Synthesis of hydroxymethyl branched [3.2.0]bicyclic nucleosides using a regioselective oxetane ring-formation.

Article abstract of DOI:10.1039/b307667a

Two [3.2.0]bicyclic nucleosides, 35 and 34, with one and two hydroxymethyl substituents, respectively, have been efficiently synthesized. A protected (3'-C-vinyl-beta-D-allofuranosyl)thymine derivative 28 was easily prepared from diacetone-D-glucose and the thymine moiety was protected with a BOM-group. After the introduction of a leaving group in the 2'-position, the subsequent nucleoside 31 was used as the substrate for a stereoselective dihydroxylation and a regioselective oxetane ring-formation to give after deprotection the bicyclic nucleoside 34. The surprisingly efficient formation of an oxetane was first discovered by serendipity on a corresponding methylfuranoside derivative. The allo-configured bicyclic nucleoside 34 was easily shortened to a ribo-configured analogue 35 by a diol-cleaving reaction and subsequent reduction. Both 34 and 35 are conformationally restricted in the important intermediate 04'-endo conformation.

Full text of DOI:10.1039/b307667a

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Synthesis of hydroxymethyl branched [3.2.0]bicyclic nucleosides
using a regioselective oxetane ring-formation
Nanna K. Christensen, Ann Katrine L. Andersen, Trine R. Schultz and Poul Nielsen*
Nucleic Acid Center†, Department of Chemistry, University of Southern Denmark,
5230 Odense M, Denmark. E-mail: pon@chem.sdu.dk
Received 7th July 2003, Accepted 11th September 2003
First published as an Advance Article on the web 2nd October 2003
Two [3.2.0]bicyclic nucleosides, 35 and 34, with one and two hydroxymethyl substituents, respectively, have been
efficiently synthesized. A protected (3Ј-C-vinyl-β--allofuranosyl)thymine derivative 28 was easily prepared from
diacetone--glucose and the thymine moiety was protected with a BOM-group. After the introduction of a leaving
group in the 2Ј-position, the subsequent nucleoside 31 was used as the substrate for a stereoselective dihydroxylation
and a regioselective oxetane ring-formation to give after deprotection the bicyclic nucleoside 34. The surprisingly
efficient formation of an oxetane was first discovered by serendipity on a corresponding methylfuranoside derivative.
The allo-configured bicyclic nucleoside 34 was easily shortened to a ribo-configured analogue 35 by a diol-cleaving
reaction and subsequent reduction. Both 34 and 35 are conformationally restricted in the important intermediate
O4Ј-endo conformation.
RNA in fully modified sequences.8,9 Both nucleosides have
Introduction
been found by NMR and molecular modelling to be conform-
Conformationally restricted nucleosides have been intensively
ationally restricted in an O4Ј-endo conformation.9,10 This has
investigated as building blocks for oligonucleotides (ON’s) with
also been confirmed in duplex structures containing single
potential therapeutic and diagnostic applications.1,2 Especially
incorporations of 1 or 2, by NMR11,12 as well as by X-ray
nucleic acid analogues based on nucleoside monomers with the
crystallography.13 The O4Ј-endo conformation is a perfect
carbohydrate moieties strongly restricted in bi- or tricyclic
intermediate between the C2Ј-endo conformations found in
systems3 have attracted significant attention with tricyclo-
archetypical B-type duplexes and the C3Ј-endo conformations
DNA4 and LNA (locked nucleic acid)5 as prime examples.
found in archetypical A-type duplexes.14 DNA:RNA duplexes
Thus, both have demonstrated improved recognition of com-
can be viewed as intermediate structures in which the DNA
plementary RNA, the latter with unprecedented affinity, and
strand possesses mixtures of C2Ј-endo and C3Ј-endo conform-
both have demonstrated promising in vitro and in vivo results as
ations.15,16 This structural feature is regarded as pivotal for the
potential antisense therapeutics.2,6,7 Also 2Ј,3Ј-linked bicyclic
cleavage of the RNA strand in DNA:RNA duplexes by RNase
nucleosides have been investigated. Thus, the [3.3.0]bicyclic
H,16 an ability that is recognised as crucial in the development
nucleoside 18 and the smaller [3.2.0]bicyclic nucleoside 29
of antisense therapeutics.2 Modified nucleic acid analogues
(Fig. 1) were synthesised by convergent strategies and incorpor-
where the nucleoside monomers are restricted in O4Ј-endo
ated into ON’s with improved recognition of complementary
conformations can be viewed as mimics of the DNA strand
in DNA:RNA duplexes.12 As such, ON’s of 2Ј-F-arabino-
modified nucleosides have been found to adopt O4Ј-endo
conformations when hybridised to RNA compliments, and
RNase H has been found to degrade the RNA strands.15,17
Nonetheless, this ability to recruit RNase H has not yet been
found for ON’s containing neither 1 nor 2.18
Inspired by the results with 1 and 2, the design of nucleoside
analogues containing the [3.3.0]bicyclic skeleton of 1 has been
accomplished including a 2Ј-OMe substituted analogue of 119
and a tricyclic nucleoside being conformationally locked in an
S-type conformation due to an additional bond between the
C-5Ј and the C-7Ј positions.20 When incorporated into ON’s,
however, both of these demonstrated large decreases in affinity
towards both DNA and RNA complements.19,20 Also the
hydroxymethyl substituted nucleoside 3, as well as its 7Ј-epimer,
has been conveniently obtained (Fig. 1).21 An ON containing
3 in a fully modified sequence displays the same enhanced
recognition of complementary RNA as observed for 1, whereas
the most intriguing property of the corresponding sequence of
the 7Ј-epimer was a strong self-association.21 Furthermore, the
7Ј-hydroxymethyl group of 3 has been used as a branching
point for the construction of Y-shaped branched ON’s,22 and
the hydroxymethyl group of the 7Ј-epimer has been used for the
Fig. 1 2Ј,3Ј-Linked bi- and tricyclic nucleosides.
introduction of charged N-methylpiperazinocarbonyl moieties
into the major groove of nucleic acid duplexes.23 In another
attempt to introduce further conformational restriction into 1,
† Nucleic Acid Center is funded by the Danish National Research
Foundation for studies on nucleic acid chemical biology.
we prepared the tricyclic nucleoside 4.10 This nucleoside
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 7 3 8 – 3 7 4 8
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
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analogue was proved by NMR and molecular modelling to
adopt a similar O4Ј-endo conformation but with the 5Ј-OH
group in an unfavourable position, and it has not been
incorporated into ON’s.10
Also the bicyclic nucleoside 2 inspires the design of novel
nucleoside analogues containing the same [3.2.0]bicyclic
skeleton. Nevertheless, the formation of an oxetane ring
demands other synthetic methods and so far, only the 3Ј-deoxy-
3Ј-azido analogue of 2 has been obtained.24 In the present
paper, however, we introduce two novel hydroxymethyl sub-
stituted analogues of 2 which we expect to adopt the same
O4Ј-endo conformation as found for 29 as well as for its
3Ј-deoxy-3Ј-azido analogue.24 In the synthesis, we take
advantage of a stereoselective dihydroxylation and an efficient
regioselective oxetane ring formation that was discovered by
serendipity in the search for an improved synthesis of 4 and
other tricyclic nucleoside analogues.
Scheme 2 Reagents and conditions: i, 48% 10, 42% 11 (ref. 10); ii, 29%
(two steps, ref. 10).
hydroxylation of the major anomer 12 afforded the diol 14 as
the major product in a (>5 : 1) epimeric mixture (Scheme 3). The
high stereoselectivity in the dihydroxylation was expected
from the similar result obtained with 8 and 9, and therefore,
a resulting (R)-configuration of C-7 in 14 was assumed.
Treatment of 14 with sodium hydride, however, did not afford a
five-membered oxolane ring but instead the four-membered
oxetane ring in the product 15, which was isolated as a pure
compound in a good yield after chromatographic purification.
The [3.2.0]bicyclic structure was deduced from NMR. Thus,
especially the OH-signal appearing as a triplet and not a duplet
verified the presence of a primary alcohol. Furthermore, the
3JH1–H2 coupling constant of 2.8 Hz was similar to what has
been observed before for the similar [3.2.0]bicyclic system in 2
and derivatives thereof (2.3 to 2.7 Hz),9,24 contrary to what has
been observed in [3.3.0]bicyclic systems as in 1, 10 and the
tricyclic nucleoside 4 (3.7 to 4.7 Hz).8,10 The two H-8 signals
were not split, a fact consistent with an exocyclic position of
C-8, and finally in 13C NMR, a significant shift downfield was
observed for the C-7 signals (71.5 ppm in 14 and 82.9 ppm in
15) and not for the C-8 signals. Further evidence was obtained
after esterification of 15 to give the methanesulfonic ester 16.
Thus, the H-8 signals performed a larger shift downfield than
the H-7 signal. An NOE-experiment on 16, for which the
H-NMR signals were conveniently separated, showed a very
clear and large mutual contact between H-5 and H-7 (9–11%)
confirming the retention of the (R)-configuration of C-7 and a
[3.2.0]bicyclic system. The trityl group of 15 was also removed
using a method reported to be chemoselective for trityl ethers in
the presence of other acid labile functionalities including
acetals as in methylfuranosides and isopropylidene protecting
Results
1,2-Di-O-isopropylidene-3-C-vinyl-β--allofuranose 5 (Scheme
1) was synthesised in three steps from diacetone--glucose.10,25
Thus, in our former preparation of the tricyclic nucleoside 4
and its α-anomer, 5 has been converted via 6 to the methyl-
furanoside 7.10 A double esterification afforded the anomeric
bis-methanesulfonic esters 8 and 9.10 Both 8 and 9 were treated
with osmium tetroxide and then, immediately, sodium hydride
to give the tricyclic products 10 and 11, respectively, as the only
major products (Scheme 2).10 These results were deduced to
the preorganisation of the intermediate diol for forming first
the six-membered ring from a nucleophilic attack of the
secondary alcohol of C-7 to C-6 and then, after the subsequent
twist in the molecule, the five-membered ring via a nucleophilic
attack from the primary alcohol of C-8 to the secondary C-2
position.10
Scheme 1 Reagents and conditions: i, 63% (ref. 10); ii, 20% HCl in aq.
MeOH, 76% 7 (ref. 10), 77% 19; iii, 44% 8 and 39% 9 (ref. 10), TrCl then
MsCl, pyr., 52% 12 and 31% 13, MsCl, pyr., 55% 20 and 34% 21;
iv, NaH, BnBr, DMF, 99%.
In order to reverse this reaction sequence and improve the
synthesis of the tricyclic nucleoside 4 or, alternatively, to obtain
other ring-systems, e.g. the 7Ј-epimeric tricyclic nucleoside, we
decided to follow a different protecting group strategy for the
two alcohols of 7 and to differentiate between the two ring-
closing reactions. Thus, the methylfuranoside 7 was selectively
tritylated and in situ mesylated to give after separation
the anomers 12 and 13 in a 1.6 : 1 ratio (Scheme 1). Di-
Scheme 3 Reagents and conditions: i, OsO4, NMO, pyr., aq. t-BuOH,
77% 14, 49% 22; ii, NaH, DMF, 81% 15, 91% 23; iii, MsCl, pyr., 85%;
iv, HCOOH, Et2O, 33%; v, BzCl, pyr., 94%, vi, thymine, N,O-
bis(trimethylsilyl)acetamide, TMS-OTf, CH3CN, 35% 25 and 34% 26.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 7 3 8 – 3 7 4 8
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groups.26 However, treatment of 15 with formic acid in diethyl
ether afforded the product 17 in only 33% yield.
to a reaction of the C-6 oxygen. However, this possibility is
excluded by the NMR data. Thus, almost identical chemical
shifts of the two H-6 atoms were observed indicating an
exocylic position of C-6. A stronger indication was given by
comparing the 13C chemical shifts of the benzylic methylene
groups. Thus, when comparing differently benzyl ether
protected hexofuranosides such as the di- versus the tri-O-
benzylated series of compounds presented here and in other
series28 with solely 3-O-benzylated compounds,28 the chemical
shifts for the tertiary 3-O benzyl, the secondary 5-O benzyl and
the primary 6-O benzyl methylene groups were consequently
near 67–68 ppm, 72 ppm and 73.5 ppm, respectively. For
compounds 25 and 26, no benzylic CH2 signal around 72 ppm
was observed.
The surprisingly regioselective ringclosure forming 15 can be
rationalised from simple modelling. Thus, the free rotation
around the C-3-C-7 bond in 14 is hindered by the large and
bulky ring-substituent at the C-4 position (Fig. 2). Therefore,
the primary hydroxy group (or the corresponding oxyanion) at
C-8 cannot reach a position for possible nucleophilic attack at
the C-2 position. Subsequently, only the secondary hydroxy
group at C-7 can perform the ring-closing reaction. In other
words, the molecule is preorganised for the formation of an
oxetane ring.
At this stage, the strategy was reconsidered again. After all,
problems performing Vorbrüggen type nucleobase couplings on
constrained bicyclic carbohydrate precursors have been
observed before.29 Instead, we decided to perform the nucleo-
base coupling before the ring-closing reaction. This would
demand a protecting group for the nucleobase during the ring-
closure in order to avoid an alternative nucleophilic attack from
the thymine C-2 oxygen. Nevertheless, this strategy seemed rea-
sonable, as the stereoselective dihydroxylation as well as the
regioselective ring-closure should be controlled in a similar
manner on a nucleoside substrate. As the first step, tribenzylic
ether 18 was converted to the anomeric mixture of acetates 27
in a high yield (Scheme 4). A standard Vorbrüggen type coup-
ling27 using thymine as the nucleobase afforded the β-con-
figured nucleoside 28 as the only product, as expected due to the
Fig. 2 Steric preorganisation for the formation of the oxetane ring.
This unexpected but very efficient route towards a con-
strained bicyclic system opened the possibility of constructing
potentially very useful [3.2.0]bicyclic nucleosides, i.e. single
or double hydroxymethyl substituted derivatives of 2 for
potentially branched and/or functionalised ON’s. However, the
present synthetic strategy had to be reconsidered at this stage
and, obviously, the differentiated protecting pattern of the C-5
and C-6 hydroxy functionalities was both inconvenient and
unnecessary. Therefore, we decided to simply use benzyl ethers
for all positions, and compound 5 was treated with an excess
of sodium hydride and benzyl bromide to give 18 in a very
high yield (Scheme 1). The mixture of methylfuranosides 19
was subsequently obtained and esterification afforded after
separation the two anomers 20 and 21 in the same 1.6 : 1 ratio
as obtained with 12 and 13. Dihydroxylation of the major
product 20 afforded again one major product 22 in a reasonable
yield (Scheme 3). Also a separate minor fraction was obtained
containing a small amount of the C-7 epimer, and overall, an
approximately 5 : 1 ratio of the two epimers was determined. As
expected, the usual basic treatment of diol 22 afforded again a
product, 23, containing on oxetane moiety. Thus, the NMR
3
data were very similar to the data for 15 including a JH1–H2
coupling constant of 2.8 Hz. In order to perform a nucleobase
coupling on the bicyclic methylfuranoside skeleton, the primary
alcohol at C-8 was protected as its benzoate 24. Again,
1H-NMR verified the structure, as a large shift of the H-8
signals was observed, and an NOE experiment indicated a
strong mutual contact between H-5 and H-7.
In an attempt to perform a Vorbrüggen type coupling of
thymine,27 compound 24 was treated with thymine, BSA as a
silylating agent and TMS-triflate as a Lewis acid catalyst
(Scheme 3). Two products 25 and 26 were obtained, the one
being a trimethylsilyl protected analogue of the other. Clearly,
the products were not nucleosides and the methylfuranoside
structure was intact. Instead, the oxetane ring has been opened
by the Lewis acid as indicated by NMR confirming a secondary
2-OH group in one of the two products, and displaying larger
3JH1–H2 coupling constants of 5.1 and 5.6 Hz. Furthermore, only
two benzyl ethers remained in the products. A reasonable
mechanistic explanation would be a reaction between the C-5
oxygen atom and the C-7 carbon atom being activated by the
Lewis acid and the oxetane ring strain. Subsequently, the
oxetane is opened and the 5-O-benzyl bond is cleaved giving
the benzylic cation and, after the aqueous work-up, benzylic
alcohol, as well as the new five-membered ring in 25 and 26.
Alternatively, a six-membered ring could have been formed due
Scheme 4 Reagents and conditions: i, a, 80% aq. AcOH, b, Ac2O, pyr.,
90%; ii, thymine, N,O-bis(trimethylsilyl)acetamide, TMS-OTf, CH3CN,
81%; iii, BOM-Cl, pyr.; iv, MeONa, MeOH, 75% (2 steps); v, MsCl,
pyr., 91%; vi, OsO4, NMO, aq. THF, 31% (see Scheme 5); vii, NaH,
DMF, 88%; viii, H2, Pd(OH)2–C, EtOH, 100%; ix, a, NaIO4, aq.
dioxane, b, NaBH4, aq. dioxane, 89%.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 7 3 8 – 3 7 4 8
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anchimeric assistance of the 2-O acetyl group.27,30 This was
confirmed by the JH1Ј–H2Ј coupling constant of 7.4 Hz consist-
easily solved as already described in the literature34 by treating
3
the mixture with concentrated sodium hydroxide to afford
the pure compound 34. Finally, this double hydroxymethyl
functionalised bicyclic nucleoside was conveniently converted
to a singly functionalised analogue 35 in a high yield by
cleavage of the vicinal diol moiety with sodium periodate
and subsequent reduction of the aldehyde with sodium boro-
hydride. As a last experiment, also the 7Ј-(S)-configurated diol
32a was subjected to the same reaction conditions as used in the
ring-closure of 32, but only a slow conversion and a mixture of
several products was observed.
ent with a β-ribo-configured nucleoside in a C2Ј-endo conform-
ation.30,31 The nucleobase was conveniently protected with the
benzyloxymethyl (BOM) group to give 29 and then
deacetylated to give 30. Conversion to the methanesulfonic
ester 31 revealed a new substrate for the dihydroxylation/ring-
closing sequence. The dihydroxylation, however, afforded sev-
eral products which were separated by chromatography
(Schemes 4 and 5). As expected, 32 was isolated as the major
product in a reasonable 31% yield in addition to 28% of
recovered starting material 31. Furthermore, the 7Ј-epimer 32a
was isolated in a 7% yield revealing a 4.5 : 1 ratio of epimers
comparable to the ratio found for the dihydroxylations of 12
and 20. In addition, dihydroxylation of the nucleobase was
demonstrated. Thus, the isomeric mixtures 32b and 32c were
isolated in 7% and 6% yields, respectively. In another experi-
ment, the yield of 32 was slightly improved to give 37% yield
after a longer reaction time. However, no starting material was
re-isolated. Thus, based on this recovery of 31, the yield of 32
following the better procedure (Scheme 5) was 43%. All
attempts to improve the yield and to diminish the undesirable
dihydroxylation of the nucleobase by the addition of pyridine
and/or the shift of solvent mixture were unsuccessful. Osmium
tetroxide mediated dihydroxylation of a thymine moiety has
been seen before for comparable, though not N-3 protected,
substrates both as the intended reaction32 and as the cause for
unintended side-products.33 With other very similar sub-
strates,20,33 including the 3Ј-C-allyl ribofuranoside derivative
used in the synthesis of 3,21 this problem was not observed.
Discussion
In summary, the [3.2.0]bicyclic nucleoside 34 was conveniently
obtained in 12 steps and 14% overall yield from diacetone--
glucose. Thus, from the highly chiral carbohydrate framework
given in the -glucose, all the key reactions were performed
with high stereoselectivity; the Grignard reaction,10,25 the
nucleobase-coupling and the dihydroxylation. The latter reac-
tion, however, was the limiting step resulting in several by-
products and a tedious separation. This reaction certainly
deserves future improvements concerning alternative methods
and/or alternative N-3 protecting groups.
The oxetane ring-formation was remarkably regioselective
giving no detectable by-products such as the alternative five-
membered oxolane ring in a [3.3.0]bicyclic product. This very
efficient ring-closure was successful with the nucleoside
substrate 32 as well as with the methylfuranosides 14 and 22,
and appears to rely completely on the preorganisation that is
incorporated in the substrate by the large C-4 (or C-4Ј) ring
substituent (Fig. 2). Future studies might demonstrate whether
this ring-closure is also compatible with smaller C-4 sub-
stituents, or in other words, whether the derivative 35 could
be obtained directly from a ribo-nucleoside substrate, i.e. a
derivative of 32 without the C-6Ј carbon which should be easily
available. Thus, in the preparation of 2, the 3Ј-C-vinyl sub-
stituted ribo-nucleoside derivative, i.e. 28 without the C-6Ј
moiety, has been easily obtained starting from 1,2-di-O-isopro-
pylidine-α--xylofuranose.9 It is an open question, whether the
C-5 moiety as an unsubstituted CH2OBn moiety would induce
sufficient preorganisation for oxetane ring-formation. None-
theless, the present efficient preparation of 35 from 34 does not
necessitate a new parallel (and hardly better) preparation of
this compound.
On the other hand, the oxetane formation seems to rely also
on the substituent attached to the attacking alcohol. Thus, in
another attempt on preparing 2, oxetane ring-formation on a
derivative of 14/22 without both the C-6Ј and the C-8Ј moieties
(i.e. the attacking alcohol being primary) has proven impos-
sible.35 Thus, no sufficient preorganisation for oxetane ring-
formation was given in that substrate, and instead, the oxetane
ring in 2 was obtained by a nucleophilic attack from the 2Ј-OH
group on an arabino-configured nucleoside towards a 3Ј-C-
CH2OMs moiety.9 Thus, as is also indicated in Fig. 2, the
preorganisation for oxetane ring-formation where the C-7
hydroxy function acts as the nucleophile presumably relies also
on the presence of the hydroxymethyl moiety (or another
substituent) on C-7. On the other hand, the formation of an
oxetane ring following attack from a primary towards a
secondary centre has been seen before, for example in the
formation of Taxol and derivatives thereof.36 The conclusion,
however, that the present ring-closure of 32 (and of 14 and 22)
is based on a perfect preorganisation, is also confirmed by the
fact that the 7Ј-epimer 32a cannot efficiently form an oxetane
ring. This is probably due to a significant and inconvenient
steric interaction between the C-5Ј and the C-7Ј/C-8Ј-diol
moieties, and subsequently, a completely different conform-
ational organisation of the reactive groups.
Scheme 5 Reagents and conditions: (See Scheme 4) 31% 32, 7% 32a, 7%
32b, 6% 32c.
The usual treatment of 32 with sodium hydride gave in a high
(88%) yield the protected bicyclic nucleoside 33 (Scheme 4).
Subsequent hydrogenation removed the benzylic ethers and the
BOM group to give quantitatively the functionalised bicyclic
nucleoside 34. The configuration of 33 and 34 was proven by
NMR. Thus, the 3JH1Ј–H2Ј coupling constants of 2.1 and 2.6 Hz,
respectively, were consistent with the [3.2.0]bicyclic structure
and an O4Ј-endo conformation.9,12,24 Again, the OH-signal in 33
appeared as a triplet verifying a primary alcohol on C-8Ј, and
an NOE-experiment confirmed a large mutual contact between
H-5Ј and H-7Ј. Finally, the characteristic 13C downfield shift of
11 ppm was observed when comparing the C-7Ј-signals of 32
and 33. In another batch, the hydrogenation afforded a mixture
of 34 and a compound with an N-3-hydroxymethyl moiety due
to incomplete removal of the BOM group. This problem was
In the near future, the bicyclic nucleosides 34 and 35 will be
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 7 3 8 – 3 7 4 8
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appropriately protected and incorporated into ON’s. This is
expected to be relatively straightforward as the necessary
synthetic methods are well established for similar nucleoside
derivatives containing constrained bicyclic carbohydrate
moieties and/or free hydroxy groups that demand protecting
groups.9,20,21 Thus, after protection of primary hydroxy group(s)
of 34 and 35, the nucleosides will be converted to 5Ј-O-DMT
protected 3Ј-O phosphoramidites for standard 3Ј to 5Ј incorpor-
ation of the nucleosides into ON’s. The nucleic acid recognition
behaviour of ON’s containing 34 and/or 35 in comparison with
the ON’s containing the original [3.2.0]bicyclic nucleoside 2 will
throw light on the possibility of influencing the hydration
pattern of a DNA:RNA duplex. Thus, ON’s containing the
strong O4Ј-endo conformational mimic 2 demonstrated large
increases in both DNA and RNA recognition, and this might
be improved by the combination of O4Ј-endo conformations
and additional hydrophilic groups that will be introduced by
34 and 35. The ability of the stable hybrids between RNA
sequences and ON’s containing 2 to be recognised by RNase H
has not yet been fully explored.18 Thus, these hybrids should be
excellent mimics of the natural DNA:RNA substrate due to the
O4Ј-endo conformation mimicking the intermediate between
C2Ј-endo and 3Ј-endo conformations.12 These hybrids might
therefore be recognised by RNase H in mixed sequences or
in gapmers containing even very small gaps of natural 2Ј-
deoxynucleosides.37,38 Whether the additional hydroxymethyl
groups in 34 and 35 will influence this envisioned ability of 2,
should also be explored.
Finally, 34 or 35 might be very convenient building blocks for
the construction of branched ON’s in the same way as formerly
described with 3.22 Nevertheless, 35 might be a more convenient
branching point than 3 being a smaller entity and more strongly
conformationally restricted. The hydroxy group should be
perfectly positioned in the major groove of a nucleic acid
duplex. While 35 could be used for Y-shaped branched ON’s,
34 might be a very useful building block for X-shaped branched
ON’s. Furthermore, the additional hydroxy functionalities of
both 34 and 35 could be used for the incorporation of other
groups as recently shown with 3 and a 4Ј-C-hydroxymethyl
nucleoside,23 or even as building blocks for DNA-templated
synthesis.39
The incorporation of the two novel bicyclic nucleosides into
ON’s is in progress in our laboratory. We expect these nucleic
acid analogues to reveal important information about the
conformational behaviour and the hydration pattern of nucleic
acid duplexes, and to further establish the scopes of the crucial
RNase H degradation of target RNA-sequences. Hereby, these
bicyclic nucleoside monomers might demonstrate favourable
properties towards the development of therapeutic ON’s.
Furthermore, we envision potential applications of these
nucleoside monomers as branching points for the introduction
of other chemical entities into nucleic acids and for the
construction of novel chemical architectures thereby revealing
attractive tools for nanobiotechnology.
Experimental
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 obtained on a Varian Gemini 2000 spectrometer. FAB
mass spectra were recorded in positive ion mode on a Kratos
MS50TC spectrometer, Plasma Desorption mass spectra on an
Applied Biosystems BIO-ION 20R spectrometer, and MALDI
mass spectra were recorded on an Ionspec Ultima Fourier
Transform mass spectrometer. Microanalyses were performed
at The Microanalytical Laboratory, Department of Chemistry,
University of Copenhagen. Assignments of NMR spectra are
1
1
based on H,1H-COSY, H,13C-COSY and/or DEPT spectra
and follow standard carbohydrate and nucleoside style; i.e. the
carbon atom next to a nucleobase is assigned C-1Ј; the number-
ing continues from C-3 (or C-3Ј) to C-7 (C-7Ј) and C-8 (C-8Ј) as
depicted in Scheme 3 for all compounds except 35 where the
numbering continues from C-3Ј to C-6Ј and C-7Ј. However,
compound names for bicyclic compounds are given according
to the von Baeyer nomenclature.
Preparation of methyl 3,5-di-O-benzyl-2-O-methylsulfonyl-6-O-
triphenylmethyl-3-C-vinyl-D-allofuranoside 12 and 13
To a stirred solution of 7 (1.00 g, 2.50 mmol) in anhydrous
pyridine (7.5 cm3) at room temperature was added trityl
chloride (1.046 g, 3.75 mmol). The reaction mixture was stirred
for 3 days at room temperature and then for 2 h at 40 ЊC. After
cooling to 0 ЊC, methane sulfonylchloride (0.76 cm3, 10.0 mmol)
was added and the reaction mixture was stirred for 1 h at room
temperature and quenched with a saturated aqueous solution
of NaHCO3 (75 cm3). The mixture was extracted with
dichloromethane (2 × 200 cm3) and the combined extracts were
dried (MgSO4). The solvent was removed by distillation under
reduced pressure and the residue purified by chromatography
over silica with petroleum ether–ethyl acetate (85 : 15) as eluent
to give the two pure products as solids;
As an additional advantage, 35 (13 steps, 12% overall yield)
and 34 are more conveniently prepared than the related deriv-
ative 3 (16 steps, 5% overall yield ϩ 6% of the 7Ј-epimer).8,21
Even more importantly, the present synthetic strategy should
be useful also in the construction of the similar nucleoside
analogues containing other nucleobases. Thus, in contrast to
the synthesis of 1, 2 and 3, the present strategy does not take
advantage of the so-called anhydro approach40 where the C-2
carbonyl moiety of a pyrimidine nucleobase is used for convert-
ing the C-2Ј configuration thereby changing ribo- to arabino-
configured nucleosides. Therefore, also the purine analogues of
34 and 35 should be easily available.
The surprisingly efficient present synthesis of an otherwise
inaccessible oxetane ring might be used in the preparation
of different classes of molecules such as conformationally
restricted carbohydrate derivatives related to 15.41 On the other
hand, the original target nucleosides, i.e. the tricyclic nucleoside
4, and especially its 7Ј-epimer, must be prepared by a different
strategy.
Methyl 3,5-di-O-benzyl-2-O-methylsulfonyl-6-O-triphenyl-
methyl-3-C-vinyl-ꢀ-D-allofuranoside 12
(936 mg, 52%) (Found: C, 71.38; H, 5.75; S, 4.62. C43H44O8S
requires C, 71.65; H, 6.15; S, 4.45%); δH (300 MHz; CDCl3;
Me4Si) 7.48–7.45 (6H, m, Ph), 7.33–7.21 (19H, m, Bn, Ph), 5.94
(1H, dd, J 17.6, 11.3 Hz, H-7), 5.53 (1H, d, J 17.6 Hz, H-8),
5.32 (1H, d, J 11.3 Hz, H-8), 5.10 (1H, d, J 4.0 Hz, H-1), 5.06
(1H, d, J 4.0 Hz, H-2), 4.70 (1H, d, J 11.0 Hz, Bn), 4.64 (2H, br
s, Bn), 4.49 (1H, d, J 7.6 Hz, H-4), 4.42 (1H, d, J 11.0 Hz, Bn),
3.62 (1H, m, H-5), 3.44 (1H, dd, J 10.2, 3.2 Hz, H-6), 3.33 (1H,
dd, J 10.2, 5.2 Hz, H-6), 3.23 (3H, s, OCH3), 2.98 (3H, s,
SO2CH3); δC (75 MHz; CDCl3; Me4Si) 144.0 (Ph), 138.9, 138.2
(Bn), 132.7 (C-7), 128.9, 128.4, 127.8, 127.8, 127.6, 127.4, 127.1,
127.0 (Bn, Ph), 118.8 (C-8), 105.9 (C-1), 86.7 (Ph3C), 85.3
(C-3), 84.4 (C-4, C-2), 78.5 (C-5), 71.9 (Bn), 67.2 (Bn), 62.9
(C-6), 56.1 (OCH3), 38.9 (SO2CH3); m/z (FAB) 743 (M ϩ Na).
Conclusion
A surprisingly efficient and selective oxetane ring-formation has
been found by serendipity with methylfuranoside derivatives
and, subsequently, used successfully on a nucleoside substrate.
Hereby, two hydroxymethyl substituted [3.2.0]bicyclic nucleo-
sides have been conveniently obtained. These are conform-
ationally restricted in O4Ј-endo conformations and should
therefore perfectly match the structure of DNA:RNA hybrids.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 7 3 8 – 3 7 4 8
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Methyl 3,5-di-O-benzyl-2-O-methylsulfonyl-6-O-triphenyl-
methyl-3-C-vinyl-ꢁ-D-allofuranoside 13
2.14 (1H, t, J 7.7 Hz, 8-OH); δC (75 MHz; CDCl3; Me4Si) 144.0
(Ph), 138.0, 137.2 (Bn), 129.0, 128.8, 128.7, 128.4, 128.1, 127.8,
127.7, 127.6, 127.3, 127.1 (Bn, Ph), 104.1 (C-1), 86.7 (Ph3C),
85.7 (C-3), 84.6 (C-2), 82.9 (C-7), 77.1 (C-5), 74.6 (C-4), 71.6
(Bn), 68.0 (Bn), 62.4 (C-6, C-8), 57.5 (OCH3); m/z (FAB) 681
(M ϩ Na).
(556 mg, 31%); δH (300 MHz; CDCl3; Me4Si) 7.48–7.44 (6H, m,
Ph), 7.33–7.20 (19H, m, Bn, Ph), 5.91 (1H, dd, J 17.6, 11.2 Hz,
H-7), 5.34 (1H, d, J 17.6 Hz, H-8), 5.28 (1H, d, J 11.2 Hz, H-8),
5.03 (1H, d, J 4.8 Hz, H-1), 4.99 (1H, d, J 4.8 Hz, H-2), 4.73
(1H, d, J 12.0 Hz, Bn), 4.68 (1H, d, J 11.5 Hz, Bn), 4.62 (1H, d,
J 12.0 Hz, Bn), 4.57 (1H, d, J 7.3 Hz, H-4), 4.42 (1H, d, J 11.5
Hz, Bn), 3.62 (1H, m, H-5), 3.45 (1H, m, H-6), 3.43 (3H, s,
OCH3), 3.31 (1H, dd, J 10.2, 5.1 Hz, H-6), 2.99 (3H, s,
SO2CH3); δC (75 MHz; CDCl3; Me4Si) 144.1 (Ph), 139.1, 138.3
(Bn), 135.7 (C-7), 128.9, 128.8, 128.3, 128.2, 127.8, 127.7, 127.5,
127.3, 127.2, 127.0 (Bn, Ph), 117.8 (C-8), 100.6 (C-1), 86.7
(Ph3C), 83.1, 81.2, 79.5 (C-2, C-3, C-4), 77.9 (C-5), 71.9 (Bn),
67.2 (Bn), 62.8 (C-6), 55.7 (OCH3), 39.0 (SO2CH3); m/z (FAB)
743 (M ϩ Na).
Preparation of (1R,2R,4R,5S,7R)-1-benzyloxy-2-(1(R)-benzyl-
oxy-2-triphenylmethoxy)ethyl-4-methoxy-7-methylsulfonyloxy-
methyl-3,6-dioxabicyclo[3.2.0]heptane 16
To a stirred solution of 15 (131 mg, 0.199 mmol) in anhydrous
pyridine (1.0 cm3) at 0 ЊC was added dropwise methane
sulfonylchloride (0.04 cm3, 0.5 mmol). The reaction mixture
was stirred at room temperature for 1 h, quenched with ice-cold
water (15 cm3) and extracted with dichloromethane (3 × 15 cm3).
The combined extracts were washed with a saturated aqueous
solution of NaHCO3 (20 cm3) and then dried (MgSO4). The
solvent was removed by distillation under reduced pressure
and the residue purified by chromatography over silica with
dichloromethane–methanol (99 : 1) as eluent to give the
product as an oil (124 mg, 85%); δH (300 MHz; CDCl3; Me4Si)
7.50–7.47 (6H, m, Ph), 7.34–7.23 (19H, m, Bn), 4.97 (1H, dd,
J 6.9, 4.0 Hz, H-7), 4.92 (1H, d, J 2.8 Hz, H-2), 4.75 (1H, d,
J 2.8 Hz, H-1), 4.71 (1H, d, J 11.3 Hz, Bn), 4.59 (1H, d, J 11.3
Hz, Bn), 4.56 (1H, m, H-8), 4.53 (1H, d, J 11.6 Hz, Bn), 4.42
(1H, d, J 11.6 Hz, Bn), 4.41 (1H, d, J 7.2 Hz, H-4), 4.37 (1H, dd,
J 11.6, 4.0 Hz, H-8), 3.96 (1H, m, H-5), 3.60 (1H, dd, J 10.4, 2.5
Hz, H-6), 3.48 (3H, s, OCH3), 3.40 (1H, dd, J 10.4, 4.2 Hz,
H-6), 2.80 (3H, s, SO2CH3); δC (75 MHz; CDCl3; Me4Si) 143.9
(Ph), 137.9, 137.0 (Bn), 128.9, 128.8, 128.6, 128.5, 128.1, 127.9,
127.8, 127.6, 127.2, 127.2 (Bn, Ph), 103.9 (C-1), 86.8 (Ph3C),
85.1 (C-3), 84.8 (C-2), 81.0 (C-7), 77.1 (C-5), 74.4 (C-4), 71.7
(Bn), 69.3 (C-8), 67.9 (Bn), 62.3 (C-6), 57.6 (OCH3), 37.3
(SO2CH3); m/z (FAB) 759 (M ϩ Na).
Preparation of methyl 3,5-di-O-benzyl-3-C-(1(R),2-dihydroxy-
ethyl)-2-O-methylsulfonyl-6-O-triphenylmethyl-ꢀ-D-allofurano-
side 14
To a solution of 12 (596 mg, 0.827 mmol) in tert-butanol
(9.2 cm3) was added water (0.50 cm3) pyridine (0.46 cm3),
N-methylmorpholine-N-oxide (673 mg, 5.80 mmol) and a 2.5
w/w-% solution of osmium tetroxide in tert-butanol (0.046
cm3). The solution was stirred under reflux at 76 ЊC for 12 h and
quenched at room temperature with a 20% aqueous solution of
Na2S2O5 (3.4 cm3). The mixture was diluted with water (30 cm3)
and extracted with dichloromethane (2 × 100 cm3). The com-
bined extracts were washed with a saturated aqueous solution
of NaHCO3 (75 cm3) and then dried (MgSO4). The solvent was
removed by distillation under reduced pressure and the residue
purified by chromatography over silica with dichloromethane–
methanol (99 : 1) as eluent to give the product as a clear oil (482
mg, 77%) which was used without further purification in the
next step; δH (300 MHz; CDCl3; Me4Si) (major isomer) 7.52–
7.42 (6H, m, Ph), 7.33–7.21 (19H, m, Bn, Ph), 5.27 (1H, d, J 3.3
Hz, H-2), 5.08 (1H, d, J 3.3 Hz, H-1), 4.73–4.64 (4H, m, Bn,
H-4), 4.31 (1H, d, J 10.5 Hz, Bn), 4.05 (1H, m, H-7), 3.97 (1H,
m, H-5), 3.78 (1H, dd, J 11.8, 5.4 Hz, H-8), 3.62–3.57 (2H, m,
H-6, H-8), 3.37 (1H, dd, J 10.4, 3.8 Hz, H-6), 3.20 (3H, s,
OCH3), 3.03 (3H, s, SO2CH3); δC (75 MHz; CDCl3; Me4Si)
(major isomer) 143.91 (Ph), 138.0, 137.5 (Bn), 128.9, 128.7,
128.6, 128.6, 128.4, 128.1, 128.0, 127.9, 127.6, 127.2, 127.1 (Bn,
Ph), 107.4 (C-1), 87.0, 86.6 (Ph3C, C-3), 83.9, 81.5 (C-2, C-4),
77.5 (C-5), 71.5, 71.4 (Bn, C-7), 67.7 (Bn), 63.2, 61.2 (C-6, C-8),
56.4 (OCH3), 38.4 (SO2CH3); m/z (PDMS) 777 (M ϩ Na).
Preparation of (1R,2R,4R,5S,7R)-1-benzyloxy-2-(1(R)-benzy-
loxy-2-hydroxy)ethyl-7-hydroxymethyl-4-methoxy-3,6-dioxa-
bicyclo[3.2.0]heptane 17
A solution of 15 (90 mg, 0.137 mmol) in anhydrous diethyl
ether (0.37 cm3) was stirred at 0 ЊC, formic acid (0.30 cm3) was
added, and the mixture was stirred at 0 ЊC for 1 h. The reaction
mixture was quenched with water (5 cm3) and neutralised
with NaHCO3. The mixture was extracted with diethyl ether
(2 × 10 cm3) and the combined extracts were dried (MgSO4).
The solvent was removed by distillation under reduced pressure
and the residue was purified by chromatography over silica with
dichloromethane–methanol (97 : 3) as eluent to give the
product as a clear oil (19 mg, 33%); δH (300 MHz; CDCl3;
Me4Si) 7.35–7.26 (10H, m, Bn), 4.95 (1H, d, J 2.8 Hz, H-2), 4.82
(1H, d, J 2.8 Hz, H-1), 4.79 (1H, m, H-7), 4.68 (1H, d, J 11.2
Hz, Bn), 4.64 (1H, d, J 11.2 Hz, Bn), 4.57 (1H, d, J 11.4 Hz,
Bn), 4.52 (1H, d, J 11.4 Hz, Bn), 4.30 (1H, d, J 8.5 Hz, H-4),
4.03 (1H, m, H-6), 3.86–3.39 (4H, m, H-5, H-6, H-8), 3.58 (3H,
s, OCH3); δC (75 MHz; CDCl3; Me4Si) 137.6, 136.9 (Bn), 128.7,
128.6, 128.6, 128.3, 128.1, 128.0, 128.0, 127.9, 127.7, 127.7 (Bn),
104.3 (C-1), 85.6 (C-3), 83.9 (C-2), 83.0 (C-7), 77.6 (C-5), 74.2
(C-4), 71.9 (Bn), 68.4 (Bn), 62.3 (C-8), 61.0 (C-6), 57.8 (OCH3);
m/z (FAB) 439 (M ϩ Na).
Preparation of (1R,2R,4R,5S,7R)-1-benzyloxy-2-(1(R)-benzy-
loxy-2-triphenylmethoxy)ethyl-7-hydroxymethyl-4-methoxy-3,6-
dioxabicyclo[3.2.0]heptane 15
A solution of 14 (458 mg, 0.608 mmol) in anhydrous DMF
(1.0 cm3) was stirred at 0 ЊC and a 60% oily dispersion of NaH
(38 mg, 0.91 mmol) was added. The reaction mixture was
stirred at room temperature for 2 days, and then quenched with
a saturated aqueous solution of NaHCO3 (20 cm3) and
extracted with dichloromethane (2 × 50 cm3). The combined
extracts were dried (MgSO4) and the solvent was removed by
distillation under reduced pressure. The residue was purified by
chromatography over silica with dichloromethane–methanol
(99 : 1) as eluent to give the product as a clear oil (325 mg, 80%)
(Found: C, 75.20; H, 6.46. C42H42O7, 0.5H2O requires C, 75.54;
H, 6.49%); δH (300 MHz; CDCl3; Me4Si) 7.55–7.48 (6H, m, Ph),
7.35–7.23 (19H, m, Bn), 4.92 (1H, d, J 2.8 Hz, H-2), 4.76 (1H,
d, J 2.8 Hz, H-1), 4.74 (1H, m, H-7), 4.71 (1H, d, J 11.1 Hz,
Bn), 4.63 (1H, d, J 11.1 Hz, Bn), 4.54 (1H, d, J 11.4 Hz, Bn),
4.44 (1H, d, J 8.8 Hz, H-4), 4.40 (1H, d, J 11.4 Hz, Bn), 3.99
(1H, m, H-5), 3.79–3.84 (2H, m, H-8), 3.63 (1H, dd, J 10.4, 2.4
Hz, H-6), 3.47 (3H, s, OCH3), 3.41(1H, dd, J 10.4, 4.2 Hz, H-6),
Preparation of 1,2-O-isopropylidene-3,5,6-tri-O-benzyl-3-C-
vinyl-ꢁ-D-allofuranose 18
A 60% oily dispersion of NaH (869 mg, 21.73 mmol) was
suspended in anhydrous DMF (6 cm3) and the suspension was
stirred at 0 ЊC. A solution of 5 (1.015 g, 4.12 mmol) in
anhydrous DMF (7 cm3) was added dropwise over 30 min. The
mixture was stirred at 50 ЊC for 1 h and then cooled to 0 ЊC.
Benzyl bromide (2.45 cm3, 20.63 mmol) was added dropwise
and the reaction mixture was stirred at room temperature for
20 h. The solvent was removed by distillation under reduced
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 7 3 8 – 3 7 4 8
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pressure and the residue was added to a saturated aqueous solu-
tion of NaHCO3 (50 cm3) and extracted with dichloromethane
(3 × 50 cm3). The combined extracts were dried (Na2SO4) and
the solvent was removed by distillation under reduced pressure.
The residue was purified by chromatography over silica with
petroleum ether–ethyl acetate (7 : 3) as eluent to give the prod-
uct as a clear oil (2.105 g, 99%) (Found: C, 74.02; H, 7.15.
C32H36O6 requires C, 74.40, H; 7.02%); δH (300 MHz; CDCl3;
Me4Si) 7.35–7.15 (15H, m, Bn), 5.88 (1H, dd, J 11.4, 17.8 Hz,
H-7), 5.80 (1H, d, J 3.7 Hz, H-1), 5.40 (1H, d, J 11.4 Hz, H-8),
5.27 (1H, d, J 17.8 Hz, H-8), 4.81 (1H, d, J 11.7 Hz, Bn),
4.71–4.53 (6H, m, H-2, 5 × Bn), 4.36 (1H, d, J 7.0 Hz, H-4),
3.85–3.79 (2H, m, H-5, H-6), 3.07 (1H, m, H-6), 1.60 (3H, s,
CH3), 1.38 (3H, s, CH3); δC (75 MHz; CDCl3; Me4Si) 139.0,
138.8, 138.7 (Bn), 135.9 (C-7), 128.4, 128.2, 128.2, 127.7, 127.7,
127.6, 127.5, 127.3, 127.2 (Bn), 118.2 (C-8), 112.9 (C(CH3)2),
104.0 (C-1), 85.6 (C-3), 81.8 (C-2), 80.8 (C-4), 77.3 (C-5), 73.3
(Bn), 72.2, 72.0 (Bn, C-6), 67.2 (Bn), 26.9, 26.7 (C(CH3)2); m/z
(FAB) 539 (M ϩ Na).
Me4Si) 7.36–7.23 (15H, m, Bn), 6.04 (1H, dd, J 17.7, 11.2 Hz,
H-7), 5.59 (1H, d, J 17.7 Hz, H-8), 5.42 (1H, d, J 11.2 Hz, H-8),
5.16 (1H, d, J 3.4 Hz, H-1), 5.09 (1H, d, J 3.4 Hz, H-2), 4.75
(1H, d, J 11.3 Hz, Bn), 4.65 (2H, s, Bn), 4.57 (2H, s, Bn), 4.49
(1H, d, J 11.3 Hz, Bn), 4.42 (1H, d, J 7.4 Hz, H-4), 3.82 (1H, m,
H-6), 3.71–3.64 (2H, m, H-5, H-6), 3.42 (3H, s, OCH3), 2.99
(3H, s, SO2CH3); δC (75 MHz; CDCl3; Me4Si) 138.7, 138.2,
138.1 (Bn), 132.9 (C-7), 128.3, 128.2, 127.7, 127.6, 127.6, 127.5,
127.2, 126.9 (Bn), 118.8 (C-8), 106.0 (C-1), 85.0 (C-3), 84.1
(C-2), 83.9 (C-4), 78.3 (C-5), 73.4, 71.9 (Bn), 69.7 (C-6), 67.1
(Bn), 56.3 (OCH3), 38.9 (SO2CH3); m/z (FAB) 567 (M–H).
Methyl 2-O-methylsulfonyl-3,5,6-tri-O-benzyl-3-C-vinyl-ꢁ-D-
allofuranoside 21
(2.073 g, 34%) (Found: C, 65.57; H, 6.64; S, 5.43. C31H36O8S
requires C, 65.47; H, 6.38; S, 5.64%); δH (300 MHz; CDCl3;
Me4Si) 7.32–7.23 (15H, m, Bn), 6.01 (1H, dd, J 17.8, 11.1 Hz,
H-7), 5.42 (1H, d, J 17.8 Hz, H-8), 5.39 (1H, d, J 11.1 Hz, H-8),
5.28–5.07 (2H, m, H-1, H-2), 4.81–4.50 (6H, m, Bn), 4.44 (1H,
d, J 6.9 Hz, H-4), 3.82–3.65 (3H, m, H-5, H-6), 3.48 (3H, s,
OCH3), 3.00 (3H, s, SO2CH3); δC (75 MHz; CDCl3; Me4Si)
138.9, 138.3, 138.2 (Bn), 135.6 (C-7), 128.3, 128.2, 128.1, 127.6,
127.6, 127.6, 127.4, 127.2, 127.1 (Bn), 118.0 (C-8), 100.7 (C-1),
83.2 (C-3), 81.3 (C-4), 79.3 (C-2), 77.7 (C-5), 73.4, 72.1 (Bn),
70.5 (C-6), 67.2 (Bn), 55.8 (OCH3), 39.1 (SO2CH3).
Preparation of methyl 3,5,6-tri-O-benzyl-3-C-vinyl-D-
allofuranoside 19
A solution of 18 (7.31 g, 14.15 mmol) in methanol (35 cm3) and
water (10 cm3) was stirred at 0 ЊC and a 20% solution of HCl in
anhydrous methanol (280 cm3) added. The reaction mixture
was stirred at room temperature for 2.5 h and then quenched
by the addition of NaHCO3(s) and then water (800 cm3). The
mixture was extracted with dichloromethane (3 × 250 cm3), the
combined extracts were dried (MgSO4) and the solvent was
removed by distillation under reduced pressure. The residue was
purified by chromatography over silica with dichloromethane as
eluent to give the product as a clear oil (5.359 g, 77%) as a
mixture of α- and β-anomers in a 1 : 1 ratio (Found: C, 73.14;
H, 7.08. C30H34O6 requires C, 73.45; H, 6.99%); δH (300 MHz;
CDCl3; Me4Si) 7.39–7.23 (m, Bn), 6.15 (dd, J 17.5, 11.2 Hz,
H-7), 6.06 (dd, J 17.5, 10.9 Hz, H-7), 5.49 (dd, J 17.7, 1.1 Hz,
H-8), 5.42–5.30 (m, H-8), 4.91 (d, J 4.8 Hz, H-1α), 4.87 (d, J 3.2
Hz, H-1β), 4.86–4.78 (m, Bn), 4.67–4.34 (m, Bn, H-4), 4.13–
4.06 (m, H-2), 3.92–3.83 (m, H-6), 3.75–3.62 (m, H-6, H-5),
3.47 (s, OCH3), 3.41 (s, OCH3), 3.13 (d, J 10.6 Hz, OH), 3.07 (d,
J 7.5 Hz, OH); δC (75 MHz; CDCl3; Me4Si) 139.4, 138.5, 138.4,
138.3, 138.3 (Bn), 135.6, 133.4 (C-7), 128.5, 128.4, 128.4, 128.3,
128.2, 127.9, 127.8, 127.8, 127.7, 127.4, 127.1, 126.8 (Bn), 117.7,
116.4 (C-8), 109.2 (C-1β), 101.7 (C-1α), 85.6, 83.0 (C-3), 81.9,
81.7 (C-4), 79.6 (C-2), 78.2, 78.0 (C-5), 76.3 (C-2), 73.5, 72.3,
71.9, 70.5, 70.0 (Bn, C-6), 66.5, 66.4 (Bn), 56.2, 55.3 (OCH3);
m/z (FAB) 513 (M ϩ Na).
Preparation of methyl 2-O-methylsulfonyl-3-C-(1(R),2-di-
hydroxyethyl)-3,5,6-tri-O-benzyl-ꢀ-D-allofuranoside 22
A solution of the methylfuranoside 20 (3.24 g, 5.70 mmol) in
tert-butanol (45 cm3) and water (3 cm3) was stirred at room
temperature and pyridine (4.5 cm3), N-methylmorpholine-
N-oxide (4.69 g, 40.03 mmol) and a 2.5 w/w-% solution of
osmium tetroxide in tert-butanol (0.40 cm3, 1.28 mmol) were
added. The reaction mixture was stirred with reflux at 76 ЊC for
16 h and then quenched by the addition of a 20% aqueous
solution of Na2S2O5 (50 cm3) and then water (50 cm3). The
mixture was extracted with dichloromethane (3 × 100 cm3) and
the combined extracts were washed with a saturated aqueous
solution of NaHCO3 (100 cm3) and dried (MgSO4). The solvent
was removed by distillation under reduced pressure and the
residue was then co-evaporated with toluene (20 cm3) and puri-
fied by chromatography over silica with dichloromethane–
methanol (99 : 1) as eluent to give the product as a clear oil
(1.318 g, 38%), which was used without further purification in
the next step, as well as a fraction containing an epimeric mix-
ture (372 mg, 11%); δH (300 MHz; CDCl3; Me4Si) 7.39–7.26
(15H, m, Bn), 5.29 (1H, d, J 3.1 Hz, H-2), 5.14 (1H, d, J 3.1 Hz,
H-1), 4.78 (1H, d, J 11.0 Hz, Bn), 4.70–4.55 (5H, m, H-4, Bn),
4.46 (1H, d, J 11.0 Hz, Bn), 4.07 (1H, m, H-7), 4.00 (1H, m,
H-5), 3.90 (1H, dd, J 10.7, 2.4, H-6), 3.77 (1H, dd, J 11.6, 5.5
Hz, H-8), 3.71 (1H, dd, J 10.7, 3.8 Hz, H-6), 3.61 (1H, dd, J
11.6, 4.2 Hz, H-8), 3.43 (3H, s, OCH3), 3.04 (3H, s, SO2CH3),
2.04 (1H, br s, OH), 1.65 (1H, br s, OH); δC (75 MHz; CDCl3;
Me4Si) 138.0, 137.8, 137.3 (Bn), 128.6, 128.5, 128.4, 128.1,
128.1, 127.8, 127.7, 127.5 (Bn), 107.5 (C-1), 87.0 (C-3), 83.8
(C-2), 81.3 (C-4), 77.4 (C-5), 73.5, 71.8 (Bn), 71.5 (C-7), 68.0,
67.7 (Bn, C-6), 63.2 (C-8), 56.6 (OCH3), 38.5 (SO2CH3); m/z
(FAB) 625 (M ϩ Na).
Preparation of methyl 2-O-methylsulfonyl-3,5,6-tri-O-benzyl-3-
C-vinyl-D-allofuranoside 20 and 21
A solution of the methylfuranosides 19 (5.28 g, 10.76 mmol)
in anhydrous pyridine (20 cm3) was stirred at 0 ЊC. Methane
sulfonylchloride (1.25 cm3, 16.15 mmol) was added dropwise
and the reaction mixture was stirred at 0 ЊC for 2.5 h and then at
room temperature. The reaction was quenched by the addition
of ice and water (100 cm3) and extracted with CH2Cl2 (3 × 100
cm3). The combined extracts were washed with a saturated
aqueous solution of NaHCO3 (200 cm3) and dried (MgSO4).
The solvent was removed by distillation under reduced pressure
and the residue was then co-evaporated with toluene (10 cm3)
and purified by chromatography over silica with petroleum
ether–ethyl acetate (4 : 1) as eluent to give the two products as
clear oils.
Preparation of (1R,2R,4R,5S,7R)-1-benzyloxy-2-(1(R),2-
di(benzyloxy)ethyl)-7-hydroxymethyl-4-methoxy-3,6-dioxa-
bicyclo[3.2.0]heptane 23
A solution of 22 (1.29 g, 2.15 mmol) in anhydrous DMF
(10 cm3) was stirred at 0 ЊC and a 60% oily dispersion of NaH
(155 mg, 3.87 mmol) was added. The reaction mixture was
stirred at room temperature for 6 h and then quenched by the
addition of a saturated aqueous solution of NaHCO3 (75 cm3).
The mixture was extracted with dichloromethane (3 × 75 cm3)
and the combined extracts were dried MgSO4(s). The solvent
Methyl 2-O-methylsulfonyl-3,5,6-tri-O-benzyl-3-C-vinyl-ꢀ-D-
allofuranoside 20
(3.353 g, 55%) (Found: C, 65.54; H, 6.45; S, 5.55. C31H36O8S
requires C, 65.47; H, 6.38; S, 5.64%); δH (300 MHz; CDCl3;
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 7 3 8 – 3 7 4 8
3744

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was removed by distillation under reduced pressure and the
residue was then co-evaporated with xylene (10 cm3) and puri-
fied by chromatography over silica with dichloromethane–
methanol (99 : 1) as eluent to give the pure product as a clear oil
(1.011 g, 91%); δH (300 MHz; CDCl3; Me4Si) 7.38–7.25 (15H,
m, Bn), 4.93 (1H, d, J 2.8 Hz, H-2), 4.83–4.74 (3H, m, H-1, H-7,
Bn), 4.67–4.46 (5H, Bn) 4.30 (1H, d, J 8.6 Hz, H-4), 4.04–3.95
(2H, m, H-5, H-6), 3.89–3.85 (2H, m, H-8), 3.76 (1H, dd, J 10.5,
4.2 Hz, H-6), 3.54 (3H, s, OCH3), 2.14 (1H, t, J 6.8 Hz, OH);
δC (75 MHz; CDCl3; Me4Si) 138.2, 137.9, 137.0 (Bn), 128.6,
128.4, 128.3, 128.0, 127.8, 127.7, 127.6, 127.6, 127.5 (Bn), 104.1
(C-1), 85.8 (C-3), 84.4 (C-2), 82.9 (C-7), 77.1 (C-5), 74.4 (C-4),
73.5, 72.0 (Bn), 69.5 (C-6), 67.9 (Bn), 62.4 (C-8), 57.6 (OCH3);
HiRes MALDI FT-MS m/z (M ϩ Na) found/calcd. 529.2176/
529.2197.
m, H-5), 3.69–3.67 (2H, m, H-6), 3.43 (3H, s, OCH3), 0.14 (9H,
s, Si(CH3)3); δC (75 MHz; CDCl3; Me4Si) 166.5 (Bz), 138.4,
138.0 (Bn), 132.7, 130.3, 129.8, 128.3, 128.3, 128.1, 127.8, 127.6,
127.3, 126.4 (Bz, Bn), 104.9 (C-1), 94.6 (C-3), 84.2 (C-5), 84.0
(C-4), 81.6 (C-7), 75.2 (C-2), 73.6 (Bn), 69.8 (C-6), 66.7 (Bn),
64.9 (C-8), 55.2 (OCH3), Ϫ0.1 (Si(CH3)3); m/z (FAB) 615
(M ϩ Na).
26 (31 mg, 34%); δH (300 MHz; CDCl3; Me4Si) 8.11–8.07
(2H, m, Bz), 7.57–7.22 (13H, m, Bz, Bn), 4.98 (1H, d, J 5.6 Hz,
H-1), 4.91 (1H, dd, J 12.4, 2.3 Hz, H-8), 4.79 (1H dd, J 8.0, 2.3
Hz, H-7), 4.72 (1H, d, J 12.2 Hz, Bn), 4.66–4.56 (5H, m, H-2,
H-4, Bn), 4.50 (1H, dd, J 12.2, 8.0 Hz, H-8), 4.03 (1H, m, H-5),
3.68–3.66 (2H, m, H-6), 3.47 (3H, s, OCH3), 3.00 (1H, d, J 9.9
Hz, OH); δC (75 MHz; CDCl3; Me4Si) 166.9 (Bz), 138.2, 137.9
(Bn), 133.0, 129.8, 128.4, 128.4, 128.3, 127.8, 127.7, 127.4, 126.5
(Bz, Bn), 104.4 (C-1), 94.6 (C-3), 85.2 (C-4), 84.5 (C-5), 81.4
(C-7), 74.6 (C-2), 73.6 (Bn), 69.7 (C-6), 66.8 (Bn), 64.8 (C-8),
55.6 (OCH3); m/z (FAB) 521 (M ϩ H).
Preparation of (1R,2R,4R,5S,7R)-1-benzyloxy-7-benzoyloxy-
methyl-2-(1(R),2-di(benzyloxy)ethyl)-4-methoxy-3,6-dioxa-
bicyclo[3.2.0]heptane 24
Preparation of 1,2-di-O-acetyl-3,5,6-tri-O-benzyl-3-C-vinyl-D-
allofuranose 27
A solution of 23 (933 mg, 1.84 mmol) in anhydrous pyridine
(5 cm3) was stirred at 0 ЊC and benzoyl chloride (0.27 cm3, 2.33
mmol) was added. The reaction mixture was stirred at room
temperature for 20 min. and then quenched by the addition of a
saturated aqueous solution of NaHCO3 (30 cm3). The mixture
was extracted with dichloromethane (3 × 30 cm3) and the com-
bined extracts were dried (MgSO4). The solvent was removed
by distillation under reduced pressure and the residue was then
co-evaporated with toluene (5 cm3) and purified by chromato-
graphy over silica with dichloromethane–methanol (99 : 1) as
eluent to give the pure product as a clear oil (1.055 g, 94%)
(Found: C, 72.26; H, 5.95% C37H38O8 requires C, 72.77; H,
6.27%); δH (300 MHz; CDCl3; Me4Si) 8.00–7.97 (2H, m, Bz),
7.52 (1H, m, Bz), 7.39–7.18 (17H, m, Bz, Bn), 5.09 (1H, dd,
J 7.4, 3.2 Hz, H-7), 4.98 (1H, d, J 2.8 Hz, H-2), 4.81 (1H, d,
J 2.8 Hz, H-1), 4.77–4.69 (2H, m, H-8, Bn), 4.64–4.52 (6H, m,
H-8, Bn), 4.31 (1H, d, J 7.9 Hz, H-4), 4.05 (1H, ddd, J 7.9, 4.7,
3.0 Hz, H-5), 3.95 (1H, dd, J 10.7, 3.0 Hz, H-6), 3.76 (1H, dd,
J 10.7, 4.7 Hz, H-6), 3.54 (3H, s, OCH3); δC (75 MHz; CDCl3;
Me4Si) 166.3 (Bz), 138.2, 137.8, 137.2 (Bn), 132.9, 129.7 (Bz),
128.4, 128.3, 128.3, 128.2, 128.0, 127.8, 127.7, 127.6, 127.3 (Bn,
Bz), 104.1 (C-1), 85.2 (C-3), 84.8 (C-2), 81.5 (C-7), 76.9 (C-5),
75.0 (C-4), 73.4, 72.2 (Bn), 69.6 (C-6), 67.9 (Bn), 64.8 (C-8),
57.7 (OCH3); m/z (FAB) 633 (M ϩ Na).
A solution of 18 (8.45 g, 16.4 mmol) in 80% aqueous acetic acid
(55 cm3) was stirred at 90 ЊC for 16 h. The solvent was removed
under reduced pressure, and the residue was co-evaporated
successively with ethanol (3 × 20 cm3), toluene (3 × 20 cm3)
and anhydrous pyridine (3 × 25 cm3), and then re-dissolved in
anhydrous pyridine (30 cm3). Acetic anhydride (23 cm3) was
added and the solution was stirred at room temperature for
20 h. A mixture of water and ice (20 cm3) was added and the
resulting mixture was extracted with dichloromethane (3 × 100
cm3). The organic phase was washed with a saturated aqueous
solution of NaHCO3 (2 × 50 cm3) and dried (MgSO4). The
solvent was removed by distillation under reduced pressure and
the residue was purified by chromatography over silica with
petroleum ether–ethyl acetate (4 : 1 v/v) as eluent to give the
anomeric mixture as a clear oil (8.31 g, 90%); δH (300 MHz;
CDCl3; Me4Si) 7.32–7.25 (m, Bn), 6.39 (d, J 4.4 Hz, H-1), 6.17
(d, J 2.5 Hz, H-1), 6.07–6.01 (m, H-7), 5.62 (d, J 2.5 Hz, H-2),
5.56–5.36 (m, H-2, H-8), 4.82–4.77 (m, Bn), 4.62–4.44 (m, Bn,
H-4), 3.83–3.64 (m, H-6, H-5), 2.08–2.04 (m, COCH3); δC (75
MHz; CDCl ; Me Si) 169.8, 169.4 (C᎐O) 138.9, 138.6, 138.3,
᎐
3
4
138.3, 138.2 (Bn), 134.3, 133.1 (C-7), 128.4, 128.3, 128.3, 128.1,
128.0, 127.9, 127.6, 127.5, 127.5, 127.4, 127.4, 127.2, 127.2,
126.9, 126.8 (Bn), 118.8, 118.5 (C-8), 99.1, 93.5 (C-1), 85.1,
85.0, 84.5 84.1, 78.1, 77.7, 77.6, 73.4, 73.2, 72.2, 72.0, 70.3, 67.3,
67.2 (C-2, C-3, C-4, C-5, Bn), 21.1, 21.0, 20.8, 20.5 (COCH3);
HiRes MALDI FT-MS m/z (M ϩ Na) found/calcd. 583.2322/
583.2302.
Preparation of (1R,3R,4S,5R,6S,8R)-5-benzyloxy-8-benzyloxy-
methyl-6-benzoyloxymethyl-3-methoxy-4-trimethylsilyloxy-2,7-
dioxabicyclo[3.3.0]octane 25 and (1R,3R,4S,5R,6S,8R)-5-
benzyloxy-8-benzyloxymethyl-6-benzoyloxymethyl-4-hydroxy-3-
methoxy-2,7-dioxabicyclo[3.3.0]octane 26
Preparation of 1-(2Ј-O-acetyl-3Ј,5Ј,6Ј-tri-O-benzyl-3Ј-C-vinyl-ꢀ-
D-allofuranosyl)thymine 28
Compound 24 (106.3 mg, 0.174 mmol) and thymine (66 mg,
0.52 mmol) were dried and then dissolved in anhydrous
acetonitrile (1.5 cm3). The solution was stirred at room temper-
ature and bis(trimethylsilyl)acetamide (0.4 cm3, 1.62 mmol)
added, followed by stirring with reflux at 81 ЊC for 15 min. The
reaction mixture was cooled to 0 ЊC and TMS-triflate (0.13 cm3,
0.72 mmol) was added followed by stirring at 65 ЊC for 1 h. The
reaction was quenched by the addition of a saturated aqueous
solution of NaHCO3 (6 cm3) and water (10 cm3), and the
mixture was extracted with dichloromethane (3 × 10 cm3). The
combined extracts were dried (MgSO4), and the solvent was
removed by distillation under reduced pressure. The residue was
purified by chromatography over silica with dichloromethane–
methanol (49 : 1) as eluent to give the products as clear oils as
well as starting material (11 mg, 10%);
To a stirred solution of the anomeric mixture 27 (8.31 g, 14.8
mmol) and thymine (3.74 g, 29.6 mmol) in anhydrous
acetonitrile (200 cm3) was added bis(trimethylsilyl)acetamide
(18.3 cm3, 74.1 mmol). The reaction mixture was stirred at
reflux for 30 min. and cooled to 0 ЊC. TMS-triflate (4.60 cm3,
25.2 mmol) was added dropwise and the solution was stirred
at 50 ЊC for 16 h. The mixture was allowed to cool to room
temperature and the reaction was quenched with a saturated
aqueous solution of NaHCO3 (200 cm3) followed by extraction
with dichloromethane (3 × 200 cm3). The organic phase was
washed with a saturated aqueous solution of NaHCO3 (2 × 200
cm3) and dried (MgSO4). The solvent was removed by distil-
lation under reduced pressure and the residue was purified
by dry column vacuum chromatography42 over silica with
petroleum ether–ethyl acetate (2 : 1 v/v) as eluent to give the
product as a white foam (7.51 g, 81%); (Found: C, 68.96; H,
6.08; N, 4.24. C36H38N2O8 requires C, 68.99; H, 6.11; N, 4.47%);
δH (300 MHz; CDCl3; Me4Si) 8.71 (1H, s, NH), 7.37–7.22 (16H,
25 (36 mg, 35%); δH (300 MHz; CDCl3; Me4Si) 8.11–8.08
(2H, m, Bz), 7.53 (1H, m, Bz), 7.43–7.23 (12H, m, Bz, Bn), 4.94
(1H, dd, J 8.4, 1.7 Hz, H-7), 4.83 (1H, d, J 5.1 Hz, H-1), 4.75
(1H, dd, J 12.3, 1.7 Hz, H-8), 4.65–4.60 (3H, m, H-4, Bn),
4.59–4.54 (2H, m, Bn), 4.52–4.47 (2H, m, H-2, H-8), 4.08 (1H,
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 7 3 8 – 3 7 4 8
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m, Bn, H-6), 6.25 (1H, d, J 7.4 Hz, H-1Ј), 6.05 (1H, dd, J 17.9,
11.0 Hz, H-7Ј), 5.84 (1H, d, J 7.4 Hz, H-2Ј), 5.40 (1H, d, J 11.1
Hz, H-8Ј), 5.39 (1H, d, J 17.8 Hz, H-8Ј), 4.87–4.48 (6H, m, Bn),
4.45 (1H, d, J 5.9 Hz, H-4Ј), 3.90–3.85 (1H, m, H-5Ј), 3.79–3.70
(2H, m, H-6Ј), 2.09 (3H, s, COCH3), 1.60 (3H, s, CH3); δC (75
(Bn), 70.5 (BOM), 68.8 (C-6Ј), 67.1 (Bn), 38.9 (SO2CH3), 12.7
(CH3); HiRes MALDI FT-MS m/z (M ϩ Na) found/calcd.
805.2768/805.2765.
Preparation of 3-N-benzyloxymethyl-1-(2Ј-O-methylsulfonyl-3-
C-(1(R),2-dihydroxyethyl)-3Ј,5Ј,6Ј-tri-O-benzyl-ꢀ-D-allofurano-
syl)thymine 32
MHz; CDCl ; Me Si) 170.0 (C᎐O), 163.4 (C-4), 150.6 (C-2),
᎐
3
4
138.7, 137.8, 137.7 (Bn), 135.7 (C-6), 132.7 (C-7Ј), 128.5, 128.4,
128.3, 128.0, 127.7, 127.6, 127.4, 126.8, 126.8 (Bn), 119.1 (C-8Ј),
111.3 (C-5), 85.3, 85.0, 84.7 (C-1Ј, C-3Ј, C-4Ј), 79.02 (C-5Ј),
74.5 (C-2Ј), 73.4, 71.8 (Bn), 69.3 (C-6Ј), 67.2 (Bn), 20.7
(COCH3), 12.0 (CH3); HiRes MALDI FT-MS m/z (M ϩ Na)
found/calcd. 649.2528/649.2520.
To a solution of 31 (1.982 g, 2.53 mmol) in THF and water (1 : 1
v/v, 30 cm3) was added N-methylmorpholine-N-oxide (0.445 g,
3.80 mmol) and a 2.5 w/w-% solution of osmium tetroxide in
tert-butanol (1.27 cm3, 0.101 mmol) and the mixture was stirred
at 50 ЊC for 19 h. The reaction was quenched by adding a
saturated aqueous solution of sodium hydrogensulfite (6 cm3)
and the mixture was stirred for 30 min. at room temperature.
The solvent was partly removed by distillation under reduced
pressure followed by extraction with ethyl acetate (4 × 50 cm3).
The organic phase was dried (MgSO4) and the solvent was
removed by distillation under reduced pressure. The residue was
purified by chromatography over silica with dichloromethane–
methanol (99.5 : 0.5 v/v) as eluent to give the starting material,
the main product and three side-products. The compounds
were eluted as follows:
Preparation of 3-N-benzyloxymethyl-1-(3Ј,5Ј,6Ј-tri-O-benzyl-3Ј-
C-vinyl-ꢀ-D-allofuranosyl)thymine 30
To a stirred solution of 28 (7.51 g, 12.0 mmol) in anhydrous
acetonitrile (130 cm3) was added benzyloxymethyl chloride
(2.08 cm3, 15.0 mmol) and DBU (2.24 cm3, 15.0 mmol). After
stirring at room temperature for 18 h, the solvent was removed
by distillation under reduced pressure and the residue was
re-dissolved in anhydrous methanol (125 cm3). Sodium
methoxide (1.29 g, 24.0 mmol) was added and the mixture was
stirred at room temperature for 16 h. The mixture was neutral-
ised with acetic acid and the solvent was partly removed by
distillation under reduced pressure followed by extraction with
dichloromethane (2 × 250 cm3). The organic phase was washed
with a saturated aqueous solution of NaHCO3 (3 × 200 cm3)
and dried (MgSO4). The solvent was removed by distillation
under reduced pressure and the residue was purified by dry
column vacuum chromatography42 over silica with petroleum
ether–ethyl acetate (3 : 1 v/v) as eluent to give the product as
a white foam (6.32 g, 75%); δH (300 MHz; CDCl3; Me4Si)
7.38–7.23 (20H, m, Bn), 7.07 (1H, d, J 1.0 Hz, H-6), 6.18 (1H,
dd, J 17.6, 11.0 Hz, H-7Ј), 5.98 (d, J 7.7 Hz, H-1Ј), 5.52–5.41
(4H, m, H-8Ј, BOM), 4.87–4.49 (9H, m, Bn, H-4Ј), 4.30 (1H,
dd, J 10.3, 7.7 Hz, H-2Ј), 3.89 (1H, m, H-5Ј), 3.81–3.69 (2H, m,
H-6Ј), 2.93 (1H, d, J 10.3 Hz, 2Ј-OH), 1.64 (3H, d, J 1.0 Hz,
CH3); δC (75 MHz; CDCl3; Me4Si) 163.2 (C-4), 151.6 (C-2),
137.9, 137.7, 137.6 (Bn), 134.7 (C-6), 132.1 (C-7Ј), 128.5, 128.4,
128.3, 128.0, 127.8, 127.8, 127.7, 127.6, 127.3, 126.9 (Bn), 118.6
(C-8Ј), 110.6 (C-5), 88.8 (C-1Ј), 84.3 (C-3Ј), 81.7 (C-4Ј), 79.0
(C-5Ј), 76.4 (C-2Ј), 73.5, 72.1, 72.1 (Bn), 70.6 (BOM), 68.7
(C-6Ј), 66.2 (Bn), 12.7 (CH3); HiRes MALDI FT-MS m/z
(M ϩ Na) found/calcd. 727.3001/727.2990.
Starting material 31
(0.554 g, 28%).
3-N-Benzyloxymethyl-5,6-dihydroxy-5-methyl-1-(2Ј-O-methyl-
sulfonyl-3Ј,5Ј,6Ј-tri-O-benzyl-3Ј-C-vinyl-ꢀ-D-allofurano-
syl)pyrimidine-2,4-dion 32b
(0.144 g, 7%); δH (300 MHz; CDCl3; Me4Si) (major isomer)
7.37–7.24 (20H, m, Bn), 6.33 (1H, d, J 8.2 Hz, H-1Ј), 6.05 (1H,
dd, J 17.8, 11.2 Hz, H-7Ј), 5.63 (1H, d, J 17.7 Hz, H-8Ј), 5.51
(1H, d, J 11.2 Hz, H-8Ј), 5.40 (1H, d, J 8.2 Hz, H-2Ј), 5.35 (1H,
d, J 10.3 Hz, BOM), 5.24 (1H, d, J 10.3 Hz, BOM), 4.97 (1H, d,
J 2.2 Hz, H-6), 4.93–4.39 (9H, m, Bn, H-4Ј), 3.87 (1H, m, H-5Ј),
3.76–3.67 (2H, m, H-6Ј), 3.42 (1H, s, 5-OH), 3.01 (1H, d, J 2.2
Hz, 6-OH), 2.82 (3H, s, SO2CH3), 2.32 (1H, dd, J 7.0, 5.3 Hz,
8Ј-OH), 1.31 (3H, s, CH3); δC (75 MHz; CDCl3; Me4Si) (major
isomer) 173.7 (C-4), 151.1 (C-2), 138.7, 137.5, 137.4 (Bn), 131.7
(C-7Ј), 128.7, 128.6, 128.5, 128.4, 128.3, 128.3, 128.2, 128.0,
127.9, 127.8, 127.6, 127.5, 127.3, 126.8, 126.7 (Bn), 120.4 (C-8Ј),
84.5 (C-3Ј), 84.1 (C-4Ј), 83.8 (C-1Ј), 80.8 (C-2Ј), 80.2 (C-5Ј),
77.3 (C-6), 73.5, 72.4 (Bn), 72.0 (C-5), 71.4 (Bn), 70.5 (BOM),
69.3 (C-6Ј), 67.1 (Bn), 39.0 (SO2CH3), 22.2 (CH3); HiRes
MALDI FT-MS m/z (M ϩ Na) found/calcd. 839.2857/
839.2820.
Preparation of 3-N-benzyloxymethyl-1-(2Ј-O-methylsulfonyl-
3Ј,5Ј,6Ј-tri-O-benzyl-3Ј-C-vinyl-ꢀ-D-allofuranosyl)thymine 31
Compound 30 (6.28 g, 8.92 mmol) was dissolved in anhydrous
pyridine (140 cm3) and cooled to 0 ЊC. Methane sulfonylchlor-
ide (1.73 cm3, 22.3 mmol) was added dropwise and the solution
was stirred at room temperature for 75 min. The reaction was
quenched with water and ice (200 cm3) and the mixture was
extracted with dichloromethane (3 × 400 cm3). The organic
phase was washed with a saturated aqueous solution of
NaHCO3 (300 cm3), dried (MgSO4) and the solvent was
removed by distillation under reduced pressure. The residue was
purified by column chromatography over silica with dichloro-
methane–methanol (99 : 1 v/v) as eluent to give the product as
a white foam (6.37 g, 91%); δH (300 MHz; CDCl3; Me4Si) 7.37–
7.25 (20H, m, Bn), 7.16 (1H, d, J 1.0 Hz, H-6), 6.28 (1H, d, J 7.1
Hz, H-1Ј), 6.08 (1H, dd, J 17.8, 11.0 Hz, H-7Ј), 5.59–5.47 (5H,
m, H-2Ј, H-8Ј, BOM), 4.87–4.49 (9H, m, Bn, H-4Ј), 3.95 (1H,
m, H-5Ј), 3.78–3.69 (2H, m, H-6Ј), 2.96 (3H, s, SO2CH3), 1.61
(3H, d, J 1.0 Hz, CH3); δC (75 MHz; CDCl3; Me4Si) 163.2 (C-4),
151.3 (C-2), 138.3, 137.8, 137.6, 137.6 (Bn), 134.7 (C-6), 131.8
(C-7Ј), 128.6, 128.4, 128.4, 128.3, 128.2, 127.8, 127.7, 127.6,
127.5, 126.9, 126.7 (Bn), 120.1 (C-8Ј), 110.9 (C-5), 86.6 (C-1Ј),
84.4 (C-3Ј), 84.2 (C-4Ј), 79.5 (C-2Ј), 79.0 (C-5Ј), 73.4, 71.9, 71.8
3-N-Benzyloxymethyl-1-(2Ј-O-methylsulfonyl-3-C-(1(S ),2-
dihydroxyethyl)-3Ј,5Ј,6Ј-tri-O-benzyl-ꢀ-D-allofuranosyl)thymine
32a
(0.141 g, 7%); δH (300 MHz; CDCl3; Me4Si) 7.37–7.23 (20H, m,
Bn), 6.96 (1H, d, J 0.9 Hz, H-6), 6.03 (1H, d, J 7.1 Hz, H-2Ј),
5.66 (1H, d, J 7.1 Hz, H-1Ј), 5.50 (1H, d, J 9.9 Hz, BOM), 5.44
(1H, d, J 9.9 Hz, BOM), 5.06–4.48 (8H, m, Bn), 4.78 (1H, d,
J 9.1 Hz, H-4Ј), 4.36 (1H, m, H-5Ј), 4.18 (1H, d, J 4.1 Hz,
7Ј-OH), 4.04–3.99 (1H, m, H-7Ј), 3.94 (1H, dd, J 11.0, 2.4 Hz,
H-6Ј), 3.88–3.85 (2H, m, H-8Ј), 3.74 (1H, dd, J 11.1, 2.9 Hz,
H-6Ј), 3.00 (3H, s, SO2CH3), 2.32 (1H, dd, J 7.0, 5.3 Hz, 8Ј-OH),
1.87 (3H, d, J 0.9 Hz, CH3); δC (75 MHz; CDCl3; Me4Si) 163.3
(C-4), 151.1 (C-2), 138.3, 138.2, 137.8 (Bn), 136.0 (C-6), 128.8,
128.6, 128.5, 128.4, 128.3, 128.2, 127.9, 127.8, 127.8, 127.6,
127.3 (Bn), 110.6 (C-5), 92.8 (C-1Ј), 85.4 (C-3Ј), 79.2 (C-4Ј),
78.4 (C-5Ј), 75.4 (C-2Ј), 73.4 (Bn), 73.0 (C-7Ј), 72.0, 71.7 (Bn),
70.3 (BOM), 68.2, 66.9 (C-6Ј, Bn), 61.9 (C-8Ј), 38.6 (SO2CH3),
12.9 (CH3); HiRes MALDI FT-MS m/z (M ϩ Na) found/calcd.
839.2811/839.2820.
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3-N-Benzyloxymethyl-1-(2Ј-O-methylsulfonyl-3-C-(1(R),2-
dihydroxyethyl)-3Ј,5Ј,6Ј-tri-O-benzyl-ꢀ-D-allofuranosyl)thymine
32
J 2.6 Hz, H-1Ј), 5.00 (1H, t, J 4.1 Hz, H-7Ј), 4.86 (1H, d, J 2.6
Hz, H-2Ј), 4.02 (1H, m, H-5Ј), 3.92–3.86 (2H, m, H-8Ј), 3.76–
3.71 (2H, m, H-4Ј, H-6Ј), 3.59 (1H, dd, J 11.7, 5.5 Hz, H-6Ј),
1.80 (3H, s, CH3); δC (75 MHz; CD3OD; Me4Si) 166.3 (C-4),
152.0 (C-2), 139.9 (C-6), 110.0 (C-5), 89.7 (C-2Ј), 86.5 (C-7Ј),
85.0 (C-1Ј), 82.2 (C-4Ј), 81.6 (C-3Ј), 71.9 (C-5Ј), 65.2, 63.4
(C-6Ј, C-8Ј), 12.4 (CH3); HiRes MALDI FT-MS m/z (M ϩ Na)
found/calcd. 353.0967/353.0955.
(0.631 g, 31%); δH (300 MHz; CDCl3; Me4Si) 7.38–7.22 (21H,
m, Bn, H-6), 6.20 (1H, d, J 6.7 Hz, H-1Ј), 5.52 (2H, s, BOM),
5.45 (1H, d, J 6.7 Hz, H-2Ј), 4.88–4.55 (9H, m, Bn, H-4Ј), 4.34
(1H, m, H-5Ј), 4.27 (1H, m, H-7Ј), 3.89–3.71 (5H, m, H-6Ј,
H-8Ј, 7Ј-OH), 2.83 (3H, s, SO2CH3), 2.13 (1H, t, J 6.1 Hz,
8Ј-OH), 1.94 (3H, s, CH3); δC (75 MHz; CDCl3; Me4Si) 163.2
(C-4), 151.5 (C-2), 137.8, 137.6, 137.5, 137.1 (Bn), 134.2 (C-6),
128.6, 128.4, 128.4, 128.3, 128.0, 127.9, 127.8, 127.7, 127.6,
126.8, (Bn), 111.5 (C-5), 86.3 (C-1Ј), 81.9 (C-3Ј), 78.7 (C-4Ј),
77.2, 77.2 (C-2Ј, C-5Ј), 73.6, 73.4, 71.8 (Bn), 71.7 (C-7Ј), 70.6
(BOM), 68.4 (C-6Ј), 67.6 (Bn), 61.8 (C-8Ј), 38.6 (SO2CH3), 13.4
(CH3); HiRes MALDI FT-MS m/z (M ϩ Na) found/calcd.
839.2844/839.2820.
Preparation of (1R,2R,4R,5S,7R)-2,7-di(hydroxymethyl)-1-
hydroxy-4-(thymin-1-yl)-3,6-dioxabicyclo[3.2.0]heptane 35
To a solution of 34 (0.0125 g, 0.0378 mmol) in dioxane and
water (1 : 1 v/v, 1 cm3) was added a solution of sodium period-
ate (0.0081 g, 0.0378 mmol) in water (0.08 cm3). The mixture
was stirred for 50 min. at room temperature, and subsequently,
ethanol was added until the precipitation of sodium iodate was
observed. The solution was filtered, sodium borohydride
(0.0013 g, 0.0344 mmol) was added and the mixture was stirred
at room temperature for 35 min. The mixture was filtered and
neutralised with 10% aqueous acetic acid, and the solvent was
removed by distillation under reduced pressure. The residue was
purified by chromatography over silica with dichloromethane–
methanol (95 : 5 v/v) as eluent to give the product as a white
solid (0.0101 g, 89%); δH (300 MHz; CD3OD; Me4Si) 7.82 (1H,
d, J 0.9, H-6), 5.93 (1H, d, J 2.8 Hz, H-1Ј), 4.92 (1H, d, J 2.8
Hz, H-2Ј), 4.90 (1H, t, J 4.2 Hz, H-6Ј), 4.04–3.83 (5H, m, H-4Ј,
H-5Ј, H-7Ј), 1.88 (3H, d, J 0.9, CH3); δC (75 MHz; CD3OD;
Me4Si) 166.3 (C-4), 152.0 (C-2), 140.0 (C-6), 110.0 (C-5), 89.7,
86.0, 85.2, 84.3, 81.2 (C-1Ј, C-2Ј, C-3Ј, C-4Ј, C-6Ј), 63.2, 61.0
(C-5Ј, C-7Ј), 12.4 (CH3); HiRes MALDI FT-MS m/z (M ϩ Na)
found/calcd. 323.0849/323.0850.
3-N-Benzyloxymethyl-5,6-dihydroxy-5-methyl-1-(2Ј-O-methyl-
sulfonyl-3-C-(1,2-dihydroxyethyl)-3Ј,5Ј,6Ј-tri-O-benzyl-ꢀ-D-
allofuranosyl)pyrimidine-2,4-dion 32c
(0.131 g, 6%); HiRes MALDI FT-MS m/z (M ϩ Na) found/
calcd. 873.2891/873.2875.
Preparation of (1R,2R,4R,5S,7R)-1-benzyloxy-2-(1(R),2-
di(benzyloxy)ethyl)-7-hydroxymethyl-4-(3-N-(benzyloxy-
methyl)thymin-1-yl)-3,6-dioxabicyclo[3.2.0]heptane 33
A stirred solution of 32 (0.362 g, 0.443 mmol) in anhydrous
DMF (2.5 cm3) was cooled to 0 ЊC and a 60% oily dispersion of
NaH (0.027 g, 0.67 mmol) was added. The mixture was stirred
at room temperature for 20 h. The reaction was quenched
with a saturated aqueous solution of NaHCO3 (12 cm3) and
extracted with dichloromethane (3 × 30 cm3). The organic
phase was dried (MgSO4) and the solvent was removed by distil-
lation under reduced pressure. The residue was purified by
column chromatography over silica with dichloromethane–
methanol (99.5 : 0.5 v/v) as eluent to give the product as a white
foam (0.280 g, 88%); δH (300 MHz; CDCl3; Me4Si) 7.48 (1H, s,
H-6), 7.39–7.25 (20H, m, Bn), 5.96 (1H, d, J 2.1 Hz, H-1Ј), 5.47
(2H, s, BOM), 5.19 (1H, d, J 2.1 Hz, H-2Ј), 4.94 (1H, t, J 5.0
Hz, H-7Ј), 4.85–4.52 (8H, m, Bn), 4.39 (1H, d, J 7.7 Hz, H-4Ј),
4.12 (1H, m, H-5Ј), 3.93–3.88 (3H, m, H-6Ј, H-8Ј), 3.73 (1H, dd,
J 10.5, 5.0 Hz, H-6Ј), 2.09 (1H, t, J 6.8 Hz, 8Ј-OH), 1.88 (3H, s,
CH3); δC (75 MHz; CDCl3; Me4Si) 163.2 (C-4), 150.8 (C-2),
137.8, 137.7, 136.4, 136.1 (Bn, C6), 128.7, 128.6, 128.4, 128.3,
128.0, 127.8, 127.7, 127.7, 127.6, 127.5 (Bn), 109.2 (C-5), 85.2
(C-3Ј), 83.9 (C-2Ј), 83.8 (C-1Ј), 83.1 (C-7Ј), 76.6 (C-5Ј), 75.3
(C-4Ј), 73.6, 72.6, 72.1 (Bn), 70.5 (BOM), 69.6 (C-6Ј), 68.2 (Bn),
62.2 (C-8Ј), 13.3 (CH3); HiRes MALDI FT-MS m/z (M ϩ Na)
found/calcd. 743.2918/743.2939.
Acknowledgements
The Danish Natural Science Research Council and the Danish
National Research Foundation are thanked for financial
support. Ms Birthe Haack is thanked for invaluable synthetic
assistance.
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