Analogues of a locked nucleic acid with three-carbon 2′,4′- linkages: Synthesis by ring-closing metathesis and influence on nucleic acid duplex stability and structure

Article abstract of DOI:10.1021/jo061225g

(Chemical Equation Presented) Two bicyclic 2′-deoxynucleoside analogues containing a saturated and an unsaturated three-carbon 2′,4′-linkage, respectively, have been synthesized using a ring-closing metathesis-based linear strategy starting from uridine.

Full text of DOI:10.1021/jo061225g

Analogues of a Locked Nucleic Acid with Three-Carbon
2′,4′-Linkages: Synthesis by Ring-Closing Metathesis and Influence
on Nucleic Acid Duplex Stability and Structure
Nanna Albæk, Michael Petersen, and Poul Nielsen*
Nucleic Acid Center,† Department of Chemistry, UniVersity of Southern Denmark,
5230 Odense M, Denmark
pon@chem.sdu.dk
ReceiVed June 14, 2006
Two bicyclic 2′-deoxynucleoside analogues containing a saturated and an unsaturated three-carbon 2′,4′-
linkage, respectively, have been synthesized using a ring-closing metathesis-based linear strategy starting
from uridine. Both analogues have been incorporated into oligodeoxynucleotide sequences and increased
the stability of DNA:RNA hybrid duplexes (∆Tm ∼ 2.5-5.0 °C per modification) and decreased the
stability of dsDNA duplexes (∆Tm ∼ 2.5-1.0 °C per modification). CD spectroscopy revealed that the
bicyclic nucleosides induced formation of A-type-like duplexes albeit to a lesser degree than found for
locked nucleic acid (LNA) monomers. From the CD data and UV melting analysis, we propose that the
2′-oxygen atom of the bicyclic moiety is essential for the formation of stabilized A-type-like dsDNA but
not for the formation of a stabilized A-type DNA:RNA hybrid.
Introduction
restriction of the carbohydrate moieties into the N-type (or C-3′-
endo) conformations (Figure 1) leading to the formation of
A-type or A-type-like duplexes.3-7 The prime example is LNA
(locked nucleic acid), defined as a nucleic acid containing one
or several replacements of the natural nucleosides with the
bicyclic nucleosides 1.3 In 1, a 2′,4′-connection locks the sugar
The molecular recognition between two complementary
nucleic acid sequences forming a duplex structure is a key
subject for investigations in chemical biology and biomimetic
chemistry.1 The search for small chemical perturbations leading
to an increased thermal stability of a duplex without compro-
mising the nature and selectivity of the Watson-Crick base
pairing has been very successful by the introduction of confor-
mationally restricted nucleoside building blocks.2 The most
dramatic increases in stability have been obtained with a strong
(2) (a) Herdewijn, P. Biochim. Biophys. Acta 1999, 1489, 167-179. (b)
Meldgaard, M.; Wengel, J. J. Chem. Soc., Perkin Trans. 1 2000, 3539-
3554. (c) Leumann, C. J. Bioorg. Med. Chem. 2002, 10, 841-854.
(3) (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.
† Nucleic Acid Center is funded by the Danish National Research Foundation
for studies on nucleic acid chemical biology.
(1) (a) Kool, E. T. Chem. ReV. 1997, 97, 1473-1487. (b) Hill, D. J.;
Mio, M. J.; Prince, R. B.; Hughes, T. S.; Moore, J. S. Chem. ReV. 2001,
101, 3893-4011. (c) Kurreck, J. Eur. J. Biochem. 2003, 270, 1628-1644.
10.1021/jo061225g CCC: $33.50 © 2006 American Chemical Society
Published on Web 09/08/2006
J. Org. Chem. 2006, 71, 7731-7740
7731

Albæk et al.
crystallography,12 and it was shown that the LNA nucleosides
drive their neighboring 2′-deoxynucleosides into N-type con-
formations leading to overall A-type or A-type-like duplexes
even with only three LNA nucleosides in a nonamer duplex.11
The features of LNA have led to a number of closely related
analogues including stereoisomers of LNA13,14 and analogues
in which the 2′,4′-bridge has been altered (Figure 1). Both the
sulfur analogue, 2, and the amino analogue, 3, give virtually
the same increases in thermal stability as the parent oxo-
compound 1 (∆Tm’s for each modification in an oligodeoxy-
nucleotide (ODN) range between +3 and +5 °C with DNA
complements and between +4 and +8 °C with RNA comple-
ments).15 The 2′-nitrogen of amino-LNA 3 has been used for
attaching other chemical groups into the stabilized A-type
duplex.16 Nucleosides with three atoms in the 2′,4′-linkage have
also been reported, and 4, named ENA, displays a stabilization
of nucleic acid duplexes formed with complementary RNA that
is equal or slightly lower than that found for LNA (∆Tm’s
between +3.5 and +5.5 °C in a mixed-sequence context).17,18
The closely related analogue 5 where the additional methylene
group was inserted between the 2′-carbon and the oxygen
displayed somewhat smaller increases compared to LNA
(∆Tm’s between +2 and +3 °C with RNA complements).19
With complementary DNA, 4 shows only a small stabilization
(∆Tm’s between +0.5 and +2 °C)17,18 whereas 5 shows almost
unchanged melting temperatures compared to a native DNA
duplex (∆Tm’s between -0.5 and +1 °C).19 Two nucleosides
with a four-atom 2′,4′-linkage have also been synthesized,
and in the case of 6, a destabilization of duplexes with both
DNA and RNA complements was seen (one measured ∆Tm of
-2.0 °C with a DNA and of -0.5 °C with an RNA complement,
respectively).18 Exchanging one carbon atom with oxygen led
to 7, which recently showed a moderate stabilization of a duplex
with complementary RNA (∆Tm’s between +1 and +2 °C) and
FIGURE 1. (a) Low-energy conformations of 2′-deoxynucleosides.
(b) Bicyclic nucleosides with 2′,4′-linkages and locked N-type con-
formations.
conformation of the building block into a perfect N-type
conformation (Figure 1).3 Unprecedented stability of duplexes
has been observed with short LNA oligonucleotides containing
entirely LNA nucleosides or mixmers of both LNA and
unmodified ribo- or 2′-deoxyribonucleosides with complemen-
tary sequences being RNA, DNA, or other LNA sequences.3
The increase in thermal stability compared to unmodified DNA
duplexes ranges from +3 to +8 °C for each incorporation of
an LNA nucleoside.3 Owing to this high potential in the
recognition of complementary natural DNA and RNA se-
quences, LNA sequences are now in use as diagnostic materials
and under study as therapeutic compounds following the
antisense approach8 or as LNAzymes.9 The intriguing properties
of LNA have inspired structural research by NMR10,11 and X-ray
(10) (a) Bondensgaard, K.; Petersen, M.; Singh, S. K.; Rajwanshi, V.
K.; Kumar, R.; Wengel, J.; Jacobsen, J. P. Chem.-Eur. J. 2000, 6, 2687-
2695. (b) Nielsen, K. E.; Rasmussen, J.; Kumar, R.; Wengel, J.; Jacobsen,
J. P. Bioconjugate Chem. 2004, 15, 449-457.
(11) Petersen, M.; Bondensgaard, K.; Wengel, J.; Jacobsen, J. P. J. Am.
Chem. Soc. 2002, 124, 5974-5982.
(12) Egli, M.; Minasov, G.; Teplova, M.; Kumar, R.; Wengel, J. Chem.
Commun. 2001, 651-652.
(13) (a) Rajwanshi, V. K.; Håkansson, A. E.; Sørensen, M. D.; Pitsch,
S.; Singh, S. K.; Kumar, R.; Nielsen, P.; Wengel, J. Angew. Chem., Int.
Ed. 2000, 39, 1656-1659. (b) Nielsen, P.; Christensen, N. K.; Dalskov, J.
K. Chem.-Eur. J. 2002, 8, 712-722. (c) Christensen, N. K.; Bryld, T.;
Sørensen, M. D.; Arar, K.; Wengel, J.; Nielsen, P. Chem. Commun. 2004,
282-283.
(14) R-L-LNA can be incorporated into mixmers with 2′-deoxynucleo-
sides leading to duplexes with complementary DNA and RNA that are only
slightly less stabilized than those for the corresponding LNA sequences:
Sørensen, M. D.; Kværnø, L.; Bryld, T.; Håkansson, A. E.; Verbeure, B.;
Gaubert, G.; Herdewijn, P.; Wengel, J. J. Am. Chem. Soc. 2002, 124, 2164-
2176.
(15) (a) Singh, S. K.; Kumar, R.; Wengel, J. J. Org. Chem. 1998, 63,
10035-10039. (b) Kumar, R.; Singh, S. K.; Koshkin, A. A.; Rajwanshi,
V. K.; Meldgaard, M.; Wengel, J. Bioorg. Med. Chem. Lett. 1998, 8, 2219-
2222.
(16) (a) Sørensen, M. D.; Petersen, M.; Wengel, J. Chem. Commun. 2003,
2130-2131. (b) Hrdlicka, P. J.; Babu, B. R.; Sørensen, M. D.; Harrit, N.;
Wengel, J. J. Am. Chem. Soc. 2005, 127, 13293-13299.
(17) Morita, K.; Hasegawa, C.; Kaneko, M.; Tsutsumi, S.; Sone, J.;
Ishikawa, T.; Imanishi, T.; Koizumi, M. Bioorg. Med. Chem. Lett. 2002,
12, 73-76.
(18) Morita, K.; Takagi, M.; Hasegawa, C.; Kaneko, M.; Tsutsumi, S.;
Sone, J.; Ishikawa, T.; Imanishi, T.; Koizumi, M. Bioorg. Med. Chem. 2003,
11, 2211-2226.
(19) (a) Wang, G.; Girardet, J.-L.; Gunic, E. Tetrahedron 1999, 55,
7707-7724. (b) Wang, G.; Gunic, E.; Girardet, J.-L.; Stoisavljevic, V.
Bioorg. Med. Chem. Lett. 1999, 9, 1147-1150.
(4) Other bicyclic nucleosides restricted in N-type conformations have
been introduced into nucleic acid duplexes: (a) Altmann, K.-H.; Kesselring,
R.; Francotte, E.; Rihs, G. Tetrahedron Lett. 1994, 35, 2331-2334. (b)
Wang, G.; Stoisavljevic, V. Nucleosides, Nucleotides Nucleic Acids 2000,
19, 1413-1425. (c) Pradeepkumar, P. I.; Cheruku, P.; Plashkevych, O.;
Acharya, P.; Gohil, S.; Chattopadhyaya, J. J. Am. Chem. Soc. 2004, 126,
11484-11499.
(5) A large series of 2′-O-alkyl and 2′-F-ribonucleoside analogues have
been examined and in general have been found to adopt N-type conforma-
tions and in oligonucleotides have been found to lead to an increased affinity
for complementary RNA: Manoharan, M. Biochim. Biophys. Acta 1999,
1489, 117-130.
(6) For other N-type-mimicking nucleic acid analogues leading to in-
creased RNA recognition, see: (a) Hendrix, C.; Rosemeyer, H.; Verheggen,
I.; Seela, F.; Van Aerschot, A.; Herdewijn, P. Chem.-Eur. J. 1997, 3, 110-
120. (b) Gryaznov, S. M. Biochim. Biophys. Acta 1999, 1489, 131-140.
(7) For nucleic acid analogues with strong conformational restriction in
S-type or E-type conformations using other bi- or tricyclic nucleosides,
see: (a) Tarko¨y, M.; Bolli, M.; Schweizer, B.; Leumann, C. HelV. Chim.
Acta 1993, 76, 481-510. (b) Altmann, K.-H.; Imwinkelried, R.; Kesselring,
R.; Rihs, G. Tetrahedron Lett. 1994, 35, 7625-7628. (c) Nielsen, P.;
Pfundheller, H. M.; Wengel, J. Chem. Commun. 1997, 825-826. (d)
Christensen, N. K.; Petersen, M.; Nielsen, P.; Jacobsen, J. P.; Olsen, C. E.;
Wengel, J. J. Am. Chem. Soc. 1998, 120, 5458-5463. (e) Kværnø, L.;
Wightman, R. H.; Wengel, J. J. Org. Chem. 2001, 66, 5106-5112. (f)
Renneberg, D.; Leumann, C. J. J. Am. Chem. Soc. 2002, 124, 5993-6002.
(g) Obika, S.; Sekiguchi, M.; Somjing, R.; Imanishi, T. Angew. Chem., Int.
Ed. 2005, 44, 1944-1947.
(8) Petersen, M.; Wengel, J. Trends Biotechnol. 2003, 21, 74-81.
(9) Vester, B.; Lundberg, L. B.; Sørensen, M. D.; Babu, B. R.;
Douthwaite, S.; Wengel, J. J. Am. Chem. Soc. 2002, 124, 13682-13683.
7732 J. Org. Chem., Vol. 71, No. 20, 2006

Analogues of a Nucleic Acid with Three-Carbon Linkages
a corresponding destabilization with complementary DNA
(∆Tm’s between -1 and -2 °C).20
possible route, and we decided to start with the deoxygenative
introduction of the 2′-C-allyl group. The protecting group most
often used for the 3′- and 5′-hydroxy groups in ribonucleosides
while introducing the 2′-C-allyl group28 or a plethora of 2′-O-
alkyl groups5 is the elegantly selective bidentate TIPDS group
developed by Markiewicz.30 However, we decided to use the
less commonly used selective protection of the same hydroxy
groups with the tert-butyldimethylsilyl (TBS) group as reported
by Hakimelahi et al.31 This allows us to perform the same
desired transformation in the 2′-position,29 and more importantly,
it subsequently allows a selective acidic deprotection of the 5′-
position.23 Thus, the 3′-O-TBS group can be maintained as a
permanent protection during the entire synthesis. We expected
that the 4′-C-vinyl group could be introduced on the 5′-O-
unprotected nucleoside through a series of steps involving
oxidation, aldol condensation to introduce a hydroxymethyl
group,3,19,32 selective protection,3,23,27,32 oxidation, and a Wittig
reaction.18,23,27 The nucleoside would subsequently be set up to
combine the allyl and the vinyl groups in an RCM reaction,
leading after deprotection to 9 (base ) U) and after hydrogena-
tion to 8 (base ) U).
The interesting carbocyclic analogue of LNA/ENA, 8 (base
) T), was mentioned briefly in a review almost a decade ago.21
Therein, no synthetic details and only one melting temperature
were given claiming a decrease in thermal stability of -2.3 °C
for one central incorporation of 8 in a 17-mer ODN against a
complementary RNA sequence.21 In terms of the results obtained
with other LNA analogues, especially 4 and 5 with an equal
length of the 2′,4′-linkage, this result seems somewhat surprising,
and we decided to synthesize 8 to explore whether a direct
correlation between the constitution of the 2′,4′-linkage in the
bicyclic nucleoside, including the presence and position of any
oxygen atom, and the influence in thermal stability of duplexes
can be revealed. We here present the synthesis of 8 using a
ring-closing metathesis-based strategy. Besides 8, this strategy
gives access to the equally interesting unsaturated bicyclic
nucleoside 9. Both compounds, analyzed with uracil as the
nucleobase, enlighten the scope for conformational restriction
and present additional data on the importance of the 2′-oxygen
atom in nucleic acid duplexes.
The 3′,5′-di-O-TBS-protected uridine 10 (Scheme 1) was
synthesized from uridine in one step using the literature
method.31 The method is robust leading to the product 10 in
90% isolated yield after a chromatographic purification to
remove small amounts of the 2′,5′-protected isomer. Compound
10 was in two steps converted to the 2′-C-allyl derivative 1229
in 51% overall yield using a 2′-O-phenoxythiocarbonyl inter-
mediate 11 and a radical reaction with allyltributyltin.28,29 The
removal of the 5′-O-TBS group was achieved most selectively
in a slow treatment with 80% acetic acid at room temperature23
over 3 days giving 13 in 75% yield. Attempts to shorten the
reaction time by heating the reaction mixture or using other
acids resulted in removal of both TBS groups. The primary
hydroxy group in 13 was oxidized with Dess-Martin periodi-
nane,33 and the resulting aldehyde was reacted with formalde-
hyde in an aldol condensation. After reduction with sodium
borohydride, the diol 14 was isolated in 52% overall yield from
13. This method for introducing a 4′-hydroxymethyl group has
been previously reported on a range of nucleoside substrates
usually giving comparable yields.32 A protection of the 5′-
hydroxy group was necessary to allow the introduction of a 4′-
C-vinyl group. However, in all similar molecules, it has been
shown that the 4′-C-hydroxymethyl group is the more reactive
of the two primary hydroxy groups.23,27,32 Eventually, a three-
step procedure was devised, where protection of the 4′-hydroxy-
methyl was followed by protection of the 5′-hydroxy group and
finally by a removal of the 4′-hydroxymethyl protecting group.
Different approaches were attempted using a DMT, a TBS, or
a benzoyl group in the first protection step. In our hands,
benzoylation followed by purification, TBS protection, and
deprotection of the benzoyl group gave the best result. Obvi-
ously, a resulting nucleoside with TBS groups in both the 3′-O
Results
Chemical Synthesis. As mentioned above, we planned to
introduce the additional 2′,4′-bridge into the desired nucleosides
via a ring-closing metathesis (RCM) reaction. This type of
reaction has been successfully used on nucleoside and nucleotide
substrates by us22,23 and others24 for making varying sizes of
rings, including a bicyclic nucleoside with a conformationally
restricting 3′,4′-linkage.25 The RCM transformation leading to
our target nucleosides 8 and 9 requires a 2′-deoxynucleoside
substrate with a vinyl group in the 4′-C position and an allyl
group in the 2′-C position. Because efficient methods for
introducing both of these two alkyl groups into nucleosides
(though never in the same molecule) have been presented in
the literature,23,26-29 we decided to follow a linear synthetic
strategy starting from uridine. Hence, this should be the shortest
(20) Hari, Y.; Obika, S.; Ohnishi, R.; Eguchi, K.; Osaki, T.; Ohishi, H.;
Imanishi, T. Bioorg. Med. Chem. 2006, 14, 1029-1038.
(21) Freier, S. M.; Altmann, K.-H. Nucleic Acids Res. 1997, 25, 4429-
4443.
(22) (a) Sørensen, A. M.; Nielsen, P. Org. Lett. 2000, 2, 4217-4219.
(b) Ravn, J.; Nielsen, P. J. Chem. Soc., Perkin Trans. 1 2001, 985-993.
(c) Albæk, N.; Ravn, J.; Freitag, M.; Thomasen, H.; Christensen, N. K.;
Petersen, M.; Nielsen, P. Nucleosides, Nucleotides Nucleic Acids 2003, 22,
723-725. (d) Børsting, P.; Freitag, M.; Nielsen, P. Tetrahedron 2004, 60,
10955-10966. (e) Børsting, P.; Christensen, M. S.; Steffansen, S. I.; Nielsen,
P. Tetrahedron 2006, 62, 1139-1149.
(23) Kirchhoff, C.; Nielsen, P. Tetrahedron Lett. 2003, 44, 6475-6478.
(24) (a) Montembault, M.; Bourgougnon, N.; Lebreton, J. Tetrahedron
Lett. 2002, 43, 8091-8094. (b) Chen, X.; Wiemer, D. F. J. Org. Chem.
2003, 68, 6597-6604. (c) Gillaizeau, I.; Lagoja, I. M.; Nolan, S. P.;
Aucagne, V.; Rozenski, J.; Herdewijn, P.; Agrofoglio, L. A. Eur. J. Org.
Chem. 2003, 666-671. (d) Ewing, D. F.; Glac¸on, V.; Mackenzie, G.; Postel,
D.; Len, C. Tetrahedron 2003, 59, 941-945. (e) Busca, P.; Etheve-
Quelquejeu, M.; Vale´ry, J.-M. Tetrahedron Lett. 2003, 44, 9131-9134. (f)
Fang, Z.; Hong, J. H. Org. Lett. 2004, 6, 993-995.
(25) Freitag, M.; Thomasen, H.; Christensen, N. K.; Petersen, M.; Nielsen,
P. Tetrahedron 2004, 60, 3775-3786.
(30) Markiewicz, W. T. J. Chem. Res. (S) 1979, 24-25.
(31) Hakimelahi, G. H.; Proba, Z. A.; Ogilvie, K. K. Can. J. Chem. 1982,
60, 1106-1113.
(26) Sugimoto, I.; Shuto, S.; Matsuda, A. J. Org. Chem. 1999, 64, 7153-
7157.
(32) (a) Jones, G. H.; Taniguchi, M.; Tegg, D.; Moffatt, J. G. J. Org.
Chem. 1979, 44, 1309-1317. (b) O-Yang, C.; Wu, H. Y.; Fraser-Smith, E.
B.; Walker, K. A. M. Tetrahedron Lett. 1992, 33, 37-40. (c) Thrane, H.;
Fensholdt, J.; Regner, M.; Wengel, J. Tetrahedron 1995, 51, 10389-10402.
(d) Wang, G.; Seifert, W. E. Tetrahedron Lett. 1996, 37, 6515-6518. (e)
Nomura, M.; Shuto, S.; Tanaka, M.; Sasaki, T.; Mori, S.; Shigeta, S.;
Matsuda, A. J. Med. Chem. 1999, 42, 2901-2908.
(27) Detmer, I.; Summerer, D.; Marx, A. Eur. J. Org. Chem. 2003, 1837-
1846.
(28) (a) Cicero, D. O.; Neuner, P. J. S.; Franzese, O.; D’Onofrio, C.;
Iribarren, A. M. Bioorg. Med. Chem. Lett. 1994, 4, 861-866. (b) Grøtli,
M.; Undheim, K. Acta Chem. Scand. 1995, 49, 217-224.
(29) Tronchet, J. M. J.; Grand, E.; Zsely, M.; Giovannini, R.; Geoffroy,
M. Carbohydr. Lett. 1998, 3, 161-168.
(33) Dess, D. B.; Martin, J. C. J. Am. Chem. Soc. 1991, 113, 7277-7287.
J. Org. Chem, Vol. 71, No. 20, 2006 7733

Albæk et al.
SCHEME 1. Synthesis of Bicyclic Nucleosidesa
FIGURE 2. Optimized geometry of 8 and 9 obtained by ab initio
calculations.
CHPh,34 in refluxing dichloromethane, and the resulting bicyclic
nucleoside 18 was isolated in an excellent 96% yield. The fact
that the ring closure was performed confirmed the 2′,4′-cis
positioning of the olefins respective to the furanose ring and
thereby the 4′-configuration of 15 and 16. The silyl protecting
groups of 18 were removed with TBAF to give the target
nucleoside 9 in 98% yield. Hydrogenation of the double bond
in 9 was performed in methanol using Adams’ catalyst, and
the saturated target bicyclic nucleoside 8 was isolated in
quantitative yield. The structures of the target nucleosides 8 and
9 were confirmed by NMR and mass spectrometry, and a more
thorough conformational study was performed (see below). To
prepare nucleosides 8 and 9 for automated oligonucleotide
synthesis, both were in two steps converted to the appropriately
protected phosphoramidites. Thus, the primary 5′-hydroxy
groups were protected with the DMT group by standard
protocols, and afterward, the 3′-O-phosphoramidites 19 and 20
were obtained in acceptable yields.
Conformational Analysis. The 1H NMR spectra of 8 and 9
3
show the three-bond coupling constant JH1′H2′ ) 0 as found
for all bicyclic nucleosides with 2′,4′-linkages. This clearly
indicates a locked N-type conformation. On the other hand, no
significant structural information about the carbocyclic ring can
a Reagents and conditions: (a) PhOC(S)Cl, DMAP, CH3CN (79%); (b)
allyltributyltin, AIBN, toluene (65%); (c) 80% CH3COOH (75%); (d) (i)
Dess-Martin periodinane, CH2Cl2, (ii) HCHO, 2 M NaOH, dioxane, then
NaBH4 (52%); (e) BzCl, pyridine, CH3CN (65%); (f) (i) TBSCl, imidazole,
DMF, (ii) NaOCH3, CH3OH (71%); (g) (i) Dess-Martin periodinane,
CH2Cl2 (93%), (ii) BuLi, Ph3PBrCH3, THF (95%); (h) Grubbs second-
generation catalyst, CH2Cl2 (96%); (j) TBAF, THF (98%); (k) from 9, H2,
PtO2, CH3OH (100%); (l) (i) DMTCl, pyridine, CH3CN (78%), (ii)
NC(CH2)2OP(Cl)N(iPr)2, EtN(iPr)2, CH2Cl2 (57%); (m) (i) DMTCl, Et3N,
CH2Cl2 (78%), (ii) NC(CH2)2OP(Cl)N(iPr)2, EtN(iPr)2, CH2Cl2 (63%). TBS
) tert-butyldimethylsilyl. DMT ) 4,4′-dimethoxytrityl. CE ) 2-cyanoethyl.
1
be taken from the H NMR spectrum of 8 because of a severe
overlap of signals. Ab initio calculations revealed the lowest-
energy conformations of the bicyclic nucleosides 8 and 9, as
shown in Figure 2. Both nucleosides are locked in N-type
conformations but with slightly different sugar puckering (Table
1). The cyclohexane ring of 8 adopts a perfect chair conforma-
tion, and the cyclohexene ring of 9 is in a near-envelope
conformation with five atoms in a plane. Table 1 compares the
pseudorotation angles, P, and puckering amplitudes35 for various
bicyclic nucleosides with a locked N-type conformation. X-ray
data were used when available, i.e., for 1, 4, and 7, whereas ab
initio calculations were performed on 5 and 6 in the same way
as those with 8 and 9. Ab initio calculations of 1 and 4 give
results (data not shown) in accordance with X-ray data,
validating the calculations. All the monomers are locked in
N-type conformations with P in the range from 11° to 21°,
except for 9 with P ) 27°. The puckering amplitude strictly
follows the size of the additional ring: for two-atom 2′,4′-
linkages, νmax is around 57°; for three-atom 2′,4′-linkages, νmax
and 5′-O positions gives the advantage of a combined removal
of both on the final nucleoside. Initial attempts to perform
regioselective benzoylation of the diol gave only one mono-
protected isomer 15 as well as the dibenzoyl derivative.
Therefore, a procedure using only 0.9 equiv of benzoyl chloride
was chosen. No dibenzoyl isomer was formed, and 15 was
isolated in 65% yield along with 28% recovered starting material
14, i.e., a 90% effective yield of 15. The TBS protection of the
remaining hydroxy group using TBSCl and imidazole followed
by removal of the benzoyl group with sodium methoxide gave
16 in 71% overall yield. Oxidation with Dess-Martin perio-
dinane gave the aldehyde, which was reacted in a Wittig
reaction, and the diene 17 was isolated in a very good 88%
yield over the two steps. The two terminal alkenes in 17 were
reacted in a ring-closing metathesis (RCM) reaction using
Grubbs’ second-generation catalyst, ((Mes)2Im)(Cy3P)Cl2Rud
(34) Scholl, M.; Ding, S.; Lee, C. W.; Grubbs, R. H. Org. Lett. 1999, 1,
953-956.
(35) For definitions and nomenclature regarding nucleoside and nucleic
acid structure and conformations, see: Saenger, W. Principles of Nucleic
Acid Structure; Springer: New York, 1984.
7734 J. Org. Chem., Vol. 71, No. 20, 2006

Analogues of a Nucleic Acid with Three-Carbon Linkages
TABLE 1. Sugar Puckering Data for Different Bicyclic
Nucleosides with 2′,4′-Linkagesa
pseudorotation angle puckering amplitude
2′,4′-linkage
4′-CH2O-2′
P
νmax
1
4
17° b
15° b
20° c
15° c
12° d
20° e
15° e
27° e
19° f
17° g
14° h
57° b
57° b
58° c
48° c
48° d
48° e
46° e
49° e
38° f
38° g
38° h
4′-CH2CH2O-2′
5
8
9
6
7
4′-CH2OCH2-2′
4′-CH2CH2CH2-2′
4′-CHdCHCH2-2′
4′-CH2CH2CH2O-2′
4′-CH2OCH2O-2′
A-type RNA
a Data obtained from literature X-ray data or ab initio calculated energy-
minimized structures. b Two different conformations are in the unit cell:
X-ray data, base ) T, ref 18. c X-ray data, base ) A, ref 18. d X-ray data,
base ) isobutyl-G, ref 18. e Ab initio data from the present study, base )
U. f Ab initio data from the present study, base ) T. g X-ray data, base )
T, ref 20. h Taken from ref 35.
TABLE 2. Thermal Stability Data of Modified Duplexes
complementary DNA complementary RNA
5′-dGCATATCAC-3′ 5′-rGCAUAUCAC-3′
ODN sequencesa
Tm (∆Tm)/°Cb
Tm (∆Tm)/°Cb
21 5′-dGTGATATGC-3′
22 5′-dGTGAXATGC-3′
23 5′-dGXGAXAXGC-3′
24 5′-dGTGAYATGC-3′
25 5′-dGYGAYAYGC-3′
30.0
27.5
29.0 (-1.0)
27.0 (-1.0)
29.0 (-1.0)
23.0 (-2.3)
32.0 (+4.5)
38.0 (+3.5)
31.5 (+4.0)
34.5 (+2.3)
a Oligodeoxynucleotide sequences with X ) 8 (incorporation of 20) and
Y ) 9 (incorporation of 19). b Melting 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, and 0.1 mM EDTA, pH 7.0, using 1.5 µM concentrations of
each strand. Values in brackets show the changes in Tm values per
modification compared with the reference strand 21.
is around 48°; and for four-atom 2′,4′-linkages, νmax is around
38° (the value for ribose/deoxyribose).
Synthesis and Hybridization Properties of Oligonucle-
otides. The two phosphoramidites 19 and 20 were used as
building blocks in the synthesis of oligonucleotides using a
standard automated solid-phase method. Both phosphoramidites
19 and 20 were coupled in high yields and incorporated into
9-mer ODN sequences with either one or three modifications
(Table 2). For the study, we have chosen the same 9-mer
sequence as that employed in the first study of LNA and other
nucleic acid analogues which will ease comparison.3,15 The
composition of the modified oligonucleotides was verified by
MALDI MS. The hybridization of the modified oligonucleotides
toward complementary DNA and RNA sequences was studied
by thermal denaturation experiments. The melting temperatures
of the formed duplexes were determined and compared with
the unmodified duplexes (Table 2). These experiments show
that a single incorporation of the bicyclic nucleoside 8 in an
ODN increases the stability of the duplex formed with RNA
with 4.5 °C. In the case of three modifications, a stabilization
of 3.5 °C per modification is seen. The similar incorporations
of the other bicyclic nucleoside containing an unsaturated 2′,4′-
linkage 9 lead to slightly smaller increases in thermal stability
of 4.0 and 2.3 °C per modification, for one and three modifica-
tions, respectively. Hybridization of the same modified se-
quences with a DNA complement demonstrates a general
FIGURE 3. CD spectra of duplexes formed by the oligonucleotides
21-28 and their DNA and RNA complements. (A) Modified DNA:
RNA duplexes. (B) Modified dsDNA duplexes. Sequences 26 and
27 correspond to the LNA sequences 5′-dGTGATLATGC-3′ and 5′-
dGTLGATLATLGC-3′, respectively, where TL is the LNA thymidine
monomer 1. Sequence 28 corresponds to the RNA sequence 5′-
rGUGAUAUGC-3′.
destabilization in the range of -1 °C for each of the incorpora-
tions, even though a more pronounced destabilization of -2.3
°C for three incorporations of 9 was observed.
Circular Dichroism Spectroscopy. CD spectra were re-
corded for all duplexes of the study (Table 2) as well as for the
corresponding duplexes with one or three incorporations of LNA
1 (base ) T) and a dsRNA duplex (Figure 3A). A- and B-type
duplex geometries have distinctly different CD features. A-type
duplexes give an intense negative band at ∼210 nm and a
positive one at ∼260 nm of approximately equal magnitude as
exemplified by the dsRNA in Figure 3. B-type duplexes give
much less of a CD signal than A-type duplexes, as there is no
negative signal at ∼210 nm and negative and positive bands of
relatively low intensity at ∼250 nm and ∼275 nm, respec-
J. Org. Chem, Vol. 71, No. 20, 2006 7735

Albæk et al.
tively.36 The DNA:RNA duplex with three LNA modifications
clearly displays the A-type characteristics found for the RNA:
RNA duplex (compare 27 and 28, Figure 3A). Thus, the negative
band at 210 nm is intense for both duplexes and small for the
unmodified DNA:RNA duplex (21). For the duplexes modified
with 8 or 9, this band is of lower intensity than that observed
with LNA and clearly decreases in intensity in the order
LNA > 8 > 9 > DNA for both one (i.e., 26, 22, and 24) and
three incorporations (i.e., 27, 23, and 25) in the DNA:RNA
duplex. A similar picture can be seen with the positive band
around 260 nm. Thus, three LNA modifications result in the
complete shift of a DNA:RNA duplex toward an A-type duplex,
whereas this effect is significantly smaller with three incorpora-
tions of 8 and even smaller with 9.
is of no influence. Little variation in the pseudorotation angle
(11°-27°) is observed describing near E envelope conforma-
3
tions in all cases. In the case of an 3E envelope sugar
conformation, the puckering amplitude describes the deviation
of the C-3′ atom from the plane of the remaining sugar atoms.
Changes in the puckering amplitude and the position of C-3′
lead to changes in the δ backbone angle, which has a value of
∼65°, ∼75°, and ∼80° for the two-, three-, and four-atom 2′,4′-
linkages, respectively (data not shown). At first glance, the
stabilization of duplexes follows the length of the 2′,4′-linkage
and thereby the puckering amplitude or value of δ. We note,
however, that the difference in duplex stabilization between
LNA (1, two-atom linkage) and ENA (4, three-atom linkage)
is small if existent at all.
The unmodified dsDNA duplex is a pure B-type duplex,
whereas all the modified dsDNA duplexes seem to be changed
toward an intermediate duplex form (Figure 3B). The deviation
from the standard B-type geometry is clearly followed in the
decreasing bands at 210 and 250 nm, and it follows the same
trend as that seen for the DNA:RNA duplexes. Thus, the
duplexes containing 9 (i.e., 24 and 25) are less altered than the
duplexes containing 8 (i.e., 22 and 23) which on the other hand
are considerably less altered than the LNA-containing duplexes
(i.e., 26 and 27). As expected, three modifications lead to larger
deviations than one modification for each of the three different
bicyclic nucleic acid analogues.
In a direct comparison of the influence of different three-
atom linkages, 8 shows an influence on duplex stability
comparable to what has been reported for its oxa-analogues 4
and 5. Hence, in comparable though not identical sequences, 8
leads to an increase in the stability of a DNA:RNA hybrid
similar to that seen for 417,18 (i.e., ∆Tm’s between +3.5 and
+5.5 °C per modification). In comparison, 519 as well as our
unsaturated analogue 9 lead to slightly smaller increases in
stability (∆Tm’s between +2 and +4 °C per modification). It
should, of course, be taken into account that the sequence
contexts are different. The sequence used in the present study
of 8 and 9 is the same 9-mer sequence as that used in our
original study of LNA 1 and later in the study of sulfur and
amino analogues 2 and 3. The melting temperatures were in all
cases somewhat higher for 1, 2, or 3 (45-50 °C) than for 8
(38 °C, 23, Table 2). The same sequence has not been studied
with ENA 4. In the original study of ENA, however, a direct
comparison with LNA in equal oligopyrimidine sequences
revealed almost identical melting temperatures.17 In the context
of a dsDNA duplex, the modifications with a three-atom 2′,4′-
linkage range from a considerable stabilization reported for
4,17,18 and a neutral influence reported for 5,19 to slight
destabilizations seen for both 8 and 9 (Table 2). Again, a direct
comparison of ENA and LNA (4 and 1) revealed similar
increases in Tm.17 The general observation with the bicyclic
nucleosides with three-atom 2′,4′-linkages is, therefore, that the
variation is large ranging from the unprecedented duplex
stabilization similar to that seen with two-atom 2′,4′-linkages
(LNA and analogues, 1-3) to the more or less neutral stability
similar to that generally seen with the four-atom 2′,4′-linkages
(6 and 7). From the data in hand, it therefore appears that the
atomic constitution of the 2′,4′-linkage is more important than
its length. When comparing the different three-atom linkers, the
exact position of the oxygen emerges as a very important feature
for duplex stabilization, and more so in dsDNA duplexes than
in DNA:RNA hybrid duplexes.
Discussion
The synthesis of 8 and 9 was easily accomplished using the
linear strategy from uridine. Thus, the ring-closing key step by
an RCM reaction was extremely efficient in producing the
conformationally restricting cyclohexene ring. The drawback
of the linear strategy is, of course, that the access to the
analogues of 8 and 9 with other nucleobases relies on repeating
the complete series of steps starting from the other natural
ribonucleosides. However, the cytosine analogues should be
readily accessible from 18 through a standard U to C conversion.
The olefin of 9 opens the opportunity of preparing a range of
analogues of 8 that are functionalized on the carbocyclic ring.
The observed increase in RNA affinity obtained with 8
(base ) U) is significant and surprising with regards to the brief
report on 8 (base ) T) given in the literature21 which claims
decreased RNA affinity. The discrepancy might be explained
by different sequence contexts including different nucleobases
of the bicyclic nucleoside, although this seems unlikely. The
fact that the nucleosides 8 and 9 presented in our study are
uridine and not thymidine analogues should be taken into
account but points in the opposite direction. Thus, even higher
melting temperatures should be expected for the corresponding
thymidine analogues due to the well-known positive influence
of the 5-methyl group (∼1 °C per methyl group).
It has been suggested that a major feature responsible for the
ability of LNA-DNA mixmers to form stabilized A-type
duplexes with complementary RNA (and A-type-like duplexes
with complementary DNA) is the conformational steering of
neighboring 2′-deoxynucleotides toward N-type sugar puckers.11
As indicated from the CD spectra (Figure 3), this effect is less
pronounced when the LNA monomers are replaced with 8 and
even further reduced by replacement with 9. Thus, the confor-
mational steering is, apparently, reduced by the hydrophobic
nature of the 2′,4′-linkage in 8 and 9. It is noteworthy that ENA
and LNA increase the thermal stability of duplexes equally, and
from CD spectra, it also appears that they perturb the duplex
With the present data for the two new members of the series
of bicyclic nucleosides with 2′,4′-linkages and locked N-type
conformations (Figure 1), the overall relation between duplex
structure and stability vs the nature of the bicyclic building
blocks can be further enlightened. When comparing the sugar
puckering data (Table 1), it is clear that the length of the 2′,4′-
linkage directly determines the puckering amplitude of the
bicyclic nucleoside, whereas the constitution of the 2′,4′-linkage
(36) Rodger, A.; Norde´n, B. Circular dichroism and linear dichroism;
Oxford University Press: Oxford, 1997.
7736 J. Org. Chem., Vol. 71, No. 20, 2006

Analogues of a Nucleic Acid with Three-Carbon Linkages
structure comparably.18 Overall, for LNA, ENA, 8, and 9, there
is a strong correlation between duplex structure and stability,
i.e., the more conformational steering (and consequently A-type
characteristics of a given duplex), the higher the thermal
stability. It is interesting that the degree of conformational
steering depends on the presence of a 2′-oxygen atom. Con-
sidering specific duplex types, a larger difference in melting
temperature is observed for dsDNA duplexes than for DNA:
RNA duplexes going from LNA and ENA to 8 and 9. In the
case of DNA:RNA duplexes, the locked N-type conformation
leads to a general gain in thermal stability, whereas the presence
of a 2′-oxygen is crucial for optimizing the effect of the locked
N-type conformation in dsDNA. In other words, the 2′-oxygen
is more important for conformational steering and duplex
stability in the case of dsDNA than for DNA:RNA.
A dsDNA duplex has a B-type geometry with a narrow minor
groove which is extensively hydrated (spine of hydration),37
whereas a DNA:RNA duplex possesses a geometry intermediate
between A- and B-type, albeit closer to A- than B-type, with a
fairly wide minor groove. As a result, the change in duplex
geometry upon inclusion of N-type nucleotides is larger for a
dsDNA duplex (B-type to intermediate geometry) than for a
DNA:RNA duplex (intermediate to A-type geometry). It is
possible that the 2′-oxygen is important for maintaining a water
network upon the widening of the minor groove in going from
a B-type to an intermediate duplex geometry. In this case, it
would imply that the cooperative conformational steering of
neighboring nucleotides into N-type sugar puckers is driven by
the hydration of the minor groove. The hybridization properties
of oligonucleotides with 8 and 9 incorporated toward RNA
complements show that the hydrophobic 2′,4′-linkers are equally
as well tolerated as the hydrophilic linkers of LNA and ENA
in this context. This could imply that minor groove hydration
is less important for A-type duplexes.
The difference in duplex stabilization between 8 and 9 is
difficult to explain. The CD spectra show that incorporation of
8 leads to a larger change in global duplex structure toward
A-type than the incorporation of 9. This could possibly be due
to differential influence on the minor groove hydration by 8
and 9, but we also note that the sugar pucker of 9 (P ) 27°) is
slightly different compared to the other nucleic acid analogues
discussed in this paper (Table 1).
In summary, the RNA-selective recognition of oligonucle-
otides containing 8 might be a useful property. Thus, the RNA
affinity is almost retained compared to LNA and ENA, whereas
the DNA affinity is reduced below the level of unmodified
ODNs. This difference in RNA affinity over DNA affinity is
larger than that for any of the other bicyclic nucleic acid
analogues with 2′,4′-linkages. If the nuclease-mediated degrada-
tion of oligonucleotides with 8 or 9 incorporated, as it might
be expected, is reduced to the same extent as that reported for
ENA,18 8 and 9 could be promising candidates in the develop-
ment of antisense oligonucleotides. The relatively short and
efficient synthetic route is a significant advantage in this
perspective.
of 13% from uridine, using a linear strategy with a very efficient
RCM reaction as the key step. Both were easily incorporated
into ODNs and were found to stabilize DNA:RNA duplexes
significantly and to destabilize dsDNA duplexes slightly. Com-
parison with other 2′,4′-linked nucleoside analogues leads to
the conclusion that the position of a hydrophilic oxygen atom
is more important in an A-type-like dsDNA duplex than in an
A-type DNA:RNA duplex. It also indicates that the duplex
enhancing property of this class of bicyclic nucleic acid building
blocks in general, and of LNA in particular, might be related
to water binding in the minor groove. Dependent on their
pharmacokinetic properties, both the saturated carbocyclic LNA
analogue 8 and its unsaturated equivalent 9 could be promising
candidates in antisense research. The RNA-selective recognition
mode can also be an advantage in diagnostic applications. The
unsaturated moiety of 9 gives the opportunity for an easy
synthetic access to a plethora of functionalized derivatives of 8
and 9.
Experimental Section
Synthesis of 2′-Deoxy-2′-C-allyl-3′-O-(tert-butyldimethylsilyl)-
uridine (13). A solution of 12 (0.766 g, 1.54 mmol) in 80% CH3-
COOH (20 mL) was stirred for 3 days. After neutralization with a
saturated aqueous solution of NaHCO3, the solution was extracted
with CH2Cl2 (3 × 120 mL). The combined extracts were washed
with a saturated aqueous solution of NaHCO3 (80 mL) and dried
(MgSO4). The solvent was removed under reduced pressure, and
the residue was purified by silica gel column chromatography
(EtOAc-petroleum ether 2:3 v/v) to give the product 13 (0.450 g,
75%) as a white solid: Rf 0.3 (EtOAc); 1H NMR (300 MHz, CDCl3)
δ 9.00 (s, 1H, NH), 7.56 (d, 1H, J ) 8.0 Hz, H-6), 5.81 (d, 1H,
J ) 8.6 Hz, H-1′), 5.77-5.65 (m, 2H, H-5, CHdCH2), 5.06 (dd,
1H, J ) 1.4 Hz, J ) 17.3 Hz, CHdCH2), 4.99 (d, 1H, J ) 10.1
Hz, CHdCH2), 4.39 (dd, 1H, J ) 1.7 Hz, J ) 5.6 Hz, H-3′), 4.00
(m, 1H, H-4′), 3.88 (dt, 1H, J ) 3.2 Hz, J ) 11.9 Hz, H-5′), 3.76
(ddd, 1H, J ) 2.8 Hz, J ) 6.0 Hz, J ) 11.9 Hz, H-5′), 2.83 (br s,
1H, OH), 2.56 (m, 1H, H-2′), 2.43 (m, 1H, CH2CHdCH2), 2.14
(m, 1H, CH2CHdCH2), 0.92 (s, 9H, SiC(CH3)3), 0.09 (s, 6H,
SiCH3); 13C NMR (75 MHz, CDCl3) δ 163.2 (C-4), 150.4 (C-2),
141.9 (C-6), 135.2 (CHCH2), 116.8 (CHdCH2), 102.6 (C-5), 91.6
(C-1′), 87.6 (C-4′), 73.8 (C-3′), 62.7 (C-5′), 47.6 (C-2′), 29.2 (CH2-
CHdCH2), 25.7 (C(CH3)3), 18.0 (C(CH3)3), -4.5 (CH3Si), -4.9
(CH3Si); MALDI MS m/z (405.2 [M + Na]+, C18H30O5N2Si - Na+
calcd 405.2).
Synthesis of 2′-Deoxy-2′-C-allyl-4′-C-hydroxymethyl-3′-O-
(tert-butyldimethylsilyl)uridine (14). To a stirred solution of 13
(3.88 g, 10.1 mmol) in anhydrous CH2Cl2 (150 mL) was added
Dess-Martin periodinane (4.45 g, 10.7 mmol). After 2 h, the
reaction mixture was filtered through Celite and the sinter was rinsed
with EtOAc (100 mL). The combined organic phases were washed
with a mixture of a saturated aqueous solution of NaHCO3 and a
saturated aqueous solution of Na2S2O3 (1:1, 120 mL), and the water
phase was extracted with EtOAc (160 mL). The combined organic
phases were dried (MgSO4) and evaporated under reduced pressure
to give the aldehyde as a white foam. A solution of the foam in
dioxane (100 mL) was added to HCHO (37%, 2.2 mL, 25 mmol)
and a 2 M, aqueous solution of NaOH (6.5 mL, 13 mmol). After
stirring for 17 h, the mixture was cooled to 0 °C and NaBH4 (1.071
g, 28.3 mmol) was added. The reaction mixture was stirred at room
temperature for 5 h, and then we added a mixture of CH3COOH
and pyridine (1:4, 10 mL) and H2O (100 mL). The resulting mixture
was extracted with CHCl3 (3 × 150 mL), and the combined extracts
were washed with a saturated aqueous solution of NaHCO3 (50
mL), dried (MgSO4), and evaporated under reduced pressure. The
residue was purified by silica gel column chromatography (EtOAc)
to give 14 (2.168 g, 52%) as a white solid: Rf 0.2 (EtOAc)
Conclusion
Two different modified nucleosides with a three-carbon
2′,4′-linkage were synthesized in the same good overall yield
(37) Egli, M.; Tereshko, V.; Teplova, M.; Minasov, G.; Joachimiak, A.;
Sanishvili, R.; Weeks, C. M.; Miller, M. R.; Maier, M. A.; An, H.; Cook,
P. D.; Manoharan, M. Biopolymers 2000, 48, 234-252.
J. Org. Chem, Vol. 71, No. 20, 2006 7737

Albæk et al.
(Found: C, 54.69; H, 8.18; N, 6.56. C19H32O6N2Si‚1/2H2O requires
C, 54.13; H, 7.89; N, 6.65%); 1H NMR (300 MHz, CDCl3) δ 8.17
(s, 1H, NH), 7.31 (d, 1H, J ) 8.3 Hz, H-6), 5.75 (d, 1H, J ) 8.8
Hz, H-1′), 5.72 (d, 1H, J ) 8.3 Hz, H-5), 5.58 (m, 1H, CHdCH2),
5.06 (dd, 1H, J ) 0.9 Hz, J ) 17.1 Hz, CHdCH2), 4.98 (d, 1H,
J ) 10.1 Hz, CHdCH2), 4.60 (d, 1H, J ) 6.6 Hz, H-3′), 3.86 (dd,
1H, J ) 3.5 Hz, J ) 12.2 Hz, CH2O), 3.78 (d, 1H, J ) 3.9 Hz,
J ) 11.5 Hz, CH2O), 3.67 (dd, 1H, J ) 7.0 Hz, J ) 11.5 Hz,
CH2O), 3.57 (dd, 1H, J ) 9.8 Hz, J ) 12.2 Hz, CH2O), 2.83-2.76
(m, 2H, OH, H-2′), 2.38 (m, 1H, CH2CHdCH2), 2.30-2.16 (m,
2H, CH2CHdCH2, OH), 0.95 (s, 9H, SiC(CH3)3), 0.13 (s, 6H,
SiCH3); 13C NMR (75 MHz, CDCl3) δ 163.3 (C-4), 150.5 (C-2),
141.9 (C-6), 135.0 (CHdCH2), 117.1 (CHdCH2), 102.7 (C-5), 91.3,
88.8 (C-1′, C-4′), 74.8 (C-3′), 64.2, 63.8 (C-5′, CH2O), 47.2 (C-
2′), 30.4 (CH2CHdCH2), 25.9 (C(CH3)3), 18.1 (C(CH3)3), -4.4
(CH3Si), -4.6 (CH3Si); HRMALDI MS m/z (435.1922 [M + Na]+,
C19H32O6N2Si - Na+ calcd 435.1923).
J ) 10.7 Hz, H-5′), 3.71 (d, 1H, J ) 10.7 Hz, H-5′), 3.52 (d, 1H,
J ) 12.1 Hz, CH2OH), 2.44-2.30 (m, 2H, H-2′, CH2CHdCH2),
2.18 (m, 1H, CH2CHdCH2), 0.95 (s, 9H, SiC(CH3)3), 0.94 (s, 9H,
SiC(CH3)3), 0.13 (s, 3H, SiCH3), 0.12 (s, 3H, SiCH3), 0.10 (s, 3H,
SiCH3), 0.10 (s, 3H, SiCH3); 13C NMR (75 MHz, CDCl3) δ 162.9
(C-4), 150.3 (C-2), 140.1 (C-6), 134.8 (CHdCH2), 116.8 (CHd
CH2), 102.8 (C-5), 88.4, 87.5 (C-1′, C-4′), 75.3 (C-3′), 65.4, 63.7
(C-5′, CH2O), 49.4 (C-2′), 29.3 (CH2CHdCH2), 25.9 (C(CH3)3),
25.8 (C(CH3)3), 18.3 (C(CH3)3), 18.1 (C(CH3)3), -4.3 (CH3Si),
-4.4 (CH3Si), -5.5 (CH3Si), -5.6 (CH3Si); HRMALDI MS m/z
(549.2776 [M + Na]+, C25H46O6N2Si2 - Na+ calcd 549.2787).
Synthesis of 2′-Deoxy-2′-C-allyl-3′,5′-di-O-(tert-butyldimethyl-
silyl)-4′-C-vinyluridine (17). To a stirred solution of 16 (0.668 g,
1.27 mmol) in anhydrous CH2Cl2 (17 mL) was added Dess-Martin
periodinane (0.659 g, 1.55 mmol). After 1 1/2 h, the mixture was
filtered through Celite and the sinter was rinsed with EtOAc (100
mL). The combined organic phases were washed with a mixture
of a saturated aqueous solution of NaHCO3 and a saturated aqueous
solution of Na2S2O3 (1:1, 40 mL), and the water phase was extracted
with CH2Cl2 (2 × 75 mL). The combined organic phases were dried
(MgSO4) and evaporated under reduced pressure. The residue was
purified by silica gel column chromatography (EtOAc-petroleum
ether 1:1 v/v) to give the aldehyde (0.619 g, 93%) as a white
foam: 1H NMR (300 MHz, CDCl3) δ 9.53 (s, 1H, CHO), 8.96 (s,
1H, NH), 7.77 (d, 1H, J ) 8.1 Hz, H-6), 6.42 (d, 1H, J ) 9.4 Hz,
H-1′), 5.72 (d, 1H, J ) 8.1 Hz, H-5), 5.64 (m, 1H, CHdCH2),
5.05 (dd, 1H, J ) 1.4 Hz, J ) 17.1 Hz, CHdCH2), 4.98 (d, 1H,
J ) 10.3 Hz, CHdCH2), 4.50 (d, 1H, J ) 5.0 Hz, H-3′), 4.04 (d,
1H, J ) 11.4 Hz, H-5′), 3.68 (d, 1H, J ) 11.4 Hz, H-5′), 2.51-
2.14 (m, 3H, H-2′, CH2CHdCH2), 0.94 (s, 9H, SiC(CH3)3), 0.90
(s, 9H, SiC(CH3)3), 0.14 (s, 3H, SiCH3), 0.14 (s, 3H, SiCH3), 0.07
(s, 3H, SiCH3), 0.02 (s, 3H, SiCH3); 13C NMR (75 MHz, CDCl3)
δ 200.5 (CHO), 162.9 (C-4), 150.4 (C-2), 140.0 (C-6), 134.4 (CHd
CH2), 117.0 (CHdCH2), 103.1 (C-5), 91.9, 89.3 (C-1′, C-4′), 77.9
(C-3′), 64.2 (C-5′), 49.4 (C-2′), 28.6 (CH2CHdCH2), 25.8 (C(CH3)3),
25.8 (C(CH3)3), 18.3 (C(CH3)3), 18.0 (C(CH3)3), -4.0 (CH3Si),
-4.7 (CH3Si), -5.5 (CH3Si), -5.6 (CH3Si). A suspension of Ph3-
PBrCH3 (0.513 g, 1.44 mmol) in anhydrous THF (4 mL) was stirred
at -78 °C under an Ar atmosphere, and BuLi (1.6 M in hexanes,
0.85 mL, 1.36 mmol) was added. The mixture was stirred for 15
min at -78 °C and then at 0 °C. A solution of the aldehyde (0.194
g, 0.37 mmol) in THF (2 mL) was added. The reaction mixture
was stirred at 0 °C for 45 min, and after an additional 2 h at room
temperature, a saturated aqueous solution of NH4Cl (7 mL) was
added. The resulting mixture was extracted with Et2O (3 × 15 mL),
and the combined extracts were washed with brine (10 mL), dried
(MgSO4), and evaporated under reduced pressure. Purification by
silica gel column chromatography (EtOAc-petroleum ether 1:3 v/v)
Synthesis of 2′-Deoxy-2′-C-allyl-4′-C-benzoyloxymethyl-3′-O-
(tert-butyldimethylsilyl)uridine (15). A stirred solution of 14
(2.151 g, 5.21 mmol) in anhydrous CH3CN (25 mL) and anhydrous
pyridine (25 mL) was cooled to 0 °C, and BzCl (0.50 mL, 4.3
mmol) was added. The reaction mixture was stirred at room
temperature, and after 1.75 h H2O (50 mL) was added. The resulting
mixture was extracted with CHCl3 (3 × 100 mL), and the combined
extracts were washed with a saturated aqueous solution of NaHCO3
(50 mL), dried (MgSO4), and evaporated under reduced pressure.
The residue was purified by silica gel column chromatography
(EtOAc-petroleum ether 2:3 v/v) to give 15 (1.737 g, 65%) as a
white foam as well as recovered starting material 14 (0.588 g, 28%).
Data for 15: Rf 0.3 (EtOAc); 1H NMR (300 MHz, CDCl3) δ 8.90
(s, 1H, NH), 8.04 (d, 2H, J ) 7.2 Hz, Ph), 7.55 (m, 1H, Ph), 7.37
(m, 2H, Ph), 7.27 (d, 1H, J ) 8.1 Hz, H-6), 5.81 (d, 1H, J ) 9.1
Hz, H-1′), 5.71 (d, 1H, J ) 8.1 Hz, H-5), 5.61 (m, 1H, CHdCH2),
5.06 (d, 1H, J ) 17.1 Hz, CHdCH2), 4.98 (d, 1H, J ) 10.2 Hz,
CHdCH2), 4.69 (d, 1H, J ) 6.2 Hz, H-3′), 4.55 (d, 1H, J ) 12.2
Hz, CH2OBz), 4.47 (d, 1H, J ) 12.2 Hz, CH2OBz), 3.80 (d, 1H,
J ) 11.8 Hz, H-5′), 3.69 (d, 1H, J ) 11.8 Hz, H-5′), 3.26 (br s,
1H, OH), 2.75 (m, 1H, H-2′), 2.42 (m, 1H, CH2CHdCH2), 2.22
(m, 1H, CH2CHdCH2), 0.97 (s, 9H, SiC(CH3)3), 0.18 (s, 3H,
SiCH3), 0.14 (s, 3H, SiCH3); 13C NMR (75 MHz, CDCl3) δ 167.2
(CO), 162.9 (C-4), 150.3 (C-2), 141.9 (C-6), 135.0 (CHdCH2),
133.3 (Ph), 129.8 (Ph), 129.6 (Ph), 128.4 (Ph), 117.1 (CHdCH2),
102.7 (C-5), 91.7, 87.3 (C-1′, C-4′), 74.7 (C-3′), 64.9, 63.7 (C-5′,
CH2O), 46.8 (C-2′), 30.2 (CH2CHdCH2), 25.9 (C(CH3)3), 18.2
(C(CH3)3), -4.3 (CH3Si), -4.6 (CH3Si); HRMALDI MS m/z
(539.2192 [M + Na]+, C26H36O7N2Si - Na+ calcd 539.2184).
Synthesis of 2′-Deoxy-2′-C-allyl-4′-C-hydroxymethyl-3′,5′-di-
O-(tert-butyldimethylsilyl)uridine (16). To a stirred solution of
15 (1.718 g, 3.33 mmol) in anhydrous DMF (50 mL) was added
imidazole (1.714 g, 25.6 mmol) and TBSCl (1.691 g, 11.2 mmol).
The mixture was stirred for 15 h. H2O (60 mL) was added, and the
mixture was extracted with Et2O (3 × 150 mL). The combined
extracts were washed with a saturated aqueous solution of NaHCO3
(60 mL) and brine (60 mL), dried (MgSO4), and evaporated under
reduced pressure. The residue was dissolved in anhydrous CH3OH
(50 mL), and NaOCH3 (0.773 g, 14.3 mmol) was added. The
reaction mixture was stirred at 35 °C for 10 h and then at room
temperature for 1 day. The reaction was quenched with 80% CH3-
COOH (10 mL) and extracted with CH2Cl2 (3 × 100 mL), and the
combined extracts were washed with a saturated aqueous solution
of NaHCO3 (60 mL), dried (MgSO4), and evaporated under reduced
pressure. The residue was purified by silica gel column chroma-
tography (EtOAc-petroleum ether 1:3 v/v) to give 16 (1.243 g,
71%) as a white foam: Rf 0.5 (EtOAc); 1H NMR (300 MHz, CDCl3)
δ 8.00 (br s, 1H, NH), 7.60 (d, 1H, J ) 8.2 Hz, H-6), 6.11 (d, 1H,
J ) 9.1 Hz, H-1′), 5.70 (dd, 1H, J ) 2.1 Hz, J ) 8.2 Hz, H-5),
5.60 (m, 1H, CHdCH2), 5.03 (dd, 1H, J ) 1.1 Hz, J ) 17.0 Hz,
CHdCH2), 4.95 (d, 1H, J ) 10.3 Hz, CHdCH2), 4.42 (d, 1H, J )
5.7 Hz, H-3′), 3.87 (d, 1H, J ) 12.1 Hz, CH2OH), 3.78 (d, 1H,
1
gave 17 (0.184, 95%) as a white foam: Rf 0.6 (EtOAc); H NMR
(300 MHz, CDCl3) δ 8.75 (s, 1H, NH), 7.80 (d, 1H, J ) 8.2 Hz,
H-6), 6.09 (d, 1H, J ) 6.5 Hz, H-1′), 5.86 (dd, 1H, J ) 10.9 Hz,
J ) 17.4 Hz, CCHdCH2), 5.74-5.63 (m, 2H, H-5, CH2CHdCH2),
5.47 (dd, 1H, J ) 1.7 Hz, J ) 17.4 Hz, CCHdCH2), 5.23 (dd, 1H,
J ) 1.7 Hz, J ) 10.9 Hz, CCHdCH2), 5.05-4.96 (m, 2H, CH2-
CHdCH2), 4.50 (d, 1H, J ) 6.3 Hz, H-3′), 3.64-3.52 (m, 2H,
H-5′), 2.40-2.29 (m, 2H, H-2′, CH2CHdCH2), 2.13 (m, 1H, CH2-
CHdCH2), 0.94 (s, 9H, SiC(CH3)3), 0.93 (s, 9H, SiC(CH3)3), 0.11
(s, 3H, SiCH3), 0.11 (s, 3H, SiCH3), 0.10 (s, 3H, SiCH3), 0.08 (s,
3H, SiCH3); 13C NMR (75 MHz, CDCl3) δ 163.0 (C-4), 150.1 (C-
2), 140.5 (C-6), 135.6, 135.2 (CHdCH2), 116.5, 116.1 (CHdCH2),
102.6 (C-5), 89.0, 87.4 (C-1′, C-4′), 73.4 (C-3′), 66.4 (C-5′), 49.7
(C-2′), 30.5 (CH2CHdCH2), 25.9 (C(CH3)3), 25.9 (C(CH3)3), 18.3,
18.1 (C(CH3)3), -4.3 (CH3Si), -4.4 (CH3Si), -5.5 (CH3Si), -5.5
(CH3Si); MALDI MS m/z (547.2 [M + Na]+, C26H46O5N2Si2 -
Na+ calcd 547.2).
Synthesis of (1R,5R,6R,8S)-8-tert-Butyldimethylsilyloxy-1-tert-
butyldimethylsilyloxymethyl-6-(uracil-1-yl)-7-oxabicyclo[3.2.1]-
oct-2-ene (18). To a stirred solution of 17 (0.277 g, 0.53 mmol) in
anhydrous CH2Cl2 (15 mL) was added Grubbs’ second-generation
7738 J. Org. Chem., Vol. 71, No. 20, 2006

Analogues of a Nucleic Acid with Three-Carbon Linkages
catalyst (((Mes)2Im)(Cy3P)Cl2RudCHPh)34 (0.022 g, 0.026 mmol),
and the solution was heated to reflux for 25 h. The mixture was
evaporated under reduced pressure, and the residue was purified
by silica gel column chromatography (EtOAc-petroleum ether 1:4
v/v) to give 18 (0.251 g, 96%) as a white foam: Rf 0.6 (EtOAc);
1H NMR (300 MHz, CDCl3) δ 8.25 (s, 1H, NH), 8.12 (d, 1H, J )
8.2 Hz, H-6), 5.91 (m, 1H, CHdCHCH2), 5.65 (d, 1H, J ) 8.2
Hz, H-5), 5.61 (s, 1H, H-1′), 5.41 (d, 1H, J ) 9.3 Hz, CHdCHCH2),
4.34 (d, 1H, J ) 5.0 Hz, H-3′), 3.80 (d, 1H, J ) 11.6 Hz, H-5′),
3.65 (d, 1H, J ) 11.6 Hz, H-5′), 2.55 (m, 1H, CHdCHCH2), 2.40-
2.30 (m, 2H, H-2′, CHdCHCH2), 0.95 (s, 9H, SiC(CH3)3), 0.85
(s, 9H, SiC(CH3)3), 0.13 (s, 3H, SiCH3), 0.12 (s, 3H, SiCH3), 0.07
(s, 3H, SiCH3), 0.06 (s, 3H, SiCH3); 13C NMR (75 MHz, CDCl3)
δ 163.5 (C-4), 150.2 (C-2), 140.2 (C-6), 130.7, 126.6 (CHdCH),
101.0 (C-5), 88.9, 81.8 (C-1′, C-4′), 65.1, 61.8 (C-3′, C-5′), 44.8
(C-2′), 28.5 (CH2CHdCH), 25.9 (C(CH3)3), 25.5 (C(CH3)3), 18.3,
17.9 (C(CH3)3), -4.7 (CH3Si), -5.1 (CH3Si), -5.5 (CH3Si), -5.6
(CH3Si); HRMALDI MS m/z (517.2505 [M + Na]+, C24H42O5N2-
Si2 - Na+ calcd 517.2525).
CHdCHCH2), 5.64 (s, 1H, H-1′), 5.48-5.45 (m, 2H, H-5, CHd
CHCH2), 4.50 (dd, 1H, J ) 5.6 Hz, J ) 7.7 Hz, H-3′), 3.80 (s, 6H,
OCH3), 3.54 (d, 1H, J ) 11.1 Hz, H-5′), 3.36 (d, 1H, J ) 11.1 Hz,
H-5′), 2.64-2.54 (m, 2H, CHdCHCH2, H-2′), 2.43 (m, 1H, CHd
CHCH2); 13C NMR (75 MHz, CDCl3) δ 163.6 (C-4), 158.6 (Ar),
150.3 (C-2), 144.5 (Ar), 140.1 (C-6), 135.4, 135.3, 131.4, 130.1,
128.1, 128.0, 127.2, 127.1, 113.3 (Ar, CHdCH), 101.2 (C-5), 89.2,
86.9, 81.0 (C-1′, C-4′, Ar3C), 66.0, 62.2 (C-3′, C-5′), 55.2 (OCH3),
44.2 (C-2′), 28.0 (CHdCHCH2). To a stirred solution of the DMT-
protected intermediate (74 mg, 0.13 mmol) in anhydrous CH2Cl2
(1 mL) was added N,N-diisopropylethylamine (0.1 mL, 0.58 mmol)
and 2-cyanoethyl N,N-diisopropylphosphoramidochloridite (0.05
mL, 0.22 mmol). The mixture was stirred for 19 h, and then we
added CH2Cl2 (4 mL). The mixture was washed with a saturated
aqueous solution of NaHCO3. The aqueous phase was extracted
with CH2Cl2 (5 mL), and the combined organic phases were dried
(Na2SO4) and evaporated under reduced pressure. Purification by
silica gel column chromatography (EtOAc-petroleum ether 1:4 v/v
with 1% NEt3) gave 19 (57 mg, 57%) as a white foam: Rf 0.4
(EtOAc); 31P (CDCl3) δ 149.57.
Synthesis of (1R,5R,6R,8S)-8-Hydroxy-1-hydroxymethyl-6-
(uracil-1-yl)-7-oxabicyclo[3.2.1]oct-2-ene (9). A 1 M solution of
TBAF in THF (1.1 mL, 1.1 mmol) was added to a stirred solution
of 18 (0.247 g, 0.50 mmol) in anhydrous THF (9 mL), and after 3
h, an additional amount of the TBAF solution (0.14 mmol) was
added. After stirring for another 2 h, the mixture was evaporated
under reduced pressure and the residue was purified by silica gel
column chromatography (petroleum ether, then CH3OH (1-5%)
in CH2Cl2) to give 9 (0.130 g, 98%) as a white solid: Rf 0.1 (2 ×
Synthesis of (1R,5R,6R,8S)-8-(2-Cyanoethoxy(diisopropylami-
no)phosphinoxy)-1-(4,4′-dimethoxytrityloxymethyl)-6-(uracil-1-
yl)-7-oxabicyclo[3.2.1]octane (20). To a stirred solution of 8 (62
mg, 0.23 mmol) in anhydrous CH2Cl2 (1 mL) was added Et3N (0.07
mL, 0.50 mmol) and DMTCl (83 mg, 0.24 mmol). The mixture
was stirred for 4 h and then evaporated under reduced pressure.
The residue was purified by silica gel column chromatography to
give the DMT-protected product (101 mg, 78%) as a white foam:
1
1
EtOAc); H NMR (300 MHz, DMSO) δ 11.27 (s, 1H, NH), 7.96
Rf 0.4 (EtOAc); H NMR (300 MHz, CDCl3) δ 8.38 (d, 1H, J )
(d, 1H, J ) 8.1 Hz, H-6), 5.82 (m, 1H, CHdCHCH2), 5.55 (d, 1H,
J ) 8.1 Hz, H-5), 5.48 (d, 1H, J ) 9.6 Hz, CHdCHCH2), 5.41 (s,
1H, H-1′), 5.15 (d, 1H, J ) 4.8 Hz, 3′-OH), 5.09 (t, 1H, J ) 5.4
Hz, 5′-OH), 4.12 (t, 1H, J ) 4.8 Hz, H-3′), 3.59 (dd, 1H, J ) 5.4
Hz, J ) 12.5 Hz, H-5′), 3.50 (dd, 1H, J ) 5.4 Hz, J ) 12.5 Hz,
H-5′), 2.47 (m, 1H, CHdCHCH2), 2.31 (m, 1H, H-2′), 2.08 (m,
1H, CHdCHCH2); 13C NMR (75 MHz, DMSO) δ 163.4 (C-4),
150.3 (C-2), 140.2 (C-6), 130.2, 127.3 (CHdCH), 100.5 (C-5), 88.0,
81.2 (C-1′, C-4′), 64.4, 60.4 (C-3′, C-5′), 43.7 (C-2′), 28.2 (CH2-
CHdCH); HRMALDI MS m/z (289.0795 [M + Na]+, C12H14O5N2
- Na+ calcd 289.0795).
Synthesis of (1R,5R,6R,8S)-8-(Hydroxy)-1-(hydroxymethyl)-
6-(uracil-1-yl)-7-oxabicyclo[3.2.1]octane (8). To a stirred solution
of 9 (60 mg, 0.23 mmol) in CH3OH (1 mL) was added PtO2 (21
mg, 0.09 mmol). The mixture was degassed with N2 and then placed
under a hydrogen atmosphere. The reaction mixture was stirred
for 1 1/2 h and then filtered through Celite. The filtrate was
evaporated under reduced pressure to give 8 (62 mg, 100%) as a
white foam: Rf 0.1 (EtOAc); 1H NMR (300 MHz, DMSO) δ 11.24
(s, 1H, NH), 8.36 (d, 1H, J ) 8.0 Hz, H-6), 5.63 (s, 1H, H-1′),
5.53 (d, 1H, J ) 8.0 Hz, H-5), 5.25-5.18 (m, 2H, OH), 4.07 (m,
1H, H-3′), 3.48-3.43 (m, 2H, H-5′), 2.22 (m, 1H, H-2′), 1.90 (m,
1H, CH2), 1.68-1.14 (m, 5H, CH2); 13C NMR (75 MHz, DMSO)
δ 163.4 (C-4), 150.2 (C-2), 140.4 (C-6), 99.7 (C-5), 86.5, 86.0 (C-
1′, C-4′), 64.2, 61.6 (C-3′, C-5′), 44.9 (C-2′), 25.5, 23.0, 17.4 (CH2-
CH2CH2); HRMALDI MS m/z (291.0951 [M + Na]+, C12H16O5N2
- Na+ calcd 291.0951).
Synthesis of (1R,5R,6R,8S)-8-(2-Cyanoethoxy(diisopropylami-
no)phosphinoxy)-1-(4,4′-dimethoxytrityloxymethyl)-6-(uracil-1-
yl)-7-oxabicyclo[3.2.1]oct-2-ene (19). DMTCl (76 mg, 0.22 mmol)
was added to a stirred solution of 9 (48 mg, 0.18 mmol) in
anhydrous pyridine (0.5 mL) and anhydrous CH3CN (0.5 mL), and
the mixture was stirred for 22 h. Additional DMTCl (13 mg, 0.04
mmol) was added, and after stirring for 1 h, the solvent was
evaporated under reduced pressure. The residue was purified by
silica gel column chromatography (CH3OH (0-5%), pyridine (1%)
in CH2Cl2) to give the DMT-protected product (79 mg, 78%) as a
colorless oil: Rf 0.3 (EtOAc); 1H NMR (300 MHz, CDCl3) δ 8.62
(s, 1H, NH), 8.20 (d, 1H, J ) 8.0 Hz, H-6), 7.46-7.19 (m, 9H,
Ar), 6.85 (dd, 4H, J ) 8.9 Hz, J ) 1.3 Hz, Ar), 5.99 (m, 1H,
8.0 Hz, H-6), 7.99 (br s, 1H, NH), 7.46-7.12 (m, 9H, Ar), 6.88-
6.78 (m, 4H, Ar), 5.78 (s, 1H, H-1′), 5.33 (d, 1H, J ) 8.0 Hz,
H-5), 4.25 (m, 1H, H-3′), 3.80 (s, 6H, OCH3), 3.51 (d, 1H, J )
11.4 Hz, H-5′), 3.28 (d, 1H, J ) 11.4 Hz, H-5′), 2.42 (m, 1H, H-2′),
1.88 (m, 1H, CH2), 1.75-1.50 (m, 3H, CH2), 1.30-1.15 (m, 2H,
CH2); HRESI MS m/z (593.2254 [M + Na]+, C33H34O7N2 - Na+
calcd 593.2258). To a stirred solution of the DMT-protected
intermediate (100 mg, 0.18 mmol) in anhydrous CH2Cl2 (1.2 mL)
was added N,N-diisopropylethylamine (0.11 mL, 0.64 mmol) and
2-cyanoethyl N,N-diisopropylphosphoramidochloridite (0.06 mL,
0.25 mmol). After 4 1/2 h, more 2-cyanoethyl N,N-diisopropyl-
phosphoramidochloridite (0.03 mL, 0.13 mmol) was added, and
after stirring for 6 h, CH2Cl2 (10 mL) was added. The mixture was
washed with a saturated aqueous solution of NaHCO3 (8 mL), and
the aqueous phase was extracted with CH2Cl2 (10 mL). The
combined organic phases were dried (Na2SO4) and evaporated under
reduced pressure. Purification by silica gel column chromatography
(EtOAc-petroleum ether 1:4 v/v with 0.5% pyridine) gave 20 (85
mg, 63%) as a white foam: Rf 0.5, 0.6 (EtOAc); 31P (CDCl3) δ
150.42, 149.54; HRESI MS m/z (793.3330 [M + Na]+, C42H51O8N4P
- Na+ calcd 793.3337).
Synthesis of Oligodeoxynuclotides. Oligonucleotide synthesis
was carried out on an automated DNA synthesizer following the
phosphoramidite approach. Synthesis of oligonucleotides 21-25
was performed on a 0.2 µmol scale by using the amidites 19 and
20 as well as the corresponding commercial 2-cyanoethyl phos-
phoramidites of the natural 2′-deoxynucleosides. The synthesis fol-
lowed the regular protocol for the DNA synthesizer. However, for
19 and 20, a prolonged coupling time of 10 min was used. Coupling
yields for all 2-cyanoethyl phosphoramidites were >98%. After a
final detritylation, the oligonucleotides were removed from the uni-
versal solid support by treatment with concentrated ammonia at
55 °C for 20 h affording the >95% pure oligonucleotides. MALDI-
MS [M - H]- gave the following results (found/calcd): 22 (2776.0/
2780); 23 (2827.8/2828); 24 (2775.7/2778); 25 (2822.8/2826).
Melting Experiments. UV melting experiments were carried
out on a UV spectrometer. 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 concentra-
tions of the two complementary sequences. The increase in
J. Org. Chem, Vol. 71, No. 20, 2006 7739

Albæk et al.
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 temperature was determined as the local maximum of the
first derivatives of the absorbance vs temperature curve. All melting
curves were found to be reversible. All determinations are averages
of triplicates.
buffer as that used in the UV melting experiments was used with
3.0 µM concentrations of the two complementary sequences.
Quantum Mechanical Calculations. Ab initio quantum me-
chanical calculations were performed with the Gaussian 03
program.38 Full geometry optimizations (HF/6-31G*) were carried
out for the selected nucleosides as mentioned in the text. To ensure
that no hydrogen bonds were formed between the 3′-hydroxy group
and the nucleobase, the ꢀ torsion angle was constrained to -75°.
The â, γ, and ø angles were not constrained, and in the optimized
structures, there are no steric interactions among the 5′-hydroxy
group, the sugar, and the nucleobase.
CD Spectroscopy. CD spectra were recorded at the Department
of Chemistry, University of Copenhagen. The same medium salt
(38) 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.; Al-Laham,
M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.;
Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A. Gaussian
03, revision C.02; Gaussian, Inc.: Wallingford, CT, 2004.
Acknowledgment. The Danish National Research Founda-
tion, the Danish Natural Science Research Council, the Danish
Center for Scientific Computing, and Møllerens Fond are
thanked for financial support. Birthe Haack and Lene Jørgensen
are thanked for synthetic and technical assistance.
Supporting Information Available: The general introduction
to the Experimental Section as well as the experimental details for
the compounds 11 and 12. This material is available free of charge
via the Internet at http://pubs.acs.org.
JO061225G
7740 J. Org. Chem., Vol. 71, No. 20, 2006