Analogues of LNA (Locked Nucleic Acid): Synthesis of the 2′-thio-LNA ribothymidine and 5-methylcytidine phosphoramidites

Article abstract of DOI:10.1055/s-2004-815959

The bicyclic 2′-thio-LNA ribothymidine phosphoramidite (13) has been synthesised using a strategy making the synthesis of 2′-thio-LNA convergent with the syntheses of LNA and 2′-amino-LNA. The key step was the formation of anhydro-nucleoside 4 that preven

Full text of DOI:10.1055/s-2004-815959

578
PAPER
Analogues of LNA (Locked Nucleic Acid): Synthesis of the 2 -Thio-LNA
Ribothymidine and 5-Methylcytidine Phosphoramidites
S
ynthesis of 2 -T
h
a
io-LNA Th
n
ymine
a
nd 5-
i
Methy
e
lcytosine l Sejer Pedersen,1 Troels Koch*
Santaris Pharma A/S, Bøge Allé 3, 2970 Hørsholm, Denmark
Fax +4545179898; E-mail: tk@santaris.com
Received 2 October 2003; revised 12 January 2004
O
B
O
B
O
B
Abstract: The bicyclic 2 -thio-LNA ribothymidine phosphoramid-
ite (13) has been synthesised using a strategy making the synthesis
of 2 -thio-LNA convergent with the syntheses of LNA and 2 -ami-
no-LNA. The key step was the formation of anhydro-nucleoside 4
that prevented unwanted oxetane (3) formation during debenzyla-
tion of the 3 -OBn group, while concomitantly furnishing the nec-
essary inversion of configuration at the C2 position. No oxetane
was observed even under basic conditions. We believe that this is
due to the rigid tricyclic anhydro-nucleoside limiting the conforma-
tional freedom of the furanose, thereby preventing the formation of
a strained tetracyclic system. After removal of the benzyl protection
group the liberated 3 -OH group (5) was easily benzoylated (6) and
the anhydro-nucleoside ring opened to give the threo configured nu-
cleoside 7. The 2 -thio-LNA ribothymidine phosphoramidite was
synthesised in 9 steps from anhydro-nucleoside 4 in 30% yield. The
2 -thio-LNA 5-methylcytidine phosphoramidite was synthesised
from nucleoside 12 using standard protocols.
O
O
O
O
O
O
P
O
O
P
N
O
P
S
R
O
O
O
O
LNA
2'-Amino-LNA
2'-Thio-LNA
Figure 1 B = Nucleobase or nucleobase analogue
There is no convergence between the published synthesis
of 2 -thio-LNA15,17 and the synthesis of LNA.22 In the
published procedure there are problems with respect to
synthesis on larger scales and several of the steps are low
yielding. In this paper we describe the synthesis of the 2 -
thio-LNA ribothymidine phosphoramidite building block
that is convergent with the published procedure of LNA22
and 2 -amino-LNA.23 The key feature in the synthesis is
the prevention of oxetane formation (3, Scheme 1) during
debenzylation of the 3 -hydroxyl group by locking the
carbohydrate moiety as the tricyclic anhydro-nucleoside
(4, Scheme 2). Using this novel strategy oxetane forma-
tion was prevented during the debenzylation while obtain-
ing inversion of configuration at C2 , producing the threo
configured nucleoside 7, after benzoyl protection of the
3 -OH position and opening of the anhydro-nucleoside
with aqueous acid.
Key words: nucleosides, bicyclic compounds, antisense agents,
hydrogenations, bioorganic chemistry
Locked Nucleic Acid (LNA2) is a class of conformation-
ally restricted oligonucleotide analogues comprising bicy-
clic 2 ,4 -O-CH2-furanosyl residues (Figure 1).3–5 This
class of oligonucleotides have many promising properties
in the fields of diagnostics and therapeutics.6–14 Wengel
and co-workers were the first to report syntheses of LNA
analogues in which the oxygen in the original 2 ,4 -O-
CH2-furanosyl bicyclic structure had been substituted
with sulfur (2 ,4 -S-CH2-, 2 -thio-LNA) and nitrogen
(2 ,4 -NH-CH2-, 2 -amino-LNA).15–17 Both analogues
(Figure 1) showed hybridisation properties toward native
nucleic acids comparable to those of the parent LNA.15,16
The strategy employed in the synthesis of 2 -amino-LNA
proved to be inefficient for the preparation of 2 -thio-
LNA.23 Although a wide range of conditions were inves-
tigated it proved impossible to debenzylate the 3 -OH po-
sition after the introduction of sulfur. Clearly, it was
necessary to debenzylate prior to the introduction of sulfur
at the 2 -position. Initially, we attempted debenzylating
the trimesylate 1 (Scheme 1). Under neutral conditions
the major product was the undesired oxetane 3. However,
if the hydrogenation was performed in glacial acetic acid
nucleoside 2 was the exclusive product.
Incorporation of thiols, sulfides and disulfides in nucleo-
sides can lead to a number of interesting biological prop-
erties of the corresponding oligonucleotides.18–20 For
example the in vivo distribution of oligonucleotides as
well as resistance towards exonuclease degradation can be
improved significantly.21
Unfortunately, attempts to purify nucleoside 2 by chroma-
tography led to a substantial loss of the target molecule,
due to on-column formation of 3, giving only a low yield
of 2 (ca. 25%), even when acidic chromatography condi-
tions were employed. Thus, it was clear that another strat-
egy had to be devised in which both oxetane formation
was avoided, and where the 3 -OBn group was debenzy-
lated prior to introduction of sulfur. We envisaged that de-
benzylation of the rigid tricyclic anhydro-nucleoside 4
(Scheme 2), would prevent the formation of a strained tet-
racyclic oxetane system.
As part of our ongoing study of the individual LNA ana-
logues we turned our attention to 2 -thio-LNA to study
what properties it can confer to antisense oligonucleo-
tides.
SYNTHESIS 2004, No. 4, pp 0578–0582
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x
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x
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Advanced online publication: 09.02.2004
DOI: 10.1055/s-2004-815959; Art ID: T10403SS
© Georg Thieme Verlag Stuttgart · New York

PAPER
Synthesis of 2′-Thio-LNA Thymine and 5-Methylcytosine
579
T
T
cleoside 7 in 79% yield from nucleoside 4. In spite of the
basic conditions employed for the benzoylation of nucleo-
side 5, no oxetane formation was observed. Presumably
the rigid molecular framework of anhydro-nucleoside 5
makes it impossible to form a strained tetracyclic oxetane.
MsO
MsO
MsO
i
O
O
MsO
BnO
OMs
HO
OMs
1
2
T
MsO
The obtained threo configured nucleoside 7 was subse-
quently triflated, to give crude nucleoside 8, that was
treated with Na2S in DMF producing 2 -thio-LNA nucleo-
side 9 in 72% yield from 7. In the next reaction the 5 -me-
sylate was substituted with sodium benzoate to give the
dibenzoate 10 in 81% yield, followed by hydrolysis of
both benzoyl esters to yield the fully deprotected 2 -thio-
LNA nucleoside 11 in a 76% yield. The 2 -thio-LNA ri-
bothymidine phosphoramidite (13) was finally obtained
by protection of the 5 -hydroxyl group with 4,4-dimethoxy-
trityl chloride, and phosphitylation of the 3 -hydroxyl
group according to standard procedures24 in an over-
all yield of 88% from 11. Starting from 3-O-benzyl-4-C-
hydroxymethyl-1,2-O-isopropylidene- -D-ribofuranose
Wengel and co-workers prepared the 2 -thio-LNA ribou-
ridine phosphoramidite in an overall yield of 3%.3,15,17
The method for the preparation of 2 -thio-LNA ribothymi-
dine presented herein constitutes a significant improve-
ment when compared to the original procedure producing
the 2 -thio-LNA ribothymidine phosphoramidite 13 in an
overall yield of 21% starting from the same material. The
2 -thio-LNA 5-methylcytidine phosphoramidite was syn-
thesised starting from nucleoside 12 using standard meth-
ods.23
O
O
OMs
3
Scheme 1 Undesired oxetane formation. Reagents and conditions:
i) Pd(OH)2/C, H2, concd HOAc (ca. 25%).
Indeed, no oxetane formation was observed when catalyt-
ic hydrogenation in glacial acetic acid was employed for
debenzylation. However, the conditions resulted in a 1:1
mixture of the desired nucleoside 5 and a nucleoside
where undesired reduction of the nucleobase had oc-
curred.
To prevent reduction of the nucleobase a range of differ-
ent solvent combinations under neutral conditions using
either Pd/C or Pd(OH)2/C were investigated. Reduction
with H2 and 10 mol% Pd on carbon in a 1:1 mixture of ac-
etone and MeOH was identified as the optimal conditions,
yielding almost exclusively 5 (Scheme 2). However, due
to the very polar nature of 5 work-up and purification was
difficult. In order to obtain the product the palladium cat-
alyst had to be refluxed in DMF and chromatography of
the product was not feasible. A ‘one-pot’ strategy turned
out to be the solution to this problem. Thus, concentration
of the reaction mixture from the debenzylation produced
5 that was dissolved in DMF. Benzoyl chloride and pyri-
dine were added and the reaction was stirred at ambient
temperature overnight and quenched with MeOH. After
removal of excess MeOH in vacuo, aqueous H2SO4 was
added and the reaction mixture was heated to 80 °C over-
night. Chromatography of the product mixture gave nu-
An efficient procedure for the preparation of the 2 -thio-
LNA ribothymidine phosphoramidite, convergent with
the syntheses of LNA and 2 -amino-LNA has been pre-
sented. The key step is the formation of the 2 -anhydro-
nucleoside 5 by which the configuration at the 2 -position
is inverted and oxetane formation is prevented. The pre-
sented synthetic route to 2 -thio-LNA ribothymidine was
O
O
O
MsO
MsO
MsO
MsO
MsO
MsO
N
O
N
N
O
N
N
O
N
ii
iii
i
O
O
O
BnO
OH
OBz
4
5
6
T
TfO T
MsO
MsO
HO
MsO
MsO
RO
T
iv
v
O
O
O
S
R1O
OBz
OBz
9 R=Ms, R1=Bz
10 R=R1=Bz
7
8
vi
vii
viii
ix
11 R=R1=OH
12 R=DMT, R1=OH
13 R=DMT, R1=PN2
Scheme 2 Synthesis of the 2 -thio-LNA ribothymidine phosphoramidite. Reagents and conditions: i) Pd/C, H2, acetone, MeOH; ii) BzCl, py-
ridine, DMF; iii) 0.25 M H2SO4 (aq), DMF, 80 °C (79% from 4; 3 steps); iv) Tf2O, DMAP, CH2Cl2, 0 °C; v) Na2S, DMF (72% from 7; 2 steps);
vi) NaOBz, DMF, 100 °C (81%); vii) NH3, MeOH (76%); viii) DMT-Cl, pyridine (88%); ix) P(OCH2CH2CN)[N(i-Pr)2]2, 4,5-dicyanoimidazo-
le, CH2Cl2 (99%). DMT = 4,4 -dimethoxytrityl, PN2 = 2-cyanoethoxy(diisopropylamino)phosphinoyl.
Synthesis 2004, No. 4, 578–582 © Thieme Stuttgart · New York

580
D. S. Pedersen, T. Koch
PAPER
1H NMR (DMSO-d6): = 11.39 (br s, 1 H, NH), 8.10–8.08 (m, 2 H,
Ph), 7.74–7.70 (m, 1 H, Ph), 7.60–7.56 (m, 2 H, Ph), 7.51 (d, J = 1.1
Hz, 1 H, H6), 6.35 (d, J = 4.9 Hz, 1 H, H1 ), 6.32 (d, J = 5.3 Hz, 1
H, 2 -OH), 5.61 (d, J = 4.0 Hz, 1 H, H3 ), 4.69 (d, J = 10.8 Hz, 1 H),
4.59 (m, 1 H, H2 ), 4.55 (d, J = 10.8 Hz, 1 H), 4.52 (d, J = 10.8 Hz,
1 H), 4.46 (d, J = 10.6 Hz, 1 H, H5 , H1 ), 3.28 (s, 3 H, Ms), 3.23
(s, 3 H, Ms), 1.81 (s, 3 H, CH3).
extended to provide the 2 -thio-LNA 5-methylcytidine
phosphoramidite using standard protocols.23 The proper-
ties of 2 -thio-LNA containing oligonucleotides are cur-
rently being evaluated and the results will be reported in
due course.
For reactions conducted under anhydrous conditions glassware was
dried overnight in an oven at 150 °C and was allowed to cool in a
dessicator over anhyd KOH. Anhydrous reactions were carried out
under an atmosphere of Ar using anhydrous solvents. Solvents were
HPLC grade, of which DMF, pyridine, MeOH and CH2Cl2 were
dried over molecular sieves (4 Å from Grace Davison) and THF was
freshly distilled from Na·benzophenone to a water content below 20
ppm. TLC was run on Merck silica 60 F254 aluminium sheets. Dry
Column Vacuum Chromatography (DCVC) was performed accord-
ing to the published procedure.25 Nucleoside 4 was synthesised as
previously published.23 1H and 13C NMR spectra were recorded at
400 MHz and 100 MHz, respectively, with solvents as internal stan-
dard ( H: CDCl3 7.26 ppm, DMSO-d6 2.50; C: CDCl3 77.0 ppm,
DMSO-d6 39.4 ppm). 31P NMR was run at 121 MHz with 85%
H3PO4 as external standard. Coupling constants (J) were calculated
using Mestre-C 2.3a software.26 Assignments of NMR spectra are
based on 2D spectra and follow the standard carbohydrate/nucleo-
side nomenclature (the carbon atom of the 4 -C-substituent is num-
bered C1 ) even though the systematic compound names of the
bicyclic nucleoside derivatives are given according to the von Baey-
er nomenclature. Elemental analyses were obtained from the Uni-
versity of Copenhagen, Microanalytical Department.
13C NMR (DMSO-d6): = 164.5, 163.6 [C4, PhC(O)], 150.3 (C2),
137.7 (C6), 133.8, 129.6, 128.7, 128.6 (Ph), 108.1 (C5), 84.8 (C1 ),
81.1 (C4 ), 78.0 (C3 ), 73.2 (C2 ), 68.0, 67.1 (C5 , C1 ), 36.7, 36.6
(2 × Ms), 11.9 (CH3).
ESI–MS: m/z [MH]+ calcd: 549.1; found: 549.0.
Anal. Calcd for C20H24N2O12S2·0.33 H2O: C, 44.34; H, 4.65; N,
4.85. Found: C, 44.32; H, 4.58; N, 4.77.
(1R,3R,4R,7R)-7-Benzoyloxy-1-methansulfonyloxymethyl-3-
(thymin-1-yl)-2-oxa-5-thiabicyclo[2:2:1]heptane (9)
1-(3-O-Benzoyl-5-O-methanesulfonyl-4-C-methanesulfonyloxym-
ethyl- -D-threo-pentofuranosyl)thymine (7) (10.00 g, 18.23 mmol)
was dissolved in CH2Cl2 (500 mL) and cooled to 0 °C. Pyridine (15
mL) and DMAP (8.91 g, 72.9 mmol) were added followed by drop-
wise addition of trifluoromethanesulfonic anhydride (10.30 g, 36.5
mmol, 6.0 mL). After 1 h the reaction was quenched with sat. aq
NaHCO3 (500 mL) and transferred to a separatory funnel. The or-
ganic layer was washed with 1.0 M aq HCl (500 mL), sat. aq
NaHCO3 (500 mL) and brine (500 mL). The organic layer was
evaporated in vacuo with toluene (100 mL) to give 1-(3-O-benzoyl-
5-O-methanesulfonyl-4-C-methanesulfonyloxymethyl-2-O-trifluo-
romethanesulfonyl- -D-threo-pentofuranosyl)thymine (8) as a yel-
low powder.
1-(3-O-Benzoyl-5-O-methanesulfonyl-4-C-methanesulfonyloxy-
methyl- -d-threo-pentofuranosyl)thymine (7)
The crude nucleoside 8 was dissolved in DMF (250 mL) and Na2S
(1.57 g, 20.1 mmol) was added to give a dark green slurry. After 3
h the reaction was quenched with half sat. aq NaHCO3 (500 mL)
and extracted with CH2Cl2 (500 + 2 × 250 mL). The combined or-
ganic layers were washed with brine (500 mL), dried (Na2SO4), fil-
tered and concentrated in vacuo to give a yellow liquid. Residual
solvent was removed overnight on a high vacuum pump to give a
yellow gum that was purified by Dry Column Vacuum Chromatog-
raphy (id = 6 cm: 50 mL fractions; 50–100% EtOAc in n-heptane:
10% increments; 2–20% MeOH in EtOAc; 2% increments) to give
nucleoside 9 (6.15 g, 72%) as a yellow foam; Rf 0.27 (20% n-hep-
tane in EtOAc).
1H NMR (CDCl3) = 8.70 (br s, 1 H, NH), 8.01–7.99 (m, 2 H, Ph),
7.67 (d, J = 1.1 Hz, 1 H, H6), 7.65–7.61 (m, 1 H, Ph), 7.50–7.46 (m,
2 H, Ph), 5.98 (s, 1 H, H1 ), 5.34 (d, J = 2.4 Hz, 1 H, H3 ), 4.66 (d,
J = 11.7 Hz, 1 H, H5 a), 4.53 (d, J = 11.5 Hz, 1 H, H5 b), 4.12 [m
(overlapping with residual EtOAc), 1 H, H2 ), 3.15–3.13 (m, 4 H,
H1 a, Ms), 3.06 (d, J = 10.6 Hz, 1 H, H1 b), 1.98 (d, J = 1.1 Hz, 3
H, CH3).
Anhydro-nucleoside 423 (30.0 g, 58.1 mmol) was heated to 70 °C in
a mixture of MeOH (1000 mL) and acetone (1000 mL) until a clear
solution was obtained and the solution was allowed to reach r.t. The
reaction flask was flushed with Ar and Pd/C (10 wt.% Pd on carbon,
6.2 g, 5.8 mmol) was added. The mixture was stirred vigorously un-
der an atmosphere of hydrogen gas (balloon). After 23 h the slurry
was filtered through a pad of celite. The catalyst was recovered from
the celite and refluxed in DMF (1000 mL) for 1 h. The hot DMF
slurry was filtered through a pad of celite and the organic layers
were combined and evaporated in vacuo to give nucleoside 5 as a
yellow powder. Residual solvents were removed with the help of a
high vacuum pump overnight.
The crude nucleoside 5 (23 g) was heated to 70 °C in DMF (300
mL) to give a clear yellow solution that was allowed to cool to r.t.
Benzoyl chloride (81.7 g, 581 mmol, 67.4 mL) was added followed
by pyridine (70 mL). After 18 h the reaction was quenched with
MeOH (200 mL) and excess MeOH was removed in vacuo.
To the dark brown solution of nucleoside 6 aq H2SO4 (0.25 M, 400
mL) was added. The solution was heated to 80 °C on an oil bath (At
approx 50 °C precipitation occurs. The solution becomes clear
again at 80 °C). After 22 h at 80 °C the solution was allowed to cool
to r.t. The reaction mixture was transferred to a separatory funnel
with EtOAc (1000 mL). The organic layer was washed with sat. aq
NaHCO3 (2 × 1000 mL). The combined aqueous layers were ex-
tracted with EtOAc (1000 + 500 mL). The organic layers were com-
bined and washed with sat. aq NaHCO3 (1000 mL), dried (Na2SO4),
filtered and evaporated in vacuo to give a yellow liquid. Residual
solvents were removed on a high vacuum pump overnight to give a
yellow syrup. The product was purified by Dry Column Vacuum
Chromatography (id = 10 cm; 100 mL fractions; 50–100% EtOAc
in n-heptane; 10% increments; 2-24% MeOH in EtOAc; 2% incre-
ments). Fractions containing the product were combined and evap-
orated in vacuo giving nucleoside 7 (25.1 g, 79%) as a white foam;
Rf 0.54 (5% MeOH in EtOAc).
13C NMR (CDCl3): = 165.2, 163.5 [C4, PhC(O)], 149.9 (C2),
134.1, 133.9, 129.8, 128.7, 128.3 (C6, Ph), 110.7 (C5), 91.1 (C1 ),
86.8 (C4 ), 72.6 (C3 ), 65.8 (C5 ), 50.5 (C2 ), 37.9 (Ms), 35.1 (C1 ),
12.5 (CH3).
ESI–MS: m/z [MH]+ calcd: 469.1; found: 469.0.
Anal. Calcd for C19H20N2O8S2·0.33 EtOAc: C, 49.21; H, 4.72; N,
5.47. Found: C, 49.25; H, 4.64; N, 5.48.
(1R,3R,4R,7R)-7-Benzoyloxy-1-benzoyloxymethyl-3-(thymin-1-
yl)-2-oxa-5-thiabicyclo[2:2:1]heptane (10)
Nucleoside 9 (1.92 g, 4.1 mmol) was dissolved in DMF (110 mL).
Sodium benzoate (1.2 g, 8.2 mmol) was added and the mixture was
heated to 100 °C for 24 h. The reaction mixture was transferred to a
separatory funnel with half sat. brine (200 mL) and extracted with
EtOAc (3 × 100 mL). The combined organic layers were dried
Synthesis 2004, No. 4, 578–582 © Thieme Stuttgart · New York

PAPER
Synthesis of 2′-Thio-LNA Thymine and 5-Methylcytosine
581
(Na2SO4), filtered and evaporated in vacuo to give a brown liquid.
The product was put on a high vacuum pump to remove residual sol-
vent. The resulting brown gum was purified by Dry Column Vacu-
um Chromatography (id = 4 cm; 50 mL fractions; 0–100% EtOAc
in n-heptane; 10% increments; 2–10% MeOH in EtOAc; 2% incre-
ments) to give nucleoside 10 (1.64 g, 81%) as a slightly yellow
foam; Rf 0.57 (20% n-heptane in EtOAc).
1H NMR (CDCl3): = 9.02 (br s, 1 H, NH), 8.07–7.99 (m, 4 H, Ph),
7.62–7.58 (m, 2 H, Ph), 7.47–7.42 (m, 5 H, Ph, H6), 5.95 (s, 1 H,
H1 ), 5.46 (d, J = 2.2 Hz, 1 H, H3 ), 4.93 (d, J = 12.8 Hz, 1 H, H5 a),
4.60 (d, J = 12.8 Hz, 1 H, H5 b), 4.17 (d, J = 2.2 Hz, 1 H, H2 ), 3.27
(d, J = 10.6 Hz, 1 H, H1 a), 3.16 (d, J = 10.6 Hz, 1 H, H1 b), 1.55
(d, J = 1.1 Hz, 3 H, CH3).
(d, J = 10.8 Hz, 1 H, H1 b), 2.69 (d, J = 9.2 Hz, 1 H, 3 -OH), 1.42
(s, 3 H, CH3).
13C NMR (CDCl3): = 158.7 (C4), 150.1 (C2), 144.1, 135.2, 135.1,
130.1, 129.1, 128.1, 128.0, 127.1, 127.0, 113.3 (C6, 3 × Ph), 110.0
(C5), 90.2 [C(Ph)3], 89.6 (C1 ), 87.0 (C4 ), 71.7 (C3 ), 60.9 (C5 ),
55.2 (C2 ), 34.7 (C1 ), 12.2 (CH3).
ESI-MS: m/z [M – H]+ calcd: 587.2; found 587.1.
Anal. Calcd for C32H32N2O7S·0.5 H2O: C, 64.31; H, 5.57; N, 4.69.
Found: C, 64.22; H, 5.67; N, 4.47.
(1R,3R,4R,7R)-7-(2-Cyanoethoxy(diisopropylamino)phosphi-
noxy)-1-(4,4’-dimethoxytrityloxymethyl)-3-(thymin-1-yl)-2-
oxa-5-thiabicyclo[2.2.1]heptane (13)
13C NMR (CDCl3): = 165.8, 165.1, 163.7 [C4, 2 × PhC(O)], 150.0
(C2), 133.9, 133.7, 133.6, 129.8, 129.6, 129.0, 128.8, 128.6, 128.5
(C6, 2 × Ph), 110.3 (C5), 91.3 (C1 ), 87.5 (C4 ), 72.9 (C3 ), 61.3
(C5 ), 50.6 (C2 ), 35.6 (C1 ), 12.3 (CH3).
According to the published method24 nucleoside 12 (0.78 g, 1.33
mmol) was dissolved in CH2Cl2 (5 cm3) and a 1.0 M solution of 4,5-
dicyanoimidazole in MeCN (0.93 mL, 0.93 mmol) was added fol-
lowed by dropwise addition of 2-cyanoethyl-N,N,N’,N’-tetraisopro-
pylphosphorodiamidite (0.44 mL, 1.33 mmol). After 2 h the
reaction was transferred to a separatory funnel with CH2Cl2 (40 mL)
and washed with sat. aq NaHCO3 (2 × 25 mL) and brine (25 mL).
The organic layer was dried (Na2SO4), filtered and evaporated in
vacuo to give nucleoside 13 (1.04 g, 99%) as a white foam; Rf 0.29,
0.37, two diastereoisomers (20% n-heptane in EtOAc).
ESI–MS: m/z [MH]+ calcd: 495.1; found: 495.1.
Anal. Calcd for C25H22N2O7S: C, 60.72; H, 4.48; N, 5.66. Found: C,
60.34; H, 4.49; N, 5.35.
(1R,3R,4R,7R)-7-Hydroxy-1-hydroxymethyl-3-(thymin-1-yl)-2-
oxa-5-thiabicyclo[2:2:1]heptane (11)
Nucleoside 10 (1.50 g, 3.0 mmol) was dissolved in MeOH saturated
with NH3 (50 mL). The reaction flask was sealed and stirred at am-
bient temperature for 20 h. The reaction mixture was concentrated
in vacuo to give a yellow gum that was purified by Dry Column
Vacuum Chromatography (id = 4 cm; 50 mL fractions; 0–16%
MeOH in EtOAc; 1% increments) giving nucleoside 11 (0.65 g,
76%) as clear needles; Rf 0.31 (10% MeOH in EtOAc).
1H NMR (DMSO-d6) = 11.32 (br s, 1 H, NH), 7.96 (d, J = 1.1 Hz,
1 H, H6), 5.95 (s, 1 H, H6), 5.70 (d, J = 4.2 Hz, 1 H, 3 -OH), 5.62
(s, 1 H, H1 ), 4.49 (t, J = 5.3 Hz, 1 H, 5 -OH), 4.20 (dd, J = 4.1, 2.1
Hz, 1 H, H3 ), 3.77–3.67 (m, 2 H, H5 ), 3.42 (d, J = 2.0 Hz, 1 H,
H2 ), 2.83 (d, J = 10.1 Hz, 1 H, H1 a), 2.64 (d, J = 10.1 Hz, 1 H,
H1 b), 1.75 (d, J = 1.1 Hz, 3 H, CH3).
31P NMR (DMSO-d6): = 150.39, 150.26.
ESI–MS: m/z [MH]+ calcd:789.3; found: 789.3.
References
(1) New address: Daniel Sejer Pedersen, Cambridge University,
University Chemical Laboratory, Lensfield Road,
Cambridge CB2 1EW, UK.
(2) LNA is defined as an oligonucleotide containing one or more
2 -O,4 -C-methylene- -D-ribofuranosyl nucleotide mono-
mers (LNA monomers).3,5 For clarity the terms 2 -amino-
LNA and 2 -thio-LNA are used herein for the LNA
analogues, where the 2 -oxygen has been substituted by a
nitrogen atom or a sulfur atom.
(3) Koshkin, A. A.; Singh, S. K.; Nielsen, P.; Rajwanshi, V. K.;
Kumar, R.; Meldgaard, M.; Olsen, C. E.; Wengel, J.
Tetrahedron 1998, 54, 3607.
(4) Obika, S.; Nanbu, D.; Hari, Y.; Andoh, J.-I.; Morio, K.; Doi,
T.; Imanishi, T. Tetrahedron Lett. 1998, 39, 5401.
(5) Singh, S. K.; Nielsen, P.; Koshkin, A. A.; Wengel, J. Chem.
Commun. 1998, 455.
13C NMR (DMSO-d6): = 163.8 (C4), 150.0 (C2), 135.3 (C6),
107.5 (C5), 90.2, 89.6 (C1 , C4 ), 69.4 (C3 ), 58.0 (C5 ), 52.1 (C2 ),
34.6 (C1 ), 12.4 (CH3).
ESI–MS: m/z [MH]+ calcd: 287.1; found: 287.1.
Anal. Calcd for C11H14N2O5S: C, 46.15; H, 4.93; N, 9.78. Found: C,
46.35; H, 4.91; N, 9.54.
(1R,3R,4R,7R)-1-(4,4 -Dimethoxytrityloxymethyl)-7-hydroxy-
5-methyl-3-(thymin-1-yl)-2-oxa-5-thiabicyclo[2:2:1]heptane
(12)
(6) Braasch, D. A.; Corey, R. Chem. Biol. 2000, 55, 1.
(7) Braasch, D. A.; Corey, D. R. Nucleic Acids Res. 2002, 30,
5160.
Nucleoside 11 (0.60 g, 2.1 mmol) was dissolved in pyridine (10
mL). 4,4 -Dimethoxytrityl chloride (0.88 g, 2.6 mmol) was added
and the reaction mixture was stirred at ambient temperature for 3 h.
The reaction mixture was transferred to a separatory funnel with
water (100 mL) and extracted with EtOAc (100 + 2 × 50 mL). The
combined organic layers were washed with sat. aq NaHCO3 (100
mL), brine (100 mL) and evaporated to dryness in vacuo to give a
viscous yellow liquid. The product was redissolved in toluene (50
mL) and concentrated in vacuo to give a yellow foam. The foam
was dried on a high vacuum pump overnight and purified by Dry
Column Vacuum Chromatography (id = 4 cm; 50 mL fractions; 10–
100% EtOAc in n-heptane; 10% increments) giving nucleoside 12
(1.08 g, 88%) as a white foam; Rf 0.24 (20% n-heptane in EtOAc).
(8) Childs, J. L.; Disney, M. D.; Turner, D. H. Proc. Natl. Acad.
Sci. U.S.A. 2002, 99, 11091.
(9) Crinelli, R.; Bianchi, M.; Gentilini, L.; Magnani, M. Nucleic
Acids Res. 2002, 30, 2435.
(10) Elayadi, A. N.; Braasch, D. A.; Corey, D. R. Biochemistry
2002, 41, 9973.
(11) Fluiter, K.; ten Asbroek, A. L. M. A.; de Wissel, M. B.;
Jakobs, M. E.; Wissenbach, M.; Olsson, H.; Olsson, O.;
Oerum, H.; Baas, F. Nucleic Acids Res. 2003, 31, 953.
(12) Hertoghs, K. M.; Ellis, J. H.; Catchpole, I. R. Nucleic Acids
Res. 2003, 31, 5817.
(13) Kurreck, J.; Wyszko, E.; Gillen, C.; Erdmann, V. A. Nucleic
Acids Res. 2002, 30, 1911.
1H NMR (CDCl3): = 8.96 (br s, 1 H, NH), 7.74 (d, J = 1.1 Hz, 1
H, H6), 7.46–7.44 (m, 2 H, Ph), 7.35–7.22 (m, 9 H, Ph), 7.19–7.15
(m, 2 H, Ph), 6.86–6.80 (m, 2 H, Ph), 5.82 (s, 1 H, H1 ), 4.55 (dd,
J = 9.3, 2.1 Hz, 1 H, H3 ), 3.79 (s, 6 H, OCH3), 3.71 (d, J = 2.0 Hz,
1 H, H2 ), 3.50 (s, 2 H, H5 ), 2.81 (d, J = 10.8 Hz, 1 H, H1 a), 2.77
(14) Petersen, M.; Wengel, J. Trends Biotechnol. 2003, 21, 74.
(15) Kumar, R.; Singh, S. K.; Koshkin, A. A.; Rajwanshi, V. K.;
Meldgaard, M.; Wengel, J. Bioorg. Med. Chem. Lett. 1998,
8, 2219.
Synthesis 2004, No. 4, 578–582 © Thieme Stuttgart · New York

582
D. S. Pedersen, T. Koch
PAPER
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(23) Rosenbohm, C.; Christensen, S. M.; Sørensen, M. D.;
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(19) Covés, J.; Le Hir de Fallois, L.; Le Pape, L.; Décout, J.-L.;
Fontecave, M. Biochemistry 1996, 35, 8595.
(20) Roy, B.; Chambert, S.; Lepoivre, M.; Aubertin, A.-M.;
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Synthesis 2004, No. 4, 578–582 © Thieme Stuttgart · New York