Synthesis of the 3′-C-hydroxymethyl-branched locked nucleic acid thymidine monomer

Article abstract of DOI:10.1002/ejoc.200800716

A 3′-C-hydroxymethyl-branched Locked Nucleic Acid (LNA) monomer 3 was synthesized from diacetone-α-D-glucose taking advantage of a stereoselective Grignard reaction for the introduction of a vinyl group, an aldol/Cannizzarro sequence for introducing the 4

Full text of DOI:10.1002/ejoc.200800716

FULL PAPER
DOI: 10.1002/ejoc.200800716
Synthesis of the 3Ј-C-Hydroxymethyl-Branched Locked Nucleic Acid
Thymidine Monomer
Surender Kumar,[a,b] Pawan K. Sharma,[a,b] Paul C. Stein,[a] and Poul Nielsen*[a]
Keywords: Locked nucleic acid / Nucleosides / Oligonucleotides / Ruthenium oxidation
A 3Ј-C-hydroxymethyl-branched Locked Nucleic Acid (LNA)
monomer 3 was synthesized from diacetone-α- -glucose tak-
ing advantage of a stereoselective Grignard reaction for the
introduction of a vinyl group, an aldol/Cannizzarro sequence
for introducing the 4Ј-substituent, oxidative cleavage of the
vinyl group using a RuO4-based protocol, Vorbrüggen-type
nucleobase coupling and finally the ring closure by ether for-
mation giving the bicyclic skeleton.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2008)
D
Introduction
Conformationally restricted nucleosides have been very
successful for the construction of oligonucleotides that are
preorganised for high-affinity nucleic acid recognition.[1]
Especially nucleoside analogues with bicyclic carbohydrate
moieties have been designed and applied as the monomers
in conformationally restricted oligonucleotide sequences,
which have displayed very promising results as compounds
with improved recognition of complementary RNA and
DNA sequences.[1] The prime example is Locked Nucleic
Acid (LNA, Figure 1), in which the bicyclic nucleoside
monomer 1 is a perfect mimic of the N-type nucleoside con-
formation.[2,3] This leads to the formation of thermally ex-
tremely stable A-type nucleic acid duplexes between LNA
and complementary DNA and RNA sequences. The incor-
poration of just one or a few LNA monomers into an oligo-
deoxynucleotide (ODN) strongly increases the thermal sta-
bility of the duplexes due to conformational steering of
neighbouring 2Ј-deoxynucleotides towards N-type confor-
mations.[3,4] LNA has demonstrated very promising results
as potential antisense therapeutics.[3,5]
Another approach in nucleic acid chemistry and nano-
technology is the introduction of branched nucleoside
monomers as conjugation sites in order to introduce vari-
ous decorations onto the nucleic acid duplex or to obtain
branched nucleic acid nanostructures. An example of a
building block is the 3Ј-C-hydroxymethyl-branched nucleo-
side monomer 2 (Figure 1), which have been incorporated
into ODNs and found to improve the enzymatic stability of
the ODN and to be more or less neutral concerning the
Figure 1. The general LNA nucleoside monomer 1 and the N-type
conformation of LNA. 3Ј-C-(Hydroxymethyl)thymidine 2 and the
3Ј-C-hydroxymethyl LNA thymidine monomer 3 as well as their
incorporation in nucleic acids as S-type and N-type mimics, respec-
tively. T = thymin-1-yl.
binding affinity for complementary nucleic acid se-
quences.[6,7] Also other 3Ј-C-modifications have been pre-
pared, and in general neutral or slightly decreased binding
affinities were found.[7,8] A 3Ј-C-alkyl substituent is ex-
pected to be oriented in a pseudoequatorial position, driv-
ing the nucleoside monomer towards an S-type conforma-
tion.[6–8] The 3Ј-C-substituents point into the major groove
of nucleic acid duplexes and are reasonably well tolerated
in the duplex structure.[6–8]
Due to the conformationally locked nature of the LNA
monomer in an N-type conformation, a 3Ј-C-substituent of
LNA is forced into an axial position and into a very dif-
ferent orientation as compared to a 3Ј-C-substituted 2Ј-de-
oxynucleotide (Figure 1). Therefore, it was appealing to
study this structural feature in the form of a 3Ј-C-hy-
droxymethyl LNA monomer. Longer alkyl groups in the
same position have been recently studied in ODNs leading
[a] Nucleic Acid Center, Department of Physics and Chemistry,
University of Southern Denmark,
5230 Odense M, Denmark
Fax: +45-66158780
E-mail: pon@ifk.sdu.dk
[b] Department of Chemistry, Kurukshetra University,
Kurukshetra 136119, India
Eur. J. Org. Chem. 2008, 5715–5722
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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S. Kumar, P. K. Sharma, P. C. Stein, P. Nielsen
FULL PAPER
to some decrease in affinity for complementary sequences optimised synthesis of LNA monomers.[16] We attempted
as compared to LNA.[9] Foreseeing that the smaller and less this as a selective reaction using one equivalent of NaOBz,
hydrophobic 3Ј-hydroxymethyl group might be better ac- expecting the formation of a monobenzoyl furanoside.
commodated into duplexes, we decided to attempt the syn- However, the bicyclic furanoside 9 was obtained under the
thesis of the 3Ј-C-hydroxymethyl LNA nucleoside 3.[10] Be- reaction conditions used. The constitution of 9 was proved
sides the potential as a direct building block for ODN syn- by NMR-studies. The application of HMBC-spectra proved
thesis in order to obtain simple 3Ј-functionalized LNA, al- the presence of the 3-O-benzyl group, the removal of the
ternative incorporations into nucleic acid nanostructures, 3-benzyloxymethylene group and the formation of a five-
for instance branched LNA, as well as biological properties membered ring. The formation of this bicyclic product
such as antiviral or anticancer therapeutic potential of the might be based on the preorganisation for forming a five-
monomer can be envisioned. Recently, we have shown the membered ring from the benzyl ether oxygen to the mesyl-
synthesis of
a
complementary 6Ј-C-hydroxymethyl- activated carbon and the destabilisation of the benzyl ether
branched LNA thymidine monomer using a mercury cycli- in the presence of the benzoate ion. Therefore, we decided
sation.[11]
to follow another strategy, whereby the vinyl group would
be converted into a hydroxymethyl group at a later stage of
the reaction sequence after the 4Ј-alkylation, and where the
5-O-mesyl group is avoided.
Results and Discussion
A convergent synthetic strategy was chosen for the syn-
thesis of the target hydroxymethyl LNA nucleoside 3 in or-
der to obtain the desired configuration at C-3Ј. A retrosyn-
thetic analysis revealed the introductions of the 3Ј-C-sub-
stituent and the 4Ј-C-substituent, as well as the ring closure
to form the bicyclic system, to be the key steps. It is well
known, that insertion of the nucleobase can not be per-
formed on a preformed bicyclo[2.2.1] skeleton,[12] wherefore
a ring-closing reaction from the 2Ј-hydroxy group to an ac-
tivated 4Ј-hydroxymethyl group was planned to be the last
of the key steps. Diacetone-α--glucose was chosen as a
cheap and commercially available starting material, on
Scheme 1. Reagents and conditions: i. ref.[13]
, 68%; ii. a)
which the 3Ј-C-substituent can be easily introduced.[13]
A
RuCl3·xH2O, NaIO4, H2O, EtOAc, CH3CN; b) NaBH4, H2O,
THF; c) NaIO4; d) NaBH4, 74%; iii. a) BnBr, NaH, DMF; b) 80%
aq. CH3COOH, 74%; iv. a) NaIO4, H2O, THF; b) HCHO, NaOH,
93%; v. MsCl, pyridine, quantitative; vi. NaOBz, DMF, 82%.
vinyl group was envisioned as a precursor for the hy-
droxymethyl group based on our recently developed ruthe-
nium-mediated protocol.[14] Either before or after the oxi-
dative cleavage of the vinyl group, the 4Ј-C-hydroxymethyl
group can be introduced by a known aldol condensation/
Cannizzarro sequence.[15]
Following this analysis, diacetone-α--glucose was con-
Starting from compound 4 conversion to the diol 10 was
verted into the known 3-C-vinyl derivative 4 in three stan- performed in two standard steps in 94% yield (Scheme 2).
dard steps (oxidation, stereoselective Grignard and ben- Selective benzylation of diol 10 was achieved using sodium
zylation) in 68% yield (Scheme 1).[13] As the first strategy, hydride and 1.2 equiv. of benzyl bromide to give 5-O-benzyl
we decided to perform the cleavage reaction before inserting derivative 11 in 60% yield along with the 4-epimer 12 in
the 4Ј-C-hydroxymethyl group. Oxidative cleavage of the 28% yield. Mesylation of the free hydroxy group of 3,5-di-
double bond of 4 was achieved by the ruthenium-mediated O-benzylfuranose 11 gave the corresponding mesyl deriva-
protocol based on RuO4 formed in situ, oxidative cleavage tive, which was converted into a 3-hydroxymethyl derivative
and a final reduction[14] to give the hydroxymethyl deriva- 13 by the same ruthenium-mediated protocol for the oxidat-
tive 5 in 74% yield. The dibenzylfuranose 6, was obtained ive cleavage as above in 54% yield from 11. Benzoylation
in another 74% yield after benzylation using a standard of the primary hydroxy group of 13, followed by acetolysis
procedure and selective cleavage of the 5,6-O-isopropylid- and acetylation, afforded the anomeric mixture of di-O-ace-
ene ring with 80% aqueous acetic acid. Oxidative cleavage tates, which was subsequently used as a glycosyl donor in a
of the diol 6 using NaIO4 and subsequent aldol condensa- modified Vorbrüggen[17] reaction. However, the major
tion between the resulting aldehyde and formaldehyde fol- product obtained in 20% yield after the chromatographic
lowed by an in situ Cannizzarro reaction afforded the diol separation of a crude mixture was characterized as the bicy-
7 in 93% yield. The mesylation of the two primary hydroxy clic nucleoside 14. It seems that the preorganisation of a 3-
groups was achieved using excess of methanesulfonyl chlo- C-benzyloxy group in proximity to the 4-C-mesyloxymethyl
ride to give derivative 8 in quantitative yield. We decided to group by Lewis acid activation lead to the formation of an
investigate a nucleophilic displacement of the mesyl group oxetane ring. Approx. 10% of a crude compound indicated
by NaOBz – a methodology that has been applied in an by MS to be a nucleoside with two thymines was also ob-
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© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2008, 5715–5722

3Ј-C-Branched Locked Nucleic Acid Thymidine Monomer
tained. This could be due to the opening of the oxetane in
14 by a second thymine – a reaction observed before with
a similar bicyclic system.[18] It was hereby clear that the acti-
vation of alcohols with mesyl groups should be avoided un-
til needed for the final ring-closing step.
Scheme 3. Reagents and conditions: i. a) BzCl, pyridine; b)
RuCl3·xH2O, NaIO4, H2O, EtOAc, CH3CN; c) NaBH4, H2O,
THF; d) NaIO4; e) NaBH4; 71%; ii. BnBr, NaH, DMF, 75%; iii.
a) 80% aq. CH3COOH; b) Ac2O, pyridine, 91%; iv. Thymine, BSA,
TMS-triflate, CH3CN, 93%; v. NaOCH3, MeOH, 86%; vi. a)
MsCl, CH2Cl2, pyridine; b) NaH, 1,4-dioxane, 72%; vii. H2,
Pd(OH)2/C, EtOH, quantitative.
Scheme 2. Reagents and conditions: i. a) H5IO6, EtOAc; b) HCHO,
NaOH, NaBH4, 94%; ii. NaH, BnBr, DMF, 60% 11 and 28% 12;
iii. a) MsCl, Pyridine; b) RuCl3·xH2O, NaIO4, H2O, EtOAc,
CH3CN; c) NaBH4, H2O, THF; d) NaIO4; e) NaBH4; 54%; iv. a)
BzCl, pyridine; b) 80% aq. CH3COOH; c) Ac2O, pyridine; d) thy-
mine, BSA, TMS-triflate, CH3CN, 20%.
In the third strategy, the free hydroxy group of 11 was
protected as a benzoyl ester followed by ruthenium-medi-
ated oxidative cleavage[14] of the vinyl group. This resulted
in the formation of 3-C-(hydroxymethyl)furanose 15 in 71%
yield from 11 (Scheme 3). In order to be able to deprotect
the 4Ј-C-hydroxymethyl group selectively, we decided to in-
troduce another convenient benzyl group for the 3Ј-C-hy-
droxymethyl group, even though differentiation between the
two primary positions of the final product was lost. Ben-
zylation of the primary hydroxy group afforded 16 in 75%
yield, which was treated with 80% aqueous acetic acid to
remove the 1,2-O-isopropylidene protective group followed
by acetylation of the resulting two hydroxy groups to give
Conclusions
We have successfully synthesized a 3Ј-C-hydroxymethyl-
branched LNA nucleoside analogue 3 in an overall yield of
11% after 19 steps, using a convergent synthesis starting
from diacetone-α--glucose as a cheap starting material.
This nucleoside has a broad potential as a monomer for the
functionalisation of LNA.
Experimental Section
General: All reagents were obtained from commercial suppliers and
the anomeric mixture of diacetates 17 in 91% yield. Coup- used without further purification except dichloromethane which
was distilled. Reactions were performed under an atmosphere of
nitrogen when anhydrous solvents were used. All reactions were
followed on TLC plates made of aluminium sheets with silica gel
60 F254 from Merck. The plates were visualized under UV-light
and then developed in a 5% solution of H2SO4 in MeOH. Column
chromatography was carried out on glass columns using silica gel
60 (0.040–0.063 mm) which was purchased from Merck. NMR
ling between 17 and thymine using the modified
Vorbrüggen conditions[17] provided nucleoside 18 in 93%
yield. Cleavage of the acetyl and benzoyl esters in a single
transesterification step using sodium methoxide in meth-
anol resulted in the formation of nucleoside 19 in 86%
yield. The selective mesylation of 19 followed by the ring
formation using sodium hydride in anhydrous 1,4-dioxane
resulted in the formation of bicyclic nucleoside 20 in 72%
yield. Debenzylation using 20% palladium hydroxide over
1
spectra were recorded at 200, 300 or 500 MHz for H NMR and
75 or 125 MHz for 13C NMR spectroscopy. Chemical shifts are in
1
ppm relative to tetramethylsilane as internal standard (for H and
carbon under hydrogen atmosphere proceeded efficiently to 13C NMR). Assignments of the NMR spectroscopic data were
based on 2D spectra (COSY, HETCOR, HSQC and/or HMBC)
and follow standard carbohydrate and nucleoside nomenclature
and numbering, i.e. the carbon atom next to a nucleobase is as-
signed C-1Ј, etc. In nucleosides, C-1ЈЈ designates the carbon atom
in the 3Ј-branch and C-5ЈЈ the carbon atom in the 4Ј-branch; other-
wise, C-1Ј designates the carbon atom in the 3-branch and C-5Ј the
carbon atom in the 4-branch. Compound names for the bi- and
tricyclic compounds are given according to the von Baeyer no-
menclature. High-resolution MALDI mass determinations were
performed on an Ionspec Ultima Fourier transform mass spec-
trometer.
yield the target branched LNA nucleoside monomer 3 in
1
quantitative yield. The constitution of 3 was proved by H
3
NMR spectroscopy showing e.g. JH1Ј–H2Ј = 0 Hz and
thereby the locked N-type conformation of an LNA-mono-
mer.
We are currently investigating different ways for the dif-
ferentiation and protection of the two primary alcohols and
for the phosphitylation of the tertiary alcohol in order to
make a phosphoramidite building block of the 3Ј-branched
LNA-monomer 3 for oligonucleotide synthesis.
Eur. J. Org. Chem. 2008, 5715–5722
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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S. Kumar, P. K. Sharma, P. C. Stein, P. Nielsen
FULL PAPER
3-O-Benzyl-1,2:5,6-di-O-isopropylidene-3-C-(hydroxymethyl)-α-
D
-
reduced pressure and dissolved in ethyl acetate (100 mL). The or-
allofuranose (5): A solution of sodium periodate (321 mg, ganic phase was washed with water (2ϫ50 mL), dried (Na2SO4)
1.50 mmol) in water (2 mL) was stirred at 0 °C and RuCl3·xH2O
(18 mg, 0.07 mmol) was added. In another flask, benzylated com-
pound 4 (376 mg, 1.0 mmol) was dissolved in a mixture of ethyl
acetate (5 mL) and acetonitrile (5 mL) and the mixture was stirred
for 5 min at 0 °C. The aqueous solution of RuCl3·xH2O and so-
dium periodate was added and the slurry was stirred for 4 min.
The reaction was quenched by the addition of a saturated aqueous
solution of Na2S2O3 (5 mL). Phases were separated and the aque-
ous layer was extracted with ethyl acetate (3ϫ4 mL). The com-
bined organic phase was dried (Na2SO4) and concentrated under
reduced pressure. The residue was dissolved in a mixture of THF
(3 mL) and water (3 mL). Sodium borohydride (76 mg, 2.0 mmol)
was added, and a flocculent black powder began to separate after
5 min. The mixture was stirred for 20 min at room temperature,
and concentrated under reduced pressure. The residue was coevap-
orated with toluene and purified by silica gel column chromatog-
raphy (0–45% ethyl acetate in petroleum ether) to give 6 (8.73 g,
74%) as a white solid: Rf = 0.25 (1:1, ethyl acetate/petroleum ether).
1H NMR (300 MHz, CDCl3): δ = 7.40–7.25 (m, 10 H, Ph), 5.68
(d, J = 3.9 Hz, 1 H, 1-H), 4.73–4.67 (m, 2 H, 3-OCH2Ph), 4.61,
4.50 (AB, J = 12.0 Hz, 2 H, 1Ј-OCH2Ph), 4.45 (d, J = 3.9 Hz, 1 H,
2-H), 4.05 (d, J = 8.7 Hz, 1 H, 4-H), 3.88 (m, 1 H, 5-H), 3.77–3.62
(m, 4 H, 1Ј-H, 6-H), 3.31 (d, J = 2.7 Hz, 1 H, 5Ј-OH), 2.33 (m, 1
H, 6Ј-OH), 1.59 (s, 3 H, CH3), 1.34 (s, 3 H, CH3) ppm. 13C NMR
(75 MHz, CDCl3): δ = 138.3, 136.8, 128.7, 128.4, 128.3, 128.1,
127.7, 127.6 (Ph), 113.1 [C(CH3)2], 104.0 (C-1), 84.9 (C-3), 79.9 (C-
2), 79.4 (C-4), 74.1 (1Ј-OCH2Ph), 70.3, 68.1, 67.9, 64.7 (3-OCH2Ph,
C-5, C-1Ј, C-6), 26.8 (CH3) ppm. HR-MALDI MS: m/z 453.1870
water (3 mL) was added, and the mixture was extracted with ([M + Na]+, C24H30O7Na+ calcd. 453.1884).
dichloromethane (3 ϫ4 mL). The combined organic phase was
3-O-Benzyl-3-C-(benzyloxymethyl)-4-C-(hydroxymethyl)-1,2-di-O-
isopropylidene-α- -ribofuranose (7): To a stirred solution of 6
washed with a saturated aqueous solution of NaHCO3 (3ϫ3 mL),
dried (Na2SO4) and concentrated under reduced pressure. The resi-
due was dissolved in THF (3 mL) and water (3 mL) at 0–5 °C. So-
dium periodate (430 mg, 2.0 mmol) was added in small portions
and the solution was stirred for 2 h at room temperature. The reac-
tion mixture was diluted with water (3 mL). The mixture was ex-
tracted with ethyl acetate (3ϫ4 mL), and the combined organic
phase was dried (Na2SO4), and concentrated under reduced pres-
sure. The residue was redissolved in a mixture of THF (3 mL) and
water (3 mL), and sodium borohydride (76 mg, 1.0 mmol) was
added. The reaction mixture was stirred at room temperature for
1 h, water (4 mL) was added, and the mixture was extracted with
dichloromethane (3 ϫ4 mL). The combined organic phase was
washed with a saturated aqueous solution of NaHCO3 (3ϫ2 mL),
dried (Na2SO4) and concentrated under reduced pressure. The resi-
due was purified by silica gel column chromatography (0–1.5 %
methanol in dichloromethane) to give 5 (281 mg, 74%) as a colour-
less oil: Rf = 0.50 (19:1, CH2Cl2/CH3OH). 1H NMR (300 MHz,
CDCl3): δ = 7.41–7.25 (m, 5 H, Ph), 5.76 (d, J = 3.9 Hz, 1 H, 1-
H), 4.74 (s, 2 H, CH2Ph), 4.49 (d, J = 3.9 Hz, 1 H, 2-H), 4.37 (m,
1 H, 5-H), 4.18 (d, J = 6.9 Hz, 1 H, 4-H), 4.17–4.02 (m, 2 H, 6-
H), 3.90 (dd, J = 12.0, 6.0 Hz, 1 H, 1Ј-H), 3.80 (dd, J = 12.0,
6.3 Hz, 1 H, 1Ј-H), 3.07 (dd, J = 6.0, 6.3 Hz, 1 H, OH), 1.59 (s, 3
H, CH3), 1.43 (s, 3 H, CH3), 1.36 (s, 3 H, CH3), 1.34 (s, 3 H, CH3)
ppm. 13C NMR (75 MHz, CDCl3): δ = 138.7, 128.3, 127.6, 127.6
(Ph), 112.9 (C(CH3)2), 110.0 [C(CH3)2], 104.1 (C-1), 84.7 (C-3),
82.0 (C-2), 80.2 (C-6), 73.8 (C-4), 67.2 (3Ј-OCH2Ph), 66.8 (C-5),
61.4 (C-1Ј), 27.0 (CH3), 26.8 (CH3), 26.4 (CH3), 24.9 (CH3) ppm.
HR-MALDI MS: m/z 403.1720 ([M + Na]+, C20H28O7Na+ calcd.
403.1727).
D
(8.69 g, 20.20 mmol) in a mixture of THF and water (66 mL, 1:1
v/v) at 0 °C was added sodium periodate (4.76 g, 22.22 mmol) in
small portions over a period of 30 min, and the mixture was stirred
for 3 h. The precipitate was filtered off and washed with diethyl
ether (3 ϫ 20 mL). The phases were separated and the aqueous
phase was extracted with diethyl ether (3ϫ20 mL). The combined
organic phase was washed with brine (2ϫ10 mL), concentrated un-
der reduced pressure and redissolved in 1,4-dioxane (20 mL).
Formaldehyde (37% solution, 5 mL) and NaOH (2 , 21 mL) were
added and the reaction mixture was stirred overnight. Dichloro-
methane (50 mL) was added and the phases were separated. The
aqueous phase was extracted with dichloromethane (3ϫ50 mL).
The combined organic phase was washed with brine (2ϫ50 mL),
dried (Na2SO4) and concentrated under reduced pressure.
Crystallization from CH2Cl2/petroleum ether afforded 7 (8.04 g,
93%) as white solid: Rf = 0.25 (1:1, ethyl acetate/petroleum ether).
1H NMR (300 MHz, CDCl3): δ = 7.39–7.24 (m, 10 H, Ph), 5.75
(d, J = 4.5 Hz, 1 H, 1-H), 4.74, 4.63 (AB, J = 10.8 Hz, 2 H, 3-
OCH2Ph), 4.57–4.52 (m, 3 H, 2-H, 1Ј-OCH2Ph), 4.14–4.02 (m, 2
H, 5Ј-H), 3.82–3.72 (m, 4 H, 5-H, 1Ј-H), 2.86 (m, 1 H, 5-OH), 2.33
(m, 1 H, 5Ј-OH), 1.63 (s, 3 H, CH3), 1.31 (s, 3 H, CH3) ppm. 13C
NMR (75 MHz, CDCl3): δ = 138.4, 136.8, 128.7, 128.4, 128.3,
128.1, 127.7, 127.4 (Ph), 113.0 [C(CH3)2], 104.4 (C-1), 87.4, 86.1
(C-3, C-4), 81.2 (C-2), 74.0 (1Ј-OCH2Ph), 69.3, 68.0, 62.9, 62.6 (3-
OCH2Ph, C-1Ј, C-5, C-5Ј), 26.3 (CH3), 25.9 (CH3) ppm. HR-
MALDI MS: m/z 453.1869 ([M + Na]+, C24H30O7Na+ calcd.
453.1884).
3-O-Benzyl-3-C-(benzyloxymethyl)-1,2-di-O-isopropylidene-5-O-
(methylsulfonyl)-4-C-(methylsulfonyloxymethyl)-α-
D-ribopentofura-
3-O-Benzyl-3-C-(benzyloxymethyl)-1,2-di-O-isopropylidene-α-
D-allo- nose (8): To a solution of 7 (8.0 g, 18.60 mmol) in anhydrous
furanose (6): To a solution of 5 (10.49 g, 27.60 mmol) in anhydrous
DMF (75 mL) at 0 °C was added a 60% oil dispersion of NaH
dichloromethane (10 mL) and anhydrous pyridine (7.5 mL,
93.01 mmol) at 0 °C, methanesulfonyl chloride (3.20 mL,
(1.66 g, 41.40 mmol) and the mixture was stirred for 2 h. Benzyl 40.93 mmol) was added dropwise, and the reaction mixture was
bromide (6.60 mL, 55.21 mmol) was added dropwise, and the reac-
tion mixture was stirred overnight at room temperature. The reac-
tion was quenched by slow addition of water (150 mL) at 0 °C and
the resulting mixture was extracted with ethyl acetate (3ϫ75 mL).
The combined organic phase was washed successively with a satu-
rated aqueous solution of NaHCO3 (100 mL), brine (100 mL) and
water (100 mL). The organic layer was dried (Na2SO4) and concen-
trated under reduced pressure, and the residue was coevaporated
with toluene (3ϫ25 mL). The residue was dissolved in 80% aque-
ous acetic acid (70 mL) and the reaction mixture was stirred for
45 h at room temperature. The mixture was concentrated under
stirred at room temperature for 1.5 h. A saturated aqueous solution
of NaHCO3 (20 mL) was added and the mixture was stirred for
15 min. The reaction mixture was diluted with dichloromethane
(20 mL) and the phases were separated. The organic phase was
washed with 1  HCl (3ϫ10 mL) and a saturated aqueous solution
of NaHCO3 (5 mL), dried (Na2SO4) and evaporated under reduced
pressure to give 8 (10.90 g, quantitative) as a white foam: Rf = 0.28
1
(1:1, ethyl acetate/petroleum ether). H NMR (300 MHz, CDCl3):
δ = 7.40–7.25 (m, 10 H, Ph), 5.79 (d, J = 3.9 Hz, 1 H, 1-H), 4.83,
4.62 (AB, J = 11.7 Hz, 2 H), 4.74, 4.60 (AB, J = 11.1 Hz, 2 H, 3-
OCH2Ph, 5Ј-H), 4.70, (d, J = 3.9 Hz, 1 H, 2-H), 4.58, 4.51 (AB, J
5718
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© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2008, 5715–5722

3Ј-C-Branched Locked Nucleic Acid Thymidine Monomer
= 11.4 Hz, 2 H, 1Ј-OCH2Ph), 4.39, 4.34 (AB, J = 9.9 Hz, 2 H, 5-
H), 3.81, 3.79 (AB, J = 9.9 Hz, 2 H, 1Ј-H), 2.99 (s, 3 H, SO2CH3),
2.98 (s, 3 H, SO2CH3), 1.70 (s, 3 H, CH3), 1.30 (s, 3 H, CH3) ppm.
13C NMR (75 MHz, CDCl3): δ = 138.0, 136.8, 128.7, 128.5, 128.3,
128.0, 127.8, 127.4 (Ph), 113.5 [C(CH3)2], 105.2 (C-1), 85.6, 85.1
(C-3, C-4), 82.1 (C-2), 74.0 (1Ј-OCH2Ph), 69.2, 68.8, 68.5, 65.9 (3-
OCH2Ph, C-1Ј, C-2, C-5Ј, C-5), 38.0 (SO2CH3), 37.4 (SO2CH3),
26.3 (CH3), 25.9 (CH3) ppm. HR-MALDI MS: m/z 609.1441 ([M
+ Na]+, C26H34O11S2Na+ calcd. 609.1435).
MALDI MS: m/z 359.1478 ([M + Na]+, C18H24O6Na+ calcd.
359.1465).
3,5-Di-O-benzyl-4-C-(hydroxymethyl)-1,2-di-O-isopropylidene-3-C-
vinyl-α-
D-ribofuranose (11) and 3-O-Benzyl-4-C-(benzyloxymethyl)-
1,2-di-O-isopropylidene-3-C-vinyl-α-
D-ribofuranose (12): The diol 10
(29.61 g, 88.13 mmol) was dissolved in anhydrous DMF (400 mL)
and stirred at –20 °C. A 60 % oily dispersion of NaH (4.23 g,
105.8 mmol) was added and the mixture was stirred at –20 °C for
30 min. Benzyl bromide (12.56 mL, 105.8 mmol) was added drop-
wise, and the reaction mixture was stirred for 30 min at –20 °C. The
reaction was quenched by the dropwise addition of water (5 mL)
followed by stirring for 15 min. The reaction mixture was concen-
trated under reduced pressure and the residue was partitioned be-
tween ethyl acetate (200 mL) and water (100 mL). The aqueous
phase was extracted with ethyl acetate (2ϫ50 mL). The combined
organic phase was dried (Na2SO4) and concentrated under reduced
pressure. The residue was purified by silica gel column chromatog-
raphy (0–45% ethyl acetate in petroleum ether) to give the two
products 11 and 12 as clear viscous oils. 11 (22.37 g, 60%): Rf =
0.45 (1:1, petroleum ether/ethyl acetate). 1H NMR (300 MHz,
CDCl3): δ = 7.36–7.24 (m, 10 H, Ph), 5.89 (d, J = 3.9 Hz, 1 H, 1-
H), 5.84 (dd, J = 11.1, 18.0 Hz, 1 H, 1Ј-H), 5.40 (d, J = 11.1 Hz,
1 H, 2Ј-H), 5.27 (d, J = 18.0 Hz, 1 H, 2Ј-H), 4.72, 4.56 (AB, J =
11.4 Hz, 2 H, CH2Ph), 4.70 (d, J = 3.9 Hz, 1 H, 2-H), 4.57, 4.48
(AB, J = 12.0 Hz, 2 H, CH2Ph), 4.45–4.11 (m, 2 H, 5Ј-H), 3.54 (s,
2 H, 5-H), 2.46 (t, J = 6.9 Hz, 1 H, 5Ј-OH), 1.67 (s, 3 H, CH3),
1.36 (s, 3 H, CH3) ppm. 13C NMR (75 MHz, CDCl3): δ = 135.9
(C-1Ј), 138.5, 138.4, 128.4, 128.3, 127.6, 127.5, 127.4, 127.2 (Ph),
117.5 (C-2Ј), 113.3 [C(CH3)2], 104.8 (C-1), 88.6, 87.2 (C-3, C-4),
82.5 (C-2), 73.6 (5-OCH2Ph), 71.3 (C-5), 67.4 (3-OCH2Ph), 62.1
(C-5Ј), 26.5 (CH3), 26.1 (CH3) ppm. HR-MALDI MS: m/z
449.1940 ([M + Na]+, C25H30O6Na+ calcd. 449.1935). 12 (10.51 g,
28 %): Rf = 0.42 (1:1, ethyl acetate/petroleum ether). 1H NMR
(300 MHz, CDCl3): δ = 7.36–7.24 (m, 10 H, Ph), 5.96 (dd, J =
11.4, 18.0 Hz, 1 H, 1Ј-H), 5.88 (d, J = 4.2 Hz, 1 H, 1-H), 5.47 (d,
J = 11.4 Hz, 1 H, 2Ј-H), 5.32 (d, J = 18.0 Hz, 1 H, 2Ј-H), 4.72 (d,
J = 4.2 Hz, 1 H, 2-H), 4.70, 4.58 (AB, J = 11.1 Hz, 2 H, CH2Ph),
4.60, 4.53 (AB, J = 12.0 Hz, 2 H, CH2Ph), 4.20, 4.11 (AB, J =
10.5 Hz, 2 H, 5Ј-H), 3.78–3.65 (m, 2 H, 5-H), 2.56 (dd, J = 6.0,
7.2 Hz, 1 H, 5-OH), 1.52 (s, 3 H, CH3), 1.35 (s, 3 H, CH3) ppm.
13C NMR (75 MHz, CDCl3): δ = 136.2 (C-1Ј), 138.8, 138.2, 128.4,
128.3, 128.0, 127.7, 127.4, 127.1 (Ph), 117.6 (C-2Ј), 113.2
[C(CH3)2], 104.7 (C-1), 88.4, 86.4 (C-3, C-4), 82.5 (C-2), 73.8 (5Ј-
OCH2Ph), 71.4 (C-5Ј), 67.3 (3-OCH2Ph), 65.0 (C-5Ј), 26.4 (CH3),
26.2 (CH3) ppm. HR-MALDI MS: m/z 449.1915 ([M + Na]+,
C25H30O6Na+ calcd. 449.1935).
(1S,2S,6R,8R)-1-(Benzyloxy)-8-(methylsulfonyloxymethyl)-4,4-di-
methyl-3,5,7,10-tetraoxatricyclo[6.3.0.02.6]undecane (9): Sodium
benzoate (12.3 mg, 0.09 mmol) was added to a solution of com-
pound 8 (50 mg, 0.09 mmol) in DMF (2 mL). The mixture was
stirred for 5 h at 100 °C, cooled to room temperature, and filtered.
The solvent was evaporated under reduced pressure, and the resi-
due was suspended in ethyl acetate (5 mL), washed with water
(3ϫ2 mL), dried (Na2SO4), and concentrated under reduced pres-
sure. The residue was purified by silica gel column chromatography
(0–25% ethyl acetate in petroleum ether) to give 9 (28 mg, 82%) as
a white solid: Rf = 0.15 (1:1, ethyl acetate/petroleum ether). 1H
NMR (500 MHz, CDCl3): δ = 7.45–7.30 (m, 5 H, Ph), 5.96 (d, J
= 3.6 Hz, 1 H, 1-H), 4.82 (d, J = 11.8 Hz, 1 H, 5Ј-H), 4.73–4.69
(m, 2 H, CH2Ph, 2-H), 4.49 (d, J = 11.8 Hz, 1 H, 5Ј-H), 4.44 (d, J
= 10.5 Hz, 1 H, CH2Ph), 4.15 (d, J = 10.5 Hz, 1 H, 1Ј-H), 4.06 (d,
J = 10.5 Hz, 1 H, 5-H), 3.97 (d, J = 10.5 Hz, 1 H, 1Ј-H), 3.88 (d,
J = 10.5 Hz, 1 H, 5-H), 3.06 (s, 3 H, SO2CH3), 1.68 (s, 3 H, CH3),
1.38 (s, 3 H, CH3) ppm. 13C NMR (125 MHz, CDCl3): δ = 137.4,
128.7, 128.3, 127.8 (Ph), 114.2 [C(CH3)2], 105.8 (C-1), 93.1 (C-4),
92.1 (C-3), 81.4 (C-2), 76.4 (C-5Ј), 75.6 (C-1Ј), 71.7 (C-5), 68.7
(CH2Ph), 38.0 (SO2CH3), 26.8 (CH3), 26.2 (CH3) ppm. HR-
MALDI MS: m/z 423.1078 ([M + Na]+, C18H24O8SNa+ calcd.
423.1084).
3-O-Benzyl-4-C-(hydroxymethyl)-1,2-di-O-isopropylidene-3-C-vinyl-
α-D-ribofuranose (10): The furanose 4 (32.00 g, 85.1 mmol) was dis-
solved in anhydrous ethyl acetate (400 mL), and H5IO6 (23.29 g,
102.1 mmol) was added. The mixture was stirred at room tempera-
ture for 1.5 h and then filtered through a layer of celite. The com-
bined filtrates were concentrated under reduced pressure, and the
residue was dissolved in THF (250 mL). An aqueous solution of
formaldehyde [24 mL, 37% (w/v) containing 10% CH3OH] and an
aqueous solution of NaOH (2 , 96.6 mL, 193.2 mmol) were added
dropwise, and the reaction mixture was stirred at room temperature
for 24 h. The mixture was cooled to 0 °C, NaBH4 (4.85 g,
127.7 mmol) was added, and the mixture was stirred at room tem-
perature for 1 h. The mixture was neutralised with 4  acetic acid
and extracted with ethyl acetate (3ϫ100 mL). The combined or-
ganic phase was washed with a saturated aqueous solution of
NaHCO3 (3ϫ50 mL), dried (Na2SO4) and concentrated under re-
duced pressure. The residue was purified by silica gel column
chromatography (0–45% ethyl acetate in petroleum ether) to give
3,5-Di-O-benzyl-3-C-(hydroxymethyl)-1,2-di-O-isopropylidene-4-C-
(methylsulfonyloxymethyl)-α-D-ribofuranose (13): To a solution of
11 (409 mg, 0.96 mmol) in anhydrous dichloromethane (1 mL) and
anhydrous pyridine (0.40 mL, 4.80 mmol) at 0 °C, methanesulfonyl
chloride (82.0 µL, 1.06 mmol) was added dropwise and the reaction
mixture was stirred at room temperature for 1.5 h. A saturated
10 (26.73 g, 94%) as a colourless viscous oil: Rf = 0.20 (1:1, ethyl
1
acetate/petroleum ether). H NMR (300 MHz, CDCl3): δ = 7.36– aqueous solution of NaHCO3 (5 mL) was added and the mixture
7.26 (m, 5 H, Ph), 5.93 (dd, J = 11.4, 18.0 Hz, 1 H, 1Ј-H), 5.88 (d,
J = 3.9 Hz, 1 H, 1-H), 5.50 (d, J = 11.4 Hz, 1 H, 2Ј-H), 5.32 (d, J
= 18.0 Hz, 1 H, 2Ј-H), 4.73 (d, J = 3.9 Hz, 1 H, 2-H), 4.71, 4.57
(AB, J = 10.5 Hz, 2 H, CH2Ph), 4.33 (dd, J = 6.3, 11.7 Hz, 1 H,
5Ј-H), 4.07 (dd, J = 7.5, 11.7 Hz, 1 H, 5Ј-H), 3.76–3.68 (m, 2 H,
was stirred for 15 min. The reaction mixture was diluted with
dichloromethane (10 mL) and the phases were separated. The or-
ganic phase was washed with 1  HCl (3ϫ4 mL) and a saturated
aqueous solution of NaHCO3 (5 mL), dried (Na2SO4) and concen-
trated under reduced pressure to give the mesylated derivative in
5-H), 2.58 (m, 1 H, OH), 2.45 (m, 1 H, OH), 1.67 (s, 3 H, CH3), quantitative yield as a white foam. In a separate flask, a solution
1.37 (s, 3 H, CH3) ppm. 13C NMR (75 MHz, CDCl3): δ = 135.7
(C-1Ј), 138.0, 128.6, 127.9, 127.5 (Ph), 117.7 (C-2Ј), 113.2
[C(CH3)2], 104.7 (C-1), 87.8, 87.1 (C-3, C-4), 82.3 (C-2), 67.7
of sodium periodate (64 mg, 0.30 mmol) in water (1 mL) at 0 °C
was added RuCl3·xH2O (3.50 mg, 0.01 mmol). The mesylated com-
pound (100 mg, 0.20 mmol) was dissolved in a mixture of ethyl
(CH2Ph), 65.5, 63.8 (C-5, C-5Ј), 26.4 (CH3), 25.9 (CH3) ppm. HR- acetate (3 mL) and acetonitrile (3 mL) and the mixture was stirred
Eur. J. Org. Chem. 2008, 5715–5722
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5719

S. Kumar, P. K. Sharma, P. C. Stein, P. Nielsen
FULL PAPER
for 5 min at 0 °C. To this solution, the aqueous solution of
RuCl3·xH2O and sodium periodate was added in one portion and
the slurry was stirred for 5 min. The reaction was quenched by the
addition of a saturated aqueous solution of Na2S2O3 (5 mL). The
phases were separated and the aqueous layer was extracted with
ethyl acetate (3ϫ5 mL). The combined organic phase was dried
(Na2SO4) and concentrated under reduced pressure. The residue
was dissolved in a mixture of THF (2.5 mL) and water (2.5 mL).
Sodium borohydride (15 mg, 0.40 mmol) was added, and a floccu-
lent black powder began to separate after 5 min. The mixture was
stirred for 20 min at room temperature, water (10 mL) was added,
and the mixture was extracted with CH2Cl2 (3ϫ10 mL). The com-
bined organic phase was washed with a saturated aqueous solution
of NaHCO3 (3ϫ5 mL), dried (Na2SO4) and concentrated under
reduced pressure. The residue was dissolved in THF (2.5 mL) and
water (2.5 mL) at 0–5 °C. Sodium periodate (85 mg, 0.40 mmol)
was added in small portions, and the solution was stirred for 2 h
at room temperature. The reaction mixture was diluted with water
(10 mL). The mixture was extracted with ethyl acetate (3ϫ5 mL).
The combined organic phase was dried (Na2SO4), and concen-
trated under reduced pressure. The residue was redissolved in a
mixture of THF (2.5 mL) and water (2.5 mL), and sodium borohy-
dride (15 mg, 0.40 mmol) was added. The reaction mixture was
stirred at room temperature for 1 h, water (10 mL) was added, and
the mixture was extracted with CH2Cl2 (3ϫ10 mL). The combined
organic phase was washed with a saturated aqueous solution of
NaHCO3 (3ϫ5 mL), dried (Na2SO4) and concentrated under re-
duced pressure. The residue was purified by silica gel column
chromatography (0–30% ethyl acetate in petroleum ether) to give
13 (54 mg, 54%) as a white solid: Rf = 0.35 (1:1, ethyl acetate/
0.29 mmol). The reaction mixture was stirred under reflux for
30 min. After cooling to 0 °C, trimethylsilyl triflate (21 µL,
0.12 mmol) was added dropwise, and the solution was stirred for
24 h at 60 °C. The reaction was quenched by the addition of a cold
saturated aqueous solution of NaHCO3 (10 mL), and the resulting
mixture was extracted with dichloromethane (3ϫ5 mL). The com-
bined extracts were washed with a saturated aqueous solution of
NaHCO3 (2ϫ5 mL) and brine (2 ϫ5 mL), dried (Na2SO4) and
concentrated under reduced pressure. The residue was purified by
silica gel column chromatography (0–45% ethyl acetate in petro-
leum ether) to give nucleoside 14 (10.6 mg, 20%) as a white foam:
Rf = 0.40 (3:1, ethyl acetate/petroleum ether). 1H NMR (300 MHz,
CDCl3): δ = 8.40 (br. s, 1 H, NH), 7.99–7.96 (m, 2 H, Ph), 7.65–
7.24 (m, 9 H, Ph, 6-H), 6.68 (d, J = 6.9 Hz, 1 H, 1Ј-H), 5.17 (d, J
= 6.9 Hz, 1 H, 2Ј-H), 4.84 (d, J = 12.3 Hz, 1 H), 4.76 (d, J =
7.5 Hz, 1 H), 4.65–4.40 (m, 4 H), 3.97 (d, J = 10.5 Hz, 1 H), 3.73
(d, J = 10.5 Hz, 1 H), 2.09 (s, 3 H, COCH3), 1.75 (d, J = 0.9 Hz,
3 H, CH3) ppm. 13C NMR (75 MHz, CDCl3): δ = 170.4 (COCH3),
165.8 (COPh), 163.2 (C-4), 150.5 (C-2), 136.8 (C-6), 134.8, 133.6,
129.7, 128.8, 128.7, 128.6, 128.4, 127.9 (Ph), 112.4 (C-5), 91.3, 86.3,
85.4, 76.9, 75.2, 74.0, 68.2, 63.2 (C-1Ј, C-2Ј, C-3Ј, C-4Ј, C-5Ј, C-
1ЈЈ, C-5ЈЈ, CH2Ph), 20.5 (COCH3), 12.6 (CH3) ppm. HR-MALDI
MS: m/z 559.1676 ([M + Na]+, C28H28N2O9Na+ calcd. 559.1687).
4-C-(Benzoyloxymethyl)-3,5-di-O-benzyl-3-C-(hydroxymethyl)-1,2-
di-O-isopropylidene-α-D-ribofuranose (15): To a stirred solution of
11 (1.12 g, 2.63 mmol) in anhydrous pyridine (10 mL) at 0 °C was
added benzoyl chloride (0.46 mL, 3.94 mmol). The reaction mix-
ture was stirred at room temperature for 2 h. The mixture was con-
centrated under reduced pressure and coevaporated with toluene
(3 ϫ 5 mL). The residue was partitioned between ethyl acetate
(15 mL) and water (9 mL). The organic phase was washed with a
saturated aqueous solution of NaHCO3 (3 ϫ 5 mL), dried
(Na2SO4), and concentrated under reduced pressure to give the
benzoylated compound as an oily residue. In a separate flask, a
solution of sodium periodate (844 mg, 3.94 mmol) in water (5 mL)
was stirred at 0 °C, and RuCl3·xH2O (46.50 mg, 0.18 mmol) was
added. The benzoylated compound was dissolved in a mixture of
ethyl acetate (15 mL) and acetonitrile (15 mL), and the mixture was
stirred for 5 min at 0 °C. The aqueous solution of RuCl3·xH2O and
sodium periodate was added and the slurry was stirred for 4 min.
The reaction was quenched by the addition of a saturated aqueous
1
petroleum ether). H NMR (300 MHz, CDCl3): δ = 7.36–7.25 (m,
10 H, Ph), 5.87 (d, J = 4.2 Hz, 1 H, 1-H), 4.97 (d, J = 11.7 Hz, 1
H), 4.75–4.53 (m, 5 H), 4.34 (d, J = 11.7 Hz, 1 H), 3.86–3.62 (m,
5 H), 3.07 (s, 3 H, SO2CH3), 1.54 (s, 3 H, CH3), 1.36 (s, 3 H, CH3)
ppm. 13C NMR (75 MHz, CDCl3): δ = 138.0, 136.5, 128.9, 128.8,
128.5, 128.3, 127.9, 127.5 (Ph), 113.1 [C(CH3)2], 105.2 (C-1), 87.0,
86.5, 81.8, 74.6, 69.9, 67.8, 67.8, 62.6 (C-2, C-3, C-4, C-5, C-1Ј, C-
5Ј, 2ϫ CH2Ph), 38.3 (SO2CH3), 26.0 (CH3), 25.6 (CH3) ppm. HR-
MALDI MS: m/z 531.1641 ([M + Na]+, C25H32O9SNa+ calcd.
531.1659).
(1S,3R,4R,5S)-4-(Acetyloxy)-5-(benzoyloxymethyl)-1-(benzyloxy-
methyl)-3-(thymin-1-yl)-2,6-dioxabicyclo[3.2.0]heptane (14): To a solution of Na2S2O3 (25 mL). The phases were separated and the
stirred solution of 13 (50 mg, 0.10 mmol) in anhydrous pyridine
(2 mL) at 0 °C was added benzoyl chloride (24 µL, 0.20 mmol). The
reaction mixture was stirred at room temperature for 2 h, concen-
trated under reduced pressure and coevaporated with toluene
(3 ϫ 5 mL). The residue was partitioned between ethyl acetate
(5 mL) and water (3 mL). The organic phase was washed with a
saturated aqueous solution of NaHCO3 (3 ϫ 5 mL), dried
(Na2SO4), and concentrated under reduced pressure. The residue
was dissolved in 80% aqueous acetic acid (2 mL) and the solution
was stirred at 90 °C for 24 h. The mixture was concentrated under
reduced pressure, and the residue was coevaporated with 99% etha-
nol (3 ϫ 5 mL), toluene (3 ϫ 5 mL) and anhydrous pyridine
(2ϫ5 mL), and redissolved in anhydrous pyridine (2 mL). Acetic
anhydride (0.5 mL) was added, and the solution was stirred at
room temperature for 20 h. The reaction was quenched with ice-
cold water (10 mL) and extracted with dichloromethane
(2ϫ10 mL). The combined extracts were washed with a saturated
aqueous solution of NaHCO3 (3ϫ5 mL), dried (Na2SO4) and con-
centrated under reduced pressure. To a stirred solution of the resi-
aqueous phase was extracted with ethyl acetate (3ϫ15 mL). The
combined organic phase was dried (Na2SO4) and concentrated un-
der reduced pressure. The residue was dissolved in a mixture of
THF (12.5 mL) and water (12.5 mL). Sodium borohydride
(200 mg, 5.26 mmol) was added, and a flocculent black powder be-
gan to separate after 5 min. The mixture was stirred for 20 min at
room temperature, water (10 mL) was added, and the mixture was
extracted with CH2Cl2 (3ϫ20 mL). The combined organic phase
was washed with a saturated aqueous solution of NaHCO3
(3ϫ15 mL), dried (Na2SO4) and concentrated under reduced pres-
sure. The residue was dissolved in THF (12.5 mL) and water
(12.5 mL) at 0–5 °C. Sodium periodate (1.13 g, 5.26 mmol) was
added in small portions, and the solution was stirred for 2 h at
room temperature. The reaction mixture was diluted with water
(15 mL), and extracted with ethyl acetate (3ϫ20 mL). The com-
bined organic phase was dried (Na2SO4), and concentrated under
reduced pressure. The residue was redissolved in a mixture of THF
(12.5 mL) and water (12.5 mL), and sodium borohydride (200 mg,
2.63 mmol) was added. The reaction mixture was stirred at room
due and thymine (15 mg, 0.12 mmol) in anhydrous acetonitrile temperature for 1 h, water (15 mL) was added, and the mixture was
(1 mL) was added N,O-bis(trimethylsilyl)acetamide (71.0 µL, extracted with CH2Cl2 (3ϫ20 mL). The combined organic phase
5720
www.eurjoc.org
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2008, 5715–5722

3Ј-C-Branched Locked Nucleic Acid Thymidine Monomer
was washed with a saturated aqueous solution of NaHCO3
(3ϫ10 mL), dried (Na2SO4) and concentrated under reduced pres-
sure. The residue was purified by silica gel column chromatography
(0–22% ethyl acetate in petroleum ether) to give 15 (540 mg, 71%)
as a clear viscous oil: Rf = 0.45 (1:1, ethyl acetate/petroleum ether).
1H NMR (300 MHz, CDCl3): δ = 7.96–7.94 (m, 2 H, Ph), 7.54–
7.50 (m, 1 H, Ph), 7.41–7.24 (m, 12 H, Ph), 5.89 (d, J = 4.2 Hz, 1
H, 1-H), 5.06 (d, J = 12.6 Hz, 1 H, 5Ј-H), 4.82 (d, J = 10.8 Hz, 1
H, CH2Ph), 4.71–4.65 (m, 2 H, CH2Ph), 4.63 (d, J = 4.2 Hz, 1 H,
2-H), 4.55 (d, J = 12.0 Hz, 1 H, CH2Ph), 4.45 (d, J = 12.6 Hz, 1
H, 5Ј-H), 3.89 (d, J = 6.9 Hz, 2 H, 1Ј-H), 3.88–3.83 (m, 2 H, 5-H),
3.67 (t, J = 6.9 Hz, 1 H, 1Ј-OH), 1.66 (s, 3 H, CH3), 1.34 (s, 3 H,
CH3) ppm. 13C NMR (75 MHz, CDCl3): δ = 4.2 Hz, H-1), 5.02 (d,
1 H, J = 12.0 Hz), 4.82–4.65 (m, 4 H), 4.45–4.25 (m, 4 H), 3.85–
3.65 (m, 3 H), 3.58 (d, 1 H, J = 9.9 Hz), 1.62 (s, 3 H, CH3), 1.32
(s, 3 H, CH3). 13C NMR (75 MHz, CDCl3): δ = 166.4 (CO), 139.1,
137.8, 137.4, 132.7, 130.6, 129.8, 128.6, 128.4, 128.3, 128.2, 128.1,
128.0, 127.7, 127.2, 127.0, 126.9 (Ph), 113.5 [C(CH3)2], 104.9 (C-
1), 87.3, 85.5, 82.8, 73.7, 73.5, 69.8, 68.7, 68.0, 64.8 (C-2, C-3, C-
4, C-5, C-5Ј, C-1Ј, 3ϫ CH2Ph), 26.7 (CH3), 26.5 (CH3) ppm. HR-
MALDI MS: m/z 647.2596 ([M + Na]+, C38H40O8Na+ calcd.
647.2615).
= 12.0 Hz, 2 H), 4.39, 4.25 (AB, J = 11.4 Hz, 2 H), 3.98–3.67 (m,
4 H), 2.07 (s, 3 H, COCH3), 1.42 (s, 3 H, CH3) ppm. 13C NMR
(75 MHz, CDCl3): δ = 170.3 (COCH3), 166.2 (COPh), 163.6 (C-4),
150.8 (C-2), 138.2 (C-6), 136.8, 136.7, 136.1, 133.1, 130.0, 129.8,
128.9, 128.7, 128.6, 128.5, 128.4, 128.2, 128.1, 127.8, 127.7, 127.6,
127.5 (Ph), 111.3 (C-5), 88.6, 84.1, 76.5, 73.8, 73.8, 73.6, 71.0, 67.8,
67.0, 66.6 (C-1Ј, C-2Ј, C-3Ј, C-4Ј, C-5Ј, C-1ЈЈ, C-5ЈЈ, 3ϫ CH2Ph),
20.8 (COCH3), 12.0 (CH3) ppm. HR-MALDI MS: m/z 757.2760
([M + Na]+, C42H42N2O10Na+ calcd. 757.2732).
1-[3,5-Di-O-benzyl-3-C-(benzyloxymethyl)-4-C-(hydroxymethyl)-β-
D
-ribofuranosyl]thymine (19): Sodium methoxide (134 mg,
2.48 mmol) was added to a solution of 18 (455 mg, 0.62 mmol) in
anhydrous methanol (5 mL), and the reaction mixture was stirred
for 2 h at room temperature. Excess of sodium methoxide was neu-
tralized with dilute aqueous hydrochloric acid. The mixture was
extracted with dichloromethane (2ϫ20 mL) and the combined ex-
tract was washed with a saturated aqueous solution of NaHCO3
(3ϫ15 mL), dried (Na2SO4), and concentrated under reduced pres-
sure. The residue was purified by silica gel column chromatography
(0–2% methanol in dichloromethane) to give nucleoside 19 as a
white foam (312 mg, 86%): Rf = 0.50 (9:1, CH2Cl2/CH3OH). 1H
NMR (300 MHz, CDCl3): δ = 8.50 (br. s, 1 H, NH), 7.62 (s, 1 H,
6-H), 7.38–7.18 (m, 15 H, Ph), 6.04 (d, J = 7.8 Hz, 1 H, 1Ј-H),
5.06, 4.87 (AB, J = 11.1 Hz, 2 H), 4.53, 4.36 (AB, J = 10.5 Hz, 2
H), 4.30–4.25 (m, 1 H), 4.27 (d, J = 7.8 Hz, 1 H, 2Ј-H), 4.20–4.10
(m, 2 H), 4.00 (dd, J = 4.2, 10.8 Hz, 1 H, 5ЈЈ-H), 3.86–3.82 (m, 1
H), 3.81, 3.72 (AB, J = 10.5 Hz, 2 H), 3.58 (dd, J = 8.7, 10.8 Hz,
1 H, 5ЈЈ-H), 3.50 (br. s, 1 H, 2Ј-OH), 2.87 (dd, J = 4.2, 8.7 Hz, 1
H, 5ЈЈ-OH), 1.48 (s, 3 H, CH3) ppm. 13C NMR (75 MHz, CDCl3):
δ = 163.9 (C-4), 151.6 (C-2), 136.8 (C-6), 138.7, 136.3, 136.2, 128.8,
128.7, 128.6, 128.5, 128.4, 128.3, 128.1, 127.9, 127.6, 127.5 (Ph),
111.1 (C-5), 90.0, 87.2, 84.1, 78.7, 73.8, 73.6, 72.7, 68.1, 67.0, 63.7
(C-1Ј, C-2Ј, C-3Ј, C-4Ј, C-5Ј, C-1ЈЈ, C-5ЈЈ, 3ϫ CH2Ph), 12.1 (CH3)
ppm. HR-MALDI MS: m/z 611.2379 ([M + Na]+, C33H36N2-
O8Na+ calcd. 611.2364).
1,2-Di-O-acetyl-4-C-(benzoyloxymethyl)-3,5-di-O-benzyl-3-C-(ben-
zyloxymethyl)-(α/β)-D-ribofuranose (17): Compound 16 (1.78 g,
2.85 mmol) was dissolved in 80% aqueous acetic acid (20 mL) and
the mixture was stirred for 3 h at 90 °C. The reaction mixture was
concentrated under reduced pressure and coevaporated with anhy-
drous ethanol (4 mL), toluene (4 mL) and pyridine (4 mL). The
residue was redissolved in anhydrous pyridine (20 mL) and acetic
anhydride (4.6 mL) was added dropwise. The reaction mixture was
stirred overnight at room temperature and quenched by the ad-
dition of ice-water (10 mL). The mixture was extracted with dichlo-
romethane (3 ϫ 10 mL) and the combined organic phase was
washed with a saturated aqueous solution of NaHCO3, dried
(Na2SO4) and concentrated under reduced pressure. The residue
was purified by silica gel column chromatography (0–16% ethyl
acetate in petroleum ether) to give 17 as a mixture of anomers
(1.73 g, 91%; β:α ≈2.5:1) as a clear viscous oil: Rf = 0.60 (1:1, petro-
(1S,3R,4R,7S)-1,7-Bis(benzyloxymethyl)-7-(benzyloxy)-3-(thymin-
1-yl)-2,5-dioxabicyclo[2.2.1]heptane (20): A solution of 19 (526 mg,
0.89 mmol) in anhydrous dichloromethane (3 mL) and anhydrous
pyridine (0.29 mL) was cooled to –40 °C. Methanesulfonyl chloride
(104 µL, 1.34 mmol) was added dropwise, and the mixture was
stirred at –40 °C to 0 °C for 7 h. Water (3 mL) was added followed
by a saturated aqueous solution of NaHCO3 (10 mL). The resulting
mixture was extracted with dichloromethane (3ϫ5 mL) and the
organic phase was dried (Na2SO4) and concentrated under reduced
pressure. The residue was dissolved in anhydrous 1,4-dioxane
(4 mL) and the solution was stirred at 10 °C. A 60% oil dispersion
of NaH (90 mg, 2.23 mmol) was added in one portion. The reac-
tion mixture was stirred at 50 °C for 12 h and then quenched by
the addition of a saturated aqueous solution of NH4Cl (2 mL).
The reaction mixture was extracted with CH2Cl2 (4ϫ5 mL). The
combined organic phase was dried (Na2SO4) and concentrated un-
der reduced pressure. The residue was purified by silica gel column
chromatography (0–3 % methanol in dichloromethane) affording
nucleoside 20 (369 mg, 72 %) as a white foam: Rf = 0.30 (19:1,
1
leum ether/ethyl acetate). H NMR (300 MHz, CDCl3): δ = 7.97–
7.93 (m, 2 H, Ph), 7.58–7.45 (m, 1 H, Ph), 7.40–7.23 (m, 17 H, Ph),
6.45 (d, J = 5.4 Hz, 1α-H), 6.28 (d, J = 3.0 Hz, 1β-H), 5.81 (d, J
= 3.0 Hz, 2β-H), 5.60 (d, J = 5.4 Hz, 2α-H), 4.83–4.60 (m), 4.55–
4.25 (m), 4.05–3.55 (m), 2.03, 2.00, 1.98, 1.97 (each s, CH3) ppm.
HR-MALDI MS: m/z 691.2481 ([M + Na]+, C39H40O10Na+ calcd.
691.2514).
1-[2-O-Acetyl-4-C-(benzoyloxymethyl)-3,5-di-O-benzyl-3-C-(benzyl-
oxymethyl)-β-D-ribofuranosyl]thymine (18): To a stirred solution of
17 (1.69 g, 2.52 mmol) and thymine (636 mg, 5.05 mmol) in anhy-
drous CH3CN (20 mL) was added N,O-bis(trimethylsilyl)acetamide
(3.12 mL, 12.62 mmol). The reaction mixture was stirred for
30 min. After cooling to 0 °C, trimethylsilyl triflate (1.37 mL,
7.57 mmol) was added dropwise, and the solution was stirred over-
night at 70 °C. After cooling to room temperature, the reaction
mixture was diluted with ethyl acetate (20 mL) and washed with a
saturated aqueous solution of NaHCO3 (3 ϫ10 mL). The com-
bined organic phase was dried (Na2SO4) and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography (0–2% methanol in dichloromethane) to give nu-
1
CH2Cl2/CH3OH). H NMR (300 MHz, CDCl3): δ = 8.22 (s, 1 H,
NH), 7.50 (d, J = 0.9 Hz, 1 H, 6-H), 7.36–7.11 (m, 15 H, Ph), 5.54
(s, 1 H, 1Ј-H), 5.39 (s, 1 H, 2Ј-H), 4.77, 4.70 (AB, J = 11.1 Hz, 2
H), 4.57 (s, 2 H), 4.24, 3.99 (AB, J = 7.5 Hz, 2 H), 4.20 (s, 2 H),
3.82–3.68 (m, 4 H), 1.87 (d, J = 0.9 Hz, 3 H, CH3) ppm. 13C NMR
cleoside 18 (1.72 g, 93%) as a white foam: Rf = 0.40 (19:1, CH2Cl2/
1
CH3OH). H NMR (300 MHz, CDCl3): δ = 8.37 (br. s, 1 H, NH), (75 MHz, CDCl3): δ = 163.5 (C-4), 149.8 (C-2), 138.3 (C-6), 137.7,
8.00–7.98 (m, 2 H, Ph), 7.66 (s, 1 H, 6-H), 7.55–7.25 (m, 18 H, Ph),
6.52 (d, J = 8.1 Hz, 1 H, 1Ј-H), 5.69 (d, J = 8.1 Hz, 1 H, 2Ј-H), 127.4 (Ph), 109.4 (C-5), 90.1 (C-4Ј), 89.0 (C-1Ј), 84.8 (C-3Ј), 78.1
4.90, 4.81 (AB, J = 10.5 Hz, 2 H), 4.74 (s, 2 H), 4.52, 4.32 (AB, J (C-2Ј), 74.6, 73.8, 73.8, 70.6, 69.3, 65.1 (C-5ЈЈ, C-1ЈЈ, C-5Ј, 3ϫ
137.0, 134.5, 128.6, 128.6, 128.4, 128.0, 128.0, 127.8, 127.7, 127.6,
Eur. J. Org. Chem. 2008, 5715–5722
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5721

S. Kumar, P. K. Sharma, P. C. Stein, P. Nielsen
FULL PAPER
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CH2Ph), 12.6 (CH3) ppm. HR-MALDI MS: m/z 593.2246 ([M +
Na]+, C33H34N2O7Na+ calcd. 593.2258).
(1S,3R,4R,7S)-7-Hydroxy-1,7-bis(hydroxymethyl)-3-(thymin-1-yl)-
2,5-dioxabicyclo[2.2.1]heptane (3): To a stirred solution of nucleo-
side 20 (502 mg, 0.88 mmol) in ethanol (70 mL) was added 20%
palladium hydroxide over carbon (500 mg). The mixture was de-
gassed several times with argon and placed under a hydrogen atmo-
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[5]
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[7]
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1
yield) as a white foam: Rf = 0.20 (9:1, CH2Cl2/MeOH). H NMR
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(300 MHz, CD3OD): δ = 7.77 (d, J = 0.9 Hz, 1 H, 6-H), 5.41 (s, 1
H, 1Ј-H), 4.69 (s, 1 H, 2Ј-H), 4.13 (d, J = 8.1 Hz, 1 H), 3.90–3.88
(m, 3 H), 3.75, 3.59 (AB, J = 12.3 Hz, 2 H), 1.91 (s, 3 H, CH3)
ppm. 13C NMR (75 MHz, CD3OD): δ = 166.6 (C-4), 152.1 (C-2),
136.7 (C-6), 110.7 (C-5), 91.4 (C-4Ј), 89.6 (C-1Ј), 82.1, 81.9 (C-2Ј,
C-3Ј), 74.5, 61.9, 57.9 (C-5ЈЈ, C-1ЈЈ, C-5Ј), 12.5 (CH3) ppm. HR-
MALDI MS: m/z 323.0864 ([M + Na]+, C12H16N2O7Na+ calcd.
323.0850).
[9]
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Acknowledgments
[11]
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The work was supported by the Sixth Framework Program Marie
Curie Host Fellowships for Early Stage Research Training under
contract MEST-CT-2004-504018, by the Danish National Research
Council, by the Danish National Research Foundation, and by
Møllerens Fond. Mrs. Birthe Haack is thanked for technical assist-
ance. Furthermore, the Nucleic Acid Center is funded by the
Danish National Research Foundation for studies on nucleic acid
chemical biology.
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Received: July 18, 2008
Published Online: October 17, 2008
5722
www.eurjoc.org
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2008, 5715–5722