A ring-closing metathesis based synthesis of bicyclic nucleosides locked in S-type conformations by hydroxyl functionalised 3′,4′-trans linkages

Article abstract of DOI:10.1016/j.tet.2004.03.019

A [4.3.0]bicyclic nucleoside that contains an unsaturated hydroxylated 3′,4′-trans linkage has been efficiently synthesised. Thus, from diacetone-D-glucose as the starting material, stereoselective Grignard reactions for the introduction of allyl groups,

Full text of DOI:10.1016/j.tet.2004.03.019

Tetrahedron 60 (2004) 3775–3786
A ring-closing metathesis based synthesis of bicyclic nucleosides
locked in S-type conformations by hydroxyl functionalised
30,40-trans linkages
*
Morten Freitag, Helena Thomasen, Nanna K. Christensen, Michael Petersen and Poul Nielsen
Department of Chemistry, Nucleic Acid Center,† University of Southern Denmark, 5230 Odense M, Denmark
Received 7 January 2004; revised 18 February 2004; accepted 4 March 2004
Abstract—A [4.3.0]bicyclic nucleoside that contains an unsaturated hydroxylated 30,40-trans linkage has been efficiently synthesised. Thus,
from diacetone-D-glucose as the starting material, stereoselective Grignard reactions for the introduction of allyl groups, a nucleobase
coupling and, subsequently, a ring-closing metathesis (RCM)-reaction were applied as the key reactions. The cyclohexene moiety introduced
in this nucleoside reveals a large potential for further derivatisation, and as the first example, a stereoselective dihydroxylation followed by
deprotection afforded a multihydroxylated bicyclic nucleoside. The configuration and conformational behaviour was determined by NMR
spectroscopy and ab initio calculations, and both this bicyclic nucleoside and its unsaturated analogue were found to be strongly restricted in
S-type conformations.
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
The biological importance of the conformational equi-
librium of nucleosides between N-type and S-type confor-
mations following the pseudorotational cycle1,2 has
motivated the preparation of a significant number of
synthetic nucleoside analogues mimicking these confor-
mational ranges. Thus, nucleosides with conformationally
restricted bi- or tricyclic carbohydrate parts have been
intensively studied as building blocks in nucleic acid
analogues,3–5 for the study of enzymes and receptors with
nucleoside or nucleotide substrates,6–9 and/or for potential
antiviral agents.6,8–11 As a prime example, nucleosides with
the [2.2.1]bicyclic core structure 1 are locked in an N-type
conformation due to a 20,40-linkage (Fig. 1),12,13 and
Figure 1. Selected bi- and tricyclic nucleoside analogues mimicking N-type
(1, 2) and S-type conformations (3–6). T¼thymin-1-yl.
oligonucleotides containing the monomer 1 (R¼OH) have
been defined as LNA (locked nucleic acid).12 LNA has
the intermediate E-type conformations have been pre-
demonstrated high affinity recognition of DNA and RNA12,14
sented5,9,11,18–20 and used in similar studies.9
and, subsequently, has shown promising results towards
potential therapeutic applications.14 Other N-type mimick-
Bi- or tricyclic nucleoside analogues that are conformation-
ing nucleosides based on the same structure (e.g. 1,
ally restricted towards S-type conformations have also been
R¼N3)15,16 or on bicarbocyclic structures6–8,17 (e.g., 2,
obtained.4,5 Thus, nucleosides with the bicarbocyclic
R¼H, OH, or N3) have been used in the study of different
structure 3 have been used in the studies of several enzymes
receptors and enzymes, including the HIV reverse tran-
such as HIV reverse transcriptase as well as adenosine
scriptase.6 Also bicyclic nucleosides that are restricted in
receptors.6,7 Oligonucleotides containing the monomer 3
(R1¼H, R2¼OH), however, demonstrated slightly
decreased recognition of DNA and RNA complements.21
Similar results have been obtained with other bicyclic
Keywords: Ring-closing metathesis; Stereoselective dihydroxylation;
Nucleosides; Conformational restriction.
*
Corresponding author. Tel.: þ45-65502565; fax: þ45-66158780;
nucleoside monomers that are strongly restricted in S-type
conformations due to a 10,30-cis linkage22 or a 20,30-trans
linkage.18 However, the reason for the induced duplex
e-mail address: pon@chem.sdu.dk
Nucleic Acid Center is funded by the Danish National Research
Foundation for studies on nucleic acid chemical biology.
†
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2004.03.019

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M. Freitag et al. / Tetrahedron 60 (2004) 3775–3786
destabilisation might, in all three cases, be steric influence
from the additional ring structures or distortion of duplex
h0yd0ration patterns. On the other hand, the pioneering
3 ,5 -linked bicyclic nucleoside 4 (R¼H) has demonstrated
slightly increased but varying affinities for DNA and RNA
complements when incorporated into oligonucleotides.23,24
The nucleoside monomers 0are restricted towards an S-type
conformation but the C4 –C50 bond, described by the
standard torsion angle g,2 is restricted in the ap range
which is not favourable for duplex formation by standard
Watson–Crick base-pairing.23,24 This has been significantly
improved, however, by the introduction of an additional
cyclopropane ring in a tricyclic nucleoside which, on the
other hand, is no longer a true S-type mimic.25
We have recently introduced a new synthetic strategy based
on ring-closing metathesis (RCM) towards 4 and its ana-
logues26–28 including the ribo-configured analogue (4,
R¼OH),26 and a tricyclic nucleoside 5.27 Oligonucleotides
containing 5, however, displayed a significant destabilisa-
tion of duplexes with complementary DNA and RNA.27
This result, in combination with the results of 4,23,24
demonstrates that the design of a nucleoside monomer for
oligonucleotides that are preorganised for duplex formation
should not combine a locked S-type conformation with g in
an inappropriate angle. Therefore, we have focused 0on the
design of bicyclic nucleoside analogues with 30,4 -trans
linkages combining a locked S-type conformation with an
unrestricted C40 –C50-bond. The first 30,40-trans linked
bicyclic nucleoside structure 6 (R¼OH or OCH3) was
introduced recently as a perfect S-type mimic.29,30 0In the
same communication,30 we introduced a novel 30,4 -trans
linked nucleoside by a synthetic strategy that is based on
RCM31 and comparable with the one used successfully in
the preparation of 04 0and 5. This synthesis leading to a
nucleoside with a 3 ,4 -trans fused cyclohexene ring with a
large potential for further modification is hereby described
in detail. Furthermore, the potential of this [4.3.0]-
bicyclic core structure is exemplified by a stereoselective
dihydroxylation and, subsequently, a highly hydroxylated
and hydrophilic nucleoside analogue that is conformation-
ally locked in an S-type conformation.
Scheme 1. Reagents and conditions: (a) NaH, BnBr, DMF, 92% (Ref. 32);
(b) H5IO6, EtOAc; (c) H2CO, NaOH, THF, H2O then NaBH4, 86% (2 steps);
(d) NaH, BnBr, DMF, 10 61% and 11 22%; (e) PCC, CH2Cl2;
(f) vinylMgBr, THF, 12 17% and 13 63% (2 steps); (g) A (2 mol%),
CH2Cl2, 14 76%, 15 93%; (h) 80% aq. AcOH, then Ac2O, pyridine, 47%;
(i) BzCl, pyridine, 98%.
2. Results
were only indicated at this stage. Thus, a comparable ratio
has been obtained on a similar substrate without the 3-allyl
group, i.e., the O-benzylation of 3-O-benzyl-4-C-hydroxy-
methyl-1,2-di-O-isopropylidene-a-D-ribofuranose.34 When
2.1. Chemical synthesis
As a convenient and cheap starting material, diacetone-D-
glucose was converted by oxidation and a stereoselective
Grignard reaction to give the 30-C-allyl derivative 7
(Scheme 1).32 This was further protected as a benzyl ether
to give 8.33 Treatment with periodic acid to give in situ
regio-selective cleavage of the primary acetonide and
subsequent cleavage of the diol was followed by an aldol
condensation of the resulting aldehyde with formaldehyde
and a Cannizzarro reaction to give the diol 9 in a high yield.
Differentiation between the two primary alcohols of 9 was
possible probably because of a weak steric shielding of
the a-phase of the bicyclic system. Thus, the benzylation
afforded a 3:1 ratio of bis-benzylic ethers of which 10 was
obtained as the major isomer after chromatographic
separation. The full assignments of 10 and its 4-epimer 11
00
1
exploring the H NMR data given for that case, the H-1
signals‡ of both isomers were seen to be shifted downfield
compared to the H-5 signals.34 We deduce this phenomenon
to the deshielding by the electronegative 3-O atom. For 10,
the highest chemical shifts were observed for the signal
coupling to an OH-signal hereby confirming the 5-O-
benzylation, whereas in the 4-epimer 11 the situation is
opposite. The 13C NMR data supports this argument
perfectly. Thus, the C-5 signal has shifted from 63.2 ppm
in 9 to 68.7 ppm in 10 confirming00the alkylation of the C-5
hydroxyl group, whereas the C-1 signal has only shifted
‡
For the numbering of the different carbon atoms, see the depictions in
Scheme 1 and 2, as well as the introduction to Section 5.

M. Freitag et al. / Tetrahedron 60 (2004) 3775–3786
3777
from 63.5 ppm to 61.5 ppm. For 11, the C-100 signal has
shifted to 71.6 ppm and the C-5 signal remains almost
unchanged at 63.6 ppm.
Subsequently, 10 was oxidised using a simple and mild
chromium(VI) mediated oxidation followed by another
Grignard reaction to give the two epimers 12 and 13 in an
approx. 1:4 ratio and in a high yield after chromatographic
separation (Scheme 1). The configurations of the two
epimers were not determined at this stage but we deduced
this problem to be much easier solved after RCM-mediated
ring-closure. Thus, both epimers were used as substrates for
an RCM reaction using the second generation Grubbs’
catalyst A (Scheme 1).35 The major isomer 13 afforded
smoothly the cyclohexene analogue 15 in 93% yield
whereas 12 after longer reaction time and higher loading
of A (3 mol% instead of 2 mol%) gave 14 in 76% yield. The
1
configurations of 14 and 15 were determined by H NMR
spectroscopy hereby giving a determination also of 12 and
13 and a further confirmation of the configurations of 10 and
11. Thus, from NOE-difference spectra strong mutual
contacts0 between H-5 and H-2 as well as between H-5 and
one H-4 confirmed the C-4 configuration of 15, and strong
mutual contacts between H-5 and H-10 on0the cyclohexene
ring confirmed the (S)-configuration of C-1 . Analogously, a
significantly smaller contact between H-10 and H-5
indicated the (R)-configuration of C-100 in 14. A very large
difference in chemical shift for the H-1 atoms (5.26 ppm in
14 and0 3.83 ppm in 15) further verified this determination,
as H-1 in 14 is expected to be strongly deshielded by the
3-O atom.
Problems with Lewis-acid promoted nucleobase coupling
reactions have been observed before with constrained
bicyclic carbohydrate substrates.36,37 Nevertheless, we
decided to use the major of the two tricyclic products 15
in our first investigation towards the preparation of the
bicyclic nucleoside targets. Thus, we attempted the standard
conversion using acetic acid followed by basic acetylation
to give an anomeric mixture of bicyclic furanosyl acetates.
However, instead of this mixture we obtained one major
compound which from NMR spectroscopy was proved to be
the cyclohexene 16. This result can be deduced to the high
ring strain of the bicyclic furanose due to the 3,4-trans fused
cyclohexene ring. Thus, the intermediate free aldose
derivative simply prefers its open form, and subsequently,
the hydroxyaldehyde undergoes an a-ketol rearrangement
to a more thermodynamically stable hydroxyketone deriva-
tive. Finally, acetylation of the primary and secondary
alcohols gives 16. This observation of ring strain gives a
final confirmation of the determination of C-4 configuration
(i.e., of the epimers 10 and 11).
Scheme 2. Reagents and conditions: (a) i, 80% aq. AcOH, ii, Ac2O,
pyridine, 92% (2 steps); (b) thymine, N,O-bis(trimethylsilyl)acetamide,
TMS-OTf, CH3CN, 93%; (c)
A (2 mol%), ClCH2CH2Cl, 90%;
(d) NaOCH3, CH3OH, 87%; (e) BCl3, hexanes, CH2Cl2, 22 55% and 24
21% (from 21), 24 69% (from 23); (f) NaOCH3, CH3OH, 48%;
(g) NaOCH3, CH3OH, reflux, 69%; (h) OsO4, NMO, THF, H2O, 49%;
(i) NaOCH3, CH3OH, 75%; (j) H2, Pd(OH)2–C, MeOH, 95%.
high yield due to anchimeric assistance from the 20-O-acetyl
group.40,41 This was confirmed by the large coupling
3
0
0
constant JH1 H2 ¼8.5 Hz. A RCM reaction using the same
catalyst as before afforded smoothly the bicyclic nucleoside
20 in 90% yield. The structure of this compound was
confirmed by MS showing the expected loss of the mass of
ethylene and NMR as the large coupling constant
3
0
0
JH1 H2 ¼7.6 Hz again confirms the nucleoside to be
b-configured and restricted in an S-type conformation.42
A basic treatment of 20 using methanolic ammonia or,
alternatively, sodium methoxide afforded a selective
cleavage of only the acetate ester to give 21. A subsequent
Lewis acid mediated debenzylation of 21 using boron
trichloride afforded both the benzoate 22 and, surprisingly,
the fully deprotected bicyclic nucleoside 24. Finally, this
target nucleoside 24 was further obtained after a new
treatment of 22 with sodium methoxide. Hereby 24 was
obtained in 41% combined yield over the three steps from
A better route towards the 30,40-trans linked bicyclic
nucleosides was hereafter initiated and, obviously, the
nucleobase coupling should be performed before the RCM
reaction. Thus, the compatibility of RCM reactions and
nucleosides has been demonstrated several times
before.31,38,39 Therefore, 13 was protected as its benzoic
ester to give 17 in a high yield (Scheme 1) followed by
hydrolysis and acetylation to give the anomeric mixture 18
(Scheme 2). Standard Vorbru¨ggen type coupling of thymine
to this substrate gave exclusively the b-nucleoside 19 in a

3778
M. Freitag et al. / Tetrahedron 60 (2004) 3775–3786
20. In order to obtain a better overall yield of 24, a stronger
treatment of 20 with sodium methoxide hydrolysed both
esters simultaneously to give 23, which subsequently was
debenzylated using boron trichloride to give 24 in 46% yield
over the two steps. The structure of 24 was confirmed by
NMR spectroscopy. Thus, comparison of the spectra of 23
and 24 with the spectra of 15 revealed all expected
similarities including the relatively low chemical shift of
H-100.
yield over the two steps as a fully deprotected multi-
hydroxylated bicyclic nucleoside derivative. The absolute
configuration of 27 was hereafter determined from NMR-
studies (see below).
2.2. Determination of the configuration and
conformation of 27
To determine the configuration at C-200 and C-300 and the
conformation of the [4.3.0]bicyclic skeleton in 27, NMR
investigations and molecular modelling were performed.
The NMR-signals of 27 were determined from 2D-experi-
ments and the assignment of the two different H-400 signals
was performed after an NOE-difference experiment. Thus,
strong mutua0l0 contacts were observed betwe0e0 n H-50 and
The double bond now incorporated in the 30,40-linkage of 24
opens the opportunity for further functionalisation. As the
first example, the protected nucleoside 20 was used as the
substrate in a dihydroxylation reaction. After treatment with
osmium tetraoxide and N-methylmorpholine-N-oxide as a
co-oxidant, the diol 25 was obtained in a medium 49% yield
as an approximately 16:1 ratio of diastereomers. However,
based on the recovery of 33% starting material this
corresponds to a 73% yield. With a longer reaction time,
the isolated yield of 25 could be slightly improved to 53%
yield but with only 10% starting material recovered this
displayed no improvement. Also a small amount of a bis-
dihydroxylated product 25a on which also the nucleobase
has been dihydroxylated was isolated in 10% yield, or with a
longer reaction time, 16% yield. This problem has been
observed before for comparable nucleoside substrates.20,28
The configurations of the two diastereomers of 25 were not
determined at this stage and the stereoselective outcome of
the dihydroxylation process was not obviously predicted
from simple modelling. Nevertheless, the mixture was
deprotected to give only the pure main diastereomer 26 and
further debenzylated via hydrogenation to give 27 in 71%
3
only one H-4 hereafter determined to be H-4up. The JHH
coupling constants of 27 were determined as given in
Table 1. As no significant changes for any of the coupling
constants were observed in the temperature range from 250
to 50 8C, we assume that 27 exists in only one conformer
and not in a dynamic equilibrium between two or more
conformers. All subsequent modelling was performed under
0
this assumption. The Karplus relationships between JH1 H2
0
,
00
00
00
00
00
00
00
00
JH1 H2 , JH2 H3 , JH3 H4 up, and JH3 H4 down and the corre-
sponding torsion angles were derived for both of the
possible configurations.1,43 For each Karplus relationship
there are, typically, four possible torsion angles correspond-
ing to the given coupling constant. However, the restraints
of the covalent geometry reduces this to two allowed torsion
00
angles for uH1 H2 and uH2 H3 , and considering the large
00
00
00
00
00
00
JH3 H4 coupling constant and the fact that H-4up and
00
H-4
0a0 re geminal, just one allowed geometry of the
00
fragment for each possible
up/down
down
H300 –C3 –C400 –H4
configuration of 27. The possible torsion angles for one of
these, the ‘up’-isomer (i.e., the 200(R),300(R)-isomer as
depicted in Scheme 2), are included in Table 1.
Table 1. Measured coupling constants for 27 together with possible torsions
angles of the ‘up’-isomer
Possible torsion anglesa
For each of the two possible configurations of 27, the four
possible combinations of torsion angles in the cyclohexane
ring were included as restraints in a simulated annealing
(SA) protocol, thus yielding eight calculations (Table 2). An
SA protocol was employed to assure that the global
minimum was found for each possible combination of
torsion angles. Only the calculation for one of the eight
possible combinations (1) yielded geometries in accordance
with all experimental observations hereby establishing
the configuration of 27 to be the hydroxylation anti to the
J, (Hz)
Allowedb
Not allowedb
H1000H2000
H1 H200
H200H300
H300H4up
6.9
2.0
4.7
10.7
5.3
1478
768, 1278
368, 2388
21668
08, 21478
21068, 2508
1348, 21368
1618
00
H300H4
2508
358, 1288, 21388
down
a
Possible torsion angles as determined by analysis of Karplus
relationships.
Torsion angles allowed or not allowed by the covalent geometry of the
nucleoside.
b
Table 2. Summary of modelling performed on either configuration of nucleoside 27a
Torsion angle combinationb
EAMBER (kcal/mol)
˚
Sum of restraint violations (A)
‘up’-isomer
1
2
3
4
1
2
3
4
1.4
44
14
24
33
42
36
24
39
56
46
51
63
56
62
92
‘down’-isomer
a
Shown is the torsion angle combination employed as restraints in calculations, the sum of restraint violations and the force field energy (EAMBER) returned in
calculations.
b
00
00
00
00
00
00
00
00
The torsion angle combinations are as follows (given as uH1 H2 , uH2 H3 , uH3 H4 up, uH3 H4 down); up-isomer 1: 768, 368, 21668, 2508; 2: 768, 2388, 21668,
2508; 3: 1278, 368, 21668, 2508; 4: 1278, 2388, 21668, 2508; down-isomer 1: 668, 388, 21598, 2388; 2: 668, 2368, 21598, 2388; 3: 2658, 388, 21598,
2388; 4: 2658, 2368, 21598, 2388.

M. Freitag et al. / Tetrahedron 60 (2004) 3775–3786
3779
100-O-substituent. In the calculations, no constraints were
be potentially used in the construction of similar bicyclic
nucleosides with other nucleobases.
0
0
employed for the furanose ring, so the value of uH1 H2
obtained should serve as to validate the conformation
0
0
determined. Indeed, we observe uH1 H2 ¼1518 which is in
All the bicyclic nucleosides obtained are restricted in S-type
3
0
0
good agreement with the value of 1478 obtained by analysis
0
conformations as indicated by their large JH1 H2 coupling
constants.29,30,42 Thus, both 24 and 27 are locked S-type
mimics, and we determined the pseudorotation angle P of 27
to be 2078 by molecular modelling. Furthermore, the
flexibility of the C40 –C50 bond is indicated to be unchanged
compared to natural nucleosides. The strong restriction in
an 0S-type conformation combined with flexibility of the
C4 –C50 bond is expected to be very favourable in the
preparation of oligonucleotides with strong recognition of
complementary nucleic acids in the formation of B-type
duplexes and triplexes. Thus, the first bicyclic nucleosides
to be incorporated into oligodeoxynucleotides23,24 had the
C40 –C50 bond restricted in unnatural torsion angles and,
hence, the affinity towards complementary nucleic acids
were very dependent on the nucleotide sequences. Further-
more, the flexibility of the C40 –C50 bond in combination
with a locked N-type conformation might be a key factor for
the impressing hybridisation behaviour of LNA. In order to
obtain a bicyclic deoxynucleoside analogue better suited for
B-type duplex formation, the preparation of 20-deoxy-
genated analogues of 24 and 27 is in progress. The
additional hydroxy functionalities on the cyclohexane ring
might, on the other hand, favour duplex formation, and
especially duplex hydration, by their hydrophilic nature.
Thus, the cyclohexane is expected to be positioned at the brim
of the minor groove in a B-type duplex, and hydrophilicity
might be a significant advantage over the rather hydrophobic
bicyclic structures investigated before, e.g., 2, 3, 4 (and its
tricyclic analogue), 6 and others.5,18–21,23–25,29,30 Finally, 24,
27and related compounds of the same[4.3.0]bicyclic skeleton
might be useful molecules for the studies of protein/
nucleoside(tide) interactions.
0
0
0
of the Karplus relationship between JH1 H2 and uH1 H2
(Table 1).
Thus, we have also determined the conformation of the
bicyclic ring system. The furanose ring is locked in a C-30-
exo (S-type) conformation (3E, P¼2078, Fmax¼46.78)1,2
while the cyclohexane ring is found in a very slightly
twisted chair conformation. In our modelling procedure, we
have included no constraints for the g and 1 backbone
torsion angles or for the glycosidic angle, x, as no relevant
experimental information is available. However, to assess if
the nucleoside 27 might be compatible with the standard
genus of backbone angles of right-handed nucleic acid
duplexes, we performed an ab initio geometry optimisation
(HF 3-21G level) with a nucleoside structure where the g, 1
and x torsion angles were initially located in the þsc, ap,
and anti ranges, respectively. As this calculation returned a
geometry optimised structure with the g, 1 and x angles in
identical ranges, it appears that there are no apparent
arguments against incorporating 27 or preferably a 20-deoxy
derivative thereof into a nucleic acid duplex. In Figure 2 is
given a view of 27 obtained from the ab initio geometry
optimisation.
4. Conclusion
Figure 2. Stereoview of the structure of 27 obtained from an ab initio
calculation. Hydrogens of the hydroxyl functionalities have been removed.
In conclusion, we have synthesised 0a bicyclic nucleoside
structure with a functionalised 30,4 -trans linkage and a
locked S-type conformation. We expect this general bicyclic
nucleoside structure to be an important tool in the study of
biological versus conformational behaviour of nucleoside
and nucleotides as well as in the development of
conformationally restricted oligonucleotides with high
affinity for complementary nucleic acids and thereby
potential therapeutic, diagnostic and biotechnological
applications. The construction of other nucleosides based
on the key intermediate 20 is in progress.
3. Discussion
In summary, the fully protected bicyclic nucleoside 20 has
been synthesised in 11 steps and a satisfying 18% overall
yield from diacetone-D-glucose. The general strategy was
only slightly hampered, however, by the demand for
separation of isomers after the benzylation of 9 as well as
after the Grignard reaction giving 12 and 13. Nevertheless,
all RCM reactions afforded the constrained bicyclic
carbohydrate or nucleoside derivatives. The ring strain of
the RCM-generated cyclohexene ring was illuminated by
the acidic ring-opening of the furanose in 15. Simple
deprotection of 20 afforded 24, but the nucleoside 20 is also
a key intermediate towards the construction of other
bicyclic nucleosides e.g., saturated or 20-deoxygenated
derivatives. Alternatively, the olefinic moiety gives the
potential for further derivatisation e.g., mono- or dihydrox-
ylation, amination and/or the attachment of other groups.
Hereby, a plethora of conformationally restricted nucleoside
analogues might be constructed from 20, and herein, we
have shown the first example, the multihydroxylated
bicyclic nucleoside 27. Furthermore, the precursor 18 can
5. Experimental
5.1. General
All commercial reagents were used as supplied. When
necessary, reactions were performed under an atmosphere
of nitrogen. Column chromatography was carried out on
glass columns using silica gel 60 (0.040–0.063 mm). NMR
spectra were recorded on a Varian Gemini 2000 spectro-
1
meter or at a Varian Unity 500 spectrometer. H NMR

3780
M. Freitag et al. / Tetrahedron 60 (2004) 3775–3786
spectra were recorded at 300 or 500 MHz, 13C NMR spectra
were recorded at 75.5 MHz. Values for d are in ppm relative
to tetramethylsilane as internal standard. Fast-atom bom-
bardment mass spectra (FAB-MS) were recorded in positive
ion mode on a Kratos MS50TC spectrometer and MALDI
mass spectra were recorded on an Ionspec Ultima Fourier
Transform mass spectrometer. Microanalyses were per-
formed at The Microanalytical Laboratory, Department of
Chemistry, University of Copenhagen. Assignments of
NMR spectra when given are based on 1H,1H-COSY,
1H,13C-COSY and/or DEPT spectra and follow standard
carbohydrate and nucleoside styl0e; i.e., the carbon atom next
to a nucleobase is assigned C-1 ; in the compounds 9–13,
17–18, the numberin0g0 continues from C-3 to C-10 (20 and 30)
and from C-4 to C-1 (200 and 0300); in 0t0he0n0 ucleoside 19 the
numbering 0c00ontinues from C-3 to C-1 (2 and 300) and from
C-40 to C-1 (2000 and 3000); in compounds 14–15 the carbons
in the cyclohexene ring are numbered from C-4 to C-10, 20,
30 and 40 next to C-3; in the bicyclic nucleosides 20–27 0the
carbons in00the cyclohexene ring are numbered from C-4 to
C-100, 200, 3 and 400 next to C-30. However, compound names
for bi- and tricyclic compounds are given according to the
von Baeyer nomenclature. 1H NOE difference spectra were
recorded for compounds 14, 15, 24 and 27.
The diol 9 (4.66 g, 13.3 mmol) was dissolved in anhydrous
DMF (20 mL) and stirred at 25 8C. A 60% oily dispersion
of NaH (638 mg, 16.0 mmol) was added in small portions.
Benzylbromide (1.90 mL, 16.0 mmol) was added dropwise
and the mixture was stirred at room temperature for 1 h. The
reaction mixture was poured on ice and water (12 mL) and
extracted with ethyl acetate. The combined organic frac-
tions were dried (Na2SO4) and evaporated to dryness under
reduced pressure. The residue was purified by silica gel
column chromatography using petrol ether–ethyl acetate
(3:1) as eluent to give the two products as clear oils.
10: (3.59 g, 61%) (Found: C, 70.85; H, 7.39% C26H32O6
1
requires C, 70.89; H, 7.39%); H NMR (CDCl3) d 7.34–
7.23 (10H, m, 2£Ph), 5.90 (1H, m, H-20), 5,72 (1H, d, J¼
4.2 Hz, H-1), 5.19–5.14 (2H, m, H-30), 4.71, 4.66 (2H, AB
system, J¼11.6 Hz, Bn), 4.62, 4.54 (2H, AB system, J¼
11.8 Hz, Bn), 4.53 (1H, d, J¼4.2 Hz, H-2), 4.30 (1H, dd,
J¼4.5, 11.6 Hz, H-100) 3.91 (1H, dd, J¼9.1, 11.6 Hz, H-100),
3.72, 3.57 (2H, AB system, J¼9.9 Hz, H-5), 2.79 (1H, d0d,
J¼7.2, 15.1 Hz, H-10), 2.49 (1H, dd, J¼6.3, 15.1 Hz, H-1 ),
2.25 (1H, dd, J¼4.5, 9.1 Hz, 100-OH), 1.63 (3H, s, CH3),
1.31 (3H, s, CH3); 13C NMR (CDCl3) d 138.4, 137.9 (Ph),
132.2 (C-20), 128.3, 128.2, 127.7, 127.6, 127.3, 127.1 (Ph),
119.2 (C-30), 112.7 (C(CH3)2), 103.7 (C-1), 88.4, 85.5 (C-3,
C-4), 83.4 (C-2),0 73.7 (Bn), 68.7 (C-5), 66.7 (Bn), 61.5
(C-100), 36.3 (C-1 ), 26.2, 25.8 (C(CH3)2); m/z (FAB) 441
(MþH).
5.1.1. Preparation of 3-C-allyl-3-O-benzyl-4-C-hydroxy-
methyl-1,2-di-O-isopropylidene-a-D-ribofuranose (9).
The furanose 8 (6.08 g, 15.6 mmol) was dissolved in
anhydrous ethyl acetate (150 mL), and H5IO6 (4.26 g,
18.7 mmol) was added. The mixture was stirred at room
temperature for 1.5 h and then filtered through a layer of
celite. The combined filtrates were evaporated to dryness
under reduced pressure, and the residue was dissolved in
anhydrous THF (50 mL). An aqueous solution of formal-
dehyde (4.40 mL, 49.5 mmol, 36% (w/v) containing 10%
MeOH) and an aqueous solution of NaOH (2 M, 17.7 mL,
35.4 mmol) were added dropwise, and the reaction mixture
was stirred at room temperature for 24 h. The mixture was
cooled to 0 8C, NaBH4 (0.88 g, 23.4 mmol) was added, and
the mixture was stirred at room temperature for 1 h. The
mixture was neutralised with 4 M acetic acid and extracted
with ethyl acetate. The combined organic phases were
washed with a saturated aqueous solution of NaHCO3, dried
(MgSO4) and evaporated to dryness under reduced pressure.
The residue was purified by silica gel column chromato-
graphy using petrol ether–ethyl acetate (7:3) as eluent to give
the product as a clear oil (4.67 g, 86%) (Found: C, 64.99; H,
11: (1.26 g, 22%) (Found: C, 70.39; H, 7.36% C26H32O6 1/
1
4H2O requires C, 70.17; H, 7.37%); H NMR (CDCl3) d
7.33–7.23 (10H, m, 2£Ph), 5.95 (1H, m, H-20), 5.71 (1H, m,
H-1), 5.29 5.18 (2H, m, H-30), 4.69 (2H, m, Bn), 4.59–4.45
(3H, m, Ph, H-2), 4.16–4.02 (2H, m, H-100), 3.92–3.80 (2H,
m, H-5), 2.84 (1H, dd, J¼7.3, 15.3 Hz, H-10), 2.58–2.50
(2H, m, H-10, 5-OH), 1.51 (3H, s, CH3), 1.30 (3H,0s, CH3);
13C NMR (CDCl3) d 138.5, 137.9 (Ph), 132.2 (C-2 ), 128.3,
128.1, 127.9, 127.6, 127.2, 127.1 (Ph), 119.4 (C-30), 112.5
(C(CH3)2), 103.6 (C-1), 88.0, 85.1 (C-3, C-4), 83.5 (C-2),
73.7 (Bn), 71.6 (C-100), 66.6 (Bn), 63.6 (C-5), 36.1 (C-10),
26.2, 25.8 (C(CH3)2); m/z (FAB) 463 (MþNa).
5.1.3. Preparation of 3-C-allyl-3,5-di-O-benzyl-4-C-
(1(R)-hydroxyallyl)-1,2-di-O-isopropylidene-a-D-ribo-
furanose (12) and 3-C-allyl-3,5-di-O-benzyl-4-C-(1(S)-
hydroxyallyl)-1,2-di-O-isopropylidene-a-D-ribofuranose
(13). The bis-benzylic ether 10 (4.47 g, 10.1 mmol) was
dissolved in anhydrous CH2Cl2 (250 mL) and PCC (6.56 g,
30.4 mmol) was added. The reaction mixture was stirred at
room temperature for 42 h and diluted with ethyl acetate
(25 mL). After stirring for another 30 min the mixture was
filtered though a layer of silica and the filter was rinsed with
ethyl acetate. The combined filtrates were evaporated to
dryness under reduced pressure and the residue was
dissolved in anhydrous THF (100 mL). The solution was
cooled to 0 8C and a 1 M solution of vinylMgBr in THF
(18.3 mL) was added dropwise. The reaction mixture was
stirred at room temperature for 17 h, poured on ice and
water (50 mL) and neutralised with 4 M acetic acid. The
mixture was concentrated under reduced pressure and
extracted with CH2Cl2. The combined organic fractions
were dried (Na2SO4) and evaporated to dryness under
reduced pressure. The residue was purified by silica gel
1
7.49% C19H26O6 requires C, 65.13; H, 7.48%); H NMR
(CDCl3) d 7.39–7.26 (5H, m, Ph), 5.96 (1H, m, H-20), 5.72
(1H, d, J¼4.2 Hz, H-1), 5.30–5.20 (2H, m, H-30), 4.72, 4.65
(2H, AB system, J¼10.4 Hz, Bn), 4.54 (1H, d, J¼4.3 Hz,
H-2), 4.14 (2H, br s, H-100), 3.86 (2H, br s, H-5), 2.77 (1H, d0d,
J¼8.1, 15.0 Hz, H-10), 2.62 (1H, dd, J¼5.6, 15.0 Hz, H-1 ),
1.65 (3H, s, CH3), 1.32 (3H, s, CH3); 13C NMR (CDCl3) d
138.0 (Ph), 131.9 (C-20), 128.4, 127.7, 127.6, 127.6, 127.4
(Ph), 119.6 (C-30), 112.6 (C(CH3)2), 104.1 (C-1), 87.5, 85.9
(C-30, C-4), 83.1 (C-2), 67.1 (Bn), 63.5, 63.2 (C-5, C-100), 36.7
(C-1 ), 26.1, 25.6 (C(CH3)2); m/z (FAB) 373 (MþNa).
5.1.2. Preparation of 3-C-allyl-3,5-di-O-benzyl-4-C-
hydroxymethyl-1,2-di-O-isopropylidene-a-D-ribofura-
nose (10) and 3-C-allyl-3-O-benzyl-4-C-benzyloxy-
methyl-1,2-di-O-isopropylidene-a-D-ribofuranose (11).

M. Freitag et al. / Tetrahedron 60 (2004) 3775–3786
3781
column chromatography using petrol ether–ethyl acetate
(9:1) as eluent to give the two products as clear oils.
tricyclo[6.4.0.03.7]dodec-10-ene (15). The bis-allylic com-
pound 13 (259 mg, 0.56 mmol) was dissolved in anhydrous
CH2Cl2 (25 mL) and Grubb’s catalyst
A (9.4 mg,
12: (0.79 g, 17%) (Found: C, 72.23; H, 7.51% C28H34O6
1
1.1£1025 mol, 2 mol%) added. The reaction mixture was
stirred at reflux for 24 h. The mixture was evaporated under
reduced pressure and the residue was purified by silica gel
column chromatography using petrol ether–ethyl acetate
(2:1) as eluent to give the product as off-white crystals
requires C, 72.08; H, 7.34%); H NMR (CDCl3) d 7.37–
7.24 (10H, m, 2£Ph), 6.09–5.91 (2H, m, H-20, H-200), 5.81
(1H, d, J¼4.400Hz, H-1), 5.32 (1H, m, H-300), 5.19–5.09 (3H,
m, H-30, H-1 ), 5.00 (1H, m, H-300), 4.75, 4.70 (2H, AB
system, J¼10.9 Hz, Bn), 4.56 (1H, d, J¼4.4 Hz, H-2), 4.43
(2H, br s, Bn), 3.56, 3.53 (2H, AB system, J¼10.7 Hz, H-5),
3.26 (1H, d, J¼1.4 Hz, 100-OH), 2.85 (1H, dd, J¼7.7,
15.1 Hz, H-10), 2.68 (1H, dd, J¼6.1, 15.1 Hz, H-10), 1.63
(3H, s, CH3), 1,33 (3H, s, CH3); 13C NMR (CDCl3) d 138.3,
137.9 (Ph), 136.7, 133.0 (C-20, C-200), 1280.3, 128.2, 127.7,
127.5, 127.4 (Ph), 118.9, 114.2 (C-3 , C-300), 113.2
(C(CH3)2), 104.1 (C-1), 89.7 86.0 (C-3, C-4), 83.6 (C-2),
73.5 (Bn), 71.3 (C-100), 69.8 (C-5), 67.1 (Bn), 37.3 (C-10),
26.1, 26.0 (C(CH3)2); m/z (FAB) 489 (MþNa).
1
(228 mg, 93%); H NMR (CDCl3) d 7.39–7.21 (10H, m,
2£Ph), 6.12 (1H, d, J¼5.0 Hz, H-1), 6.02 (1H, m, H-20),
5.70 (1H, m, H-30), 4.94 (1H, d, J¼5.1 Hz, H-2), 4.66, 4.39
(2H, AB system, J¼10.0 Hz, Bn), 4.51, 4.44 (2H, AB
system, J¼11.9 Hz, Bn), 4.18 (1H, d, J¼10.9 Hz, 10-OH),
3.83 (1H, m, H-10), 3.44, 3.39 (2H, AB sys0tem, J¼10.3 Hz,
H-5), 2.96 (1H, dd, J¼5.5, 18.6 Hz, H-4 ), 2.21 (1H, m,
40-H), 1.43 (3H, s, CH3), 1.35 (3H, s, CH3); 13C NMR
(CDCl3) d 137.7, 137.2 (Ph), 132.6 (C-20), 128.5, 128.4,
127.9, 127.8, 127.6 (Ph), 123.7 (C-30), 115.8 (C(CH3)2),
105.8 (C-1), 90.6, 82.03 (C-3, C-4), 84.9 (C-2), 75.8 (C-5),
73.9 (Bn), 68.7 (C-1 ), 67.1 (Bn), 29.1 (C-40), 27.4, 26.7
(C(CH3)2); m/z (FAB) 461 (MþNa).
13: (2.95 g, 63%) (Found: C, 72.01; H, 7.46% C28H34O6
1
requires C, 72.08; H, 7.34%); H NMR (CDCl3) d 7.40–
7.22 (10H, m, 2£Ph), 6.24 (1H, m, H-200), 6.04 (1H, m,
H-2000), 5.81 (1H, d, J¼4.0 Hz, H-1), 5.41–5.13 (5H, m, H-100,
H-3 , H-30), 4.82, 4.72 (2H, AB system, J¼10.3 Hz, Bn),
4.52 (1H, d, J¼4.4 Hz, H-2), 4.52, 4.45 (2H, AB system,
J¼11.9 Hz, Bn), 3.88, 3.68 (2H, AB system, J¼11.4 Hz,
H-5), 3.30 (1H, d, J¼1.6 Hz, 100-OH), 2.90 (1H, dd,0J¼7.8,
15.0 Hz, H-10), 2.78 (1H, dd, J¼6.0, 15.0 Hz, H-1 ), 1.60
(3H, s, CH3), 1.34 (3H, s, CH3); 13C NMR (CDCl3) d 138.2,
137.8 (Ph), 133.1 (C-20), 128.5,0128.2, 127.8, 127.7, 127.6,
127.4 (Ph), 118.8, 114.6 (C-3 , C-300), 112.9 (C(CH3)2),
104.7 (C-010 ), 88.8, 87.0 (C-3, C-4), 83.7 (C-2), 73.7 (Bn),
72.3 (C-1 ), 68.8 (C-5), 67.5 (Bn), 37.9 (C-10), 26.3, 26.1
(C(CH3)2); m/z (FAB) 489 (MþNa).
5.1.6. Preparation of 3-acetyloxy-5-acetyloxymethylcar-
bonyl-5-benzyloxy-4-benzyloxymethyl-4-hydroxycyclo-
hexene (16). The alcohol 15 (94.8 mg, 0.22 mmol) was
dissolved in an 80% aqueous solution of acetic acid
(1.0 mL). The reaction mixture was stirred at 90 8C for 6 h
and then concentrated under reduced pressure. The residue
was co-evaporated with anhydrous ethanol, toluene and
pyridine and then redissolved in anhydrous pyridine
(0.4 mL). Acetic anhydride (0.33 mL, 3.49 mmol) was
added dropwise and the reaction mixture was stirred at
room temperature for 48 h and then quenched by the
addition of ice and water (2 mL). The mixture was extracted
with CH2Cl2 and the combined organic phases were washed
with a saturated aqueous solution of NaHCO3, dried
(MgSO4) and evaporated to dryness under reduced pressure.
The residue was purified by silica gel chromatography using
petrol ether–ethyl acetate (4:1) as eluent to give the product
as a clear oil (48.9 mg, 47%) (Found: C, 66.18; H, 6.26%
5.1.4. Preparation of (1S,3R,7R,8S,12R)-8-benzyloxy-1-
benzyloxymethyl-5,5-dimethyl-12-hydroxy-2,4,6-trioxa-
tricyclo[6.4.0.03.7]dodec-10-ene (14). The bis-allylic
compound 12 (53.5 mg, 0.12 mmol) was dissolved in
anhydrous CH2Cl2 (5 mL) and Grubb’s catalyst
A
(2.0 mg, 2.4£1026 mol, 2 mol%) added. The reaction
mixture was stirred at reflux for 48 h, another 1 mol% of
the catalyst was added and the mixture was stirred for
another 48 h. The mixture was evaporated under reduced
pressure and the residue was purified by silica gel column
chromatography using petrol ether–ethyl acetate (2:1) as
eluent to give the product as off-white crystals (39.9 mg,
76%) (Found: C, 70.87; H, 7.18% C26H30O6 1/4H2O
C27H30O8 1/2H2O requires C, 65.98; H, 6.36%); H NMR
1
(CDCl3) d 7.41–7.30 (10H, m, 2£Ph), 5.83–5.78 (2H, m,
H-1, H-2), 5.34 (1H, br s, H-3), 5.26, 5.09 (2H, AB system,
J¼17.5 Hz, 4-CH2), 4.52–4.42 (4H, m, Bn), 3.45, 3.35 (2H,
AB system, J¼10.1 Hz, 5-COCH2O), 3.37 (1H, s, 4-OH),
2.79–2.53 (2H, m, H-6), 2.15 (3H, s, COCH3), 2.06 (3H,
s, COCH3); 13C NMR (CDCl3) d 203.3 (5-CO), 170.6
(CH3CO), 170.2 (CH3CO), 137.6, 137.1, 128.3, 128.2,
127.7, 127.7, 127.7, 127.5, 127.4 (Ph), 126.1 (C-2), 124.9
(C-1), 83.8, 75.6 (C-4, C-5), 73.7 (Bn), 70.6 (COCH2O),
69.4 (C-3), 68.1 (4-CH2O), 66.1 (Bn), 26.9 (C-6), 21.0
(CH3CO), 20.5 (CH3CO); m/z (FAB) 505 (MþNa).
1
requires C, 70.49; H, 6.83%); H NMR (CDCl3) d 7.37–
7.15 (10H, m, 2£Ph), 6.03 (1H, d, J¼5.0 Hz, H-1), 5.69–
5.58 (2H, m, H-20, H-30), 5.26 (1H, br s, H-10), 4.99 (1H, d,
J¼5.0 Hz, H-2), 4.73, 4.37 (2H, AB system, J¼10.7 Hz,
Bn), 4.53, 4.44 (2H, AB system, J¼11.9 Hz, Bn), 3.82, 3.73
(1H, AB system, J¼11.0 Hz, H-5), 2.86 (1H, dd, J¼4.0,
18.3 Hz, H-40), 2.38 (1H, br s, OH), 2.24 (1H, m, H-40), 1.34
(3H, s, CH3), 1.31 (3H, s, CH3); 13C NMR (CDCl3) d 139.1,
137.7 (Ph), 132.5, 124.9 (C-20, C-30), 128.4, 127.9, 127.7,
127.5, 127.0, 126.8 (Ph), 115.6 (C(CH3)2), 106.0 (C-1),
92.6, 82.2 (C-3, C-4), 85.5 (C-2), 74.0 (Bn), 73.4 (C-5), 73.0
(C-10), 67.1 (Bn), 29.2 (C-40), 27.6, 26.5 (C(CH3)2).
5.1.7. Preparation of 3-C-allyl-3,5-di-O-benzyl-4-C-
(1(S)-benzoyloxy)allyl-1,2-di-O-isopropyliden-a-D-ribo-
furanose (17). The alcohol 13 (3.73 g, 7.99 mmol) was
dissolved in anhydrous pyridine (30 mL) and cooled to 0 8C.
Benzoyl chloride (2.78 mL, 24.0 mmol) was added, and the
reaction mixture was stirred at room temperature for 21 h.
Another portion of benzoyl chloride (0.93 mL, 8.0 mmol)
was added, and the reaction mixture was stirred for another
4 h and then concentrated under reduced pressure. The
residue was dissolved in CH2Cl2 and extracted with a
5.1.5. Preparation of (1S,3R,7R,8S,12S)-8-Benzyloxy-1-
benzyloxymethyl-5,5-dimethyl-12-hydroxy-2,4,6-trioxa-

3782
M. Freitag et al. / Tetrahedron 60 (2004) 3775–3786
saturated aqueous solution of NaHCO3 and brine. The
combined organic phases were dried (MgSO4) and evapo-
rated to dryness under reduced pressure. The residue was
purified by silica gel chromatography using petrol ether–
ethyl acetate (9:1) as eluent to give the product as a clear oil
(4.48 g, 98%) (Found: C, 73.35; H, 6.74% C35H38O7
(20 mL) and the mixture was extracted with CH2Cl2. The
combined organic phases were washed with a saturated
aqueous solution of NaHCO3 and brine, dried (MgSO4) and
evaporated to dryness under reduced pressure. The residue
was purified by silica gel chromatography using petrol
ether–ethyl acetate (4:1) as eluent to give the product as a
white solid (1.05 g, 93%) (Found: C, 68.45; H, 6.13; N,
3.91% C39H40O9N2 requires C, 68.81; H, 5.92; N, 4.12%);
1H NMR (CDCl3) d 8.60 (1H, br s, NH), 8.00–7.98 (m, 2H,
Ph), 7.71 (1H, d, J¼0.9 Hz, H-6), 7.64–7.59 (1H, m, Ph),
7.50–7.26 (m, 12H, Ph), 6.43 (1H, d, J¼8.5 Hz, H-10), 6.22
(1H, m, H-1000), 60.002 (1H, ddd, J¼17.6, 11.1, 4.1 Hz, H-2000),
5.85 (1H, m, H-200), 5.88 (1H, d, J¼8.5 Hz, H-20), 5.21–4.59
(6H, m, Ph, H-3 , H-3000), 4.75, 4.67 (2H, AB system, J0¼
11.5 Hz, Bn), 4.01, 3.95 (2H, AB system, J¼11.0 Hz, H-5 ),
2.98 (1H, dd, J¼16.0, 6.5 Hz, H-100), 2.83 (1H, dd, J¼16.0,
1
requires C, 73.66; H, 6.71%); H NMR (CDCl3) d 7.77–
7.74 (2H, m, Ph), 7.49–7.22 (9H, m, 2£Ph), 7.09–6.97 (4H,
m, 2£Ph), 6.63 (1H, m, H-100), 6.35 (1H, ddd, J¼17.4, 11.0,
3.6 Hz, H-200), 5.91 (1H, m, H-20), 5.90 (1H, d, J¼4.6 Hz,
H-1), 5.20–5.01 (4H, m, H-30, H-300), 4.71, 4.54 (2H, AB
system, J¼10.9 Hz, Bn), 4.63–4.47 (2H, m, Bn), 4.56 (1H,
d, J¼4.6 Hz, H-2), 4.04, 3.85 (2H, AB system, J¼10.8 Hz,
H-5), 2.85 (1H, dd,0 J¼15.6, 7.7 Hz, H-10), 2.73 (1H, dd,
J¼15.6, 5.8 Hz, H-1 ), 1.65 (3H, s, CH3), 1.36 (3H, s, CH3);
13C NMR (CDCl3) d 164.6 (CO), 138.0, 137.9 (Ph), 134.4
(C-200), 133.6 (C-20), 132.3, 130.3, 129.6, 128.3, 127.9,
127.7, 127.7, 127.6, 127.3, 126.8 (Ph), 118.3, 115.0 (C-30,
C-300), 113.5 (C(CH3)2), 104.8 (C-1), 88.7, 85.6 (C-3, C-4),
85.0 (C-2), 73.9 (Bn), 73.4 (C-100), 70.1 (C-5), 67.0 (Bn),
37.2 (C-10), 26.6, 26.4 (C(CH3)2); m/z (FAB) 593 (MþH).
7.6 Hz, H-100), 2.09 (3H, s, CH3CO), 1.41 (3H, s, CH3); 13
C
NMR (CDCl3) d 169.8 (CH3CO), 163.8, 163.6 (C-2, CO),
150.9 (C-4), 137.7, 136.5, 136.0 (Ph), 133.2, 132.9, 132.7
(C-6, C-200, C-2000), 129.7, 129.6, 128.8, 128.5, 128.3, 128.3,
127.9, 127.7, 127.4 (Ph), 118.4, 116.4 (C-300, C-3000)0, 111.2
(C-5), 89.3, 83.2 (C-30, C-40), 83.6 (C-10), 78.0 (C-2 ), 73.8
(Bn), 73.2 (C-1000), 70.7 (C-50), 68.0 (Bn), 34.9 (C 100), 20.8
(CH3CO), 11.8 (CH3); m/z (FAB) 703 (MþNa).
5.1.8. Preparation of 1,2-di-O-acetyl-3-C-allyl-3,5-di-O-
benzyl-4-C-(1(S)-benzoyloxy)allyl-(a/b)-D-ribofuranose
(18). The isopropylidene protected furanose 17 (4.48 g,
7.85 mmol) was dissolved in 80% aqueous acetic acid
(38 mL) and stirred for 90 min. at 90 8C. The reaction
mixture was concentrated under reduced pressure and co-
evaporated with anhydrous ethanol, toluene and pyridine.
The residue was redissolved in anhydrous pyridine (20 mL)
and acetic anhydride (11 mL) was added dropwise. The
reaction mixture was stirred at room temperature for 48 h
and the reaction was then quenched by the addition of ice
and water. The mixture was extracted with CH2Cl2 and the
combined organic phases were washed with a saturated
aqueous solution of NaHCO3, dried (MgSO4) and evapo-
rated to dryness under reduced pressure. The residue was
purified by silica gel chromatography using CH2Cl2–
methanol (99.5:0.5) as eluent to give the product as a
mixture of anomers (4.46 g, 92%; b:a ,5:1) (Found: C,
70.68; H, 6.08% C36H38O9 requires C, 70.34; H, 6.23%); 1H
NMR (CDCl3) d 7.95–7.92 (m), 7.57–7.52 (m), 7.45–7.22
(m), 6.42 (d, J¼5.2 Hz), 6.30 (d, J¼3.9 Hz), 6.28–5.86 (m),
5.58 (d, J¼5.1 Hz), 5.21–4.56 (m), 4.01–3.96 (m), 3.77–
3.69 (m), 2.91–2.81 (m), 2.07–1.93 (m); 13C NMR (CDCl3)
d (for the major b anomer) 169.9, 169.4 (CH3CO), 164.1
(CO), 138.0, 137.6 (Ph), 133.0, 129.6, 128.4, 128.3, 128.1,
127.7, 127.5, 127.3, 127.3 (Ph, C-20, C-200), 118.3, 116.7
(C-30, C-300), 98.7 (C-1), 90.9, 84.9 (C-3, C-4), 79.8 (C-2),
74.0, 73.7, 70.5, 67.5 (Ph, C-5, C-100), 35.8 (C-10), 21.1, 20.9
(C(CH3)2); m/z (FAB) 637 (MþH).
5.1.10. Preparation of (1S,5S,6S,8R,9R)-9-acetyloxy-5-
benzoyloxy-1-benzyloxy-6-benzyloxymethyl-8-(thymin-
1-yl)-7-oxabicyclo[4.3.0]non-3-ene (20). A solution of the
protected nucleoside 19 (1.01 g, 1.48 mmol) in anhydrous
ClCH2CH2Cl (15 mL) was added the precatalyst A (19 mg,
22 mmol, 2 mol%) and the reaction mixture was stirred at
reflux for 25 h and then concentrated under reduced
pressure. The residue was purified by silica gel chromato-
graphy using CH2Cl2–methanol (99:1) as eluent to give the
product as an off-white solid (873 mg, 90%) (Found: C,
67.97; H, 5.72; N, 4.30% C37H36O9N2 requires C, 68.09; H,
1
5.56; N, 4.29%); H NMR (CDCl3) d 8.59 (1H, m, NH),
7.80 (1H, s, H-6), 7.55–7.31 (13H, m, Ph) 7.10–7.04 (2H,
m, Ph), 6.71 (1H, d, J¼7.4 Hz, H-10), 060 .17 (1H, d, J¼7.4 Hz,
H-20), 6.00–5.96 (2H, m, H-200, H-3 ), 5.32 (1H, s, H-100),
4.78 (1H, d, J¼9.1 Hz, Bn), 4.65 (2H, s, Bn), 4.32 (1H, d,
J¼9.1 Hz, Bn), 3.81, 3.67 (2H, AB system, J¼10.8 Hz,
H-50), 2.96 (1H, dd, J¼18.9, 4.6 Hz, H-400), 2.47 (1H, d, J¼
18.9 Hz, H-400), 2.14 (3H, s, CH3CO), 1.54 (3H, s, CH3); 13
C
NMR (CDCl3) d 170.6 (CH3CO), 166.6, 163.6 (C-2, CO),
150.8 (C-4), 137.9, 136.4, 136.0 (Ph), 132.6, 129.8, 129.5,
128.8, 128.6, 128.5, 128.3, 128.1, 127.9, 127.8, 127.4, 1260.5
(Ph, C-200, C-300, C-6), 110.7 (C-5), 87.0, 80.8 (C 30, C-4 ),
86.90(0 C-10), 78.3 (C-20), 74.7, 73.9 (Ph, C 50), 67.8 (Bn), 67.1
(C-1 ), 27.7 (C-400), 20.8 (CH3CO), 12.1 (CH3); m/z
(MALDI) 675 (MþNa).
5.1.9. Preparation of 1-(2-O-acetyl-3-C-allyl-3,5-di-O-
benzyl-4-C-(1(S)-benzoyloxy)allyl-b-D-ribofuranosyl)-
thymine (19). A solution of the bis-acetate 18 (1.02 g,
1.65 mmol) and thymine (417 mg, 3.30 mmol) in anhydrous
CH3CN (17 mL) was stirred at room temperature and
N,O-bis(trimethylsilyl)acetamide (2.05 mL, 8.26 mmol)
was added dropwise. The reaction mixture was stirred at
reflux for 30 min. and cooled to 0 8C. TMS-OTf (0.51 mL,
2.81 mmol) was added dropwise and the reaction mixture
was stirred at 40 8C for 17 h. The reaction was quenched by
the addition of ice-cold aqueous solution of NaHCO3
5.1.11. Preparation of (1S,5S,6S,8R,9R)-5-benzoyloxy-1-
benzyloxy-6-benzyloxymethyl-9-hydroxy-8-(thymin-1-
yl)-7-oxabicyclo[4.3.0]non-3-ene (21). A solution of 20
(301 mg, 0.46 mmol) in methanol (13 mL) was added
NaOMe (37.0 mg, 0.69 mmol) and the reaction mixture
was stirred at room temperature for 3 h. The reaction
mixture was concentrated under reduced pressure. The
residue was purified by silica gel chromatography using
petrol ether–ethyl acetate (2:3) as eluent to give the product
as a white solid (243 mg, 87%) (Found: C, 68.21; H, 5.67;
N, 4.67% C35H34O8N2·1/4H2O requires C, 68.34; H, 5.65;

M. Freitag et al. / Tetrahedron 60 (2004) 3775–3786
3783
1
N, 4.55%); H NMR (CDCl3) d 9.39 (1H, br s, NH), 7.73
OH), 3.43, 3.41 (2H, AB system, J¼11.1 Hz, H-50), 3.07
(1H, dd, J¼18.6, 5,4 Hz, H-400), 2.15 (1H, d, J¼18.6 Hz,
H-400), 1.80 (3H, s, CH3); 13C NMR (CDCl3) d 164.1, 152.2
(1H, s, H-6), 7.65 (2H, d, J¼7.6 Hz, Ph), 7.51–7.23 (m,
11H, Ph),007.11 (2H, t, J¼7.7 Hz, Ph), 6.02–5.94 (2H, m,
H-200, H-3 ), 5.95 (1H, d, J¼5.4 Hz, H-10), 5.63 (1H, d, J¼
3.1 Hz, H-100),0 5.15 (1H, d, J¼9.5 Hz, Bn), 4.85 (1H, d,
J¼5.7 Hz, H-2 ), 4.71 (1H, br s, 20-OH), 4.51, 4.44 (2H, AB
system, J¼11.5 Hz, Bn), 4.33 (1H, d, J¼9.5 Hz, Bn), 3.61,
3.55 (2H, AB system, J¼10.8 Hz, H-50), 3.14 (1H, dd,
J¼18.8, 4.5 Hz, H-400), 2.23 (1H, d, J¼18.8 Hz, H-400), 1.78
(s, 3H, CH3); 13C NMR (CDCl3) d 166.7, 164.0 (C-2, CO),
152.1 (C-4), 138.9, 136.5, 136.4 (Ph), 132.6, 129.8, 128.6,
128.2, 128.2, 128.1, 127.4, 127.3,0 126.7 (Ph, C-200,0 C-3000),
129.9 (C-6), 109.7 (C-5), 94.6 (C-1 ), 88.4, 81.9 (C-3 , C 4 ),
81.2 (C-2000), 73.9 (Bn), 73.3 (C-50), 67.7 (Bn), 67.2 (C 100),
27.6 (C-4 ), 12.4 (CH3); m/z (FAB) 611 (MþH).
00
(C-1, C-4), 137.5 (Ph), 136.5, 136.4 (Ph, C-6), 131.9 (C 2 ),
00
128.5, 128.5, 128.4, 128.2, 128.0, 127.30(Ph),0 123.1 (C-3 ),
0
109.6 (C-5), 94.9 (C-10), 88.5, 84.5 (C-3 , C040 ), 82.0 (C 2 ),
73.8 (Bn), 72.9 (C-50), 68.2, 68.1 (Ph, C-1 ), 27.7 (C-400),
12.4 (CH3); m/z (FAB) 507 (MþH).
5.1.14. Preparation of (1S,5S,6S,8R,9R)-1,5,9-tri-
hydroxy-6-hydroxymethyl-8-(thymin-1-yl)-7-oxabicyclo-
[4.3.0]non-3-ene (24). Method A: A solution of 22
(35.0 mg, 0.8 mmol) in methanol (2.3 mL) was added
NaOMe (6.50 mg, 0.12 mmol) and stirred at room tempera-
ture for 1 h. An additional amount of NaOMe (9.00 mg,
0.17 mmol) was added and the reaction mixture was stirred
at 50 8C for 2 h. The reaction mixture was concentrated
under reduced pressure and the residue was purified by silica
gel chromatography using CH2Cl2–methanol (9:1) as eluent
to give the product as a white solid (12.6 mg, 48%).
5.1.12. Preparation of (1S,5S,6S,8R,9R)-5-benzoyloxy-
1,9-dihydroxy-6-hydroxymethyl-8-(thymin-1-yl)-7-oxa-
bicyclo[4.3.0]non-3-ene (22). A solution of 21 (91.0 mg,
0.15 mmol) in anhydrous CH2Cl2 (2.50 mL) was stirred at
278 8C. A solution of BCl3 in hexane (1 M, 0.40 mL,
0.40 mmol) was added dropwise and the reaction mixture
was stirred at 278 8C for 4 h. An additional amount of BCl3
(0.40 mL, 0.40 mmol) was added and the reaction mixture
was allowed to reach room temperature and stirred for
another 16 h. The reaction was quenched by the addition of
methanol (2 mL) and water (0.1 mL) and the mixture was
stirred for 1 h and concentrated under reduced pressure. The
residue was purified by silica gel chromatography using
CH2Cl2–methanol (19:1) as eluent to give the product as
a white solid as well as the debenzoylated product 24
(10.5 mg, 21%).
Method B: A solution of 23 (22.5 mg, 0.045 mmol) in
anhydrous CH2Cl2 (0.90 mL) was stirred at 278 8C. A
solution of BCl3 in hexane (1 M, 0.20 mL, 0.20 mmol) was
added dropwise and the reaction mixture was stirred at
278 8C for 4 h. An additional amount of BCl3 (0.15 mL,
0.15 mmol) was added and the reaction mixture was
allowed to reach room temperature and stirred for another
16 h. The reaction was quenched by the addition of
methanol (0.7 mL) and water (0.1 mL) and the mixture
was stirred for 1 h and concentrated under reduced pressure.
The residue was purified by silica gel chromatography using
CH2Cl2–methanol (9:1) as eluent to give the product as a
1
1
22: (35.6 mg, 55%); H NMR (CDCl3) d 9.85 (1H, br s,
white solid (10.2 mg, 69%); H NMR (DMSO-d6) d 11.31
NH), 8.02 (2H, d, J¼7.300Hz, Ph), 7.56–7.38 (4H, m, H-6),
6.07–5.96 (2H, m, H-2 , H-300), 50.76 (1H, d, J¼4.1 Hz,
H-100), 5.67 (1H, d, J¼6.7 Hz, H-1 ), 4.94 (1H, m, H-20),
4.26 (1H, br s, 20 OH), 3.93–3.59 (4H, m, H-50, 50-OH,
30-OH), 2.58 (1H, dd, J¼17.6, 4.2 Hz, H-400), 2.34 (1H, d,
J¼17.6 Hz, H-400), 1.75 (3H, s, CH3); 13C NMR (CDCl3) d
165.9, 164.2 (C-2, CO), 151.4 (C-4), 139.5 (C-6), 133.3,
129.8, 129.7, 129.6, 128.5 (Ph), 130.6, 0125.2 (C-200, C-3000),
110.6 (C-050 ), 95.4 (C-10), 86.4, 77.2 (C-3 , C-40), 75.0 (C-2 ),
67.4 (C-1 ), 64.7 (C-50), 32.9 (C-400), 12.2 (CH3); m/z (FAB)
431 (MþH).
(1H, br s, NH), 8.17 (1H, br s, H-6), 6.10 (1H, d, J¼7.4 Hz,
H-10), 5.81 5.71 (3H, m, H-200, H-3000, 30-OH), 5.48 (1H, br s,
50-OH), 5.29 (1H, d, J¼6.3 Hz, 2 -OH), 5.10 (1H, d, J¼
8.9 Hz, 100-OH), 4.64 (1H, t, J¼6.7 Hz, H-20), 3.76 (1H, m,
H-100), 3.56–3.35 (2H, m, H-50), 2.32–2.26 (2H, m, H-400),
1.79 (3H, s, CH3); 13C NMR (DMSO-d6) d 163.6, 151.4
(C-2, C-4), 137.4 (C-6), 129.9, 126.2 (C-200, C-300), 1090.2
(C-5), 88.1 (C-10), 850.1, 78.3 (C-30, C-40), 74.3 (C-2 ),
67.1 (C-100), 64.9 (C-5 ), 32.4 (C-400), 12.2 (CH3); HiRes
MALDI FT-MS m/z (MþNa) found/calcd 349.1000/
349.1006.
5.1.13. Preparation of (1S,5S,6S,8R,9R)-1-benzyloxy-6-
benzyloxymethyl-5,9-dihydroxy-8-(thymin-1-yl)-7-oxa-
bicyclo[4.3.0]non-3-ene (23). A solution of 20 (250 mg,
0.38 mmol) in methanol (11 mL) was added NaOMe
(41 mg, 0.76 mmol) and stirred at reflux for 24 h. An
additional amount of NaOMe (41 mg, 0.76 mmol) was
added and the reaction mixture was stirred at reflux for
another 24 h. The reaction mixture was concentrated under
reduced pressure and the residue was purified by silica gel
chromatography using petrol ether–ethyl acetate (1:3) as
eluent to give the product as a white solid (133 mg, 69%);
1H NMR (CDCl3) d 9.78 (1H, br s, NH), 7.82 (1H, d,
J¼1.3 Hz, H-6), 7.38–7.16 (10H, m, Ph), 6.06 (1H0,0 m, H-
200), 5.89 (1H, d, J¼5.3 Hz, H-10), 5.81 (1H, m, H-3 ), 5.10
(1H, d, J¼9.4 Hz, Bn), 4.92 (1H, br s, OH), 4.78 (1H, d,
J¼5.3 Hz, H-20), 4.43, 4.33 (2H, AB system, J¼16.7 Hz,
Bn), 4.31 (1H, d, J¼9.4 Hz, Bn), 4.06–3.99 (2H, m, H-100,
5.1.15. Preparation of (1S,3R,4R,5S,6S,8R,9R)-9-acetyl-
oxy-5-benzoyloxy-1-benzyloxy-6-benzyloxymethyl-3,4-
dihydroxy-8-(thymin-1-yl)-7-oxabicyclo[4.3.0]nonane
(25). A solution of 20 (640 mg, 0.980 mmol) in a mixture of
THF and water (1:1, 10 mL) was added N-methylmorpho-
line-N-oxide (172 mg, 1.47 mmol) and a 2.5% solution of
OsO4 in tert-butanol (0.49 mL, 0.039 mmol). The reaction
mixture was stirred at 50 8C for 7 h and then quenched by
the addition of a saturated aqueous solution of NaHSO3
(3 mL). The mixture was stirred for 30 min at room
temperature and then partly concentrated under reduced
pressure. The aqueous residue was extracted with ethyl
acetate, and the combined organic extracts were dried
(Na2SO4). The residue was purified by silica gel chroma-
tography using CH2Cl2–methanol (49:1) as eluent to give
the product as a white solid and a (16:1)-mixture of
stereoisomers (330 mg, 49%), in addition to a side product

3784
M. Freitag et al. / Tetrahedron 60 (2004) 3775–3786
00
25a as a white solid (71 mg, 10%) and unreacted starting
material 20 (208 mg, 33%).
J¼6.5, 14.5 Hz, H-4
), 1.62 (1H, dd, J¼8.6, 14.5 Hz,
down
00
H-4 ), 1.58 (3H, s, CH3); 13C NMR (DMSO-d6) d 163.6
up
(C-4), 151.4 (C-2), 138.3, 137.9, 136.7 (C-6, Ph), 128.5,
128.4, 128.3, 127.7, 1027.6, 0127.2 (Ph), 109.3 (C-5), 88.7
(C-10), 85.8, 85.3 (C-3 , C-040 ), 76.3, 740.05, 74.4, 73.1, 72.5,
66.6, 64.4 (C-20, C-50, C-1 , C-200, C-3 , Bn), 29.4 (C-400),
11.9 (CH3); m/z (MALDI) 563 (MþNa).
1
25: H NMR (CDCl3) d (Major isomer) 8.30 (1H, s, NH),
7.81 (1H, s, H-6), 7.81–7.79 (2H, 0m, Ph), 7.54–7.24 (13H,
m, Ph), 6.68 (1H, d, J¼7.7 Hz, H-1 ), 6.17 (1H, d, J¼7.7 Hz,
H-20), 4.99 (1H, d, J¼10.1 Hz, B0n0 ), 4.78 (1H, d, J¼11.6 Hz,
Bn), 4.65 (1H, d, J¼4.5 Hz, H-1 ), 4.61 (1H, d, J¼11.6 Hz,
Bn), 4.54 (1H, d, J¼11.1 Hz, H-50), 4.40 (1H, d, J¼10.1 Hz,
Bn), 4.38 (1H, m, H-300), 4.33 (1H, m, H-200), 3.88 (1H, br s,
5.1.17. Preparation of (1S,3R,4R,5S,6S,8R,9R)-6-hydroxy-
methyl-1,3,4,5,9-pentahydroxy-8-(thymin-1-yl)-7-oxabi-
cyclo[4.3.0]nonane (27). A degassed solution of 26 (19 mg,
36 mmol) in anhydrous methanol (3 mL) was added 20%
Pd(OH)2/C (9 mg, 12 mmol) and stirred at room tempera-
ture. The mixture was bubbled with a stream of hydrogen
for 10 min and then stirred under an atmosphere of hydro-
gen for 24 h. The reaction mixture was filtered through a
layer of celite which was rinsed with methanol. The residue
was concentrated under reduced pressure to give the product
OH), 3.84 (1H, d, J¼11.1 Hz, H-50), 3.31 (1H, br s, OH),
00
3.01 (1H, dd, J¼9.1, 15.7 Hz, H-4
), 2.16 (1H, d, J¼
down
00
15.7 Hz, H-4 ), 2.11 (3H, s, CH3CO), 1.50 (3H, s, CH3);
up
13C NMR (CDCl3) d 170.2 (CH3CO), 167.8, 163.5
(C-4, CO), 150.8 (C-2) 137.3, 136.4, 133.3, 129.9, 129.3,
128.7, 128.5, 128.4, 128.2, 127.9, 1207.6, 127.5 (C-6, Ph),
110.9 (C-5), 87.1, 86.9, 82.7 (C-1 , C-30, C-40), 78.2,
77.8 (C-20, C-100) 73.8, 73.7, 73.5, 67.4, 65.5 (C-50, C-200,
C-300, Bn), 29.8 (C-400), 20.8 (CH3CO), 12.0 (CH3); HiRes
MALDI FT-MS m/z (MþNa) found/calcd 709.2350/
709.2368.
1
as a white solid (12.1 mg, 95%); mp 168–72 8C; H NMR
(DMSO-d6) d 11.28 (1H, br s, NH), 8.09 (1H, s, H-6), 6.20
(1H, br s, 30-0O0 H), 6.02 (1H, d, J¼7.5 Hz, H-10), 5.45 (1H, d,
J¼7.8 Hz, 1 -OH), 5.27 (1H, br s, 20-OH), 4.97 (1H, br s,
5000-OH), 4.90 (1H, br s, 200-OH), 4.63–4.59 (2H, m, H-20,
3 -OH), 4.25 (1H, m, H-300), 3.98 (1H, d, J¼12.0 Hz, H-50),
3.86 (1H, d, J¼3.5 Hz, H-200), 3.72 (1H, d, J¼6.1 Hz, H-100),
3.63 (1H, dd,00J¼3.9, 12.0 Hz, H-50), 1.96 (1H, dd, J¼5.5,
12.7 Hz, H-4down), 1.78 (3H, s, CH3), 1.44 (1H, t, J¼
12.7 Hz, H-4u00p); 13C NMR (DMSO-d6) d 163.8 (C-4), 151.6
(C-2), 137.7 (C-6), 109.0 (C-5), 89.2 (C-10), 84.5, 81.2
(C-30, C-40), 74.1, 73.9, 73.4, 64.4, 63.3 (C-20, C-50, C-100,
C-200, C-300), 33.3 (C-400), 12.3 (CH3); 1H NMR (CD3OD) d
8.07 (1H, d, J¼1.3 Hz, H-6), 6.05 (1H, d, J¼6.9 Hz, H-10),
4.71 (1H, d, J¼6.9 Hz, H-20), 4.44 (1H, m, H-300), 4.09 (1H,
dd, J¼2.0, 4.7 Hz, H-200), 4.05 (1H, d, J¼2.0 Hz, H-100), 3.99
(1S,3R,4R,5S,6S,8R,9R)-9-Acetyloxy-5-benzoyloxy-1-
benzyloxy-6-benzyloxymethyl-3,4-dihydroxy-8-(5,6-
dihydroxypyrimidine-2,4-dion-1-yl)-7-oxabicyclo-
[4.3.0]nonane 25: 1H NMR (CDCl3) d (Major isomer)
7.82–7.78 (3H, m, Ph, NH), 7.52–7.21 (13H, m, Ph), 6.50
(1H, d, J¼8.0 Hz, H-10), 6.10 (1H, d, J¼8.0 Hz, H-20), 5.51
(1H, d, J¼2.2 Hz, H-6), 4.99 (1H, d, J¼10.5 Hz, Bn), 4.71
(1H, d, J¼11.0 Hz, Bn), 4.59 (1H, d, J¼4.3 Hz, H-1000), 4.51
(1H, d, J¼11.0 Hz, Bn), 4.43 (2H, d, J¼11.3 Hz, H-5 ), 4.40
(1H, m, H-300), 4.39 (1H, d, J¼10.5 Hz, Bn), 4.28 (1H, m,
H-200), 4.02 (1H, d, J¼2.8 Hz, 200-OH), 3.71 (1H, d, J¼
2.2 Hz, 6-OH), 3.68 (1H, d, J¼11.3 Hz, H-50), 3.37 (1H, s,
OH), 3.39 (1H, s, OH), 2.99 (1H, dd, J¼9.7 Hz, 15.8 Hz,
(1H, d, J¼12.5 Hz, H-50), 3.98 (1H, d, J¼12.5 Hz, H-50),
00
00
00
H-4
), 2.17 (1H, m, H-4 ), 2.11 (3H, s, CH3CO), 1.30
2.17 (1H, dd, J¼5.3, 12.8 Hz, H-4
), 1.89 (3H, d, J¼
down
up
down
00
(3H, s, CH3); 13C NMR (CDCl3) d 173.5 (CH3CO), 171.5,
167.9 (C-4, CO), 150.9 (C-2), 137.4, 136.2, 133.2, 130.0,
129.9, 129.5, 129.5, 128.8, 128.7, 0128.5, 128.3, 127.8, 127.5
(Ph), 86.2, 85.5, 82.4 (C-10, C-3 , C-40), 78.3, 78.20, 78.1,
74.0, 73.9,0073.7, 72.2, 67.3, 65.4 (C-5, C-6, C-10, C-2 , C-50,
C-100, C-2 , C-300, Bn), 29.7 (C-400), 21.9 (CH3), 20.9
(CH3CO); HiRes MALDI FT-MS m/z (MþNa) found/
calcd 743.2439/743.2423.
1.3 Hz, CH3), 1.60 (1H, dd, J¼10.7, 12.8 Hz, H-4 ); 13C
up
NMR (CD3OD) d 153.5 (C-4), 145.8 (C-2), 139.7 (C-6),
111.3 (C-5), 93.2 (C-10), 86.6, 82.8 (C-30, C-40), 76.9 (C-20),
74.900(C-200), 74.7 (C-100), 66.4 (C-300), 64.4 (C-50), 34.3
(C-4 ), 12.5 (CH3); HiRes MALDI FT-MS m/z (MþNa)
found/calcd 383.1044/383.1061.
5.2. Measurement of three-bond coupling constants of 27
5.1.16. Preparation of (1S,3R,4R,5S,6S,8R,9R)-1-benzyl-
oxy-6-benzyloxymethyl-3,4,5,9-tetrahydroxy-8-(thymin-
1-yl)-7-oxabicyclo[4.3.0]nonane (26). A solution of 25
(72 mg, 0.10 mmol) in methanol (2.0 mL) was added
NaOMe (28 mg, 0.50 mmol) and stirred at 65 8C for 9 h.
The reaction mixture was concentrated under reduced
pressure and the residue was purified by silica gel
chroma-tography using CH2Cl2–methanol (49:1) as eluent
to give the product as a white solid (43 mg, 75%); 1H NMR
(DMSO-d6) d 11.34 (1H, s, NH), 7.76 (1H, s, H-6), 7.45–
7.29 (10H, m, Ph), 6.24 (1H, d, J¼7.6 Hz, H-10), 5.85 (1H,
d, J¼4.5 Hz, 20-OH), 5.36 (1H, d, J¼10.8 Hz, Bn), 5.11
(1H, d, J¼4.9 Hz, 200-OH), 4.92 00(1H, dd, J¼4.5, 7.6 Hz,
H-20), 4.83 (1H, d, J¼6.3 Hz, 3 -OH), 4.59 (1H, d, J¼
10.8 Hz, Bn), 4.53 (2H0,0 s, Bn), 4.16 (1H, d, J¼11.4 Hz,
H-50), 4.10 (1H, m, H-3 ), 4.01 (1H, d, J¼9.9 Hz, 100-OH),
3.86 (1H, dd, J¼4.9, 7.3 Hz, H-200), 3.79 (1H, d, J¼11.4 Hz,
H-50), 3.57 (1H, dd, J¼2.7, 9.9 Hz, H-100), 2.71 (1H, dd,
1D 1H NMR spectra of the nucleoside studied were acquired
on a Varian Unity 500 MHz spectrometer. The nucleoside
27 was dissolved in CD3OD and spectra were obtained in
the temperature range from 250 to þ50 8C. Coupling
constants were measured as the splitting of multiplet
components, thereby limiting the accuracy to within 10%
of the linewidth (,0.1 Hz).
5.3. Karplus relationships
3
Karplus relationships correlating JHH and torsion angles
were constructed employing a state-of-art generalised
Karplus equation for nucleosides and nucleotides developed
by Altona and co-workers:43–45
3
3
X
X
JHHðuÞ ¼
CmcosðmuÞ þ
SnsinðnuÞ
m¼0
n¼1

M. Freitag et al. / Tetrahedron 60 (2004) 3775–3786
3785
Kumar, R.; Meldgaard, M.; Olsen, C. E.; Wengel, J.
Tetrahedron 1998, 54, 3607.
where the electronegativity of the HCCH-fragment sub-
stituents is accounted for in the coefficients Cm and Sn:
13. Obika, S.; Nanbu, D.; Hari, Y.; Andoh, J.; Morio, K.; Doi, T.;
Imanishi, T. Tetrahedron Lett. 1998, 39, 5401.
5.4. Force field calculations
14. Petersen, M.; Wengel, J. Trends Biotech. 2003, 21, 74.
15. Obika, S.; Andoh, J.; Sugimoto, T.; Miyashita, K.; Imanishi, T.
Tetrahedron Lett. 1999, 40, 6465.
Briefly described, the torsion angle restraints were
employed as flat-well potentials in the calculations, with
the flat-well part being the value obtained from Karplus
relationships ^58, and force constants of 50 kcal/(mol rad2).
In the simulated annealing protocol, each structure was
initially restrained energy minimised before being subjected
to 40 ps molecular dynamics (40,000 steps of 1 fs) with the
temperature being lowered from 2000 to 250 K. Finally,
each structure was restrained energy minimised again. In
this manner, 10 structures were generated for each of the
eight calculations (as detailed in Table 2) by randomly
varying initial atomic velocities.
16. Olsen, A. G.; Rajwanshi, V. K.; Nielsen, C.; Wengel, J.
J. Chem. Soc., Perkin Trans. 1 2000, 3610.
17. Kim, H. S.; Jacobson, K. A. Org. Lett. 2003, 5, 1665.
18. Nielsen, P.; Pfundheller, H. M.; Olsen, C. E.; Wengel, J.
J. Chem. Soc., Perkin Trans. 1 1997, 3423.
19. Christensen, N. K.; Petersen, M.; Nielsen, P.; Jacobsen, J. P.;
Olsen, C. E.; Wengel, J. J. Am. Chem. Soc. 1998, 120, 5458.
20. Christensen, N. K.; Andersen, A. K. L.; Schultz, T. R.;
Nielsen, P. Org. Biomol. Chem. 2003, 1, 3738.
21. Altmann, K.-H.; Imwinkelried, R.; Kesselring, R.; Rihs, G.
Tetrahedron Lett. 1994, 35, 7625.
5.5. Ab initio calculation
22. Kværnø, L.; Wightman, R. H.; Wengel, J. J. Org. Chem. 2001,
66, 5106.
¨
23. Tarkoy, M.; Bolli, M.; Schweizer, B.; Leumann, C. Helv.
The ab initio quantum mechanical calculation was
performed using the Gaussian94 program.46 The geometry
optimisation was carried out at the 3-21G* level using the
restricted Hartree–Fock procedure.
Chim. Acta 1993, 76, 481.
24. Bolli, M.; Litten, J. C.; Schu¨tz, R.; Leumann, C. J. Chem. Biol.
1996, 3, 197.
25. Renneberg, D.; Leumann, C. J. J. Am. Chem. Soc. 2002, 124,
5993.
26. Ravn, J.; Nielsen, P. J. Chem. Soc., Perkin Trans. 1 2001, 985.
27. Ravn, J.; Thorup, N.; Nielsen, P. J. Chem. Soc., Perkin Trans.
1 2001, 1855.
Acknowledgements
The Danish National Research Foundation is thanked for
financial support. Ms. Birthe Haack and Ms. Elinborg
Nygaard are thanked for synthetic assistance.
28. Ravn, J.; Freitag, M.; Nielsen, P. Org. Biomol. Chem. 2003, 1,
811.
29. Obika, S.; Sekiguchi, M.; Osaki, T.; Shibata, N.; Masaki, M.;
Hari, Y.; Imanishi, T. Tetrahedron Lett. 2002, 43, 4365.
30. Thomasen, H.; Meldgaard, M.; Freitag, M.; Petersen, M.;
Wengel, J.; Nielsen, P. Chem. Commun. 2002, 1888.
31. A less restricted bicyclic nucleoside with a 30,40-cis linkage has
been recently obtained by an RCM-strategy Montembault, M.;
Bourgourgnon, N.; Lebreton, J. Tetrahedron Lett. 2002, 43,
8091.
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