, [M – H]–, C14H17N2O6): m/z calcd 309.1086,
found: 309.1092.
HRMS (ESI-TOF, [M – H]–, C20H32N2O6Si): m/z calcd 423.1971,
found: 423.1985.
(1S,5S,7S,11R)-N-[9-(5,11-Dihydroxy-2,8-dioxatricyclo-
[5.3.1.01,5]undec-3-yl)-9H-purin-6-yl]benzamide (8)
(1R,3S,5S,7S,11R)-N-{9-[11-(tert-Butyl{dimethyl}silyl)oxy]-5-
trimethylsilyloxy-2,8-dioxatricyclo[5.3.1.01,5]undec-3-yl]-9H-
purin-6-yl}benzamide (6)
To a solution of 6 (410 mg, 0.67 mmol) in THF (10 mL) was added
Bu4NF 3H2O (1.1 g, 2.2 mmol). After sirring for 4 h at 40 °C, the
mixture was evaporated and the residue purified by FC (40 g SiO2,
CH2Cl2 + 10% MeOH) to give 8 (190 mg, 67%) as a white foam.
TLC (5% MeOH in CH2Cl2): Rf 0.22; [ ]D +26.5 (c = 0.5, MeOH).
To a suspension of N6-benzoyladenine (40 mg, 0.324 mmol) in
ClCH2CH2Cl (1 mL) was added BSA (0.12 mL, 0.65 mmol). Upon
stirring for 2 h at 80 °C a clear solution was formed. To this solution
was added a solution of 4 (37 mg, 0.108 mmol) in ClCH2CH2Cl (1
mL), followed by TMSOTf (0.05 mL, 0.21 mmol). This mixture
was kept at 80 °C for 7 h. The solution was diluted with EtOAc (30
mL) and washed with aq sat. NaHCO3 (20 mL). The aqueous phase
was extracted with EtOAc, (2 30 mL), the combined organic lay-
ers were dried (MgSO4) and evaporated. FC (8 g SiO2, hexane–
EtOAc, 1:3) provided the -(3S) nucleoside 6 (38 mg, 58%) as a
white foam together with the corresponding de-trimethylsilyl prod-
uct (10 mg, 17%). TLC (hexane–EtOAc, 1:3): Rf 0.39.
IR (CHCl3): 3027s, 2673m, 2359m, 2071m, 1699s, 1653s, 1588m,
1540m, 1506m, 1456m, 1117s, 973s, 893s cm–1.
1H NMR (300 MHz, CDCl3): = 9.13 (s, 1 H, NH), 8.80 [d, J = 4.4
Hz, 1 H, H-C(2)], 8.12 [s, 1 H, H-C(8)], 8.1–7.9 (m, 2 Harom), 7.7–
7.5 (m, 3 Harom), 6.52 [d, J = 9.19 Hz, 1 H, H-C(3 )], 4.43–4.35 [m,
1 H, H-C(9 )], 4.23 [d, J = 5.15 Hz, 1 H, H-C(7 )], 3.94–3.75 [m, 2
H, H-C(9 ), H-C(11 )], 3.24 [m, 2 H, 2 H-C(4 )], 3.0–2.5 [m, 3 H,
OH, H-C(10 ), H-C(6 )], 2.25–1.94 [2 H, H-C(10 ), H-C(6 )].
IR (CHCl3): 3401w, 2956m, 2360w, 1700s, 1613s, 1583m, 1454s,
1297w, 1256s, 1229s, 1122s, 908m, 853s cm–1.
13C NMR (75 MHz, CDCl3): = 151.31 (s, C-4), 150.99 (s, C-2),
144.08 (d, C-8), 143.49 (s, C-6), 132.45, 128.26, 127.95 (Carom),
129.15 (s, C-5), 94.98 (s, C-1 ), 87.84 (d, C-3 ), 81.90 (s, C-5 ),
80.34 (d, C-7 ), 80.27 (d, C-11 ), 60.29 (t, C-9 ), 49.19 (t, C-6 ),
40.81 (t, C-4 ), 32.00 (t, C-10 ).
HRMS (ESI-TOF, [M – H]–, C21H20N5O5): m/z calcd 422.1465,
found: 422.1469.
1H NMR (300 MHz, CDCl3): = 8.82 [s, 1 H, H-C(2)], 8.38 [s, 1 H,
H-C(8)], 8.05 (d, J = 7.3 Hz, 2 Harom), 7.61–7.53 (m, 3 Harom), 6.62
[d, J = 7.0 Hz, 1 H, H-C(3 )], 4.14 [dd, J = 1.1, 5.2 Hz, 1 H, H-
C(7 )], 4.06–3.8 [2 m, 2 H, 2 H-C(9 )], 3.69 [s, 1 H, H-C(11 )], 3.15
[dd, J = 7.7, 14.0 Hz, 1 H, H-C(4 )], 2.57–2.5 [m, 2 H, H-C(4 ), H-
C(6 )], 2.24–2.00 [m, 3 H, 2 H-C(10 ), H-C(6 )], 0.98 [s, 9 H,
(CH3)3CSi], 0.20, 0.17 [2 s, 6 H, (CH3)2Si], 0.04 [s, 9 H, (CH3)3Si].
(1R,3S,5S,7S,11R)-1-[5-Hydroxy-11-(9-phenyl-9H-xanthen-9-
yloxy)-2,8-dioxatricyclo[5.3.1.01,5]undec-3-yl]-5-methyl-1H-
pyrimidine-2,4-dione (9)
1H NMR-difference-NOE (300 MHz, CDCl3): = 8.38 H-C(8)
6.62 H-C(3 ), 2.20 H-C(10 ).
To a solution of 7 (210 mg, 0.68 mmol) in anhyd pyridine (16 mL)
was added under argon 9-chloro-9-phenylxanthene (800 mg, 2.72
mmol) and the mixture was stirred at r.t. for 18 h. After that time the
mixture was diluted with EtOAc (20 mL) and extracted with aq sat.
NaHCO3 (30 mL). The aqueous phase was washed with EtOAc (40
mL) and the combined organic layers were dried (Na2SO4) and
evaporated. FC (10 g SiO2, EtOAc–hexane, 3:1 + 1% Et3N) gave 9
(308 mg, 80%) as a white foam. TLC (EtOAc–hexane, 3:1 + 1%
Et3N): Rf 0.21.
13C NMR (75 MHz, CDCl3): = 152.58 (s, C-2), 151.32 (s, C-4),
149.12 (s, C-6), 141.81 (d, C-8), 132.68, 128.86, 128.66, 127.76,
127.20 (Carom), 120.15 (s, C-5), 96.79 (s, C-1 ), 88.71 (d, C-3 ),
85.30 (s, C-5 ), 81.31 (d, C-7 ), 80.38 (d, C-11 ), 60.90 (t, C-9 ),
49.89 (t, C-6 ), 41.03 (t, C-4 ), 32.84 (t, C-10 ), 25.86, 18.08 [2 q,
(CH3)3CSi], 1.82 [q, (CH3)2Si], 1.48 [q, (CH3)3Si].
MS (–ESI-TOF): m/z (%) = 608.27 (100, [M – H]–), 423.19 (30),
262.07 (80).
HRMS (ESI-TOF, [M – H]–, C30H42N5O5Si2): m/z calcd 608.2725,
found 608.2724.
IR (CHCl3): 3391w, 3026w, 1688s, 1603w, 1575w, 1478m, 1448m,
1320w, 1279w, 1125m, 1071m, 908m, 786s cm–1.
1H NMR (300 MHz, CDCl3): = 8.77 (s, 1 H, NH), 7.40–7.00 (m,
13 Harom), 6.00 [dd, J = 6.8, 2.4 Hz, 1 H, H-C(3 )], 4.55 (s, 1 H, OH),
4.0 [m, 1 H, H-C(9 )], 3.57 (dd, J = 6.76, 11.0 Hz, 1 H, H-C(9 )],
Synthesis 2002, No. 6, 789–796 ISSN 0039-7881 © Thieme Stuttgart · New York

SPECIAL TOPIC
A Novel Conformationally Constrained -Nucleoside Analogue
795
3.34 [s, 1 H, H-C(7 )], 3.34–3.13 [m, 2 H, H-C(4 ), H-C(11 )], 2.63
[d, J = 14.7 Hz, 1 H, H-C(4 )], 2.45 [dd, J = 15.4, 5.2 Hz, 1 H, H-
C(10 )], 1.96–1.91 [m, 5 H, H-C(6 ), H-C(10 ), CH3], 1.70 [m, 1 H,
H-C(6 )].
13C NMR (75 MHz, CDCl3): = 163.81 (s, C-4), 151.85 (Carom),
150.7 (s, C-2), 150.25, 147.51 (Carom), 139.71 (d, C-6), 130.31,
130.11, 129.97, 129.82, 128.11, 127.15, 126.96, 123.60, 123.50,
123.31, 122.48, 116.87, 116.49 (Carom), 110.81 (s, C-5), 93.64 (s, C-
1 ), 93.45 (d, C-3 ), 83.11 (d, C-7 ), 82.87 (s, C-11 ), 78.61 (s, C-5 ),
76.59 (s, Carom), 60.21 (t, C-9 ), 48.84 (t, C-6 ), 41.67 (t, C-4 ), 33.09
(t, C-10 ), 12.35 (q, CH3).
IR (CHCl3): 3686w, 3624m, 3024s, 2977m, 2435w, 1401s, 1685w,
1580, 1523s, 1424s, 1220s, 1047s, 799s cm–1.
1H NMR (300 MHz, CDCl3): = 8.93 (s, 1 H, NH), 7.58 [s, 1 H, H-
C(6)], 7.40–7.06 (m, 13 Harom), 6.66 [dd, J = 8.1, 1.8 Hz, 0.5 H, H-
C(3 )], 6.51 [d, J = 6.3, 0.5 H, H-C(3 )], 3.9–3.5 [m, 6 H, 2 H-C(9),
2 CH, OCH2CH2CN], 3.3–2.9 [m, 3 H, H-C(7 ), H-C(11 ), H-C(4 )],
2.6–2.3 [m, 4 H, H-C(10 ), H-C(4 ), OCH2CH2CN], 1.9–1.75 [m, 5
H, 2 H-C(6), CH3], 1.2–1.0 [m, 12 H, 2 (CH3)2CH].
13C NMR (75 MHz, CDCl3): = 164.18, 163.93 (2 s, C-4), 151.55,
150.7 (s, C-2), 147.53–147.23 (4 Carom), 137.77 (d, C-6), 135.92,
135.89, 131.03, 130.97, 130.82, 130.41, 130.37, 130.32, 129.79,
129.76, 129.74, 129.69, 128.02, 128.00, 127.02, 126.95, 126.85,
126.79, 123.74, 123.61, 123.60, 123.37, 123.,8, 123.19, 122.26,
122.3, 116.64, 116.15 (Carom), 116.09, 116.05 (2 s, CN), 109.55,
109.32 (2 d), 108.90 (d), 95.06 (s), 89.29, 88.74 (2 d), 88.66, 88.00
(2 s), 81.85, 81.72 (2 d), 79.32, 79.19 (2 d), 78.60, 78.33 (2 d),
60.17, 60.05 (2 t), 57.93, 57.51 (2 t), 46.66, 46.26 (2 t), 43.43, 43.26
(2 d), 39.68, 39.55 (2 t), 33.13, 33.05 (2 t), 24.59, 24.39, 24.22,
23.84 (4 q), 20.54, 20.36 (2 t), 12.35, 12.28 (2 q).
1H NMR-difference-NOE (500 MHz, CDCl3): = 2.63 [ H-C(4 )]
4.55 (OH), 3.13 [ H-C(4 )] 6.00 [H-C(3 )], 6.00 [H-C(3 )]
7.2 Harom
.
HRMS (ESI-TOF, [M – H]–, C33H29N2O7): m/z calcd 565.1957,
found: 565.1974.
(1R,3S,5S,7S,11R)-N-{9-[5-Hydroxy-11-(9-phenyl-9H-xanthen-
9-yloxy)-2,8-dioxatricyclo[5.3.1.01,5]undec-3-yl]-9H-purin-6-
yl}benzamide (10)
31P NMR (162 MHz, CDCl3): = 148.20, 145.62.
To a solution of 8 (60 mg, 0.14 mmol) in pyridine (0.7 mL) was add-
ed 9-chloro-9-phenylxanthene (420 mg, 1.4 mmol), and the mixture
was stirred at r.t for 18 h. After dilution with EtOAc (20 mL), the
mixture was washed with aq sat. NaHCO3 (30 mL) and the aqueous
phase was extracted with EtOAc (2 40 mL). The combined organ-
ic layers were dried (Na2SO4) and evaporated. FC (8 g SiO2,
EtOAc–hexane, 3:1 + 1% Et3N) provided 10 (75 mg, 79%) as a
white foam. TLC (EtOAc–hexane, 3:1 + 1% Et3N): Rf 0.26.
MS (–ESI-TOF): 765.26 (30, [M – H]–), 728.26 (50), 514.28 (45),
380.18 (100).
HRMS (ESI-TOF, [M – H]–, C42H47N4O8P): m/z calcd 765.3074,
found: 765.3053.
(1R,3S,5S,7S,11R)-N-{9-[5-(2-Cyanoethoxydiisopropylamino-
phosphinoxy)-11-(9-phenyl-9H-xanthen-9-yloxy)-2,8-dioxatri-
cyclo[5.3.1.01,5]undec-3-yl]-9H-purin-6-yl}benzamide (12)
To a solution of 10 (70 mg, 0.1 mmol) in THF (2 mL) was added i-
Pr2NEt (0.1 mL, 0.6 mmol) and chloro(diisopropylamino)- -cyano-
ethoxyphosphine (0.07 mL, 0.3 mmol). After stirring at r.t. for 8 h,
the mixture was diluted with EtOAc (20 mL) and washed with aq
sat. NaHCO3 (15 mL). The aqueous phase was extracted with
EtOAc (2 20 mL). The combined organic phases were dried
(Na2SO4) and evaporated. FC (14 g SiO2, EtOAc–hexane, 2:1 + 1%
Et3N) followed by precipitation from hexane (4 °C) afforded 12 (75
mg, 85%) as a mixture of isomers in the form of a white solid; TLC
(EtOAc–hexane, 3:1 + 1% Et3N): Rf 0.59/0.52.
IR (CHCl3): 3675w, 3026w, 2916w, 1700w, 1615m, 1478m, 1456s,
1319m, 1229m, 1118m, 1072w cm–1.
1H NMR (300 MHz, CDCl3): = 9.11 (s, 1 H, NH), 8.77 [s, 1 H, H-
C(2)], 8.02 [d, J = 7.0 Hz, 2 Harom], 7.98 [s, 1 H, H-C(8)], 7.6–6.9
(m, 16 Harom), 6.42 [dd, J = 2.9, 9.5 Hz, 1 H, H-C(3 )], 4.21 [dd,
J = 4.0, 11.4 Hz, 1 H, H-C(9 )], 3.60 (dd, J = 11.4, 6.6 Hz, 1 H, H-
C(9 )], 3.51–3.28 [m, 2 H, H-C(4 ), H-C(11 )], 3.22 [d, J = 5.2 Hz,
1 H, H-C(7 )], 2.91 [dd, J = 2.6, 14.7 Hz, 1 H, H-C(4 )], 2.56 [dd,
J = 5.5, 15.4 Hz, 1 H, H-C(10 )], 2.11–2.05 [m, 2 H, H-C(10 ), H-
C(6 )], 1.65 [dd, J = 7.0, 11.7 Hz, 1 H, H-C(6 )].
13C NMR (75 MHz, CDCl3): = 164.72 (s, CO), 152.02 (s, C-4),
151.55 (s, C-2), 147.47 (d, C-8), 143.30 (s, C-6), 133.38, 132.98,
130.18, 130.07, 129.98, 129.87, 129.40, 129.03, 128.88, 128.84,
128.62, 128.03, 127.99, 127.92, 127.73, 127.25, 127.04, 126.51,
126.45, 126.40, 126.38, 126.34, 125.29, 123.53, 125.43, 116.94,
116.58, 116.18 (Carom, s, C-5), 94.02 (d, C-3 ), 88.26 (s, C-1 ), 83.19
(s, C-5 ), 82.97 (d, C-11 ), 78.73 (d, C-7 ), 77.26 (s, Carom), 60.17 (t,
C-9 ), 49.21 (t, C-6 ), 41.57 (t, C(4 ), 29.70 (t, C-10 ).
IR (CHCl3): 3407w, 3020w, 2971m, 1707m, 1613s, 1585m, 1479w,
1455s, 1321w, 1297m, 1200s, 1124m, 1077m, 941w, 909s, 823w
cm–1.
1H NMR (300 MHz, CDCl3): = 9.04 (s, 1 H, NH), 8.85 [s, 1 H, H-
C(2)], 8.28 [s, 1 H, H-C(8)], 8.02 (d, J = 7.0 Hz, 2 Harom), 7.7–7.0
(m, 16 Harom), 6.85 [d, J = 6.25 Hz, 1 H, H-C(3 )], 4.1–3.9 [m, 1 H,
H-C(9 )], 3.9–3.75 [m, 1 H, H-C(9 )], 3.74–3.20 [m, 7 H, H-C(4 ),
H-C(11 ), H-C(7 ), 2 (CH3)2CH, OCH2], 2.65–2.4 [m, 4 H, H-C(4 ),
H-C(10 ), CH2CN], 2.05–1.7 [m, 3 H, H-C(10 ), 2 H-C(6 )], 1.1–
0.84 [m, 12 H, (CH3)2CH)].
HRMS (ESI-TOF, [M – H]–, C40H32N5O6): m/z calcd 678.2378,
found: 678.2358.
13C NMR (75 MHz, CDCl3): = 152.66 (s, C-2), 151.59 (s, C-4),
147.62 (s, C-6), 141.57 (d, C-8), 132.65, 130.97, 130.48, 129.86,
128.85, 128.11, 127.78, 127.03, 123.70, 123.44, 123.36 (Carom),
122.32 (s, C-5), 116.79, 116.19 (2 s), 96.05 (s), 88.05, 87.97 (2 d),
81.66 (d), 79.57 (d), 78.43 (s), 76.58 (s), 60.34, 57.86, 57.57, 57.55,
57.45 (5 t), 43.52, 43.34 (2 d), 39.87, 39.78, 33.43 (3 t), 24.74,
24.65, 24.29, 24.19 (4 q), 20.48, 20.37 (2 t).
(1R,3S,5S,7S,11R)-1-{5-[(2 cyanoethoxy)(diisopropylamino)-
phosphinoxy]-11-(9-phenyl-9H-xanthen-9-yloxy)-2,8-dioxatri-
cyclo[5.3.1.01,5]undec-3-yl}-5-methyl-1H-pyrimidine-2,4-dione
(11)
A stirred solution of 9 (170 mg, 0.3 mmol) in MeCN (2 mL) was
treated at r.t. with i-Pr2NEt (0.4 mL, 2.4 mmol) and chloro(diisopro-
pylamino)- -cyanoethoxyphosphine (0.26 mL, 1.2 mmol). After 3
h, the mixture was diluted with EtOAc (40 mL) and washed with aq
sat. NaHCO3 (30 mL). The aqueous phase was extracted with
EtOAc (2 20 mL), the combined organic layers dried (Na2SO4)
and evaporated. FC (10 g SiO2, EtOAc–hexane, 3:1 + 1% Et3N) fol-
lowed by precipitation from hexane (4 °C) afforded 11 (210 mg,
90%, mixture of isomers) as a white solid. TLC (EtOAc–hexane 3:1
+ 1% Et3N): Rf 0.44/0.49.
31P NMR (162 MHz, CDCl3): = 147.34, 145.60.
HRMS (ESI-TOF, [M – H]–, C49H50N7O7P): m/z calcd 878.3428,
found: 878.3431.
Synthesis of Oligonucleotides 13–18
Oligonucleotides 13–18 were prepared on a Pharmacia Gene As-
sembler Special DNA synthesizer. All unmodified phosphoramidite
building blocks were from Glen Research. N6-Benzoyl- -deoxyad-
Synthesis 2002, No. 6, 789–796 ISSN 0039-7881 © Thieme Stuttgart · New York

796
B. M. Keller, C. J. Leumann
SPECIAL TOPIC
enosine was from R.I. Chemicals. -Deoxythymidine as well as the
-nucleoside phosphoramidite building blocks were prepared as de-
scribed.20 The assembly of oligonucleotides was performed accord-
ing to the standard phosphoramidite protocol (trityl off mode) with
the exception of a prolonged coupling time (15–20 min), and an in-
creased concentration of the phosphoramidite solutions (0.2 M in
MeCN, 20-fold excess per coupling) for the modified building
blocks 11 and 12. In the coupling step, 1H-tetrazole was replaced by
the more active (S-benzylthio)-1H-tetrazole (0.25 M in MeCN) or
5-(ethylthio)-1H-tetrazole (0.25 M in MeCN). As solid support the
universal solid support from CT Gen was used. After synthesis, the
solid support was suspended in conc. NH4OH (ca. 1 mL) and left at
65 °C for 72–96 h. The crude oligonucleotides were purified by an-
ion-exchange HPLC (Macherey-Nagel, nucleogen DEAE 60/7) and
desalted over SEP-PAK C-18 cartridges (Waters).
(5) Morvan, F.; Rayner, B.; Imbach, J.-L.; Lee, M.; Hartley, J.
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Design 1991, 6, 521.
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Keith, G.; Paoletti, J.; Malvy, C. Antisense Res. Dev. 1992,
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78, 2077.
(10) Egger, A.; Hunziker, J.; Rihs, G.; Leumann, C. Helv. Chim.
Acta 1998, 81, 734.
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M. J. Am. Chem. Soc. 1982, 104, 5564.
Acknowledgements
(13) Vorbrüggen, H.; Bennua, B. Chem. Ber. 1981, 114, 1279.
(14) Chattopadhyaya, J. B.; Reese, C. B. J. Chem. Soc., Chem.
Commun. 1978, 639.
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L.; Lamond, A. I. J. Chem. Soc., Perkin Trans.1 1994, 419.
(16) Bolli, M.; Lubini, P.; Tarköy, M.; Leumann, C. In
Carbohydrate Modifications in Antisense Research, ACS
Symposium Series 580; Sanghvi, Y. S.; Cook, P. D., Eds.;
ACS: Washington, 1994, 100.
We would like to thank our colleague Philippe Renaud for inspiring
advice concerning the radical cyclization reaction described herein.
Financial support of this work from the Swiss National Science
Foundation is gratefully acknowledged. B. M. K. thanks the ‘Sti-
pendienfonds der Schweizerischen Chemischen Industrie’ for a re-
search grant.
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Synthesis 2002, No. 6, 789–796 ISSN 0039-7881 © Thieme Stuttgart · New York