Convenient Syntheses of 7-Hydroxy-1-(hydroxymethyl)-3-(thymin-1-yl)-2,5-dioxabicyclo[2.2.1]heptanes: α-L-Ribo- and α-L-Xylo-Configured LNA Nucleosides

Article abstract of DOI:10.1021/jo000232g

Synthesis of the diastereoisomeric LNA (locked nucleic acid) nucleosides 1-(2-O,4-C-methylene-α-L-ribofuranosyl)thymine (6) and 1-(2-O,4-C-methylene-α-L-xylofuranosyl)thymine (13) are reported via convenient reaction cascades from di-O-p-toluenesulfonyl and tri-O-methanesulfonyl nucleoside derivatives 3, 7, and 10.

Full text of DOI:10.1021/jo000232g

J . Org. Chem. 2000, 65, 5161-5166
5161
Con ven ien t Syn th eses of 7-Hyd r oxy-1-(h yd r oxym eth yl)-
3-(th ym in -1-yl)-2,5-d ioxa bicyclo[2.2.1]h ep ta n es: r-L-Ribo- a n d
r-L-Xylo-Con figu r ed LNA Nu cleosid es
Anders E. Håkansson,† Alexei A. Koshkin,‡ Mads D. Sørensen,† and J esper Wengel*,†,§
Center for Synthetic Bioorganic Chemistry, Department of Chemistry, University of Copenhagen,
Universitetsparken 5, DK-2100 Copenhagen, Denmark, and Exiqon A/ S, Bygstubben 9,
DK-2950 Vedbæk, Denmark
jwe@chem.sdu.dk
Received February 18, 2000
Synthesis of the diastereoisomeric LNA (locked nucleic acid) nucleosides 1-(2-O,4-C-methylene-R-
L-ribofuranosyl)thymine (6) and 1-(2-O,4-C-methylene-R-L-xylofuranosyl)thymine (13) are reported
via convenient reaction cascades from di-O-p-toluenesulfonyl and tri-O-methanesulfonyl nucleoside
derivatives 3, 7, and 10.
“LNA” (locked nucleic acid, â-D-ribo isomer, Figure 1)
is a novel nucleic acid analogue1-3 displaying unprec-
edented binding affinity toward complementary DNA and
RNA. The furanose ring of an LNA nucleoside monomer,
being part of a dioxabicyclo[2.2.1]heptane skeleton, is
efficiently locked in an N-type conformation (C3′-endo/
3E conformation). We have very recently studied the
properties of diastereoisomeric forms of LNA (Figure 1),
i.e. “R-L-LNA” (R-L-ribo isomer),4,5 “xylo-LNA” (â-D-xylo
isomer),4 and “R-L-xylo-LNA” (R-L-xylo isomer).5 Espe-
cially the fact4,5 that R-L-LNA displays nucleic acid
binding properties closely approaching those of parent
LNA highlights the importance of developing efficient
synthetic procedures for the diastereoisomeric LNA
nucleosides. Whereas synthesis of the monomeric thy-
mine LNA and xylo-LNA nucleosides has been reported
earlier,1,6 we herein describe the first syntheses of the
thymine R-L-LNA and R-L-xylo-LNA nucleosides 6 and
13, respectively.
The starting material for synthesis of the R-L-LNA
thymine nucleoside 6 (Scheme 1) was the 4′-C-hydroxy-
methyl nucleoside 1 which was obtained from 1,2:5,6-di-
O-isopropylidine-R-D-glucofuranose as described earlier.6
Selective monoprotection of 1 by reaction with 1.1 equiv
F igu r e 1. Four synthesized diastereoisomeric LNAs.1-5
of 4,4′-dimethoxytrityl chloride ((DMT)Cl) afforded nu-
cleoside diol 2 in limited 31% yield as the major product.
According to analytical TLC, a slightly more polar DMT-
containing byproduct as well as a less polar DMT-
containing byproduct were formed in minor amounts. The
preferential formation of the desired mono DMT-protect-
ed product 2 can be explained by sterical hindrance, i.e.
the positioning of both a benzyloxy and a thymine moiety
at the same face of the furanose ring in starting material
1. The structure of 2 was conclusively assigned on the
basis of the unambiguous structure of the cyclized
nucleosides, e.g. 4 and 6. To prepare for ring closure, the
di-O-tosylated derivative 3 was prepared in 63% yield by
treatment with a 10-fold excess of p-toluenesulfonyl
chloride (TsCl) and a catalytic amount of 4-(N,N-di-
methylamino)pyridine (DMAP) in pyridine. Conversion
of nucleoside 3 directly to the desired 2,5-dioxabicyclo-
[2.2.1]heptane derivative 4 proceeded very satisfactory
in 81% yield in a mixture of 2 M aqueous NaOH and H2O:
EtOH (1:1; heating under reflux for 24 h). This conversion
involves epimerization at the 2′-carbon atom and subse-
quent cyclization and is anticipated to proceed via the
three integrated reaction steps shown in Figure 2. Thus,
† University of Copenhagen.
‡ Exiqon A/S.
§ Present address: Department of Chemistry, University of South-
ern Denmark, DK-5230 Odense M, Denmark.
(1) (a) Singh, S. K.; Nielsen, P.; Koshkin, A. A.; Wengel, J . Chem.
Commun. 1998, 455. (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. (c) Koshkin, A. A.; Nielsen, P.; Meldgaard,
M.; Rajwanshi, V. K.; Singh, S. K.; Wengel, J . J . Am. Chem. Soc. 1998,
120, 13252.
(2) We have defined LNA as an oligonucleotide containing one or
more 2′-O,4′-C-methylene-â-D-ribofuranosyl nucleotide monomer(s).
The natural â-D-ribo configuration is assigned to LNA (and LNA
nucleosides) as the positioning of the 1-nitrogen and 2′-, 3′- and 5′-
oxygen atoms follows that of RNA.
(3) Obika, S.; Nanbu, D.; Hari, Y.; Andoh, J .; Morio, K.; Doi, T.;
Imanishi, T. Tetrahedron Lett. 1998, 39, 5401.
(4) (a) Rajwanshi, V. K.; Håkansson, A. E.; Dahl, B. M.; Wengel, J .
Chem. Commun. 1999, 1395. (b) Rajwanshi, V. K.; Håkansson, A. E.;
Kumar, R.; Wengel, J . Chem. Commun. 1999, 2073.
(5) 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.
(6) Rajwanshi, V. K.; Kumar, R.; Hansen, M. K.; Wengel, J . J . Chem.
Soc., Perkin Trans. 1 1999, 1407.
10.1021/jo000232g CCC: $19.00 © 2000 American Chemical Society
Published on Web 08/03/2000

5162 J . Org. Chem., Vol. 65, No. 17, 2000
Håkansson et al.
Sch em e 1a
a
Reagents: (i) DMTCl, AgNO3, THF, pyridine (31%); (ii) TsCl, DMAP, pyridine (63%); (iii) 2 M NaOH, EtOH, H2O (81%); (iv) HCOONH4,
10% Pd/C, MeOH (5: 80%; 6: 17%); (v) MsCl, pyridine (92%); (vi) 6 M NaOH, EtOH, H2O (58%); (vii) DMTCl, pyridine (93%); (viii)
H2/(Pd(OH)2/C)/EtOH (98%); (ix) DMTCl, pyridine (98%).
furanosyl)thymine (6), the parent R-L-LNA thymine
nucleoside (Scheme 1). Conversion of DMT-derivative 5
to the desired 3′-O-phosphitylated building block has
been described elsewhere.4a
Alternatively, the tri-O-mesyl derivative 7 was ob-
tained in 92% yield from nucleoside triol 1 by reaction
with excess methanesulfonyl chloride (MsCl) in anhy-
drous pyridine. Treatment of nucleoside 7 with a mixture
of 6 M NaOH and H2O:EtOH (1:1; heating under reflux
for 43 h) afforded the desired bicyclic 3′-O-benzylated
derivative 8 in 58% yield (Scheme 1). An analogous
reaction cascade such as the one described above is
anticipated to be involved, in this case supplemented by
the conversion of the 5′-mesyloxy group into a hydroxy
group.11 DMT protection of nucleoside 8 afforded in 93%
compound 4 which above has been shown to be a
convenient intermediate in the synthesis of nucleoside
5. In another approach toward 5, debenzylation of
nucleoside 8 to give diol 6 was attempted first unsuc-
cessfully using ammonium formate/(10% Pd/C)/MeOH
(reflux, 53 h) and then successfully in 98% yield using
H2/(Pd(OH)2/C)/EtOH (room temperature, 24 h). Subse-
quent selective DMT protection of the primary hydroxy
group of nucleoside 6 afforded in 98% yield the desired
nucleoside intermediate 5 (see above). The two alterna-
tive routes from nucleoside 1 to intermediate 5 (via 2, 3,
and 4 or via 7, 8, and 6) involve the same number of steps
but especially the straightforward synthesis of tri-O-
mesylate 7 from 1 in 92% yield makes this route the
preferred one, not only for synthesis of 5 but also for
synthesis of the unprotected R-L-LNA nucleoside 6. It
should be mentioned that the cyclization efficiency (see
also the subsequent conversion of 10 to 11) seems
comparable for 2′-O-tosyl and 2′-O-mesyl derivatives,
and, e.g., the tri-O-tosyl derivative of 7 would likely afford
the 5′-O-tosyl derivative of 8. However, due to the ease
of conversion of a 5′-O-mesyl LNA derivative into the
desired 5′-OH derivative,11 we choose to proceed via the
tri-O-mesyl derivatives 7 and 10.
F igu r e 2. Suggested mechanism for the conversion of nucleo-
side 3 into the bicyclic derivative 4.
a reaction cascade involving 2-2′-anhydro intermediate
formation by intramolecular nucleophilic attack, hydroly-
sis of this intermediate to give a putative 2′-OH inter-
mediate with inverted configuration at the 2′-carbon
atom, and a second intramolecular nucleophilic attack
on the 5′-tosyloxy group from the inverted 2′-OH group
offers a probable mechanism for the successful stereose-
lective conversion of compound 3 to 4. This convenient
synthesis of nucleoside 4 stresses the usefulness of
anhydro-intermediate formation7 in pyrimidine nucleo-
side chemistry as also illustrated by recent syntheses of
nucleosides containing other bicyclic furanose moieties.8,9
For automated synthesis of R-L-LNA the phosphor-
amidite approach10 with 5′-O-DMT-protected 3′-O-phos-
phitylated building blocks was used.4 It was therefore
very convenient that hydrogenolysis of nucleoside 4
chemoselectively furnished the debenzylated 5′-O-DMT-
protected derivative 5 in 80% yield together with a small
amount (17% yield) of 1-(2-O,4-C-methylene-R-L-ribo-
In Scheme 2, an analogous approach for synthesis of
the R-L-xylo-LNA thymine nucleoside 13 is described
starting from 5′-O-mesyl-4′-C-mesyloxymethyl-D-ribo-
furanosyl nucleoside 9.11 2′-O-Mesylation afforded in 97%
yield the tri-O-mesyl nucleoside 10, and concomitant
treatment with a mixture of 2 M aqueous NaOH and
H2O:EtOH (1:1; heating under reflux for 40 h) yielded a
(7) Yung, N. C.; Fox, J . J . J . Am. Chem. Soc. 1961, 83, 3060.
(8) Nielsen, P.; Pfundheller, H. M.; Olsen, C. E.; Wengel, J . J . Chem.
Soc., Perkin Trans. 1 1997, 3423.
(9) Miah, A.; Reese, C. B.; Song, Q.; Sturdy, Z.; Niedle, S.; Simpson,
I. J .; Read, M.; Rayner, E. J . Chem. Soc., Perkin Trans. 1 1998, 3277.
(10) Caruthers, M. H. Acc. Chem. Res. 1991, 24, 278.
(11) Koshkin, A. A. Manuscript in preparation.

Synthesis of Diastereomeric LNA Nucleosides
J . Org. Chem., Vol. 65, No. 17, 2000 5163
Sch em e 2a
a
Reagents: (i) MsCl, pyridine (97%); (ii) 2 M NaOH, EtOH, H2O (11: 35%; 12: 32%); (iii) KOH, EtOH (79%); (iv) H2, 20% Pd(OH)2/C,
EtOH (93%); (v) DMTCl, pyridine (95%); (vi) NC(CH2)2OP(Cl)N(i-Pr)2, EtN(i-Pr)2, CH2Cl2 (60%).
mixture of 5′-O-mesyl nucleoside 11 (35% yield) and the
desired 5′-hydroxy nucleoside 12 (32% yield). Under more
vigorous conditions (KOH in EtOH, 60 °C, 12 h) it was
possible to achieve efficient conversion of 11 to 12 (79%
yield). Debenzylation of 3′-O-benzyl nucleoside 12 to give
1-(2-O,4-C-methylene-R-L-xylofuranosyl)thymine (13), the
parent R-L-xylo-LNA thymine nucleoside, proceeded in
93% yield with H2 and 20% Pd(OH)2/C in EtOH. To
prepare for automated synthesis of R-L-xylo-LNA oligo-
mers, the primary hydroxy group of nucleoside diol 13
was selectively DMT-protected to give nucleoside 14 in
95% yield by reaction with DMTCl in pyridine followed
by 3′-O-phosphitylation in 60% yield using 2-cyanoethyl
N,N-diisopropylphosphoramidochloridite and N,N-diiso-
propylethylamine in CH2Cl2, yielding the desired phos-
phoramidite derivative 15.12
By virtue of their 2,5-dioxabicyclo[2.2.1]heptane skel-
eton the conformations of the furanose rings of the
diastereoisomeric LNA nucleosides 6 and 13 are ef-
ficiently locked. Molecular modeling (HyperChem pro-
gram, Polak-Ribiere algorithm) show that the furanose
ring of the R-L-LNA nucleosides 6 and the R-L-xylo-LNA
nucleoside 13 adopt an S-type conformation (C3′-exo/3E
conformation). For structural verification, NOE experi-
ments were performed on the unprotected nucleosides 6
and 13. For compound 6, mutual NOEs between H-6/H-
5′′ (6%/11%; H-5′′ designates the methylene hydrogens
in the 4′-C-branch), H-1′/H-2′ (9%/8%), and H-1′/H-3′
(17%/11%), as well as NOEs in 3′-OH (2% when irradiat-
ing H-5′′) and in H-3′ (3% when irradiating H-5′) support
the assigned R-L-ribo configuration and C3′-exo confor-
mation. For compound 13, mutual NOEs between H-6/
H-5′′ (3%/11%) and H-1′/H-2′ (8%/8%), as well as NOEs
in 3′-OH (3% when irradiating H-5′) and in H-2′ (3%
when irradiating 3′-OH) support the assigned R-L-xylo
configuration and C3′-exo conformation.
the methods devised have allowed discovery of the
remarkable hybridization properties of R-L-LNA and
should in addition allow synthesis of a variety of novel
nucleosides for biological screening.
Exp er im en ta l Section
Gen er a l P r oced u r e. Reactions were conducted under an
atmosphere of nitrogen when anhydrous solvents were used.
Column chromatography was carried out on glass columns
using Silica gel 60 (0.040-0.063 mm). After drying organic
phases using Na2SO4, filtration was performed. Petroleum
ether of distillation range 60-80 °C was used. Chemical shifts
are reported in parts per million relative to tetramethylsilane
1
as internal standard for H (300 or 400 MHz) and 13C (75.5 or
100.6 MHz) and relative to 85% H3PO4 (121.5 MHz) as external
standard for 31P. Assignments of NMR spectra when given are
based on 2D spectra and follow standard nucleoside style; i.e.
the carbon atom next to the nucleobase is assigned C-1′ etc.
(C-5′′ designates the carbon atom in the 4′-C-branch). However,
compound names in the Experimental Section for the bicyclic
compounds are given according to the von Baeyer nomencla-
ture. Fast-atom bombardment mass spectra (FAB-MS) were
recorded in positive ion mode. Microanalyses were performed
at The Microanalytical Laboratory, Department of Chemistry,
University of Copenhagen.
1-(3-O-Ben zyl-4-C-(4,4′-d im eth oxytr ityloxym eth yl)-â-D-
xylofu r a n osyl)th ym in e (2). To a solution of 1-(3-O-benzyl-
4-C-hydroxymethyl-â-D-xylofuranosyl)thymine (1)6 (5.38 g, 14.2
mmol) in anhydrous tetrahydrofuran (THF) (400 mL) was
added AgNO3 (2.66 g, 15.7 mmol) followed by anhydrous
pyridine (5.7 mL) and 4,4′-dimethoxytrityl chloride ((DMT)-
Cl, 5.30 g, 15.6 mmol). The mixture was stirred in the dark
for 18 h at room temperature whereupon the reaction was
quenched by addition of a saturated aqueous solution of
NaHCO3, and the resulting mixture was extracted with CH2-
Cl2. The combined organic phase was evaporated to dryness
under reduced pressure, and the residue was coevaporated
with toluene and was purified by silica gel column chroma-
tography using CH2Cl2/MeOH/pyridine (99:0.5:0.5 (v/v/v)) as
eluent affording nucleoside 2 (3.13 g, 31%) as a white foam
after evaporation of the solvents and coevaporation with
In conclusion, convenient syntheses of the diastereo-
isomeric LNA nucleosides 1-(2-O,4-C-methylene-R-L-ri-
bofuranosyl)thymine (6) and 1-(2-O,4-C-methylene-R-L-
xylofuranosyl)thymine (13), and protected derivatives
thereof, have been developed. The syntheses involve
efficient reaction cascades from di-O-tosyl and tri-O-
mesyl nucleoside derivatives with stereochemical inver-
sion at the 2′-carbon atoms which limits the synthetic
utility to nucleosides of the pyrimidine series. However,
1
toluene. H NMR ((CD3)2SO) δ 11.34 (s, 1H, NH), 7.86 (s, 1H,
6-H), 6.85-7.40 (m, 18H, m, DMT, Bn), 5.93 (d, 1H, J 7.3 Hz,
1′-H), 5.80 (d, 1H, J 5.5 Hz, 2′-OH), 4.82 (t, 1H, J 5.3 Hz, 5′-
OH), 4.63 (d, 1H, J 12.0 Hz, Bn), 4.42-4.46 (m, 2H, Bn, 2′-H),
4.14 (d, 1H, J 7.3 Hz, 3′-H), 3.72 (s, 6H, DMT), 3.65-3.69 (m,
1H, 5′-H), 3.50-3.47 (m, 1H, 5′-H), 3.09 (d, 1H, J 9.7 Hz, 5′′-
H), 2.98 (d, 1H, J 9.7 Hz, 5′′-H), 1.78 (s, 3H, CH3). 13C NMR
((CD3)2SO) δ 164.1 (C-4), 158.4, 145.1, 138.5, 137.0, 135.9,
135.7, 130.1, 130.1, 129.2, 128.5, 128.5, 128.2, 128.1, 127.7,
127.6, 127.0, 125.7, 113.5 (DMT, Bn, C-6), 151.4 (C-2), 110.1
(C-5), 85.8, 85.2, 84.6, 83.5 (C-1′, C-3′, C-4′, DMT), 76.8 (C-2′),
72.3 (Bn), 65.2 (C-5′′), 62.1 (C-5′), 55.4 (DMT), 12.6 (CH3).
(12) Amidite 15 has successfully been used for synthesis of R-L-xylo-
LNA5 on an automated DNA synthesizer using the same conditions
described for synthesis of R-L-LNA.4a

5164 J . Org. Chem., Vol. 65, No. 17, 2000
Håkansson et al.
1-(3-O-Ben zyl-4-C-(4,4′-d im eth oxytr ityloxym eth yl)-2,5-
d i-O-(p-tolu en esu lfon yl)-â-D-xylofu r a n osyl)th ym in e (3).
To a solution of nucleoside 2 (2.79 g, 3.9 mmol) in anhydrous
pyridine (10 mL) was added p-toluenesulfonyl chloride (TsCl,
6.50 g, 34 mmol) and a catalytic amount of DMAP. The mixture
was stirred in the dark for 24 h at room temperature
whereupon the reaction was quenched by addition of a
saturated aqueous solution of NaHCO3 (25 mL). The resulting
mixture was extracted with CH2Cl2, and the combined organic
phase was washed successively with saturated aqueous solu-
tions of NaHCO3 and sodium chloride, dried (Na2SO4), and
evaporated to dryness under reduced pressure. The residue
was purified by silica gel column chromatography using CH2-
Cl2/MeOH/pyridine (99:0.5:0.5 (v/v/v)) as eluent, yielding
nucleoside 3 (2.40 g, 63%) as a yellowish foam after evapora-
tion of the solvents. FAB-MS m/z 989 [M + H]+, 1011.4 [M +
Na]+; 1H NMR ((CD3)2SO) δ 11.29 (s, 1H, NH), 8.58 (s, 1H,
6-H), 6.84-7.66 (m, 26H, DMT, Bn, Ts), 6.10 (d, 1H, J 7.7 Hz,
1′-H), 5.27 (t, 1H, J 7.2 Hz, 2′-H), 4.44 (d, 1H, J 6.8 Hz, 3′-H),
4.39 (d, 1H, J 11.4 Hz, Bn), 4.24 (d, 1H, J 11.5 Hz, Bn), 4.18
(d, 1H, J 10.4 Hz, 5′-H), 4.13 (d, 1H, J 10.3 Hz, 5′-H), 3.73 (s,
3H, DMT), 3.73 (s, 3H, DMT), 3.14 (d, 1H, J 10.3 Hz, 5′′-H),
3.07 (d, 1H, J 10.3 Hz, 5′′-H), 2.35 (s, 3H, Ts), 2.34 (s, 3H, Ts),
1.72 (s, 3H, CH3); 13C NMR ((CD3)2SO) δ 163.2 (C-4), 158.2,
145.9, 145.1, 144.3, 136.8, 135.0, 134.9, 134.8, 131.8, 131.6,
130.2, 130.0, 129.7, 128.2, 127.9, 127.8, 127.6, 127.5, 127.5,
127.4, 126.8, 113.3 (DMT, C-6, 2 × Ts, Bn), 150.2 (C-2), 110.8
(C-5), 95.0, 86.2 (DMT, C-4′), 82.2, 81.9 (C-1′, C-2′), 81.2 (C-
3′), 72.9 (Bn), 79 (C-5′′), 64 (C-5′), 55.1 (DMT), 21.2, 21.2 (2 ×
Ts), 12.0 (CH3).
(1S,3R,4S,7R)-7-Ben zyloxy-1-(4,4′-d im eth oxytr ityloxy-
m eth yl)-3-(th ym in -1-yl)-2,5-dioxabicyclo[2.2.1]h eptan e (4).
To a solution of nucleoside 3 (3.87 g, 3.92 mmol) in a mixture
of EtOH and H2O (100 mL, 1:1 (v/v)) was added a solution of
aqueous NaOH (2 M, 8 mL). The mixture was heated under
reflux for 24 h and, after cooling to room temperature,
extracted with CH2Cl2. The combined organic phase was
washed with a saturated aqueous solution of NaHCO3, dried
(Na2SO4), and evaporated to dryness under reduced pressure.
The residue was purified by silica gel column chromatography
using CH2Cl2/MeOH/pyridine (99:0.5:0.5 (v/v/v)) as eluent,
affording nucleoside 4 (2.10 g, 81%) as a white foam after
evaporation of the solvents. FAB-MS m/z 663 [M + H]+; 1H
NMR ((CD3)2SO) δ 11.40 (s, 1H, NH), 7.62 (s, 1H, 6-H), 6.84-
7.43 (m, 18H, DMT, Bn), 6.04 (s, 1H, 1′-H), 4.70 (d, 1H, J 11.9
Hz, Bn), 4.64 (s, 1H, 2′-H), 4.60 (d, 1H, J 12.1 Hz, Bn), 4.57 (s,
1H, 3′-H), 4.11 (d, 1H, J 8.2 Hz, 5′-H), 3.88 (d, 1H, J 8.6 Hz,
5′-H), 3.73 (s, 3H, DMT), 3.72 (s, 3H, DMT), 3.36 (d, 1H, J
11.0 Hz, 5′′-H), 3.26 (d, 1H, J 10.8 Hz, 5′′-H), 1.84 (s, 3H, CH3);
13C NMR ((CD3)2SO) δ 163.8 (C-4), 158.2, 158.1, 144.7, 137.7,
135.9, 135.2, 135.1, 129.8, 129.7, 128.3, 127.9, 127.7, 127.7,
127.4, 126.7, 113.35 (DMT, Bn, C-6) 150.3 (C-2), 108.1 (C-5),
88.4, 85.5 (C-4′, DMT), 86.4 (C-1′), 79.5 (C-2′), 76.3 (C-3′), 72.6
(C-5′), 71.2 (Bn), 58.9 (C-5′′), 55.1 (DMT), 12.4 (CH3).
2), 108.0 (C-5), 89.2, 85.4 (C-4′, DMT), 86.4 (C-1′), 78.9 (C-2′),
72.9 (C-3′), 72.3 (C-5′), 59.9 (C-5′′), 55.1 (DMT), 12.5 (CH3).
1
Nu cleosid e 6: FAB-MS m/z 271 [M + H]+; H NMR ((CD3)2-
SO) δ 11.34 (s, 1H, NH), 7.63 (s, 1H, 6-H), 5.97 (d, 1H, J 4.3
Hz, 3′-OH), 5.96 (s, 1H, 1′-H), 4.94 (t, 1H, J 5.8 Hz, 5′′-OH),
4.27 (d, 1H, J 4.1 Hz, 3′-H), 4.20 (s, 1H, 2′-H), 3.94 (d, 1H, J
8.4 Hz, 5′-H), 3.92 (d, 1H, J 8.4 Hz, 5′-H), 3.73 (d, 2H, J 5.6
Hz, 5′′-H), 1.93 (s, 3H, CH3); 13C NMR ((CD3)2SO) δ 163.9 (C-
4), 150.4 (C-2), 136.0 (C-6), 107.9 (C-5), 91.0 (C-4′), 86.5 (C-
1′), 78.9 (C-2′), 72.5 (C-3′), 72.0 (C-5′), 57.5 (C-5′′), 12.3 (CH3).
The enantiomer of 6 has been published earlier.13
1-(3-O-Ben zyl-2,5-d i-O-m eth a n esu lfon yl-4-C-(m eth a n e-
su lfon yloxym eth yl)-â-D-xylofu r a n osyl)th ym in e (7). To a
solution of nucleoside 16 (1.63 g, 4.31 mmol) in anhydrous
pyridine (8 mL) at 0 °C was added drop by drop methane-
sulfonyl chloride (MsCl, 1.2 mL, 15.5 mmol). The mixture was
stirred for 2 h at 0 °C and then at 2 h at room temperature.
The reaction mixture was cooled to 0 °C, and H2O (4 mL) was
added followed by a saturated aqueous solution of NaHCO3
(10 mL). Extraction was performed with CH2Cl2, and the
organic phase was dried (Na2SO4) and evaporated to dryness
under reduced pressure. The residue was purified by silica gel
column chromatography using CH2Cl2/MeOH (99:1 (v/v)) as
eluent, affording nucleoside 7 (2.41 g, 92%) as a white solid
material after evaporation of the solvents. FAB-MS m/z 613
1
[M + H]+; H NMR (CDCl3) δ 9.53 (br s, 1H, NH), 7.27-7.40
(m, 6H, Bn, 6-H), 6.16 (d, 1H, J 3.7 Hz, 1′-H), 5.30 (t, 1H, J
3.6 Hz, 2′-H), 4.76 (d, 1H, J 11.4 Hz, Bn), 4.66 (d, 1H, J 11.4
Hz, Bn), 4.61 (d, 1H, J 11.2 Hz), 4.44 (d, 1H, J 3.3 Hz, 3′-H),
4.39 (d, 1H, J 11.2), 4.34 (d, 1H, J 10.8 Hz), 4.26 (d, 1H, J
10.8 Hz), 3.19 (s, 3H, Ms), 3.06 (s, 3H, Ms), 3.02 (s, 3H, Ms),
1.86 (s, 3H, CH3); 13C NMR (CDCl3) δ 163.3 (C-4), 150.6 (C-2),
135.6, 134.6 (C-6, Bn), 128.7, 128.3 (Bn), 112.2 (C-5), 87.9 (C-
1′), 85.0 (C-4′), 83.1 (C-2′), 80.9 (C-3′), 73.3 (Bn), 66.6, 66.2 (C-
5′, C-5′′), 38.6, 37.6, 37.6 (3 × Ms), 12.2 (CH3).
(1R,3R,4S,7R)-1-(Hyd r oxym eth yl)-7-ben zyloxy-3-(th y-
m in -1-yl)-2,5-d ioxa bicyclo[2.2.1]h ep ta n e (8). To a solution
of nucleoside 7 (522 mg, 0.85 mmol) in a mixture of EtOH and
H2O (16 mL, 1:1 (v/v)) was added aqueous NaOH (6 M, 1.5
mL). After heating under reflux for 43 h, the mixture was
evaporated to dryness under reduced pressure, the residue was
dissolved in CH2Cl2 (10 mL), and washing was performed using
a saturated aqueous solution of NaHCO3. The organic phase
was dried (Na2SO4) and evaporated to dryness under reduced
pressure. The residue was purified by silica gel column
chromatography using CH2Cl2/MeOH (97.5:2.5 (v/v)) as eluent
affording nucleoside 8 (179 mg, 58%) as a white solid material
after evaporation of the solvents. FAB-MS m/z 361 [M + H]+;
1H NMR ((CD3)2SO) δ 11.37 (s, 1H, NH), 7.63 (s, 1H, 6-H),
7.27-7.39 (m, 5H, Bn), 5.87 (s, 1H, 1′-H), 5.07 (br s, 1H, 5′′-
OH), 4.69 (d, 1H, J 11.9 Hz), 4.65 (d, 1H, J 11.9 Hz), 4.51 (s,
1H, 2′-H), 4.33 (s, 1H, 3′-H), 4.01 (d, 1H, J 8.6 Hz), 3.93 (d,
1H, J 8.4 Hz), 3.78 (d, 1H, J 13.0 Hz), 3.74 (d, 1H, J 12.8 Hz),
1.83 (s, 1H, CH3); 13C NMR ((CD3)2SO) δ 163.8 (C-4), 150.3
(C-2), 138.0, 135.8 (C-6, Bn), 128.3, 127.7, 127.5 (Bn), 108.0
(C-5), 90.2 (C-4′), 86.5 (C-1′), 79.3 (C-3′), 76.5 (C-2′), 72.5, 71.2,
57.2 (Bn, C-5′, C-5′′), 12.3 (CH3).
(1S,3R,4S,7R)-1-(4,4′-Dim eth oxytr ityloxym eth yl)-7-h y-
d r oxy-3-(th ym in -1-yl)-2,5-d ioxa bicyclo[2.2.1]h ep ta n e (5)
a n d (1R,3R,4S,7R)-7-Hyd r oxy-1-(h yd r oxyoxym et h yl)-3-
(th ym in -1-yl)-2,5-d ioxa bicyclo[2.2.1]h ep ta n e (6). To a so-
lution of nucleoside 4 (1.09 g, 1.65 mmol) in MeOH (90 mL)
was added ammonium formate (0.33 g, 5.29 mmol). A catalytic
amount of 10% Pd/C suspended in MeOH (10 mL) was added,
and the mixture was heated for 2 h under reflux. After cooling
to room temperature, the mixture was evaporated to dryness
under reduced pressure and the residue was purified by silica
gel column chromatography using CH2Cl2/MeOH/pyridine
(97.5:2:0.5 (v/v/v)) as eluent yielding nucleoside 5 (0.76 g, 80%)
and nucleoside 6 (148 mg, 17%) as white solid materials after
evaporation of the solvents. Nu cleosid e 5: FAB-MS m/z 573
Alter n a tive P r ep a r a tion of 4. Nucleoside 8 (179 mg, 0.5.
mmol) was coevaporated with anhydrous pyridine (2 × 20 mL),
and the residue was dissolved in anhydrous pyridine (20 mL).
(DMT)Cl (409 mg, 1,21 mmol) was added, and the reaction
mixture was stirred for 24 h at room temperature. A saturated
aqueous solution of NaHCO3 (45 mL) was added, and the
resulting mixture was extracted with CH2Cl2. The combined
organic phase was dried (MgSO4) and evaporated to dryness
under reduced pressure followed by coevaporation with toluene
twice. The residue was purified by silica gel column chroma-
tography using CH2Cl2/MeOH/pyridine (2% MeOH, 0.5% py-
ridine (v/v/v)) as eluent to afford nucleoside 4 (308 mg, 93%)
as a yellowish solid material. All analytical data were identical
to those reported above for 4.
1
[M + H]+; H NMR ((CD3)2SO) δ 11.39 (s, 1H, NH), 7.62 (s,
1H, 6-H), 6.89-7.44 (m, 13H, DMT), 5.97 (s, 1H, 1′-H), 5.93
(d, 1H, J 4.3 Hz, 3′-OH), 4.44 (d, 1H, J 4.3 Hz, 3′-H), 4.23 (s,
1H, 2′-H), 4.13 (d, 1H, J 8.4 Hz, 5′-H), 3.92 (d, 1H, J 8.4 Hz,
5′-H), 3.74 (s, 6H, DMT), 3.31 (s, 2H, 5′′-H), 1.86 (s, 3H, CH3);
13C NMR ((CD3)2SO) δ 163.9 (C-4), 158.2, 144.8, 135.8, 135.4,
135.3, 129.8, 127.9, 127.7, 126.8, 113.3 (DMT, C-6), 150.4 (C-
Alter n a tive P r ep a r a tion of 6. To a stirred solution of
nucleoside 8 (179 mg, 0.50 mmol) in EtOH (7 mL) at room
(13) Nielsen, P.; Wengel, J . Chem. Commun. 1998, 2645.

Synthesis of Diastereomeric LNA Nucleosides
J . Org. Chem., Vol. 65, No. 17, 2000 5165
temperature was added 20% Pd(OH)2/C (50 mg). The mixture
was degassed and subjected to an atmosphere of hydrogen.
After 24 h at room temperature, the mixture was filtered
through Celite. The Celite was washed with MeOH, and the
combined filtrate was evaporated to dryness under reduced
pressure to give nucleoside 6 (132 mg, 98%) as a white solid
material. All analytical data were identical to those reported
above for 6.
Alter n a tive P r ep a r a tion of 5. Nucleoside 6 (132 mg, 0.49
mmol) was coevaporated with anhydrous pyridine (2 × 3 mL),
and the residue was dissolved in anhydrous pyridine (5 mL).
(DMT)Cl (331 mg, 0.98 mmol) was added, and the mixture was
stirred at room temperature for 22 h. The mixture was poured
into a mixture of petroleum ether/CH2Cl2/H2O (30 mL, 1:1:1
(v/v)). The organic phase was separated and washed with a
saturated aqueous solution of NaHCO3 and dried (Na2SO4).
The solvent was removed under reduced pressure, and the
residue was purified by silica gel column chromatography
(CH2Cl2/MeOH, 0-1% MeOH (v/v)) to give nucleoside 5 as a
white foam (273 mg, 98%). All analytical data were identical
to those reported above for 5.
(CDCl3) δ 163.7, 150.2, 136.2, 135.5, 128.7, 128.5, 128.0, 109.9,
90.0, 85.9, 81.7, 75.9, 73.1, 65.1, 37.6, 12.6. Nu cleosid e 12:
1
FAB-MS m/z 361 [M + H]+; H NMR (CDCl3) δ 9.49 (s, 1H,
NH), 7.52 (d, 1H, J 1.3 Hz, 6-H), 7.26-7.39 (m, 5H, Bn), 6.24
(s, 1H, 1′-H), 4.71 (d, 1H, J 11.9 Hz), 4.65 (d, 1H, J 12.1 Hz),
4.47 (dd, 1H, J 2.4 Hz, J 0.9 Hz, 2′-H), 4.22 (d, 1H, J 2.4 Hz,
3′-H), 4.05 (m, 3H, 2 × 5′′-H, 5′-Ha), 3.91 (dd, 1H, J 12.3 and
J 8.8 Hz, 5′-Hb), 2.83 (t, 1H, J 6.6 Hz, 5′-OH), 1.92 (s, 3H, CH3);
13C NMR (CDCl3) δ 164.2 (C-4), 150.5 (C-2), 136.7, 136.0 (C-6,
Bn), 128.7, 128.4, 128.0 (Bn), 109.6 (C-5), 89.9 (C-1′), 88.7 (C-
4′), 81.9 (C-3′), 76.1 (C-2′), 73.9 (C-5′′), 73.0 (Bn), 59.0 (C-5′),
12.7 (CH3).
(1R,3R,4S,7S)-7-Hydr oxy-1-(h ydr oxym eth yl)-3-(th ym in -
1-yl)-2,5-d ioxa bicyclo[2.2.1]h ep ta n e (13). Compound 12 (87
mg, 0.24 mmol) was dissolved in EtOH (2.5 mL) and 20% Pd-
(OH)2/C (25 mg) was added. The mixture was degassed several
times with N2 and placed under an atmosphere of hydrogen.
After stirring for 14 h at room temperature, the mixture was
filtered through Celite and the solvent was evaporated under
reduced pressure to give compound 13 as a white solid material
1
(61 mg, 93%). FAB-MS m/z 271 [M + H]+, 289 [M + 19]; H
1-(3-O-Ben zyl-2,5-d i-O-m eth a n esu lfon yl-4-C-(m eth a n e-
su lfon yloxym eth yl)-â-D-r ibofu r an osyl)th ym in e (10). Com-
pound 911 (497 mg, 0.93 mmol) was coevaporated with anhy-
drous pyridine (2 × 2.5 mL), and the residue was dissolved in
anhydrous pyridine (5 mL). To this stirred solution at 0 °C
was added MsCl (0.14 mL, 1.86 mmol), and the mixture was
then stirred first at 0 °C for 10 min and then at room
temperature for 5 h. The mixture was cooled to 0 °C, and H2O
(10 mL) was added. The mixture was extracted with CH2Cl2,
and the combined organic phase was washed with a saturated
aqueous solution of NaHCO3 and dried (Na2SO4). The solvent
was removed under reduced pressure, and the residue was
purified by silica gel column chromatography (2% MeOH in
CH2Cl2 (v/v)) to give tri-O-mesylate 10 as a white solid material
(550 mg, 97%). 1H NMR (CDCl3) δ 9.66 (s, 1H, NH), 7.17-
7.38 (m, 6H, 6-H, Bn), 5.80 (d, 1H, J 2.3 Hz, 1′-H), 5.57 (dd,
1H, J 6.4 and 2.3 Hz, 2′-H), 4.75 (d, 1H, J 10.8 Hz), 4.73 (d,
1H, J 6.4 Hz, 3′-H), 4.60 (d, 1H, J 11.1 Hz), 4.54 (d, 1H, J 11.7
Hz), 4.41 (d, 1H, J 10.8 Hz), 4.35 (d, 1H, J 10.8 Hz), 4.33 (d,
1H, J 11.4 Hz), 3.13 (s, 3H, Ms), 3.01 (s, 3H, Ms), 2.98 (s, 3H,
Ms), 1.91 (s, 3H, CH3); 13C NMR (CDCl3) δ 163.7 (C-4), 150.3
(C-2), 137.9, 136.2 (C-6, Bn), 128.6, 128.5, 128.4 (Bn), 111.7
(C-5), 93.3 (C-1′), 84.2 (C-4′), 77.6, 76.8, 74.1, 68.1, 67.5 (Bn,
C-2′, C-3′, C-5′, C-5′′), 38.5, 37.5, 37.4 (3 × Ms), 12.1 (CH3).
Anal. Calcd for C21H28N2O13S3: C, 41.2; H, 4.6; N, 4.6. Found:
C, 40.7; H, 4.3; N, 4.5.
NMR ((CD3)2SO) δ 11.34 (s, 1H, NH), 7.64 (d, 1H, J 1.1 Hz,
6-H), 6.10 (s, 1H, 1′-H), 5.91 (d, 1H, J 3.8 Hz, 3′-OH), 4.94 (t,
1H, J 5.5 Hz, 5′-OH), 4.32 (m, 1H, 3′-H), 4.18 (dd, 1H, J 0.7
Hz, J 2.6 Hz, 2′-H), 4.04 (s, 2H, 5′′-H), 3.72 (d, 2H, J 4.4 Hz,
5′-H), 1.83 (d, 3H, J 0.9 Hz, CH3); 13C NMR ((CD3)2SO) δ 163.9
(C-4), 150.4 (C-2), 136.5 (C-6), 108.0 (C-5), 89.2, 89.1 (C-1′,
C-4′), 77.3 (C-2′), 74.7 (C-3′), 73.6 (C-5′′), 57.3 (C-5′), 12.3 (CH3).
Anal. Calcd For C11H14N2O6‚0.2H2O: C, 48.3; H, 5.3; N, 10.2.
Found: C, 48.2; H, 5.3; N, 9.8.
(1S,3R,4S,7S)-1-(4,4′-Dim eth oxytr ityloxym eth yl)-7-h y-
dr oxy-3-(th ym in -1-yl)-2,5-dioxabicyclo[2.2.1]h eptan e (14).
Compound 13 (280 mg, 1.04 mmol) was coevaporated with
anhydrous pyridine (5 × 6 mL), and the residue was dissolved
in anhydrous pyridine (8 mL). (DMT)Cl (678 mg, 2.04 mmol)
was added, and the mixture was stirred at room temperature
for 8 h. The mixture was poured into a mixture of petroleum
ether/CH2Cl2/H2O (30 mL, 1:1:1 (v/v)). The organic phase was
separated and washed with a saturated aqueous solution of
NaHCO3 and dried (Na2SO4). The solvent was removed under
reduced pressure and the residue was purified by silica gel
column chromatography (CH2Cl2/MeOH/pyridine, 0-2% MeOH,
0.5% pyridine (v/v/v)) to give nucleoside 14 as a white solid
material (563 mg, 95%). FAB-MS m/z 573 [M + H]+; 1H NMR
(CDCl3) δ 9.62 (s, 1H), 7.57 (d, 1H, J 1.2 Hz), 7.19-7.43 (9H,
m), 6.81-6.86 (4H, m), 6.31 (s, 1H), 4.52 (m, 2H), 4.33 (1H, br
s), 4.08 (d, 1H, J 9.1 Hz), 3.98 (d, 1H, J 8.8 Hz), 3.78 (s, 6H),
3.57 (d, 1H, J 10.3 Hz), 3.51 (d, 1H, J 10.3 Hz), 1.94 (d, 3H, J
1.2 Hz); 13C NMR (CDCl3) δ 164.0, 158.5, 150.4, 149.3, 144.1,
136.1, 136.0, 135.0, 129.9, 127.8, 126.9, 123.8, 109.4, 90.1, 88.2,
86.6, 77.8, 76.1, 74.2, 59.7, 12.7.
(1S ,3R ,4S ,7S )-7-Be n zyloxy-1-(m e t h a n e su lfon yloxy-
m eth yl)-3-(th ym in -1-yl)-2,5-dioxabicyclo[2.2.1]h eptan e (11)
a n d
(1R,3R,4S,7S)-7-Ben zyloxy-1-(h yd r oxym eth yl)-3-
(th ym in -1-yl)-2,5-d ioxa bicyclo[2.2.1]h ep ta n e (12). Com-
pound 10 (1.63 g, 2.66 mmol) was dissolved in a mixture of
96% aqueous EtOH and H2O (1:1 (v/v), 60 mL). A 2 M amount
of aqueous NaOH (10 mL, 20 mmol) was added, and the
mixture was heated at 85 °C for 40 h. Analytical TLC revealed
the formation of two major products both with lower mobility
than 10. The mixture was evaporated to half volume under
reduced pressure, and the resulting mixture was extracted
with CH2Cl2. The combined organic phase was washed with a
saturated aqueous solution of NaHCO3 and dried (Na2SO4).
The solvent was removed under reduced pressure, and the
residue was purified by silica gel column chromatography (0-
1% MeOH in CH2Cl2 (v/v)) to give nucleosides 11 (408 mg, 35%)
and 12 (304 mg, 32%) as white solid materials. Con ver sion
of 11 to 12. Compound 11 (138 mg, 0.31 mmol) was dissolved
in EtOH (24 mL), and KOH (177 mg, 3.10 mmol) was added.
The mixture was heated at 60 °C for 12 h. The solvent was
removed under reduced pressure, and the residue was purified
by silica gel column chromatography (0-1% MeOH in CH2Cl2
(v/v)) to give nucleoside 12 as a white solid material (87 mg,
(1S,3R,4S,7S)-7-(2-Cya n oet h oxy(d iisop r op yla m in o)-
p h osp h in oxy)-1-(4,4′-d im eth oxytr ityloxym eth yl)-3-(th y-
m in -1-yl)-2,5-d ioxa bicyclo[2.2.1]h ep ta n e (15). Compound
14 (200 mg, 0.35 mmol) was coevaporated with anhydrous
acetonitrile (3 × 2 mL) and dissolved in anhydrous CH2Cl2 (4
mL). The solution was stirred under N2 and cooled to 0 °C.
N,N-Diisopropylethylamine (DIPEA, 0.18 mL, 1.05 mmol) and
2-cyanoethyl N,N-diisopropylphosphoramidochloridite (0.17
mL, 0.51 mmol) were added. The mixture was heated to room
temperature, and stirring was continued for 12 h. After 2, 4,
and 6 h, respectively, additional DIPEA (0.18 mL, 1.05 mmol)
and 2-cyanoethyl N,N-diisopropylphosphoramidochloridite (0.17
mL, 0.51 mmol) were added. After an additional 2 h, MeOH
(0.05 mL) was added, and the mixture was diluted with CH2-
Cl2 (30 mL) and washed with a saturated aqueous solution of
NaHCO3 and dried (Na2SO4). The solvent was removed under
reduced pressure, and the residue was purified by silica gel
column chromatography (EtOAc/n-hexane/NEt3, 45:45:10 (v/
v/v)) to give a white foam after evaporation under reduced
pressure. This residue was dissolved in anhydrous toluene (0.5
mL), and the product was precipitated by addition drop by drop
of this solution to petroleum ether (50 mL, cooled to -30 °C)
under vigorous stirring. The precipitate was collected by
filtration and dried to give compound 15 as a white solid
1
79%). Nu cleosid e 11: FAB-MS m/z 439 [M + H]+; H NMR
(CDCl3) δ 9.95 (s, 1H), 7.28-7.54 (m, 6H), 6.27 (s, 1H), 4.71
(d, 1H, J 11.7 Hz), 4.64 (d, 1H, J 12.0 Hz), 4.57 (d, 1H, J 11.4
Hz), 4.49 (d, 1H, J 11.7 Hz), 4.26 (s, 1H), 4.10 (d, 1H, J 9.1
Hz), 4.05 (d, 1H, J 8.8 Hz), 3.05 (s, 3H), 1.94 (s, 3H); 13C NMR

5166 J . Org. Chem., Vol. 65, No. 17, 2000
Håkansson et al.
Su p p or tin g In for m a tion Ava ila ble: Figures showing 13
material (161 mg, 60%). FAB-MS m/z 773 [M + H]+; 31P NMR
((CD3)2SO) δ 150.8, 150.5.
C
NMR spectra for compounds 2-7 and 10-14 and the 31P NMR
spectrum for compound 15. This information is available free
of charge via the Internet at http://pubs.acs.org.
Ack n ow led gm en t. The Danish Natural Science
Research Council, The Danish Technical Research
Council, and Exiqon A/S are thanked for financial
support.
J O000232G