Dinucleotides containing two allyl groups by combinations of allyl phosphotriesters, 5-allyl-, 2′-O-allyl- and 2′-arabino-O-allyl uridine derivatives as substrates for ring-closing metathesis

Article abstract of DOI:10.1016/j.tet.2004.09.023

Five different dinucleotides, each containing two allyl groups in various positions, were prepared and studied as substrates for ring-closing metathesis reactions. These dinucleotides were designed from appropriate nucleoside building blocks combining four different positions for the allyl group; the allyl phosphotriester linkage, 5-allyl-2′-deoxyuridine, and ribo- as well as arabino-configured 2′-O-allyluridine. Thus, convenient procedures for these building blocks were developed. From the dinucleotides, two new cyclic nucleotide structures were obtained; one connecting two adjacent nucleobase moieties and the other forming an unsaturated four-carbon linkage between the phosphate moiety and the adjacent pyrimidine nucleobase. The latter cyclic dinucleotide was also prepared with a saturated four-carbon linkage using a tandem ring-closing metathesis-hydrogenation procedure. This compound was found to be significantly more stable towards a nucleophilic ring-opening than its unsaturated counterpart.

Full text of DOI:10.1016/j.tet.2004.09.023

Tetrahedron 60 (2004) 10955–10966
Dinucleotides containing two allyl groups by combinations of allyl
phosphotriesters, 5-allyl-, 20-O-allyl- and 20-arabino-O-allyl
uridine derivatives as substrates for ring-closing metathesis
*
Philip Børsting, Morten Freitag and Poul Nielsen
Nucleic Acid Center,† Department of Chemistry, University of Southern Denmark, 5230 Odense M, Denmark
Received 9 July 2004; revised 24 August 2004; accepted 9 September 2004
Available online 30 September 2004
Abstract—Five different dinucleotides, each containing two allyl groups in various positions, were prepared and studied as substrates for
ring-closing metathesis reactions. These dinucleotides were designed from 0appropriate nucleoside building blocks combining four diffe0rent
positions for the allyl group; the allyl phosphotriester linkage, 5-allyl-2 -deoxyuridine, and ribo- as well as arabino-configured 2 -O-
allyluridine. Thus, convenient procedures for these building blocks were developed. From the dinucleotides, two new cyclic nucleotide
structures were obtained; one connecting two adjacent nucleobase moieties and the other forming an unsaturated four-carbon linkage
between the phosphate moiety and the adjacent pyrimidine nucleobase. The latter cyclic dinucleotide was also prepared with a saturated four-
carbon linkage using a tandem ring-closing metathesis–hydrogenation procedure. This compound was found to be significantly more stable
towards a nucleophilic ring-opening than its unsaturated counterpart.
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
In the studies of nucleic acid chemical biology, a significant
number of conformationally restricted nucleic acid frag-
ments have been designed.12 As a relatively new approach,
however, the relationship between structure and function of
nucleic acid secondary and tertiary structures13 has
motivated the preparation of nucleic acids with covalent
intra- and interstrand linkages.14 Sekine and co-workers
have introduced a number of cyclic nucleotide and
dinucleotide structures for mimicking nucleic acid second-
ary structures.15 In these cases, linkages have been
established between the 20-position and the nucleobase in
the two adjacent nucleosides in a dinucleotide or between
the nucleobase and the adjacent phosphate moiety.15
Recently, another example of a conformationally restricted
dinucleotide for this purpose with a linkage between the
phosphate and the adjacent 50-position has been intro-
duced.16 In all these cases, the covalent linkages have been
obtained by conventional phosphoramidite chemistry,17
disulfide bond formation and/or peptide/amide bond
formation.14–16 We have concentrated on the development
of a general methodology based on RCM for the construc-
tion of this type of conformationally restricted nucleic acid
fragments. Thus, we have synthesised a series of diastereo-
meric dinucleotides with phosphotriester internucleotide
linkages containing seven-membered rings.7 These were
prepared by an RCM reaction on a dinucleotide substrate in
which two terminal double bonds have been introduced by a
50-C-vinyl moiety and an allyl phosphotriester linkage.7 In a
The application of ring-closing olefin metathesis (RCM) for
the preparation of medium and large rings has been a major
tool in organic and bioorganic chemistry.1 In particular, the
introduction of highly efficient and functional group tolerant
ruthenium-based catalysts by Grubbs and co-workers has
made metathesis technology generally attractive.2 A large
number of heterocyclic rings have been constructed by
RCM,1,3 and recently, the application of RCM for the
synthesis of phosphorus heterocycles has been specifically
reviewed.4 In a bioorganic context, cyclic peptide or peptide
mimetic structures have been achieved by RCM reactions.5
In view of this, we have focused on the application of RCM
in nucleic acid chemistry,6–10 and as a result, conformation-
ally restricted bi- and tricyclic nucleoside monomers6 as
well as di- and trinucleotides with large cyclic structures
have been achieved.7–10 In addition, other research groups
have recently prepared different nucleoside derivatives by
RCM based strategies.11
Keywords: Ring-closing metathesis; Nucleosides; Dinucleotides; Confor-
mational restriction; Nucleic acid secondary structures.
* Corresponding author. Tel.: C45 65502565; fax: C45 66158780;
e-mail: 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.2004.09.023

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P. Børsting et al. / Tetrahedron 60 (2004) 10955–10966
similar way, a ring connecting the phosphotriester linkage in
a dinucleotide with a nucleobase has been demonstrated,8,9
and two adjacent phorphortriester linkages have been linked
in a cyclic trinucleotide structure.8 Thus, a tandem RCM
and hydrogenation procedure efficiently mediated the
preparation of cyclic dinucleotides with a butylene nucleo-
base phosphotriester connection, which was unobtainable
by a conventional hydrogenation method.9 In this paper,
some of these results are reported with all experimental
details, and the general study on exploring the scope of ring-
closing metathesis in the construction of cyclic dinucleo-
tides with large rings is continued. Thus, four different
positions of terminal double bonds were investigated, the
appropriate nucleoside building blocks were prepared, and a
selection of the possible dinucleotides were synthesised and
investigated as substrates for RCM. Hereby, two cyclic
dinucleotide structures were obtained.
Scheme 1. Reagents and conditions: (a) DMTCl, AgNO3, pyridine, 84%;
(b) TBDMSCl, AgNO3, pyridine, 86%; (c) p-TsOH, CH2Cl2, CH3OH, 0 8C,
88%; (d) TBDMSCl, AgNO3, pyridine, 42%; (e) CEOP(N(i-Pr)2)2, 4,5-
dicyanoimidazole, CH2Cl2, CH3CN, 75%. DMTZ4,40-dimethoxytrityl,
TBDMSZtert-butyldimethylsilyl, CEZ2-cyanoethyl.
2. Results and discussion
Four different positions for the allyl groups were deduced
for incorporation into dinucleotides; the allyl phosphotri-
ester linkage A, 5-allyl-20-deoxyuridine B, 20-O-allyluridine
C, and 20-arabino-O-allyluridine D (Fig. 1). Among these,
A and a 5-allyluridine counterpart of B have been applied in
our earlier studies.7,8 From these four different allyl
positions, a series0 of 12 different dinucleotides might be
constructed; (50–3 ) AB, AC, AD, BB, BC, BD, CB, CC,
CD, DB, DC and DD, as A cannot logically be placed in the
30-end of the dinucleotides. Nevertheless, we decided to
make only a section of five of these dinucleotides
representing all of the four different allyl positions in at
least two dinucleotides each. Thus, AB,9 AD, BB, CB and
CD were produced and investigated as substrates for RCM
reactions.
Hereby, the appropriate building block for incorporation of
A into dinucleotides by standard phosphoramidite chem-
istry17 was formed. For the 5-allyluracil alternative B,
5-allyl-20-deoxyuridine 2 was synthesised from 20-deoxy-
uridine in two steps using a known procedure based on the
formation of an organomercuri-intermediate followed by a
transmetallation with Li2PdCl4 and allylation with
allylchloride in methanol.19 Subsequently, 2 was converted
to the 30-protected derivative 5 in three steps, that is,
selective 50-O-tritylation to give 3, 30-O-silylation to give 4,
both reactions accelerated by silver nitrate, and finally
acidic detritylation to give the product 5 in 64% yield over
the three steps (Scheme 1). Hereby, the building block for
incorporating B in the 30-end of the dinucleotides was
formed.9 Furthermore, 2 was selectively protected at the
50-position as a silyl ether to give 6 and converted to the
30-phosphoramidite 7 using a phosphordiamidite reagent
and 4,5-dicyanoimidazole20 as an activating reagent. This
formed the building block for incorporating B in the 50-end
of the dinucleotides.
In order to make dinucleotides with an allyl phosphotriester
linkage A, the phosphoramidite 1 (Scheme 1) was formed in
two steps from thymidine as shown in the literature.7,18
The formation of 20-O-allyl-ribonucleosides, C, is well
described in the literature.21–24 Thus, Sproat et al.
synthesised 20-O-allyluridine21 via a neutral palladium(0)
catalysed allylation method25 combined with a 50,30-TIPDS
protection of the two alcohols and a 4-O-(2,6-dichloro-
phenyl) protection of the uracil moiety.21 Later, it was
demonstrated that the allylation can be performed with a
simpler and cheaper 3-N-(4-tert-butylbenzoyl) group as
protection of the uracil moiety.22 Recently, also a 4-O-(2,6-
dimethylphenyl) protection of the uracil has been success-
fully applied in the same reaction.23 On the other hand, a
conventional Williamson type allylation, that is, allyl
bromide and sodium hydride, has been used with 50-O-
MMT-30-O-TBDMS-uridine as substrate.24 Thus, an allyla-
tion can be performed without a protecting group for the
uracil, and we decided to attempt the same reaction with
TIPDS-protected uridine 8 (Scheme 2). However, this led to
only 20% of the 20-O-allyl derivative due to a partial basic
cleavage of the silyl ether moieties. Subsequently, we
Figure 1. The four different positions for allyl groups in this study.

P. Børsting et al. / Tetrahedron 60 (2004) 10955–10966
10957
the same procedure for allylation as applied with the ribo-
configured uridine. Thus, the known TIPDS-protected
arabino-configured uridine 1429 was treated with benzoyl
chloride using the same phase transfer conditions as before
but in this case only 34% yield of the 3-N-benzoylated
product 15 was obtained due to a formation of a significant
amount of the 3-N,20-O-dibenzoylated product. Therefore,
an in situ trimethylsilyl protection of the 20-O-position was
accomplished before a conventional N-benzoylation to give,
after mild acidic desilylation, 15 in 81% yield over the three
steps (Scheme 2). The palladium based allylation22,25
afforded 16, and after complete deprotection, the target 20-
O-allyl-arabinouridine 17 was obtained in 76% yield from
15. Hereby, 17 has been obtained in approximately 53%
overall yield from arabinouridine, that is, 45% from uridine
counting the two step conversion of the 20-configuration.30
In comparison, the reported method,28 albeit in fewer steps,
gave 17 in only approximately 17% overall yield from
uridine. Finally, 17 was reprotected as its bis-silyl ether 18
in 83% yield and selectively deprotected using mild acid to
give 19 in 73% yield as0the appropriate building block for
incorporating D in the 3 -end of the dinucleotides.
The five different protected dinucleotides 20–24 were made
by the same general method using standard phosphorami-
dite chemistry17 (Schemes 3–5). Thus, the appropriate
50-alcohols and 30-phosphoramidites were coupled with 1H-
tetrazole as the activator followed by oxidation with
tBuOOH. This oxidation reagent was efficient and easily
handled, and the alternative standard reagent iodine was
avoided due to the presence of double bonds. The general
procedure afforded the dinucleotides in 60% to 100% yields.
In all cases, the expected mixtures of two phosphorus
epimers were obtained in approximately equimolar ratios as
Scheme 2. Reagents and conditions: (a) BzCl, Bu4NBr, CH2Cl2, aq
Na2CO3, 77%; (b) allylethylcarbonate, tris(benzylidenacetone)dipalla-
dium(0), bis(diphenylphosphino)butane, THF, 92%, reflux; (c) i. TBAF,
THF, ii. aq NH3, CH3OH, 95%; (d) TBDMSCl, AgNO3, pyridine, 79%; (e)
CEOP(Cl)N(i-Pr)2, EtN(i-Pr)2, CH2Cl2, 65%; (f) i. TMSCl, Et3N, CH2Cl2,
ii. BzCl, EtN(i-Pr)2, pyridine, iii. p-TsOH, CH2Cl2, 81%; (g) as (b), (h) as
(c), 75%, two steps; (i) TBDMSCl, imidazole, DMF, 83%; (j) 80% aq
AcOH, 73%. TBDMSZtert-butyldimethylsilyl, TMSZtrimethylsilyl,
CEZ2-cyanoethyl.
1
estimated from H and 31P NMR.
decided to apply the neutral palladium(0) catalysed
allylation method25 with 8. Thus, in this first attempt,
nucleobas0e protection was avoided and, as expected, only
the 3-N,2 -O-dialkylated product, or even the 3-N-mono-
alkylated product, was obtained. Therefore, we decided to
follow the procedure by Sproat and co-workers22 but with a
conventional 3-N-benzoyl protection of the uracil instead of
the 3-N-(4-tert-butylbenzoyl) group. Thus, the TIPDS-
protected uridine 8 was treated with benzoyl chloride in a
known phase transfer reaction with DCM/Na2CO3(aq) and
tetrabutylammonium bromide as phase transfer catalyst to
give 9 in 77% yield (Scheme 2).26,27 The palladium(0)
catalysed allylation procedure25 applied with 9 afforded 10
in 92% yield. Subsequently, desilylation with TBAF in THF
followed by debenzoylation with 25% ammonia in methanol
gave 20-O-allyluridine 11 in 95% yield, that is a con-
veniently 67% overall yield from 8. Finally, 11 was
reprotected as a silyl ether at the 50-position to give 12
and converted to the 30-phosphoramidite 13 by standard
methods (Scheme 2). Hereby, the building block for
incorporating C in the 50-end of the dinucleotides was
formed.
Subsequently, all the protected dinucleotides 20–24 were
investigated as substrates for RCM reactions using 5–
15 mol% of Grubbs second-generation catalyst X (MesZ
2,4,6-trimethylphenyl, CyZcyclohexyl),2 in dichloro-
methane or 1,2-dichloroethane under reflux. We only
investigated this catalyst, as this is by far the most generally
successful according to the literature1–4 as well as the most
successfully applied in our lab for the synthesis of other
dinucleotides with large rings.7–10 We did not make a
thorough investigation of a range of solvents, as dichloro-
methane and 1,2-dichloroethane have been the only
successful solvents in our previous studies on nucleoside
and nucleotide substrates.6–10
The formation of 20-O-allyl-arabinouridine, D, has also been
described in the literature.28 However, only the conven-
tional Williamson ether formation has been demonstrated
using 50,30-bis-O-tetrahydropyranyl protected arabinouri-
dine without protection of the uracil moiety to afford the 20-
O-allyl product in 40% yield.28 Therefore, we decided to use
Of the five different dinucleotide substrates, however,
neither 20, 21 or 22 were good substrates for RCM reactions
under these conditions (Scheme 3). In the case of 21 the
substrate was converted to a highly polar material indicating
polymerisation products, whereas both 20 and 22 could be
re-isolated from the reaction mixtures. In the latter case,

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P. Børsting et al. / Tetrahedron 60 (2004) 10955–10966
with an approximately 1:10 ratio of phosphorus epimers.
Nevertheless, the formation of a ring-closed product was
also confirmed by MS showing the expected loss of
ethylene, and by H NMR demonstrating the conversion
1
of terminal to internal double bonds.
Finally, the dinucleotide 249 turned out to be the most
efficient substrate for RCM. Thus, a treatment with 5 mol%
of the catalyst in dichloromethane afforded 26 in 58% yield
as a mixture of two phosphorus epimers in an equimolar
ratio as deduced from 31P NMR (Scheme 5). Again the
1
product was also confirmed by MS and H NMR showing
the loss of ethylene and the conversion of terminal to
internal double bonds. Thus, this RCM reaction was
performed very smoothly, as it has also been demonstrated
for its 30-ribonucleosidic analogue.8 Thus, 26 was the most
easily obtained of the five projected cyclic dinucleotides,
and subsequently, 26 has been further investigated for its
stability towards ammonia.9 Thus, ammonia is a standard
reagent used in the final deprotection step in oligonucleotide
synthesis, and as an allylic phosphotriester, 26 was expected
to be labile towards nucleophiles. Thus, in an analytical
experiment treating 26 with 32% aqueous ammonia for 24 h
at room temperature, the allylic phosphotriester was found
to react completely with ammonia at the allylic CH2 group
located next to the phosphate to give a zwitterionic
ammonia adduct 27 as a dinucleotide with an achiral
phosphordiester linkage in which also both TBDMS ethers
were hydrolysed.9 In order to increase the stability of the
phosphotriester, we decided to prepare the saturated
analogue of 26. Thus, 24 was applied in a tandem RCM
and hydrogenation reaction31 by performing the RCM
reaction as described earlier followed by hydrogenation of
the reaction mixture in a Parr-bomb with 1000 psi H2 at
50 8C. This gave the cyclic dinucleotide 28 in 63% yield as
an equimolar mixture of two phosphorus epimers.9 The
protecting groups were subsequently removed by an acidic
treatment to give the cyclic dinucleotide 29 in a quantitative
yield. This dinucleotide was considerably more stable
towards ammonia, as experiments showed that treatment
with 32% NH3(aq) at room temperature for 24 h resulted in
only 10% conversion to the dinucleotide 30 as indicated by
31P NMR and MS. A harsher treatment with 32% NH3(aq) at
55 8C for 5 days resulted, however, in a complete
conversion to the ammonia adduct 30.
Scheme 3. Reagents and conditions: (a) i. 1H-tetrazole, CH2Cl2, CH3CN, ii.
t-BuOOH, CH2Cl2, CH3CN, toluene, 100%; (b) 1H-tetrazole, CH3CN, ii. t-
BuOOH, CH2Cl2, toluene, 85%; (c) 1H-tetrazole, CH2Cl2, CH3CN, ii. t-
BuOOH, CH2Cl2, CH3CN, toluene, 78%; (d) 5–15 mol% X, CH2Cl2/
ClCH2CH2Cl/THF, reflux. TBDMSZtert-butyldimethylsilyl, CEZ2-
cyanoethyl.
In summary, the present study has demonstrated that not all
the five dinucleotide substrates were found to be substrates
for RCM reactions. It could be argued, of course, that a
longer range of catalysts and solvents should be included in
the study. However, Grubbs second-generation catalyst X
was in all cases tested by us6–10,32 found to be superior to the
first-generation catalyst ((Cy3P)2RuCl2CHPh).2 The
recentlycommercialisedHoveyda–Grubbssecond-generation
catalyst33 was not available, when most of the presented
experiments were conducted. On the other hand, not even
this catalyst has been found to be superior to X with
dinucleotide substrates in our lab.32 In the case of 21 Grubbs
first-generation catalyst has been attempted, but only
polymerisation was observed once again. Concerning
solvents, we have attempted toluene in other studies6–8 but
this solvent has been incompatible with our nucleotide
substrates. Furthermore, the major subject of this study has
however, solubility problems forced us to investigate other
solvents; toluene, ethyl acetate, acetonitrile/dichloro-
methane and THF, and with the latter, traces of a cyclised
product was indicated from MS. On the other hand, the
dinucleotide 23 was slowly converted to a ring-closed
product 25 (Scheme 4). The best result was obtained with
5 mol% of the catalyst in dichloromethane at reflux from
which 25 was isolated in 23% yield as a mixture of four
stereoisomers in an approximately 1:1:4:6 ratio as deduced
from the 31P NMR spectrum. This indicates that both E and
Z-configured products were obtained and also that the ratios
are dependent on the configuration of the phosphorus and
that the two isomers in the substrate reacted in different
rates. Thus, 27% of the starting material was re-isolated

P. Børsting et al. / Tetrahedron 60 (2004) 10955–10966
10959
Scheme 4. Reagents and conditions: (a) i. 1H-tetrazole, CH3CN, ii. t-BuOOH, CH3CN, toluene, 60%; (b) 5 mol% X, CH2Cl2, 23%, reflux. TBDMSZtert-
butyldimethylsilyl, CEZ2-cyanoethyl.
been to explore and compare different potential positions for
introducing allyl groups into nucleotide building blocks and
to compare different dinucleotides as RCM substrates. Thus,
if a given substrate reacts very slowly with X or seems to
prefer a polymerisation metathesis instead of a ring-closing
reaction, no perspectives were seen in exploring a long
range of reaction conditions. Therefore, we must conclude
that only two of the five substrates, 23 and 24 could be
transferred into the envisioned cyclic dinucleotides. Thus,
the cyclic structures 25 and 26/29 can be used in the design
of oligonucleotides that are conformationally restricted and
potentially useful for targeting and mimicking nucleic acid
secondary structures.
When comparing the four different building blocks A–D in
Figure 1, all four have been very conveniently obtained and
incorporated into dinucleotides. Thus, as an interesting spin-
off from this study, very convenient procedures for
preparing the two epimeric 20-O-allyluridine 11 and 20-O-
allyl-arabinouridine 17 have been developed. Especially in
the latter case, our procedure has surpassed the existing
literature method. However, the efficient RCM reactions
have been obtained only with A and B. Thus, taking in
account our former studies,7,8 the allyl group on the
phosphotriester linkage is very reactive towards the catalyst
and very well-positioned for performing ring-closing
reactions. The 20-O-ribo position of the allyl group, C, on
Scheme 5. Reagents and conditions: (a) 1H-tetrazole, CH3CN, then t-BuOOH, toluene, 64%; (b) 5 mol% X, CH2Cl2, reflux, 58%; (c) 32% aq NH3; (d) From
24, 5 mol% X, CH2Cl2, reflux, then 1000 psi H2, 50 8C, 63%; (e) 90% aq TFA, 100%. TBDMSZtert-butyldimethylsilyl.

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P. Børsting et al. / Tetrahedron 60 (2004) 10955–10966
the other hand, seems to be problematic. However, in other
studies by us32 and others,11e this position has been used to
form cyclic nucleotide structures with large rings, and the
reasons for the failure of 20 and 22 might be found in the
particular conformational properties of these substrates.
Similarly, the 20-O-arabino position of the allyl group, D,
has failed as demonstrated by the dinucleotides 21 and 22.
Nevertheless, reactivity between this allyl group and the
catalyst is proven by the fact, that 21 is converted by
polymerisation, probably by cross metathesis. Therefore,
the reason for 21 being a bad substrate for RCM should be
found in its conformational limitations making it impossible
for the two double bonds (and their ruthenium [2C
2]adducts) to approach and react in an intramolecular
reaction. The 5-position of the pyrimidine, B, seems to be a
good position for reaction with the catalyst X, and the
possibility of making the very large ring structure in
compound 25, the largest of the projected rings in this study,
might be partly driven by an ability to stack between the two
pyrimidines. Furthermore, the planar geometry of these
probably reduces any steric problems compared to the other
positions for allyl groups. Nevertheless, also in this case, the
reactivity is limited by geometry as illustrated by the fact
that different reactivity was observed for the two phos-
phorus epimers. Thus, even though the selection of five
dinucleotides that we choose for RCM studies led to the
examination of each of the four allyl positions A–D, we
cannot exclude the possibility that very efficient substrates
and subsequent cyclic dinucleotides could be found with
other combinations of A–D.
4. Experimentals
4.1. 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-
meter. 1H NMR spectra were recorded at 300 MHz, 13C NMR
spectra at 75.5 MHz, and 31P NMR spectra at 121.5 MHz.
Values for d are in ppm relative to tetramethylsilane as
internal standard or 85% H3PO4 as external standard. Fast-
atom bombardment 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
performed at The Microanalytical Laboratory, Department
of Chemistry, University of Copenhagen. Assignments of
NMR spectra are based on 2D spectra and follow standard
nucleoside style; that is, the carbon next to the nucleobase is
assigned C-10. For dinucleotides, the upper (50) nucleotide is
depicted U1 or T1 and the lower nucleoside U2 or T2.
4.1.1. Preparation of 5-allyl-20-deoxy-50-O-dimethoxy-
trityluridine (3). 5-Allyl-20-deoxyuridine 219 (0.700 g,
2.61 mmol) was coevaporated twice with anhydrous
pyridine and redissolved in anhydrous pyridine (40 mL) in
a darkened flask. AgNO3 (0.557 g, 3.28 mmol) was added
and the mixture was stirred for 5 min. DMTCl (1.099 g,
3.24 mmol) was added and the reaction mixture was stirred
for 4 h, concentrated under reduced pressure and coevapo-
rated with toluene. The residue was dissolved in ethyl
acetate (50 mL), washed with a saturated aqueous solution
of NaHCO3 (50 mL) and brine (2!50 mL), dried (Na2SO4)
and concentrated under reduced pressure. The residue was
purified by column chromatography (1–4% CH3OH and 1%
pyridine in CH2Cl2) to give the product as a white foam
(1.280 g, 86%) (Found C, 68.59; H, 6.07; N, 4.75%;
C33H34N2O7$1/2H2O requires C, 68.38; H, 6.08; N, 4.83%);
mpZ79–82 8C; Rf 0.23 (5% CH3OH in CH2Cl2); 1H NMR
(CDCl3) d 2.24–2.44 (2H, m, H-20), 2.51–2.77 (2H, m,
5-CH2), 3.33–3.43 (2H, m, H-50), 3.78 (6H, s, OCH3), 4.06
(1H, m, H-40), 4.53 (1H, m, H-30), 4.76–4.82 (2H, m,
CH]CH2), 5.60 (1H, m, CH]CH2), 6.41 (1H, m, H-10),
6.83 (4H, m, Ph), 7.16–7.41 (9H, m, Ph), 7.47 (1H, s, H-6),
9.330(1H, s, NH); 13C NMR (CDCl3) d 30.5 (5-CH2), 40.8
(C-2 ), 55.2 (OCH3), 63.5 (C-50), 72.4 (C-30), 84.8 (C-10),
86.1 (Ph), 86.8 (C-40), 113.23 (Ph), 113.71 (C-5), 116.65
(CH]CH2), 125.3, 127.1, 127.9, 128.1, 128.2, 129.0,
130.1, 134.3, 135.4 (Ph), 135.4 (CH]CH2), 136.1 (C-6),
144.3 (Ph), 150.4 (C-2), 158.6 (Ph), 163.1 (C-4); m/z FAB
570 (M).
As evident from the present results, the butylene nucleobase
to phosphotriester connection obtained in 29 has been
efficiently obtained from the tandem RCM hydrogenation
strategy. Further effort can now be put into separating the
two phosphorus epimers of this compound and incorporat-
ing these into oligonucleotides. The stability of 29 towards
ammonia supports these plans. Of the long range of possible
cyclic dinucleotides investigated by this study, 29 is the
most easily obtained and the obvious choice for further
studies. Furthermore, the synthesis of other cyclic dinucleo-
tides, constructed from allyl phosphotriester linkages and/or
other positions for terminal alkene moieties10,32 are in
progress.
3. Conclusion
Five different dinucleotides combining four nucleotide
building blocks with different allyl substituents have been
explored as substrates for RCM reactions. Two cyclic
dinucleotide structures were obtained and the one further
elaborated to give a stable butylene connection between a
nucleobase and a phosphate internucleotide linkage. Even
though not all projected cyclisations could be performed in
practice, more general knowledge within the scopes and
limitation of the RCM based methodology towards
conformationally restricted nucleic acid fragments has
been obtained. Thus, we envision a large potential for
constructing a plethora of cyclic dinucleotide structures
mimicking a large range of nucleic acid secondary
structures, and we expect the present RCM methodology
to be a general future tool in nucleic acid chemical biology.
4.1.2. Preparation of 5-allyl-30-O-tert-butyldimethylsilyl-
20-deoxy-50-O-dimethoxytrityluridine (4). Compound 3
(0.490 g, 0.86 mmol) was dissolved in anhydrous pyridine
(30 mL) in a darkened flask. AgNO3 (0.165 g, 0.97 mmol)
was added and the mixture was stirred for 5 min. TBDMSCl
(0.145 g, 0.96 mmol) was added and the reaction mixture
was stirred overnight, concentrated under reduced pressure
and coevaporated with toluene. The residue was dissolved
in ethyl acetate (50 mL), washed with a saturated aqueous

P. Børsting et al. / Tetrahedron 60 (2004) 10955–10966
10961
solution of NaHCO3 (50 mL) and brine (50 mL), dried
(Na2SO4) and concentrated under reduced pressure. The
residue was purified by column chromatography (1–5%
CH3OH and 1% pyridine in CH2Cl2) to give the product as a
white foam (0.495, 86%) (Found C, 66.96; H, 7.09; N,
3.84%; C39H48N2O7Si H2O requires C, 66.64; H, 7.16; N,
3.98%); mpZ60–64 8C; Rf 0.60 (5% CH3OH in CH2Cl2);
1H NMR (CDCl3) d 0.02–0.16 (6H, m, Si(CH3)2), 0.81–0.97
(9H, m, C(CH3)3), 2.17–2.39 (2H, m, H-20), 2.60–2.88 (2H,
m, 5-CH2), 3.28–3.48 (2H, m, H-50), 3.82 (6H, s, OCH3),
4.00 (1H, m, H-40), 4.52 (1H, m, H-30), 4.80–4.86 (2H, m,
CH]CH2), 5.67 (1H, m, CH]CH2), 6.38 (1H, t, JZ
6.7 Hz, H-10), 6.82 (4H, m, Ph), 7.16–7.51 (9H, m, Ph), 7.55
(1H, s, H-6), 8.98 (1H, s, NH); 13C NMR (CDCl3) d K4.9,
K4.7 (Si(CH3)2), 17.9 (C(CH3)3), 25.7 (C(CH3)3), 30.6
(C-20) 41.3 (5-CH2), 55.2 (OCH3), 62.8 (C-50), 72.1 (C-30),
84.8 (C-10), 86.7 (Ph), 87.4 (C-40), 113.2 (Ph), 113.48 (C-5),
116.6 (CH]CH2), 127.1, 127.7, 127.8, 127.9, 128.1, 128.2,
129.0, 129.1, 130.0, 134.4, 135.4 (Ph), 135.5 (CH]CH2),
136.1 (C-6), 144.3 (Ph), 150.1 (C-2), 158.6 (Ph), 163.0
(C-4); m/z FAB 684 (M).
(MgSO4) and concentrated under reduced pressure. The
residue was purified by dry column chromatography (2–4%
CH3OH in CH2Cl2) to give the product as white foam
(0.597 g, 42%) (Found C, 55.96; H, 8.02; N, 7.19%;
C18H30N2O5Si$1/4H2O requires C, 55.86; H, 7.94; N,
7.23%); Rf 0.48 (10% CH3OH in CH2Cl2); 1H NMR
(CDCl3) d 0.07–0.10 (6H, m, Si(CH3)2), 0.91 (9H, s,
C(CH3)3), 2.08 (1H, m, H-20), 2.40 (1H, m, H-200), 3.05–
3.07 (2H, m, 5-CH2), 3.81–3.83 (2H, m, H-50), 4.04 (1H, m,
H-40), 4.40 (1H, m, H-30), 5.06–5.12 (2H, m, CH]CH2),
5.86 (1H, m, CH]CH2), 6.34 (1H, m, H-10), 7.42 (1H, s,
H-6), 9.12 (1H, s, NH); 13C NMR (CDCl3) d K5.3, K5.1
(Si(CH3)2), 18.5 (C(CH3)3), 26.1 (C(CH3)3), 31.1 (C-20),
41.1 (5-CH2), 63.8 (C-50), 72.7 (C-30), 85.3 (C-10), 87.2
(C-40), 113.5 (C-5), 116.5 (CH]CH2), 134.7 (CH]CH2),
136.2 (C-6), 150.5 (C-2), 163.2 (C-4); HiRes MALDI
FT-MS m/z (MCNa) found/calcd 405.1836/405.1816.
4.1.5. Preparation of 5-allyl-50-O-(tert-butyldimethylsi-
lyl)uridine-30-O-(N,N-diisopropyl)-(2-cyanoethyl)phos-
phoramidite (7). Compound 6 (0.152 g, 0.40 mmol) was
dissolved in anhydrous CH2Cl2 (2.5 mL) and a 0.5 M
solution of 4,5-dicyanoimidazole (0.55 mL, 0.28 mmol) in
CH3CN was added. 2-Cyanoethyl-N,N,N0,N0-tetraisopropyl-
phosphordiamidite (0.126 g, 0.42 mmol) was added drop-
wise over 5 min and the reaction mixture was stirred at room
temperature for 30 min. The mixture was diluted with
CH2Cl2 (50 mL) and washed with a saturated aqueous
solution of NaHCO3 (25 mL) and brine (25 mL). The
combined aqueous phases were extracted with CH2Cl2
(30 mL) and the combined organic phases were dried
(Na2SO4) and concentrated under reduced pressure. The
residue was purified by dry column chromatography (50–
60% ethyl acetate and 1% triethylamine in petrol ether) to
give the product as a colourless oil and an epimeric mixture
(0.173 g, 75%); Rf 0.36, 0.49 (75% ethyl acetate in petrol
4.1.3. Preparation of 5-allyl-30-O-tert-butyldimethylsilyl-
20-deoxyuridine (5). Compound 4 (0.450 g, 0.66 mmol)
was dissolved in a mixture of CH3OH and CH2Cl2 (2:3 v/v,
50 mL) and the solution was stirred at 0 8C. A solution of p-
toluenesulfonic acid monohydrate (0.155 g, 0.82 mmol) in a
mixture of CH3OH and CH2Cl2 (2:3 v/v, 5 mL) was added
over 5 min. The reaction mixture was stirred for 30 min and
quenched by the addition of saturated aqueous ammonia
(0.5 mL). The mixture was concentrated under reduced
pressure and the residue was dissolved in CH2Cl2 (50 mL),
washed with water (3!50 mL), dried (MgSO4) and
concentrated under reduced pressure. The residue was
purified by column chromatography (1–3% CH3OH in
CH2Cl2) to give the product as a white foam (0.220 g, 88%);
Rf 0.22 (5% CH3OH in CH2Cl2); 1H NMR (CDCl3) d 0.01–
0.13 (6H, m, Si(CH3)2), 0.83–0.96 (9H, m, C(CH3)3), 2.19–
2.41 (2H, m, H-20), 3.06–3.09 (2H, m, 5-CH2), 3.72–3.76
(2H, m, H-50), 3.92 (1H, m, H-40), 4.50 (1H, m, H-30), 5.11–
5.18 (2H, m, CH]CH2), 5.85 (1H, m, CH]CH2), 6.15
(1H, t, JZ6.7 Hz, H-10), 7.34 (1H, s, H-6), 9.01 (1H, s, NH);
13C NMR (CHCl3) d K4.9, K4.7 Si(CH3)2), 18.0
(C(CH3)3), 25.7 (C(CH3)3), 30.5 (C-20), 40.4 (5-CH2),
62.0 (C-50), 71.6 (C-30), 87.2 (C-10), 87.6 (C-40), 113.5
(C-5), 117.6 (CH]CH2), 134.2 (CH]CH2), 137.6 (C-6),
150.2 (C-2), 163.1 (C-4); HiRes MALDI FT-MS m/z (MC
Na) found/calcd 405.1828/405.1822.
1
ether); H NMR (CDCl3) d 0.10–0.11 (6H, m, Si(CH3)2),
0.88–0.98 (9H, m, C(CH3)3), 1.17–1.22 (12H, m,
CH(CH3)), 2.01–2.11 (1H, m, H-20), 2.39–2.55 (1H, m,
H-200), 2.61–2.66 (2H, m, CH2CN), 3.06–3.08 (2H, m,
5-CH2), 3.56–3.91 (6H, m, H-50, CH2OP, 2!CH(CH3)),
4.09–4.18 (1H, m, H-40), 4.48–4.54 (1H, m, H-30), 5.06–
5.13 (2H, m, CH]CH2), 5.80–5.91 (1H, m, CH]CH2),
6.30–6.34 (1H, m, H-10), 7.41, 7.45 (1H, 2 s, H-6), 8.27 (1H,
br s, NH); 31P NMR (CDCl3) d 149.45, 149.89; m/z FAB
583 (MCH).
4.1.6. Preparation of 20-O-allyl-3-N-benzoyl-30,50-O,O-
(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)uridine (10).
Compound 927 (0.496 g, 0.840 mmol) and allylethylcarbo-
nate34 (0.351 g, 2.70 mmol) was dissolved in anhydrous
THF (4 mL). A solution of tris(dibenzylidenacetone)dipal-
ladium(0) (0.012 g,0.013 mmol, 1.5 mol%) andbis(diphenyl-
phosphino)butane (0.038 g, 0.09 mmol, 10 mol%) in
anhydrous THF (1 mL) was added. The reaction mixture
was stirred at reflux for 45 min and then concentrated under
reduced pressure. The residue was purified by column
chromatography (4–50% ethyl acetate in petrol ether) to
give the product as a white foam (0.487 g, 92%) (found: C,
59.19; H, 7.42; N, 4.43% C31H46N2O8Si2 requires: C, 59.02;
H, 7.35; N, 4.44%); Rf 0.71 (75% ethyl acetate in petrol
ether); 1H NMR (CDCl3) d 0.85–1.26 (28H, m,
SiCH(CH3)2), 3.92 (1H, s, H-40), 4.00 (1H, d, JZ14.0 Hz,
4.1.4. Preparation of 5-allyl-50-O-tert-butyldimethylsilyl-
20-deoxyuridine (6). 5-Allyl-20-deoxyuridine 219 (0.979 g,
3.65 mmol) was coevaporated with anhydrous pyridine and
redissolved in anhydrous pyridine (36 mL) in a darkened
flask. AgNO3 (0.503 g, 2.96 mmol) was added and the
mixture was stirred for 5 min. TBDMSCl (0.235 g,
1.56 mmol) was added and the reaction mixture was stirred
for 2 h. Another portion of TBDMSCl (0.339 g, 2.25 mmol)
was added in 3 parts during 3 h, and the reaction was stirred
for 5 h and quenched by the addition of CH3OH (1 mL). The
reaction mixture was concentrated under reduced pressure
and coevaporated with toluene. The residue was dissolved
in CH2Cl2 (80 mL) and washed with a saturated aqueous
solution of NaHCO3 (40 mL) and brine (40 mL), dried

10962
P. Børsting et al. / Tetrahedron 60 (2004) 10955–10966
H-50), 4.21–4.33 (5H, m, H-20, H-30, H-500, 20-OCH2), 5.15
(1H, dd, JZ1.5, 12.4 Hz, CH]CH2), 5.31 (1H, dd, JZ1.5,
17 Hz, CH]CH2), 5.79 (1H, d, JZ8.2 Hz, H-5), 5.76 (1H,
s, H-10), 5.87 (1H, m, CH]CH2), 7.48–7.95 (m, 5H, Ph),
8.04 (1H, d, JZ8.2 Hz, H-6); 13C NMR (CDCl3) d 12.4,
12.8, 13.1, 13.4 (SiCH(CH3)2), 16.7, 16.8, 17.0, 17.1, 17.3,
17.3, 17.4, 17.5, 17.9 (SiCH(CH3)2), 59.4 (C-50), 67.9
(C-30), 71.2 (20-OCH2), 81.1 (C-40), 82.0 (C-20), 89.2 (C-10),
101.45 (C-5), 117.4 (CH]CH2), 129.2, 130.5, 131.3, 134.1
(Ph), 135.2 (CH]CH2), 139.2 (C-6), 148.9 (C-2), 162.2
(C-4), 168.7 (C]O); m/z FAB 631 (MCH).
NMR (CDCl3) d K5.6 (Si(CH3)2), 18.4 (C(CH3)3), 25.9
(C(CH3)3), 61.3 (C-50), 67.9 (C-30), 71.4 (20-OCH2), 81.5
(C-40), 84.6 (C-20), 87.3 (C-10), 102.0 (C-5), 118.7
(CH]CH2), 133.3 (CH]CH2), 140.0 (C-6), 150.1 (C-2),
163.3 (C-4); HiRes MALDI FT-MS m/z (MCNa) found/
calcd 421.1750/421.1765.
4.1.9. Preparation of 20-O-allyl-50-O-(tert-butyldimethyl-
silyl)uridine-30-O-(N,N-diisopropyl)-(2-cyanoethyl)phos-
phoramidite (13). Compound 12 (0.123 g, 0.31 mmol) was
dissolved in anhydrous CH2Cl2 (1.5 mL) and stirred at 0 8C.
N,N-Diisopropylethylamine (0.30 mL, 0.227 g, 1.76 mmol)
was added and chloro-N,N-diisopropylamino-(2-cyano-
ethyl)phosphine (0.20 g, 0.85 mmol) was added. The
reaction mixture was stirred at 0 8C for 10 min and at
room temperature for 5 h. The reaction mixture was cooled
to 0 8C and another portion of chloro-N,N-diisopropyl-
amino-(2-cyanoethyl)phosphine (0.20 g, 0.85 mmol) was
added. The mixture was stirred at room temperature for
1.5 h and then quenched by the addition of water (1 mL).
The mixture was diluted with ethyl acetate (10 mL) and
washed with a 5% aqueous solution of NaHCO3 (2!
25 mL) and brine (25 mL), dried (Na2SO4) and concentrated
under reduced pressure. The residue was purified by column
chromatography (0.5% triethylamine and 50% ethyl acetate
in petrol ether) to give the product as yellow foam and an
epimeric mixture (0.120 g, 65%); Rf 0.36 (75% ethyl acetate
4.1.7. Preparation of 20-O-allyluridine (11). Compound 10
(2.40 g, 3.80 mmol) was dissolved in THF (10 mL) and the
solution was stirred at room temperature. A 1 M solution of
TBAF in THF (8.6 mL, 8.6 mmol) was added over 5 min
and the mixture was stirred for 30 min. A mixture of
pyridine, CH3OH and water (3:1:1 v/v, 10 mL) was added
and the combined mixture was poured on 13 g amberlite
IR-120w in pyridine, CH3OH and water (3:1:1 v/v, 60 mL).
The suspension was stirred for 30 min and filtered. The
filtrate was concentrated under reduced pressure and the
residue was dissolved in CH3OH (50 mL) and added 25%
aqueous ammonia (10 mL). The reaction mixture was
stirred for 1 h 30 min, concentrated under reduced pressure
and coevaporated with anhydrous ethanol. The residue was
purified by dry column chromatography (0–10% CH3OH in
CH2Cl2) to give the product as a hygroscopic white foam
(1.030 g, 95%); Rf 0.55 (10% CH3OH in CH2Cl2); 1H NMR
(CD3OD) d 3.71–3.89 (2H, m, H-50), 3.90–4.02 (2H, m,
H-20, H-40), 4.20–4.24 (3H, m, H-30, 20-OCH2), 5.13–5.34
(2H, m, CH]CH2), 5.68 (1H, d, JZ7.9 Hz, H-5), 5.87–
5.98 (1H, m, CH]CH2), 5.95 (1H, d, JZ3.8 Hz, H-10), 8.07
(1H, d, JZ7.9 Hz, H-6); 13C NMR (CD3OD) d 61.7 (C-50),
70.0 (C-30), 72.4 (20-OCH2), 82.5 (C-40), 86.2 (C-20), 89.2
(C-10), 102.5 (C-5), 117.9 (CH]CH2), 135.7 (CH]CH2),
142.5 (C-6), 152.2 (C-2), 166.2 (C-4); HiRes MALDI
FT-MS m/z (MCNa) found/calcd 307.0898/307.0901.
1
in petrol ether); H NMR (CDCl3) d 0.10–0.13 (6H, m,
Si(CH3)2), 0.94–0.97 (9H, m, C(CH3)3), 1.11–1.23 (12H, m,
CH(CH3)2), 2.59–2.66 (2H, m, CH2CN), 3.59–3.70 (4H, m,
CH2OP, CH(CH3)2), 3.70–3.84 (2H, m, H-50), 3.84–4.01
(2H, m, 20-OCH2), 4.17–4.32 (3H, m, H-20, H-30, H-40),
5.16–5.33 (2H, m, CH]CH2), 5.64–5.68 (1H, m, H-5),
5.85–6.92 (1H, m, CH]CH2), 6.02–6.06 (1H, m, H-10),
7.94–7.98 (2H, m, H-6); 31P NMR (CDCl3) d 150.91,
150.97; m/z FAB 599 (MCH).
4.1.10. Preparation of 3-N-benzoyl-1-(30,50-O,O-(1,1,3,3-
tetraisopropyldisiloxan-1,3-diyl)-b-D-arabinofuranosyl)
uracil (15). Compound 1429 (5.33 g, 11.0 mmol) was
dissolved in anhydrous CH2Cl2 (100 mL), stirred at 0 8C
and added triethylamine (7.63 mL, 54.8 mmol). TMSCl
(4.17 mL, 32.9 mmol) was added over 5 min and the
reaction mixture was stirred at room temperature for 2 h.
The mixture was cooled to 0 8C and added a 1.0 M aqueous
solution of NaHCO3 (100 mL). The organic phase was
separated, dried (Na2SO4) and concentrated under reduced
pressure. The residue was redissolved in anhydrous pyridine
(50 mL) and stirred at 0 8C. N,N-diisopropylethylamin
(5.62 mL, 32.9 mmol) was added and the mixture was
stirred for 10 min. Benzoyl chloride (3.81 mL, 32.9 mmol)
was added and the reaction mixture was stirred at room
temperature for 17 h. The reaction mixture was cooled to
0 8C and a 1.0 M aqueous solution of NaHCO3 (100mL) was
added. The organic phase was dried (Na2SO4), concentrated
under reduced pressure and coevaporated with toluene (3!
10 mL). The residue was redissolved in CH2Cl2 (100 mL)
and stirred at 0 8C. A solution of p-toluenesulfonic acid
(4.72 g, 27.4 mmol) in THF (50 mL) was added and the
reaction mixture was stirred for 10 min. Triethylamine
(3.05 mL, 21.9 mmol) was added and the mixture was
stirred for 15 min. A 1.0 M aqueous solution of NaHCO3
(100mL) was added, and the organic phase was separated,
4.1.8. Preparation of 20-O-allyl-50-O-(tert-butyldimethyl-
silyl)uridine (12). Compound 11 (0.312 g, 1.10 mmol) was
dissolved in anhydrous pyridine (9 mL). TBDMSCl
(0.213 g, 1.41 mmol) and AgNO3 (0.740 g, 4.36 mmol)
were added, and the reaction mixture was stirred at room
temperature for 24 h. The reaction was quenched by the
addition of CH3OH (1 mL) and the mixture was concen-
trated under reduced pressure and coevaporated with
toluene. The residue was dissolved in ethyl acetate
(75 mL) and washed with a saturated aqueous solution of
NaHCO3 (50 mL) and brine (50 mL). The aqueous phase
was extracted with ethyl acetate (50 mL), and the combined
organic phases were dried (Na2SO4) and concentrated under
reduced pressure. The residue was purified by dry column
chromatography (0–6% CH3OH in CH2Cl2) to give the
product as a white foam (0.344 g, 79%) (Found C, 54.61; H,
7.70; N, 6.81%; C18H30N2O6Si requires C, 54.25; H, 7.59;
N, 7.03%); Rf 0.67 (10% CH3OH in ethyl acetate); 1H NMR
(CDCl3) d 0.12 (6H, s, Si(CH3)2), 0.93 (9H s, C(CH3)3),
2.64 (1H, d, JZ7.9 Hz, 30-OH), 3.84–3.92 (2H, m, H-500),
4.01–4.07 (2H, m, 20-OCH2), 4.20–4.24 (2H, m, H-20, H-4 ),
4.40 (1H, m, H-30), 5.23–5.33 (2H, m, CH]CH2), 5.67 (1H,
d, JZ8.3 Hz, H-5), 5.89 (1H, m, CH]CH2), 5.98 (1H, s,
H-10), 8.06 (1H, d, JZ8.3 Hz, H-6), 9.07 (1H, s, NH); 13C

P. Børsting et al. / Tetrahedron 60 (2004) 10955–10966
10963
dried (Na2SO4) and concentrated under reduced pressure.
The residue was purified by column chromatography (5–
40% ethyl acetate in petrol ether) to give the product as a
white foam (5.21 g, 81%); mpZ50–53 8C; 1H NMR
(CDCl3) d 0.97–1.13 (28H, m, CH(CH3)2), 3.31 (1H, d,
JZ4.6 Hz, 20-OH), 3.73 (1H, m, H-40), 4.05 (1H, dd, JZ
2.5, 13.5 Hz, H-50), 4.12 (1H, dd, JZ1.4, 13.5 Hz, H-50),
4.16 (1H, t, JZ8.6 Hz, H-30), 4.42 (1H, m, H-200), 5.75 (1H,
d, JZ8.2 Hz, H-5), 6.03 (1H, d, JZ6.2 Hz, H-1 ), 7.80 (1H,
d, JZ8.2 Hz, H-6), 7.93–7.42 (5H, m, Ph); 13C NMR
(CDCl3) d 12.2, 12.8, 12.9, 13.3 (CH(CH3)2), 16.7, 16.8,
16.9, 17.1, 17.2, 17.3, 17.4 (CH(CH3)2), 60.0 (C-50), 72.4
(C-30), 75.4 (C-20), 80.7 (C-40), 101.3 (C-5), 128.3, 130.3,
131.3, 134.9 (Ph), 140.2 (C-6), 149.9 (C-2), 162.1 (C-4),
168.5 (C]O).
H-20, H-40, H-50), 4.29 (1H, t, JZ5.2 Hz, H-30), 5.11–5.20
(2H, m, CH]CH2), 5.65–5.81 (2H, m, CH]CH2, H-5),
6.26 (1H, JZ5.3 Hz, H-10), 7.65 (1H, d, JZ8.4 Hz, H-6),
8.91 (1H, br s, NH); 13C NMR (CDCl3) d K5.5, K5.4,
K4.9, K4.5 (Si(CH3)2), 17.9, 18.4 (C(CH3)3), 25.5, 25.6,
25.9 (C(CH3)3), 61.0 (C-50), 72.3 (C-30), 73.6 (20-OCH2),
83.2, 83.7, 83.9 (C-10, C-20, C-30), 101.2 (C-5), 117.6
(CH]CH2), 133.3 (CH]CH2), 141.9 (C-6), 150.3 (C-2),
163.2 (C-4); m/z MALDI (MCNa) 535.
4.1.13. Preparation of 1-(20-O-allyl-30-O-(tert-butyldi-
methylsilyl)-b-D-arabinofuranosyl)uracil (19). Com-
pound 18 (0.701 g, 1.37 mmol) was dissolved in 80%
aqueous acetic acid (20 mL) and the reaction mixture was
stirred at 60 8C for 2 h 30 min. The reaction mixture
was concentrated under reduced pressure and the residue
was purified by column chromatography (0–2% CH3OH in
CH2Cl2) to give the product as a white foam (0.399 g, 73%);
4.1.11. Preparation of 1-(20-O-allyl-b-D-arabinofurano-
syl)uracil (17). Compound 15 (5.21 g, 8.82 mmol) was
dissolved in anhydrous THF (35 mL) and added allylethyl-
carbonate34 (1.51 mL, 13.2 mmol), tris(dibenzylideneace-
1
mpZ131–132 8C; H NMR (CDCl3) d 0.11–0.16 (6H, m,
Si(CH3)2), 0.87–0.94 (9H, m C(CH3)3), 2.23 (1H, m,
50-OH), 3.78–3.98 (6H, m, 20-OCH2), 4.24 (1H, t, JZ
3.8 Hz, H-30), 5.15–5.21 (2H, m, CH]CH2), 5.71 (1H, d,
JZ8.2 Hz, H-5), 5.75 (1H, m, CH]CH2), 6.27 (1H, d, JZ
4.6 Hz, H-10), 7.62 (1H, d, JZ8.2 Hz, H-6), 8.71 (1H, br s,
NH); 13C NMR (CDCl3) d K4.9, K4.6 (Si(CH3)2), 17.9
(C(CH3)3), 25.6 (C(CH3)3), 61.4 (C-50), 72.3 (C-30), 75.1
(20-OCH2), 83.3, 84.3, 84.4 (C-10, C-20, C-40), 101.2 (C-5),
118.3 (CH]CH2), 132.9 (CH]CH2), 142.0 (C-6), 150.2
(C-2), 163.0 (C-4); HiRes MALDI FT-MS m/z (MCNa)
found/calcd 421.1751/421.1765.
tone)dipalladium(0)
(0.121 g,
0.132 mmol)
and
1,4-bis(diphenylphosphino)butane (0.376 g, 0.88 mmol).
The reaction mixture was stirred at reflux for 3 h and then
concentrated under reduced pressure. The crude compound
16 was dissolved in THF (50 mL) and a 1.0 M solution of
TBAF in THF (19.4 mL, 19.4 mmol) was added over 5 min.
The reaction mixture was stirred for 15 min and then
concentrated under reduced pressure. The residue was
dissolved in CH3OH (35 mL) and the solution was stirred at
0 8C. NH3(g) was bubbled through the solution for 45 min.
The mixture was concentrated under reduced pressure and
the residue was dissolved in a mixture of pyridine, CH3OH
and water (3:1:1 v/v, 80 mL). The mixture was stirred with
Dowexw (50 W!2) for 2 h and the resin was removed by
filtration. The filtrate was concentrated under reduced
pressure and coevaporated with toluene. The residue was
purified by column chromatography (0–7% CH3OH in
CH2Cl2) to give the product as white solid (1.90 g, 76%);
4.1.14. Preparation of (5-allyl-30-O-tert-butyldimethylsi-
lyl-20-deoxyuridin-50-yl) (20-O-allyl-50-O-tert-butyldi-
methylsilyluridin-30-yl) 2-cyanoethylphosphate (20).
Compound 13 (0.103 g, 0.17 mmol) and compound 5
(0.073 g, 0.19 mmol) were coevaporated with anhydrous
CH2Cl2 and dissolved in anhydrous CH2Cl2 (6 mL). A
0.45 M solution of 1H-tetrazole in CH3CN (1.9 mL,
0.86 mmol) was added and the reaction mixture was stirred
for 1 h. The reaction was quenched by the addition of
CH3OH (1 mL) and the mixture was concentrated under
reduced pressure. The residue (crude phosphite, Rf 0.41
(75% ethyl acetate in petrolether)) was dissolved in a
mixture of CH2Cl2 and CH3CN (3:1 v/v, 8 mL) and cooled
to 0 8C. A 3 M solution of t-BuOOH in toluene (0.3 mL,
0.9 mmol) was added and the reaction mixture was stirred
for 1 h at 0 8C and for 2 h at room temperature. The reaction
was quenched by the addition of CH3OH (1 mL) and the
mixture was concentrated under reduced pressure. The
residue was purified by column chromatography (50% ethyl
acetate in petrol ether and 10% CH3OH in CH2Cl2) to give
the product as a white foam (0.156 g, 100%); Rf 0.27 (75%
1
mpZ167–168 8C; H NMR (CD3OD) d 3.34–3.83 (2H, m,
H-50), 3.87 (1H, m, H-40), 3.97 (1H, m, 20-OCH2), 4.10 (1H,
m, H-20), 4.15 (1H, m, 20-OCH2), 4.20 (1H, t, JZ4.8 Hz,
H-40), 5.11–5.26 (2H, m, CH]CH2), 5.69 (1H, d, JZ
8.2 Hz, H-5), 5.97 (1H, m, CH]CH2), 6.26 (1H, d, JZ
5.2 Hz, H-10), 7.85 (1H, d, JZ80.2 Hz, H-6); 13C NMR
(CD3OD) d 61.8 (C-50), 72.6 (C-3 ), 74.9 (20-OCH2), 84.6
(C-20), 85.1 (C-40), 85.7 (C-10), 101.3 (C-5), 117.2
(CH]CH2), 135.2 (CH]CH2), 144.1 (C-6), 152.2 (C-2),
166.2 (C-4).
4.1.12. Preparation of 1-(20-O-allyl-30,50-O,O-bis(tert-
butyldimethylsilyl)-b-D-arabinofuranosyl)uracil (18).
Compound 17 (0.200 g, 0.70 mmol) was dissolved in
anhydrous DMF (7 mL) and added imidazole (0.480 g,
7.04 mmol) and TBDMSCl (0.530 g, 3.52 mmol). The
reaction mixture was stirred for 24 h at room temperature,
concentrated under reduced pressure and coevaporated with
xylene (2.5 mL). The residue was dissolved in CH2Cl2
(20 mL), washed with water (2!20 mL), dried (Na2SO4)
and concentrated under reduced pressure. The residue was
purified by column chromatography (0–2% CH3OH in
CH2Cl2) to give the product as a colourless oil (0.298 g,
1
ethyl acetate in petrol ether); H NMR (CD3CN) d 0.10–
0.12 (12H, m, Si(CH3)2), 0.81–0.92 (18H, m, C(CH3)3),
2.18–2.26 (2H, m, T2-H-20), 2.78–2.82 (4H, m, 5-CH2,
CH2CN), 3.83–4.43 (12H, m, U1-H-50, U1-H-40, U1-H-20,
U1-20-OCH2, T2-H-50, T2-H-40, T2-H-30, CH2OP), 4.87–
4.92 (1H, m, U1-H-30), 5.01–5.29 (4H, m, 2!CH]CH2),
5.63–5.67 (1H, m, H-5), 5.78–5.94 (2H, m, 2!CH]CH2),
5.99–6.02 (1H, m, U1-H-10), 6.15–6.21 (1H, m, T2-H-10),
7.31 (½H, s, T2-H-6), 7.38 (½H, s, T2-H-6), 7.72–7.75 (1H,
m, H-6), 8.91–8.95 (2H, br s, 2!NH); 31P NMR (CD3CN) d
K1.74, K1.49; m/z ESI 918 (MCNa), 896 (MCH).
1
83%); H NMR (CDCl3) d 0.09–0.13 (12H, m, Si(CH3)2),
0.87–0.93 (18H, m, C(CH3)3), 3.74–4.01 (6H, m, 20-OCH2,

10964
P. Børsting et al. / Tetrahedron 60 (2004) 10955–10966
4.1.15. Preparation of allyl (20-O-allyl-30-O-(tert-butyldi-
methylsilyl)arabinouridin-50-yl) (50-O-tert-butyldi-
methylsilylthymidin-30-yl) phosphate (21). Compound
19 (0.081 g, 0.20 mmol) was coevaporated with anhydrous
CH3CN and dissolved in anhydrous CH3CN (3 mL). A
0.45 M solution of 1H-tetrazole in CH3CN (0.68 mL,
0.30 mmol) was added and the mixture for stirred for
10 min. A solution of 1 (0.252 g, 0.46 mmol) in anhydrous
CH3CN (3 mL) was added and the reaction mixture was
stirred for 2 h. The reaction was quenched by the addition of
CH3OH (0.5 mL) and the reaction mixture was concentrated
under reduced pressure and coevaporated with CH2Cl2. The
residue was dissolved in anhydrous CH2Cl2 (4 mL) and
cooled to 0 8C. A 3 M solution of t-BuOOH in toluene
(0.14 mL, 0.41 mmol) was added dropwise and the reaction
mixture was stirred at room temperature for 1 h. The
reaction was quenched by the addition of CH3OH (1 mL)
and the mixture was concentrated under reduced pressure.
The residue was purified by column chromatography (0–2%
CH3OH in CH2Cl2) to give the product as a white foam and
CH]CH2), 5.92–5.97 (1H, m, U1-H-10), 6.21–6.23 (1H,
m, U2-H-10), 7.56–7.72 (2H, m, 2!H-6), 11.41–11.47 (2H,
m, 2!NH); 31P NMR (DMSO-d6) d K1.12; HiRes MALDI
FT-MS m/z (MCNa) found/calcd 934.3442/934.3462.
4.1.17. Preparation of (5-allyl-20-deoxy-30-O-tert-butyl-
dimethylsilyluridin-50-yl) (5-allyl-20-deoxy-50-O-tert-
butyldimethylsilyluridin-30-yl) 2-cyanoethylphosphate
(23). Compound 7 (0.156 g, 0.268 mmol) and compound 5
(0.111 g, 0.290 mmol) were coevaporated twice with
anhydrous CH3CN and dissolved in anhydrous CH3CN
(3 mL). A 0.45 M solution of 1H-tetrazole in CH3CN
(3.0 mL, 1.35 mmol) was added and the reaction mixture
was stirred for 30 min. The reaction was quenched by the
addition of CH3OH (0.6 mL) and the mixture was
concentrated under reduced pressure. The residue (crude
phosphite, Rf 0.23 (75% ethyl acetate in petrol ether)) was
dissolved in anhydrous CH3CN (3 mL) and a 3 M solution
of t-BuOOH in toluene (0.45 mL, 1.35 mmol) was added.
The reaction mixture was stirred for 1.5 h and added another
portion of the 3 M solution of t-BuOOH in toluene
(0.23 mL, 0.69 mmol). The reaction mixture was stirred
for 30 min and the reaction was quenched by the addition of
CH3OH (0.5 mL). The mixture was concentrated under
reduced pressure, and the residue was dissolved in CH2Cl2
(50 mL), washed with a saturated aqueous solution of
NaHCO3 (25 mL), dried (Na2SO4) and concentrated under
reduced pressure. The residue was purified by dry column
chromatography (50–0% petrol ether in ethyl acetate) to
give the product as a white foam and an epimeric mixture
(0.142 g, 60%); (Found C, 53.01; H, 7.32; N, 7.42%;
C39H62N5O12PSi2 requires C, 53.23; H, 7.10; N, 7.96%); Rf
0.36 (75% ethyl acetate in petrolether); 1H NMR (CDCl3) d
0.10–0.11 (12H, m, Si(CH3)2), 0.89–0.91 (18H, m,
C(CH3)3), 2.08–2.68 (4H, m, T1-H-20, T2-H-20), 2.75–
2.80 (2H, m, CH2CN), 3.00–3.08 (4H, T1-5-CH2, T2-5-
CH2), 3.80–4.03 (4H, m, T2-H-50, T2-H-40, T1-H-40), 4.20–
4.30 (5H, m, T1-H-50, T2-H-30, CH2OP), 4.41–4.45 (1H, m,
T1-H-30), 5.04–5.17 (4H, m, 2!CH]CH2), 5.85–5.89 (2H,
m, 2!CH]CH2), 6.10–6.17 (1H, m, T1-H-10), 6.27–6.32
(1H, m, T2-H-10), 7.20–7.22 (1H, m, H-6), 7.39–7.41 (1H,
m, H-6), 9.00–9.13 (2H, m, 2!NH); 31P NMR (CDCl3) d
K1.58, K1.44; HiRes MALDI FT-MS m/z (MCNa) found/
calcd 902.3581/902.3563.
1
an epimeric mixture (0.148 g, 85%); mpZ70–71 8C; H
NMR (CDCl3) d 0.08–0.13 (12H, m, Si(CH3)2), 0.90–0.92
(18H, m, C(CH3)3), 1.90–1.94 (3H, br s, T1-CH3), 2.00–
2.06 (1H, m, T1-H-20), 2.52–2.58 (1H, m, T1-H-20), 3.90–
4.28 (10H, m, U2-20-OCH2, U2-H-50, U2-H-40, U2-H-30,
U2-H-20, T1-H-50, T1-H-40), 4.56-4.61 (2H, m, CH2OP),
4.98-5.02 (1H, m, T1-H-30), 5.14-5.21 (2H, m, U2-
CH]CH2), 5.29–5.43 (2H, m, T1-CH]CH2), 5.68–5.80
(2H, m, U2-CH]CH2, U2-H-5), 5.95–6.01 (1H, m, T1-
CH]CH2), 6.28–6.32 (1H, m, U2-H-10), 6.33–6.39 (1H, m,
T1-H-10), 7.48–7.52 (1H, m, T1-H-6), 7.61–7.69 (1H, m,
U2-H-6), 9.53–9.69 (2H, m, 2!NH); 31P NMR (CDCl3) d
K0.59; HiRes MALDI FT-MS m/z (MCNa) found/calcd
879.3420/879.3404.
4.1.16. Preparation of (20-O-allyl-30-O-(tert-butyldi-
methylsilyl)arabinouridin-50-yl) (20-O-allyl-50-O-tert-
butyldimethylsilyluridin-30-yl) 2-cyanoethylphosphate
(22). Compound 13 (0.140 g, 0.23 mmol) and compound
19 (0.111 g, 0.28 mmol) were coevaporated twice with
anhydrous CH3CN and redissolved in anhydrous CH3CN
(7 mL). A 0.45 M solution of 1H-tetrazole in CH3CN
(2.6 mL, 1.17 mmol) was added and the reaction mixture
was stirred for 2 h. The reaction was quenched by the
addition of CH3OH (0.5 mL) and the mixture was
concentrated under reduced pressure. The residue was
dissolved in a mixture of CH2Cl2 and CH3CN (1:1 v/v,
16 mL) and stirred at 0 8C. A 3 M solution of t-BuOOH in
toluene (0.38 mL, 1.14 mmol) was added and the reaction
mixture was stirred at room temperature for 90 min. The
reaction was quenched by the addition of CH3OH (0.5 mL)
and the mixture was concentrated under reduced pressure.
The residue was purified by column chromatography (ethyl
acetate) and precipitated twice from a mixture of ethyl
acetate and petrol ether to give the product as a white
powder and an epimeric mixture (0.181 g, 78%); Rf 0.27
4.1.18. Preparation of allyl (5-allyl-20-deoxy-30-O-tert-
butyldimethylsilyluridin-50-yl) (50-O-tert-butyldimethyl-
silylthymidin-30-yl) phosphate (24). Compound 5
(0.188 g, 0.49 mmol) and compound
1
(0.520 g,
0.96 mmol) were coevaporated twice with anhydrous
CH3CN (10 mL) and dissolved in anhydrous CH3CN
(25 mL). A 0.45 M solution of 1H-tetrazole in CH3CN
(5.3 mL, 2.38 mmol) was added over 5 min and the reaction
mixture was stirred for 1.5 h at room temperature. A 3 M
solution of t-BuOOH in toluene (0.82 mL, 2.46 mmol) was
added, and the mixture was stirred for another 1.5 h. The
reaction was quenched by the addition of CH3OH
(0.25 mL), and the mixture was concentrated under reduced
pressure. The residue was dissolved in ethyl acetate (50 mL)
and washed with a saturated aqueous solution of NaHCO3
(2!20 mL) and brine (20 mL). The aqueous phase was
extracted with ethyl acetate (30 mL) and the combined
organic phases were dried (Na2SO4) and concentrated under
1
(75% ethyl acetate in petrol ether); H NMR (DMSO-d6) d
0.08–0.12 (12H, m, Si(CH3)2), 0.86–0.90 (18H, m,
C(CH3)3), 2.92–2.96 (2H, m, CH2CN), 3.77–4.28 (15H,
m, U1-H-500, U1-H-40, U1-H0-20, U2-H-50, U2-H-40, U2-H-
30, U2-H-2 , CH2OP, U1-2 -OCH2, U2-20-OCH2), 4.82–
4.86 (1H, m, U1-H-30), 5.09–5.25 (4H, m, 2!CH]CH2),
5.59–5.68 (2H, m, 2!H-5), 5.70–5.90 (2H, m, 2!

P. Børsting et al. / Tetrahedron 60 (2004) 10955–10966
10965
reduced pressure. The residue was purified by column
chromatography (20–70% ethyl acetate in petrol ether) to
give the product as a white foam and an epimeric mixture
(0.266 g, 64%); mpZ74–76 8C; Rf 0.34 (75% ethyl acetate
T1-H-20, T2-H-20), 2.24–2.33 (1H, m, T1-H-200), 2.92–3.09
(2H, m, T2-5-CH2), 3.40–3.43 (2H, m, CH2N), 3.51–3.59
(2H, m, T1-H-50), 3.75–4.31 (5H, m, T2-H-50, T1-H-40, T2-
H-40, T2-H-30), 4.60–4.70 (1H, m, T1-H-30), 5.20–5.32 (2H,
m, 2!OH), 5.69–5.78 (1H, m, CH]CHCH2N), 5.89–5.97
(1H, m, CH]CHCH2N), 6.14–6.18 (1H, m, T1-H-10), 6.25
(1H, t, JZ7.4 Hz, T2-H-10), 7.68 (1H, s, T2-H-6), 7.70 (1H,
s, T1-H-6); 31P NMR (DMSO-d6) d K0.86; MALDI-MS m/
z: 601.27 (MHC), 623.19 (MNaC).
1
in petrol ether); H NMR (CDCl3) d 0.01–0.12 (12H, m,
Si(CH3)2), 0.83–0.92 (18H, m, C(CH3)3), 1.90–1.94 (3H, m,
CH3), 2.08–2.19 (1H, m, T1-H-20), 2.21–2.29 (2H, m, T2-
H-20), 2.50–2.58 (1H, m, T1-H-200), 3.08–3.11 (2H, m, T2-
5-CH2), 3.85–3.92 (T1-H-50), 4.01–4.03 (1H, m, T2-H-40),
4.20–4.29 (3H, m, T2-H-50, T1-H-40), 4.41–4.44 (1H, m,
T2-H-30), 4.55–4.60 (2H, m, CH2OP), 5.00–5.04 (1H, T1-
H-30), 5.09–5.17 (2H, m, T2-CH]CH2), 5.28–5.41 (2H, m,
T1–CH]CH2), 5.84–6.00 (2H, m, 2!CH]CH2), 6.17–6.25
(1H, m, T2-H-10), 6.32–6.37(1H, m, T1-H-10), 7.24–7.28 (1H,
m, T2-H-6), 7.46–7.48 (1H, m, T1-H-6), 8.76–8.87 (2H, m,
2!NH); 31P NMR (CDCl3) d K0.61; HiRes MALDI FT-MS
m/z (MCNa) found/calcd 863.3454/863.3454.
4.1.22. Preparation of cyclic 50-O,30-O-bis-TBDMS
protected dTpU containing a phosphate-to-5 butanelin-
ker (28). Compound 24 (0.255 g, 0.303 mmol) was
dissolved in anhydrous CH2Cl2 (30 mL) and Grubbs
second-generation catalyst X2 (14.0 mg, 16 mmol,
5.4 mol%) was added. The reaction mixture was stirred at
reflux for 3.5 h and then bubbled with hydrogen for 5 min. The
mixture was placed in an autoclave at 1000 psi hydrogen at
50 8C overnight. The mixture was concentrated under reduced
pressure and the residue was purified by column chromato-
graphy (20–0% petrol ether in ethyl acetate) to give the
product as a white foam and an epimeric mixture (0.156 g,
4.1.19. Preparation of cyclic 50-O,30-O-bis-TBDMS pro-
tected dUpU containing a 5-to-5 (E/Z)-2-butenelinker
(25). Compound 23 (26 mg, 0.030 mmol) was dissolved in
anhydrous CH2Cl2 (3 mL). Grubbs second-generation
catalyst X2 (1.4 mg, 1.6 mmol) was added and the reaction
mixture was stirred at reflux for 22 h. The mixture was
concentrated under reduced pressure, and the residue was
purified by column chromatography (2–8% CH3OH in
CH2Cl2) to give the product as a white powder and a mixture
of diastereomers (6 mg, 23%) as well as starting material 23
(7 mg, 27%); Rf 0.28 (10% CH3OH in CH2Cl2); 31P NMR
(CDCl3) d K2.01 (1), K0.90 (0.13), K0.56 (0.61), 0.02
(0.17); HiRes MALDI FT-MS m/z (MCNa) found/calcd
874.3199/874.3250.
1
63.0%); Rf 0.29 (1% CH3OH in ethyl acetate); H NMR
(CDCl3) d 0.10–0.13 (12H, m, Si(CH3)2), 0.89–0.93 (18H, m,
C(CH3)3), 1.55–1.85 (4H, m, CH2CH2CH2CH2OP), 1.92–
1.94 (3H, m, CH3), 2.10–2.58 (6H, m, T1-H-20, T2-H-20, T2-
5-CH2), 3.85–4.01 (4H, m, T1-H-50, CH2OP), 4.09–4.44 (3H,
m, T2-H-40, T2-H30, T1-H-40), 4.45–4.60 (2H, m, T2-H-50),
4.95–5.05 (1H, m, T1-H-30), 6.29–6.40 (2H, m, T2-H-10, T1-
H-10), 7.39–7.52 (2H, m, T1-H-6, T2-H-6), 9.13–9.24 (2H, m,
2!NH); 31P NMR (CDCl3) d K0.81, 1.29; HiRes MALDI
FT-MS m/z (MCNa) found/calcd 837.3336/837.3298.
4.1.20. Preparation of cyclic 50-O,30-O-bis-TBDMS
protected dTpU containing a phosphate-to-5 (E/Z)-2-
butenelinker (26). Compound 24 (0.052 g, 0.062 mmol)
was dissolved in anhydrous CH2Cl2 (6 mL). Grubbs second-
generation catalyst X2 (2.8 mg, 3.2 mmol, 5.2 mol%) was
added and the reaction mixture was stirred at reflux for 2 h.
The mixture was concentrated under reduced pressure and
the residue was purified by column chromatography (10–0%
petrol ether in ethyl acetate) to give the product as a white
foam and an epimeric mixture (29 mg, 58%); Rf 0.29 (ethyl
acetate); 1H NMR (CDCl3) d 0.10–0.13 (12H, m, Si(CH3)2),
0.89–0.94 (18H, m, C(CH3)3), 1.90–1.94 (3H, m, T1-CH3),
2.06–2.30 (3H, m, T2-H-20, T1-H-20), 2.50–2.60 (1H, m,
T1-H-200), 3.10–3.14 (2H, m, T2-5-CH2), 3.80–3.96 (2H, m,
T1-H-500), 3.98–4.49 (5H, m, T1-H-40, T2-H-50, T2-H-40,
T2-H-3 ), 4.51–4.90 (2H, m, CH2OP), 5.01–5.05 (1H, m,
T1-H-30), 5.84–5.89 (1H, m, CH]CHCH2OP), 5.91–5.98
(1H, m, CH]CHCH2OP), 6.36–6.42 (2H, m, T1-H-10, T2-
H-10), 7.37–7.51 (2H, m, T1-H-6, T2-H-6), 9.02–9.20 (2H,
m, NH); 31P NMR (CDCl3) d K0.65, 2.01. HiRes MALDI
FTKMS m/z (MCNa) found/calcd 835.3148/835.3141; IR
(KBr) n cmK1: 3441, 3065, 2954, 2930, 2857, 1694, 1471,
1276, 1100, 1006, 987, 837, 780.
4.1.23. Preparation of cyclic dTpU containing a phos-
phate-to-5 butanelinker (29). Compound 28 (0.145 g,
0.178 mmol) was dissolved in a 90% aqueous solution of
trifluoroacetic acid (5 mL) and stirred for 3 h. The mixture
was concentrated under reduced pressure and coevaporated
with 99.9% CH3CH2OH (3!5 mL) and with CH3OH
(5 mL) to give the product as a white powder and an
1
epimeric mixture (0.104 g, 100%); H NMR (CD3OD) d
1.56–1.75 (4H, m, CH2CH2CH2CH2OP), 1.77–1.80 (3H, s,
CH3), 2.08–2.50 (6H, m, T1-H-20, T2-H-20, T2-5-CH2),
3.69–3.73 (2H, m, T1-H-500), 3.90–4.42 (7H, m, T1-H-40,
T2-H-50, T2-H-40, T2-H-3 , POCH2), 4.99–5.02 (1H, m,
T1-H-30), 6.18–6.28 (2H, m, T1-H-10, T2-H-10), 7.40 (½H,
s, T2-H-6), 7.50 (½H, s, T2-H-6), 7.69–7.70 (1H, m, T1-H-
6); 31P NMR (CD3OD) d K1.05, 0.01 (1H and 31P NMR
data from DMSO-d6 has been published in Ref. 9); HiRes
MALDI FT-MS m/z (MCNa) found/calcd 609.1584/609,
1568; IR (KBr) n cmK1: 3435, 1690, 1472, 1277, 1022, 784.
4.1.24. Analytical formation of Tp-5-(4-aminobutanyl)-
20-deoxyuridine (30). Compound 29 (2 mg) was dissolved
in 32% aqueous NH3 (1 mL) for 5 days at 55 8C. The
reaction mixture was concentrated under reduced pressure
to give the product as a white foam; 1H NMR (DMSO-d6) d
1.51–1.56 (4H, m, CH2CH2CH2CH2N), 1.78 (3H, s, CH3),
2.03–2.10 (2H, m, T2-H-20), 2.11–2.19 (2H, m, T2-5-CH2),
2.23-2.28 (2H, m, T1-H-20), 2.81–2.84 (2H, m, CH2N),
3.58–3.62 (2H, m, T1-H-500), 3.81–3.88 (2H, m, T2-H-50),
3.94–3.96 (2H, m, T1-H-4 , T2-H-400), 4.30–4.32 (1H, m,
T2-H-30), 4.63–4.67 (1H, m, T1-H-3 ), 5.31 (1H, br, OH),
4.1.21. Analytical formation of Tp-5-(4-amino-2-bute-
nyl)-20-deoxyuridine (27). Compound 26 (2 mg) was
dissolved in 32% aqueous NH3 (1 mL) for 24 h at room
temperature. The reaction mixture was concentrated under
1
reduced pressure to give the product as a white foam; H
NMR (DMSO-d6) d 1.78 (3H, s, CH3), 1.91–2.18 (3H, m,

10966
P. Børsting et al. / Tetrahedron 60 (2004) 10955–10966
6.16 (1H, t, JZ6.7 Hz, T1-H-10), 6.26 (1H, t, JZ7.0 Hz,
T2-H-10), 7.70 (1H, s, T1-H-6), 7.73 (1H, s, T2-H-6), 8.2–
9.2 (3H, br, NH3); 31P NMR (DMSO-d6) d K0.82; MALDI-
MS m/z: 603 (MC), 625 (MNaC); IR (KBr) n cmK1: 3400,
2255, 2128, 1684, 1469, 1277, 1203, 1026, 825, 763.
´
M.; Valery, J.-M. Tetrahedron Lett. 2003, 44, 9131. (f) Fang,
Z.; Hong, J. H. Org. Lett. 2004, 6, 993.
12. For reviews, see (a) Kool, E. T. Chem. Rev. 1997, 97, 1473. (b)
Meldgaard, M.; Wengel, J. J. Chem. Soc., Perkin Trans. 1
2000, 3539. (c) Leumann, C. J. Bioorg. Med. Chem. 2002, 10,
841. (d) Petersen, M.; Wengel, J. Trends Biotech. 2003, 21, 74.
13. For a review on nucleic acid structures, see Belmont, P.;
Constant, J.-F.; Demeunynck, M. Chem. Soc. Rev. 2001, 30, 70.
14. (a) Allerson, C. R.; Verdine, G. L. Chem. Bio. 1995, 2, 667. (b)
Osborne, S. E.; Cain, R. J.; Glick, G. D. J. Am. Chem. Soc.
1997, 119, 1171. (c) Alefelder, S.; Siggurdsson, S. T. Bioorg.
Med. Chem. 2000, 8, 269. (d) Kowalczyk, A.; Carmical, J. R.;
Zou, Y.; Van Houten, B.; Lloyd, R. S.; Harris, C. M.; Harris,
T. M. Biochemistry 2002, 41, 3109.
Acknowledgements
The Danish National Research Foundation and The Danish
Natural Science Research Council are thanked for financial
support.
References and notes
15. (a) Seio, K.; Wada, T.; Sakamoto, K.; Yokoyama, S.; Sekine,
M. J. Org. Chem. 1998, 63, 1429. (b) Seio, K.; Wada, T.;
Sekine, M. Helv. Chim. Acta 2000, 83, 162. (c) Seio, K.; Wada,
T.; Sakamoto, K.; Yokoyama, S.; Sekine, M. J. Org. Chem.
1996, 61, 1500. (d) Sekine, M.; Kurasawa, O.; Shohda, K.;
Seio, K.; Wada, T. J. Org. Chem. 2000, 65, 3571. (e) Sekine,
M.; Kurasawa, O.; Shohda, K.; Seio, K.; Wada, T. J. Org.
Chem. 2000, 65, 6515. (f) Sekine, M.; Kurasawa, O.; Shohda,
K.; Seio, K.; Wada, T. Eur. J. Org. Chem. 2001, 1989.
1. For reviews on RCM, see (a) Grubbs, R. H.; Chang, S.
Tetrahedron 1998, 54, 4413. (b) Jørgensen, M.; Hadwiger, P.;
Madsen, R.; Stu¨tz, A. E.; Wrodnigg, T. M. Curr. Org. Chem.
2000, 4, 565. (c) Fu¨rstner, A. Angew. Chem. Int. Ed. 2000, 39,
3012. (d) Schrock, R. R.; Hoveyda, A. H. Angew. Chem. Int.
Ed. 2003, 42, 4592.
2. (a) Scholl, M.; Ding, S.; Lee, C. W.; Grubbs, R. H. Org. Lett.
1999, 1, 953. (b) Trnka, T. M.; Grubbs, R. H. Acc. Chem. Res.
2001, 34, 18.
´
16. Le Clezio, I.; Escudier, J.-M.; Vigroux, A. Org. Lett. 2003, 5,
161.
3. Deiters, A.; Martin, S. F. Chem. Rev. 2004, 104, 2199.
4. McReynolds, M. D.; Dougherty, J. M.; Hanson, P. R. Chem.
Rev. 2004, 104, 2239.
17. (a) Caruthers, M. H. Acc. Chem. Res. 1991, 24, 278. (b)
Beaucage, S. L.; Iyer, R. P. Tetrahedron 1992, 48, 2223.
18. Bhat, B.; Leonard, N. J.; Robinson, H.; Wang, A. H.-J. J. Am.
Chem. Soc. 1996, 118, 10744.
5. (a) Blackwell, H. E.; Sadowsky, J. D.; Howard, R. J.;
Sampson, J. N.; Chao, J. A.; Steinmetz, W. E.; O0Leary,
D. J.; Grubbs, R. H. J. Org. Chem. 2001, 66, 5291. (b)
Reichwein, J. F.; Versluis, C.; Liskamp, R. M. J. J. Org. Chem.
2000, 65, 6187. (c) Colombo, L.; Di Giacomo, M.; Vinci, V.;
Colombo, M.; Manzoni, L.; Scolastico, C. Tetrahedron 2003,
59, 4501. (d) Harris, P. W. R.; Brimble, M. A.; Gluckman,
P. D. Org. Lett. 2003, 5, 1847. (e) Barrett, A. G. M.; Hennessy,
19. Ruth, J. L.; Bergstrom, D. E. J. Org. Chem. 1978, 43, 2870.
20. Vargeese, C.; Carter, J.; Yegge, J.; Krivjansky, S.; Settle, A.;
Kropp, E.; Peterson, K.; Pieken, W. Nucleic Acids Res. 1998,
26, 1046–1050.
21. Sproat, B. S.; Iribarren, A.; Beijer, B.; Pieles, U.; Lamond,
A. I. Nucleosides Nucleotides 1991, 10, 25.
22. Beijer, B.; Grøtli, M.; Douglas, M. E.; Sproat, B. S. Nucleo-
sides Nucleotides 1994, 13, 1905.
´
¨
A. J.; Le Vezouet, R.; Procopiou, P. A.; Seale, P. W.;
Stefaniak, S.; Upton, R. J.; White, A. J. P.; Williams, D. J.
J. Org. Chem. 2004, 69, 1028. (f) Brouwer, A. J.; Liskamp,
R. M. J. J. Org. Chem. 2004, 69, 3662. (g) Ijsselstijn, M.;
Aguilera, B.; van der Marel, G. A.; van Boom, J. H.; van Delft,
F. L.; Schoemaker, H. E.; Overkleeft, H. S.; Rutjes, F.P.J.T.;
Overhand, M. Tetrahedron Lett. 2004, 45, 4379.
23. Okamoto, I.; Shohda, K.; Seio, K.; Sekine, M. J. Org. Chem.
2003, 68, 9971.
24. Wu, J. C.; Xi, Z.; Gioeli, C.; Chattopahyaya, J. Tetrahedron
1991, 47, 2237.
25. Lakhmiri, R.; Lhoste, P.; Sinou, D. Tetrahedron Lett. 1989, 30,
4669.
6. (a) Ravn, J.; Nielsen, P. J. Chem. Soc., Perkin Trans. 1 2001,
985. (b) Ravn, J.; Thorup, N.; Nielsen, P. J. Chem. Soc., Perkin
Trans. 1 2001, 1855. (c) Thomasen, H.; Meldgaard, M.;
Freitag, M.; Petersen, M.; Wengel, J.; Nielsen, P. Chem.
Commun. 2002, 1888. (d) Freitag, M.; Thomasen, H.;
Christensen, N. K.; Petersen, M.; Nielsen, P. Tetrahedron
2004, 60, 3775.
26. Sekine, M. J. Org. Chem. 1989, 54, 2321.
27. Matsuda, A.; Yasuoka, J.; Sasaki, T.; Ueda, T. J. Med. Chem.
1991, 34, 999.
28. (a) Manfredini, S.; Baraldi, P. G.; Bazzanini, R.; Simoni, D.;
Balzarini, J.; De Clercq, E. Bioorg. Med. Chem. Lett. 1997, 7,
473. (b) Manfredini, S.; Baraldi, P. G.; Durini, E.; Vertuani, S.;
Balzarini, J.; De Clercq, E.; Karlsson, A.; Buzzoni, V.;
Thelander, L. J. Med. Chem. 1999, 42, 3243.
29. Markiewicz, W. Chem. Scripta 1986, 26, 123.
30. (a) Hampton, A.; Nichol, A. W. Biochemistry 1966, 5, 2076. (b)
McGee, D. P. C.; Vargeese, V.-S. J. Org. Chem. 1996, 61, 781.
31. Louie, J.; Bielawski, C. W.; Grubbs, R. H. J. Am. Chem. Soc.
2001, 123, 11312.
7. (a) Sørensen, A. M.; Nielsen, P. Org. Lett. 2000, 2, 4217. (b)
Sørensen, A. M.; Nielsen, K. E.; Vogg, B.; Jacobsen, J. P.;
Nielsen, P. Tetrahedron 2001, 57, 10191.
8. Børsting, P.; Sørensen, A. M.; Nielsen, P. Synthesis 2002, 797.
9. Børsting, P.; Nielsen, P. Chem. Commun. 2002, 2140.
10. Kirchhoff, C.; Nielsen, P. Tetrahedron Lett. 2003, 44, 6475.
11. (a) Montembault, M.; Bourgourgnon, N.; Lebreton, J. Tetra-
hedron Lett. 2002, 43, 8091. (b) Chen, X.; Wiemer, D. F.
J. Org. Chem. 2003, 68, 6597. (c) Gillaizeau, I.; Lagoja, I. M.;
Nolan, S. P.; Aucagne, V.; Rozenski, J.; Herdewijn, P.;
Agrofoglio, L. A. Eur. J. Org. Chem. 2003, 601. (d) Ewing,
D. F.; Glac¸on, V.; Mackenzie, G.; Postel, D.; Len, C.
Tetrahedron 2003, 59, 941. (e) Busca, P.; Etheve-Quelquejeu,
32. Børsting, P.; Mikkelsen, B. H.; Nielsen, P., manuscript in
preparation.
33. Garber, S. B.; Kingsbury, J. S.; Gray, B. L.; Hoveyda, A. H.
J. Am. Chem. Soc. 2000, 34, 8169. Available with this product
name from Materia/Sigma-Aldrich.
34. Cravotto, G.; Giovenzana, G. B.; Sisti, M.; Palmisano, G.
Tetrahedron 1998, 54, 1639.