Synthesis of dinucleotides with 2′-C to phosphate connections by ring-closing metathesis

Article abstract of DOI:10.1016/j.tet.2005.10.074

Four different nucleosides with olefinic 2′-modifications were prepared; 2′-C-methylene, 2′-C-(propen-1-yl), 2′-C-allyl and 2′-O-allyl uridines, respectively. These were incorporated into dinucleotides with allyl phosphate or vinyl phosphonate linkages. Hence, six different dinucleotides were studied as substrates for RCM reactions, and from four of these, cyclic dinucleotides with connections between 2′-C and phosphorus of 3-6 atoms were obtained.

Full text of DOI:10.1016/j.tet.2005.10.074

Tetrahedron 62 (2006) 1139–1149
0
Synthesis of dinucleotides with 2 -C to phosphate connections
by ring-closing metathesis
Philip Børsting, Mikkel S. Christensen, Signe I. Steffansen and Poul Nielsen
*
†
Nucleic Acid Center, Department of Chemistry, University of Southern Denmark, 5230 Odense M, Denmark
Received 17 August 2005; revised 12 October 2005; accepted 27 October 2005
Available online 18 November 2005
0
0
0
0
Abstract—Four different nucleosides with olefinic 2 -modifications were prepared; 2 -C-methylene, 2 -C-(propen-1-yl), 2 -C-allyl and
0
2 -O-allyl uridines, respectively. These were incorporated into dinucleotides with allyl phosphate or vinyl phosphonate linkages. Hence, six
different dinucleotides were studied as substrates for RCM reactions, and from four of these, cyclic dinucleotides with connections between
0
2 -C and phosphorus of 3–6 atoms were obtained.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
have been prepared by the use of RCM or related cross-
6
metathesis methods.
,7
The ring-closing metathesis (RCM) reaction is generally
acknowledged as an extremely powerfull synthetic method
for introducing medium to large rings tolerating most
We have introduced RCM in nucleic acid chemistry as
a general synthetic strategy for the introduction of
conformationally restricting internucleotide connections in
1
functional groups. The method relies on efficient catalysts
being able to induce a series of [2C2] cycloadditions
8
,9
appropriate dinucleotide substrates.
Hereby, dinucleo-
1
,2
combining two olefinic moieties into a new cyclic olefin.
Two of the most useful catalysts for the purpose are the
tides and, subsequently, oligonucleotides with artificial
bends can be obtained as potential tools in targeting and
modeling secondary nucleic acid structures like bulges or
2
,3
4
ruthenium-based A and B (MesZ2,4,6-trimethylphenyl,
CyZcyclohexyl).
1
0
three-way-junctions. The dinucleotide substrates studied
so far have involved nucleoside building blocks with
0
11
terminal alkenes like 5 -C-vinylthymidine, 5 -C-allyl-
0
1
thymidine, 4 -C-vinylthymidine, 2 -O-allyluridine,
2
0
12
0
9
0
9
-O-allylarabinouridine, 5-allyluridine
8,13
0
2
deoxyuridine
and 5-allyl-2 -
in combination with allyl phosphortriester
9
,13
linkages. In several cases, cyclic dinucleotides have been
obtained in high yields by RCM on these substrates. Also a
tandem RCM-hydrogenation protocol has been applied
for the direct introduction of saturated linkages avoiding
the E/Z-isomeri often involved in the production of large
9
,13
cycloalkene structures.
Among many successful applications, the RCM method-
ology has been used in peptide chemistry for the preparation
of cyclic peptide analogues, for example, conformationally
In the present study, we focus on the scope for preparing
0
connections between the 2 -C position and the adjacent
phosphorus in dinucleotides—in other words the prepara-
5
0
0
restricted b-turn mimics. Also nucleosides and nucleotides
tion of 2 -3 -fused bicyclic nucleotides with varying ring
sizes. Hence, a series of different nucleosides with olefinic
0
Keywords: Ring-closing metathesis; Nucleosides; Dinucleotides; Confor-
mational restriction.
2 -modifications are prepared and incorporated into di-
nucleotides with allyl phosphate or vinyl phosphonate
*
Corresponding author. Tel.: C45 6550 2535; fax: C45 6615 8780;
e-mail: pon@chem.sdu.dk
Nucleic Acid Center is funded by the Danish National Research
Foundation for studies on nucleic acid chemical biology.
0
linkages. Subsequently, 2 -C to P connections of 1–6 atoms
can be formed by RCM reactions. The most successfully
prepared of these cyclic dinucleotides (containing a 5 atom
†
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.10.074

1140
P. Børsting et al. / Tetrahedron 62 (2006) 1139–1149
0
2
-C-(CH ) O–P connection) has been presented in a
2 4
separate publication including the incorporation of the
dinucleotide into oligonucleotides and the subsequent
impact on nucleic acid duplexes, bulged duplexes and
1
4
three-way junctions. In the present paper, we cover the
complete synthetic effort investigating the scope of RCM to
0
produce 2 -C to phosphorus connections in dinucleotides.
2
. Results and discussion
0
containing 2 -modifications were prepared. These represent
Four different 5 -O-protected nucleosides with olefin-
0
four distances, from 0 to 3 bonds, between the sugar and the
olefin moiety; a 2 -C-methylene group, a 2 -C-(propen-1-yl)
0
0
0
0
group, a 2 -C-allyl group and a 2 -O-allyl group. All
building blocks were made from the widely used TIPDS-
protected uridine 1 as the starting material (Scheme 1). The
0
15
known 2 -methylene derivative 2 was made by an
oxidation/Wittig procedure according to recent literature
1
methods and deprotected to give the likewise well-known
6
0
gave the 5 -protected compound 4 in a good yield.
0
0
16,17
2
-deoxy-2 -C-methyleneuridine 3.
Selective silylation
Another oxidation of 1 followed by a stereoselective
Grignard reaction afforded the 2 -(propyn-1-yl) derivative
0
. We preferred to use a propyn-1-yl group over an ethynyl
5
group since a controlled reduction to give a double bond
18
could be expected. Moreover, several RCM-reactions with
methyl substituated double bonds leading to the release of
propylene have been reported. Direct introduction of a
1
vinyl-group was avoided. All though the reaction of
1
9
VinylMgBr with this substrate has been shown before,
low yields with this reagent on related substrates have been
1
1
observed. A lot of Grignard, organolithium or organo-
aluminium reactions with the present substrate have been
1
9–23
described in the literature
other alkynyl groups.
including the introduction of
However, the introduction of
1
9,22,23
the propyn-1-yl group has not been shown before. Never-
theless, our first yield of 5 (52% from the ketone) was
hampered by the formation of partly desilylated side
products as demonstrated similarly with the use of
2
0
MeMgBr. This problem was solved, however, by the use
of a CeCl assisted Grignard reaction affording 5 as the only
3
product in 69% yield. Removal of the protecting group
afforded 6 and the selective silylation gave the 5 -TBDMS
0
protected derivative 7 in a high yield. As expected, the
1
8
controlled hydrogenation using the Lindlar catalyst gave
in a very high yield the Z-configured alkene 8 as the only
product. Exact control of the reaction time was found to be
crucial, as isomerisation to give the E-configured isomer or
over reduction to give the propyl group was observed in the
reaction mixture after more than 30 min reaction time. No
Scheme 1. Reagents and conditions: (a) Ref. 16; (b) TBAF, THF, Refs.
16, 17; (c) TBDMSCl, pyridine, 83% (two steps); (d) (i) CrO , pyridine,
3
Ac
THF, 86%; (f) TBDMSCl, imidazole, DMF, 70%; (g) H
quinoline, MeOH, 95%; (h) ((i-Pr) N) POCH CH]CH
CH CN, 66%; (i) ((i-Pr) N) PCH]CH , 4,5-dicyanoimidazole, CH
4% (17), 71% (18). TBDMSZt-butyldimethylsilyl.
2
O, CH
2
Cl
2
, 93%; (ii) CH
3
C^CMgBr, CeCl
3
, THF, 69%; (e) TBAF,
, Lindlar catalyst,
, 1H-tetrazole,
Cl
2
2
2
2
2
2
,
0
3
2
2
2
2
effort towards the deoxygenation of the 2 -tertiary alcohol
was undertaken, as similar compounds are well-known to
7
0
afford the arabino-configured 2 -deoxy-products after
radical-type deoxygenations.
1
9,23,24
0
0
From the four 5 -protected 2 -olefinic uridine derivatives
a series of dinucleotides were prepared as substrates
for RCM-reactions. Hence, 4, 8, 9 and 10 were all coupled
From recently published procedures from our group, the
0
0
0
0
0
appropriately 5 -protected 2 -deoxy-2 -C-allyl and 2 -O-
with the allylated 5 -phosphoramidite 12 (obtained from
0
14
9
allyl derivatives 9 and 10 were also obtained from the
same starting material 1 (Scheme 1) via well-known
methodologies.
3 -O-TBDMS-thymidine 11, Scheme 1) to give the four
dinucleotides 13–16 in good yields (Schemes 2 and 3).
Similarly, two representative vinyl phosphonate dinucleotides
2
5,26

P. Børsting et al. / Tetrahedron 62 (2006) 1139–1149
1141
0
were approached. However, these were realized from 3 -
amidites. Thus, 4 and 9 were reacted with bis(diisopropyla-
The next substrate 14 was more reactive (Scheme 2). Thus,
treatment of 14 with 2!10 mol% of A in refluxing DCM for
5 h afforded the product 21 in 26% yield. The product was
confirmed by mass spectrometry showing the mass to be
decreased by 42 compared to the substrate according to the
0
mino)vinylphosphinate to give the 3 -amidites 17 and 18 in
0
reasonable yields (Scheme 1). These were coupled with 3 -O-
TBDMS-thymidine 11 to give the two dinucleotides 19 and 20
3
1
loss of propylene. P NMR showed a 3:2 mixture of
phosphorus epimers as also found for the starting material
(Scheme 4).
14. Hence, the relatively low yield cannot be addressed to
different reactivity of the two epimers. The mixture of
epimers 21 was separated using simple column chromato-
graphy to give the two epimers in 16 and 10% yield,
1
respectively. The two H NMR spectra being very similar
demonstrated both the lack of signals from a terminal alkene
1
and the propylene methyl group. From the H NMR spectra
3
of the major of the two epimers, a JHC]CH coupling
constant of 11.9 Hz was observed confirming the new cyclic
alkene to be Z-configurated. A tandem RCM-hydrogenation
1
3
protocol with 14 as the substrate was also performed.
Thus, after subjecting the starting material for the RCM-
reaction, the reaction mixture was stirred in a 70 atm
hydrogen atmosphere in which the catalyst A worked as a
hydrogenation catalyst. After a chromatographic separation,
the two saturated cyclic phosphorus epimers of 22 were
isolated in low yields, 8 and 3%, respectively. Nevertheless,
the constitution of the products was clearly verified from
3
1
MS and NMR. In the P NMR it was clear that the chemical
shifts of the two epimers were more similar indicating the
more flexible nature of the saturated cyclic structure 22
compared to the unsaturated counterpart 21.
The dinucleotide 15 was a much more efficient substrate for
1
4
an RCM-reaction than 13 and 14 (Scheme 3). Thus, the
cyclic product 23 was obtained in a good yield, and so was
the saturated product 24. Thus, after separation, the two
phosphorus epimers of 24 were obtained in 48 and 17%
yield, respectively. These were desilylated, and the exact
phosphorus configurations were established by a combi-
nation of advanced NMR and molecular modeling. Finally,
the major isomer was incorporated into oligodeoxynucleo-
tides. This part of the study including the discovery of a
stabilised artificial three-way junction has been published
1
4
separately in a recent paper.
The dinucleotide 16 was also efficient as a substrate for an
RCM-reaction (Scheme 3). A treatment of 16 with 7 mol%
A in DCM afforded in a 45% yield the mixture of cyclic
dinucleotides 25 in a 3:1 ratio of phosphorus epimers. A
similar treatment with B, on the other hand, did not afford
any product. Again, the product was confirmed by NMR and
MS confirming the loss of ethylene. A tandem RCM-
hydrogenation afforded the saturated cyclic dinucleotide 26
as a 5:3 mixture of epimers in 62% yield. The starting
material was a 3:2 epimeric mixture, and therefore, one of
the isomers (the major) seems to react slightly faster than
the other. The higher yield of the tandem reaction
corresponds to a relatively better reaction of the slower
reacting epimer in that case. The mixture of dinucleotides
26 was not separated but deprotected using an acidic
treatment to give the epimeric mixture 27 in 59% yield. The
stability of the phosphortriester moiety towards basic
conditions was examined, and 27 was found to be
unchanged after a treatment with saturated ammonia at
room temperature for 24 h. This was similar to the findings
Scheme 2. Reagents and conditions: (a) (i) 1H-tetrazole, CH
ii) t-BuOOH, toluene, CH CN, 97% (13), 100% (14); (b) Grubbs
Cl 26% (21); (c) A, CH Cl then
3
CN,
(
2
3
nd gen. catalyst A, CH
0 atm 50 8C, 11%.
2
2
,
2
2
,
7
2
H ,
The four dinucleotides 13–16 were studied as substrates for
RCM-reactions using Grubbs second generation catalyst A.
Dinucleotide 13, however, did not afford any product and
the starting material was recovered (Scheme 2). The
reaction was attempted with varying amounts of A in
refluxing DCM as well as in a microwave reactor at 100 8C.
Also, the Hoveyda–Grubbs catalyst B was used under the
same conditions but with no ring-closed product formed.

1142
P. Børsting et al. / Tetrahedron 62 (2006) 1139–1149
1
4
Scheme 3. Reagents and conditions: (a) (i) 1H-tetrazole, CH
3
CN, (ii) t-BuOOH, toluene, CH CN, 85% (15); 95% (16), (b) Grubbs 2nd gen. catalyst A,
3
14
14
CH Cl , 76% (23), 45% (25); (c) A, CH Cl , then 70 atm H , 50 8C, 65% (24), 62% (26); (d) 90% TFA (aqueous), 59%.
2 2 2 2 2
Scheme 4. Reagents and conditions: (a) 4,5-dicyanoimidazole, CH
70 atm H , 50 8C, 57%; (d) A, 20% CuCl, CH Cl , 35%.
3 2 2 2 2
CN, 54% (19), 71% (20); (b) Grubbs 2nd gen. catalyst A, CH Cl ; (c) A, CH Cl , then
2
2
2

P. Børsting et al. / Tetrahedron 62 (2006) 1139–1149
1143
1
4
for the major isomer of 24 and therefore, the incorporation
of this product into oligonucleotides should also be
relatively straightforward.
found to be superior to the first generation catalyst ((Cy P) -
3 2
1
RuCl CHPh) and the Hoveyda–Grubbs second generation
,2
2
catalystBwasattemptedandnotfoundtobeagoodalternative
in this study. Concerning solvents, we have attempted toluene
8
,11
Finally, the possibility of using the vinyl phosphonate moiety
as an alternative starting point for RCM-reactions was
explored. This has two obvious advantages. First, the potential
ring size of the cyclic dinucleotides decreases, and therefore,
the conformational strain of the product should increase.
Secondly, the base lability of the phosphortriester moiety and
the potential troubles with the incorporation of the cyclic
dinucleotides into oligonucleotides (as already seen with one
in other studies but this solvent has been incompatible with
our nucleotide substrates. Furthermore, the major subject of
this study was to explore the scope of making large rings
0
between a 2 -olefin moiety and the adjacent phosphorus, in
other words to find the optimally formed rings.
The most conveniently prepared ring was the nine-
membered ring in 23/24. In other words, the 2 -C-allyl-2 -
0
deoxy moiety was the most reactive among the 2 -C
0
1
4
0
connected olefins. The 2 -O-allyl moiety was also reactive,
of the epimers of 24) should be avoided. Hence, a
phosphonate diester should be significantly less labile. As
the first substrate for this study, however, dinucleotide 19 did
not afford any product when treated with 5 mol% A in neither
DCMnortoluene(Scheme 4).Thereactionwasalsoattempted
under a 70 atm pressure of nitrogen over night followed by a
treatment of 70 atm hydrogen in order to perform a tandem
RCM-hydrogenation. However, the product isolated in 57%
yield displayed a mass increased by 4 Da corresponding to the
double hydrogenated product 28. The NMR spectra indicated
a mixture of four diastereomers corresponding to the
unselective hydrogenation of the methylene group as well as
a pair of phosphorus epimers. These results in combination
with the failed attempts of cyclisation of 13 clearly
demonstrated the inability of the precatalyst A to initiate a
0
and 25/26 were obtained in reasonable yields. This is in
contrary to our recent results where dinucleotides consisting
of a 2 -O-allyluridine moiety with either a 5-allyl-
0
2 -deoxyuridine or a 2 -O-allyl-arabinouridine moiety
0
were unreactive towards A. Thus, those results must be
0
9
due to the potential ring-sizes being unfavourable and
particular conformational properties of the substrates.
9
Among the two conveniently formed ring systems, 23/24
1
4
and 25/26, the former was selected for further studies. It
was shown that the major of the two phosphorus epimers of
24 after deprotection could be successfully incorporated
into oligonucleotides. In these, the cyclic moiety was
demonstrated to induce a significant distortion of a duplex
but also a slight stabilization of a three-way junction. This is
formed between an oligonucletide with the cyclic moiety in
the middle of the sequence and a complementary RNA
strand with a stem-loop sequence opposite the cyclic
0
catalytic metathesis cycle with a 2 -methylene nucleoside—
most probably for steric reasons. On the other hand, when
converted to a hydrogen donor, the catalyst is able to induce a
hydrogenation of the same position. Another substrate, which
is less sterically crowded, 20, was also studied. However,
neither this substrate was active when subjected to 5 mol% A
in DCM. However, using the known phosphine scavenger
CuCl in combination with 5 mol% A was also attempted
with 20, and in this case, a cyclised product 29 was obtained in
5% yield as a 3:1 mixture of phosphorus epimers. It has,
1
4
moiety. We expect also 27 to be easily incorporated into
oligonucleotides. Thus, the stability of 27 against aqueous
ammonia was found to be good, and this is crucial if
standard procedures for automated oligonucleotide syn-
thesis should be followed. Comparing 26/27 and 24,
completely different conformational behaviour of the
2
7
3
however, been impossible in our hands to improve this yield
by other reaction conditions.
resulting oligonucleotides can be expected. Thus, the
2
0
conformation of the uridine moiety into a N-type
conformation as usually found for 2 -O-alkylated nucleo-
-oxygen atom in 26/27 should take the nucleoside
From the present results it is clear that not all the
dinucleotide substrates studied were found to be efficient
for RCM reactions. The best formed rings have been the
0
0
29
sides due to the gauche effect of the 2 -oxygen. However,
the ring is larger and perhaps more flexible in 27. Hence, the
H NMR data of 27 indicated that none of the two
larger 9- and 10-membered rings in compounds 23–26. The
2
0
the sterically demanding catalyst. This would explain the
1
-C-methylene group might be inaccessible for
phosphorus epimers seems to be restricted towards an
0
complete inactivity of the substrates 13 and 19. The result
that 19 do react with the ‘Ru-H ’ intermediate is explained
from the lower sterical demand of this intermediate. It is
also clear that the vinyl phosphonate group is significantly
less reactive compared to the allyl phosphate group. This is
of course expected as unsaturated esters are generally less
N-type conformation. Very different J
0
coupling
H1 H2
constants (3.8 and 7.7 Hz, respectively) were found and
only the former indicates a slight preference for an N-type
conformation. For 24, on the other hand, an S-type
2
1
4
conformation of the uridine moiety was found as
0
0
completely expected for a 2 -deoxy-2 -alkylated nucleoside
due to the tendency for carbon substituents to prefer a
pseudoequatorial orientation. Efforts towards the separation
of the phosphorus epimers of 27 and the incorporation of
these into oligonucleotides are in progress.
1
,7
susceptible for metathesis reactions. On the other hand,
vinyl phosphonates have been used before, also in cross
0
The lower reactivity was in that case
metathesis reactions involving a vinyl 3 -nucleoside
phosphonate.
7
,28
demonstrated by the inability of the substrate to undergo
homodimerisation by cross metathesis. Nevertheless, we
demonstrated an RCM reaction of vinyl phosphonate 20.
2
8
Also the cyclic nucleotide with a phosphonate containing
ring 29 might be incorporated into oligonucleotides. Hence,
this structure is expected to be more stable compared to the
phosphortriester moieties in the other dinucleotides,
especially after an eventual hydrogenation. On the other
hand, the yield of the ring-closure must be improved.
It could be argued, of course, that a longer range of catalysts
andsolventsshouldbeincludedinthestudy.However, Grubbs
second generation catalyst A was in all cases tested by us

1144
P. Børsting et al. / Tetrahedron 62 (2006) 1139–1149
Finally, the incorporation of 22 can be completed. However,
in this case the efficiency of the ring-closure was too low,
and we will prefer in the near future to make another ring-
system with the same ring-size, for example, from a
substrate with a vinyl group instead of a propen-1-yl
6 h, concentrated under reduced pressure and co-evaporated
with anhydrous toluene. The residue was dissolved in DCM
(50 mL) and washed with a saturated aqueous solution
of NaHCO (50 mL) and brine (50 mL). The combined
3
aqueous phases were extracted with DCM (50 mL) and the
combined organic phases were dried (MgSO ) and
0
group and, preferably, without a 2 -hydroxy moiety.
4
concentrated under reduced pressure. The residue was
purified by dry column chromatography (1–4% methanol in
DCM) to give the product as a white foam (0.248 g, 83%)
3. Conclusions
(Found C, 52.01; H, 7.35; N, 7.42; C H N O Si$H O
requires C, 51.63; H, 7.48; N, 7.48); R 0.19 (5% methanol
16 26 2 5 2
0
Four different cyclic dinucleotides with 2 -C to P
connections have been prepared in varying yields. The
most efficiently obtained cyclic dinucleotide, containing a
f
1
in DCM); H NMR (CDCl ) d 0.10–0.11 (6H, s, Si(CH ) ),
3
3 2
0
3.93–4.00 (2H, m, H-5 ), 4.76 (1H, m, H-3 ), 5.45–5.55 (2H,
0.91 (9H, s, C(CH ) ), 2.70 (1H, s, OH), 3.87 (1H, m, H-4 ),
3
3
0
0
0
2
-C-(CH ) O–P connection, was chosen for incorporation
4
2
into oligonucleotides. In general, dinucleotide substrates
with allyl phosphates were much more readily applied than
with vinyl phosphonates, and the reactivity of the 2 -C
olefins increased by the decrease of sterical demand near the
sugar moiety. In the future, the scope of RCM in the
preparation of oligonucleotides targeting secondary nucleic
acid structures will be further explored.
m, CH]CH ), 5.69 (1H, d, JZ8.1 Hz, H-5), 6.69 (1H, m,
2
0
NMR (CDCl ) d K5.4, K5.3 (Si(CH ) ), 18.5 (C(CH ) ),
13
H-1 ), 7.60 (1H, d, JZ8.1 Hz, H-6), 9.33 (1H, s, NH);
C
0
3
3 2
3 3
0
84.8 (C-1 ), 102.9 (C-5), 113.8 (C]CH ), 141.0 (C-6),
0
0
26.0 (C(CH ) ), 62.0 (C-5 ), 70.7 (C-3 ), 84.3 (C-4 ),
3
3
0
148.8 (C-2 ), 150.9 (C-2), 163.5 (C-4); HiRes MALDI
2
0
FT-MS m/z (MCNa ) found/calcd 377.1511/ 377.1503.
C
0 0 0
.1.2. Preparation of 1-(2 -C-(propyn-1-yl)-3 ,5 -O-
(1,1,3,3-tetraisopropyldisiloxandiyl)-b-D-arabino-
4
4. Experimental
furanosyl)uracil (5). A mixture of CrO (1.1 g, 12 mmol)
3
4
.1. General
and DCM (20 mL) was stirred at 0 8C. Pyridine (1.6 mL)
was added dropwise and the mixture was stirred at room
temperature. Acetic anhydride (1 mL) and a solution of
compound 1 (1.1 g, 2.3 mmol) in DCM (5 mL) were added,
and the reaction mixture was stirred for 2 h. Ethyl acetate
(50 mL) was added and the mixture was filtered through a
layer of silica. The filter was rinsed with more ethyl acetate
and the combined filtrates were concentrated under reduced
pressure to give the ketone (1.0 g, 93%). CeCl $7H O
All commercial reagents were used as supplied. When
necessary, reactions were performed under an atmosphere
of nitrogen. An EMRYSe Creator was used for microwave
assisted synthesis. 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
1
spectrometer. H NMR spectra were recorded at 300 MHz,
C NMR spectra at 75.5 MHz, and P NMR spectra at
3
2
1
3
31
(5.0 g, 13.3 mmol) was dried at 140 8C under reduced
pressure for 12 h and added anhydrous THF (15 mL). The
mixture was stirred for 2 h at room temperature and then at
121.5 MHz. Values for d are in ppm relative to tetra-
methylsilane as internal standard or 85% H PO as external
3
4
standard. Microanalyses were performed at The Micro-
analytical Laboratory, Department of Chemistry, University
of Copenhagen. Assignments of NMR spectra are based on
K78 8C. A 0.5 M solution of CH C^CMgBr in THF
3
(20 mL, 10 mmol) was added dropwise and the mixture was
stirred for 1.5 h. A solution of the ketone (1.0 g, 2.1 mmol)
in THF (2.5 mL) was cooled to K78 8C and added dropwise
to the stirred solution of Grignard reagent. The mixture was
stirred for 9 h and then added acetic acid. The mixture was
extracted with ethyl acetate (4!20 mL). The combined
organic phases were washed with an aqueous solution of
NaHCO , dried (MgSO ) and concentrated under reduced
3 4
pressure. The residue was purified by column chromatog-
raphy (0–4% methanol in DCM) to give the product as a
2
carbon next to the nucleobase is assigned C-1 . For
D spectra and follow standard nucleoside style; that is, the
0
dinucleotides, the upper (5 ) nucleotide is depicted U and
the lower nucleoside T.
0
0
0
4
.1.1. Preparation of 5 -O-tert-butyldimethylsilyl-2 -
0
15
deoxy-2 -methyleneuridine (4). Compound 2 (0.452 g,
.936 mmol) was dissolved in THF (10 mL) and a 1 M
0
solution of TBAF in THF (3 mL, 3 mmol) was added. The
reaction mixture was stirred for 20 min and a mixture of
pyridine, methanol and water (5 mL, 3:1:1) was added. A
mixture of Dowex 50 W!2 (4 g) in pyridine, methanol and
water (25 mL, 3:1:1) was added and the suspension was
stirred for 30 min and filtered. The filter was washed with a
mixture of pyridine, methanol and water (25 mL, 3:1:1), and
the combined filtrates were concentrated under reduced
pressure. The residue was purified by dry column
chromatography (17% ethanol in ethyl acetate) to give the
crude compound 3. This material (0.337 g) was dissolved in
anhydrous pyridine (9 mL) and TBDMSCl (0.146 g,
white foam (0.76 g, 69%); R
f
0.47 (10% methanol in DCM);
) d 1.00–1.10 (28H, s, SiCH(CH ), 1.92
) 3.05 (1H, s, OH), 3.90–4.20 (4H, m, H-
3 , H-4 , H-5 ), 5.69 (1H, dd, JZ2.0, 8.0 Hz, H-5), 6.02 (1H,
1
H NMR (CDCl
(3H, s, C^CCH
)
3 2
3
3
0
0
0
0
13
s, H-1 ), 7.87 (1H, d, JZ8.0 Hz, H-6), 8.70 (1H, s, NH);
NMR (CDCl ) d 4.02 (C^CCH ), 12.5, 12.9, 13.1, 13.5
), 16.7, 16.9, 17.0, 17.1, 17.2, 17.4, 17.4, 17.5
C
3
3
(SiCH(CH
(SiCH(CH
76.5 (C^CCH
)
)
3
2
2
0
0
), 60.0 (C-5 ), 73.0 (C-3 ), 75.8 (C^CCH
),
), 81.4 (C-4 ), 85.0 (C-2 ), 89.6 (C-1 ), 101.8
3
3
3
0
0
0
(C-5), 140.0 (C-6), 151.4 (C-2), 163.2 (C-4); HiRes MALDI
FT-MS m/z (MCNa ) found/calcd 547.2244/547.2266.
C
0
furanosyl)uracil (6). Compound 5 (1.03 g, 1.97 mmol)
0
.97 mmol) was added. The reaction mixture was stirred
for 2 h and an additional amount of TBDMSCl (0.056 g,
.37 mmol) was added. The reaction mixture was stirred for
4.1.3. Preparation of 1-(2 -C-(propyn-1-yl)-arabino-
0
was dissolved in THF (10 mL) and a solution of TBAF$H O
2

P. Børsting et al. / Tetrahedron 62 (2006) 1139–1149
1145
0
0
0
(
1.10 g, 3.94 mmol) in THF (4 mL) was added. The solution
was stirred for 20 min and added acetic acid (0.2 mL,
mmol) and silica gel (4.5 g). The mixture was concen-
4.22 (1H, m, H-3 ), 4.24 (1H, m, H-4 ), 4.95 (1H, s, 2 -OH),
5.64 (1H, dd, JZ1.8, 8.4 Hz, H-5), 5.69 (1H, m,
4
CH]CHCH3), 6.03 (1H, dq, JZ7.1, 11.6 Hz,
0
H-6), 8.72 (1H, s, NH); C NMR (CDCl ) d K5.5, K5.4
trated under reduced pressure, and the residue purified by
column chromatography (0–10% methanol in DCM) to give
the product as a white foam (0.575 g, 86%); R 0.33 (20%
methanol in DCM); H NMR (DMSO-d ) d 1.83 (3H, s,
6
C^CCH ) 3.53–3.67 (2H, m, H-5 ), 3.70–3.85 (2H, m, H-
3
3
5
7
(
(
CH]CHCH ), 6.24 (1H, s, H-1 ), 7.67 (1H, d, JZ8.4 Hz,
3
1
3
3
(Si(CH ) ), 15.2 (CH]CHCH ), 18.4 (C(CH ) ), 25.8
3 2
f
3
3 3
0
1
0
(C-4 ), 88.1 (C-1 ), 101.0 (C-5), 123.8 (CH]CHCH ),
0
(C(CH ) ), 63.8 (C-5 ), 78.6 (C-3 ), 81.1 (C-2 ), 84.4
3 3
0
0
136.0 (CH]CHCH ), 142.0 (C-6), 150.8 (C-2), 163.1 (C-
0
3
0
.67 (1H, br s, OH), 6.05 (1H, s, H-1 ), 6.23 (1H, br s, OH),
0
, H-4 ), 5.07 (1H, br s, OH), 5.57 (1H, d, JZ8.2 Hz, H-5),
3
0
C
4); HiRes MALDI FT-MS m/z (MCNa ) found/calcd
421.1753/421.1770.
1
3
.68 (1H, d, JZ8.2 Hz, H-6), 11.26 (1H, s, NH); C NMR
0
C^CCH ), 77.0 (C-3 ), 77.2 (C^CCH ), 83.6 (C-2 ),
DMSO-d ) d 3.60 (C^CCH ), 60.7 (C-5 ), 75.9
6
3
0
4.2 (C-4 ), 86.9 (C-1 ), 100.3 (C-5), 142.4 (C-6), 150.6 (C-
0
0
silylthymidin-5 -yl)diisopropylamino phosphine (12).
3
3
4.1.6. Preparation of allyloxy(3 -O-tert-butyldimethyl-
0
Compound 11 (0.795 g, 2.23 mmol) was dissolved in
0
), 163.2 (C-4); HiRes MALDI FT-MS m/z (MCNa )
0
8
2
C
found/calcd 305.0752/305.0750.
anhydrous CH CN (4 mL). Allyloxy-bis(diisopropylami-
3
no)phosphine (0.97 g, 3.35 mmol) was added, followed by
addition of a 0.45 M solution of 1H-tetrazole in CH CN
0
C-(propyn-1-yl)arabinofuranosyl)uracil (7). Compound 6
0
4
.1.4. Preparation of 1-(5 -O-tert-butyldimethylsilyl-2 -
3
(7.5 mL, 3.4 mmol) over a period of 5 min. The reaction
mixture was stirred for 1 h, filtrated and added DCM
(40 mL). The solution was washed with a saturated aqueous
solution of NaHCO3 (20 mL) and brine (4 mL), dried
(Na SO ) and concentrated under reduced pressure. The
(
(
0.565 g, 1.72 mmol) was dissolved in anhydrous DMF
10 mL) and imidazole (0.28 g, 4.0 mmol) was added. A
solution of TBDMSCl (0.30 g, 2.0 mmol) in DMF (1.0 mL)
was added dropwise, and the reaction mixture was stirred
for 1 h. An additional amount of TBDMSCl (0.05 g,
2
4
residue was purified by dry column chromatography
(0–100% ethyl acetate and 0.5% Et N in petrol ether) to
0
.33 mmol) dissolved in DMF (0.5 mL) was added and
3
the reaction mixture was stirred for 7 h and quenched by the
addition of ethanol (2 mL). Water (30 mL) was added and
the mixture was extracted with diethylether (4!10 mL).
give the product as an oil with an epimeric mixture (0.800 g,
66%); R 0.60 (75% ethyl acetate in petrol ether); H NMR
1
f
(CDCl ) d 0.09 (6H, br s, Si(CH ) ), 0.90 (9H, br s,
3
3 2
The combined organic phases were dried (MgSO ) and
4
C(CH ) ), 1.17–1.28 (12H, m, CH(CH ) ), 1.91–1.93 (6H,
3 3 3 2
0
0
3.54–3.95 (3H, m, CH(CH ) , H-5 ), 3.99–4.06 (1H, m,
concentrated under reduced pressure. The residue was
purified by column chromatography (0–10% methanol in
DCM) to give the product as a white foam (0.476 g, 70%);
m, CH ), 2.00–2.14 (1H, m, H-2 ), 2.22–2.29 (1H, m, H-2 ),
3
0
H-3 ), 4.11–4.22 (2H, m, CH OP), 4.42–4.49 (1H, m, H-4 ),
3
2
0
5.12–5.31 (2H, m, CH]CH ), 5.88–5.97 (1H, m,
0
2
1
R 0.35 (9% methanol in DCM); H NMR (CDCl ) d 0.16
f
3
2
0
7.75 (1/2H, s, H-6), 8.85 (1H, br s, NH); P NMR (CDCl )
(
6H, s, Si(CH ) ), 0.94 (9H, s, C(CH ) ), 1.93 (3H, s,
3
CH]CH ), 6.31–6.38 (1H, m, H-1 ), 7.58 (1/2H, s, H-6),
2
2
3 3
0
H-5 ), 4.01 (1H, m, H-5 ), 4.06 (1H, m, H-3 ), 4.14 (1H, m,
31
C^CCH ), 2.74 (1H, d, JZ2.9 Hz, 3 -OH), 3.86 (1H, m,
3
3
0
0
0
0
C
d 148.92, 149.04. ESI FT-MS m/z (2MCNa ) 1109.467.
0
H-4 ), 4.98 (1H, s, 2 -OH), 5.65 (1H, d, JZ8.1 Hz, H-5),
0
NH); C NMR (CDCl ) d K5.5, K5.4 (Si(CH ) ), 18.5
0
2 -deoxy-2 -methyleneuridin-3 -yl)(3 -O-tert-butyldi-
6
.25 (1H, s, H-1 ), 7.76 (1H, d, JZ8.1 Hz, H-6), 8.81 (1H, s,
4.1.7. Preparation of allyl(5 -O-tert-butyldimethylsilyl-
1
3
0
methylsilylthymidin-5 -yl)phosphate (13). Compound 4
0
0
0
3
3 2
0
0
(0.050 g, 0.141 mmol) and compound 12 (0.145 g,
(
7
8
C(CH ) ), 26.0 (C(CH ) ), 4.03 (C^CCH ), 63.0 (C-5 ),
3
3
3
3 3
0
7.5 (C-1 ), 87.8 (C-2 ), 101.3 (C-5), 141.5 (C-6), 150.8
0
3.3 (C^CCH ), 77.1 (C^CCH ), 77.4 (C-3 ), 83.4 (C-4 ),
3
3
0
0
0.282 mmol) were dried and co-evaporated twice from
anhydrous CH CN. The mixture was redissolved in
C
(C-2), 163.2 (C-4); HiRes MALDI FT-MS m/z (MCNa )
found/calcd 419.1595/419.1613.
3
anhydrous CH CN (2 mL) and a 0.45 M solution of 1H-
3
tetrazole in CH CN (0.32 mL, 0.144 mmol) was added over
3
0
C-(cis-propen-1-yl)-arabinofuranosyl)uracil (8). Lindlar
0
4
.1.5. Preparation of 1-(5 -O-tert-butyldimethylsilyl-2 -
a period of 5 min. The reaction mixture was stirred at room
temperature for 90 min. A 3 M solution of t-BuOOH in
toluene (0.2 mL, 0.60 mmol) was added and the reaction
mixture was stirred for 30 min and quenched by the addition
of methanol (0.5 mL). The solution was concentrated under
reduced pressure and the residue was purified by column
chromatography (75–100% ethyl acetate in petrol ether) to
give the product as a white foam with an epimeric mixture
catalyst (0.117 g) was added freshly distilled methanol
(
6.0 mL) and freshly distilled quinoline (0.46 mL). The
mixture was stirred under an atmosphere of hydrogen for
h. A solution of compound 7 (0.464 g, 1.17 mmol) in
1
methanol (6.0 mL) was added and the mixture was stirred
for 30 min The hydrogen atmosphere was replaced with
nitrogen and the mixture was filtered through a layer of
Celite. The filtrate was concentrated under reduced pressure
and the residue was dissolved in DCM (10 mL) and added
silica gel. This mixture was concentrated under reduced
pressure and purified by column chromatography (0–10%
(0.111 g, 97%); R 0.22 (75% ethyl acetate in petrol ether);
f
1
H NMR (CDCl ) d 0.08–0.10 (12H, m, Si(CH ) ),
3
3 2
0.89–0.90 (18H, m, C(CH ) ), 1.93–1.95 (3H, m, T-CH ),
3
3
3
0
3.99–4.04 (1H, m, T-H4 ), 4.15–4.32 (4H, m, T-H3 , U-H4 ,
0
2.12–2.32 (2H, m, T-H2 ), 3.86–3.95 (2H, m, U-H5 ),
0
T-H5 ), 4.38–4.43 (1H, m, U-H3 ), 4.56–4.64 (2H, m,
0
0
0
CH OP), 5.29–5.42 (4H, m, C]CH , CH]CH ),
0
methanol in DCM) to give the product as a white foam
(
1
0.442 g, 95%); R 0.40 (9% methanol in DCM); H NMR
f
2
2
2
(
1
CDCl ) d 0.19 (6H, s, Si(CH ) ), 0.96 (9H, s, C(CH ) ),
3
5.69–5.78 (1H, m, U-H5), 5.84–6.01 (1H, m, CH]CH ),
2
6.28–6.33 (1H, m, T-H1 ), 6.76–6.79 (1H, m, U-H1 ),
7.38–7.47 (1H, m, T-H6), 7.54–7.62 (1H, m, U-H6),
3 2
3 3
0
0
.89 (3H, dd, JZ1.6, 7.1 Hz, CH]CHCH ), 2.24 (1H, d,
3
0
0
0
JZ1.4 Hz, 3 -OH), 3.87 (1H, m, H-5 ), 4.02 (1H, m, H-5 ),

1146
P. Børsting et al. / Tetrahedron 62 (2006) 1139–1149
1
.48–9.50 (1H, m, NH), 9.67–9.71 (1H, m, NH); C NMR
3
0
9
(1H, m, T-H1 ), 7.34 (3/5H, d, JZ1.1 Hz, T-H6), 7.44
(
CDCl ): d K5.5, K5.4, K4.8, K4.5, 12.6, 18.0, 18.4, 25.6,
3
(2/5H, d, JZ1.1 Hz, T-H6), 7.82 (2/5H, d, JZ8.3 Hz, U-H6),
7.84 (3/5H, d, JZ8.3 Hz, U-H6), 9.00–9.24 (2H, m, NH);
2
8
1
1
0
8
5.8, 25.9, 40.8, 62.5, 67.0, 68.9, 69.0, 71.5, 71.5, 76.2,
4.0, 84.1, 84.1, 85.0, 85.1, 85.2, 85.3, 103.3, 111.4, 111.5,
17.3, 119.4, 128.4, 130.2, 133.4, 135.7, 141.1, 144.7,
3
1
P NMR (CDCl ) d K0.77 (3/5), K0.61 (2/5); HiRes
3
C
MALDI FT-MS m/z (MCNa ) found/calcd 879.3434/
879.3404.
3
1
50.5, 151.0, 163.4, 164.1; P NMR (CDCl ) d K0.12,
3
C
.00; HiRes MALDI FT-MS m/z (MCNa ) found/calcd
35.3118/835.3141.
0
-deoxy-2 -methyleneuridine-3 -yl)diisopropylamino phos-
4.1.10. Preparation of vinyl(5 -O-tert-butyldimethylsilyl-
2
0
phinate (17). Compound 4 was dissolved in anhydrous
0
0
0
-(cis-propen-1-yl)-2 -arabinouridin-3 -yl)(3 -O-tert-
4
2
.1.8. Preparation of allyl(5 -O-tert-butyldimethylsilyl-
0
0
0
0
DCM (3 mL) and a 1 M solution of 4,5-dicyanoimidazole
in CH CN (0.62 mL, 0.62 mmol) was added. A solution
0
butyldimethylsilylthymidin-5 -yl)phosphate (14). Com-
pound 8 (0.044 g, 0.11 mmol) was dissolved in anhydrous
CH CN and a 0.50 M solution of compound 12 in CH CN
(
CH CN (0.25 mL) was added. The reaction mixture was
stirred for 30 min and added another portion of the 0.50 M
solution of 12 (0.10 mL). The mixture was stirred for 2 h
and added a 3 M solution of t-BuOOH in toluene (0.12 mL,
3
of bis(diisopropylamino)vinylphosphinate (0.214 g,
0.828 mmol) in DCM (1.5 mL) was added over 10 min
and the reaction mixture was stirred for 2.5 h. An additional
amount of bis(diisopropylamino)vinylphosphinate (0.078 g,
0.30 mmol) dissolved in DCM (0.5 mL) was added and the
reaction mixture was stirred for 2 h. The reaction mixture
was diluted with ethyl acetate (30 mL) and washed with a
3
3
0.25 mL) followed by a 0.45 M solution of 1H-tetrazole in
3
0
.4 mmol). The mixture was stirred for 45 min and added
ethyl acetate (6 mL). The mixture was washed with brine
5 mL), dried (Na SO ) and concentrated under reduced
saturated aqueous solution of NaHCO (2!30 mL) and
3
brine (30 mL), dried (Na SO ) and concentrated under
2
4
(
reduced pressure. The residue was purified by dry column
chromatography (10–40% ethyl acetate and 1% pyridine in
petrol ether) to give the product as a colourless oil with an
2
4
pressure. The residue was purified by column chromatog-
raphy (0–100% ethyl acetate in petrol ether) to give the
product as a white foam with an epimeric mixture (0.061 g,
epimeric mixture (0.227 g, 74%); R 0.62 (50% ethyl acetate
f
1
1
in petrol ether); H NMR (CDCl ) d 0.07–0.12 (6H, m,
6
5%); R 0.43 (ethyl acetate); H NMR (CDCl ) d 0.07–0.19
f
3
3
(
12H, m, Si(CH ) ), 0.89–0.95 (18H, m, C(CH ) ),
3 3
Si(CH ) ), 0.89–0.92 (9H, m, C(CH ) ), 1.03–1.32 (12H, m,
3 3
3
2
3 2
1
2
.82–1.85 (3H, CH]CHCH ), 1.92–1.94 (3H, m, T-CH ),
3
CH(CH ) ), 3.47–3.57 (2H, m, CH(CH ) ), 3.82–4.02 (3H,
3 2 3 2
m, H-5 , H-4 ), 4.85–4.97 (1H, m, H-3 ), 5.22–5.55 (2H, m,
3
0
U-H3 , T-H5 , U-H5 ), 4.53–4.65 (2H, m, CH OP),
0
0
0
0
0
C]CH ), 5.60–5.84 (3H, H-5, PCH]CH ), 6.15–6.35 (1H,
.04–2.32 (2H, m, T-H2 ), 3.95–4.45 (7H, m, U-H4 , T-H3 ,
0
.74–4.78 (1H, m, T-H4 ), 5.11–5.14 (1H, m, OH), 5.26–
0
0
2
2
2
0
.43 (2H, CH]CH ), 5.56–5.65 (1H, CH]CHCH ), 5.76–
0
4
5
5
m, PCH]CH ), 6.69–6.75 (1H, m, H-1 ), 7.55 (1/2H, d, JZ
2
8.2 Hz, H-6), 7.74 (1/2H, d, JZ8.2 Hz, H-6), 8.55–8.65
2
3
3
(1H, br, NH); P NMR (CDCl ) d 118.64, 120.22.
1
.98 (2H, CH]CH , CH]CHCH ), 5.69–5.78 (1H, m,
2
3
3
0
0
U-H5), 6.17–6.33 (2H, m, T-H1 , U-H1 ), 7.31–7.39 (1H, m,
T-H6), 7.60–7.64 (1H, m, U-H6), 9.00–9.20 (2H, m, NH);
P NMR (CDCl ) d K0.80 (3/5), K0.53 (2/5); HiRes
0
4.1.11. Preparation of vinyl(5 -O-tert-butyldimethylsilyl-
3
1
0
0
0
0
3
2 -deoxy-2 -C-allyluridine-3 -yl)-N,N -diisopropylamino
phosphinate (18). Compound 9 (0.100 g, 0.261 mmol) was
dissolved in DCM (3 mL) and a 1 M solution of 4,5-
C
MALDI FT-MS m/z (MCNa ) found/calcd 879.3416/
8
79.3404.
dicyanoimidazole in CH CN (0.55 mL, 0.55 mmol) was
3
0
methylsilyluridin-3 -yl)(3 -O-tert-butyldimethylsilyl-
0
4
.1.9. Preparation of allyl(2 -O-allyl-5 -O-tert-butyldi-
added. Bis(diisopropylamino)vinylphosphinate (0.135 g,
0.522 mmol) was added dropwise over a period of 5 min,
and the reaction mixture was stirred for 4 h at room
temperature. An additional amount of bis(diisopropyl-
amino)vinylphosphinate (0.135 g, 0.522 mmol) was added
and the mixture was stirred for 6 h. An additional amount of
0
0
0
9
thymidin-5 -yl)phosphate(16). Compound 10 (0.890 g,
.23 mmol) and compound 12 (1.86 g, 3.42 mmol) were co-
evaporated with CH CN and redissolved in CH CN
2
3
3
(
37 mL). A 0.45 M solution of 1H-tetrazole in CH CN
3
(
8.0 mL, 3.6 mmol) was added over a period of 10 min and
the 1 M solution of 4,5-dicyanoimidazole in CH CN
3
the reaction mixture was stirred for 100 min. A 3 M solution
of t-BuOOH in toluene (3.7 mL, 11.1 mmol) was added in
small portions over 5 min. The reaction mixture was stirred
for 145 min and added methanol (2.0 mL). The mixture was
concentrated under reduced pressure, and the residue was
purified by dry column chromatography (0–100% ethyl
acetate in petrol ether) to give the product as a white foam
(0.55 mL, 0.55 mmol) was added and the reaction mixture
was stirred at room temperature for 23 h. The reaction
mixture was diluted with DCM (40 mL) and washed with a
saturated aqueous solution of NaHCO (2!20 mL) and
3
brine (30 mL), dried (Na SO ) and concentrated under
2
4
reduced pressure. The residue was purified by dry column
chromatography (0–30% ethyl acetate and 1% pyridine in
petrol ether) to give the product as a white foam with an
with an epimeric mixture (1.81 g, 95%); R 0.41 (75% ethyl
f
1
acetate in petrol ether); H NMR (CDCl ) d 0.06–0.16 (6H,
epimeric mixture (0.100 g, 71%); R 0.50 (50% ethyl acetate
f
3
1
in petrol ether); H NMR (CDCl ) d 0.09–0.12 (6H, m,
m, Si(CH ) ), 0.86–0.93 (18H, m, C(CH ) ), 1.91–1.95 (3H,
3
2
3 3
3
0
U-H5 ), 4.00–4.03 (1H, m, T-H4 ), 4.08–4.13 (1H, m, U-H2 ),
m, T-CH ), 2.09–2.32 (2H, m, T-H2 ), 3.75–3.98 (2H, m,
3
Si(CH ) ), 0.91–0.93 (9H, m, C(CH ) ), 1.04–1.33 (12H, m,
3 2
3 3
0
.16–4.19 (2H, m, 2 -OCH ), 4.22–4.26 (2H, m, T-H5 ),
0
0
0
(2H, m, CH(CH ) ), 3.78–3.95 (2H, m, H-5 ), 4.17–4.22
0
CH(CH ) ), 2.05–2.55 (3H, m, H-2 , 2 –CH ), 3.41–3.58
3
2
2
0
.28–4.33 (1H, m, U-H4 ), 4.38–4.45 (1H, m, T-H3 ), 4.53–
0
0
(1H, m, H-4 ), 3.32–4.46 (1H, m, H-3 ), 4.95–5.07 (2H, m,
4
4
4
2
3 2
0
.65 (2H, m, CH OP), 4.85–4.93 (1H, m, U-H3 ), 5.15–5.40
0
0
CH]CH2), 5.60–6.02 (4H, m, H-5, CH]CH2,
0
0
2
0
(
(
4H, m, 2!CH]CH ), 5.67–5.73 (1H, m, U-H5), 5.75–6.00
2
2H, m, 2!CH]CH ), 6.10–6.14 (1H, m, U-H1 ), 6.26–6.33
2
PCH]CH ), 6.10–6.55 (2H, m, H-1 , PCH]CH ), 7.87
2
2
0
(1/2H, d, JZ8.1 Hz, H-6), 7.92 (1/2H, d, JZ8.1 Hz, H-6),

P. Børsting et al. / Tetrahedron 62 (2006) 1139–1149
1147
3
.20–8.35 (1H, m, NH); P NMR (CDCl ) d 111.46,
1
8
4.1.14. Preparation of 3R- and 3S-(1R,8S,9R,11R)-3-
(3(S)-(tert-butyldimethylsilyl)oxy-5(R)-(thymin-1-yl)-
tetrahydrofuran-2(R)-yl)methoxy-8-hydroxy-3-oxo-11-
3
1
21.14.
(
1
tert-butyldimethylsilyl)oxymethyl-9-(thymin-
-yl)-2,4,10-trioxa-3-phosphabicyclo[6.3.0]undec-6(Z)-
0
-deoxy-2 -C-methyleneuridin-3 -yl)(3 -O-tert-butyldi-
methylsilylthymidin-5 -yl)phosphonate (19). Compound
1
4
2
.1.12. Preparation of vinyl(5 -O-tert-butyldimethylsilyl-
0
0
0
0
ene (21). Compound 14 (30 mg, 0.035 mmol) and Grubbs
second generation catalyst A (3 mg, 3.5 mmol) were
dissolved in DCM (1.8 mL), and the reaction mixture was
refluxed for 2 h. Another portion of A (3 mg) dissolved in
DCM (0.1 mL) was added and the reaction mixture was
stirred for another 4 h. The reaction was purified by column
chromatography (0–100% ethyl acetate in petrol ether) to
give the product as two separate epimers as white solids as
0
7 (0.084 g, 0.17 mmol) and compound 11 (0.068 g,
0
redissolved in anhydrous CH CN (8.5 mL). A 1 M solution
.191 mmol) were co-evaporated with DCM (10 mL) and
3
of 4,5-dicyanoimidazole in CH CN (0.17 mL, 0.17 mmol)
3
was added over 5 min and the reaction mixture was stirred
for 1 h (resulting phosphonite; R 0.40 (75% ethyl acetate in
petrol ether)). A 3 M solution of t-BuOOH in toluene
f
well as recovered starting material (4.3 mg, 14%);
Compound 21a (4.5 mg, 16%) R 0.22 (ethyl acetate); H
NMR (CDCl ) d 0.05–0.15 (12H, m, Si(CH ) ), 0.85–0.95
3 2
(0.27 mL, 0.81 mmol) was added over 5 min. The reaction
mixture was stirred for 30 min and added methanol
0.5 mL). The mixture was diluted with ethyl acetate
50 mL) and washed with a saturated aqueous solution of
1
f
3
(
(
(18H, m, C(CH ) ), 1.96 (3H, s, T-CH ), 2.00–2.32 (2H, m,
T-H2 ), 3.86–4.58 (8H, m, T-H4 , U-H4 , T-H3 , U-H3 ,
3 3 3
0
0
0
T-H5 , U-H5 ), 4.81 (1H, s, OH), 4.87–5.00 (2H, m,
0
0
0
NaHCO (50 mL) and brine (50 mL), dried (Na SO ) and
4
0
3
2
concentrated under reduced pressure. The residue was
purified by column chromatography (50–100% ethyl acetate
in petrol ether) to give the product as a white foam with an
POCH CH]CH), 5.68 (1H, d, JZ8.1 Hz, U-H5), 6.02
2
(1H, d, JZ11.9 Hz, POCH CH]CH), 6.15 (1H, dt, JZ7.5,
2
0
1
1.9 Hz, POCH CH]CH), 6.22 (1H, s, U-H1 ), 6.28 (1H,
2
epimeric mixture (0.072 g, 54%); R 0.18 (75% ethyl acetate
f
0
.1 Hz, U-H6), 8.50 (1H, br s, NH), 8.78 (1H, br s, NH);
m, T-H1 ), 7.33 (1H, d, JZ0.9 Hz, T-H6), 7.77 (1H, d, JZ
8
NMR (CDCl ) d K0.12; HiRes MALDI FT-MS m/z
1
in petrol ether); H NMR (CDCl ) d 0.08–0.10 (12H, m,
3
31
P
Si(CH ) ), 0.88–0.92 (18H, m, C(CH ) ), 1.92–1.94 (3H, m,
3
2
3 3
0
0
3
T-CH ), 2.07–2.30 (2H, m, H-2 ), 3.80–4.45 (6H, m, U-H5 ,
3
(MCNa) found/calcd 837.2944/837.2942. Compound 21b
(2.8 mg, 10%) R 0.12 (Ethyl acetate); P NMR (CDCl ) d
0
0
0
0
T-H5 , T-H4 , T-H3 ), 4.63–4.69 (1H, m, U-H4 ), 5.38–5.65
2H, m, C]CH ), 5.66–5.72 (1H, m, H-5), 5.81–5.85 (1H,
m, H-3 ), 6.00–6.53 (4H, m, PCH]CH , T-H1 ), 6.75–6.77
31
f
3
(
2
K1.55; HiRes MALDI FT-MS m/z (MCNa) found/calcd
37.2919/837.2942.
0
0
2
8
0
(
1H, m, U-H1 ), 7.32–7.35 (1H, m, T-H6), 7.52 (2/5H, d,
JZ8.0 Hz, H-6), 7.59 (3/5H, d, JZ8.0 Hz, H-6), 8.60–8.75
3
1H, m, NH), 8.80–8.90 (1H, m, NH); P NMR (CDCl3)
1
4.1.15. Preparation of 3R- and 3S-(1R,8S,9R,11R)-3-(3(S)-
tert-butyldimethylsilyl)oxy-5(R)-(thymin-1-yl)tetrahydro-
(
d 19.92 (2/5), 20.19 (3/5); HiRes MALDI FT-MS m/z
(
C
MCNa ) found/calcd 805.3035/805.3035.
furan-2(R)-yl)methoxy-8-hydroxy-3-oxo-11-(tert-butyldi-
methylsilyl)oxymethyl-9-(thymin-1-yl)-2,4,10-trioxa-3-phos-
phabicyclo[6.3.0]undecane (22). Compound 14 (22 mg,
0.025 mmol) and Grubbs second generation catalyst A
(3.8 mg, 4.5 mmol) was dissolved in DCM (1.3 mL), and the
reaction mixture was refluxed for 2 h. The reaction mixture
(
0
-deoxy-2 -C-allyluridin-3 -yl)(3 -O-tert-butyldimethyl-
4
2
.1.13. Preparation of vinyl(5 -O-tert-butyldimethylsilyl-
0
silylthymidin-5 -yl)phosphonate (20). Compound 11
0
0
0
0
(
0
0.118 g, 0.331 mmol) and compound 18 (0.180,
.333 mmol) were co-evaporated from CH CN (10 mL)
and redissolved in CH CN (10 mL). A 0.45 M solution of
was stirred in a 70 atm H atmosphere at 50 8C for 13 h. The
2
reaction was purified by column chromatography (0–100%
ethyl acetate in petrol ether) to give the product as two
separate epimers as white solids as well as recovered
starting material (1.0 mg, 5%); Compound 22a (0.6 mg,
3
3
1
H-tetrazole (3.7 mL, 1.66 mmol) in CH CN was added
3
dropwise over 5 min. The reaction mixture was stirred for
h, added methanol (1 mL) and concentrated under reduced
pressure. The residue was redissolved in CH CN (7 mL) and
3
1
3
3%) R
HiRes MALDI FT-MS m/z (MCNa) found/calcd 839.3090/
839.3099. Compound 22b (1.6 mg, 8%) R 0.10 (ethyl
f
0.20 (ethyl acetate); P NMR (CDCl ) d 5.83;
3
3
cooled to 0 8C. A 3 M solution of t-BuOOH in toluene
(
f
1
0.55 mL, 1.7 mmol) was added dropwise over 5 min and
acetate); H NMR (CDCl
0.85–0.95 (18H, m, C(CH
3
) d 0.05–0.15 (12H, m, Si(CH
3
)
), 1.96–1.99 (3H, s, T-CH
2
3
),
),
the reaction mixture was stirred for 2 h at room temperature
and concentrated under reduced pressure. The residue was
purified by dry column chromatography (10–100% ethyl
acetate in petrol ether) to give the product as a white powder
3
0
)
3
1.90–2.40 (6H, m, T-H2 , POCH
0
CH
(10H, m, T-H4 , U-H4 , T-H3 , U-H3 , T-H5 , U-H5
CH
0
), 3.76–4.44
0
2
2
2
0
0
0
POCH CH ), 5.02 (1H, s, OH), 5.63 (1H, d, JZ8.2 Hz,
2 2
0
0
with an epimeric mixture (0.061 g, 23%); R 0.15 (75%
f
U-H5), 6.04 (1H, s, U-H1 ), 6.24 (1H, m, T-H1 ), 7.35 (1H,
1
ethyl acetate in petrol ether); H NMR (MeOD-d ) d 0.12–
s, T-H6), 7.67 (1H, d, JZ8.2 Hz, U-H6), 8.12 (2H, br s,
4
3
NH); P NMR (CDCl ) d K0.85; HiRes MALDI FT-MS
1
0
1
2
.15 (12H, m, Si(CH ) ), 0.92–0.94 (18H, m, C(CH ) ),
2
3
3
3 3
0
0
-CH ), 3.73–3.83 (2H, m, U-H5 ), 3.85–4.02 (2H, m,
.89–1.92 (3H, m, T-CH ), 2.02–2.50 (5H, T-H2 , U-H2 ,
3
m/z (MCNa) found/calcd 839.3070/839.3099.
0
0
2
0
1H, m, U-H3 ), 4.90–5.02 (2H, m, CH]CH ), 5.52–5.73
0
0
0
T-H5 ), 4.06–4.13 (3H, m, T-H4 , U-H4 ,T-H3 ), 4.36–4.43
(
(
6
7
2
8
4.1.16. Preparation of 3R- and 3S-(1R,10R,11R,13R)-3-
(3(S)-(tert-butyldimethylsilyl)oxy-5(R)-(thymin-1-yl)tetra-
hydrofuran-2(R)-yl)methoxy-3-oxo-13-(tert-butyldimethyl-
silyl)oxymethyl-11-(thymin-1-yl)-2,4,9,12-tetraoxa-3-phos-
phabicyclo[8.3.0]tridec-6(Z)-ene (25). Compound 16
(0.099 g, 0.116 mmol) was dissolved in DCM (11 mL).
Grubbs second generation catalyst A (6.8 mg, 8.1 mmol)
0
2H, m, U-H5, CH]CH ), 5.93–6.01 (1H, m, U-H1 ), 6.05–
2
0
.40 (4H, m, T-H1 , PCH]CH ), 7.36–7.48 (1H, m, T-H6),
2
0
.65–7.82 (1H, m, U-H6); P NMR (MeOD-d ) d 19.87,
2
3
1
4
C
0.58; HiRes MALDI FT-MS m/z (MCNa ) found/calcd
33.3354/833.3313.

1148
P. Børsting et al. / Tetrahedron 62 (2006) 1139–1149
0
(2/5H, d, JZ3.8 Hz, U-H1 ), 5.90 (3/5H, d, JZ7.7 Hz,
was added and the reaction mixture was stirred at reflux for
4 h. The reaction mixture was concentrated under reduced
0
0
2
U-H1 ), 6.16–6.21 (1H, m, T-H1 ), 7.45 (3/5H, d, JZ
1.1 Hz, T-H6), 7.50 (2/5H, d, JZ1.0 Hz, T-H6), 7.85 (2/5H,
d, JZ8.1 Hz, U-H6), 7.93 (3/5H, d, JZ8.2 Hz, U-H6),
pressure and the residue was purified by dry column
chromatography (0–100% ethyl acetate in petrol ether) to
give the product as a foam with an epimeric mixture
3
1
11.31–11.43 (2H, m, NH); P NMR (DMSO-d ) d K1.39
6
1
C
(2/5), K1.25 (3/5); HiRes MALDI FT-MS m/z (MCNa )
found/calcd 625.1490/625.1517.
(
0.043 g, 45%); R 0.33 (ethyl acetate); H NMR (CDCl ) d
3
f
0
C(CH ) ), 1.91–1.96 (3H, s, T-CH ), 2.05–2.33 (2H, m,
.08–0.13 (12H, m, Si(CH ) ), 0.91–0.94 (18H, m,
3
2
3
3
3
0
0
U-H2 , T-H3 , T-H4 , T-H5 , U-H4 , CH CH]CH ), 4.56–
0
2 -deoxy-2 -methyluridin-3 -yl)(3 -O-tert-butyldimethyl-
T-H2 ), 3.79–3.87 (2H, m, U-H5 ), 4.10–4.45 (8H, m,
4.1.19. Preparation of ethyl(5 -O-tert-butyldimethylsilyl-
0
0
0
0
0
.64 (2H, m, CH CH]CH ), 4.87–4.93 (1H, m, U-H3 ),
0
silylthymidin-5 -yl)phosphonate (28). Compound 21
0
0
0
2
2
0
.26–5.35 (1H, m, CH]CH), 5.65–5.69 (1H, m, U-H5),
.91–5.95 (2H, m, U-H1 , CH]CH), 6.28–6.35 (1H, m,
0
4
5
5
2
2
(0.022 g, 0.028 mmol) and Grubbs second generation
catalyst A (2.4 mg, 0.003 mmol) were dissolved in DCM
(3 mL). The reaction mixture was stirred in an 70 atm N2
atmosphere at 50 8C for 16 h and then in an 70 atm H2
atmosphere at 50 8C for 16 h. The reaction mixture was
concentrated under reduced pressure and the residue was
purified by column chromatography (75–100% ethyl acetate
in petrol ether) to give the product as a white foam with an
0
0
.36–9.40 (1H, m, NH), 9.80–9.92 (1H, m, NH); P NMR
T-H1 ), 7.27–7.38 (1H, m, T-H6), 7.98–8.06 (1H, m, U-H6),
9
3
1
(
FT-MS m/z (MCNa ) found/calcd 851.3072/851.3091.
CDCl ) d K2.75 (1/4), K1.47 (3/4); HiRes MALDI
3
C
4
(
.1.17. Preparation of 3R- and 3S-(1R,10R,11R,13R)-3-
3(S)-(tert-butyldimethylsilyl)oxy-5(R)-(thymin-1-yl)-
epimeric mixture (12 mg, 57%); R 0.07 (75% ethyl acetate
f
3
in petrol ether). P NMR (CDCl ) d 33.62, 35.42, 35.84,
1
tetrahydrofuran-2(R)-yl)methoxy-3-oxo-13-(tert-butyl-
dimethylsilyl)oxymethyl-11-(thymin-1-yl)-2,4,9,12-tetra-
oxa-3-phosphabicyclo[8.3.0]tridecane (26). Compound 16
3
C
36.76; MALDI FT-MS m/z (MCNa ) 809.
(0.666 g, 0.777 mmol) and Grubbs second generation
catalyst A (32 mg, 0.038 mmol) catalyst were mixed and
4
(
.1.20. Preparation 3R- and 3S-(1R,7S,8R,10R)-3-(3(S)-
tert-butyldimethylsilyl)oxy-5(R)-(thymin-1-yl)tetra-
dissolved in DCM (77 mL). The reaction mixture was
stirred at reflux for 26 h and then in a 70 atm H -atmosphere
at 50 8C for 16 h. The mixture was concentrated under
reduced pressure and the residue was purified by column
chromatography (50–100% ethyl acetate in petrol ether and
then 1–2% methanol in ethyl acetate) to give the product as
hydrofuran-2(R)-yl)methoxy-3-oxo-10-(tert-butyldi-
methylsilyl)oxymethyl-8-(thymin-1-yl)-2,9-dioxa-3-
phosphabicyclo[5.3.0]dec-4(Z)-ene (29). Compound 20
2
(
0.030 g, 0.037 mmol) was dissolved in DCM (3.7 mL)
and Grubbs second generation catalyst A (1.6 mg,
.002 mmol) and CuCl (0.7 mg, 0.007 mmol) were
0
a foam with an epimeric mixture (0.398 g, 62%); R 0.47,
f
added. The reaction mixture was stirred at reflux for
24 h and concentrated under reduced pressure. The
residue was purified by column chromatography (0–4%
methanol in ethyl acetate) to give the product as a powder
1
0
.53 (10% methanol in ethyl acetate); H NMR (CDCl ) d
3
0
C(CH ) ), 1.53–1.74 (2H, m, OCH CH ), 1.90–2.35 (7H,
.09–0.18 (12H, m, Si(CH ) ), 0.83–1.00 (18H, m,
3
2
3
3
2
2
0
U-H2 , T-H4 , T-H5 , U-H4 , T-H3 , OCH CH ), 4.38–4.65
0
m, OCH CH , T-H2 , T-CH ), 3.80–4.32 (10H, m, U-H5 ,
2
2
0
3
with an epimeric mixture (0.010 g, 35%); R 0.52 (10%
f
0
0
0
0
2H, m, OCH CH ), 4.82–4.90 (1H, m, U-H3 ), 5.63–5.71
3
methanol in ethyl acetate); P NMR (CDCl ) d 19.07
1
2
2
3
0
1H, m, U-H5), 5.89–5.95 (1H, m, U-H1 ), 6.18 (3/8H, m,
C
(1/4), 20.23 (3/4) HiRes MALDI FT-MS m/z (MCNa )
found/calcd 805.3036/805.3014.
(
(
2
2
0
T-H1 ), 6.29 (5/8H, m, T-H1 ), 7.25–7.29 (1H, m, T-H6),
0
0
7
U-H6), 8.55–8.80 (2H, m, NH); P NMR (CDCl ) d K2.29
(
found/calcd 853.3209/853.3247.
.92 (3/8H, d, JZ8.2 Hz, U-H6), 8.00 (5/8H, d, JZ8.2 Hz,
3
1
3
Acknowledgements
C
3/8), K1.74 (5/8); HiRes MALDI FT-MS m/z (MCNa )
The Danish National Research Foundation and Møllerens
Fond are thanked for financial support. Mrs Birthe Haack is
thanked for technical assistance.
4
(
.1.18. Preparation of 3R- and 3S-(1R,10R,11R,13R)-3-
3(S)-hydroxy-5(R)-(thymin-1-yl)tetrahydrofuran-2(R)-
yl)methoxy-13-hydroxymethyl-3-oxo-11-(thymin-1-yl)-
,4,9,12-tetraoxa-3-phosphabicyclo[8.3.0]tridecane (27).
Compound 26 (0.180 g, 0.217 mmol) was dissolved in a
0% aqueous solution of TFA (3 mL) and stirred at room
2
9
References and notes
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under reduced pressure and co-evaporated with anhydrous
ethanol. The residue was purified by dry column chroma-
tography (0–20% ethanol in ethyl acetate) to give the
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2004, 60, 7117.
product as a white powder with an epimeric mixture
(
NMR (DMSO-d ) d 1.35–1.90 (7H, m, OCH CH CH ,
1
0.077 g, 59%). R 0.2 (20% ethanol in ethyl acetate); H
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f
6
2
2
2
0
U-H5 ), 3.69–4.45 (10H, m, T-H3 , T-H4 , T-H5 , U-H4 ,
T-CH ), 2.08–2.21 (2H, m, T-H2 ), 3.49–3.67 (2H, m,
3
0
0
0
0
0
U-H2 , 2!OCH ), 4.70–4.88 (1H, m, U-H-3 ), 5.36–5.45
(
8
4. Garber, S. B.; Kingsbury, J. S.; Gray, B. L.; Hoveyda, A. H. J.
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0
2
0
0
2H, m, 3 -OH, 5 -OH), 5.67 (3/5H, dd, JZ2.0 Hz, JZ
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