Synthesis and restricted furanose conformations of three novel bicyclic thy mine nucleosides: A xylo-LNA. nucleoside, a 3'-0,5'-Cmethylene-linked nucleoside, and a 2′-0,5′-C-methylene-linked nucleoside

Article abstract of DOI:10.1039/a901729a

The xylo-LNA nucleoside 1-(2-O,4-C-methylene-β-D-xyIofuranosyl)thymine (9) and the 2′-0,5′-C-methylene linked nucleoside 1-(2,6-anhydro-β-D-altrofuranosyI)thymine (28) were obtained in overall yields of 13% (8 steps) and 31% (7 steps) starting from furanose derivatives 1 and 21, respectively. In the synthesis of 3′-O,5′-C-methylene-linked nucleoside derivatives, cyclization by intramolecular attack from the 6-hydroxy group on the 3-keto functionality to give C-3-hemiketal furanose 11 and its subsequent transformation into nucleoside 15 proved very efficient. It was, however, impossible to isolate the debenzylated 2'-hydroxy, 2'-O-methyl and 2'-O-ter/-butyldimethylsiIyl derivatives 16,18 and 20, respectively, in analytically pure form. Solution-phase conformational analysis showed the bicyclic nucleosides 8,9,14,15,17 and 19 to exist predominantly in an N-type furanose conformation and bicyclic nucleotides 27 and 28 to adopt an S-type conformation.

Full text of DOI:10.1039/a901729a

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Synthesis and restricted furanose conformations of three novel
bicyclic thymine nucleosides: a xylo-LNA nucleoside, a 3Ј-O,5Ј-C-
methylene-linked nucleoside, and a 2Ј-O,5Ј-C-methylene-linked
nucleoside
Vivek K. Rajwanshi, Ravindra Kumar, Mikael Kofod-Hansen and Jesper Wengel*
Center for Synthetic Bioorganic Chemistry, Department of Chemistry,
University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen, Denmark
Received (in Cambridge) 4th March 1999, Accepted 16th April 1999
The xylo-LNA nucleoside 1-(2-O,4-C-methylene-β--xylofuranosyl)thymine (9) and the 2Ј-O,5Ј-C-methylene linked
nucleoside 1-(2,6-anhydro-β--altrofuranosyl)thymine (28) were obtained in overall yields of 13% (8 steps) and 31%
(7 steps) starting from furanose derivatives 1 and 21, respectively. In the synthesis of 3Ј-O,5Ј-C-methylene-linked
nucleoside derivatives, cyclization by intramolecular attack from the 6-hydroxy group on the 3-keto functionality to
give C-3-hemiketal furanose 11 and its subsequent transformation into nucleoside 15 proved very efficient. It was,
however, impossible to isolate the debenzylated 2Ј-hydroxy, 2Ј-O-methyl and 2Ј-O-tert-butyldimethylsilyl derivatives
16, 18 and 20, respectively, in analytically pure form. Solution-phase conformational analysis showed the bicyclic
nucleosides 8, 9, 14, 15, 17 and 19 to exist predominantly in an N-type furanose conformation and bicyclic
nucleotides 27 and 28 to adopt an S-type conformation.
Introduction
2'
Base
3'
O
Base
O
A successful approach towards oligonucleotide analogues
capable of binding with high affinity to complementary
single- stranded DNA and RNA has been the use of oligomers
containing conformationally restricted monomers.1,2 Thus,
several oligonucleotide analogues containing bi- and tricyclic
carbohydrate moieties have displayed enhanced duplex stab-
ilities compared with the unmodified reference oligo-
nucleotides.3–18 LNA (Locked Nucleic Acid; Fig. 1) is a prime
example displaying unprecedented increases in the thermal
stability (melting temperature, Tm) of duplexes towards com-
plementary single-stranded DNA and RNA (∆Tm/modifi-
cation = ϩ3 ЊC to ϩ11 ЊC).10–17 † Conformationally restricted
nucleosides are also useful as tools in evaluation of conform-
ational preferences of nucleos(t)ide converting enzymes and
thereby also of structure–activity relationships of, e.g., anti-
viral nucleosides. As an example, Marquez et al. have recently
found HIV-1 reverse transcriptase to discriminate between
two conformationally locked carbocyclic AZT triphosphate
analogues.19 Most of the research conducted in these directions
has been focused on restricting the conformation of the furan-
ose ring, or structural entities mimicking the furanose ring, into
either an S-type (e.g., C-2Ј-endo) or an N-type (e.g., C-3Ј-endo)
conformation (Fig. 1).‡ Due to the low energy barrier (~2 kcal
molϪ1 20 §) between the two dominating conformers a fast
equilibrium between the N- and the S-type states exists21–23
for unmodified monomeric nucleosides in solution. However,
the furanose moieties in RNA normally exist in an N-type
O
O
O
O
P
–O
O
O
P
OH
–O
S-type conformation
N-type conformation
O
Base
O
Base
O
O
Base
O
O
O
O
O
O
O
–O
–O
P
P
O
O
–O
P
O
LNA10-17
xylo-DNA24-27
xylo-LNA
Fig. 1
conformation leading to A-type duplexes and in DNA generally
in an S-type conformation leading to B-type duplexes. In
addition, one conformer is expected to be dominating for a
nucleoside bound in the active sites of enzymes.19
The work in this area has been based on the assumption that
complexation between an oligonucleotide analogue and its
DNA or RNA target, and between a nucleoside analogue and a
relevant enzyme, is favoured by the appropriate preorganization
of the ligand. The results obtained1–19 have shown this assump-
tion to be valid, but in order to gain further knowledge of the
natural systems and to be able to design improved therapeutic
molecules, novel analogues are needed. In this paper we
describe the synthesis of three novel classes of conformation-
ally restricted nucleoside analogues all containing a bicyclic
pentofuranose moiety and hydroxy groups positioned at the 3Ј-
and 5Ј-positions allowing the formation of 3Ј-O- to 5Ј-O-linked
oligonucleotide analogues and 5Ј-phosphorylated nucleoside
derivatives thus mimicking the natural regiochemistry. Our
interest in the xylo-LNA nucleoside 9 (Scheme 1) was stim-
ulated by a series of papers in which Seela et al. have studied
† We have defined LNA as an oligonucleotide containing one or more
2Ј-O,4Ј-C-methylene-linked bicyclic ribonucleoside monomers.
‡ A thorough introduction to the conformational aspects of nucleo-
sides is given in ref. 19. The term ‘endo’ designates the positioning of an
atom ‘above’ the reference plane of the furanose ring, e.g., the C-1Ј,
C-2Ј, C-4Ј and O atoms. The term ‘exo’ designates the positioning of an
atom below the reference plane.
§ A simple rule to evaluate the percentage S and percentage N con-
former in a two-state model is given in ref. 23: %S = 10 × J1Ј,2Ј
;
%N = 10 × J3Ј,4Ј. The problem in the context of the bicyclic nucleosides
prepared herein is that no value exists for J3Ј,4Ј for compounds 8 and 9
(no H-4Ј present) and 14, 15, 17, 19 and 20 (no H-3Ј present).
J. Chem. Soc., Perkin Trans. 1, 1999, 1407–1414
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RO
RO
O
O
BzO
BzO
O
OBn
O
OBn
HO
BnO
O
O
O
ii
O
OAc
O
ii
i
O
O
O
O
O
OAc
BnO
O
HO
O
O
O
1 R = H
3
i
10
11
12
2 R = Bz
O
iii
NH
O
O
NH
O
N
BnO
BnO
O
O
O
O
iii
OAc
iv
R1O
R2O
N
O
OBn
BnO OAc
BnO OR
14 R = Ac
15 R = H
O
13
OR3
v
4 R1 = R2 = Bz; R3 = Ac
iv
v
O
5 R1 = R2 = R3 = H
NH
NH
6 R1 = R3 = H; R2 = Ts
O
O
N
N
O
RO
RO
O
viii
O
vi
O
15
O
O
N
vii
RO OCH3
NH
RO OTBDMS
NH
O
O
O
N
DMTO
17 R = Bn
18 R = H
DMTO
19 R = Ac
20 R = H
vii
O
OBn
O
vii
vi (from 6)
vii
NH
OBn
TsO
O
N
HO
O
O
O
OH
7
8
HO OH
viii
16
O
Scheme 2 Reagents and conditions: i) 80% AcOH (96%); ii) NaH,
BnBr, DMF (93%); iii) a) 80% AcOH, b) Ac2O, pyridine (57%); iv)
N,O-bis(trimethylsilyl)acetamide, thymine, trimethylsilyl triflate,
acetonitrile (85%); v) NaOCH3, CH3OH (90%); vi) NaH, CH3I, CH2Cl2
(62%); vii) H2, 20% Pd(OH)2/C, ethanol (for 20: 50%); viii) TBDMSCl,
imidazole, DMF (85%).
NH
O
N
HO
O
OH
methodology,29,30 the thymine β-configured nucleoside 4 was
stereoselectively obtained in 85% yield by in situ silylation of
thymine and trimethylsilyl triflate-mediated coupling. Treat-
ment of compound 4 with sodium methoxide resulted in
deacylation to give nucleoside triol 5 in 89% yield. To prepare
for cyclization, we investigated the selectivity of monotosyla-
tion by reaction using 1.1 equivalent of toluene-p-sulfonyl
chloride and pyridine in anhydrous dichloromethane. As we
have reported earlier for a structurally closely related nucleo-
side,31 the 4Ј-C-hydroxymethyl group positioned at the α-face
of the furanose ring is more reactive than the 5Ј-hydroxy group
under such conditions. Accordingly, we could isolate 4Ј-C-
tosyloxymethyl nucleoside 6 as the major product (35% yield)
and one monotosylated and one ditosylated compound in only
7% and 6% yield, respectively (data not reported). To our
surprise, cyclization of 6 (sodium hydride, anhydrous DMF)
proved unsuccessful at room temperature even after stirring for
several days using either DMF or THF as solvent. However,
after DMT-protection of the 5Ј-hydroxy group [4,4Ј-dimethoxy-
trityl chloride (DMTCl) and 4-(dimethylamino)pyridine
(DMAP) in anhydrous pyridine; 75% yield of nucleoside 7]
efficient cyclization was accomplished to give the protected
bicyclic 2Ј-O,4Ј-C-methylene-β--xylofuranosyl nucleoside 8
in 96% yield. Apparently, the introduction of the bulky DMT
group induces a conformational change favourable for the
cyclization. Catalytic hydrogenation yielded the parent xylo-
LNA nucleoside 9 [1-(2Ј-O,4Ј-C-methylene-β--xylofuranosyl)-
thymine] in 82% yield by concomitant debenzylation and
detritylation (Scheme 1).
O
9
Scheme 1 Reagents and conditions: i) BzCl, pyridine (90%); ii) a) 80%
AcOH, b) Ac2O, pyridine (92%); iii) N,O-bis(trimethylsilyl)acetamide,
thymine, trimethylsilyl triflate, acetonitrile (85%); iv) NaOCH3, CH3OH
(89%); v) toluene-p-sulfonyl chloride, pyridine, CH2Cl2 (35%); vi)
DMTCl, DMAP, pyridine (75%); vii) NaH, DMF (96%); viii) H2, 10%
Pd/C, CH3OH (82%).
xylo-DNA (Fig. 1) containing one or more 2Ј-deoxy-β--
xylofuranosyl nucleotide monomers,24–27 and by the remarkable
properties of LNA. A convenient synthetic methodology
towards xylo-LNA nucleosides should allow the synthesis of
xylo-LNA (Fig. 1) containing one or more 2Ј-O,4Ј-C-methyl-
ene-β--xylofuranosyl nucleotide monomer(s) as a stereo-
isomer of LNA. As a continuation of our general interest in
conformationally restricted nucleosides and oligonucleotide
analogues, we decided also to synthesize bicyclic 3Ј-O,5Ј-C-
methylene-linked and 2Ј-O,5Ј-C-methylene-linked nucleosides
(Schemes 2 and 3).
Results and discussion
Chemical synthesis
Benzoylation of the 4-C-hydroxymethyl-β--threo-pento-
furanose 128 afforded in 90% yield the di-O-benzoyl derivative 2
which was subsequently converted into the 1,2-di-O-acetylated
furanose 3 in 92% yield by acetolysis using 80% acetic acid
followed by acetylation. Employing a modified Vorbrüggen
1408
J. Chem. Soc., Perkin Trans. 1, 1999, 1407–1414

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thymine. Deacylation to give the 2Ј- and 6Ј-hydroxy derivative
BzO
BnO
BzO
RO
25 was accomplished in 88% yield using sodium methoxide. To
prepare for ring-closure, the 2Ј,6Ј-di-O-p-tolylsulfonyl nucleo-
side 26 was synthesized in 71% yield by reaction with excess
toluene-p-sulfonyl chloride and DMAP in anhydrous dichloro-
methane. Ring-closure was subsequently attempted using
sodium hydroxide in a mixture of H2O and ethanol hoping
for initial anhydro formation leading to in situ inversion at C-2Ј
and subsequent intramolecular nucleophilic attack from the
liberated (α-face oriented) 2Ј-hydroxy group on the 6Ј-O-tosyl
group. This strategy turned out to be very successful as the
nucleoside 27 containing a novel 2Ј-O,5Ј-C-methylene-linked
bicyclic furanose skeleton was obtained in 93% yield directly
from the di-O-tosylated nucleoside 26. Debenzylation was
effected using hydrogen and 20% Pd(OH)2/C as catalyst to give
the unprotected bicyclic nucleoside 28 in 98% yield.
O
O
OAc
ii (from 22)
O
BnO OAc
BnO
O
23
21 R = H
i
22 R = Bn
iii
O
NH
R3O
R2O
O
N
O
With the synthetic methods described herein, the xylo-LNA
nucleoside 9 and the 2Ј-O,5Ј-C-methylene-linked nucleoside 28
was obtained in overall yields of 13% (8 steps) and 31% (7
steps) starting from the known furanose derivatives 1 and 21,
respectively. The methods are stereoselective, affording only one
stereoisomer in the nucleobase-coupling steps and straight-
forward C-2Ј-inversion during the synthesis of 28. The method
depicted in Scheme 1 should be applicable for both pyrimidine
and purine bases whereas the strategy towards nucleoside 28 is
limited to pyrimidine derivatives because of the probable
intermediacy of an 2,2Ј-O-anhydro compound during the C-2Ј-
inversion. In the synthesis of the 3Ј-O,5Ј-C-methylene-linked
derivatives, spontaneous cyclization by intramolecular attack
on the 3-keto functionality and the subsequent trapping as the
ribo-configured furanoside C-3-acetal proved very efficient
in analogy with our results on a similar cyclization to give 2Ј-
O,3Ј-C-ethylene-linked C-2Ј-acetal nucleosides which could
be debenzylated in high yield.34 However, after analogous
debenzylation of the 2Ј-hydroxy, 2Ј-O-methyl and 2Ј-O-tert-
butyldimethylsilyl derivatives 15, 17 and 19, respectively, the
isolation of the pure products proved very difficult probably
due to the instability of the hemiacetals on the column
materials (silica gel, basic alumina; 2Ј-O-tert-butyldimethylsilyl
derivative 20 appeared to be more stable than the 2Ј-hydroxy
and 2Ј-O-methyl nucleosides 16 and 18). Our interest in deriv-
atives 18 and 20 originates from their potential as intermediates
in the synthesis of phosphoramidite building blocks towards
the corresponding 2Ј-O-methyl-RNA and RNA oligonucleo-
tides, respectively. However, the problematic debenzylations
render the chosen strategy inconvenient. It can therefore be
summarized that quite efficient methods for synthesis of xylo-
LNA nucleosides and 2Ј-O,5Ј-C-methylene-linked bicyclic
nucleosides of the pyrimidine series have been developed and
that further derivatizations of, e.g., nucleosides 9 and 28 towards
oligonucleotide building blocks35 or functionalized nucleoside
analogues should be possible.¶
BnO OR1
24 R1 = Ac; R2 = Bn; R3 = Bz
iv
v
25 R1 = R3 = H; R2 = Bn
26 R1 = R3 = Ts; R2 = Bn
O
O
N
NH
NH
O
O
N
O
HO
O
BnO
O
O
vii
vi (from 26)
HO
BnO
28
27
Scheme 3 Reagents and conditions: i) NaH, BnBr, DMF (90%); ii) a)
80% AcOH, b) Ac2O, pyridine (74%); iii) N,O-bis(trimethylsilyl)-
acetamide, thymine, trimethylsilyl triflate, acetonitrile (81%); iv)
NaOCH3, CH3OH (88%); v) toluene-p-sulfonyl chloride, DMAP,
CH2Cl2 (71%); vi) NaOH, H2O, ethanol (93%); vii) H2, 20% Pd(OH)2/C,
ethanol (98%).
The 3Ј-O,5Ј-C-methylene-linked nucleoside derivatives were
synthesized as outlined in Scheme 2. Selective removal of the
5,6-O-isopropylidene protecting group of the known 3-ulose
1032 using 80% acetic acid at room temperature afforded
smoothly the tricyclic hemiacetal furanose derivative 11 in 96%
yield. To prepare for nucleoside coupling, 1,2-di-O-acetylated
furanose 13 was prepared from 11 via 12 in 53% yield by ben-
zylation, acetolysis and acetylation. Using the methodology
described above for the synthesis of 4, the thymine nucleoside
14 was stereoselectively obtained in 85% yield. Deacetylation
using sodium methoxide gave in 90% yield the nucleoside
derivative 15 with an unprotected 2Ј-hydroxylic functionality.
Debenzylation of 15 to afford the bicyclic ribo-configured
nucleoside triol 16 proved impossible in our hands. As judged
from analytical TLC during the debenzylation (hydrogen in
combination with various catalysts as well as transfer hydro-
genation were attempted11) a major product having the
expected Rf-value was formed during the reaction but all
attempts to isolate this product in pure form were unsuccessful.
Analogously, debenzylation of 2Ј-O-methyl and 2Ј-O-tert-butyl-
dimethylsilyl derivatives 17 and 19, respectively, proceeded
efficiently according to analytical TLC but isolation proved
troublesome in our hands and only the 2Ј-O-tert-butyldimethyl-
silyl derivative 20 could be isolated in acceptable purity (accord-
ing to NMR; microanalysis could not be obtained).
Furanose conformations
Based on molecular modelling (Hyperchem Program, MMϩ
Molecular Mechanics Force Field; handmodel building), the
nucleosides 8 and 9 were expected to adopt an N-type furanose
conformation whereas 27 and 28 were expected to adopt an
S-type furanose conformation. The situation was more dubious
for compounds 14, 15, 17, 19 and 20 as these bicyclic nucleo-
sides consist of a bicyclo[3.3.0]octane system intrinsically more
flexible compared with the bicyclo[2.2.1]heptane system of 8
and 9 and the bicyclo[3.2.1]octane system of 27 and 28. From
the coupling constants in the 1H NMR spectrum, especially the
J1Ј,2Ј- and J3Ј,4Ј-values, the furanose conformation of nucleosides
The 2Ј-O,5Ј-C-linked thymine nucleoside 28 was synthesized
as outlined in Scheme 3. Benzylation of the known furanose
2133 by reaction with benzyl bromide and sodium hydride in
anhydrous DMF (22, 90% yield) followed by acetolysis and
acetylation in 74% yield afforded the anomeric mixture 23 as a
suitable substrate to give stereoselectively the fully protected
novel thymine nucleoside 24 in 81% yield after coupling with
¶ Copies of the 13C NMR spectra for compounds 9, 20 and 28 were
enclosed with this manuscript on submission to verify the purity and
identity of these three bicyclic nucleosides.
J. Chem. Soc., Perkin Trans. 1, 1999, 1407–1414
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in solutions can be estimated.21–23 Following the prediction
from modelling, the signals for H-1Ј for the xylo-LNA nucleo-
sides 8 and 9 were found to be singlets, revealing that these
conformationally locked nucleosides exclusively adopt an N-
type conformation. For the 3Ј-O,5Ј-C-methylene-linked nucleo-
sides 14, 15, 17 and 19, the J1Ј,2Ј-values were found to be 2.1 Hz
(for 14) and 0 Hz (for 15, 17 and 19). Thus, this 3Ј-O,5Ј-C-
methylene linkage effectively introduces a novel class of N-type
conformationally restricted nucleosides. The J1Ј,2Ј-values of 2.2
and 2.3 Hz found for the 2Ј-O,5Ј-C-methylene-linked nucleo-
sides 27 and 28, respectively, and the singlet for H-4Ј found for
28 verify the expected S-type conformational preference for this
class of nucleosides. It should, however, be mentioned that the
analyses given herein are limited to the furanose puckering.
Other structural features, e.g., the orientation of the nucleo-
base and the 5Ј-hydroxy group, are likewise important aspects
of nucleoside preorganization.
methane–methanol (99:1, v/v), as eluent to give furanose 2
(7.50 g, 90%) as a yellowish oil after evaporation of the solvents
under reduced pressure; δH(CDCl3) 8.02–7.23 (15H, m), 6.08
(1H, d, J 4.2), 4.81–4.50 (7H, m), 4.22 (1H, d, J 1.0), 1.59 (3H,
s), 1.37 (3H, s); δC(CDCl3) 166.1, 165.8, 136.7, 133.1, 133.0,
129.9, 129.7, 129.6, 129.5, 128.5, 128.4, 128.3, 128.0, 127.9,
113.3, 105.4, 86.4, 85.1, 83.8, 72.3, 64.3, 63.8, 27.0, 26.4; FAB-
MS m/z 521 [M ϩ H]ϩ [Found (%): C, 69.1; H, 5.9. C30H32O8
requires C, 69.2; H, 6.2].
1,2-Di-O-acetyl-5-O-benzoyl-4-C-benzoyloxymethyl-3-O-
benzyl-L-threo-pentofuranose 3
A solution of furanose 2 (7.40 g, 0.014 mol) in 80% acetic acid
(60 cm3) was stirred for 9 h at 90 ЊC. The mixture was evapor-
ated to dryness under reduced pressure, and the residue was
coevaporated with toluene (10 cm3 × 3) and dissolved in
anhydrous pyridine (80 cm3). Acetic anhydride (5.5 cm3) was
added and the solution was stirred for 46 h at room tempera-
ture. The mixture was evaporated to dryness under reduced
pressure, and the residue was coevaporated with toluene (10
cm3 × 3) and dissolved in dichloromethane (150 cm3). The solu-
tion was washed successively with saturated aqueous sodium
hydrogen carbonate (30 cm3 × 3) and brine (30 cm3 × 3), dried
(Na2SO4), and concentrated under reduced pressure. The resi-
due was purified by silica gel column chromatography using,
first, petroleum ether–dichloromethane (1:1, v/v), and then
dichloromethane–methanol (99:1, v/v), as eluent to give the
anomeric mixture 3 (α:β = 3:1; 7.33 g, 92%) as a clear oil
after evaporation of the solvents under reduced pressure. This
oil was used in the next step without further purification;
δC(CDCl3) 169.4, 169.0, 165.8, 165.6, 137.0, 133.2, 133.1, 133.0,
129.6, 129.5, 129.2, 128.3, 127.8, 127.7, 127.4, 99.4, 92.3, 87.0,
83.2, 82.2, 80.7, 77.4, 76.9, 76.3, 73.2, 72.4, 20.9, 20.8, 20.6,
20.3; FAB-MS m/z 562 [M]ϩ.
Conclusions
Efficient and stereoselective methods have been developed for
synthesis of bicyclic nucleosides of the xylo-LNA type (e.g., 9)
and of the 2Ј-O,5Ј-C-methylene-linked type (e.g., 28). Con-
formational analysis indicates the xylo-LNA-type nucleosides,
and the 3Ј-O,5Ј-C-linked derivatives 14, 15, 17 and 19, to adopt
an N-type furanose conformation, whereas the 2Ј-O,5Ј-C-
methylene-linked nucleosides adopt an S-type furanose con-
formation. These results suggest that the members of these
novel classes of conformationally restricted nucleosides should
be evaluated as building blocks in oligonucleotide analogues
or as biologically active nucleosides, possibly after further
functionalizations. This work shows the wide spectrum of
restricted furanose conformations attainable for bicyclic
nucleoside analogues.
Experimental
General
1-(2-O-Acetyl-5-O-benzoyl-4-C-benzoyloxymethyl-3-O-benzyl-
á-L-threo-pentofuranosyl)thymine 4
To a stirred suspension of the anomeric mixture 3 (7.26 g, 0.013
mol) and thymine (3.25 g, 0.028 mol) in anhydrous acetonitrile
(80 cm3) was added N,O-bis(trimethylsilyl)acetamide (19.1 cm3,
0.077 mol). The reaction mixture was stirred at 60 ЊC for 1 h
and then cooled to 0 ЊC. Trimethylsilyl triflate (4.1 cm3, 0.023
mol) was added dropwise during 10 min and the mixture was
subsequently heated for 22 h under reflux. After cooling of the
mixture to room temperature, saturated aqueous sodium
hydrogen carbonate (30 cm3) was added and extraction was
performed using dichloromethane (100 cm3 × 3). The combined
organic phase was washed successively with saturated aqueous
sodium hydrogen carbonate (30 cm3 × 3) and brine (50
cm3 × 3), dried (Na2SO4), and concentrated under reduced
pressure. The residue was purified by silica gel column chrom-
atography using dichloromethane–methanol (0.5–2.0% meth-
anol, v/v) as eluent to give nucleoside 4 (6.88 g, 85%) as a white
solid after evaporation of the solvents under reduced pressure;
δH(CDCl3) 8.97 (1H, br s), 8.04–7.23 (16H, m), 6.37 (1H, d,
J 3.6), 5.42 (1H, t, J 3.1), 4.89–4.56 (6H, m), 4.22 (1H, d, J 2.6),
2.13 (3H, s), 1.74 (3H, d, J 0.8); δC(CDCl3) 169.9, 166.0, 165.7,
163.4, 150.4, 136.2, 135.2, 133.5, 133.4, 129.8, 129.7, 129.6,
129.5, 129.0, 128.6, 128.4, 128.2, 112.0, 87.4, 86.0, 81.3, 80.3,
72.6, 63.1, 62.9, 20.8, 12.3; FAB-MS m/z 629 [M ϩ H]ϩ [Found
(%): C, 64.4; H, 4.9; N, 4.4. C34H32N2O10ؒ0.25H2O requires C,
64.5; H, 5.1; N, 4.4].
Reactions were conducted under an atmosphere of nitrogen
when anhydrous solvents were used. Column chromatography
was carried out using glass columns and Silica gel 60 (0.040–
0.063 mm). After drying organic phases with Na2SO4, filtration
was performed. Petroleum ether of distillation range 60–80 ЊC
was used. Chemical-shift values δ are in ppm relative to tetra-
methylsilane as internal reference. J-Values are given in Hz.
The names of the bicyclic nucleosides and tricyclic furanoses in
this section are given according to the von Baeyer nomen-
clature. However, for easy comparison the assignments (when
given based on 2D NMR experiments) of the signals of the
NMR spectra are indicated using standard carbohydrate/
nucleoside numbering with the thymine moiety having the
highest priority. Microanalyses were performed at The Micro-
analytical Laboratory, Department of Chemistry, University of
Copenhagen.
5-O-Benzoyl-4-C-benzoyloxymethyl-3-O-benzyl-1,2-O-
isopropylidene-â-L-threo-pentofuranose 2
To a stirred, ice-cold solution of 3-O-benzyl-4-C-hydroxy-
methyl-1,2-O-isopropylidene-β--threo-pentofuranose 128 (5.00
g, 0.016 mol) in anhydrous pyridine (60 cm3) was added benzoyl
chloride (4.1 cm3, 0.035 mol). After stirring at room temper-
ature for 4 h, the reaction mixture was cooled to 0 ЊC, H2O (50
cm3) was added, and the mixture was extracted with dichloro-
methane (100 cm3 × 3). The combined organic phase was
washed successively with saturated aqueous sodium hydrogen
carbonate (30 cm3 × 3) and brine (20 cm3 × 3), dried (Na2SO4),
and evaporated to dryness under reduced pressure. The residue
was purified by silica gel column chromatography using, first,
petroleum ether–dichloromethane (1:1, v/v), and then dichloro-
1-(3-O-Benzyl-4-C-hydroxymethyl-á-L-threo-pentofuranosyl)-
thymine 5
To a stirred solution of nucleoside 4 (9.00 g, 0.014 mol) in
methanol (130 cm3) was added sodium methoxide (3.87 g,
0.0716 mol). The reaction mixture was stirred at room temper-
1410
J. Chem. Soc., Perkin Trans. 1, 1999, 1407–1414

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ature for 4 h and then neutralized with dilute hydrochloric acid.
The mixture was evaporated to dryness under reduced pressure
followed by coevaporation using toluene (15 cm3 × 3). The resi-
due was purified by silica gel column chromatography using
dichloromethane–methanol (4–15% methanol, v/v) as eluent to
give nucleoside triol 5 (4.82 g, 89%) as a white solid after evap-
oration of the solvents under reduced pressure; δH(CD3OD)
7.89 (1H, d, J 1.2), 7.40–7.24 (5H, m), 5.97 (1H, d, J 6.2), 4.83–
4.65 (2H, m), 4.53 (1H, t, J 6.2), 4.21 (1H, d, J 6.2), 3.84 (1H, d,
J 12.0), 3.63 (1 H, d, J 12.0), 3.59 (2H, d, J 2.6), 1.82 (3H,
d, J 1.1); δC(CD3OD) 164.4, 150.9, 137.5, 136.6, 127.5, 127.0,
126.9, 109.8, 86.7, 86.4, 82.8, 78.0, 72.1, 62.3, 61.1, 10.5;
FAB-MS m/z 379 [M ϩ H]ϩ [Found (%): C, 56.2; H, 6.0; N, 7.0.
C18H22N2O7ؒ0.25H2O requires C, 56.5; H, 5.9; N, 7.3].
163.7, 158.5, 151.0, 144.9, 144.4, 137.1, 135.8, 135.2, 135.0,
132.5, 130.1, 129.8, 128.3, 128.0, 127.8, 127.7, 126.9, 113.1,
110.0, 90.2, 87.1, 86.4, 83.3, 79.9, 72.9, 68.7, 62.2, 55.2, 21.6,
12.0; FAB-MS m/z 835 [M ϩ H]ϩ [Found (%): C, 66.0; H, 5.7;
N, 3.3. C46H46N2O11S requires C, 66.1; H, 5.5; N, 3.4].
(1R,3R,4R,7R)-7-Benzyloxy-1-(4,4Ј-dimethoxytrityloxymethyl)-
3-(thymin-1-yl)-2,5-dioxabicyclo[2.2.1]heptane 8
To a solution of nucleoside 7 (4.22 g, 5.06 mmol) in anhydrous
DMF (25 cm3) at 0 ЊC was added a 60% suspension of sodium
hydride in mineral oil (w/w; 0.607 g, 15.7 mmol, added in four
portions during 20 min) and the reaction mixture was stirred at
room temperature for 25 h, cooled to 0 ЊC and diluted with
dichloromethane–pyridine (100 cm3; 99.5:0.5, v/v). Saturated
aqueous sodium hydrogen carbonate (120 cm3) was added
whereupon extraction was performed using dichloromethane
(75 cm3 × 2). The combined organic phase was washed succes-
sively with saturated aqueous sodium hydrogen carbonate (60
cm3 × 3) and brine (40 cm3 × 3), dried (Na2SO4), and evapor-
ated to dryness under reduced pressure. The residue was
purified by silica gel column chromatography using dichloro-
methane–methanol–pyridine (0.5–1.5% methanol; 0.5% pyrid-
ine, v/v/v) as eluent to yield nucleoside 8 (3.20 g, 96%) as a white
solid material after evaporation of the solvents under reduced
pressure; δH(CDCl3) 13.24 (1H, s, NH), 7.70–7.19 (19H, m, Bn,
DMT, 6-H), 6.15 (1H, s, 1Ј-H), 4.98 (1H, s, 2Ј-H), 4.55 (1H, d,
J 11.2, Bn), 4.42 (1H, d, J 11.2, Bn), 4.40 (1H, s, 3Ј-H), 4.34
(1H, d, J 8.0, 1Љ-Ha), 4.17 (1H, d, J 8.0, 1Љ-Hb), 3.94 (2H, s,
5Ј-H2), 3.67 (3H, s, OCH3), 3.64 (3H, s, OCH3), 1.75 (3H, d,
J 0.7, CH3); δC(CDCl3) 165.0, 159.2, 151.5, 145.5, 137.4, 136.6,
136.0, 130.6, 128.7, 128.6, 128.4, 128.3, 127.3, 113.8, 108.1,
89.3, 88.6, 86.7, 80.6, 77.0, 73.8, 73.0, 59.8, 55.2, 12.7; FAB-MS
m/z 663 [M ϩ H]ϩ [Found (%): C, 70.4; H, 5.8; N, 4.0.
C39H38N2O8 requires C, 70.7; H, 5.7; N, 4.2].
1-[3-O-Benzyl-4-C-(p-tolylsulfonyloxymethyl)-â-D-xylofurano-
syl]thymine 6
To a solution of nucleoside 5 (7.25 g, 0.0192 mol) in anhydrous
pyridine (20 cm3) and dichloromethane (70 cm3) at Ϫ30 ЊC was
added dropwise during 1.5 h a solution of toluene-p-sulfonyl
chloride (4.38 g, 0.023 mol) in dichloromethane (8 cm3). The
temperature was raised to 0 ЊC for 2 h, whereupon additional
toluene-p-sulfonyl chloride (1.80 g, 0.0094 mol) was added at
Ϫ20 ЊC and the mixture was stirred for 12 h at Ϫ20 ЊC. At that
time further toluene-p-sulfonyl chloride (0.736 g, 3.86 mmol)
was added and stirring was continued at Ϫ20 ЊC for an addi-
tional 24 h. The reaction mixture was diluted with dichloro-
methane (75 cm3) and H2O (75 cm3), and extraction was
performed with dichloromethane (75 cm3 × 3). The combined
organic phase was washed successively with saturated aqueous
sodium hydrogen carbonate (30 cm3 × 3) and brine (40
cm3 × 3). The aqueous phase was extracted with ethyl acetate
(30 cm3 × 3), and these extracts were combined with the
dichloromethane extracts, dried (Na2SO4), and evaporated to
dryness under reduced pressure. The residue was purified by
silica gel column chromatography using dichloromethane–
methanol (1.5–3.5% methanol, v/v) as eluent to give nucleoside
6 (3.56 g, 35%) as a white solid after evaporation of the solvents
under reduced pressure; δH(CDCl3) 10.23 (1H, s), 7.78–7.26
(10H, m), 5.84 (1H, d, J 5.5), 4.84 (1H, d, J 11.5), 4.59 (1H, d,
J 11.5), 4.53 (1H, t, J 5.5), 4.19 (1H, d, J 5.6), 4.09 (1H,
d, J 10.6), 4.03 (1H, d, J 10.6), 3.85 (1H, d, J 12.4), 3.67 (1H, d,
J 12.4), 2.39 (3H, s), 1.78 (3H, d, J 0.6); δC(CDCl3) 164.1, 151.5,
145.3, 137.0, 136.2, 132.3, 130.0, 128.6, 128.2, 128.0, 111.0,
88.5, 85.4, 83.8, 79.8, 73.2, 69.4, 63.0, 21.6, 12.5; FAB-MS m/z
533 [M ϩ H]ϩ [Found (%): C, 56.7; H, 5.4; N, 4.9. C25H28N2O9S
requires C, 56.4; H, 5.3; N, 5.2].
(1S,3R,4R,7R)-7-Hydroxy-1-hydroxymethyl-3-(thymin-1-yl)-
2,5-dioxabicyclo[2.2.1]heptane 9
Nucleoside 8 (3.09 g, 4.66 mmol) was dissolved in methanol (40
cm3) and 10% Pd/C [3 g, suspended in methanol (20 cm3)] was
added. The mixture was degassed, and stirred under an atmos-
phere of hydrogen. After 26 h, the mixture was filtered [silica
gel, washed with dichloromethane–methanol (700 cm3; 1:3,
v/v)] and the volume of the filtrate was reduced to 25% of its
initial volume. After repeated filtration, the filtrate was evapor-
ated to dryness under reduced pressure and the residue was
subjected to column chromatography on silica gel using
dichloromethane–methanol (5–12% methanol, v/v) as eluent
which furnished nucleoside 9 (1.03 g, 82%) as a white solid
after evaporation of the solvents under reduced pressure;
δH(CD3OD) 7.73 (1H, d, J 1.1, 6-H), 5.56 (1H, s, 1Ј-H), 4.32
(1H, d, J 2.2, 2Ј-H), 4.21 (1H, d, J 2.2, 3Ј-H), 4.06 (1H, d, J 8.2,
1Љ-Ha), 4.01 (2H, s, 5Ј-H2), 3.86 (1H, d, J 8.2, 1Љ-Hb), 1.85 (3H, d,
J 1.1, CH3); δC(CD3OD) 166.8, 139.4, 108.4, 91.0, 90.3, 79.6,
74,5, 70.0, 59.0, 12.6; FAB-MS m/z 271 [M ϩ H]ϩ [Found (%):
C, 47.8; H, 5.5; N, 9.5. C11H14N2O6ؒ0.5H2O requires C, 47.3; H,
5.4; N, 10.0].
1-[3-O-Benzyl-5-O-(4,4Ј-dimethoxytrityl)-4-C-(p-tolylsulfonyl-
oxymethyl)-â-D-xylofuranosyl]thymine 7
To a solution of nucleoside 6 (3.66 g, 6.88 mmol) in anhydrous
pyridine (25 cm3) were added DMAP (0.84 g, 6.81 mmol) and
DMTCl (3.5 g, 13.2 mmol) and the mixture was stirred for 23 h
at room temperature. Additional DMAP (0.250 g, 2.06 mmol)
and DMTCl (0.700 g, 2.06 mmol) were added, and stirring
was continued for 36 h at room temperature. Ice-cold H2O
(50 cm3) was added and the reaction mixture was diluted with
dichloromethane (150 cm3). The organic phase was separated,
and washed successively with saturated aqueous sodium hydro-
gen carbonate (25 cm3 × 3) and brine (40 cm3 × 3), dried
(Na2SO4), and evaporated to dryness under reduced pressure.
The residue was purified by silica gel column chromatography
using dichloromethane–methanol–pyridine (0.75–1.5% meth-
anol; 0.5% pyridine, v/v/v) as eluent to afford nucleoside 7 (4.28
g, 75%) as a white solid after evaporation of the solvents under
reduced pressure; δH(CDCl3) 9.40 (1H, s), 7.72–6.68 (23H,
m), 5.77 (1H, d, J 4.2), 4.86 (1H, d, J 11.3), 4.49–4.43 (2H, m),
4.23–4.12 (3H, m), 3.76 (3H, s), 3.75 (3H, s), 3.45 (1H, d, J
10.2), 3.17 (1H, d, J 10.2), 2.37 (3H, s), 1.44 (3H, s); δC(CDCl3)
(1S,2R,6R,8R,9R)-1,9-Dihydroxy-4,4-dimethyl-3,5,7,11-tetra-
oxatricyclo[6.3.0.02,6]undecane 11
1,2;5,6-Di-O-isopropylidene-α--ribofuranos-3-ulose32
10
(10.0 g, 38.7 mmol) was stirred for 19 h at room temperature in
80% acetic acid. The mixture was evaporated to dryness under
reduced pressure and the residue was coevaporated with tolu-
ene (2 × 100 cm3) and acetonitrile (2 × 50 cm3) to give com-
pound 11 (8.26 g, 96%) as a clear oil; δH(CDCl3) 5.98 (1H, d,
J 3.9), 4.53–4.43 (3H, m), 4.28 (1H, dd, J 9.2 and 6.3), 3.81–3.75
(1H, dd, J 9.2 and 5.5), 3.70–3.50 (2H, br s), 1.58 (3H, s), 1.40
(3H, s); δC(CDCl3) 113.9, 110.8, 106.9, 84.0, 82.6, 73.6, 70.9,
J. Chem. Soc., Perkin Trans. 1, 1999, 1407–1414
1411

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27.0; FAB-MS m/z 219 [M ϩ H]ϩ [Found (%): C, 48.8; H, 6.4.
C9H14O6ؒ0.25H2O requires C, 48.5; H, 6.6].
white solid after filtration; δH(CDCl3) 8.65 (1H, br s), 7.60 (1H,
d, J 1.2), 7.37–7.24 (10H, m), 6.18 (1H, d, J 2.1), 5.42 (1H, d,
J 2.1), 4.70–4.39 (6H, m), 4.16 (1H, dd, J 9.5 and 7.3), 3.99 (1H,
dd, J 9.5 and 5.2), 2.09 (3H, s), 1.86 (3H, d, J 1.1); δC(CDCl3)
169.1, 163.5, 150.0, 137.1, 137.0, 135.7, 128.6, 128.5, 128.2,
128.0, 128.0, 127.5, 113.4, 111.3, 91.6, 83.0, 78.4, 75.1, 72.8,
72.6, 66.8, 20.7, 12.6; FAB-MS m/z 509.1 [M ϩ H]ϩ [Found
(%): C, 64.0; H, 5.5; N, 5.3. C27H28N2O8 requires C, 63.8; H, 5.6;
N, 5.5].
(1S,2R,6R,8R,9R)-1,9-Dibenzyloxy-4,4-dimethyl-3,5,7,11-tetra-
oxatricyclo[6.3.0.02,6]undecane 12
A solution of derivative 11 (4.45 g, 20.4 mmol) in anhydrous
DMF (100 cm3) at 0 ЊC was added dropwise during 20 min to a
mixture of NaH (3.3 g; 60% suspension in mineral oil, w/w;
81.6 mmol) and anhydrous DMF (50 cm3). The mixture was
stirred at room temperature for 45 min whereupon a solution of
benzyl bromide (10.0 cm3, 84.1 mmol) in anhydrous DMF (10
cm3) was added dropwise. After 2 h, ethyl acetate (10 cm3) was
added and the mixture was evaporated to dryness under
reduced pressure. The residue was purified by silica gel column
chromatography using ethyl acetate–petroleum ether (0–5%
ethyl acetate, v/v) as eluent. After evaporation of the solvents
under reduced pressure, compound 12 (7.56 g, 93%) was
obtained as a yellowish solid, which was used without further
purification in the next step; δH(CDCl3) 7.37–7.22 (10H, m),
6.02 (1H, d, J 3.8), 4.72–3.93 (9H, m), 1.60 (3H, s), 1.39 (3H, s);
δC(CDCl3) 137.8, 137.5, 128.4, 128.3, 128.0, 127.9, 127.7, 115.0,
113.5, 107.5, 81.7, 80.3, 78.0, 72.6, 71.5, 66.5, 27.4, 27.2; FAB-
MS m/z 399 [M ϩ H]ϩ.
(1R,4R,5R,7R,8S)-1,4-Dibenzyloxy-8-hydroxy-7-(thymin-1-yl)-
2,6-dioxabicyclo[3.3.0]octane 15
Sodium methoxide (1.32 g, 24.4 mmol) was added to a solution
of nucleoside 14 (4.10 g, 8.10 mmol) in anhydrous methanol (50
cm3) and the resulting mixture was stirred for 24 h at room
temperature. After neutralization (4 M hydrochloric acid), the
mixture was evaporated to dryness under reduced pressure and
dichloromethane (250 cm3) was added. The resulting mixture
was washed using saturated aqueous sodium hydrogen carb-
onate (2 × 100 cm3) and the combined aqueous phase was
extracted using dichloromethane (4 × 50 cm3). The combined
organic phase was dried (Na2SO4), and evaporated to dryness
under reduced pressure to give nucleoside 15 (3.46 g, 90%) as a
yellowish solid; δH(CDCl3) 10.18 (1H, br s, NH), 8.02 (1H, d,
J 1.2, H-6), 7.39–7.25 (10 H, m, 2 × Bn), 5.97 (1H, s, H-1Ј), 5.41
(1H, d, J 3.9, OH), 4.82 (1H, d, J 4.9, H-4Ј), 4.75–4.48 (6H, m,
H-5Ј, -2Ј, Bn), 4.26–4.19 (1H, dd, J 10.0 and 7.2, Ha-6Ј), 3.92–
3.87 (1H, dd, J, 10.0 and 3.7, Hb-6Ј), 1.78 (3H, d, J 1.1, CH3);
δC(CDCl3) 164.5 (C-4), 150.7 (C-2), 137.3, 137.0 (Bn), 136.4
(C-6), 128.4, 128.3, 128.0, 128.0, 127.8, 127.7 (Bn), 115.4 (C-3Ј),
109.8 (C-5), 96.0 (C-1Ј), 84.0 (C-4Ј), 79.0, 75.1 (C-2Ј, -5Ј), 73.2
(C-6Ј), 72.5 (Bn), 66.3 (Bn), 12.2 (CH3); FAB-MS m/z 467.2
[M ϩ H]ϩ [Found (%): C, 62.9; H, 5.7; N, 5.7. C25H26N2O7ؒ
0.5H2O requires C, 63.2; H, 5.5; N, 5.9].
(1S,4R,5R,7RS,8S)-7,8-Diacetoxy-1,4-dibenzyloxy-2,6-dioxa-
bicyclo[3.3.0]octane 13
Derivative 12 (10.32 g, 25.9 mmol) was stirred in 80% acetic
acid (125 cm3) for 26 h at 80 ЊC. After evaporation of the
solvents under reduced pressure and coevaporation first with
absolute ethanol (100 cm3) and then toluene (2 × 100 cm3), the
residue was dissolved in a mixture of anhydrous pyridine (90
cm3) and acetic anhydride (40 cm3). The mixture was stirred at
room temperature for 19 h and then evaporated to dryness
under reduced pressure after addition of a mixture of ice and
H2O (40 cm3). The residue was purified by silica gel column
chromatography using ethyl acetate–petroleum ether (5–10%
ethyl acetate, v/v) as eluent to afford the anomeric mixture 13
(6.60 g, 57.0%; ~2:1 ratio between diastereoisomers) as a
yellowish oil after evaporation of the solvents under reduced
pressure; δC(CDCl3) 169.3, 169.2, 168.4, 137.3, 137.3, 128.3,
128.2, 127.9, 127.8, 127.6, 127.3, 114.2, 112.9, 101.0, 96.9,
84.7, 82.3, 77.5, 76.4, 75.2, 72.5, 72.4, 72.4, 71.7, 71.6,
66.5, 66.3, 20.8, 20.6, 20.5, 20.3; FAB-MS m/z 441.4 [M Ϫ H]Ϫ
[Found (%): C, 64.7; H, 5.9. C24H26O8ؒ0.25H2O requires C, 64.5;
H, 6.0].
(1R,4R,5R,7R,8S)-1,4-Dibenzyloxy-8-methoxy-7-(thymin-1-
yl)-2,6-dioxabicyclo[3.3.0]octane 17
A solution of nucleoside 15 (900 mg, 1.93 mmol) and sodium
hydride (200 mg; 60% suspension in mineral oil, w/w; 5.00
mmol) in dichloromethane (10 cm3) was stirred for 20 min at
room temperature. Methyl iodide (0.90 cm3, 14.5 mmol) was
added and stirring at room temperature was continued for 48 h
whereupon additional sodium hydride (600 mg; 60% suspen-
sion in mineral oil, w/w; 15.0 mmol) and methyl iodide (4 cm3,
64.3 mmol) were added. After 168 h, ethyl acetate (10 cm3) was
added together with methanol (5 cm3), dichloromethane (10
cm3) and silica gel (10 g), and the mixture was evaporated to
dryness under reduced pressure. The residue was purified by
silica gel column chromatography and elution, first with ethyl
acetate–petroleum ether (10–50% ethyl acetate, v/v) and then
with methanol–dichloromethane (1:99, v/v), yielded nucleoside
17 (573 mg, 62%) as a white solid after evaporation of the sol-
vents; δH(CDCl3) 8.97 (1H, br s, NH), 7.90 (1H, d, J 1.2, H-6),
7.41–7.27 (10H, m, Bn), 6.1 (1H, s, H-1Ј), 4.71 (1H, d, J 11.2,
Bn), 4.65 (1H, d, J 4.8, H-4Ј), 4.62–4.46 (4H, m, Bn, H-5Ј), 4.22
(1H, dd, J 9.8 and 7.3, Ha-6Ј), 3.99 (1H, s, H-2Ј), 3.91 (1H, dd,
J 9.8 and 4.1, Hb-6Ј), 3.65 (3H, s, OCH3), 1.82 (3H, d, J 1.1,
CH3); δC(CDCl3) 163.9, 150.1, 137.2, 137.1, 136.5, 128.6, 128.5,
128.2, 128.1, 128.0, 114.7, 110.0, 92.2, 83.6, 79.0, 77.2, 73.0,
72.7, 66.7, 58.5, 12.4; FAB-MS m/z 481.2 [M ϩ H]ϩ [Found
(%): C, 65.1; H, 6.0; N, 5.8. C26H28N2O7 requires C, 65.0; H, 5.9;
N, 5.8].
(1S,4R,5R,7R,8S)-8-Acetoxy-1,4-dibenzyloxy-7-(thymin-1-yl)-
2,6-dioxabicyclo[3.3.0]octane 14
The bicyclic furanose 13 (218 mg, 0.49 mmol) and thymine (125
mg, 0.99 mmol) were dissolved in anhydrous acetonitrile (5
cm3). N,O-Bis(trimethylsilyl)acetamide (0.85 cm3, 3.45 mmol)
was added and the mixture was stirred for 1 h under reflux.
After cooling of the mixture to Ϫ30 ЊC, trimethylsilyl triflate
(0.25 cm3, 1.38 mmol) was added dropwise during 20 min. The
temperature was allowed to rise to room temperature and the
mixture was stirred for 40 h at 50 ЊC and subsequently for 1 h
under reflux. After cooling of the mixture to room temperature,
ice-cold saturated aqueous sodium hydrogen carbonate (5 cm3)
was added. Extraction was performed using dichloromethane
(3 × 5 cm3) and the combined organic phase was washed suc-
cessively with saturated aqueous sodium hydrogen carbonate (5
cm3) and brine (5 cm3), dried (Na2SO4), and evaporated to dry-
ness under reduced pressure. The residue was purified by silica
gel column chromatography using methanol–dichloromethane
as eluent (0–1% methanol, v/v). The fractions containing the
product were collected, and evaporated to 10% volume under
reduced pressure. Petroleum ether (10 cm3) was added and pre-
cipitation yielded the product nucleoside 14 (219 mg, 85%) as a
(1S,4R,5R,7R,8S)-8-(tert-Butyldimethylsilyloxy)-1,4-dibenzyl-
oxy-7-(thymin-1-yl)-2,6-dioxabicyclo[3.3.0]octane 19
Nucleoside 15 (500 mg, 2.04 mmol) was dissolved in anhydrous
DMF (5 cm3). tert-Butyldimethylsilyl chloride (TBDMSCl)
(485 mg, 3.2 mmol) and imidazole (292 mg, 4.3 mmol) were
1412
J. Chem. Soc., Perkin Trans. 1, 1999, 1407–1414

View Article Online
added and the mixture was stirred for 24 h at room temperature.
After addition of MeOH (1 cm3), the mixture was evaporated
to dryness under reduced pressure and the residue was purified
by silica gel column chromatography using ethyl acetate–
petroleum ether (0–12% ethyl acetate, v/v) as eluent to give
product nucleoside 19 (527 mg, 85%) as a white solid after
evaporation of the solvents; δH(CDCl3) 8.76 (1H, br s), 7.89
(1H, d, J 1.1), 7.35–7.26 (10H, m), 5.90 (1H, s), 4.73–4.43 (7H,
m), 4.14 (1H, dd, J 9.9 and 7.1), 3.88 (1H, dd, J 9.9 and 4.2),
1.80 (3H, d, J 1.1), 0.93 (9H, s), 0.24 (3H, s), 0.18 (3H, s);
δC(CDCl3) 163.8, 151.0, 137.4, 137.0, 136.5, 128.4, 128.3, 128.0,
127.7, 127.6, 127.4, 114.8, 109.7, 94.5, 83.2, 78.7, 76.0, 72.8,
72.5, 65.6, 25.6, 17.8, 12.2 (signals below 0 ppm not shown);
FAB-MS m/z 581.3 [M ϩ H]ϩ [Found (%): C, 64.1; H, 6.7; N,
4.6. C31H40N2O7Si requires C, 64.1; H, 6.9; N, 4.8].
ated to dryness under reduced pressure and the residue was
dissolved in dichloromethane (150 cm3), washed successively
with saturated aqueous sodium hydrogen carbonate (60 cm3)
and brine (30 cm3), dried (Na2SO4), and evaporated to dryness
under reduced pressure. The residue was purified by silica gel
column chromatography using petroleum ether–dichloro-
methane (1:1, v/v) as eluent to afford the anomeric mixture 23
as a clear oil (4.50 g, 74%) after evaporation of the solvents
under reduced pressure. This oil was used in the next step with-
out further purification; δC(CDCl3) 169.9, 169.2, 166.2, 138.1,
137.0, 133.2, 133.1, 133.0, 129.9, 129.7, 128.5, 128.4, 128.3,
128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 127.6, 127.5, 127.4,
98.6, 94.3, 84.7, 82.3, 82.0, 77.7, 76.5, 76.4, 76.3, 74.7, 74.1,
73.8, 73.3, 73.1, 72.8, 71.8, 70.0, 63.8, 63.2, 21.2, 20.8, 20.8,
20.6; FAB-MS m/z 547 [M Ϫ H]Ϫ.
(1S,4R,5R,7R,8S)-8-(tert-Butyldimethylsilyloxy)-1,4-dihydroxy-
7-(thymin-1-yl)-2,6-dioxabicyclo[3.3.0]octane 20
1-(2-O-Acetyl-6-O-benzoyl-3,5-di-O-benzyl-â-D-allofuranosyl)-
thymine 24
Nucleoside 19 (181 mg, 0.43 mmol) was dissolved in absolute
ethanol (3 cm3) and 20% Pd(OH)2/C (90 mg) was added. After
degassing, the solution was stirred in an atmosphere of hydro-
gen for 26 h at room temperature. The mixture was filtered and
the filtrate was evaporated to dryness under reduced pressure.
The residue was dissolved in methanol–dichloromethane (2
cm3; 1:99, v/v) and quickly filtered through a bed of silica gel.
After washing with methanol–dichloromethane (1:99, v/v),
nucleoside diol 20 (63 mg, 50%) was obtained as a white solid.
This product decomposed during prolonged column chromato-
graphic purification (silica gel or basic alumina); δH(CDCl3)
8.84 (1H, br s), 7.21 (1H, d, J 1.3), 5.76 (1H, d, J 7.0), 4.45–4.40
(3H, m), 4.31 (1H, dd, J 10.2 and 3.9), 4.06 (1H, dd, J 10.2 and
2.5), 1.94 (3H, d, J 1.3), 0.89 (9H, s), 0.08 (3H, s), 0.01 (3H, s);
δC(CDCl3) 163.1, 150.1, 136.8, 112.0, 107.9, 93.9, 85.3, 76.0,
75.7, 71.1, 25.4, 17.8, 12.3, Ϫ5.0, Ϫ5.1; FAB-MS m/z 401.2
[M ϩ H]ϩ.
To a stirred suspension of the anomeric mixture 23 (4.50 g, 8.21
mmol) and thymine (1.55 g, 12.31 mmol) in anhydrous
acetonitrile (50 cm3) was added N,O-bis(trimethylsilyl)acet-
amide (12.2 cm3, 49.3 mmol). The reaction mixture was stirred
at 60 ЊC for 1 h and then cooled to 0 ЊC. Trimethylsilyl triflate
(2.97 cm3, 16.4 mmol) was added dropwise during 10 min and
the mixture was heated for 2 h under reflux. The reaction mix-
ture was allowed to cool to room temperature and the volume
was reduced by 50% under reduced pressure. After cooling of
the mixture to 0 ЊC, saturated aqueous sodium hydrogen carb-
onate (100 cm3) was added and extraction was performed with
dichloromethane (3 × 50 cm3). The combined organic phase
was washed with brine (50 cm3), dried (Na2SO4), and evapor-
ated to dryness under reduced pressure. The residue was
purified by silica gel column chromatography using dichloro-
methane–methanol (99.5:0.5, v/v) as eluent to give nucleoside
24 as a white solid (4.06 g, 81%) after evaporation of the solv-
ents under reduced pressure; δH(CDCl3) 8.74 (1H, br s), 8.01
(2H, m), 7.61–7.11 (14H, m), 6.09 (1H, d, J 5.3), 5.32 (1H, m),
4.86 (1H, d, J 11.7), 4.65 (1H, d, J 11.7), 4.55–4.10 (7H, m),
2.10 (3H, s), 1.59 (3H, s); δC(CDCl3) 170.1, 166.1, 163.4, 150.2,
137.4, 137.0, 135.7, 133.3, 129.7, 128.6, 128.5, 128.1, 128.0,
127.9, 127.7, 127.3, 126.9, 111.7, 87.6, 82.6, 76.7, 75.3, 73.7,
73.1, 73.0, 63.3, 20.7, 12.0; FAB-MS m/z 615 [M ϩ H]ϩ [Found
(%): C, 66.4; H, 5.6; N, 4.4. C34H34N2O9 requires C, 66.4; H, 5.6;
N, 4.6].
6-O-Benzoyl-3,5-di-O-benzyl-1,2-di-O-isopropylidene-á-D-
allofuranose 22
To a stirred solution of furanose 2133 (4.60 g, 11.1 mmol) in
anhydrous DMF (20 cm3) at 0 ЊC was added a 60% suspension
of sodium hydride in mineral oil (w/w, 0.67 g, 16.7 mmol, added
in four portions during 20 min). After stirring of the mixture
for 30 min, benzyl bromide (1.99 cm3, 16.7 mmol) was added
and stirring was continued for 2 h at room temperature. The
mixture was cooled to 0 ЊC, H2O (30 cm3) was added and
extraction was performed using dichloromethane (50 cm3 × 3).
The combined organic phase was washed successively with sat-
urated aqueous sodium hydrogen carbonate (30 cm3) and brine
(20 cm3), dried (Na2SO4), and evaporated to dryness under
reduced pressure. The residue was purified by silica gel column
chromatography using ethyl acetate–petroleum ether (1:9, v/v)
as eluent to give furanose 22 as a yellowish oil (5.0 g, 90%) after
evaporation of the solvents under reduced pressure. This
oil was used in the next step without further purification;
δH(CDCl3) 7.99 (2H, m), 7.58–7.21 (13H, m), 5.77 (1H, d,
J 3.6), 4.77–4.00 (10H, m), 1.59 (3H, s), 1.35 (3H, s); δC(CDCl3)
166.2, 138.4, 137.4, 133.0, 130.1, 129.7, 128.4, 128.3, 128.2,
128.1, 127.9, 127.8, 127.7, 127.5, 113.1, 102.2, 79.2, 77.6, 76.5,
76.3, 73.7, 72.2, 64.3, 26.9; FAB-MS m/z 505 [M ϩ H]ϩ.
1-(3,5-Di-O-benzyl-â-D-allofuranosyl)thymine 25
To a stirred solution of nucleoside 24 (3.00 g, 4.88 mmol) in
methanol (50 cm3) was added sodium methoxide (0.79 g, 14.7
mmol). The reaction mixture was stirred for 14 h at room tem-
perature and subsequently neutralized with dilute hydrochloric
acid whereupon ice-cold H2O (50 cm3) was added. The resulting
mixture was extracted using ethyl acetate (3 × 100 cm3) and the
combined organic phase was evaporated to dryness under
reduced pressure. The residue was purified by silica gel column
chromatography using dichloromethane–methanol (98.5:1.5,
v/v) as eluent to give nucleoside 25 as a white solid (2.00 g, 88%)
after evaporation of the solvents under reduced pressure;
δH(CDCl3) 9.39 (1H, br s), 7.38–7.15 (11H, m), 5.80 (1H, d,
J 4.6), 4.80–3.55 (10H, m), 1.59 (3H, s); δC(CDCl3) 163.7, 150.8,
137.7, 136.8, 136.3, 128.7, 128.4, 128.2, 128.0, 127.3, 111.4,
90.4, 82.7, 78.8, 76.5, 72.9, 72.5, 72.4, 60.7, 12.0; FAB-MS m/z
469 [M ϩ H]ϩ [Found (%): C, 64.4; H, 6.1; N, 5.5. C25H28N2O7
requires C, 64.1; H, 6.0; N, 6.0].
1,2-Di-O-acetyl-6-O-benzoyl-3,5-di-O-benzyl-D-allofuranose 23
A solution of furanose 22 (5.00 g, 9.92 mmol) in 80% acetic
acid (75 cm3) was stirred for 10 h at 80 ЊC. The solvent was
removed under reduced pressure and the residue was coevapor-
ated with toluene (10 cm3 × 3) and dissolved in a mixture of
anhydrous pyridine (30 cm3) and dichloromethane (30 cm3).
Acetic anhydride (5.0 cm3) was added and the solution was
stirred for 20 h at room temperature. The mixture was evapor-
1-[3,5-Di-O-benzyl-2,6-di-O-(p-tolylsulfonyl)-â-D-allofurano-
syl]thymine 26
To a stirred solution of nucleoside 25 (0.60 g, 1.28 mmol) in
dichloromethane (70 cm3) at room temperature was added
J. Chem. Soc., Perkin Trans. 1, 1999, 1407–1414
1413

View Article Online
DMAP (0.63 g, 5.12 mmol) and toluene-p-sulfonyl chloride
(0.73 g, 3.84 mmol). After stirring of the mixture for 3 h, ice-
cold H2O (50 cm3) was added and extraction was performed
using dichloromethane (3 × 75 cm3). The combined organic
phase was dried (Na2SO4), and evaporated to dryness under
reduced pressure. The residue was purified by silica gel column
chromatography using dichloromethane–methanol (99.5:0.5,
v/v) as eluent to give nucleoside 26 as a white solid (0.71 g, 71%)
after evaporation of the solvents under reduced pressure;
δH(CDCl3) 8.83 (1H, br s), 7.73–7.12 (18H, m), 6.58 (1H, d,
J 1.2), 5.88 (1H, d, J 6.9), 5.0 (1H, m), 4.73–3.82 (9H, m), 2.40
(3H, s), 2.35 (3H, s), 1.48 (3H, d, J 0.9); δC(CDCl3) 163.1, 149.8,
145.8, 145.2, 137.1, 137.0, 135.6, 132.4, 132.3, 130.0, 128.7,
128.5, 128.3, 128.1, 128.0, 127.8, 127.2, 111.4, 86.9, 83.1, 77.7,
75.3, 73.1, 72.5, 67.4, 21.7, 11.9; FAB-MS m/z 777 [M ϩ H]ϩ
[Found (%): C, 60.6; H, 5.2; N, 3.5. C39H40N2O11S2 requires
C, 60.3; H, 5.2; N, 3.6].
Acknowledgements
The Danish Natural Science Research Council, The Danish
Technical Research Council and Exiqon A/S are thanked for
financial support.
References
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(1S,4R,5R,7R,8R)-4,8-Dibenzyloxy-7-(thymin-1-yl)-2,6-dioxa-
bicyclo[3.2.1]octane 27
To a stirred solution of nucleoside 26 (0.63 g, 0.81 mmol) in a
mixture of ethanol and H2O (40 cm3; 1:1, v/v) at room temper-
atue was added an aqueous solution of sodium hydroxide (1 M;
7 cm3). The resulting mixture was heated under reflux for 16 h
and then neutralized by addition of dilute hydrochloric acid.
The volume of the mixture was reduced to 50% and extraction
was performed using dichloromethane (50 cm3 × 3). The com-
bined organic phase was dried (Na2SO4), and evaporated to
dryness under reduced pressure. The residue was purified by
silica gel column chromatography using dichloromethane–
methanol (99:1, v/v) as eluent to give nucleoside 27 as a white
solid (0.40 g, 93%) after evaporation of the solvents under
reduced pressure; δH(CDCl3) 8.69 (1H, br s), 7.90 (1H, d, J 1.1),
7.39–7.25 (10H, m), 5.85 (1H, d, J 2.2), 4.78–4.47 (6H, m),
3.87–3.38 (4H, m), 1.87 (3H, s); δC(CDCl3) 163.9, 149.9, 137.3,
137.1, 136.8, 128.6, 128.5, 128.2, 128.1, 127.8, 127.7, 109.4,
88.6, 79.9, 79.7, 74.5, 73.5, 71.4, 70.8, 65.0, 12.5; FAB-MS m/z
451 [M ϩ H]ϩ [Found (%): C, 66.3; H, 5.7; N, 6.1. C25H26N2O6
requires C, 66.7; H, 5.8; N, 6.2].
(1S,4R,5R,7R,8R)-4,8-Dihydroxy-7-(thymin-1-yl)-2,6-
dioxabicyclo[3.2.1]octane 28
Nucleoside 27 (0.27 g, 0.60 mmol) was dissolved in absolute
ethanol (20 cm3) and 20% Pd(OH)2/C (0.25 g) was added. The
mixture was degassed and placed under an atmosphere of
hydrogen. After stirring the mixture for 26 h the catalyst was
filtered off (silica gel; washed with methanol, 400 cm3) and the
filtrate was concentrated to dryness under reduced pressure.
The residue was subjected to column chromatography on silica
gel using dichloromethane–methanol (94:6, v/v) as eluent to
give nucleoside 28 as white solid (0.16 g, 98%) after evaporation
of the solvents under reduced pressure; δH(CD3OD) 8.06
(1H, d, J 1.2, 6-H), 5.57 (1H, d, J 2.3, 1Ј-H), 4.50 (1H, m,
2Ј-H), 4.42 (1H, s, 4Ј-H), 4.03 (1H, m, 3Ј-H), 3.93–3.80 (2H,
m, 5Ј-H, 6Ј-Ha), 3.21 (1H, m, 6Ј-Hb), 1.91 (3H, d, J 1.2, CH3);
δC(CD3OD) 166.8 (C-4), 152.0 (C-2), 139.2 (C-6), 110.2 (C-5),
90.2 (C-1Ј), 87.3 (C-4Ј), 77.0 (C-2Ј), 74.7 (C-3Ј), 68.5 (C-5Ј),
67.4 (C-6Ј), 12.5 (CH3); FAB-MS m/z 271 [M ϩ H]ϩ.
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Paper 9/01729A
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J. Chem. Soc., Perkin Trans. 1, 1999, 1407–1414