Synthesis of functionalized carbocyclic locked nucleic acid analogues by ring-closing diene and enyne metathesis and their influence on nucleic acid stability and structure

Article abstract of DOI:10.1021/jo9013657

(Chemical Equation Presented) A series of bicylic 2′-deoxynucleosides that are locked in the N-type conformation due to three-carbon linkages between the 2′- and 4′ - positions have been prepared by ring-closing diene or enyne metathesis. The alkene or 1,

Full text of DOI:10.1021/jo9013657

pubs.acs.org/joc
Synthesis of Functionalized Carbocyclic Locked Nucleic Acid Analogues
by Ring-Closing Diene and Enyne Metathesis and Their Influence on
Nucleic Acid Stability and Structure
Surender Kumar, Marianne H. Hansen, Nanna Albæk, Signe I. Steffansen,
Michael Petersen, and Poul Nielsen*,‡
Nucleic Acid Center, Department of Physics and Chemistry, University of Southern Denmark, 5230 Odense
M, Denmark. ‡The Nucleic Acid Center is funded by the Danish National Research Foundation for studies on
nucleic acid chemical biology
pon@ifk.sdu.dk
Received June 26, 2009
A series of bicylic 20-deoxynucleosides that are locked in the N-type conformation due to three-
carbon linkages between the 20- and 40-positions have been prepared by ring-closing diene or enyne
metathesis. The alkene or 1,3-diene hereby introduced in the bicyclic system is further derivatized, the
latter showing the expected potential for Diels-Alder reactions. Four derivatives that are saturated
or unsaturated as well as functionalized at the 20-40-linkage are incorporated into oligodeoxynucleo-
tides, and the affinity of these for complementary RNA and DNA is studied. Substantially increased
affinity for complementary RNA is observed, especially with additional hydroxyl groups attached to
the bicyclic system. On the other hand, decreased affinity for complementary single-stranded DNA is
obtained, whereas only a very small influence on a triplex-forming oligonucleotide sequence is found.
Hence, a strong RNA-selective nucleic acid recognition is seen, and it can be concluded that the 20-
oxygen atom is less important for the formation of DNA:RNA duplexes than for the formation of
DNA:DNA duplexes. However, the lack of a 20-oxygen in the duplex formation can be partly
compensated by other hydrophilic moieties around the 20-40-linkages indicating structural water
binding to be of significant importance.
Introduction
sequences and thus very promising properties as, e.g., anti-
sense oligonucleotides (AOs),2 siRNA,3 or triplex-forming
oligonucleotides (TFOs).4 More than any other nucleic acid
Oligonucleotides that are conformationally restricted due
to bicyclic nucleoside building blocks1 have demonstrated
unique molecular recognition of complementary nucleic acid
(2) Kurreck, J. Eur. J. Biochem. 2003, 270, 1628–1644.
(3) Watts, J. K.; Deleavey, G. F.; Damha, M. J. Drug Disc. Today 2008,
13, 842–855.
*To whom correspondence should be addressed. Tel: þ45 6550 2565. Fax:
þ45 6615 8780.
(4) Buchini, S.; Leumann, C. J. Curr. Opin. Chem. Biol. 2003, 7, 717–726.
(5) (a) Singh, S. K.; Nielsen, P.; Koshkin, A. A.; Wengel, J. Chem.
Commun. 1998, 455–456. (b) Koshkin, A. A.; Singh, S. K.; Nielsen, P.;
Rajwanshi, V. K.; Kumar, R.; Meldgaard, M.; Olsen, C. E.; Wengel, J.
Tetrahedron 1998, 54, 3607–3630. (c) Obika, S.; Nanbu, D.; Hari, Y.; Andoh,
J.; Morio, K.; Doi, T.; Imanishi, T. Tetrahedron Lett. 1998, 39, 5401–5404.
(d) Koshkin, A. A.; Nielsen, P.; Meldgaard, M.; Rajwanshi, V. K.; Singh, S.
K.; Wengel, J. J. Am. Chem. Soc. 1998, 120, 13252–13253.
(1) (a) Meldgaard, M.; Wengel, J. J. Chem. Soc., Perkin Trans. 1 2000,
3539–3554. (b) Leumann, C. J. Bioorg. Med. Chem. 2002, 10, 841–854.
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6756 J. Org. Chem. 2009, 74, 6756–6769
Published on Web 08/07/2009
DOI: 10.1021/jo9013657
r
2009 American Chemical Society

Kumar et al.
JOCArticle
each LNA-nucleoside monomer is able to conformationally
tune its neighboring unmodified 20-deoxynucleosides from
S- to N-type conformations (Figure 1).7 By this means, the
overall duplex conformation is driven toward A-type or A-
type-like duplex forms by the introduction of only a few
LNA nucleosides.
The success of LNA has motivated the development of an
increasing number of chemical analogues of the original
LNA. The first were the thio and amino analogues 2 and 3
that demonstrated recognition properties almost compar-
able to those of LNA.8 The ΔTm’s for each modification in
an oligodeoxynucleotide range between þ3 and þ5 °C with
DNA complements and between þ4 and þ8 °C with RNA
complements. Furthermore, the amino group of amino-
LNA, 3, has been used as an attachment point for a large
variety of substituents organizing these on the rim of the
A-type duplex.9 The first analogue with a longer 20-40-
bridge was ENA, 4, demonstrating almost the same stabili-
zation of nucleic acid duplexes formed with complementary
RNA (ΔTm’s between þ3.5 and þ5.5 °C in a mixed sequence
context).10,11 With complementary DNA 4 shows only a
small stabilization (ΔTm’s between þ0.5 and þ2 °C).10,11 The
analogue, in which the oxygen is positioned in the neighbor-
ing position, 5, demonstrated
a
slightly lower
affinity for both complementary RNA (ΔTm’s between þ2
and þ3 °C) and complementary DNA (ΔTm’s between -0.5
and þ1 °C).12 Also, the amino analogue of ENA, 6, has been
studied and found to give similar affinities for RNA (ΔTm’s
between þ2.5 and þ4 °C) and even lower for DNA (ΔTm’s
between -0.5 and -3 °C).13 More recently, also the oxamine
analogue 7 (as well as its N-methylated and benzylated
derivatives) has been introduced.14 In the sequences studied,
the affinities for both RNA and DNA were surprisingly good
and almost similar to the results of LNA with ΔTm’s between
þ5.3 and þ6.3 °C with complementary RNA and between
þ1.0 and þ3.8 °C with complementary DNA.14 Even longer
20-40-bridges have been introduced, however, leading only to
affinities for complementary RNA comparable to what is
found for unmodified oligodeoxynucleotides and slightly de-
creased affinities for complementary DNA.11,15
FIGURE 1. (a) Low energy conformations of 20-deoxynucleotides.
(b) Bicyclic nucleosides with 20-40-linkages and locked N-type
conformations. B = a nucleobase.
In order to study the effect of constitution of the 20-40-
linkage and the importance of a 20-oxygen for duplex
analogue, locked nucleic acid (LNA) has been recognized as
the prime tool for engineering strong and specific nucleic acid
recognition.5,6 LNA constitutes only a small structural
perturbation to natural nucleic acids, and its preparation is
completely compatible with standard solid-phase DNA
synthesis. The LNA nucleoside monomer (Figure 1, 1) is a
bicyclic nucleoside that is locked in an N-type conformation
due to an oxymethylene bridge between the 20- and 40-
positions. By introducing one or more LNA-nucleoside
monomers into an otherwise unmodified oligodeoxynucleo-
tide, unprecedented recognition of complementary RNA
and DNA has been obtained. The increase in thermal
stability (ΔTm) of the formed duplexes compared to unmo-
dified duplexes ranges from þ3 to þ8 °C for each incorpora-
tion of an LNA monomer.5,6 It has been demonstrated that
(7) Petersen, M.; Bondensgaard, K.; Wengel, J.; Jacobsen, J. P. J. Am.
Chem. Soc. 2002, 124, 5974–5982.
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Meldgaard, M.; Wengel, J. Bioorg. Med. Chem. Lett. 1998, 8, 2219–2222.
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Wengel, J. J. Am. Chem. Soc. 2005, 127, 13293–13299.
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Ishikawa, T.; Imanishi, T.; Koizumi, M. Bioorg. Med. Chem. Lett. 2002,
12, 73–76.
(11) Morita, K.; Takagi, M.; Hasegawa, C.; Kaneko, M.; Tsutsumi, S.;
Sone, J.; Ishikawa, T.; Imanishi, T.; Koizumi, M. Bioorg. Med. Chem. 2003,
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Kumar et al.
JOCArticle
formation, we introduced the first carbocyclic analogues of
ENA, 8, with a saturated ring, and 9 (B = uracil) containing
an unsaturated cyclohexene ring (Figure 1).16 The synthesis
was based on ring-closing metathesis as the key step toward
the bicyclic system. When incorporated into oligodeoxynu-
cleotides, both 8 and 9 demonstrated increased stability
of DNA:RNA hybrid duplexes (ΔTm’s between þ2.5 and
þ5.0 °C per modification) comparable to what was found for
ENA sequences, whereas the stability of dsDNA duplexes
was destabilized (ΔTm ranges between -2.5 and -1.0 °C per
modification) opposite to what was found for ENA. CD
spectroscopy revealed that the bicyclic nucleosides induced
formation of A-type like duplexes, albeit to a lesser degree
than found for LNA monomers.16
(ΔTm’s between -1.0 and þ2.5 °C), whereas the 60(S)-
positioned CH3 group of 17 drives the DNA affinity further
down as compared to 15 (ΔTm’s between -3.0 and -8.5
°C).20
Among the members of this series of 20-40-bridged locked
nucleic acid analogues, several have been studied as building
blocks in triplex-forming oligonucleotides (TFOs). TFOs are
short oligonucleotides designed to target dsDNA duplexes
with purine tracts by forming Hoogsteen-type base-pairing
parallel to the purine-rich strand.4 Incorporation of LNA
monomers, 1, into the TFOs led to significant increases in
thermal stability of the triplexes with ΔTm’s up to þ10 °C
reported,21 although in most other sequences the change
around þ5 °C.22 However, the ability to form triplexes
disappears completely with fully modified LNA sequences.22
ENA, 4, has shown almost the same increases in triplex
stability, and as opposed to LNA, fully modified ENA
sequences also form stable triplexes.23 Amino-LNA, 3,
shows the same effect as LNA in partly modified TFOs.21
With various N-substituents, even more pronounced in-
creases in affinity have been observed.21 However, the
introduction of the carbocyclic analogue 8 in TFOs has
shown only almost neutral influence on the triplex stabilities
(ΔTm’s between -0.5 and þ1.3 °C).21 The incorporation of
7, on the other hand, demonstrated very high increases in
affinity for the dsDNA target with ΔTm’s between þ3 and
þ11 °C. This very positive effect is suggested to be due to a
hydrogen bond between the NH in the bridge of the bicyclic
nucleoside and its 30-O-phosphate.14
Recently, a series of branched analogues of LNA have
been introduced. We introduced a 60-branch by using a
stereoselective mercury cyclization to give the 60(R)-hydro-
xymethyl-LNA derivative 10 (B = thymine),17 whereas Seth
et al. synthesized the derivatives 11 and 12 after separation of
60-epimers (B = cytosine/uracil) and studied these in oligo-
nucleotides rendering improved RNA recognition in end-
modified sequences when compared to unmodified or MOE-
modified sequences but slightly decreased RNA recognition
when compared to corresponding LNA sequences.18 Very
recently, Chattopadhyaya and co-workers continued the
study of electrostatic effects around the 20-40-linkage by
the introduction of a number of carbocyclic derivatives as
represented by 13-17 (B = thymine), with a variety of
substitutions for the 60-position but all with a methyl group
at the 70-position (13, 14) or 80-position (15-17) pointing
into the minor groove.19,20 The 60-unsubstituted derivatives
13 (mixture of 70-epimers) and 15 (80(S)-configuration) were
reported first,19 and oligonucleotides with single incorpora-
tions demonstrated enhanced affinities for RNA, more
pronounced for the LNA analogues 13 (ΔTm’s between
þ2.5 and þ4.0 °C) than for the ENA analogue 15 (ΔTm’s
between þ0.5 and þ1.5 °C).20 Like with unbranched carbo-
cyclic analogues 8 and 9, the affinity for complementary
DNA is decreased, more so for 15 (ΔTm’s between -1.5 and
-5.5 °C) than for 13 (ΔTm’s between þ0.5 and -2.5 °C).19
The carbocyclic LNA derivative 14 has been studied as all
four 60/70-stereoisomeric combinations separately and to-
gether with 60-methylated derivatives, whereas the carbocyc-
lic ENA derivatives 16 and 17 have been studied as the two
different 60-epimers with fixed 80(S)-configuration. In gen-
eral, only very small changes of the RNA affinities
(approximately (1 °C) as compared to the data for 13 and
15, respectively, were determined.20 Concerning the DNA
affinities, larger changes (mostly decreases) were found, and
as the two extreme examples, the 60(S)-postitioned OH group
of 14 drives the DNA affinity upward compared to 13 to be
generally slightly higher that for an unmodified sequence
All of the mentioned locked nucleosides with 20-40-
bridges are locked in perfect N-type conformations as vali-
dated by their pseudorotation angles, P (obtained from
NMR, X-ray data and/or modeling), grouping in the narrow
spectrum around 12-27°.16,24,25 The puckering amplitude,
νmax,
24 on the other hand, follows the number of atoms in the
20-40-bridge; LNA and other analogues with two-atom
bridges have νmax in the range of 56-58°,5,11,16,25 ENA and
other analogues with three atom-bridges have νmax around
46-48°,11,16,25 and the nucleosides with even longer four-
atom bridges have νmax around 38°.15,16 As excellent hybri-
dization behavior of the corresponding oligonucleotides has
been found with the use of both two- and three-atom bridges,
the differences should also be found in the constitution of the
bridge, for instance in the heteroatoms present as well as in
the electrostatic surroundings as influenced by substituents
on the 20-40-bridge.
Herein, we contribute to this study by the introduction of
four new analogues of the carbocyclic derivatives 8 and 9
with either hydrophobic or hydrophilic substituents at the
(21) Højland, T.; Kumar, S.; Babu, B. R.; Umemoto, T.; Albæk, N.;
Sharma, P. K.; Nielsen, P.; Wengel, J. Org. Biomol. Chem. 2007, 5, 2375–
2379.
(22) (a) Obika, S.; Uneda, T.; Sugimoto, T.; Nanbu, D.; Minami, T.; Doi,
T.; Imanishi, T. Bioorg. Med. Chem. 2001, 9, 1001–1011. (b) Torigoe, H.;
Hari, Y.; Sekiguchi, M.; Obika, S.; Imanishi, T. J. Biol. Chem. 2001, 276,
2354–2360. (c) Sun, B.-W.; Babu, R. V.; Sørensen, M. D.; Zahrzewska, K.;
Wengel, J.; Sun, J.-S. Biochemistry 2004, 43, 4160–4169.
(16) Albæk, N.; Petersen, M.; Nielsen, P. J. Org. Chem. 2006, 71, 7731–
7740.
(17) Enderlin, G.; Nielsen, P. J. Org. Chem. 2008, 73, 6891–6894.
(18) (a) Seth, P. P.; Siwkowski, A.; Allerson, C. R.; Vasquez, G.; Lee, S.;
Prakash, T. P.; Kinberger, G.; Migawa, M. T.; Gaus, H.; Bhat, B.; Swayze,
E. E. Nucleic Acids Symp. Ser. 2008, 52, 553–554. (b) Seth, P. P.; Siwkowski,
A.; Allerson, C. R.; Vasquez, G.; Lee, S.; Prakash, T. P.; Wancewicz, E. V.;
Witchell, D.; Swayze, E. E. J. Med. Chem. 2009, 52, 10–13.
(19) Srivastava, P.; Barman, J.; Pathmasiri, W.; Plashkevych, O.; Wenska,
M.; Chattopadhyaya, J. J. Am. Chem. Soc. 2007, 129, 8362–8379.
(20) Zhou, C.; Liu, Y.; Andaloussi, M.; Badgujar, N.; Plashkevych, O.;
Chattopadhyaya, J. J. Org. Chem. 2009, 74, 118–134.
(23) Koizumi, M.; Morita, K.; Daigo, M.; Tsutsumi, S.; Abe, K.; Obika,
S.; Imanishi, T. Nucleic Acid Res. 2003, 31, 3267–3273.
(24) For definitions and nomenclature on nucleoside puckering, see:
(a) Altona, C.; Sundaralingam, M. J. Am. Chem. Soc. 1972, 94, 8205–8212.
(b) Altona, C.; Sundaralingam, M. J. Am. Chem. Soc. 1973, 95, 2333–2344.
(c) Saenger, W. Principles of Nucleic Acid Structure; Springer: New York, 1984.
(25) Plashkevych, O.; Chatterjee, S.; Honcharenko, D.; Pathmasiri, W.;
Chattopadhyaya, J. J. Org. Chem. 2007, 72, 4726–4736.
6758 J. Org. Chem. Vol. 74, No. 17, 2009

Kumar et al.
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60- or 70-positions but with free unsubstituted 80-positions,
18-21 (B = uracil). The preparation of these is based on the
ring-closing metathesis technique formerly giving 8 and 916
but this time being elaborated into ring-closing enyne
metathesis leading to the 1,3-diene system of compound
20, which has great potential for further derivatization and
so has been processed into 19 and 21.
yield. Then, the silyl groups were removed with TBAF to give
the deprotected compound 27, and the 50-hydroxyl group
was selectively protected as a 4,40-dimethoxytrityl (DMT)
ether giving 28 in 34% overall yield. Finally, the 30-O-
phosphoramidite 29 was obtained in quantitative yield.
Whereas ring-closing diene metathesis is an established
technique also in the preparation of various nucleoside
derivates,27-29 the ring-closing enyne metathesis is less used,
and only a single bicyclic nucleoside derivative prepared by
this method has been presented.30 In order to convert the key
intermediate 2216 into an appropriate substrate for enyne
metathesis, 22 was oxidized using the Dess-Martin period-
inane to give an aldehyde, which was further converted to the
alkyne 30 in 65% yield by the use of the so-called Bestmann-
Ohira reagent.31 The enyne derivative 30 was reacted with
Grubbs’ second-generation catalyst using microwave heat-
ing and provided the enyne metathesis 1,3-diene product 31
in 82% yield. Removal of the silyl groups was accomplished
in 71% yield with potassium fluoride and crown ether to give
the unprotected bicyclic nucleoside 20, which was selectively
protected with the DMT-group to give the intermediate 32 in
65% yield. Phosphitylation was achieved to give the 30-O-
phosphoramidite 33 in 60% yield.
Complete hydrogenation of the conjugated double bonds
of 31 was achieved by the use of Adams’ catalyst to give a
mixture of diastereomers 19 in 70% yield as an 8:1 ratio.
Simple modeling in combination with the experience from
the stereoselective dihydroxylation on 24 strongly indicates
the favor of the 60(R)-isomer. Due to significant overlap of
signals, however, no proof for this was given by NMR.
Again, a selective tritylation of the 50-OH was performed
giving 34 in 77% yield, and phosphitylation afforded the 30-
O-phosphoramidite 35 in 48% yield.
Finally, we decided to convert the hydrophobic 60-sub-
stituent of 20 into a hydrophilic hydroxymethyl moiety by
selective oxidative cleavage of the terminal double bond of
the 1,3-diene substrate. Catalytic amounts of OsO4 with
N-methlymorpholine-N-oxide as the cooxidant have been
reported to give moderate selectivity for similar substrates,32
but with reference to even better selectivity,33 we used the
sharpless AD-mix reagent for selective dihydroxylation of
the terminal double bond of 31 followed by oxidative
cleavage with NaIO4 to give the desired R,β-unsaturated
aldehyde. Selective reduction of the carbonyl group was
achieved using Luche conditions (NaBH4/CeCl3) to give
the hydroxymethyl derivate 36 in 54% yield over the three
steps. The primary hydroxy group was protected as its benzo-
ate ester 37, and the silyl protecting groups were removed with
potassium fluoride and crown ether to give compound 38 in
41% yield. The 50-OH group was selectively protected with the
DMT group, and the resulting compound 39 was converted
into the 30-O-phosphoramidite 40 in 45% yield.
Results
Chemical Synthesis of Bicyclic Nucleosides. The ring-clos-
ing metathesis (RCM) has become an excellent tool for the
synthesis of medium and large ring systems.26 In the field of
nucleosides, the use of RCM reactions in the preparation of
conformationally restricted bi- and tricyclic nucleosides16,27
as well as di- and trinucleotides28 has been reported by us
and others.29 The synthesis of the bicyclic nucleosides 8 and 9
(B = uracil) was performed from uridine, which was con-
verted in nine synthetic steps to the 20-deoxy-20-allyl-40-
hydroxymethyluridine derivative 22 (Scheme 1).16 Oxidation
and Wittig methylenation afforded 23, which was converted
by RCM using Grubbs’ second-generation catalyst to 24
affording after deprotection 9 and, thereafter, by hydroge-
nation the saturated bicyclic nucleoside 8.16 The cyclohexene
of 9 is an obvious point for further activation of the bicyclic
skeleton, and we decided to investigate the introduction of
hydrophilic moieties around the 20-40-bridge. Therefore, the
protected bicyclic nucleoside 24 was treated with OsO4 to
give the dihydroxy derivative 25 in 79% yield as a single
stereoisomer (Scheme 1). The 60(S),70(S)-configuration of 25
was proven by NMR (see below for the analysis of both
configuration and conformation). In order to make a deri-
vative that is appropriately protected to be a building block
for standard automated solid-phase oligonucleotide synth-
esis using the phosphoramidite approach, the free hydroxyl
groups were protected as acetate esters to give 26 in 93%
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Chun, M. W.; Jeong, L. S. Org. Biomol. Chem. 2006, 4, 4065–4067. (h) Yang,
Y.-Y.; Xu, J.; You, Z.-W.; Xu, X.; Qiu, X.-L.; Qing, F.-L. Org. Lett. 2007, 9,
5437–5440. (i) Zhong, S.; Mondon, M.; Pilard, S.; Len, C. Tetrahedron 2008,
64, 7828–7836. (j) Stauffiger, A.; Leumann, C. J. Eur. J. Org. Chem. 2009, 8,
1153–1162.
(32) Provencal, D. P.; Gardelli, C.; Lafontaine, J. A.; Leahy, J. W.
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1997, 38, 4043–4046.
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SCHEME 1. Synthesis of Bi- and Tricyclic Nucleosidesa
aReagents and conditions: (a) OsO4/NMO, THF, H2O, 100 °C, 79%; (b) Ac2O, pyridine, DMAP, 93%; (c) TBAF, THF, 42%; (d) DMT-Cl, pyridine, CH3CN,
79%; (e) NC(CH2)2OP(Cl)N(iPr)2, EtN(iPr)2, DCE, 100%; (f) (i) Dess-Martin periodinane, CH2Cl2, (ii) dimethyl 2-oxopropylphosphonate, TsN3, K2CO3,
CH3OH, CH3CN, 65%; (g) Grubbs’ second-generation catalyst, CH2Cl2, 100 °C, MW, 82%; (h) KF, 18-crown-ether-6, CH3CN, 100 °C, MW, 71%;
(i) DMT-Cl, pyridine, CH3CN, 65%; (j) NC(CH2)2OP(Cl)N(iPr)2, EtN(iPr)2, CH2Cl2, 60%; (k) H2, PtO2, CH3OH, 70%; (l) DMT-Cl, pyridine, CH3CN, 72%;
(m) NC(CH2)2OP(Cl)N(iPr)2, EtN(iPr)2, CH2Cl2, 48%; (n) (i) K3FeCN6, K2CO3, K2OsO4 2H2O, (DHQ)2PHAL, Na2SO3, t-BuOH, H2O, (ii) NaIO4, THF,
3
H2O,(iii) NaBH4,CeCl3 7H2O, CH3OH, 54%; (o) BzCl, pyridine, 53%; (p) KF, 18-crown-ether-6, CH3CN, 100 °C, MW, 41%; (q) DMT-Cl, pyridine, CH3CN,
3
77%; (r) NC(CH2)2OP(Cl)N(iPr)2, EtN(iPr)2, CH2Cl2, 45%; (s) EtO2CCtCCO2Et, toluene, 150 °C, MW, 75%. (DHQ)2PHAL = hydroquinine
1,4-phthalazinediyl diether, Ts = 4-toluenesulfonyl, DMT = 4,40-dimethoxytrityl, NMO = N-methylmorpholine N-oxide, DCE = 1,2-dichloroethane.
Finally, the bicyclic nucleoside 20 with its 1,3-diene moiety
is an obvious substrate for Diels-Alder reactions and there-
fore an easy access to further derivatization. As Diels-Alder
reactions have previously been demonstrated to work on
oligonucleotides with 1,3-dienes in the 30-end,34 also oligo-
nuncleotides contacting 20 should be potential substrates
for future studies. We decided to make a simple proof
of principle for the nucleoside, and we reacted the protec-
ted compound 31 with an alkyne (diethyl acetylenedi-
carboxylate) and achieved the tricyclic nucleoside 41 as a
single stereoisomer in 75% yield (see Scheme 1 for config-
urational analysis).
Configurational and Conformational Analyses. The config-
uration of 25 was determined from ROE contacts (Figure 2).
Contacts between H-10 and OH-70, between H-10 and H-80a
(hereafter defined to be placed below the furanose ring), and
between H-60 and H-80b unequivocally shows that the con-
figuration of C-60 and C-70 is (S,S). We explored the con-
formation of the unprotected form of 25 with ab initio
calculations. In the low energy conformation (Figure 2),
(34) Hill, K. W.; Taunton-Rigby, J.; Carter, J. D.; Kropp, E.; Vagle, K.;
Pieken, W.; McGee, D. P. C.; Husar, G. M.; Leuck, M.; Anziano, D. J.;
Sebesta, D. P. J. Org. Chem. 2001, 66, 5352–5358.
6760 J. Org. Chem. Vol. 74, No. 17, 2009

Kumar et al.
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LNA5 and other nucleic acid analogues.8,35 Eight different
modified 9-mer oligonucleotides were synthesized on solid
support by using an automated DNA synthesizer. The four
modified 30-O-phosphoramidites of the present study, 29, 33,
35, and 40 (incorporating 18-21), were all coupled in good
overall yields in combination with commercially available 20-
deoxynucleoside 30-O-phosphoramididtes. 1H-Tetrazole
was used as the activating agent for the phosphoramidites
33 and 35, whereas pyridinium hydrochloride was used as the
activator for 29 and 40. A 30 min coupling time was used for
the modified amidites. By the end of the oligonucleotide
syntheses, deprotection of all base-labile protecting groups
including the acetate and benzoate esters from 29 and 40,
respectively, as well as cleavage from the solid support was
achieved by the use of 32% aqueous ammonia. The compo-
sition and purity of all modified oligonucleotides was con-
firmed by MALDI MS and ion exchange chromatography/
RF-HPLC, respectively.
The hybridization of the modified oligonucleotides to-
ward complementary DNA and RNA was studied by
thermal denaturation experiments. The melting tempera-
tures (Tm) of the modified duplexes were determined and
compared with unmodified duplexes (Table 1). The se-
quence, 43, with one incorporation of the nucleoside 18
demonstrated a melting temperature of the duplex formed
with complementary RNA that is increased with 5.0 °C as
compared to the unmodified duplex formed between 42 and
RNA. This increase in duplex stability is the same for one
incorporation of 21 (sequence 49, ΔTm=4.9 °C), whereas
the stability increase for the DNA:RNA duplexes with one
incorporation of either 19 or 20 (sequences 45 and 47) is
slightly less pronounced (þ3.8 and þ3.2 °C, respectively).
With the incorporation of three modified nucleosides in
the DNA:RNA duplex, the increase in stability per mod-
ification is in all cases slightly smaller than with one
incorporation, i.e., between þ2.1 and þ4.7 °C, with the
most stable of all the determined duplexes being the one
formed by oligonucleotide 44 containing three incorpora-
FIGURE 2. (a) Stereoview of the lowest energy conformation
of an unprotected form of 25 (i.e., compound 18 (B=U)) and (b)
ROESY spectrum of compound 25. For atom numbering, see
Figure 1.
the pseudoration angle P of the sugar ring is 7° and the
puckering amplitude νmax is 46°. The C30-endo sugar con-
formation is in accordance with vanishing H-10-H-20 scalar
coupling constants. The six-membered ring connecting C-20
and C-40 adopts a chair conformation with C-30 displaced
slightly more than C-70 from the plane formed by the
remaining four atoms in the ring. To gauge how constrained
the bicyclic ring system of 25 is, we calculated the energy
difference between the chair and boat conformations of the
six-membered ring. The sugar ring possesses very little free-
dom in conformational space being locked in a C30-endo
conformation. In the boat conformation, C-70 is displaced
only very slightly from the plane formed by C-20, C-80, C-60,
and C-40. It appears that further displacement of C-70,
providing a proper boat conformation, would lead to a steric
clash between H-70 and O-30. Accordingly, the boat confor-
mation is rather unfavorable, and its energy is 8.7 kcal/mol
higher than that of the chair conformation. The completely
planar conformation is only 0.3 kcal/mol higher in energy
than the boat conformation. Thus, the unprotected form of
25 appears quite constrained in the conformation shown in
Figure 2.
tions of the dihydroxylated bicyclic nucleoside 18 (Tm
=
43.1 °C). Hybridization of the same modified oligonucleo-
tide sequences 43-50 with complementary DNA demon-
strated a general destabilization of the duplexes with
decreases in melting temperatures in the range of -0.5 to
-3.7 °C per modification. The destabilization is most
pronounced for the sequences with three modifications
and most pronounced for the incorporation of 20
(sequence 48). On the other hand, the incorporation of 18
led to the smallest decreases in duplex stability (sequences
43 and 44).
Circular Dichroism Spectroscopy. CD spectra for all nat-
ural and modified duplexes of the study were recorded in
order to examine the duplex geometry. A- and B-type
duplexes are known to display distinctly different CD
spectra. A-type duplexes give an intense negative band at
∼210 nm and a positive band at ∼260 nm of approximately
the same magnitude, where B-type duplexes give a negative
band at ∼250 nm and a positive band at ∼275 nm. It is well-
known that dsDNA duplexes adopt a B-type form in
solution, whereas an RNA:RNA duplex adopts an A-type.
In addition, the configuration of 41 was determined by a
ROESY spectrum. The ROE contact between H-10 and H-80a
assigned H-80a to be placed below the furanose ring and
therefore H-80b in the opposite position. Hereafter, the
contact between H-70 and H-80b and the lack of contact
between H-70 and H-80a unequivocally show that the config-
uration of C-70 is (S) as shown in Scheme 1.
Synthesis and Hybridization Properties of Oligonucleo-
tides. In order to study the affinity for complementary
nucleic acid DNA- and RNA-sequences with an easy com-
parison, we used the same 9-mer oligodeoxynucleotide se-
quence as used in our former study on the bicyclic
nucleosides 8 and 916 as well as in the original studies on
(35) Shaikh, K. I.; Kumar, S.; Lundhus, L.; Bond, A. D.; Sharma, P. K.;
Nielsen, P. J. Org. Chem. 2009, 74, 1557–1566.
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TABLE 1. Thermal Stability Data of Modified Duplexes
Tm (ΔTm)/°C b
ODN sequencesa
complementary DNA 50-dGCATATCAC-30
complementary RNA 50-rGCAUAUCAC-30
42
43
44
45
46
47
48
49
50
50-dGTGATATGC-30
50-dGTGAXATGC-30
50-dGXGAXAXGC-30
50-dGTGAYATGC-30
50-dGYGAYAYGC-30
50-dGTGAZATGC-30
50-dGZGAZAZGC-30
50-dGTGAVATGC-30
50-dGVGAVAVGC-30
30.4
29.1
29.9 (-0.5)
27.0 (-1.1)
27.6 (-2.8)
22.2 (-2.7)
27.6 (-2.8)
19.3 (-3.7)
28.4 (-2.0)
23.1 (-2.4)
34.1 (þ5.0)
43.1 (þ4.7)
32.9 (þ3.8)
37.5 (þ2.8)
32.3 (þ3.2)
35.2 (þ2.1)
34.0 (þ4.9)
37.9 (þ3.0)
aOligodeoxynucleotide sequences with X=18, Y=19, Z=20, V=21 corresponding to the incorporation of 29, 35, 33 ,and 40, respectively. bMelting
temperatures obtained from the maxima of the first derivatives of the melting curves (A260 vs temperature) recorded in a buffer containing 5 mM
Na2HPO4, 10 mM NaH2PO4, 100 mM NaCl, 0.1 mM EDTA, pH 7.0 using 1.5 μM concentrations of each strand. Values in parentheses show the
changes in Tm values per modification compared with the reference strand.
FIGURE 3. CD spectra of duplexes formed by the oligonucleotides 42-50 and their RNA-complements: (a) modified DNA:RNA duplexes
with single bicyclic modifications; (b) modified DNA:RNA duplexes with triple bicyclic modifications. Sequence 51 corresponds to the RNA
sequence 50-rGUGAUAUGC-30.
DNA:RNA duplexes adopt intermediate A/B-type structures.
An RNA:RNA duplex is therefore taken as a standard
for the A-type, and the CD-curve (see 51 in Figure 3)
clearly displays the A-type characteristics with especially an
intense negative band at 210 nm. This band is small for
the DNA:RNA duplex (42). The modified DNA:RNA du-
plexes displays some clear A-type characteristics that are
much more pronounced with three incorporations of bicyclic
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FIGURE 4. CD spectra of duplexes formed by the oligonucleotides 42-50 and their DNA complements: (a) modified dsDNA duplexes with
single bicyclic modifications; (b) modified dsDNA duplexes with triple bicyclic modifications.
nucleosides than with single incorporations (compare parts a
and b of Figure 3). The negative band at 210 nm clearly
decreases in intensity in the order RNA > 18 > 21 > 19 >
20>DNA for both one incorporation (i.e., 51>43>49 ∼ 45 ∼
47>42) and three incorporations (i.e., 51>44>50>46>48>
42) in the DNA:RNA duplex. A similar picture though less
systematic is seen with the positive band at 260 nm.
The unmodified dsDNA duplex shows clear B-type char-
acteristics (see 42 in Figure 4), whereas the modified dsDNA
duplexes seem to be changed toward an intermediate duplex
form as clearly indicated in the decreasing bands at 210 and 250
nm. Again the largest changes are seen with three incorpora-
tions as compared to one incorporation (compare parts a and b
of Figure 4), and again, the changes are most pronounced for
the modification 18 (see 44 in Figure 4b) with a negative band
at 210 nm. Another trend in the CD spectra indicating a shift
toward more A-type like duplexes of the modified ssDNA
duplexes is that the large band at 280 nm is shifting toward 270
nm by the increasing number of modifications.
did not lead to any siginificant increase in triplex stability,21
and this result is in contrary to the very triplex-stabilizing
effects of the 20-oxy analogue, ENA 4,23 as well as of LNA 1.22
Therefore, it was interesting to study whether the dihydroxy-
lated derivative 18, which introduces hydrofilicity directly
around the 20,40-linkage could change this picture. Hence,
the 30-O-phosphoramidite 29 was incorporated into the same
TFO sequences as used in our former study21 and the thermal
stability data of the triplexes formed with a DNA duplex target
are shown in Table 2. However, few differences in melting
temperatures between the modified sequences 53/54 and the
unmodified sequence 52 can be observed, and in general, the
two hydroxy funtionalities on 18 as compared to 8 does not
seem to make any significant difference on the triplex stability.
Discussion
In our previous paper, we demonstrated the efficient linear
synthesis of 9 from uridine based on ring-closing metathesis
as the key step.16 Herein, we have proved that the enyne
metathesis can be performed with equal efficiency to give the
Triplex Studies. The incorporation of our native carbocyclic
bicyclic nucleoside 8 into triplex-forming oligonucleotides
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TABLE 2. Thermal Stability Data of Modified Triplexes
complementary DNA duplex:a
30-GGT GAA AAA TTT TCT TTT CCC CCC TGA CC-50 50-CCA CTT TTT AAAAGAAAAGGGGGGACT GG-30
TFO sequences:b Tm (ΔTm)c/°C
30.3
52
53
54
50-TTT TCT TTT CCC CCC T-30
50-TTT TCX TTT CCC CCC T-30
50-TTX TCX TTX CCC CCC T-30
29.8 (-0.5)
30.1 (-0.1)
aDNA duplex with the target part underlined. bTriplex forming oligodeoxynucleotide sequences with X = 18 corresponding to the incorporation of
29. cMelting temperatures obtained from the maxima of the first derivatives of the melting curves (A260 vs temperature) recorded in a buffer containing
10 mM sodium cacodylate, 150 mM NaCl, 10 mM MgCl2, pH 6.0 using 1.0 μM concentration of the duplex and 1.5 μM concentrations of the TFO
sequences. Values in parentheses show the changes in Tm values per modification compared with the reference strand.
1,3-diene-containing bicyclic nucleoside 20. The olefins of
9 and 20 open the opportunity of preparing a range of
analogues as we have proved by simple hydrogenation to
give 8 and 19 as well as the hydroxylated analogues 18 and 21
(or at first, protected forms thereof). Finally, the potential of
20 for Diels-Alder reactions has been enlightened by the
preparation of 41. Clearly, a much larger variety of bi- and
tricyclic nucleosides could be designed by using the two key
building blocks 9 and 20 in order to explore the effects of
decoration of the 20,40-linkage and tuning the electrostatics in
the nucleic acid structure as well as for the conjugation of the
oligonucleotides to other entities. The application of Diels-
Alder reactions on oligonucleotides containing 20 will be
applied in future studies.
neighboring 20-deoxynucleotides seems from the CD spectra
to be less pronounced for our carbocyclic analogues as
compared to LNA. This confirms the trend that the more
conformational steering toward A-type duplex formation,
the higher is the thermal stability. That this steering is
directly related to the 20-oxygen atom is confirmed by
ENA 4 as well as 7 being more or less as efficient as LNA 1
in hybridizing to RNA, whereas all carbocyclic analogues
show slightly lower RNA-affinities. A partly compensation
for the lack of 20-oxygen can be obtained by other hydro-
philic groups in the bridge as seen for 18 and to some extent
for 21. This indicates again that water binding in the minor
groove has a large influence on the duplex formation, but it
should be noted that the 20-oxygen is positioned deeply into
the minor groove, whereas the 60/70-substituents are posi-
tioned more at the rim of the groove. Probably due to steric
reasons, all the 80-methylated analogues 16-17 generally
demonstrate lower RNA-affinity than the 80-unsubstituted
analogues of this study.16,20 However, it should be addressed
that different sequence contexts hamper direct comparison.
Also in the 80-methylated series, however, the introduction of
a 60-hydroxyl group improves the RNA-affinity.20
In the formation of DNA:DNA duplexes, the indicated
effects of water binding are even more pronounced. Wheras
LNA, and to a large extent ENA, reveals strong duplex
stabilization, this decreases dramatically with other analo-
gues. The new analogues shown herein all lead to decreased
duplex stability when incorporated one or three times, with
the decrease being more pronounced for the hydrophobic
analogues 19 and 20 than for the hydroxylated 18 and 21.
The hydrophobic substituents of 19 and 20 seem to decrease
the duplex stability even further as compared to 8 and 9.16
The conformational steering as indicated by CD-spectros-
copy (Figure 4) follows the trend. Thus, the presence of a
20-oxygen is crucial for the steering and therefore for the
duplex stability, and the compensation by other hydrophilic
groups is only small. Increases in DNA:DNA duplex stabi-
lity by this series of locked nucleic acid analogues has in
general only been seen with 20-oxygens like in LNA 1, ENA
4, and 7 though with native LNA being superior, and the
effect also found with 2 and 3 indicating that the smaller
ring (and higher puckering) is also playing a role. Also the
70/80-methylated carbocyclic analogues 13-14 and 15-17
are showing decreased DNA:DNA duplex stability, although
some compensation by a 60-hydroxyl group is seen for one
stereoisomer of 13.20 The larger effect of hydration in the
modified DNA:DNA duplexes as compared to the modified
DNA:RNA duplexes can be related to the more extended
structure and thereby larger surface of a B-type or B-type like
duplex than for an A-type or A-type-like duplex.
The thermal stability data of the present study continues
the line in the growing series of different bicyclic nucleoside
building blocks with 20-40-linkages. When comparing
the hybridization properties of LNA and ENA with their
20-carba analogues 13 (although 70-methylated) and 8, re-
spectively, the presence of a 20-oxygen is clearly of high
importance, most probably due to hydration. The feature
behind the ability of LNA-DNA mixmers to form the
stabilized A-type duplexes with complementary DNA and
RNA has been suggested to be the conformational steering
of the neighboring 20-deoxynucleotides toward N-type puck-
ering,7 and this effect seems related to this hydration. Herein,
we have extended this study by attaching hydrophilic/hydro-
phobic groups to 8 and 9 from our former study,16 in order to
see whether only the 20-oxygen can induce the hydration or
whether this can be obtained by other placements of an
oxygen or negatively influenced by hydrophobic groups.
Concerning the DNA:RNA duplexes of this study, the two
entirely hydrophobic bicyclic nucleosides 19 (as an 8:1
epimeric mixture), and 20 did, in fact, lead to the smallest
increases in duplex stability at the same range though slightly
smaller than their 60-unsubstituted analogues 8 and 9. Like
with 8 and 9, the saturated analogue 19 leads to slightly
higher Tm’s than the unsaturated 20. The introduction of
hydrophilic hydroxyl groups improves the RNA-affinity
with 21 being superior to 20 and 9, and with 18 being superior
to 19 and 8. Especially, the melting temperature of the
DNA:RNA duplex formed with the triple modified sequence
44 is high, 43 °C, as compared to 38 °C with 8 instead of 18.16
These trends are clearly related to the CD spectra (Figure 3),
showing that the shift in duplex form toward a more
A-type like structure is largest for 18 decreasing in the
order 18> 21>19>20. This is coherent with the former
study showing larger shifts for the saturated 8 as compared
to the unsaturated 9 but also lower shifts for both as
compared to LNA.16 Hence, the steering performed on
6764 J. Org. Chem. Vol. 74, No. 17, 2009

Kumar et al.
JOCArticle
In the formation of triplexes, the same trend is seen, as both
LNA 1, amino-LNA 3, the longer ENA 4, and 7 form very
stable triplexes, whereas our carbocyclic analogues 8 and its
hydroxylated analogue 18 do not lead to significant increases
in stability as compared to the native oligonucleotides. In
other words, the 20-heteroatom is crucial, and compensation
has not been found with the two hydroxyl groups at the ring,
although negative sterical influence by the additional groups
cannot be excluded. This is in full accordance with an
NMR study indicating that triplexes formed by LNA have a
special structure with a more efficient network of hydration.36
Apparently, this hydration is related directly to the 20-hetero-
atom, as ENA and 7 give highly stable triplexes, whereas the
carbocyclic analogues of ENA do not.
In summary, the nucleic acid recognition performed by the
carbocyclic locked nucleic acid analogues of the present
study is very RNA-selective. Only with RNA and not with
single- or double-stranded DNA has a general increase in
thermal stability been found. This might be a useful feature
for diagnostic applications that are not found for the native
LNA and other analogues.
(dd, 1H, J = 5.4, 8.7 Hz, H-60), 2.30 (m, 1H, H-20), 2.01 (m, 1H,
H-80b), 1.84 (m, 1H, H-80a), 0.92 (s, 9H, SiC(CH3)3), 0.88 (s, 9H,
SiC(CH3)3), 0.11 (s, 3H, SiCH3), 0.05 (s, 3H, SiCH3) 0.05 (s, 3H,
SiCH3), 0.02 (s, 3H, SiCH3); 13C NMR (75 MHz, CDCl3) δ
163.8 (C-4), 150.6 (C-2), 140.7 (C-6), 100.9 (C-5), 87.3, 87.1
(C-10, C-40), 68.5 (C-60), 67.1 (C-30), 66.7 (C-70), 61.0 (C-50), 44.2
(C-20), 28.4 (C-80), 26.1, 25.6 (SiC(CH3)3), 18.5, 17.8
(SiC(CH3)3), -4.7, -5.2, -5.3, -5.6 (SiCH3); MALDI MS m/z
(551.2557 [M þ Na]þ, C24H44N2O7Si2-Naþ calcd 551.2579).
Synthesis of (1R,2S,3S,5R,6R,8S)-2,3-Diacetyloxy-8-tert-bu-
tyldimethylsilyloxy-1-tert-butyldimethylsilyloxymethyl-6-(uracil-
1-yl)-7-oxabicyclo[3.2.1]octane (26). Nucleoside 25 (850 mg,
1.609 mmol) was coevaporated with anhydrous pyridine
(4 mL) and redissolved in the same solvent (14 mL). The solution
was stirred at 0 °C, and DMAP (59 mg, 0.483 mmol) and acetic
anhydride (334 μL, 3.54 mmol) were added. The solution was
stirred at 0 °C for 15 min and at room temperature for 1.5 h.
Additional acetic anhydride (152 μL, 1.61 mmol) was added,
and after 1 h, a similar portion was added. The solution was
stirred for 19 h, and a saturated aqueous solution of NaHCO3
(20 mL), and CH2Cl2 (100 mL) was added. The residue was
extracted with CH2Cl2 (3 ꢀ 125 mL) and the combined organic
extracts were washed with a saturated aqueous solution of
NaHCO3 (100 mL), dried (MgSO4), and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography (EtOAc-petroleum ether, 1:3 v/v) to give the
desired product 26 (920 mg, 93%) as a white foam: Rf 0.60
Conclusion
We have synthesized four different modified nucleosides
starting from uridine by ring-closing diene or enyne meta-
thesis. All nucleosides were successfully incorporated into
oligonucleotides, and the hybridization properties of these
were recorded. The modified nucleosides have shown an
increase in melting temperatures of þ2.1 to þ5.0 °C per
modification against RNA, a decrease of -0.5 to -3.7 °C
per modification against DNA, and very small influence on
triplex formation. This gives new information on the impor-
tance of the 20-oxygen atom for the formation of duplexes and
triplexes. Some compensation for the lack of a 20-oxygen atom
inanall carbocyclic additional ring can be obtainedwithother
hydrophilic groups attached to the ring. Importantly, the
perspective of using the 1,3-diene of20 for further conjugation
using the Diels-Alder reaction has been demonstrated.
1
(EtOAc); H NMR (300 MHz, CDCl3) δ 9.08 (br s, 1H, NH),
8.35 (d, 1H, J = 8.1 Hz, H-6), 6.08 (s, 1H, H-10), 5.67 (d, 1H, J =
8.1 Hz, H-5), 5.50 (m, 1H, H-70), 5.31 (d, 1H, J = 5.7 Hz, H-60),
4.48 (d, 1H, J = 5.1 Hz, H-30), 4.00, 3.52 (AB, 2H, J = 11.4 Hz,
H-50), 2.44 (m, 1H, H-20), 2.35 (m, 1H, H-80), 2.13 (s, 3H,
CH3CO), 2.09-2.03 (m, 4H, H-80, CH3CO), 0.94 (s, 9H, SiC-
(CH3)3), 0.91 (s, 9H, SiC(CH3)3), 0.13 (s, 3H, SiCH3), 0.12 (s, 3H,
SiCH3) 0.09 (s, 3H, SiCH3), 0.07 (s, 3H, SiCH3); 13C NMR (75
MHz, CDCl3) δ 170.3, 169.7 (CO), 163.5 (C-4), 150.2 (C-2), 140.3
(C-6), 101.0 (C-5), 87.4, 86.0 (C-10, C-40), 68.8 (C-60), 67.2 (C-30),
66.5 (C-70), 59.8 (C-50), 44.1 (C-20), 26.3 (C-80), 26.0, 25.6
(SiC(CH3)3), 21.2, 20.5 (CH3CO), 18.5, 17.8 (SiC(CH3)3), -4.6,
-5.2, -5.3, -5.6 (SiCH3); ESI MS m/z (635.2799 [M þ Na]þ,
C28H48N2O9Si2-Naþ calcd 635.2791).
Synthesis of (1R,2R,3S,5R,6R,8S)-2,3-Diacetyloxy-8-hydro-
xy-1-(4,40-dimethoxytrityloxymethyl)-6-(uracil-1-yl)-7-oxabicyclo-
[3.2.1]octane (28). Nucleoside 26 (878 mg, 1.43 mmol) was
dissolved in anhydrous THF (30 mL), and a 1 M solution of
TBAF in anhydrous THF (3.15 mL, 3.15 mmol) was added. The
mixture was stirred at room temperature for 30 min and con-
centrated under reduced pressure. The residue was subjected to
silica gel column chromatography (EtOAc-petroleum ether,
9:1 v/v) to give the crude desired product 27 (230 mg, 42%) as a
white foam, which was used without further purification in the
next step (Rf 0.10 (EtOAc); ESI MS m/z 407.1069 [MþNa]þ,
C16H20N2O9-Naþ calcd 407.1061), as well as a mixture of
deacetylated products (510 mg). Nucleoside 27 (220 mg, 0.572
mmol) was coevaporated with anhydrous pyridine (2 mL) and
redissolved in a mixture of the same solvent (2.5 mL) and
anhydrous CH3CN (2.5 mL). DMT-Cl (194 mg, 0.572 mmol)
was added and the reaction mixture was stirred at room
temperature for 22 h. An additional amount of DMT-Cl
(39 mg, 0.114 mmol) was added, and after stirring for 5 h the
mixture was concetrated under reduced pressure. The residue
was purified by silica gel column chromatography (0-1%
CH3OH and 0.5% pyridine in CH2Cl2) to give the desired
product 28 (310 mg, 79%) as a white foam: Rf 0.30 (EtOAc);
1H NMR (300 MHz, CDCl3) δ 8.77 (s, 1H, NH), 8.09 (d, 1H,
J = 8.1 Hz, H-6), 7.40-7.25 (m, 9H, Ar), 6.84 (dd, 4H, J = 1.2,
8.7 Hz, Ar), 6.04 (s, 1H, H-10), 5.47 (t, 1H, J = 5.3 Hz, H-70),
5.40 (d, 1H, J = 8.1 Hz, H-5), 5.34 (d, 1H, J = 5.3 Hz, H-60),
Experimental Section
Synthesis of (1R,2S,3S,5R,6R,8S)-2,3-Dihydroxy-8-tert-butyldi-
methylsilyloxy-1-tert-butyldimethylsilyloxymethyl-6-(uracil-1-yl)-
7-oxabicyclo[3.2.1]octane (25). Nucleoside 2416 (509 mg,
1.03 mmol) was dissolved in THF (9 mL) and H2O (9 mL) in a
microwave vial. N-methylmorpholine N-oxide (362 mg, 3.09
mmol) and a 2.5% w/w solution of OsO4 in tert-butyl alcohol
(519 μL, 0.051 mmol) were added, and the solution was stirred in
the microwave reactor at 100 °C for 20 min. A 5% aqueous
solution of Na2S2O5 (7 mL) was added, and the solution was
concentrated under reduced pressure to approximately 15 mL.
The solution was extracted with EtOAc (4 ꢀ 50 mL), and the
combined organic extracts were dried (MgSO4) and concen-
trated under reduced pressure. The residue was purified by silica
gel column chromatography (EtOAc-petroleum ether, 1:1 v/v)
to give the desired product 25 (429 mg, 79%) as a white foam:
Rf 0.60 (EtOAc); 1H NMR (400 MHz, DMSO-d6) δ 11.29 (br s,
1H, NH), 8.15 (d, 1H, J = 8.0 Hz, H-6), 6.04 (s, 1H, H-10), 5.39
(dd, 1H, J = 1.6, 8.0 Hz, H-5), 4.63 (d, 1H, J = 2.8 Hz, 70-OH),
4.60 (d, 1H, J = 8.7 Hz, 60-OH), 4.35 (d, 1H, J = 5.2 Hz, H-30),
4.15, 3.55 (AB, 2H, J = 11.8 Hz, H-50), 3.92 (m, 1H, H-70), 3.62
(36) Sørensen, J. J.; Nielsen, J. T.; Petersen, M. Nucleic Acid Res. 2004, 32,
6078–6085.
J. Org. Chem. Vol. 74, No. 17, 2009 6765

Kumar et al.
JOCArticle
4.42 (m, 1H, H-30), 3.80 (s, 6H, OCH3), 3.60, 3.45 (AB, 2H, J =
10.8 Hz, H-50), 2.80 (d, 1H, J = 3.0 Hz, OH), 2.55 (m, 1H, H-20),
2.43 (m, 1H, H-80), 2.09 (s, 3H, CH3CO), 2.02 (m, 1H, H-80), 1.88
(s, 3H, CH3CO); 13C NMR (75 MHz, CDCl3) δ 170.3, 169.5-
(CO), 163.6 (C-4), 158.8, 158.8 (Ar), 150.2 (C-2), 144.0 (Ar),
140.2 (C-6), 136.2, 130.1, 130.0, 128.1, 128.0, 127.3, 113.4 (Ar),
101.1 (C-5), 87.8, 87.6 (C-10, C-40), 84.9 (CAr3), 68.6 (C-60), 68.4
(C-30), 66.7 (C-70), 60.7 (C-50), 55.2 (OCH3), 43.9 (C-20), 25.8
(C-80), 21.2, 20.3 (CH3CO); ESI MS m/z (709.2176 [M þ Na]þ,
C37H38N2O11-Naþ calcd 709.2368).
Synthesis of (1R,2S,3S,5R,6R,8S)-2,3-Diacetyloxy-8-cyano-
ethoxy(diisopropylamino)phosphinoxy-1-(4,40-dimethoxytrityloxy-
methyl)-6-(uracil-1-yl)-7-oxabicyclo[3.2.1]octane (29). A solu-
tion of nucleoside 28 (134 mg, 0.195 mmol) in anhydrous
DCE (2.5 mL) was stirred at roon temperature. N,N-Diisopro-
pylethylamine (170 μL, 0.977 mmol) and N,N-diisopropylami-
no-2-cyanoethylphosphinochloridite (131 μL, 0.587 mmol) were
added, and the reaction mixture was stirred at room tempera-
ture for 24 h. CH2Cl2 (15 mL) was added, and the mixture was
washed with a saturated aqueous solution of NaHCO3 (15 mL).
The aqueous phase was extracted with CH2Cl2 (3 ꢀ 15 mL), and
the combined organic extracts were dried (Na2SO4) and con-
centrated under reduced pressure. The residue was purified by
silica gel column chromatography (0-0.5% CH3OH and 1%
pyridine in CH2Cl2) to give the desired product 29 (173 mg,
100%) as a white foam: Rf 0.60 (EtOAc); 31P NMR (121.5 MHz,
CDCl3) δ 151.6, 149.5; ESI MS m/z (909.3404 [M þ Na]þ,
C46H55N4O12P-Naþ calcd 909.3446).
Synthesis of (1R,5R,6R,8S)-8-(tert-butyldimethylsilyloxy)-1-
(tert-butyldimethylsilyloxymethyl)-6-(uracil-1-yl)-2-vinyl-7-oxabi-
cyclo[3.2.1]oct-2-ene (31). To a stirred solution of 30 (1.389 g,
2.67 mmol) in anhydrous CH2Cl2 (6 mL) was added Grubbs’
second-generation catalyst (((Mes)2Im)(Cy3P)Cl2RudCHPh)
(113 mg, 0.13 mmol). The solution was stirred in a microwave
reactor at 100 °C for 2 h. The mixture was concentrated under
reduced pressure, and the residue was purified by silica gel
column chromatography (EtOAc-petroleum ether, 1:4 v/v) to
give the bicyclic nucleoside 31 (1.134 g, 82%) as a white foam:
Rf 0.70 (EtOAc-petroleum ether, 1:1 v/v); 1H NMR (300 MHz,
CDCl3) δ 9.09 (br s, 1H, NH), 8.16 (d, 1H, J = 8.1 Hz, H-6),
6.09 (m, 1H, CH = CH2), 5.88 (m, 1H, H-70), 5.68-5.61 (m, 2H,
H-5, H-10), 5.28 (dd, 1H, J = 1.8, 16.5 Hz, CHdCH2), 5.0 (dd,
1H, J = 1.8, 10.5 Hz, CHdCH2), 4.46 (d, 1H, J = 5.1 Hz, H-30),
4.04, 3.65 (AB, 2H, J = 11.4 Hz, H-50), 2.62 (m, 1H, H-80),
2.40-2.33 (m, 2H, H-20, H-80), 0.95 (s, 9H, SiC(CH3)3), 0.84
(s, 9H, SiC(CH3)3), 0.14 (s, 3H, SiCH3), 0.12 (s, 3H, SiCH3),
0.06 (s, 3H, SiCH3), 0.05 (s, 3H, SiCH3); 13C NMR (75 MHz,
CDCl3) δ 163.7 (C-4), 150.3 (C-2), 140.3 (C-6), 138.5 (C-60),
133.1 (CHdCH2), 125.7 (C-70), 116.6 (CHdCH2), 101.2 (C-5),
89.2 (C-10), 83.4 (C-40), 65.2 (C-30), 60.6 (C-50), 44.6 (C-20),
28.2 (C-80), 26.1, 25.9 (SiC(CH3)3), 18.5, 17.9 (SiC(CH3)3), -4.4,
-4.9, -5.2, -5.3 (SiCH3); HRMALDI MS m/z (543.2659
[M þ Na]þ C26H44N2O5Si2-Naþ calcd 543.2681).
Synthesis of (1R,5R,6R,8S)-8-hydroxy-1-hydroxymethyl-6-
(uracil-1-yl)-2-vinyl-7-oxabicyclo[3.2.1]oct-2-ene (20). Asolution
of nucleoside 31 (529 mg, 1.02 mmol) in anhydrous CH3CN (10
mL) was added KF (0.886 g, 15.25 mmol) and 18-crown ether-6
(1.075 g, 4.07 mmol). The solution was stirred in a microwave
reactor at 100 °C for 1 h. The mixture was concentrated under
reduced pressure, and the residue was purified by silica gel column
chromatography (0-5% CH3OH in CH2Cl2) to give 20 (212 mg,
71%) as a white foam: Rf 0.40 (MeOH-dichloromethane, 1:9 v/v);
1H NMR (300 MHz, CD3OD) δ 8.27 (d, 1H, J = 8.1 Hz, H-6),
6.19 (dd, 1H, J = 10.8, 17.1 Hz, CH = CH2), 5.95 (m, 1H, H-70),
5.65 (d, 1H, J = 8.1 Hz, H-5), 5.57 (s, 1H, H-10), 5.34 (dd, 1H, J =
2.1, 17.1 Hz, CHdCH2), 5.01 (dd, 1H, J = 2.1, 10.8 Hz,
CHdCH2), 4.50 (d, 1H, J = 5.4 Hz, H-30), 4.03, 3.72 (AB, 2H,
J = 12.0 Hz, H-50), 2.67 (m, 1H, H-80), 2.45 (m, 1H, H-20), 2.33
(m, 1H, H-80); 13C NMR (75 MHz, CD3OD) δ 166.5 (C-4), 152.1
(C-2), 142.2 (C-6), 139.8 (C-60), 134.6 (CHdCH2), 126.9 (C-70),
116.3 (CHdCH2), 101.3(C-5), 90.5(C-10), 84.2(C-40), 65.9 (C-30),
60.3 (C-50), 45.2 (C-20), 28.7 (C-80); HRMALDI MS m/z
(315.0960 [M þ Na]þ, C14H16N2O5-Naþ calcd 315.0951).
Synthesis of (1R,5R,6R,8S)-1-(4,40-Dimethoxytrityoxymethyl)-
8-hydroxy-6-(uracil-1-yl)-2-vinyl-7-oxabicyclo[3.2.1]oct-2-ene
(32). DMT-Cl (79 mg, 0.23 mmol) was added to a stirred
solution of 20 (34 mg, 0.12 mmol) in anhydrous pyridine
(0.75 mL) and anhydrous CH3CN (0.75 mL). The mixture was
stirred for 22 h and concentrated under reduced pressure. The
residue was purified by silica gel column chromatography
(0-3.0% CH3OH and 0.5% pyridine in CH2Cl2) to give the
product 32 (45 mg, 65%) as a foam: Rf 0.70 (CH3OH-dichlor-
Synthesis of 20-C-Allyl-20-deoxy-30,50-di-O-(tert-butyldimethyl-
silyl)-40-C-ethynyluridine (30). To a stirred solution of nucleo-
side 22 (2.186 g, 4.16 mmol) in anhydrous CH2Cl2 (35 mL) was
added Dess-Martin periodinane (2.203 g, 5.19 mmol). The
mixture was stirred at room temperature for 2 h and then filtered
through Celite. The filter was washed with EtOAc (30 mL), and
the combined organic phases were washed with a mixture of
saturated aqueous solutions of Na2S2O3 and NaHCO3 (1:1, v/v,
40 mL). The aqueous phase was extracted with CH2Cl2 (2 ꢀ
20 mL), and the combined organic phases were dried (Na2SO4)
and concentrated under reduced pressure to give the crude
aldehyde (2.301 g). A suspension of K2CO3 (2.871 g, 20.78
mmol) and p-toluenesulfonyl azide (2.048 g, 10.39 mmol) in
anhydrous CH3CN (10 mL) was stirred at room temperature,
and dimethyl-2-oxopropylphosphonate (1.42 mL, 10.39 mmol)
was added. The mixture was stirred for 2 h, and a solution of the
aldehyde (2.301 g, 4.16 mmol) in anhydrous CH3OH (10 mL)
was added. The mixture was stirred for 24 h and concentrated
under reduced pressure. The residue was dissolved in Et2O
(20 mL) and H2O (12 mL). The aqueous layer was separated
and the organic layer washed with H2O (12 mL) and brine (12
mL). The combined organic layers were dried (Na2SO4) and
concentrated under reduced pressure. The residue was purified
by silica gel column chromatography (EtOAc-petroleum ether,
1:4 v/v) to give 30 (1.394 g, 65%) as a white foam: Rf 0.70
(EtOAc-petroleum ether, 1:1 v/v); 1H NMR (300 MHz,
CDCl3) δ 8.85 (br s, 1H, NH), 7.63 (d, 1H, J = 8.4 Hz, H-6),
6.18 (d, 1H, J = 6.6 Hz, H-10), 5.72-5.64 (m, 2H, H-5,
CHdCH2), 5.08-4.96 (m, 2H, CHdCH2), 4.39 (d, 1H, J =
6.0 Hz, H-30), 3.90, 3.76 (AB, 2H, J = 11.1 Hz, H-50), 2.56 (s, 1H,
HCꢁC), 2.46-2.32 (m, 3H, H-20, CH2CHdCH2), 0.96 (s, 9H,
SiC(CH3)3), 0.94 (s, 9H, SiC(CH3)3), 0.13 (s, 3H, SiCH3), 0.13 (s,
3H, SiCH3), 0.12 (s, 3H, SiCH3), 0.09 (s, 3H, SiCH3); 13C NMR
(75 MHz, CDCl3) δ 163.0 (C-4), 150.1 (C-2), 140.3 (C-6), 135.3
(CHdCH2), 116.9 (CH=CH2), 102.8 (C-5), 88.1 (C-10), 85.0,
80.5, 77.6 (C-40, CꢁCH), 73.7 (C-30), 67.1 (C-50), 49.3 (C-20),
30.1 (CH2CHdCH2), 26.0, 25.9 (SiC(CH3)3), 18.4, 18.3
(SiC(CH3)3), -3.9, -4.1, -5.3, -5.3 (SiCH3); HRMALDI
MS m/z (543.2684 [M þ Na]þ, C26H44N2O5Si2-Naþ calcd
543.2681).
1
omethane, 1:9 v/v); H NMR (300 MHz, CDCl3) δ 9.17 (br s,
1H, NH), 8.19 (d, 1H, J = 8.1 Hz, H-6), 7.46-7.14 (m, 9H, Ar),
6.87 (dd, 4H, J=1.8, 8.7 Hz, Ar), 6.01 (m, 1H, H-70), 5.88 (dd,
1H, J = 10.8, 17.1 Hz, CHdCH2), 5.66 (s, 1H, H-10), 5.41
(d, 1H, J=8.1 Hz, H-5), 5.29 (dd, 1H, J=1.5, 17.1 Hz, CHdCH2),
4.95 (dd, 1H, J = 1.5, 10.8 Hz, CHdCH2), 4.69 (t, 1H, J = 6.3 Hz,
H-30), 3.80 (s, 6H, OCH3), 3.71, 3.46 (AB, 2H, J = 11.1 Hz, H-50),
2.63-2.48 (m, 3H, H-80, H-20); 13C NMR (75 MHz, CDCl3)
δ 163.3 (C-4), 158.8 (Ar), 150.3 (C-2), 144.5 (Ar), 140.3 (C-6),
138.3, 135.3, 135.2 (C-60, Ar), 132.4 (CHdCH2), 130.3, 130.2, 129.1,
128.3, 128.2, 127.3 (Ar), 126.1 (C-70), 117.0 (CHdCH2), 113.5 (Ar),
101.4 (C-5), 89.4 (C-10), 87.4, 82.4 (CAr3, C-40), 67.0 (C-30), 61.1
(C-50), 55.3 (OCH3), 43.8 (C-20), 27.5 (C-80); HRMALDI MS
m/z (617.2264 [M þ Na]þ, C35H34N2O7-Naþ calcd 617.2258).
6766 J. Org. Chem. Vol. 74, No. 17, 2009

Kumar et al.
JOCArticle
Synthesis of (1R,5R,6R,8S)-8-(2-Cyanoethoxy(diisopropyl-
amino)phosphinoxy)-1-(4,40-dimethoxytrityloxymethyl)-6-(uracil-
(0.22 mL, 1.25 mmol) and 2-cyanoethyl N,N-diisopropylpho-
sphoramidochloridite (0.14 mL, 0.63 mmol). The mixture was
stirred for 15 h, and then CH2Cl2 (2 mL) was added. The mixture
was washed with a saturated aqueous solution of NaHCO3 (2
mL). The aqueous phase was extracted with CH2Cl2 (2 ꢀ 3 mL),
and the combined organic phases were dried (Na2SO4) and
concentrated under reduced pressure. The residue was purified
by silica gel column chromatography (0-1.5% CH3OH and
0.5% pyridine in CH2Cl2) to give 35 (80 mg, 48%) as a white
foam: Rf 0.80 (CH3OH--dichloromethane, 1:9 v/v); 31P NMR
(121.5 MHz, CDCl3) δ 150.2 (major isomer), 150.0 (minor
isomer), 149.9 (minor isomer), 149.5 (major isomer); ESI MS
m/z (821.3659 [M þ Na]þ, C44H55N4O8P-Naþ calcd 821.3650).
Synthesis of (1R,5R,6R,8S)-8-(tert-Butyldimethylsilyloxy)-
1-(tert-butyldimethylsilyloxymethyl)-2-hydroxymethyl-6-(uracil-
1-yl)-7-oxabicyclo[3.2.1]oct-2-ene (36). A solution of nucleoside
31 (675 mg, 1.30 mmol) in t-BuOH (20 mL) was added to a
solution of K3FeCN6 (1.282 g, 3.89 mmol), K2CO3 (537 mg,
3.89 mmol), K2OsO2(OH)4 (10 mg, 0.03 mmol), and (DHQ)2-
PHAL (101 mg, 0.13 mmol) in water (15 mL), and the resulting
mixture was stirred at room temperature for 4 h. Solid Na2SO3
(1.4 g, 11.1 mmol) was added, and the mixture was stirred for
30 min. Et2O (20 mL) was added, and the layers were separated.
The aqueous layer was extracted with Et2O (2 ꢀ 5 mL), and the
combined organic layers were washed with brine (2 ꢀ 8 mL) and
dried (Na2SO4). The mixture was concentrated under reduced
pressure, and the residue was dissolved in a mixture of THF and
H2O (1:1, 20 mL, v/v). NaIO4 (833 mg, 3.89 mmol) was added,
and the reaction mixture was stirred at room temperature for
2 h. Et2O (10 mL) was added, and the layers were separated. The
aqueous layer was extracted with Et2O (2 ꢀ 8 mL), and the
combined organic layers were washed with brine (2 ꢀ 10 mL)
and dried (Na2SO4). The mixture was concentrated under
reduced pressure to give the crude aldehyde (910 mg), which
was dissolved in CH3OH (5 mL). A 0.4 M solution of
1-yl)-2-vinyl-7-oxabicyclo[3.2.1]oct-2-ene (33). To
a stirred
solution of nucleoside 32 (50 mg, 0.08 mmol) in anhydrous
CH2Cl2 (1 mL) were added N,N-diisopropylethylamine (73 μL,
0.42 mmol) and 2-cyanoethyl N,N-diisopropylphosphoramido-
chloridite (56 μL, 0.25 mmol). The mixture was stirred for 21 h
and CH2Cl2 (2 mL) was added. The mixture was washed with a
saturated aqueous solution of NaHCO3 (2 mL). The aqueous
phase was extracted with CH2Cl2 (2 ꢀ 3 mL), and the combined
organic phases were dried (Na2SO4) and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography (0-2% CH3OH and 0.5% pyridine in
CH2Cl2) to give 33 (40 mg, 60%) as a white foam: Rf 0.80
(CH3OH-dichloromethane, 1:9 v/v); 31P NMR (121.5 MHz,
CDCl3) δ 149.7, 149.4; ESI MS m/z (817.3331 [M þ Na]þ,
C44H51N4O8P-Naþ calcd 817.3337).
Synthesis of 2(R/S)-(1R,5R,6R,8S)-2-Ethyl-8-hydroxy-1-
(hydroxymethyl)-6-(uracil-1-yl)-7-oxabicyclo[3.2.1]octane (19).
To a stirred solution of bicyclic nucleoside 20 (0.128 g, 0.44
mmol) in CH3OH (3 mL) was added PtO2 (0.053 g, 0.23 mmol),
and the mixture was stirred under hydrogen atmosphere for
24 h. The mixture was filtered through Celite, and the filtrate
was concentrated under reduced pressure. The residue was
purified by silica gel column chromatography (0-6% CH3OH
in CH2Cl2) to give 19 (as a mixture of diastereomers in 8:1 ratio,
90 mg, 70%) as a white foam: Rf 0.40 (CH3OH-dichloro-
methane, 1:9 v/v); 1H NMR (300 MHz, CD3OD) δ 8.55 (d,
1H, J = 8.1 Hz, H-6), 8.44 (d, 1H, J = 8.1 Hz, H-6), 5.72 (s, 1H,
H-10), 5.68 (s, 1H, H-10), 5.62 (d, 1H, J = 8.1 Hz, H-5), 5.60 (d,
1H, J = 8.1 Hz, H-5), 4.50 (d, 1H, J = 5.7 Hz, H-30), 4.37 (d, 1H,
J = 5.1 Hz, H-30), 3.95, 3.58 (AB, 2H, J = 11.7 Hz, H-50), 3.83,
3.51 (AB, 2H, J = 12.0 Hz, H-50), 2.34 (br s, 2H, 2ꢀH-20), 2.08-
1.14 (m, H-60, H-70, H-80, CH3CH2), 0.89-0.84 (m, 6H, 2 ꢀ
CH3); 13C NMR (major isomer) (75 MHz, CD3OD) δ 166.4 (C-
4), 150.9 (C-2), 141.6 (C-6), 99.4 (C-5), 88.2, 87.7 (C-10, C-40),
64.9 (C-30), 60.2 (C-50), 45.2 (C-20), 36.2, 23.6, 21.8, 19.8 (C-60, C-
70, C-80, CH3CH2), 10.4 (CH3); HRMALDI MS m/z (319.1271
[M þ Na]þ, C14H20N2O5-Naþ calcd 319.1265).
CeCl3 7H2O in CH3OH (3.24 mL, 1.30 mmol) and then NaBH4
3
(0.049 g, 1.29 mmol) were added, and the reaction mixture was
stirred at room temperature for 5 min. H2O (2 mL) was added,
and the mixture was extracted in Et2O (2 ꢀ 3 mL). The combined
organic layers were dried (Na2SO4) and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography (0-4% CH3OH in CH2Cl2) to give 36 (368 mg,
54%) as a white foam: Rf 0.60 (CH3OH-dichloromethane,
Synthesis of 2(R/S)-(1R,5R,6R,8S)-1-(4,40-Dimethoxytrityl-
oxymethyl)-2-ethyl-8-hydroxy-6-(uracil-1-yl)-7-oxabicyclo[3.2.1]-
octane (34). A solution of 19 (0.090 g, 0.30 mmol) in anhydrous
pyridine (1.5 mL) and anhydrous CH3CN (1.5 mL) was stirred at
room temperature and DMT-Cl (0.206 g, 0.61 mmol) was added.
The mixture was stirred for 23 h and then concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography (0-2.5% CH3OH and 0.5% pyridine in
CH2Cl2) to give 34 (as a mixture of diastereomers in a 8:1 ratio,
130 mg, 72%) as a foam: Rf 0.70 (CH3OH-dichloromethane, 1:9
v/v); 1H NMR (300 MHz, CDCl3) δ 9.07 (br s, 2H, 2 ꢀ NH), 8.29
(d, 1H, J = 8.1Hz, H-6), 8.17 (d, 1H, J = 8.1Hz, H-6), 7.46-7.15
(m, 18H, Ar), 6.88-6.82 (m, 8H, Ar), 5.78 (s, 1H, H-10), 5.73 (s,
1H, H-10), 5.32-5.23 (m, 2H, H-5), 4.52 (br s, 1H, H-30), 4.14 (br
s, 1H, H-30), 3.78 (s, 12H, OCH3), 3.60, 3.34 (AB, 2H, J = 10.8
Hz, H-50), 3.48, 3.39 (AB, 2H, J = 11.7 Hz, H-50), 2.45-2.40 (m,
H-20), 1.99-0.95 (m, H-60, H-70, H-80, CH3CH2), 0.87-0.72 (m,
CH3); 13C NMR (major isomer) (75 MHz, CDCl3) δ 163.7 (C-4),
158.8 (Ar), 150.3 (C-2), 144.5 (Ar), 140.9 (C-6), 135.6, 135.3,
130.2, 128.3, 128.2, 127.3, 127.2, 113.4 (Ar), 101.0 (C-5), 87.7,
87.5, 87.4 (C-10, C-40, CAr3), 67.4 (C-30), 62.5 (C-50), 55.4
(OCH3), 45.1 (C-20), 36.6, 23.5, 21.9, 19.9 (C-60, C-70, C-80,
CH3CH2), 11.2 (CH3); HRMALDI MS m/z (621.2587 [M þ
Na]þ, C35H38N2O7-Naþ calcd 621.2572).
1
1:9 v/v); H NMR (300 MHz, CDCl3) δ 8.93 (br s, 1H, NH),
8.17 (d, 1H, J = 8.1 Hz, H-6), 5.86 (m, 1H, H-70), 5.69-5.62 (m,
2H, H-5, H-10), 4.45 (d, 1H, J = 5.7 Hz, H-30), 4.29, 3.88 (AB, 2H,
J = 11.4 Hz, H-50), 4.03 (m, 2H, CH2OH), 2.58-2.52 (m, 1H, H-
80), 2.37-2.30 (m, 2H, H-80, H-20), 0.95 (s, 9H, SiC(CH3)3), 0.84 (s,
9H, SiC(CH3)3), 0.14 (s, 3H, SiCH3), 0.13 (s, 3H, SiCH3), 0.07 (s,
3H, SiCH3), 0.05 (s, 3H, SiCH3); 13C NMR (75 MHz, CDCl3) δ
163.5 (C-4), 150.2 (C-2), 140.4 (C-6), 138.0 (C-60), 129.1 (C-70),
101.2 (C-5), 89.2 (C-10), 83.5 (C-40), 65.5 (C-30), 64.0 (CH2OH),
59.8 (C-50), 44.5 (C-20), 27.9 (C-80), 26.1, 25.7 (SiC(CH3)3), 18.5,
18.0 (SiC(CH3)3), -4.5, -4.8, -5.1, -5.3 (SiCH3); HRMALDI
MS m/z (547.2621 [M þ Na]þ, C25H44N2O6Si2-Naþ calcd
547.2630).
Synthesis of (1R,5R,6R,8S)-2-Benzoyloxymethyl-8-(tert-butyl-
dimethylsilyloxy)-1-(tert-butyldimethylsilyloxymethyl)-6-(uracil-
1-yl)-7-oxabicyclo[3.2.1]oct-2-ene (37). Compound 36 (368 mg,
0.70 mmol) was dissolved in anhydrous pyridine (3 mL), and the
resulting mixture was stirred at 0 °C. Benzoyl chloride (0.114
mL, 0.98 mmol) was added, and the reaction mixture was stirred
at room temperature for 1 h. The reaction mixture was concen-
trated under pressure, and the residue was dissolved in EtOAc
(5 mL) and washed with a saturated aqueous solution of
NaHCO3 (2 mL). The aqueous phase was extracted with EtOAc
(2 ꢀ 2 mL), and the combined organic phases were dried
(Na2SO4) and concentrated under reduced pressure. The residue
Synthesis of 2(R/S)-(1R,5R,6R,8S)-8-(2-Cyanoethoxy(diiso-
propylamino)phosphinoxy)-1-(4,40-dimethoxytrityloxymethyl)-2-
ethyl-6-(uracil-1-yl)-7-oxabicyclo[3.2.1]octane (35). To a stirred
solution of compound 34 (125 mg, 0.21 mmol) in anhydrous
CH2Cl2 (1.5 mL) were added N,N-diisopropylethylamine
J. Org. Chem. Vol. 74, No. 17, 2009 6767

Kumar et al.
JOCArticle
was purified by silica gel column chromatography (EtOAc-
petroleum ether, 1:4 v/v) to give 37 (232 mg, 53%) as a white
foam: Rf 0.75 (EtOAc-petroleum ether, 3:1 v/v); 1H NMR (300
MHz, CDCl3) δ 9.61 (br s, 1H, NH), 8.21 (d, 1H, J = 7.8 Hz, H-
6), 8.12 (m, 2H, Ar), 7.58 (m, 1H, Ar), 7.44 (m, 2H, Ar), 6.09
(m, 1H, H-70), 5.69-5.64 (m, 2H, H-5, H-10), 4.80, 4.71 (AB, 2H,
J = 12.9 Hz, CH2OBz), 4.48 (d, 1H, J = 5.7 Hz, H-30), 4.16, 3.89
(AB, 2H, J = 11.4 Hz, H-50), 2.62 (m, 1H, H-80), 2.42-2.35 (m,
2H, H-80, H-20), 0.90 (s, 9H, SiC(CH3)3), 0.81 (s, 9H, SiC(CH3-
)3), 0.08 (s, 3H, SiCH3), 0.07 (s, 3H, SiCH3), 0.05 (s, 3H, SiCH3),
0.05 (s, 3H, SiCH3); 13C NMR (75 MHz, CDCl3) δ 193.8
(COAr), 166.0 (C-4), 150.3 (C-2), 140.6 (C-6), 133.6, 133.2,
130.3, 129.7, 128.5 (Ar, C-60), 101.1 (C-5), 89.2 (C-10), 83.3
(C-40), 65.3, 65.1 (C-30, CH2OBz), 59.8 (C-50), 44.4 (C-20), 28.0
(C-80), 26.0, 25.6 (SiC(CH3)3), 18.5, 17.9 (SiC(CH3)3), -4.5,
-5.0, -5.2, -5.4 (SiCH3); HRMALDI MS m/z (651.2898 [M þ
Na]þ, C32H48N2O7Si2-Naþ calcd 651.2892).
was stirred for 30 h and then CH2Cl2 (3 mL) added. The mixture
was washed with a saturated aqueous solution of NaHCO3
(2 mL). The aqueous phase was extracted with CH2Cl2 (2 ꢀ
3 mL), and the combined organic phases were dried (Na2SO4)
and concentrated under reduced pressure. The residue was
purified by silica gel column chromatography (0-2.5% CH3OH
and 0.5% pyridine in CH2Cl2) to give 40 (70 mg, 45%) as a white
foam: Rf 0.80 (CH3OH-dichloromethane, 7.5:92.5 v/v); 31P
NMR (121.5 MHz, CDCl3) δ 150.0, 149.5; ESI MS m/z
(925.3543 [M þ Na]þ, C50H55N4O10P-Naþ calcd 925.3548).
Synthesis of (1R,7S,9R,10R,12S)-12-(tert-Butyldimethylilyl-
oxy)-1-(tert-butyldimethylsilyloxymethyl)-5,6-bis(ethoxycarbo-
nyl)-10-(uracil-1-yl)-11-oxatricyclo[7.2.1.02.7]dodeca-2.5-diene
(41). A solution of the nucleoside 31 (160 mg, 0.31 mmol) in
anhydrous toluene (2 mL) was added diethyl acetylendicarbox-
ylate (0.490 mL, 3.07 mmol), and the solution was stirred in a
microwave reactor at 150 °C for 2 h. The mixture was con-
cenrated under reduced pressure, and the residue was purified by
silica gel column chromatography (0-2.5% CH3OH in CH2Cl2)
to give 41 (160 mg, 75%) as a white foam: Rf 0.60 (MeOH-
dichloromethane, 1:19 v/v); 1H NMR (400 MHz, CDCl3) δ 8.62
(s, 1H, NH), 8.11 (d, 1H, J = 8.0 Hz, H-6), 5.87 (d, 1H, J = 2.8
Hz, CHdC), 5.63 (d, 1H, J = 8.0 Hz, H-5), 5.50 (s, 1H, H-10),
4.41 (d, 1H, J = 4.0 Hz, H-30), 4.35-4.15 (m, 5H, 2 ꢀ CH3CH2,
H-50), 4.08 (m, 1H, H-70), 3.68 (d, 1H, J = 11.6 Hz, H-50), 3.20
(ddd, 1H, J = 4.4, 8.8, 23.6 Hz, dC-CH2a-Cd), 2.92 (ddd, 1H,
J = 2.4, 11.2, 23.6 Hz, dC-CH2b-Cd), 2.42-2.34 (m, 2H, H-
80b, H-20), 1.79 (m, 1H, H-80a), 1.38-1.20 (m, 6H, 2 ꢀ CH3CH2),
0.95 (s, 9H, SiC(CH3)3), 0.88 (s, 9H, SiC(CH3)3), 0.22 (s, 3H,
SiCH3), 0.19 (s, 3H, SiCH3) 0.10 (s, 6H, SiCH3); 13C NMR (100
MHz, CDCl3) δ 168.2, 166.6 (CO2Et), 163.3 (C-4), 150.1 (C-2),
140.1, 139.8 (C-6, C-60), 135.6, 126.4 (O2CCdCCO2), 121.4
(CHdC), 101.0 (C-5), 90.6 (C-10), 83.8 (C-40), 68.2 (C-30), 61.3
(CH3CH2), 60.7 (C-50), 43.2 (C-20), 30.5, 30.0 (C-70, C-80),
27.6 (dC-CH2-Cd), 26.2, 25.7 (SiC(CH3)3), 18.6, 17.9 (SiC-
(CH3)3), 14.2 (CH3CH2) -4.8, -4.9, -5.1, -5.3 (SiCH3);
HRMALDI MS m/z (713.3250 [M þ Na]þ, C34H54N2O9Si2-
Naþ calcd 713.3260).
Synthesis of Oligodeoxynucleotides. Oligonucleotide synth-
esis was carried out on an automated DNA synthesizer follow-
ing the phosphoramidite approach. Synthesis of oligonu-
cleotides 42-50 was performed on a 0.2 μmol scale by using
the amidites 29, 33, 35, and 40 as well as the corresponding
commercial 2-cyanoethyl phosphoramidites of the natural 20-
deoxynucleosides. The synthesis followed the regular protocol
for the DNA synthesizer. However, for 29, 33, 35, and 40, a
prolonged coupling time of 30 min was used, and for 29 and 40,
pyridinium hydrochloride was used as the activator instead of
1H-tetrazole in all other cases. Coupling yields for all 2-cya-
noethyl phosphoramidites were >98%. The 50-O-DMT-pro-
tected oligonucleotides were removed from the universal solid
support by treatment with concentrated ammonia at 55 °C for
20 h. The oligonucleotides were purified by reversed-phase
HPLC on a Waters 600 system using a Xterra prep MS C18;
10 μm; 7.8 ꢀ 150 mm column; gradient of buffer (0.05 M
triethylammonium acetate) in 75% CH3CN(aq); 0-70% buffer,
38 min; 70-100% buffer, 7 min; 100% buffer, 10 min. All
fractions containing 50-O-DMT-protected oligonucleotide
(retention time 20-30 min) were collected and concentrated. The
products were detritylated by treatment with an 80% aqueous
solution of acetic acid for 20 min, and finally isolated by pre-
cipitation with ethanol at -18 °C overnight. MALDI-MS [M -
H]- gave the following results (found/calcd): 43 (2810.2/2811.9);
44 (2925.9/2928.0); 45 (2805.6/2807.9); 46 (2919.0/2916.0); 47
(2804.0/2803.9); 48 (2907.2/2904.0); 49 (2811.8/2807.9); 50
(2918.9/2916.0); 53 (4755.0/4758.2); 54 (4873.3/4874.2).
Synthesis of (1R,5R,6R,8S)-2-Benzoyloxymethyl-8-hydroxy-
1-hydroxymethyl-6-(uracil-1-yl)-7-oxabicyclo[3.2.1]oct-2-ene (38).
To a stirred solution of compound 37 (232 mg, 0.37 mmol) in
anhydrous CH3CN (2 mL) were added KF (0.215 g, 3.70 mmol)
and 18-crown ether-6 (390 mg, 1.48 mmol), and the solution was
stirred in a microwave reactor at 100 °C for 1 h. The mixture was
concentrated under reduced pressure, and the residue was purified
by silica gel column chromatography (0-5%CH3OH in CH2Cl2)
to give 38 (61 mg, 41%) as a white foam: Rf 0.45 (MeOH-
dichloromethane, 1:9 v/v); 1H NMR (300 MHz, (DMSO-d6)
δ 11.29 (s, 1H, NH), 8.12 (d, 1H, J = 8.1 Hz, H-6), 7.95 (m,
2H, Ar), 7.67 (m, 1H, Ar), 7.53 (m, 2H, Ar), 6.03 (m, 1H, H-70),
5.58 (d, 1H, J = 8.1 Hz, H-5), 5.48 (s, 1H, H-10), 5.30 (d, 1H, J =
4.5 Hz, 30-OH), 5.23 (t, 1H, J = 4.2 Hz, 50-OH), 4.78, 4.71 (AB,
2H, J = 12.6 Hz, CH2OBz), 4.36 (t, 1H, J = 4.5 Hz, H-30), 3.94
(dd, 1H, J=4.2, 12.3Hz, H-50), 3.76 (dd, 1H, J = 4.2, 12.3 Hz, H-
50), 2.57 (m, 1H, H-80), 2.37 (m, 1H, H-20), 2.18 (m, 1H, H-80); 13C
NMR (75 MHz, DMSO-d6) δ 165.1 (COAr), 163.3 (C-4), 150.2
(C-2), 140.1 (C-6), 133.4, 133.2, 131.5, 129.6, 129.1, 128.8 (Ar, C-
60, C-70), 100.4 (C-5), 88.3(C-10), 82.4 (C-40), 64.7, 64.2, 57.7 (C-30,
CH2OBz, C-50), 43.2 (C-20), 27.5 (C-80); HRMALDI MS m/z
(423.1172 [M þ Na]þ, C20H20N2O7-Naþ calcd 423.1163).
Synthesis of (1R,5R,6R,8S)-2-Benzoyloxymethyl-1-(4,40-di-
methoxytrityloxymethyl)-8-hydroxy-6-(uracil-1-yl)-7-oxabicyclo-
[3.2.1]oct-2-ene (39). DMT-Cl (0.155 g, 0.46 mmol) was added to
a stirred solution of 38 (0.061 g, 0.15 mmol) in anhydrous
pyridine (1 mL) and anhydrous CH3CN (1 mL), and the mixture
was stirred for 24 h. The mixture was concentrated under
reduced pressure, and the residue was purified by silica gel
column chromatography (0-4% CH3OH and 0.5% pyridine
in CH2Cl2) to give 39 (82 mg, 77%) as a foam: Rf 0.50 (CH3OH-
dichloromethane, 7.5:92.5 v/v); 1H NMR (300 MHz, CDCl3) δ
9.10 (s, 1H, NH), 8.21 (d, 1H, J = 8.1 Hz, H-6), 7.79 (m, 2H, Ar),
7.57 (m, 1H, Ar), 7.42-7.16 (m, 11H, Ar), 6.84-6.76 (m, 4H, Ar),
6.13 (m, 1H, H-70), 5.67 (s, 1H, H-10), 5.41 (d, 1H, J = 8.1 Hz, H-
5), 4.70-4.66 (m, 2H, CH2OBz, H-30), 4.56 (d, 1H, J = 12.6 Hz,
CH2OBz), 3.80-3.74 (m, 7H, OCH3, H-50), 3.68 (d, 1H, J = 10.8
Hz, H-50), 2.70-2.42 (m, 3H, H-80, H-20); 13C NMR (75 MHz,
CDCl3) δ 165.8 (COAr), 163.6 (C-4), 158.8 (Ar), 150.2 (C-2), 144.3
(Ar), 140.4 (C-6), 135.1, 135.0, 133.5, 133.1, 130.2, 130.1, 129.7,
129.6, 128.5, 128.1, 127.3, 113.3 (Ar, C-60, C-70), 101.4 (C-5), 89.4
(C-10), 87.5, 82.2 (C-40, CAr3), 67.0, 65.0, 60.3 (C-30, CH2OBz, C-
50), 55.3 (OCH3), 43.8 (C-20), 27.4 (C-80); HRMALDI MS m/z
(725.2468 [M þ Na]þ, C41H38N2O9-Naþ calcd 725.2470).
Synthesis of (1R,5R,6R,8S)-2-Benzoyloxymethyl-8-(2-cyano-
ethoxy(diisopropylamino)phosphinoxy)-1-(4,40-dimethoxytrityl-
oxymethyl)-6-(uracil-1-yl)-7-oxabicyclo[3.2.1]oct-2-ene (40). To
a stirred solution of compound 39 (0.122 g, 0.17 mmol) in
anhydrous CH2Cl2 (1.5 mL) were added N,N-diisopropylethy-
lamine (0.180 mL, 1.04 mmol) and 2-cyanoethyl N,N-diisopro-
pylphosphoramidochloridite (116 μL, 0.52 mmol). The mixture
Melting Experiments. UV melting experiments were carried
out on a UV spectrometer. For the duplex experiments, the
6768 J. Org. Chem. Vol. 74, No. 17, 2009

Kumar et al.
JOCArticle
samples were dissolved in a medium salt buffer containing
Na2HPO4 (5 mM), NaH2PO4 (10 mM), NaCl (100 mM), and
EDTA (0.1 mM), pH 7.0 with 1.5 μM concentrations of the two
complementary sequences. The increase in absorbance at
260 nm as a function of time was recorded while the temperature
was increased linearly from 10 to 70 °C at a rate of 0.5 °C/min by
means of a Peltier temperature programmer. The melting tem-
perature was determined as the local maximum of the first
derivatives of the absorbance versus temperature curve. All
melting curves were found to be reversible. All determinations
are averages of duplicates. For the triplex experiments, a buffer
containing 10 mM sodium cacodylate, 150 mM NaCl, 10 mM
MgCl2, pH 6.0 using 1.0 μM concentration of the target duplex
and 1.5 μM concentrations of the TFO sequences.
CD Spectroscopy. CD spectra were recorded in the same
medium salt buffer as in the UV melting experiments with
3.0 μM concentrations of the two complementary sequences.
Quantum Mechanical Calculation. Ab initio calculations were
carried out using the Gaussian 03 program.37 Boat and chair
conformations of the six-membered ring of the unprotected
form of 25 were generated in a two-step procedure. First, a
constrained geometry optimization was performed with con-
straints ensuring idealized chair and boat conformations. The 5-
hydroxy group was constrained in a trans geometry, and the
30-hydroxy group was rotated so as not to clash with the six-
membered ring. Second, the two geometries obtained in the first
step were subjected to free geometry optimizations. Geometry
optimizations were carried out using Hartree-Fock theory
(HF) with the 6-31G* basis set. Single-point energies of opti-
mized conformations were determined using second-order Møl-
ler-Plesset theory (MP2) with the cc-pVTZ basis set.
(37) Gaussian 03, Revision D.02: Frisch, M. J.; Trucks, G. W.; Schlegel,
H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A.,
Jr.; Vreven, T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.;
Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.;
Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda,
R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.;
Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken, V.;
Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.;
Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.;
Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski,
V. G.; Dapprich, S.; , Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D.
K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.;
Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.;
Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith,
T.; M. A.; Al-Laham; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill,
P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A.;
Gaussian, Inc., Wallingford, CT, 2004.
Acknowledgment. The Danish National Research Foun-
dation, the Danish Natural Science Research Council, the
Danish Center for Scientific Computing, and Møllerens
Fond are thanked for financial support. Birthe Haack is
thanked for synthetic and technical assistance.
Supporting Information Available: General introduction to
the Experimental Section. Selected NMR spectra. Melting
curves for the triplex study. This material is available free of
charge via the Internet at http://pubs.acs.org.
J. Org. Chem. Vol. 74, No. 17, 2009 6769