Novel bicyclic nucleoside analogue (1S,5S,6S)-6-hydroxy-5-hydroxymethyl-1-(uracil-1-yl) -3,8-dioxabicyclo[3.2.1]octane: Synthesis and incorporation into oligodeoxynucleotides

Article abstract of DOI:10.1021/jo015664l

The novel bicyclic nucleoside (1S,5S,6S)-6-hydroxy-5-hydroxymethyl-1-(uracil-1-yl)-3,8-dioxabicyclo-[3.2.1] octane[2′-deoxy-1′-C,4′-C-(2-oxapropano)uridine] (15), expected to be restricted into an O4′-endo furanose conformation, was synthesized from the known 1-(3′-deoxy-β-D-psicofuranosyl)uracil 5. The phosphoramidite derivative of 15 was successfully incorporated into oligodeoxynucleotides using standard methods, and thermal denaturation studies showed moderate decreases in duplex stabilities of -2.1 and -1.5 °C per modification toward complementary DNA and RNA, respectively.

Full text of DOI:10.1021/jo015664l

5498
J . Org. Chem. 2001, 66, 5498-5503
Novel Bicyclic Nu cleosid e An a logu e (1S,5S,6S)-6-
Hyd r oxy-5-h yd r oxym eth yl-1-(u r a cil-1-yl)-3,8-d ioxa bicyclo[3.2.1]octa n e:
Syn th esis a n d In cor p or a tion in to Oligod eoxyn u cleotid es
Lisbet Kværnø† and J esper Wengel*,‡
Center for Synthetic Bioorganic Chemistry, Department of Chemistry, University of Copenhagen,
Universitetsparken 5, DK-2100 Copenhagen, Denmark, and Department of Chemistry,
University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark
jwe@chem.sdu.dk
Received April 4, 2001
The novel bicyclic nucleoside (1S,5S,6S)-6-hydroxy-5-hydroxymethyl-1-(uracil-1-yl)-3,8-dioxabicyclo-
[3.2.1]octane [2′-deoxy-1′-C,4′-C-(2-oxapropano)uridine] (15), expected to be restricted into an O4′-
endo furanose conformation, was synthesized from the known 1-(3′-deoxy-â-D-psicofuranosyl)uracil
5. The phosphoramidite derivative of 15 was successfully incorporated into oligodeoxynucleotides
using standard methods, and thermal denaturation studies showed moderate decreases in duplex
stabilities of -2.1 and -1.5 °C per modification toward complementary DNA and RNA, respectively.
In tr od u ction
The promise of the efficient inhibition of gene expres-
sion as described by the antisense approach1 has led to
the synthesis and investigation of multiple conforma-
tionally restricted nucleoside analogues. Many of the
differences in hybridization properties between chemi-
cally modified oligonucleotides can be described by one
single parameter, the pseudorotation angle, which defines
the furanose conformation of each single nucleotide
monomer.2 The C3′-endo (or in general N-type) conforma-
tion leading to A-type duplexes as seen for natural RNA
generally effects the highest duplex stabilities.3-5 Ex-
amples of such nucleotide monomers are LNA 1 (locked
nucleic acid, Figure 1), which displays unprecedented
binding affinity toward complementary DNA and RNA,6-9
and the C2′-methylene extended derivative 2 synthesized
with thymine and cytosine bases,10 which also displays
highly stable duplexes with complementary RNA.11 X-ray
studies revealed both nucleosides to be restricted into
C3′-endo conformations,10,12 while NMR studies of LNA
single strands hybridized to complementary DNA and
RNA verified the expected A-type duplexes.13,14
F igu r e 1. Structures of four bicyclic nucleotide monomers. 1
(LNA) and 2 are restricted to C3′-endo (N-type) furanose
conformations, while 3 and the novel 4 are restricted to O4′-
endo (E-type) furanose conformations.
† University of Copenhagen.
‡ University of Southern Denmark.
(1) Uhlmann, E.; Peyman, A. Chem. Rev. 1990, 90, 543-584.
(2) Altona, C.; Sunderalingam, M. J . Am. Chem. Soc. 1972, 94,
8205-8212.
The capability to activate RNase H, the enzyme which
cleaves the RNA strand in RNA/DNA heteroduplexes,
thus allowing a single antisense oligonucleotide to target
multiple RNA strands, is another desired feature for
antisense oligonucleotides. In general, oligonucleotides
preorganized into a C3′-endo conformation do not activate
RNase H.4 So far, only two types of fully modified
oligonucleotide analogues with an altered carbohydrate
part display this ability, namely arabino nucleic acids15,16
and cyclohexene nucleic acids.17 The 2′-F-arabino nucleic
(3) Freier, S. M.; Altmann, K. H. Nucleic Acids Res. 1997, 25, 4429-
4443.
(4) Cook, P. D. Nucleosides Nucleotides 1999, 18, 1141-1162.
(5) Herdewijn, P. Biochim. Biophys. Acta 1999, 1489, 167-179.
(6) Singh, S. K.; Nielsen, P.; Koshkin, A. A.; Wengel, J . Chem.
Commun. 1998, 455-456.
(7) 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.
(8) Koshkin, A. A.; Nielsen, P.; Meldgaard, M.; Rajwanshi, V. K.;
Singh, S. K.; Wengel, J . J . Am. Chem. Soc. 1998, 120, 13252-13253.
(9) Obika, S.; Nanbu, D.; Hari, Y.; Andoh, J .; Morio, K.; Doi, T.;
Imanishi, T. Tetrahedron Lett. 1998, 39, 5401-5404.
(10) Wang, G. Y.; Girardet, J . L.; Gunic, E. Tetrahedron 1999, 55,
7707-7724.
(13) Petersen, M.; Nielsen, C. B.; Nielsen, K. E.; J ensen, G. A.;
Bondensgaard, K.; Singh, S. K.; Rajwanshi, V. K.; Koshkin, A. A.; Dahl,
B. M.; Wengel, J .; J acobsen, J . P. J . Mol. Recognit. 2000, 13, 44-53.
(14) Bondensgaard, K.; Petersen, M.; Singh, S. K.; Rajwanshi, V.
K.; Kumar, R.; Wengel, J .; J acobsen, J . P. Chem.sEur. J . 2000, 6,
2687-2695.
(11) Wang, G. Y.; Gunic, E.; Girardet, J . L.; Stoisavljevic, V. Bioorg.
Med. Chem. Lett. 1999, 9, 1147-1150.
(12) Obika, S.; Nanbu, D.; Hari, Y.; Morio, K.; In, Y.; Ishida, T.;
Imanishi, T. Tetrahedron Lett. 1997, 38, 8735-8738.
10.1021/jo015664l CCC: $20.00 © 2001 American Chemical Society
Published on Web 07/19/2001

Novel Bicyclic Nucleoside Analogue
J . Org. Chem., Vol. 66, No. 16, 2001 5499
Sch em e 1a
a
Reagents and conditions: (i) ref 23, 63%; (ii) BnBr, NaH, THF, 85%; (iii) 80% aq AcOH, 94%; (iv) Dess-Martin periodinane, CH2Cl2;
(v) H2CO (37%), 1 M NaOH (aq), 1,4-dioxane, 78% (2 steps); (vi) MsCl, pyridine; (vii) H2, 20% Pd(OH)2/C, EtOH, 55% from 9; (viii) 1 M
NaOH (aq), 1,4-dioxane, 64%; (ix) NaOBz, DMF; (x) NaOMe, MeOH, 90% (2 steps); (xi) BCl3, CH2Cl2, 84%; (xii) DMTCl, pyridine, 67%;
(xiii) NC(CH2)2OP(Cl)N(i-Pr)2, EtN(i-Pr)2, CH2Cl2, 26%; (xiv) DNA synthesizer. DMT ) 4,4′-dimethoxytrityl.
acid, which also increases the binding affinity toward
complementary RNA, was shown by X-ray crystallogra-
phy to adopt an O4′-endo furanose conformation,18,19
which is a high-energy conformation and therefore is very
unusual for natural nucleic acids.2 Similar hybridization
properties toward complementary RNA, as well as higher
duplex stabilities with complementary DNA, were ob-
tained for the bicyclic monomer 3, which was shown by
molecular modeling and NMR studies likewise to be
restricted into an O4′-endo conformation.20
Resu lts a n d Discu ssion
The known 1-(3′-deoxy-â-D-psicofuranosyl)uracil 521,22
is preferentially 4,4′-dimethoxytritylated in the 6′ posi-
tion (refer to Scheme 1, structure 5, for numbering used
throughout the discussion). Using methods similar
(DMTCl in pyridine; 4,4'-dimethoxytrityl) to those previ-
ously described,23 but with the regeneration of starting
nucleoside 5 from undesired DMT-protected isomers by
treatment with 80% AcOH and repeated tritylation, a
total 63% yield of nucleoside 6 was obtained (data not
shown). The remaining hydroxy groups were selectively
benzylated using BnBr and NaH in THF to give 7 in a
reaction where intermediate workup before addition of
extra NaH and BnBr to complete the dibenzylation
appeared to minimize the formation of the tribenzylated
byproduct. The DMT group was hydrolyzed with 80%
AcOH, affording 8 in 94% yield, whereupon oxidation to
the intermediary aldehyde was accomplished with Dess-
Martin periodinane24 in CH2Cl2. A tandem aldol conden-
sation and Cannizzaro reaction using aqueous formal-
dehyde and 1 M NaOH in 1,4-dioxane furnished the diol
9 in 78% combined yield. Mesylation of the hydroxy
groups (MsCl in pyridine) proceeded smoothly, and at this
stage, a selective debenzylation of the primary hydroxy
To further investigate this unusual O4′-endo confor-
mation, we decided to synthesize the novel bicyclic
nucleoside 4 in which the furanose ring is also expected
to be restricted into an O4′-endo conformation. Although
the additional ring system is sterically rather large, the
increased duplex stabilities for the similar bicycle 2 when
incorporated into oligonucleotides suggest no serious
sterical restraints upon duplex formation for such a
bicyclic system.
(15) Damha, M. J .; Wilds, C. J .; Noronha, A.; Brukner, I.; Borkow,
G.; Arion, D.; Parniak, M. A. J . Am. Chem. Soc. 1998, 120, 12976-
12977.
(16) Noronha, A. M.; Wilds, C. J .; Lok, C. N.; Viazovkina, K.; Arion,
D.; Parniak, M. A.; Damha, M. J . Biochemistry 2000, 39, 7050-7062.
(17) Wang, J .; Verbeure, B.; Luyten, I.; Lescrinier, E.; Froeyen, M.;
Hendrix, C.; Rosemeyer, H.; Seela, F.; Van Aerschot, A.; Herdewijn,
P. J . Am. Chem. Soc. 2000, 122, 8595-8602.
(21) Holy´, A. Nucleic Acids Res. 1974, 1, 289-298.
(22) Ono, A.; Dan, A.; Matsuda, A. Bioconjugate Chem. 1993, 4, 499-
508.
(23) Kværnø, L.; Nielsen, C.; Wightman, R. H. J . Chem. Soc., Perkin
Trans. 1 2000, 2903-2906.
(24) Dess, D. B.; Martin, J . C. J . Am. Chem. Soc. 1991, 113, 7277-
7287.
(18) Berger, I.; Tereshko, V.; Ikeda, H.; Marquez, V. E.; Egli, M.
Nucleic Acids Res. 1998, 26, 2473-2480.
(19) Minasov, G.; Teplova, M.; Nielsen, P.; Wengel, J .; Egli, M.
Biochemistry 2000, 39, 3525-3532.
(20) Christensen, N. K.; Petersen, M.; Nielsen, P.; J acobsen, J . P.;
Olsen, C. E.; Wengel, J . J . Am. Chem. Soc. 1998, 120, 5458-5463.

5500 J . Org. Chem., Vol. 66, No. 16, 2001
Kværnø and Wengel
Ta ble 1. Sequ en ces Syn th esized a n d Th er m a l
group was feasible by catalytic hydrogenation using 20%
Pd(OH)2/C as the catalyst, which afforded 55% combined
yield of 11 and only 11% combined yield of the isomer
resulting from the removal of both benzyl groups (re-
ferred to as 11B in the Experimental Section). It might
be worthwhile to note that an alternative tosylation of 9
(TsCl in pyridine or TsCl and DMAP in CH2Cl2) pro-
ceeded in only 51% and 46% yields, respectively, and that
the selective debenzylation was not reproducible for the
tosylated isomer of 10. The cyclization of 11 was per-
formed by treatment with 1 M NaOH (aq) in dioxane at
90 °C overnight to give 64% yield of 12, while no trace of
simultaneous hydrolysis of the mesyl group was detect-
able. Therefore, a two-step sequence for the removal of
the mesyl group was applied, performing first a nucleo-
philic substitution with NaOBz in DMF at 100 °C to give
the intermediate benzoate 13, followed by methanolysis
using NaOMe in MeOH, yielding 14 in 90% yield (from
12). Surprisingly, hydrogenolysis of the benzyl group
using 20% Pd(OH)2/C in EtOH under an atmosphere of
hydrogen yielded an inseparable 1:1 mixture of 15
together with the debenzylated product where also the
double bond of the uracil base was hydrogenated. Alter-
natively, DMT-protection of 14 (R1 ) DMT) followed by
hydrogenation with ammonium formate as the hydrogen
donor and 10% Pd/C as the catalyst in refluxing MeOH
afforded quantitative reduction of the nucleobase with
no significant loss of the DMT protecting group. Thus,
debenzylation by Lewis acids was applied instead, which
yielded the desired nucleoside 15 in 84% yield after
treatment with BCl3 in CH2Cl2. Selective DMT protection
of the primary hydroxy group using DMTCl in pyridine
yielded 16 in 67% yield, and last, 3′-O-phosphitylation
using 2-cyanoethyl N,N-diisopropylphosphoramidochlo-
rodite and N,N-diisopropylethylamine in CH2Cl2 fur-
nished the phosphoramidite 17. The yield of the phos-
phitylation reaction was only 26%, which most likely can
be explained, at least in part, by the difficult removal of
some phosphorus-containing impurities requiring severel
chromatographic purifications.
The bicyclic structure obtained after the cyclization to
12 was verified by HMBC (heteronuclear multiple bond
correlation) NMR showing two- and three-bond C-H
correlations, because the pairs of atoms H6′′/C1′ and H1′/
C6′′ appeared to be maximally three bonds apart. An
energy-minimized structure of 15 obtained by molecular
mechanics using MacroModel v.7.025 [pseudosystematical
Monte Carlo conformational search26 (1000 steps, limit
50 kJ /mol) with water as solvent, MMFF force field27]
supported the assumption of an O4′-endo furanose con-
formation, while the six-membered ring adopted a chair-
like conformation with one oxygen pointing toward 3′-
OH (sketched in Figure 1). Thus, out of the 79 discrete
conformations found, the 73 lowest-energy conformers all
displayed an O4′-endo furanose conformation and a
chairlike conformation of the 1,4-dioxane ring (see Sup-
porting Information for further details).
Den a tu r a tion Stu d ies tow a r d Com p lem en ta r y DNA a n d
RNA Sequ en cesa
complementary complementary
DNA
RNA
entry
sequence
Tm/°C ∆Tm/°C Tm/°C ∆Tm/°C
1
2
3
4
5
5′-d(GTGATATGC)
5′-d(GTGAUATGC)
5′-d(GTGA4ATGC)
5′-d(GUGAUAUGC) 27.3
5′-d(G4GA4A4GC) 18.7
29.4
27.9
25.8
26.6
26.4
25.8
25.6
21.0
-2.1
-2.9
-0.8
-1.5
a
Tm values measured as the maximum of the first derivative
of the melting curve (A260 vs temperature) recorded in medium
salt buffer (10 mM sodium phosphate, 100 mM sodium chloride,
0.1 mM EDTA, pH 7.0) using 1.5 µM concentrations of the two
complementary strands. ∆Tm ) change in Tm value calculated per
modification. dA ) 2′-deoxyadenosine monomer; dC ) 2′-deoxy-
cytidine monomer; dG ) 2′-deoxyguanosine monomer; dU ) 2′-
deoxyuridine monomer; dT ) thymidine monomer; 4, see Figure
1. All abbreviated as d(sequence).
tion, because H3′ and H4′ appeared only to be mutually
close, thus not contradicting the result from the modeling.
3
3
Values of J 3a′,4′ and J 3b′,4′ for compounds 12-16 in the
ranges 3.8-4.4 and 10.3-11.2 Hz, respectively, preclude
an extreme S-type (C2′-endo) conformation, though.28
Phosphoramidite 17 was incorporated once and three
times as monomer 4 (Figure 1) into the mixed 9-mer
oligodeoxynucleotide sequence depicted in Table 1 (en-
tries 3 and 5, respectively). Standard conditions were
used, except for a 10 min coupling time for the amidite
17 which afforded a coupling yield of ∼98% compared
with >99% yield for the unmodified amidites (refer to
Experimental Section for further details). The oligonucle-
otides were purified by ethanol precipitation, and capil-
lary gel electrophoresis showed >90% purity of the
products, whose compositions were verified from MALDI-
MS analysis.
As depicted in Table 1, the melting temperatures (Tm
values) and the changes in Tm value per modification,
compared with those of the unmodified reference con-
taining 2′-deoxyuridine monomers in place of the bicyclic
nucleoside 4, were determined. As can be seen when
comparing the natural DNA 9-mer in entry 1 with entries
2 and 4, small decreases in the thermal stability result
from substituting the natural thymine bases with uracil,
most pronouncedly for the 9-mer with one uracil nucleo-
base when hybridized to complementary DNA. Incorpo-
ration of 4 once into the center of the 9-mer (entry 3)
effected a decrease in the duplex stability of -2.1 °C
toward complementary DNA and -0.8 °C toward comple-
mentary RNA. When 4 was incorporated three times in
the sequence (entry 5), even larger decreases of -2.9 and
-1.5 °C per modification toward complementary DNA
and RNA, respectively, were observed. This moderate
decrease in duplex stabilities can probably be ascribed
to unfavorable steric interactions of the additional six-
membered ring combined with the O4′-endo furanose
conformation adopted by 4, which is not quite as favored
for duplex formation as the C3′-endo conformation of the
structurally closely related bicycle 2 (Figure 1).
Conformational analysis by NOE difference spectra
recorded for the bicyclic derivatives 14 and 15 was not
conclusive with respect to a certain furanose conforma-
Con clu sion
(25) Mohamadi, F.; Richards, N. G. J .; Guida, W. C.; Liskamp, R.;
Lipton, M.; Caufield, C.; Chang, G.; Hendrickson, T.; Still, W. C. J .
Comput. Chem. 1990, 11, 440-467.
In conclusion, the nucleoside 15 of a novel C2′-C5′-
fused bicyclic structure was synthesized in several steps
(26) Goodman, J . M.; Still, W. C. J . Comput. Chem. 1991, 12, 1110-
1117.
(27) Halgren, T. A. J . Comput. Chem. 1996, 17, 490-519. Planar
sp2-hybridized nitrogens were chosen.
(28) Haasnoot, C. A. G.; de Leeuw, A. A.; de Leeuw, H. P. M.; Altona,
C. Org. Magn. Reson. 1981, 15, 43-51.

Novel Bicyclic Nucleoside Analogue
J . Org. Chem., Vol. 66, No. 16, 2001 5501
2.65 (1H, dd, J ) 2.6, 15.0 Hz, H3′). 13C NMR (CDCl3): δ
163.66 (C4), 158.46 (DMT), 149.82 (C2), 144.16 (DMT), 142.29
(C6), 135.18, 129.77, 128.35, 128.22, 127.76, 127.74, 127.69,
127.61, 127.47, 127.42, 126.85 (DMT, Bn), 113.03 (DMT), 99.94
(C5), 98.93 (C2′), 86.54 (Ar2PhC), 85.34 (C5′), 78.84 (C4′), 73.42
(Bn), 71.71 (C1′), 71.03 (Bn), 62.90 (C6′), 55.09 (OMe), 41.11
(C3′).
from 1-(3′-deoxy-â-D-psicofuranosyl)uracil (5). Molecular
modeling supported the assumption of a fixed O4′-endo
furanose conformation. The phosphoramidite derivative
17 was incorporated successfully one and three times into
mixed 9-mer oligodeoxynucleotides by standard phos-
phoramidite oligonucleotide synthesis. Thermal dena-
turation studies revealed moderate decreases in duplex
stabilities of -2.1 and -1.5 °C per modification toward
complementary DNA and RNA, respectively. These re-
sults underline the fact that a locked furanose conforma-
tion is not per se leading to increased duplex stabilities.
1-[1′,4′-Di-O-b en zyl-3′-d eoxy-â-D-p sicofu r a n osyl]u r a -
cil (8). Compound 7 (2.688 g, 3.68 mmol) was dissolved in CH2-
Cl2 (10 mL), 80% AcOH (50 mL, v/v) was added, and the
mixture was stirred for 1 h 30 min and evaporated to an oil
under reduced pressure. EtOH (5 mL) and saturated aq
NaHCO3 (10 mL) were added, and the suspension was
evaporated to dryness under reduced pressure and coevapo-
rated with abs EtOH (2 × 50 mL). The residue was suspended
in MeOH (50 mL), evaporated on silica gel, and purified by
silica gel column chromatography (7.5 cm × 5.5 cm), eluting
with a gradient of 2-5% MeOH in CH2Cl2 (v/v) to give
nucleoside 8 (1.387 g, 94%) as a white foam after coevaporation
with acetonitrile (50 mL): Rf (8% MeOH in CH2Cl2 (v/v)) 0.36.
1H NMR (CDCl3): δ 9.05 (1H, br s, NH), 7.95 (1H, d, J ) 8.2
Hz, H6), 7.36-7.19 (10H, m, Bn), 5.62 (1H, dd, J ) 1.8, 8.2
Hz, H5), 4.59 (1H, d, J ) 12.3 Hz, Bn), 4.53 (1H, d, J ) 11.7
Hz, Bn), 4.46 (1H, d, J ) 11.7 Hz, Bn), 4.45 (1H, d, J ) 12.3
Hz, Bn), 4.37 (1H, m, H5′), 4.12 (1H, dt, J ) 3.1, 6.2 Hz, H4′),
3.99 (1H, d, J ) 10.4 Hz, H1′), 3.83 (1H, d, J ) 10.6 Hz, H1′),
3.76 (1H, dd, J ) 3.8, 11.5 Hz, H6′), 3.65 (1H, dd, J ) 3.9,
11.4 Hz, H6′), 2.78 (1H, dd, J ) 6.4, 15.0 Hz, H3′), 2.62 (1H,
dd, J ) 2.9, 15.0 Hz, H3′), 2.42 (1H, br s, OH). 13C NMR
(CDCl3): δ 164.09 (C4), 149.76 (C2), 142.44 (C6), 137.43,
137.37, 128.37, 128.25, 127.74, 127.64, 127.49, 127.43 (Bn),
99.77 (C5), 98.93 (C2′), 86.44 (C5′), 78.44 (C4′), 73.45 (Bn),
71.56 (C1′), 71.23 (Bn), 62.30 (C6′), 41.06 (C3′).
Exp er im en ta l Section
Gen er a l. Reactions were conducted under an atmosphere
of nitrogen when anhydrous solvents were used. All reagents
were obtained from commercial suppliers and were used
without further purification. Petroleum ether of the distillation
range 60-80 °C was used. The silica gel (0.040-0.063 mm)
used for column chromatography was purchased from Merck.
After column chromatography, fractions containing product
were pooled, evaporated under reduced pressure, and dried
overnight under high vacuum to give the product. All 1H NMR
spectra were recorded at 400 MHz, all 13C NMR spectra were
recorded at 100.6 MHz (unless otherwise stated), and the 31P
spectrum was recorded at 121.5 MHz. Chemical shifts are
reported in ppm relative to either tetramethylsilane or the
1
deuterated solvent as the internal standard for H and 13C and
relative to 85% H3PO4 as the external standard for 31P.
Assignments of NMR spectra are based on 2D spectra and
follow standard carbohydrate/nucleoside nomenclature (i.e.,
the furanose skeleton numbered 1′ to 6′, see Scheme 1), even
though the systematic compound names of the bicyclic struc-
tures are given according to the von Baeyer nomenclature. For
compound 12, a long-range HMBC spectrum gave a complete
assignment of all isolated methylene groups, while the as-
signments of atoms 6′ and 6′′ may be interchanged for
compounds 9-11 and 13-16. Fast atom bombardment mass
spectra (FAB-MS) were recorded in positive ion mode.
1-[1′,4′-Di-O-b en zyl-3′-d eoxy-5′-C-h yd r oxym et h yl-â-D-
p sicofu r a n osyl]u r a cil (9). A solution of 8 (1.520 g, 3.47
mmol) in anhydrous CH2Cl2 (20 mL) was added to a suspension
of Dess-Martin periodinane (1.824 g, 4.30 mmol) in anhydrous
CH2Cl2 (20 mL), and the suspension was stirred for 40 min.
CH2Cl2 (200 mL) was added, and the mixture was poured into
a solution of Na2S2O3‚5H2O (4061 mg, 16.36 mmol) in H2O (100
mL) and swirled until the solid had dissolved. The layers were
separated, the organic phase was filtered and evaporated to
dryness under reduced pressure, and the residue was dissolved
in 1,4-dioxane (100 mL). 37% aq formaldehyde (4.0 mL) and 1
M aq NaOH (20 mL) were added, and the mixture was stirred
for 22 h. Saturated aq NaHCO3 (20 mL) was added, and the
suspension was evaporated to dryness under reduced pressure,
coevaporated with acetonitrile (50 mL), suspended in MeOH
(50 mL), and evaporated on silica gel. The residue was purified
by silica gel column chromatography (6.5 cm × 5.5 cm), eluting
with a gradient of 1-5% MeOH in CH2Cl2 (v/v) to give
compound 9 (1.274 g, 78%) as a white foam: Rf (10% MeOH
1-[1′,4′-Di-O-ben zyl-3′-deoxy-6′-O-(4,4′-dim eth oxytr ityl)-
â-D-p sicofu r a n osyl]u r a cil (7). Nucleoside 623 (945 mg, 1.69
mmol) was dissolved in anhydrous THF (20 mL) at 0 °C, NaH
(60% w/w, 356 mg, 8.9 mmol) was added, and the mixture was
stirred at 0 °C for 5 min. BnBr (0.44 mL, 3.70 mmol) was added
dropwise, and the mixture was stirred at room temperature
for 8 h. H2O (50 mL) and saturated aq NaHCO3 (50 mL) were
added, and the mixture was extracted with EtOAc (4 × 60 mL).
The combined organic phase was washed with H2O (2 × 50
mL), evaporated to dryness under reduced pressure, and
coevaporated with acetonitrile (3 × 50 mL). The crude residue
was dissolved in anhydrous THF (25 mL), NaH (60% w/w, 495
mg, 12.4 mmol) was added, and the mixture was stirred for 5
min. BnBr (0.12 mL, 1.00 mmol) was added dropwise, and the
mixture was stirred at room temperature for 15 h. H2O (50
mL) and saturated aq NaHCO3 (50 mL) were added, and the
mixture was extracted with EtOAc (4 × 60 mL). The combined
organic phase was washed with H2O (2 × 50 mL), evaporated
to dryness under reduced pressure, and coevaporated with
acetonitrile (3 × 50 mL). The residue was purified by silica
gel column chromatography (16 cm × 2.8 cm), eluting with a
gradient of 0.5:0.5-2:99-97.5 pyridine/MeOH/CH2Cl2 (v/v/v)
to give compound 7 (1.055 g, 85%) as a white foam after
coevaporation with acetonitrile (3 × 30 mL): Rf (8% MeOH in
CH2Cl2 (v/v)) 0.49. FAB-MS m/z: 740.5 [M]+, 741.5 [M + H]+.
1H NMR (CDCl3): δ 8.97 (1H, br s, NH), 7.86 (1H, d, J ) 8.4
Hz, H6), 7.36-7.19 (19H, m, Bn, DMT), 6.81-6.78 (4H, m,
DMT), 5.53 (1H, dd, J ) 2.3, 8.4 Hz, H5), 4.60 (1H, d, J )
12.3 Hz, Bn), 4.50 (1H, d, J ) 11.9 Hz, Bn), 4.45 (1H, d, J )
12.3 Hz, Bn), 4.44 (1H, d, J ) 11.7 Hz, Bn), 4.44 (1H, m, H5′),
4.07 (1H, dt, J ) 2.6, 5.9 Hz, H4′), 4.00 (1H, d, J ) 10.6 Hz,
H1′), 3.84 (1H, d, J ) 10.4 Hz, H1′), 3.78 (6H, s, OMe), 3.20
(2H, d, J ) 4.6 Hz, H6′), 2.76 (1H, dd, J ) 6.1, 15.1 Hz, H3′),
1
in CH2Cl2 (v/v)) 0.33. FAB-MS m/z: 469.1 [M + H]+. H NMR
(CDCl3): δ 9.12 (1H, br s, NH), 7.98 (1H, d, J ) 8.2 Hz, H6),
7.36-7.20 (10H, m, Bn), 5.63 (1H, d, J ) 8.2 Hz, H5), 4.59
(1H, d, J ) 11.5 Hz, Bn), 4.57 (1H, d, J ) 12.1 Hz, Bn), 4.47
(1H, d, J ) 12.1 Hz, Bn), 4.39 (1H, d, J ) 11.7 Hz, Bn), 4.17
(1H, dd, J ) 5.1, 6.4 Hz, H4′), 3.87 (1H, d, J ) 11.5 Hz, H1′),
3.86 (2H, s, H6′′), 3.80 (1H, d, J ) 10.3 Hz, H1′), 3.68 (1H, d,
J ) 11.7 Hz, H6′), 3.64 (1H, d, J ) 11.7 Hz, H6′), 3.05 (1H,
dd, J ) 6.5, 14.9 Hz, H3′), 2.68 (1H, dd, J ) 5.0, 14.9 Hz, H3′).
13C NMR (CDCl3): δ 164.08 (C4), 149.95 (C2), 142.16 (C6),
137.17, 137.04, 128.50, 128.33, 127.98, 127.81, 127.61, 127.39
(Bn), 100.06 (C5), 98.17 (C2′), 89.80 (C5′), 79.05 (C4′), 73.52
(Bn), 71.96, 71.90 (Bn, C1′), 63.89 (C6′), 63.06 (C6′′), 39.36
(C3′).
1-[1′,4′-Di-O-ben zyl-3′-d eoxy-5′-C-m eth a n esu lfon yloxy-
m eth yl-6′-O-m eth a n esu lfon yl-â-D-p sicofu r a n osyl]u r a cil
(10). Nucleoside 9 (1.000 g, 2.13 mmol) was dissolved in
anhydrous pyridine (80 mL), evaporated to approximately half
the volume, and cooled to 0 °C. MsCl (0.70 mL, 9.0 mmol) was
added, and the mixture was stirred for 1 h 20 min at 0 °C.
Saturated aq NaHCO3 (50 mL) and H2O (50 mL) were added,
and the suspension was extracted with EtOAc (4 × 50 mL).
The combined organic phase was washed with H2O (50 mL),

5502 J . Org. Chem., Vol. 66, No. 16, 2001
Kværnø and Wengel
evaporated to dryness under reduced pressure, and coevapo-
rated with acetonitrile (2 × 50 mL). The residue was purified
by silica gel column chromatography (5.7 cm × 5.5 cm), eluting
with a gradient of 40-80% EtOAc in petroleum ether (v/v) to
give compound 10 (1.146 g containing n-hexane as an impu-
rity) as a white foam: Rf (10% MeOH in CH2Cl2 (v/v)) 0.57.
(1H, d, J ) 11.7 Hz, H6′), 4.32 (1H, d, J ) 11.7 Hz, H6′), 4.10
(1H, dd, J ) 4.2, 10.5 Hz, H4′), 4.05 (1H, d, J ) 10.7 Hz, H1′),
3.94 (1H, d, J ) 11.2 Hz, H6′′), 3.58 (1H, d, J ) 11.2 Hz, H6′′),
3.36 (1H, d, J ) 10.3 Hz, H1′), 3.23 (3H, s, Ms), 2.66 (1H, dd,
J ) 4.4, 13.0 Hz, H3′), 2.48 (1H, dd, J ) 10.3, 13.4 Hz, H3′).
13C NMR (126 MHz, DMSO-d6): δ 163.24 (C4), 149.57 (C2),
139.33 (C6), 137.99, 128.29, 127.63 (Bn), 101.27 (C5), 91.87
(C2′), 82.89 (C5′), 76.97 (C4′), 71.95 (Bn), 70.30 (C1′), 68.43
(C6′), 64.99 (C6′′), 40.98 (C3′), 36.82 (Ms).
1
FAB-MS m/z: 625.6 [M + H]+. H NMR (CDCl3): δ 9.2 (1H,
br s, NH), 7.77 (1H, d, J ) 8.2 Hz, H6), 7.37-7.20 (10H, m,
Bn), 5.68 (1H, d, J ) 8.2 Hz, H5), 4.60 (1H, d, J ) 11.5 Hz,
Bn), 4.55 (1H, d, J ) 12.1 Hz, Bn), 4.52 (1H, d, J ) 12.6 Hz,
H6′), 4.47 (1H, d, J ) 12.1 Hz, Bn), 4.40 (2H, d, J ) 11.2 Hz,
Bn, H6′), 4.25-4.18 (3H, m, H4′, H6′′), 3.90 (1H, d, J ) 10.6
Hz, H1′), 3.79 (1H, d, J ) 10.4 Hz, H1′), 3.02 (1H, m, H3′),
2.99 (3H, s, Ms), 2.93 (3H, s, Ms), 2.79 (1H, dd, J ) 3.6, 15.4
Hz, H3′). 13C NMR (CDCl3): δ 163.59 (C4), 149.83 (C2), 141.52
(C6), 137.09, 136.34, 128.55, 128.36, 128.19, 127.85, 127.71,
127.54 (Bn), 100.67 (C5), 98.79 (C2′), 86.52 (C5′), 78.57 (C4′),
73.46 (Bn), 72.10 (Bn), 71.73 (C1′), 67.91 (C6′), 66.58 (C6′′),
39.63 (C3′), 37.51, 37.23 (Ms). n-Hexane was assigned as an
impurity.
(1S,5S,6S)-6-Ben zyloxy-5-h yd r oxym et h yl-1-(u r a cil-1-
yl)-3,8-d ioxa bicyclo[3.2.1]octa n e (14). Bicycle 12 (197 mg,
0.45 mmol) was dissolved in anhydrous DMF (15 mL), NaOBz
(364 mg, 2.53 mmol) was added, and the suspension was
stirred at 120 °C for 36 h. Saturated aq NaHCO3 (15 mL) and
H2O (15 mL) were added, and the mixture was extracted with
EtOAc (4 × 25 mL). The combined organic phase was washed
with H2O (15 mL), evaporated to dryness under reduced
pressure, and coevaporated with acetonitrile (2 × 25 mL) and
n-hexane/CH2Cl2 (5 × 50 mL, 4:1 (v/v)). Crude 13 (see below)
was dissolved in MeOH (25 mL), NaOMe (230 mg, 4.3 mmol)
was added, and the mixture was stirred for 1 h 30 min.
Saturated aq NaHCO3 (1 mL) was added; the suspension was
evaporated to dryness under reduced pressure, suspended in
CH2Cl2 (50 mL), and evaporated on silica gel. The residue was
purified by silica gel column chromatography (10 cm × 2.8 cm),
eluting with a gradient of 1-3% MeOH in CH2Cl2 (v/v) to give
nucleoside 14 (145 mg, 90%) as a white foam: Rf (10% MeOH
1-[4′-O-Ben zyl-3′-deoxy-5′-C-m eth an esu lfon yloxym eth yl-
6′-O-m eth a n esu lfon yl-â-D-p sicofu r a n osyl]u r a cil (11) a n d
1-[3′-Deoxy-5′-C-m et h a n esu lfon yloxym et h yl-6′-O-m et h -
a n esu lfon yl-â-D-p sicofu r a n osyl]u r a cil (11B). Compound
10 (463 mg) was dissolved in anhydrous CH2Cl2 (2.5 mL);
anhydrous EtOH (25 mL) and Pd(OH)2/C (233 mg, 20% w/w)
were added, and the mixture was evacuated with H2 several
times. The suspension was stirred under an atmosphere of
hydrogen for 7 h, evaporated to dryness under pressure,
resuspended in CH2Cl2 (50 mL), and evaporated on silica gel.
The residue was purified by silica gel column chromatography
(10.3 cm × 2.8 cm), eluting with a gradient of 1-4% MeOH in
CH2Cl2 (v/v) to give mono- and didebenzylated nucleosides 11
(252 mg, 55% from 9) and 11B (43 mg, 11% from 9),
respectively, as white foams. Physical data for 11: Rf (10%
MeOH in CH2Cl2 (v/v)) 0.46. FAB-MS m/z: 535.4 [M + H]+.
1H NMR (CDCl3): δ 9.9 (1H, br s, NH), 7.77 (1H, d, J ) 8.2
Hz, H6), 7.36-7.26 (5H, m, Bn), 5.61 (1H, d, J ) 8.3 Hz, H5),
4.63 (1H, d, J ) 11.8 Hz, H6′), 4.60 (1H, d, J ) 12.0 Hz, Bn),
4.38 (1H, d, J ) 11.5 Hz, H6′), 4.37 (1H, d, J ) 11.8 Hz, Bn),
4.28 (1H, d, J ) 10.6 Hz, H6′′), 4.26 (1H, br s, H4′), 4.20 (1H,
d, J ) 10.4 Hz, H6′′), 4.01 (1H, d, J ) 12.3 Hz, H1′), 3.85 (1H,
d, J ) 12.2 Hz, H1′), 3.25 (1H, br s, OH), 3.04 (3H, s, Ms),
3.02 (1H, m, H3′), 3.00 (3H, s, Ms), 2.86 (1H, d, J ) 14.1 Hz,
H3′). 13C NMR (CDCl3): δ 164.42 (C4), 150.08 (C2), 142.08
(C6), 136.43, 128.54, 128.19, 127.83 (Bn), 100.63 (C5), 99.84
(C2′), 86.34 (C5′), 79.03 (C4′), 71.89 (Bn), 68.27 (C6′), 66.44
(C6′′), 65.42 (C1′), 39.05 (C3′), 37.63, 37.37 (Ms). Physical data
for 11B: Rf (10% MeOH in CH2Cl2 (v/v)) 0.26. FAB-MS m/z:
445.2 [M + H]+. 1H NMR (CD3OD): δ 7.97 (1H, d, J ) 8.2 Hz,
H6), 5.69 (1H, d, J ) 8.2 Hz, H5), 4.56 (2H, s, H6′), 4.43 (1H,
dd, J ) 2.6, 6.1 Hz, H4′), 4.36 (1H, d, J ) 10.6 Hz, H6′′), 4.32
(1H, d, J ) 10.6 Hz, H6′′), 3.92 (2H, s, H1′′), 3.23 (3H, s, Ms),
3.17 (3H, s, Ms), 3.08 (1H, dd, J ) 6.1, 15.3 Hz, H3′), 2.64
(1H, dd, J ) 2.1, 15.3 Hz, H3′). 13C NMR (CD3OD): δ 166.94
(C4), 151.88 (C2), 143.74 (C6), 101.42, 101.13 (C5, C2′), 88.92
(C5′), 73.18 (C4′), 69.52, 69.17 (C6′, C6′′), 66.14 (C1′), 44.73
(C3′), 37.43 (Ms).
1
in CH2Cl2 (v/v)) 0.33. FAB-MS m/z: 361.1 [M + H]+. H NMR
(1:1 CDCl3/CD3OD (v/v)): δ 7.82 (1H, d, J ) 8.2 Hz, H6), 7.29-
7.22 (5H, m, Bn), 5.61 (1H, d, J ) 8.2 Hz, H5), 4.62 (1H, d, J
) 11.9 Hz, Bn), 4.47 (1H, d, J ) 11.9 Hz, Bn), 4.20 (1H, d, J
) 10.2 Hz, H1′), 3.99 (1H, d, J ) 11.5 Hz, H6′′), 3.98 (1H, m,
H4′), 3.63 (1H, d, J ) 12.6 Hz, H6′), 3.61 (1H, d, J ) 11.5 Hz,
H6′′), 3.49 (1H, d, J ) 12.4 Hz, H6′), 3.30 (1H, d, J ) 10.4 Hz,
H1′), 2.88 (1H, dd, J ) 4.1, 13.7 Hz, H3′), 2.35 (1H, dd, J )
10.4, 13.7 Hz, H3′). 13C NMR (1:1 CDCl3/CD3OD (v/v)):
δ
165.40 (C4), 150.47 (C2), 141.03 (C6), 138.28, 128.90, 128.33,
128.22 (Bn), 101.66 (C5), 92.81 (C2′), 86.33 (C5′), 77.65 (C4′),
73.21 (Bn), 71.53 (C1′), 66.90 (C6′′), 62.49 (C6′′), 42.11 (C3′).
(1S,5R,6S)-6-Ben zyloxy-5-ben zoyloxym eth yl-1-(u r a cil-
1-yl)-3,8-d ioxa b icyclo[3.2.1]oct a n e (13). Physical data of
the intermediary benzoate 13 purified in analytical scale by
silica gel column chromatography, eluting with a gradient of
0-10:100-90 acetone in CH2Cl2 (v/v): Rf (20% acetone in CH2-
1
Cl2 (v/v)) 0.55. H NMR (300 MHz, CDCl3): δ 9.74 (1H, br s,
NH), 7.95 (2H, dd, J ) 1.4, 8.1 Hz, Bz), 7.66 (1H, dd, J ) 1.2,
8.1 Hz, H6), 7.61-7.24 (8H, m, Bn, Bz), 5.69 (1H, d, J ) 8.1
Hz, H5), 4.75 (1H, d, J ) 12.0 Hz, Bn), 4.48 (2H, d, J ) 12.3
Hz, Bn, H6′), 4.32 (1H, d, J ) 10.8 Hz, H1′), 4.28 (1H, d, J )
12.3 Hz, H6′), 4.20 (1H, d, J ) 11.1 Hz, H6′′), 4.09 (1H, dd, J
) 4.2, 10.2 Hz, H4′), 3.72 (1H, d, J ) 11.4 Hz, H6′′), 3.37 (1H,
d, J ) 10.5 H1′), 3.05 (1H, dd, J ) 4.2, 13.8 Hz, H3′), 2.44
(1H, dd, J ) 10.5, 13.5 Hz, H3′). 13C NMR (75 MHz, CDCl3):
δ 165.66 (OCOPh), 163.61 (C4), 149.38 (C2), 139.45 (C6),
137.03 (Bn), 133.26 (Bz), 129.49, 129.09, 128.33, 128.31,
127.81, 127.65 (Bn, Bz), 101.55 (C5), 92.33 (C2′), 83.76 (C5′),
76.77 (C4′), 72.42 (Bn), 70.84 (C1′), 66.15 (C6′), 63.43 (C6′′),
40.98 (C3′).
(1S,5R,6S)-6-Ben zyloxy-5-m eth a n esu lfon yloxym eth yl-
1-(u r a cil-1-yl)-3,8-d ioxa bicyclo[3.2.1]octa n e (12). Nucleo-
side 11 (378 mg, 0.707 mmol) was dissolved in 1,4-dioxane (10
mL) and 1 M aq NaOH (5 mL), and the mixture was stirred
at 90 °C for 22 h. After cooling, saturated aq NaHCO3 (20 mL)
and H2O (20 mL) were added, and the mixture was extracted
with EtOAc (4 × 25 mL) and CH2Cl2 (2 × 15 mL). The
combined organic phase was washed with H2O (2 × 20 mL),
evaporated to dryness under reduced pressure, and coevapo-
rated with acetonitrile (25 mL). The residue was purified by
silica gel column chromatography (10 cm × 2.8 cm), eluting
with a gradient of 1-3% MeOH in CH2Cl2 (v/v) to give bicycle
12 (197 mg, 64%) as a white solid: Rf (10% MeOH in CH2Cl2
(v/v)) 0.51. FAB-MS m/z: 439.1 [M + H]+. 1H NMR (500 MHz,
DMSO-d6): δ 11.35 (1H, br s, NH), 7.63 (1H, d, J ) 7.8 Hz,
H6), 7.39-7.29 (5H, m, Bn), 5.63 (1H, d, J ) 8.3 Hz, H5), 4.60
(1H, d, J ) 12.2 Hz, Bn), 4.55 (1H, d, J ) 11.7 Hz, Bn), 4.37
(1S,5S,6S)-6-Hyd r oxy-5-h yd r oxym eth yl-1-(u r a cil-1-yl)-
3,8-d ioxa bicyclo[3.2.1]octa n e (15). Compound 14 (116 mg,
0.322 mmol) was dissolved in anhydrous CH2Cl2 (25 mL) at
-78 °C, BCl3 (3.0 mL, 1 M solution in hexanes, 3.0 mmol) was
added dropwise over the course of 15 min, and the mixture
was stirred at -78 °C for 7 h and at room temperature for 30
min. After cooling to -78 °C, MeOH (10 mL) was added, and
the mixture was stirred at room temperature overnight. The
mixture was evaporated to dryness under reduced pressure
and coevaporated with MeOH (3 × 10 mL). The residue was
purified by silica gel column chromatography (8.5 cm × 2.0
cm), eluting with a gradient of 5-10% MeOH in CH2Cl2 (v/v)
to give nucleoside 15 (73 mg, 84%) as a white solid: Rf (20%
MeOH in CH2Cl2 (v/v)) 0.51. FAB-MS m/z: 271.0 [M + H]+.
Found: m/z (FAB, NBA + PEG300 matrix) 271.0930. Calcd
1
for C11H15O6N2: 271.0930. H NMR (DMSO-d6): δ 11.29 (1H,
s, NH), 7.76 (1H, d, J ) 8.2 Hz, H6), 5.60 (1H, d, J ) 8.1 Hz,

Novel Bicyclic Nucleoside Analogue
J . Org. Chem., Vol. 66, No. 16, 2001 5503
H5), 5.23 (1H, d, J ) 5.3 Hz, OH-4′), 4.87 (1H, t, J ) 6.0 Hz,
OH-6′), 4.02 (1H, d, J ) 10.1 Hz, H1′), 4.01 (1H, m, H4′), 3.84
(1H, d, J ) 11.2 Hz, H6′′), 3.53 (1H, dd, J ) 5.9, 12.3 Hz, H6′),
3.50 (1H, d, J ) 11.2 Hz, H6′′), 3.39 (1H, dd, J ) 6.0, 12.5 Hz,
H6′), 3.23 (1H, d, J ) 10.3 Hz, H1′), 2.50 (1H, m, H3′), 2.37
(1H, dd, J ) 10.9, 13.5 Hz, H3′). 13C NMR (DMSO-d6): δ 163.43
(C4), 149.64 (C2), 139.93 (C6), 100.78 (C5), 91.28 (C2′), 85.85
(C5′), 70.50 (C1′), 69.54 (C4′), 65.54 (C6′′), 61.39 (C6′), 43.56
(C3′).
etonitrile (2 × 20 mL). The residue was purified twice by silica
gel column chromatography, eluting first with 0.5:50:49.5
pyridine/EtOAc/n-hexane (v/v/v) and then with a gradient of
0.5:25-60:74.5-39.5 pyridine/EtOAc/n-hexane (v/v/v) to give
phosphoramidite 17 (36 mg, 26%) as a white foam after
coevaporation with acetonitrile (4 × 5 mL) and CH2Cl2 (5
mL): Rf (10% MeOH in CH2Cl2 (v/v)) 0.50. FAB-MS m/z: 773.2
[M + H]+. 31P NMR (CDCl3): δ 150.59.
Oligon u cleotid e Syn th esis. All oligomers were synthe-
sized on a DNA synthesizer using the phosphoramidite ap-
proach.29 The stepwise coupling yields were determined spec-
trophotometrically at 498 nm (quantifying the released 4,4′-
dimethoxytrityl group). Standard conditions were used, except
for the phosphoramidite 17 which was “hand-coupled” [pre-
mixing a ∼0.05 M solution of amidite in anhydrous acetonitrile
(0.2 mL, 10 µmol) and a ∼0.5 M solution of tetrazole in
anhydrous acetonitrile (0.3 mL, 150 µmol) in a syringe and
via an adaptor slowly flushing this mixture through a syn-
thesis column for 10 min]. Coupling yields for the amidite 17
were ∼98%, while coupling yields for the unmodified amidites
were >99%. Cleavage from the solid support and removal of
the protecting groups was accomplished using conc aq am-
monia (55 °C for 16 h), and purification, by ethanol precipita-
tion of the oligonucleotides. Capillary gel electrophoresis of all
synthesized sequences showed >90% purity. The composition
of the modified 9-mers was verified by MALDI-MS analysis.
Found (entry 3, Table 1): m/z 2778.6 [M - H]-. Calcd: 2780.7.
Found (entry 5, Table 5): m/z 2833.6 [M - H]-. Calcd: 2836.4.
(1S ,5R ,6S )-5-(4,4′-Dim e t h oxyt r it yloxym e t h yl)-6-h y-
d r oxy-1-(u r a cil-1-yl)-3,8-d ioxa b icyclo[3.2.1]oct a n e (16).
Nucleoside 15 (73 mg, 0.269 mmol) was dissolved in anhydrous
pyridine (10 mL), evaporated to about half the volume under
reduced pressure, and cooled to 0 °C, whereupon DMTCl (184
mg, 0.541 mmol) was added. The mixture was stirred at room
temperature for 2 h 20 min, additional DMTCl (93 mg, 0.27
mmol) was added, and the mixture was stirred for 30 min.
MeOH (1 mL) and saturated aq NaHCO3 (1 mL) were added,
and the mixture was evaporated to dryness under reduced
pressure, coevaporated with acetonitrile (2 × 30 mL), sus-
pended in CH2Cl2 (20 mL), and evaporated on silica gel. The
residue was purified by silica gel column chromatography (9.8
cm × 2.0 cm), eluting with a gradient of 0.5:0-3:99.5-96.5
pyridine/MeOH/CH2Cl2 (v/v/v) to give nucleoside 16 (103 mg,
67%) as a white foam after coevaporation with acetonitrile (5
× 10 mL) and CH2Cl2 (10 mL): Rf (10% MeOH in CH2Cl2 (v/
v)) 0.43. FAB-MS m/z: 573.1 [M + H]+. 1H NMR (99:1 CDCl3/
CD3OD (v/v)): δ 10.05 (1H, br s, NH), 7.63 (1H, d, J ) 8.2 Hz,
H6), 7.36-7.14 (9H, m, DMT), 6.77-6.74 (4H, m, DMT), 5.64
(1H, d, J ) 8.2 Hz, H5), 4.23 (1H, d, J ) 10.3 Hz, H1′), 4.04
(1H, m, H4′), 4.03 (1H, d, J ) 11.7 Hz, H6′′), 3.79 (1H, d, J )
11.7 Hz, H6′′), 3.71 (6H, s, OMe), 3.32 (1H, d, J ) 10.3 Hz,
H1′), 3.18 (2H, s, H6′), 2.71 (1H, dd, J ) 3.8, 14.1 Hz, H3′),
2.56 (1H, br s, OH), 2.44 (1H, dd, J ) 11.2, 14.1 Hz, H3′). 13C
NMR (99:1 CDCl3/CD3OD (v/v)): δ 163.75 (C4), 158.35 (DMT),
149.45 (C2), 144.15 (DMT), 139.53 (C6), 135.31, 135.16, 129.85,
129.79, 127.96, 127.83, 127.64, 127.48, 126.73, 112.96, 112.80
(DMT), 101.38 (C5), 91.77 (C2′), 86.09 (CAr2Ph), 85.56 (C5′),
72.33 (C4′), 71.19 (C1′), 67.30 (C6′′), 64.25 (C6′), 55.00 (OMe),
44.27 (C3′).
Ack n ow led gm en t. Ms. Britta Dahl, Department of
Chemistry, University of Copenhagen, is thanked for
oligonucleotide synthesis. Dr. Michael Meldgaard,
Exiqon A/S, is thanked for recording the MALDI-MS
spectra. Dr. Per-Ola Norrby, Department of Chemistry,
Technical University of Denmark, is thanked for the
molecular modeling. The Danish Natural Science Re-
search Council, The Danish Technical Research Council,
and Exiqon A/S are thanked for financial support.
Su p p or tin g In for m a tion Ava ila ble: Copies of 13C NMR
spectra for 7-11, 11B, and 12-16, a copy of the 31P NMR
spectrum for 17, copies from the transcript from capillary gel
electrophoretic analysis of the oligonucleotides containing 4
(entries 3 and 5, Table 1), and Cartesian coordinates of the
lowest-energy conformer of 15 obtained by the energy-
minimization (15.cc1). This material is available free of charge
via the Internet at http://pubs.acs.org.
(1S ,5R ,6S )-6-(2-C y a n o e t h o x y (d iis o p r o p y la m in o )-
ph osph in oxy)-5-(4,4′-dim eth oxytr ityloxym eth yl)-1-(u r acil-
1-yl)-3,8-d ioxa bicyclo[3.2.1]octa n e (17). Nucleoside 16 (103
mg, 0.179 mmol) was coevaporated with anhydrous acetonitrile
(2 × 10 mL), dissolved in anhydrous CH2Cl2 (5 mL), and cooled
to 0 °C. Diisopropylethylamine (0.5 mL) was added, followed
by 2-cyanoethyl N,N-diisopropylphosphoramidochloridite (0.25
mL, 1.1 mmol), and the mixture was stirred at room temper-
ature for 3 h. H2O (2 mL) and EtOAc (100 mL) were added,
and the mixture was washed with saturated aq NaHCO3 (20
mL) and H2O (20 mL). The organic phase was evaporated to
dryness under reduced pressure and coevaporated with ac-
J O015664L
(29) Caruthers, M. H. Acc. Chem. Res. 1991, 24, 278-284.