3′-C-branched LNA-type nucleosides locked in an N-type furanose ring conformation: Synthesis, incorporation into oligocleoxynucleotides, and hybridization studies

Article abstract of DOI:10.1021/jo049159a

Three protected 3′-C-branched LNA-type phosphoramidite building blocks 17, 27, and 38, containing furanose rings locked in an N-type conformation, were synthesized from a known 3-C-allyl allofuranose derivative using strategies relying on the introduction

Full text of DOI:10.1021/jo049159a

3′-C-Br a n ch ed LNA-Typ e Nu cleosid es Lock ed in a n N-Typ e
F u r a n ose Rin g Con for m a tion : Syn th esis, In cor p or a tion in to
Oligod eoxyn u cleotid es, a n d Hybr id iza tion Stu d ies
Michael Meldgaard,† Flemming Gundorph Hansen,‡ and J esper Wengel*,‡
Department of Chemistry, University of Copenhagen, Universitetsparken 5,
DK-2100 Copenhagen, Denmark, and Nucleic Acid Center, Department of Chemistry,
University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark
jwe@chem.sdu.dk
Received May 18, 2004
Three protected 3′-C-branched LNA-type phosphoramidite building blocks 17, 27, and 38, containing
furanose rings locked in an N-type conformation, were synthesized from a known 3-C-allyl
allofuranose derivative using strategies relying on the introduction of the branching alkyl chain
before condensation with the nucleobase. Synthesis of 3′-C-hydroxypropyl derivatives proved
superior to synthesis of the 3′-C-hydroxyethyl derivatives, and the former was converted into the
corresponding 3′-C-aminopropyl derivatives. Phosphoramidites 27 and 38 were subsequently applied
on an automated DNA synthesizer leading to the introduction of three novel 3′-C-branched LNA-
type monomers X, Y, and Z into oligodeoxynucleotides and studies of their effect on the hybridization
properties. A duplex-stabilizing effect of introducing 3′-C-aminopropyl-LNA monomer Y, relative
to 3′-C-hydroxypropyl-LNA monomer X, was observed, especially at low salt conditions. This
indicates that the primary amino group of monomer Y is protonated under the hybridization
conditions applied and that positioning of this positively charged group in the major groove has a
significant duplex stabilizing effect. Monomer Y was by an on-column conjugation method further
functionalized by a glycyl unit to give monomer Z that showed a less stabilizing effect than monomer
Y.
In tr od u ction
The antisense strategy involving targeting of single-
stranded RNA has stimulated the synthesis and bio-
physical evaluation of a large number of modified oligo-
nucleotides and oligodeoxynucleotides (ONs),1-5 including
LNA (locked nucleic acid; Figure 1, monomer 1).6-10 The
conformation of the furanose moiety in an LNA monomer
is locked in an N-type (C3′-endo) conformation (P ) 17°),8
the incorporation of one or more LNA monomers into an
ON strongly increases the thermal stability of the duplex
formed with single-stranded DNA or RNA complements,6-9
* To whom correspondence should be addressed. Tel: +45 65502510.
Fax: +45 66158780.
† University of Copenhagen.
‡ University of Southern Denmark.
(1) Freier, S. M.; Altmann, K.-H. Nucleic Acid Res. 1997, 25, 4429.
(2) Leumann, C. J . Bioorg. Med. Chem. 2002, 10, 841.
(3) Kurreck, J . Eur. J . Biochem. 2003, 270, 1628.
(4) Meldgaard, M.; Wengel, J . J . Chem. Soc., Perkin Trans. 1 2000,
3539.
(5) Kværnø, L.; Wengel, J . Chem. Commun. 2001, 1419.
(6) LNA is defined as an ON containing one or more 2′-O,4′-C-
methylene-â-D-ribofuranosyl nucleotide monomer(s). Singh, S. K.;
Nielsen, P.; Koshkin, A. A.; Wengel, J . Chem. Commun. 1998, 455.
(7) Koshkin, A. A.; Singh, S. K.; Nielsen, P.; Rajwanshi, V. K.;
Kumar, R.; Meldgaard, M.; Olsen, C. E.; Wengel, J . Tetrahedron 1998,
54, 3607.
(8) Obika, S.; Nanbu, D.; Hari, Y.; Morio, K.-I.; In, Y.; Ishida, T.;
Imanishi, T. Tetrahedron Lett. 1997, 38, 8735.
(9) Petersen, M.; Wengel, J . Trends Biotechnol. 2003, 21, 74.
(10) J epsen, J . S.; Wengel, J . Curr. Opinion Drug Dis. Dev. 2004, 7,
188.
F IGURE 1. Constitution of LNA (1), 3′-C-branched-DNA (2),
3′-C-ethyl-LNA (3), and 3′-C-propyl-LNA (4) and a sketch of
the S-type furanose conformation of monomer 2 and the N-type
furanose ring conformation of monomers 3 and 4.
and LNA has been shown to be very useful for antisense
applications.10 In an effort to further investigate the
versatility of LNA monomers, three branched monomers
have been synthesized to explore the opportunity of
attaching at an internally positioned monomer in a
10.1021/jo049159a CCC: $27.50 © 2004 American Chemical Society
Published on Web 08/19/2004
6310
J . Org. Chem. 2004, 69, 6310-6322

3′-C-Branched LNA-Type Nucleosides
TABLE 1. ONs Stu d ied a n d Th er m a l Den a tu r a tion Stu d iesa
a
Tm values were measured as the maximum of the first derivative of the melting curve (A260 vs temperature) recorded in medium salt
hybridization buffer (10 mM sodium phosphate, 100 mM sodium chloride and 0.1 mM EDTA, pH 7.0) or in low salt hybridization buffer
(10 mM sodium phosphate and 0.1 mM EDTA, pH 7.0) using 1.0 µM concentrations of the two strands assuming identical extinction
coefficients of modified and unmodified nucleotides. ∆Tm values are calculated changes in Tm values per modification introduced relative
to the reference DNA-1. b,c Measured in individual experimental series.
duplex, e.g., a third ON strand, a reporter molecule, or a
positively charged group in an orientation facing the
major groove of the duplex. Especially appealing is the
option of attaching a positively charged group in order
to mediate full or partial neutralization of an ON for
increased duplex stability or improved cellular uptake.
Several ONs containing amino-functionalized side chains
attached to the furanose moiety have been studied,
including 4′-C-,11,12 3′-C-,12,13 and 2′-O-functionalized14
derivatives. However, only moderately increased thermal
stability of duplexes toward complementary DNA or RNA
targets has at best been obtained.11-14
Introduction of a 3′-C-alkyl substituent (monomer 2,
Figure 1) on the â-face of a nucleotide monomer is well
tolerated in a duplex structure as evaluated by the
thermal stability of the duplexes between such modified
ONs and a complementary sequence.15-17 Therefore, it
was appealing to study this structural feature in the
context of an LNA monomer that in its parent un-
branched form is known to very positively increase the
thermal stability of duplexes.6-9 We therefore decided to
synthesize 3′-C-branched LNA monomers (3 and 4,
Figure 1) with different 3′-C-alkyl branches. In a pre-
liminary study, the usefulness of the branched LNA
monomer 3 (R ) oxygen linked to another ON strand)
was demonstrated for the construction of a branched ON
being able to form a bimolecular triple helical structure
with a target strand.18 Synthesis of the phosphoramidite
building block for incorporation of the monomer applied
in this study is described in this report for the first time.
Furthermore, in an attempt at improving the accessibility
of 3′-C-branched LNA monomers, we have synthesized
phosphoramidite derivatives 27 and 38 containing propyl
branches and studied the effect of the corresponding
LNA-type monomers X, Y, and Z (see Table 1) on duplex
stabilities when incorporated into ONs. A 3′-C-branched
DNA monomer is known to adopt a C2′-endo-type fura-
(15) J ørgensen, P. N.; Svendsen, M. L.; Scheuer-Larsen, C.; Wengel,
J . Tetrahedron 1995, 51, 2155
(11) Kanazaki, M.; Ueno, Y.; Shuto, S.; Matsuda, A. J . Am. Chem.
Soc. 2000, 122, 2422.
(12) Pfundheller, H. M.; Bryld, T.; Olsen, C. E.; Wengel, J . Helv.
Chim. Acta 2000, 83, 128.
(13) Wang, G.; Middelton, P. J .; He, L.; Stoisavljevic, V.; Seifert, W.
E. Nucleosides Nucleotides 1997, 16, 445.
(14) Prakash, T. P.; J ohnston, J . F.; Graham, M. J .; Condon, T. P.;
Manoharan, M., Nucleic Acids Res. 2004, 32, 828.
(16) J ørgensen, P. N.; Sørensen, U. S.; Pfundheller, H. M.; Olsen,
C. E.; Wengel, J . J . Chem. Soc., Perkin Trans. 1 1997, 3275.
(17) Pfundheller, H. M.; J ørgensen, P. N.; Sørensen, U. S.; Sharma,
S. K.; Grimstrup, M.; Stro¨ch, C.; Nielsen, P.; Viswanadham, G.; Olsen,
C. E.; Wengel, J . J . Chem. Soc., Perkin Trans. 1 1998, 1409.
(18) Sørensen, M. D.; Meldgaard, M.; Raunkjær, M.; Rajwanshi, V.
K.; Wengel, K. Bioorg. Med. Chem. Lett. 2000, 10, 1853.
J . Org. Chem, Vol. 69, No. 19, 2004 6311

Meldgaard et al.
SCHEME 1a
a
Key: (a) NaH, BnBr, DMF, 95% (synthesis of furanose 5 from 3-C-allyl-1,2:5,6-di-O-isopropylidene-R-D-allofuranose;22 (b) (i) 80% aq
AcOH or 60% aq AcOH, MeOH, (ii) NaIO4, THF, H2O, (iii) HCHO, NaOH, dioxane, H2O, 59%; (c) Ac2O, DMAP, pyridine, 85%; (d) (i) 80%
aq AcOH, (ii) Ac2O, DMAP, pyridine, 88%; (e) thymine, N,O-bis(trimethylsilyl)acetamide, TMS-triflate, MeCN, 67%; (f) NaOMe, MeOH,
81%; (g) TsCl, pyridine, 70%; (h) DMTCl, pyridine, 74%; (i) NaH, DMF, 96%; (j) (i) OsO4, NaIO4, THF, H2O, (ii) NaBH4, THF/H2O, 38%;
(k) HCOONH4, Pd/C, MeOH, 83%; (l) levulinic acid anhydride, pyridine, DMAP, 88%; (m) (iPr)2NP(Cl)OCH2CH2CN, (iPr)2NEt, CH2Cl2,
41%. T ) Thymin-1-yl.
nose conformation19 (Figure 1, monomer 2), whereas 3′-
C-branched LNA monomers will adopt a locked C3′-endo-
type furanose conformation dictated by its bicyclic
constitution. Therefore, this study is also aimed at
studying the correlation between 3′-C-branching and
furanose conformation.
resulting aldehyde and formaldehyde followed by reduc-
tion under in situ Cannizzaro reaction conditions afforded
the diol 6 in 59% yield. The two primary hydroxyl groups
were acetylated using acetic anhydride in pyridine with
a catalytical amount of DMAP to give the diacetylated
furanose derivative 7 in 85% yield. Acetolysis and
subsequent acetylation afforded the tetra-O-acetyl sugar
8, which was used as a glycosyl donor in a modified
Vorbru¨ggen condensation reaction. Thus, the anomeric
mixture 8 was stirred at 60 °C for 12 h with silylated
thymine and TMS-triflate to give the nucleoside 9 (67%
yield), obtained solely as the â-anomer due to achimeric
assistance from the 2-O-acetyl group. The acylated
nucleoside was deprotected using sodium methoxide in
methanol to give nucleoside 10. For activation of the 4′-
C-hydroxymethyl moiety, a tosyl group was chosen as a
leaving group. This could be introduced selectively by
reacting 10 with tosyl chloride in analogy with the
previously described selective reactions on this posi-
tion.23-25 Thus, nucleoside 10 was reacted with p-tolu-
enesulfonyl chloride in pyridine which afforded the 4′-
C-tosyloxymethyl nucleoside 11 in 70% yield (Scheme 1).
The site of tosylation proved impossible to verify by NMR
techniques and was therefore identified by an alternative
approach taking advantage of the fact that the signals
of the 1′-H, 2′-H, and 3′-H protons appear as singlets in
the 1H NMR spectra of LNA type nucleosides.7 Thus, only
Resu lts a n d Discu ssion
Syn th esis of 3′-C-Hydr oxyeth yl LNA-Type Nu cleo-
sid es. For the introduction of the branch at C3′ of the
nucleoside, a convergent synthesis strategy was chosen
in order to obtain the desired configuration at C-3′. Thus,
3-C-allyl-1,2:5,6-di-O-isopropylidene-R-D-allofuranose was
prepared in two steps from 1,2:5,6-di-O-isopropylidene-
R-D-glucofuranose by oxidation and subsequent Grignard
addition of allylMgBr to the ulose as previously de-
scribed.20-22 Benzylation of the tertiary hydroxyl group
to give 5 was achieved in quantitative yield by reaction
of 3-C-allyl-1,2:5,6-di-O-isopropylidene-R-D-allofuranose
with sodium hydride and benzyl bromide in DMF. The
subsequent selective removal of the 5,6-O-isopropylidene
protective group using either 80% aqueous acetic acid at
rt overnight or 60% aqueous acetic acid with a catalytic
amount of methanol at 60 °C for 3 h22 afforded the 5,6-
diol. Oxidative cleavage using NaIO4 in a mixture of THF
and H2O and subsequent aldol condensation between the
(19) Koole, L. H.; Buck, H. M.; Vial, J .-L.; Chattopadhyaya, J . Acta
Chem. Scand. 1989, 43, 665,
(20) Gable, K. P.; Benz, G. A. Tetrahedron Lett. 1991, 32, 3473.
(21) Tronchet, J . M. J .; Zsely, M.; Yazji, R. N.; Barbalat-rey, F.;
Geoffroy, M. Carbohydr. Lett. 1995, 1, 343.
(23) Fensholdt, J .; Thrane, J .; Wengel, J . Tetrahedron Lett. 1995,
36, 2535.
(24) Thrane, H.; Fensholdt, J .; Regner, M.; Wengel, J . Tetrahedron
1995, 51, 10389.
(22) Patra, R.; Bar, N. C.; Roy, A.; Achari, B.; Ghosal, N.; Mandal,
S. B. Tetrahedron 1996, 52, 11265.
(25) Nielsen, K. D.; Kirpekar, F.; Roepstorff, P.; Wengel, J . Bioorg.
Med. Chem. 1995, 3, 1493.
6312 J . Org. Chem., Vol. 69, No. 19, 2004

3′-C-Branched LNA-Type Nucleosides
SCHEME 2a
a
Key: (a) (i) BH3Me2S, THF, (ii) 2 M NaOH, H2O2, 70% (synthesis of furanose 18 from 3-C-allyl-1,2:5,6-di-O-isopropylidene-R-D-
allofuranose;22 (b) (i) NaH, BnBr, DMF, (ii) AcOH/H2O (8:2), 79%; (c) (i) NaIO4, H2O, 1,4-dioxane, THF, (ii) HCHO, 1 M NaOH, 1,4-
dioxane, 85%; (d) (i) MsCl, pyridine, (ii) TFA/H2O (8:2), (iii) Ac2O, pyridine, 87%; (e) thymine, N,O-(bistrimethylsilyl)acetamide, TMS-
triflate, CH3CN, 93%; (f) H2O, KOH, 1,4-dioxane, 66%; (g) DMTCl, pyridine, 90%; (h) Pd/C, HCOONH4, MeOH, 74%; (i) Ac2O, pyridine,
90%; (j) (iPr)2NP(Cl)OCH2CH2CN, (iPr)2NEt, CH2Cl2, 64%. T ) thymin-1-yl.
by tosylation of the hydroxy group of the 4′-C-hydroxy-
methyl substituent would it be possible to obtain LNA
type derivatives. Treatment of 11 with NaH in DMF did
indeed confirm the site of tosylation as two singlets
gradually appeared in the 1H NMR spectrum as the
reaction progressed, and after 12 h the 3′-C-allyl LNA
derivative of 11 was isolated (data not shown).
pyl)ethylamine in dichloromethane afforded the desired
LNA-type phosphoramidite building block 17 (41% yield)
that was used for incorporation of the 3′-C-hydroxyethyl
LNA-type monomer 3 (Figure 1, R ) OH) into ONs.18
However, two facts prompted us to engage in the
synthesis of the 3′-propyl derivatized phosphoramidites
27 and 38 (Schemes 2 and 3). One was the low overall
yield of the synthetic route toward phosphoramidite 17,
in particular the very low yield of the step leading to the
3′-C-hydroxyethyl nucleoside 15. The second was the
somewhat disappointing lack of stabilizing effect of 3′-
C-hydroxyethyl LNA-type monomer 3 when evaluated in
a duplex toward a DNA complement in the preliminary
study.18
Syn th esis of 3′-C-Hyd r oxyp r op yl LNA-Typ e Nu -
cleosid es. 3-C-Allyl-1,2:5,6-di-O-isopropylidene-R-D-al-
lofuranose22 was converted into the corresponding 3-C-
(3-hydroxy)propyl derivative by hydroboration using
BH3Me2S in THF followed by oxidation with a mixture
of NaOH and H2O2 giving furanose 18 in 70% yield.
Benzylation of the primary and the tertiary hydroxyl
groups using sodium hydride and benzyl bromide in
DMF, followed by the selective removal of the 5,6-O-
isopropylidene protective group using 80% aqueous acetic
acid at rt for 45 h, afforded the 5,6-diol 19 in 79% yield.
The resulting 5,6-diol system was oxidatively cleaved
using NaIO4 in a mixture of 1,4-dioxane, THF, and H2O
followed by an aldol condensation between the resulting
aldehyde and formaldehyde and reduction under in situ
Cannizzaro reaction conditions to furnish the diol 20 in
85% yield. Mesylation of the two primary hydroxyl groups
DMT (4,4′-dimethoxytrityl) protection of the remaining
primary hydroxy group of nucleoside 11 was accom-
plished by reaction with 4,4′-dimethoxytrityl chloride
(DMTCl) in anhydrous pyridine furnishing nucleoside 12
in 74% yield. For the synthesis of the 3′-C-branched LNA
monomer, compound 13 was treated with NaH in DMF
to afford cyclization, which proceeded smoothly to give
the bicyclic nucleoside 13 in 96% yield. The allyl moiety
was cleaved in an oxidative manner, taking advantage
of an OsO4-catalyzed dihydroxylation and subsequent
cleavage of the newly formed diol system with NaIO4.
The resulting aldehyde was finally reduced with NaBH4
to give the hydroxyethyl branched LNA nucleoside 14 in
only 38% yield. The rather low yield in this reaction is
comparable to low yields reported for similar reactions
on related compounds.26,27 The benzyl protective group
was subsequently removed by treatment of nucleoside 14
with ammonium formate and Pd/C in refluxing methanol
to give nucleoside diol 15 in 83% yield. The primary
hydroxy group of 15 was selectively protected as its
levulinic ester by reaction with levulinic anhydride in
pyridine28,29 giving the nucleoside 16 in 88% yield (Scheme
1). Subsequent phosphitylation using 2-cyanoethyl N,N-
(diisopropyl)phosphoramidochloridite and N,N-(diisopro-
(26) Nielsen, P.; Pfundheller, H. M.; Wengel, J . Chem. Commun.
1997, 825.
(27) Nielsen, P.; Pfundheller, H. M.; Olsen, C. E.; Wengel, J . J .
Chem. Soc., Perkin Trans. 1 1997, 3423.
(28) Hassner, A.; Strand, G.; Rubinstein, M.; Patchornik, A. J . Am.
Chem. Soc. 1975, 97, 1614.
(29) Meldgaard, M.; Nielsen, N. K.; Bremner, M.; Pedersen, O. S.;
Olsen, C. E.; Wengel, J . J . Chem. Soc., Perkin Trans I, 1997, 13, 1951.
J . Org. Chem, Vol. 69, No. 19, 2004 6313

Meldgaard et al.
SCHEME 3a
a
Key: (a) MsCl, pyridine, 79%; (b) NaN3, DMF, 84%; (c) AcOH/H2O (8:2), 82%; (d) (i) NaIO4, 1,4-dioxane, H2O, THF, (ii) HCHO, 1 M
NaOH, 1,4-dioxane, 89%; (e) (i) MsCl, pyridine, (ii) TFA/H2O (8:2), (iii) Ac2O, pyridine, 67%; (f) thymine, N,O-bis(trimethylsilyl)acetamide,
TMS-triflate, CH3CN, reflux, 75%; (g) KOH, H2O, 1,4-dioxane, reflux, 63%; (h) (i) H2, Pd(OH)2/C, MeOH, (ii) Fmoc-Cl, pyridine, 51%; (i)
DMTCl, pyridine, 68%; (j) (iPr)2NP(Cl)OCH2CH2CN, (iPr)2NEt, CH2Cl2, 87%. T ) thymin-1-yl.
using methanesulfonyl chloride in pyridine followed by
removal of the 1,2-O-isopropylidene protective group with
80% trifluoroacetic acid and acetylation of the resulting
two hydroxyl groups using acetic anhydride in pyridine
gave the anomeric mixture 21 in 87% yield, which was
used as a glycosyl donor in a modified Vorbru¨ggen
condensation reaction. Thus, the anomeric mixture 21
was stirred under reflux for 23 h with silylated thymine
and TMS-triflate to give the monoacetylated nucleoside
22 in 93% yield. The nucleoside was obtained solely as
the â-anomer due to anchimeric assistance from the 2-O-
acetyl group. The acetylated nucleoside was converted
into LNA-type nucleoside 23 in 66% yield by treatment
of 22 with aqueous potassium hydroxide and 1,4-dioxane
with heating under reflux as described previously for
other LNA-type nucleosides.30 This reaction involves
hydrolytic removal of the 2′-O-acetyl group, cyclization
by intramolecular nucleophilic attack of the 2′-hydroxy
group, and removal of the 5′-O-mesyl group. The primary
hydroxyl group of nucleoside 23 was DMT protected as
described for synthesis of nucleoside 13 affording nucleo-
side 24 in 90% yield. Debenzylation using ammonium
formate and Pd/C in methanol gave diol 25 in 74% yield,
and subsequent selective acetylation of the primary
hydroxy group with acetic anhydride in pyridine afforded
the monoacetylated nucleoside 26 in 90% yield. Finally,
standard phosphitylation using 2-cyanoethyl N,N-(diiso-
propyl)phosphoramidochloridite and N,N-(diisopropyl)-
ethylamine in dichloromethane afforded the desired 3′-
C-acetoxypropyl LNA-type phosphoramidite building block
27 in 64% yield suitable for incorporation of monomer X
(Table 1) into ONs (Scheme 2).
The synthesis of the protected 3′-C-hydroxyethyl LNA-
type phosphoramidite 17 (Scheme 1) proceeded in only
1.3% overall yield, whereas the synthesis of the pro-
tected 3′-C-hydroxypropyl phosphoramidite derivative 27
(Scheme 2) proceeded in 10% overall yield. As the former
synthesis furthermore involves more synthetic steps, the
latter offers significant advantages toward synthesis of
3′-C-branched LNA-type nucleosides, and it therefore
formed the basis for various synthetic strategies toward
the corresponding 3′-C-aminopropyl LNA-type nucleo-
sides. However, attempts at introducing various N-
nucleophiles at activated 3′-C-hydroxypropyl nucleosides
(both protected 4′-C-hydroxymethyl derivatives and bi-
cyclic LNA-type derivatives) were unsuccessful in our
hands, and we therefore decided to introduce an N-
nucleophile very early in the synthetic route (Scheme 3).
Syn th esis of 3′-C-Am in op r op yl LNA-Typ e Nu cleo-
sid es. In general, a reaction sequence quite similar to
the one used for synthesis of the 3′-C-hydroxypropyl
LNA-type nucleosides (see Scheme 2) was used. Thus,
starting from furanose 6, hydroboration using BH3Me2S
in THF and oxidation using a mixture of aq NaOH and
H2O2 afforded the primary alcohol 28 in 46% yield.
Mesylation of the primary hydroxy group to give com-
pound 29 in 79% yield followed by introduction of an
azido group using NaN3 in DMF at 100 °C yielded azido
compound 30 in 84% yield. The introduction of the azido
group was verified by IR spectroscopy. Selective removal
of the 5,6-O-isopropylidene protective group to give 5,6-
diol 31 in 82% yield and subsequent oxidative cleavage
(30) Koshkin, A. A.; Fensholdt, J .; Pfundheller, H. M.; Lomholt, C.
J . Org. Chem. 2001, 66, 8504.
6314 J . Org. Chem., Vol. 69, No. 19, 2004

3′-C-Branched LNA-Type Nucleosides
using NaIO4 in a mixture of 1,4-dioxane, THF, and H2O
followed by a Cannizzaro reaction afforded the diol 32
in 89% yield. Mesylation of the two primary hydroxyl
groups, removal of the 1,2-O-isopropylidene protective
group, and acetylation of the resulting two hydroxyl
groups furnished the anomeric mixture 33 in 67% yield.
Coupling between 33 and thymine using TMS-triflate as
Lewis acid and N,O-bis(trimethylsilyl)acetamide as the
silylating agent provided nucleoside 34 in 75% yield.
Deacetylation of the 2′-O-acetyl group and cyclization by
an intramolecular nucleophilic attack of the resulting 2′-
hydroxy group with concomitant demesylation30 was
performed in a mixture of aqueous potassium hydroxide
and 1,4-dioxane at reflux affording LNA-type nucleoside
35 in 63% yield. Reduction of the azide and debenzylation
with H2 and Pd(OH)2/C in methanol followed by Fmoc
protection of the primary amino group with Fmoc-Cl in
pyridine afforded the Fmoc-protected 3′-C-aminopropyl
LNA nucleoside 36 in 51% yield. Using standard meth-
ods, the primary hydroxy group of nucleoside 36 was
DMT protected to give nucleoside 37 (68% yield) which
was followed by phosphitylation in 87% yield to furnish
the desired Fmoc-protected 3′-C-aminopropyl phosphor-
amidite building block 38 (Scheme 3).
LNA ON Syn th esis a n d Th er m a l Den a tu r a tion
Stu d ies. To test the properties of ONs containing the
novel 3′-C-branched LNA monomers X, Y, and Z, we
efficiently synthesized LNA-1, LNA-2, and LNA-3 (Table
1) using the phosphoramidite approach on an automated
DNA synthesizer (see the Experimental Section for
details). In addition, LNA-3, while still attached at the
solid support, was also used for synthesis of LNA-4
employing an on-column conjugation approach involving
selective removal of the Fmoc group and subsequent
reaction with Fmoc-protected glycine and HBTU in DMF
following a procedure (see Experimental Section for
details) published for synthesis of 5′-end-conjugated
ONs.31 The composition of the synthesized LNA ONs was
verified by MALDI-MS and the purity (>80%) by capil-
lary gel electrophoresis/reversed-phase HPLC. Hybrid-
ization data of these modified LNA ONs toward comple-
mentary single stranded DNA and RNA are shown in
Table 1. For comparison, data of the unmodified DNA
reference (DNA-1) are also shown.
N-glycyl monomer Z was incorporated once (LNA-4)
allowing direct comparison with LNA-3. It is observed
that the exact positioning of the primary amino group
plays an important role as monomer Z, contrary to
monomer Y, has no stability-increasing effect.
To study further the affinity-enhancing effect of 3′-C-
aminopropyl LNA-type monomer Y, thermal denatur-
ation studies under low salt conditions were conducted.
These data strongly suggest that monomer Y is proto-
nated at the conditions applied as its stability increasing
effect, relative to monomer T (DNA-1) and monomer X
(LNA-1), is further enhanced (see data for LNA-2 and
LNA-3). The data obtained for LNA-4 indicate in general
a loss of the positive effect observed for monomer Y,
possibly caused by inappropriate positioning of the
primary amino group in the 3′-C-branch of monomer Z.
A general trend is that introduction of the 3′-C-
branched LNA-type monomers, like unmodified LNA
monomers,6-9 increases duplex stability more toward
complementary RNA than DNA. It is therefore clear that
furanose ring conformation plays a role, but to what
degree the stability increasing effect of introducing a
primary amino group, as clearly shown for monomer Y,
is mainly due to reduced electrostatic repulsion, the
formation of specific ion pairs between the protonated
amino groups and the phosphate backbone, or the forma-
tion of stabilizing hydrogen bonds, remains to be eluci-
dated.
Con clu sion
Synthetic routes toward the three protected 3′-C-
branched LNA-type phosphoramidite building blocks 17,
27, and 38, containing furanose rings locked in an N-type
conformation, have been developed starting from a known
3-C-allyl allofuranose derivative. The branching alkyl
chain was introduced before condensation with the
nucleobase, and conversion of a hydroxypropyl branch
into an aminopropyl branch proved efficient only at a
stage prior to condensation with the thymine nucleobase.
The synthesis developed for the 3′-C-hydroxypropyl
derivatives is clearly superior to that of the correspond-
ing 3′-C-hydroxyethyl derivative. The novel phosphor-
amidites 27 and 38 were applied on an automated DNA
synthesizer leading to the introduction of three novel 3′-
C-branched LNA-type monomers X, Y, and Z into oli-
godeoxynucleotides. A duplex stabilizing effect of intro-
ducing 3′-C-aminopropyl-LNA monomer Y, relative to 3′-
C-hydroxypropyl-LNA monomer X, was observed, especial-
ly at low salt conditions. This indicates that the primary
amino group of monomer Y is protonated under the
hybridization conditions applied and that the positioning
of a positive charged group in the major groove has a
stabilizing effect. Monomer Y was by an on-column
conjugation method further functionalized by a glycyl
unit to give monomer Z that showed a smaller stabilizing
effect than monomer Y. This points to the importance of
the three-dimensional positioning of a positively charged
amino group, i.e., Ångstro¨m-scale structural control, if a
strong duplex stabilization is aimed for.
Comparison between the reference DNA-1 and LNA-
1, containing three 3′-C-hydroxypropyl LNA-type mono-
mers X and six DNA monomers under medium salt
conditions, confirm earlier results with the 3′-C-hydroxy-
ethyl LNA-type monomer (3, R ) OH, Figure 1),18 namely
that the increase in stability expected by the introduction
of the bicyclic LNA scaffold is counterbalanced by the
apparently unfavorable introduction of the 3′-C-alkyl
substituent. It appears that a 3′-C-alkyl substituent is
more unfavorable with respect to hybridization when
attached at an nucleotide adopting a 3′-endo type (N-type)
furanose conformation than when attached at a flexible
DNA-type monomer.15-18 On the contrary, a clear stabi-
lizing effect, especially toward complementary RNA, is
observed for LNA-2 containing three 3′-C-aminopropyl
LNA-type monomers Y. This effect is confirmed against
the DNA complement for the singly modified LNA-3. The
Exp er im en ta l Section
3-C-Allyl-3-O-ben zyl-1,2:5,6-d i-O-isop r op ylid en e-R-D-a l-
lofu r a n ose (5). To a stirred solution of 3-C-allyl-1,2:5,6-di-
O-isopropylidene-R-D-allofuranose22 (5.33 g, 17.8 mmol) in
(31) Mokhir, A. A.; Tetzlaff, C. N.; Herzberger, S.; Mosbacher, A.;
Richert, C. J . Comb. Chem. 2001, 3, 374.
J . Org. Chem, Vol. 69, No. 19, 2004 6315

Meldgaard et al.
anhydrous DMF (20 mL) at rt was added NaH (1.06 g, 26.61
mmol). Benzyl bromide (2.8 mL, 25.8 mmol) was added after
30 min, and the reaction mixture was stirred for 3 h. H2O (10
mL) was added, and the resulting mixture was evaporated to
dryness. The residue was dissolved in EtOAc (150 mL), washed
successively with H2O (70 mL) and NaCl (70 mL), dried (Na2-
SO4), filtered, and evaporated to dryness. Column chromatog-
raphy (20% EtOAc/petroleum ether) afforded furanose 5 (6.60
g, 16.8 mmol, 95%) as a white solid material: Rf 0.56 (EtOAc/
petroleum ether, 2:5 (v/v)); 1H NMR (CDCl3) 7.41-7.21 (m, 5H,
Bn), 6.09-5.93 (m, 1H, CH2CHCH2-), 5.63 (d, J 3.7, 1H, 1-H),
5.20-5.13 (m, 2H, CH2CHCH2-), 4.85 (d, J ) 11.2 Hz, 1H,
Bn), 4.72 (d, J ) 11.2 Hz, 1H, Bn), 4.49 (d, J ) 3.7 Hz, 1H,
2-H), 4.25-3.94 (m, 4H, 4-H, 5-H, 6-H), 2.69 (dd, J ) 6.8, 14.9
Hz, 1H, CH2CHCH2-), 2.41 (dd, J ) 7.3,14.9 Hz, 1H, CH2-
CHCH2-), 1.59, 1.39, 1.35 and 1.34 (4 × s, 4 × 3H, 4 × CH3-
isopropylidene); 13C NMR (CDCl3) 139.3, 132.7, 128.1, 127.3,
118.7, 112.7, 109.6, 103.5, 83.7, 83.0, 81.3, 73.1, 69.1, 66.9, 35.9,
27.0, 26.6, 25.4; FAB-MS m/z 391 ([M + 1]+). Anal. Calcd for
119.5, 112.9, 103.9, 86.1, 85.2, 83.1, 66.6, 63.2, 62.2, 36.2, 26.1,
25.7, 20.9, 20.8; FAB-MS m/z 435 ([M + 1]
+).
4-C-Ace t oxym e t h yl-3-C-a llyl-3-O-b e n zyl-1,2,5-t r i-O-
a cetyl-D-er yth r o-p en tofu r a n ose (8). A solution of the prod-
uct of the preceding reaction (furanose 7, 5.50 g) in 80% aq
acetic acid (36 mL) was stirred for 3 h at 90 °C. The reaction
mixture was evaporated to dryness and coevaporated with
ethanol (2 × 30 mL), anhydrous toluene (2 × 30 mL), and
anhydrous pyridine (30 mL), and the residue was dissolved
in
a mixture of anhydrous pyridine (13 mL) and acetic
anhydride (13.0 mL, 138 mmol). A catalytic amount of DMAP
was added, and the reaction mixture was stirred at rt for 12
h. Ice-cold H2O (300 mL) was added, and the resulting mixture
was extracted with dichloromethane (2 × 200 mL). The
combined organic phase was dried (Na2SO4) and evaporated
to dryness. The residue was purified by column chromatog-
raphy (EtOAc/petroleum ether, 1:10-1:5 (v/v)) to give the
anomeric mixture 8 as a clear oil (3.38 g) that was used in the
next step without further purification: Rf 0.67 (EtOAc/
petroleum ether, 1:1 (v/v)); 13C NMR (CDCl3) 170.4, 169.8,
169.3, 169.1, 137.7, 131.6, 131.3, 128.6, 128.5, 128.4, 128.3,
127.6, 127.5, 126.9, 126.7, 119.8, 119.6, 98.1, 92.8, 88.3, 87.6,
84.0, 82.6, 79.6, 76.0, 66.7, 64.7, 63.5, 63.3, 62.4, 34.8, 34.7,
21.0, 21.0, 20.8, 20.7; FAB-MS m/z 419 ([M - AcO]+).
1-(4-C-Ace t oxym e t h yl-3-C-a llyl-3-O-b e n zyl-2,5-d i-O-
a cetyl-â-D-er yth r o-p en tofu r a n osyl)th ym in e (9). The prod-
uct of the preceding reaction (anomeric mixture 8, 4.76 g) was
coevaporated with anhydrous acetonitrile (2 × 30 mL) and
dissolved in anhydrous acetonitrile (100 mL). Thymine (2.72
g, 21.6 mmol) and N,O-(bistrimethyl)acetamide (18.6 mL, 75
mmol) were added, and the reaction mixture was heated under
reflux with stirring for 1 h. The reaction mixture was cooled
to 0 °C whereupon trimethylsilyl triflate (5.0 mL, 27 mmol)
was added. After the resulting mixture was heated to 65 °C,
stirring was continued for 2 h. Saturated aq NaHCO3 (100 mL)
was added followed by extraction using dichloromethane (3 ×
70 mL). The combined organic phase was washed with satd
aq NaCl (65 mL), dried (Na2SO4), and evaporated to dryness
under reduced pressure. The residue was purified by column
chromatography (EtOAc/petroleum ether, 2:3-1:1 (v/v)) to give
nucleoside 9 as a white foam (3.60 g, 6.70 mmol, 63%
calculated from furanose 7): Rf 0.23 (EtOAc/petroleum ether,
1:1; 1H NMR (CDCl3) 9.42 (s, 1H, NH), 7.40 (s, 1H, 6-H), 7.38-
7.28 (m, 5H, Bn), 6.39 (d, J ) 8.1 Hz, 1H, 1′-H), 5.88-5.86 (m,
1H, CH2CHCH2-), 5.57 (d, J ) 8.1 Hz, 1H, 2′-H), 5.27-5.23
(m, 2H, CH2CHCH2-), 5.06 (d, J ) 11.0 Hz, 1H, Bn), 4.72 (d,
J ) 11.2 Hz, 1H, Bn), 4.55-4.30 (m, 4H, 5′-H, 5′′-H), 3.00 (dd,
J ) 5.5, 16.5 Hz, 1H, CH2CHCH2-), 2.47 (dd, J ) 8.2, 16.7
Hz, 1H, CH2CHCH2-), 2.22, 2.13, and 2.04 (3 × s, 3 × 3H, 3
× COCH3), 1.94 (s, 3H, CH3); 13C NMR (CDCl3) 170.2, 169.9
and 169.5 (CO), 163.6 (C-4), 150.8 (C-2), 137.7 (Bn), 134.8 (C-
6), 130.8 (CH2CHCH2-), 128.5, 128.4, 128.3, 127.8, 127.7,
127.4, 127.2, CH2CHCH2-, 111.4 (C-5), 86.9 (C-4′), 83.9 (C-1′),
83.3 (C-3′), 77.9 (C-2′), 67.0 (Bn), 64.3 and 64.1 (C-5′, C-5′′),
39.6 (CH2CHCH2-), 21.0, 20.9, 20.8, 20.8 (COCH3), 12.7 (CH3);
FAB-MS m/z 419 ([M - T]+). Anal. Calcd for C21H26N2O7: C,
59.55; H, 5.92; N, 5.14. Found: C, 59.34; H, 6.14; N, 5.03.
C
22H30O6: C, 67.67; H, 7.74. Found: C, 67.99; H, 7.49.
3-C-Allyl-3-O-ben zyl-4-C-h yd r oxym eth yl-1,2-O-isop r o-
p ylid en e-R-D-er yth r o-p en tofu r a n ose (6). A solution of fura-
nose 5 (6.90 g, 17.8 mmol) in 80% aq acetic acid (100 mL) and
methanol (1 mL) was stirred for 18 h. The solvent was removed
under reduced pressure, and the residue was coevaporated
with toluene (2 × 20 mL). The residue was dissolved in EtOAc
(150 mL), and washing was performed using satd aq NaHCO3
(50 mL). The organic phase was evaporated to dryness, the
residue was dissolved in THF (40 mL), and NaIO4 (5.58 g, 26.1
mmol) dissolved in H2O (40 mL) was added. The reaction was
stirred for 1 h at rt, whereupon H2O (20 mL) was added. The
resulting mixture was extracted with dichloromethane (2 ×
100 mL). The organic phase was washed with NaCl (20 mL)
and evaporated to dryness. The residue was dissolved in THF
(40 mL), formaldehyde (5 mL, 37% solution (w/w) in methanol)
and NaOH (2 M, 20 mL) were added, and the reaction mixture
was stirred for 12 h at rt. The mixture was cooled to 0 °C,
NaBH4 (1.00 g, 26 mmol) was added, and stirring was
continued at rt for 1 h. The mixture was evaporated to half
volume under reduced pressure and diluted with H2O. The
extraction was performed using EtOAc (2 × 100 mL), and the
separated organic phase was washed with satd aq NaHCO3
(2 × 30 mL), dried over Na2SO4, filtered, and evaporated to
dryness. The residue was purified by column chromatography
(EtOAc/petroleum ether, 2:5 (v/v)) to give furanose 6 as a pale
yellow oil (3.69 g, 10.6 mmol, 59%): Rf 0.18 (EtOAc/petroleum
ether, 2:5 (v/v)); 1H NMR (CDCl3) 7.39-7.15 (m, 5H, Bn), 5.97-
5.91 (m, 1H, CH2CHCH2-), 5.72 (d, J ) 4.2 Hz, 1H, 1-H),
5.29-5.20 (m, 2H, CH2CHCH2-), 4.72 (d, J ) 10.4 Hz, 1H,
Bn), 4.65 (d, J ) 10.4, 1H, Bn), 4.53 (d, J ) 4.2 Hz, 1H, 2-H),
4.10-3.87 (m, 4H, 4-H, 5-H, 6-H), 2.76 (dd, J ) 8.1, 15.0 Hz,
1H, CH2CHCH2-), 2.65 (dd, J ) 6.1, 15.0 Hz, 1H, CH2-
CHCH2-), 1.65 and 1.33 (2 × s, 2 × 3H, 2 × CH3-isopropyl-
idene); 13C NMR (CDCl3) 138.0, 131.9, 129.0, 128.4, 128.3,
128.2, 127.7, 127.5, 127.2, 125.3, 119.6, 112.7, 104.2, 87.4, 86.0,
83.1, 67.2, 63.9, 63.5, 36.9, 26.2, 25.7; FAB-MS m/z 351 ([M +
1]+). Anal. Calcd for C19H26O6: C, 64.88; H, 7.40. Found: C,
65.13; H, 7.48.
1-(3-C-Allyl-3-O-ben zyl-4-C-h ydr oxym eth yl-â-D-er yth r o-
p en tofu r a n osyl)th ym in e (10). To a stirred solution of
nucleoside 9 (1.37 g, 2.52 mmol) in methanol (17 mL) at rt
was added NaOMe (0.86, 15 mmol), and stirring was continued
for 1 h. The reaction mixture was neutralized by addition of a
2 M solution of HCl in dioxane, and the resulting mixture was
evaporated to dryness under reduced pressure. The residue
was purified by column chromatography (methanol/dichlo-
romethane, 3:100-1:20, v/v) affording nucleoside 10 (1.85 g,
4.43 mmol, 81%) as a white solid material: Rf 0.36 (methanol/
dichloromethane, 1:9 (v/v)); 1H NMR (DMSO-d6) 11.25 (s, 1H,
NH), 7.99 (s, 1H, 6-H), 7.35-7.23 (m, 5H, Bn), 5.99-5.97 (m,
2H, 1′-H, CH2CHCH2-), 5.67 (d, J ) 5.8 Hz, 1H, 2′-OH), 5.44
(t, J ) 3.7 Hz, 1H, primary OH), 5.23-5.09 (m, 3H, CH2-
4-C-Acetoxym eth yl-5-O-a cetyl-3-C-a llyl-3-O-ben zyl-1,2-
O-isop r op ylid en e-R-D-er yth r o-p en tofu r a n ose (7). To a
solution of furanose 6 (3.30 g, 9.44 mmol) in anhydrous
pyridine (13 mL) were added acetic anhydride (13.0 mL, 138
mmol) and a catalytic amount of DMAP, and the reaction
mixture was stirred for 12 h at rt. Ice-cold H2O (300 mL) was
added, and extraction was performed using dichloromethane
(2 × 50 mL). The organic phase was evaporated to dryness,
and the residue was purified by column chromatography
(EtOAc/petroleum ether, 1:1 (v/v)) affording furanose 7 as a
clear oil (3.50 g) that was used in the next step without further
purification: Rf 0.67 (EtOAc/petroleum ether, 1:1 (v/v)); 13C
NMR (CDCl3) 170.4, 170.0, 138.0, 131.1, 128.1, 127.2, 126.7,
1
1
CHCH2-, /2CH2), 4.74 (d, J ) 12.2, 1H, /2CH2), 4.54 (t, J )
6316 J . Org. Chem., Vol. 69, No. 19, 2004

3′-C-Branched LNA-Type Nucleosides
5.6 Hz, 1H, primary OH), 4.36 (dd, J ) 5.8, 8.2 Hz, 1H, 2′-H),
3.81-3.45 (m, 4H, 2 × CH2), 3.08 (dd, J ) 5.6, 16.2 Hz, 1H,
CH2CHCH2-), 2.69 (dd, J ) 8.0, 16.4 Hz, 1H, CH2CHCH2-),
1.78 (d, J ) 1.0 Hz, 3H, CH3); 13C NMR (DMSO-d6, 163.7 (C-
4), 151.3 (C-2), 139.6, 137.8 and 134.0 (Bn, C-6 and CH2-
CHCH2-), 128.1, 126.9 and 126.6 (Bn), 117.8 (CH2CHCH2-),
109.4 (C-5), 90.2 (C-3′ or C-4′), 85.1 (C-1′), 83.5 (C-3′ or C-4′),
78.8 (C-2′), 65.6 (Bn), 63.0, 62.3 (C-5′, C-5′′), 33.0 (CH2CHCH2-
), 12.4 (CH3); FAB-MS m/z 419 ([M + 1]+). Anal. Calcd for
C21H26N2O7‚0.25 H2O: C, 59.64; H, 6.32; N, 6.62. Found: C,
59.35; H, 6.32; N, 6.75.
g, 3.06 mmol, 96%) as a white foam: Rf 0.41 (methanol/
dichloromethane, 7:93 (v/v)); 1H NMR (CDCl3) 9.87 (s, 1H, NH),
7.50-5.52 (m, 19H, DMT, Bn, 6-H), 5.69-5.58 (m, 1H,
CH2CHCH2-), 5.52 (s, 1H, 1′-H), 5.14 (s, 1H, 2′-H), 4.90 (dd,
J ) 10.1, 20.9 Hz, 2H, CH2CHCH2-), 4.57 (s, 2H, CH2), 4.29
(d, J ) 7.9 Hz, 1H, CH2), 4.15 (d, J ) 7.9, 1H, CH2), 3.78 (s,
6H, DMT), 3.57 (d, J ) 11.0, 1H, CH2), 3.43 (d, J ) 11.0, 1H,
CH2), 2.38-2.36 (m, 2H, CH2CHCH2-), 2.02 (s, 3H, CH3); 13
C
NMR (CDCl3) 163.7 (C-4), 158.6 (DMT), 145.0 (C-2), 144.4-
127.0 (DMT, Bn, C-6, CH2CHCH2-), 118.2 (CH2CHCH2-),
113.2 (DMT), 110.4 (C-5), 90.7 (C-4′), 88.9 (C-1′), 86.6 (DMT),
83.4 (C-3′), 78.5 (C-2′), 74.7, 67.9 and 59.8 (Bn, C-5′, C-5′′),
55.2 (DMT), 32.7 (CH2CHCH2-), 12.8 (CH3); FAB-MS m/z 703
([M +1]+). Anal. Calcd for C42H42N2O8: C, 71.78; H, 6.02; N,
3.99. Found: C, 72.13; H, 6.36; N, 3.71.
1-(3-C-Allyl-3-O-b en zyl-4-C-t osyloxym et h yl-â-D-r ib o-
fu r a n osyl)th ym in e (11). Nucleoside 10 (1.69 g, 4.04 mmol)
was coevaporated with anhydrous toluene (2 × 10 mL) and
dissolved in anhydrous pyridine (42 mL). The mixture was
cooled to 0 °C under stirring whereupon a solution of p-
toluenesulfonyl chloride (895 mg, 4.71 mmol) in anhydrous
pyridine (4 mL) was added slowly during 1 h. After an
additional 30 min, the reaction mixture was allowed to warm
to rt, and the reaction mixture was stirred for 22 h. H2O (60
mL) was added, and extraction was performed using dichlo-
romethane (3 × 100 mL). The combined organic phase was
dried (Na2SO4) and evaporated to dryness under reduced
pressure. The residue was purified by column chromatography
(methanol/dichloromethane, 1:50 (v/v)) affording nucleoside 11
as a white foam (1.62 g, 2.83 mmol, 70%): Rf 0.41 (methanol/
(1R,3R,4R,7S)-7-Ben zyloxy-1-(4,4′-d im et h oxyt r it yl)-
oxym eth yl-7-(2-h yd r oxyeth yl)- 3-(th ym in -1-yl)-2,5-d ioxa -
bicyclo[2.2.1]h ep ta n e (14). To a stirred solution of compound
13 (2.14 g, 3.04 mmol) in THF (20 mL) at rt was added first a
solution of NaIO4 (2.45 g, 11.5 mmol) in H2O (20 mL) and a
solution of OsO4 (0.41 mL, 2.5% solution in tert-butyl alcohol,
28 µmol), and the reaction mixture was stirred for 24 h.
Ethylene glycol (8 mL, 144 mmol) was added, and the resulting
mixture was stirred for 15 min whereupon H2O (41 mL) was
added. Extraction was performed using EtOAc (400 mL), and
the organic phase was dried (Na2SO4) and evaporated to
dryness under reduced pressure. The residue was dissolved
in a mixture of THF (20 mL) and H2O (20 mL), and NaBH4
(266 mg, 6.00 mmol) was added. After being stirred at rt for
20 h, the reaction mixture was neutralized by addition of a 2
M solution of HCl in dioxane, and the mixture was extracted
using EtOAc (350 mL). The organic phase was dried (Na2SO4)
and evaporated to dryness under reduced pressure to give a
residue that was purified by column chromatography (methanol/
dichloromethane/pyridine, 1:98.75:0.25 (v/v/v)) to give com-
pound 14 (813 mg, 1.15 mmol, 38%) as a white foam: Rf 0.43
(methanol/dichloromethane, 1:9 (v/v)); 1H NMR (CDCl3) 10.15
(s, 1H, NH), 7.51-6.81 (m, 21H, DMT, Bn, 6-H), 5.50 and 5.49
(s, 2H, 1′-H and 2′-H), 4.84 (d, J ) 11.4 Hz, 1H, CH2), 4.71 (d,
J ) 11.2 Hz, 1H, CH2), 4.15 (d, J ) 7.9 Hz, 1H, CH2), 4.10 (d,
J ) 7.9 Hz, 1H, CH2), 3.78 (s, 6H, DMT), 3.74-3.70 (m, 2H,
C3′-CH2CH2OH), 3.53 (d, J ) 10.8 Hz, 1H, CH2), 3.47 (d, J )
10.8 Hz, 1H, CH2), 1.92 (s, 3H, CH3), 1.84-1.69 (m, 2H, C3′-
CH2CH2OH); 13C NMR (CDCl3) 164.2 (C-4), 158.5 (DMT), 150.8
(C-2), 144.4-125.2 (DMT, Bn, C-6), 113.1 (DMT), 110.5 (C-5),
90.7 (C-4′), 89.0 (C-1′), 86.4 (DMT), 82.7 (C-3′), 78.9 (C-2′), 73.4
(C3′-CH2CH2OH), 68.8, 59.8, 57.3 (Bn, C-5′ and C-5′′), 55.1
(DMT), 31.4 (C3′-CH2CH2OH), 12.7 (CH3); FAB-MS m/z 706.2
([M]+). Anal. Calcd for C41H42N2O9: C, 69.67; H, 5.99; N, 3.96.
Found: C, 70.04; H, 6.03; N, 3.76.
1
dichloromethane, 1:9 (v/v)); H NMR (CDCl3) 10.05 (br s, 1H,
NH), 8.59 (s, 1H, 6-H), 7.75-7.18 (m, 9H, Ts and Bn), 5.91-
5.85 (m, 1H, CH2CHCH2-), 5.40-3.74 (m, 12 H, 5′-H, 5′′-H,
Bn, 1′-H, 2′-H, CH2CHCH2-, 5′-OH and 2′-OH), 3.16-3.11 (m,
1H, CH2CHCH2-), 2.82-2.77 (m, 1H, CH2CHCH2-), 2.40 (s,
3H, Ts), 1.77 (s, 3H, CH3); 13C NMR (CDCl3) 164.4 (C-4), 150.7
(C-2), 149.0-123.8 (Bn, Ts, CH2CHCH2-, C-6), 119.3 (CH2-
CHCH2-), 110.4 (C-5), 92.5, 88.4, 83.4 and 76.6 (C-1′, C-2′,
C-3′ and C-4′), 70.1, 66.6 and 62.5 (C-5′, C-5′′ and Bn), 33.4
(CH2CHCH2-), 21.6 (Ts), 12.8 (CH3); FAB-MS m/z 573 ([M +
1]+). Anal. Calcd for C28H32N2O9S‚0.25H2O: C, 58.27; H, 5.68;
N, 4.85. Found: C, 58.25; H, 5.57; N, 4.75.
1-(3-C-Allyl-3-O-b en zyl-4-C-t osyloxym et h yl-5-O-(4,4′-
d im eth oxytr ityl)-â-D-r ibofu r a n osyl)th ym in e (12). To a
stirred solution of nucleoside 11 (1.60 g, 2.79 mmol) in
anhydrous pyridine (6.4 mL) at rt was added DMTCl (1.59 g,
4.69 mmol). Stirring was continued for 42 h, whereupon
methanol (6 mL) was added. The resulting mixture was
evaporated to dryness under reduced pressure, and the residue
was dissolved in dichloromethane (65 mL). Washing was
performed using satd aq NaHCO3 (2 × 50 mL), and the
separated organic phase was dried (Na2SO4) and evaporated
to dryness under reduced pressure. The residue was purified
by column chromatography (methanol/dichloromethane/pyri-
dine, 0:99.75:0.25-2:97.75:0.25 (v/v/v)) to give nucleoside 12
(1.81 g, 2.06 mmol, 74%) as a white foam: Rf 0.35 (methanol/
dichloromethane, 1:19 (v/v)); 13C NMR (CDCl3) 163.7 (C-4),
158.6 (DMT), 150.8 (C-2), 149.1-123.7 (DMT, Bn, Ts, CH2-
CHCH2-, C-6), 118.9 (CH2CHCH2-), 113.0 (DMT), 110.9 (C-
5), 88.2, 87.2, 83.3 and 78.8 (C-1′, C-2′, C-3′ and C-4′), 85.5
(DMT), 70.4, 66.6 and 62.9 (C-5′, C-5′′ and Bn), 55.2 (DMT),
33.1 (CH2CHCH2-), 21.6 (Ts), 10.9 (CH3); FAB-MS m/z 874
([M]+). Anal. Calcd for C49H50N2O11S‚0.5H2O: C, 66.58; H, 5.82;
N, 3.17. Found: C, 66.42; H, 5.77; N, 3.58.
(1R,3R,4R,7S)-1-(4,4′-Dim eth oxytr ityl)oxym eth yl-7-h y-
dr oxy-7-(2-h ydr oxyeth yl)-3-(th ym in -1-yl)-2,5-dioxabicyclo-
[2.2.1]h ep ta n e (15). To a stirred solution of compound 14 (214
mg, 0.30 mmol) in anhydrous methanol (6 mL) at rt were
added NH4HCO3 (100 mg, 1.59 mmol) and Pd/C (30 mg). The
reaction mixture was stirred at 70 °C for 2.5 h, cooled to rt,
filtered, and evaporated to dryness under reduced pressure.
The residue was purified by column chromatography (methanol/
dichloromethane/pyridine, 1.0:98.75:0.25-4.0:95.75:0.25 (v/v/
v)) to give the bicyclic nucleoside 15 (156 mg, 0.25 mmol, 83%)
as a white foam: Rf 0.38 (methanol/dichloromethane, 1:9 (v/
(1R,3R,4R,7S)-7-Allyl-7-ben zyloxy-1-(4,4′-d im eth oxytr i-
t yl)oxym et h yl-3-(t h ym in -1-yl)-2,5-d ioxa b icyclo[2.2.1]-
h ep ta n e (13). To a stirred solution of nucleoside 12 (2.79 g,
3.19 mmol) in anhydrous DMF (80 mL) at rt was added NaH
(638 mg, 60% suspension, 16 mmol). After the mixture was
stirred for 20 h, H2O (6 mL followed after 5 min by additional
100 mL) was added, and the resulting mixture was extracted
with EtOAc (2 × 250 mL). The combined organic phase was
dried (Na2SO4), evaporated to dryness, and coevaporated with
anhydrous toluene (2 × 40 mL). The residue was purified by
column chromatography (pyridine/methanol/dichloromethane,
0.25:1.0:98.75 (v/v/v)) yielding the bicyclic nucleoside 13 (2.15
1
v)); H NMR (CDCl3) 10.74 (s, 1H, NH), 7.48-6.81 (m, 14H,
DMT and 6-H), 5.46 (s, 1H, 1′-H), 5.17 (s, 1H, 2′-H), 4.30 (s,
1H, OH), 4.27 (s, 1H, OH), 4.37 (d, J ) 8.2 Hz, 1H, CH2), 4.25
(d, J ) 8.2 Hz, 1H, CH2), 3.88-3.82 (m, 1H, C3′-CH2CH2OH),
3.79 (s, 6H, DMT), 3.79-3.75 (m, 1H, C3′-CH2CH2OH), 3.55
1
1
(d, J ) 10.8 Hz, /2CH2), 3.31 (d, J ) 11.0 Hz, /2CH2), 1.86 (s,
3H, CH3), 1.68-1.55 (m, 2H, C3′-CH2CH2OH); 13C NMR
(CDCl3) 164.3 (C-4), 158.6 (DMT), 151.2 (C-2), 135.7-126.9
(DMT and C-6), 113.2 (DMT), 110.3 (C-5), 90.1 (C-4′), 88.3 (C-
1′), 86.4 (DMT), 80.7 (C-2′), 78.2 (C-3′), 73.6 and 59.9 (C-5′ and
C-5′′), 56.8 (C3′-CH2CH2OH), 55.2 (DMT), 31.5 (C3′-CH2CH2-
J . Org. Chem, Vol. 69, No. 19, 2004 6317

Meldgaard et al.
OH), 12.9 (CH3); FAB-MS m/z 627 ([M + 1]+). Anal. Calcd for
C34H36N2O9‚H2O: C, 66.22; H, 5.88; N, 4.54. Found: C, 66.37;
H, 5.80; N, 4.54.
ether, 7:3 (v/v); 1H NMR (CDCl3) 5.70 (d, J ) 3.8 Hz, 1H, 1-H),
4.38 (d, J ) 3.8 Hz, 1H, 2-H), 4.18-4.07 (m, 2H, 6-H), 3.95-
3.86 (m, 1H, 5-H), 3.80 (d, J ) 8.2 Hz, 1H, 4-H), 3.75-3.68
(m, 2H, C3-CH2CH2CH2OH), 2.84 (br s, 1H, OH), 2.08 (br s,
1H, OH), 1.94-1.81 (m, 2H, C3-CH2CH2CH2OH), 1.77-1.66
(m, 2H, C3-CH2CH2CH2OH), 1.59, 1.45, 1.36 (3 × s, 12H, 4 ×
CH3); 13C NMR (CDCl3) 112.7, 109.8 (2 × isopropylidine), 103.7
(C-1), 82.5 (C-4), 81.2 (C-2), 79.2, 73.3 (C-6), 68.0 (C-5), 63.0
(C3-CH2CH2CH2OH), 28.2, 26.8, 26.8, 26.6, 26.4, 25.5 (C3-
CH2CH2CH2OH, 4 × CH3); MALDI-HRMS m/z calcd 341.15707
[M + Na]+, m/z found 341.15650. Anal. Calcd for C15H26O7:
C, 56.59; H, 8.23. Found: C, 56.61; H, 8.23.
(1R,3R,4R,7S)-1-(4,4′-Dim eth oxytr ityl)oxym eth yl-7-h y-
d r oxy-7-(2-levu lin oyloxyeth yl)-3-(th ym in -1-yl)-2,5-d ioxa -
bicyclo[2.2.1]h ep ta n e (16). To a stirred solution of didyclo-
hexyl carbodiimide (0.41 g, 2.0 mmol) in anhydrous dioxane
(3.75 mL) at rt was added levulinic acid (0.41 mL, 4 mmol),
and the reaction mixture was stirred for 16 h. The mixture
was filtered, and 0.5 mL of the filtrate was added to a solution
of nucleoside 15 (103 mg, 0.17 mmol) and DMAP (5 mg) in
pyridine (2 mL). The reaction mixture was stirred at rt for 14
h whereupon H2O (1 mL) was added. After the mixture was
stirred for 5 min, satd aq NaHCO3 (5 mL) was added. The
resulting mixture was extracted with EtOAc (3 × 15 mL), and
the combined organic phase was dried (Na2SO4) and evapo-
rated to dryness under reduced pressure. The residue was
purified by column chromatography (methanol/dichloromethane/
pyridine, 1.0:98.75:0.25 (v/v/v)) to furnish the bicyclic nucleo-
side 16 (107 mg, 0.15 mmol, 88%) as a white foam: Rf 0.40
(methanol/dichloromethane, 7:93 (v/v)); 1H NMR (CDCl3) 8.61
(d, J ) 1.6 Hz, 1H), 7.47-6.83 (m, 14H), 5.47 (s, 1H), 4.82 (s,
1H), 4.22 (m, 1H), 4.19 (d, 1H, J ) 8.8 Hz), 4.05 (d, J ) 8.8
Hz, 1H), 3.99-3.97 (m, 1H), 3.80 (s, 6H), 3.53 (d, J ) 10.9 Hz,
1H), 3.38 (d, J ) 10.9 Hz, 1H), 2.94 (s, 1H), 2.70-2.41 (m,
3-O-Ben zyl-3-C-(3-b en zyloxy)p r op yl-1,2-O-isop r op yl-
id en e-R-D-a llofu r a n ose (19). To a stirred ice-cold solution
of furanose 18 (10.69 g, 33.60 mmol) in anhydrous DMF (75
mL) was added a 60% oil suspension of NaH (4.07 g, 0.10 mol).
After 3 h, benzyl bromide (16 mL, 0.13 mol) was added
dropwise, and the reaction mixture was stirred for 3 h at rt.
Ice-cold H2O (50 mL) was added whereupon extraction was
performed using EtOAc (3 × 100 mL). The combined organic
phase was washed successively with brine (70 mL), H2O (70
mL), and saturated aqueous NaHCO3 (70 mL). The combined
aqueous phase was extracted with EtOAc (50 mL), and the
combined organic phase was evaporated to dryness under
reduced pressure and coevaporated with toluene (3 × 25 mL).
The residue was dissolved in AcOH/H2O (8:2 (v/v), 250 mL)
and allowed to stir for 45 h at rt before the mixture was
evaporated to dryness under reduced pressure. The residue
was extracted with EtOAc (200 mL) and the organic phase
washed with H2O (2 × 100 mL). The organic phase was
evaporated to dryness under reduced pressure, and the residue
was coevaporated with toluene (3 × 25 mL). The residue was
purified by dry column vacuum chromatography (EtOAc/
petroleum ether, 9:11-3:1 (v/v)) to give furanose 19 (12.16 g,
79%) as a clear oil: Rf 0.40 (EtOAc/petroleum ether, 7:3 (v/
v)); 1H NMR (CDCl3) 7.38-7.25 (m, 10H, Bn), 5.69 (d, J ) 3.7
Hz, 1H, 1-H), 4.68 (dd, J ) 16.5, 10.6 Hz, 2H, CH2Ph), 4.51 (s,
2H, CH2Ph), 4.46 (d, J ) 3.7 Hz, 1H, 2-H), 4.09 (d, J ) 9.3 Hz,
1H, 4-H) 3.90-3.64 (m, 3H, 5-H, 6-H), 3.50 (t, 1H, C3-CH2-
CH2CH2OH), 2.76 (br s, 1H, OH), 2.15 (br s, 1H, OH), 2.04-
1.74 (m, 4H, C3-CH2CH2CH2OH), 1.60, 1.35 (2 × s, 2 × 3H, 2
× CH3); 13C NMR (CDCl3) 138.4, 138.1, 128.6, 128.5, 127.8,
127.7, 113.2, 104.2, 85.1, 82.5, 79.1, 73.2, 70.4, 69.7, 67.6, 64.9,
27.9, 26.9, 26.7, 24.0. MALDI-HRMS m/z calcd 481.21967 [M
+ Na]+, m/z found 481.21830. Anal. Calcd for C26H34O7: C,
68.10; H, 7.47. Found: C, 67.73; H, 7.42.
4H), 2.16 (s, 3H), 1.94 (d, J ) 0.9 Hz), 1.82-1.70 (m, 2H); 13
C
NMR (CDCl3) 206.0, 172.6, 163.5, 158.6, 149.9, 144.3-123.6,
113.2, 110.4, 89.4, 87.9, 86.6, 80.1, 78.1, 72.9, 60.2, 59.3, 55.1,
37.7, 29.7, 27.8, 27.5, 12.8; FAB-MS m/z 715 ([M + 1]+). Anal.
Calcd for C39H42N2O11‚1/2H2O: C, 64.72; H, 5.99; N, 3.87.
Found: C, 64.63; H, 5.84; N, 4.11.
(1R,3R,4R,7S)-7-(2-Levu lin oyloxy)eth yl-7-(2-cya n oeth -
oxy)(N ,N -(d iisop r op yl)a m in o)p h osp h in oxy-1-(4,4′-d i-
m eth oxytr ityl)oxym eth yl-3-(th ym in -1-yl)-2,5-dioxabicyclo-
[2.2.1]h ep ta n e (17). Nucleoside 16 (104 mg, 0.15 mmol) was
coevaporated with anhydrous acetonitrile (3 × 1 mL) and
dissolved in anhydrous dichloromethane (1 mL) at rt under
stirring. Anhydrous N,N-(diisopropyl)ethylamine (0.6 mL, 3.5
mmol) and 2-cyanoethyl N,N-(diisopropyl)phosphoramido-
chloridite (0.15 mL, 0.54 mmol) were added, and the mixture
was stirred for 12 h at rt. Additional 2-cyanoethyl N,N-
(diisopropyl)phosphoramidochloridite (0.20 mL, 0.72 mmol)
was added, and the mixture was stirred for another 6 h at rt.
H2O (1 mL) was added, and the resulting mixture was diluted
with EtOAc (20 mL). Washing was performed using succes-
sively satd aq NaHCO3 (3 × 2 mL) and NaCl (3 × 2 mL). The
separated organic phase was dried (Na2SO4) and evaporated
to dryness under reduced pressure. The residue was purified
by silica gel column chromatography (Et3N, EtOAc, petroleum
ether, 1:9:10 (v/v/v)) afforded a yellow oil, which was dissolved
in anhydrous toluene (1 mL) and precipitated by addition of
this solution into ice-cold petroleum ether (20 mL) under
vigorous stirring to give phosphoramidite 17 (0.06 mmol, 41%)
as a pale yellow oil: Rf 0.57 (Et3N, EtOAc, CH2Cl2, 2:9:9
(v/v/v)); 31P NMR (DMSO-d6) 146.42, 146.05.
1,2:5,6-Di-O-isop r op ylid en e-3-C-(3-h yd r oxy)p r op yl-R-
D-a llofu r a n ose (18). To a stirred ice-cold solution of 3-C-allyl-
1,2:5,6-di-O-isopropylidene-R-D-allofuranose22 (13.74 g, 45.70
mmol) in anhydrous THF (240 mL) was added dropwise a
solution of BH3‚Me2S (2 M solution in diethyl ether, 69 mL,
0.138 mol). After being stirred for 3 h at rt, the solution was
cooled to 0 °C, and a mixture of 2 M NaOH (69 mL) and 35%
H2O2 (14 mL) was added dropwise. The resulting mixture was
stirred for 16 h at rt and extracted with EtOAc (300 mL). The
organic phase was washed with brine (100 mL + 200 mL).
The combined aqueous phase was extracted using EtOAc (200
mL), and the combined organic phase was evaporated to
dryness under reduced pressure and coevaporated with toluene
(2 × 25 mL). The residue was purified by dry column vacuum
chromatography (EtOAc/petroleum ether, 72:28-100:0 (v/v)
methanol/EtOAc, 1:99-12:88 (v/v)) affording furanose 18
(10.18 g, 70%) as a white powder: Rf 0.17 (EtOAc/petroleum
3-O-Ben zyl-3-C-(3-ben zyloxy)p r op yl-4-C-h yd r oxym eth -
yl-1,2-O-isop r op ylid en e-R-D-er yth r o-p en tofu r a n ose (20).
To a stirred ice-cold solution of NaIO4 (1.67 g, 7.8 mmol) in
H2O/1,4-dioxane (2:1 (v/v), 90 mL) at rt was added dropwise
furanose 19 (1.93 g, 4.2 mmol) dissolved in THF (25 mL). After
3 h, ethylene glycol (1.5 mL) was added, the resulting mixture
was filtered, the filtering funnel was flushed with EtOAc (2 ×
50 mL), and the filtrate extracted with EtOAc (2 × 100 mL).
The combined organic phase was evaporated to dryness under
reduced pressure and the residue dissolved in 1,4-dioxane (50
mL). Formaldehyde (0.95 mL, 37% aq solution (w/w), 12.9
mmol) and 1 M NaOH (50 mL, 50.0 mmol) were added, and
the resulting mixture was stirred for 16 h at rt. A 17.45 M
aqueous solution of formic acid was added to neutralize the
mixture followed by evaporation to dryness under reduced
pressure. To the residue was added EtOAc (2 × 100 mL), and
the combined organic phase was evaporated to dryness under
reduced pressure and coevaporated with toluene (50 mL) to
give a residue that was purified by dry column vacuum
chromatography (EtOAc/petroleum ether, 6:4-7:3 (v/v)) yield-
ing furanose 20 (1.64 g, 85%) as a white powder: Rf 0.35
(EtOAc/petroleum ether, 7:3 (v/v)); 1H NMR (CDCl3) 7.35-7.24
(m, 10H, Bn), 5.76 (d, 1H, J ) 4.1 Hz, 1-H), 4.67 (d, J ) 10.5
Hz, 1H, CH2Ph), 4.60 (d, J ) 10.5 Hz, 1H, CH2Ph), 4.51 (s,
2H, CH2Ph), 4.50 (s, 1H, 2-H), 4.12 (s, 2H, 5-H), 3.87-3.80
(m, 2H, 5′-H), 3.51 (t, 2H, C3-CH2CH2CH2OH), 2.66 (br s, 1H,
6318 J . Org. Chem., Vol. 69, No. 19, 2004

3′-C-Branched LNA-Type Nucleosides
OH), 2.54 (br s, 1H, OH), 2.13-1.72 (m, 4H, C3-CH2CH2CH2-
OH), 1.64, 1.31 (2 × s, 2 × 3H, 2 × CH3); 13C NMR (CDCl3)
138.5, 138.2, 128.5, 127.8, 127.8, 127.7, 127.7, 127.6, 112.8,
104.0, 87.8, 86.5, 84.2, 73.2, 70.3, 66.9, 63.8, 63.5, 29.1, 26.3,
25.8, 23.7; MALDI-HRMS m/z calcd 481.21967 [M + Na]+, m/z
found 481.21750. Anal. Calcd for C26H34O7‚0.1H2O: C, 67.84;
H, 7.49. Found: C, 67.73; H, 7.51.
reaction mixture was heated under reflux for 45 h. To the
reaction mixture was added brine (200 mL), and extraction
was performed using dichloromethane (2 × 100 mL). The
combined organic phase was evaporated to dryness under
reduced pressure to give a residue that was coevaporated with
toluene (2 × 25 mL) and then purified by dry column vacuum
chromatography (MeOH/EtOAc, 0:1-1:9 (v/v)) to give the
bicyclic nucleoside 23 (944.0 mg, 66%) as a white foam: Rf
1,2-Di-O-Acetyl-3-O-ben zyl-3-C-(3-ben zyloxy)p r op yl-4-
O-m esyl-4-C-m esyloxym et h yl-D-er yth r o-p en t ofu r a n ose
(21). Furanose 20 (1.64 g, 3.60 mmol) was coevaporated with
ice-cold anhydrous pyridine (10 mL) and dissolved in anhy-
drous pyridine (15 mL), and MsCl (1.3 mL, 7.7 mmol) was
added dropwise. The solution was stirred for 1 h at rt followed
by addition of ice-cold H2O (20 mL). The resulting mixture was
extracted using CH2Cl2 (2 × 20 mL) and evaporated to dryness
under reduced pressure. The residue was coevaporated with
toluene (3 × 15 mL) and dissolved in TFA/H2O (8:2 (v/v), 15
mL), and the resulting mixture was stirred for 3 h at rt. The
mixture was evaporated to dryness under reduced pressure
and coevaporated successively with toluene (4 × 15 mL) and
pyridine (15 mL). The residue was dissolved in anhydrous
pyridine (10 mL), and Ac2O (2.0 mL, 21.2 mmol) was added.
The resulting mixture was stirred for 17 h at rt, more Ac2O
(2.0 mL, 21.2 mmol) was added, and stirring was continued
for additional 3 h. Ice (30 mL) was added, and after stirring
for 5 min the mixture was evaporated to dryness. The residue
was coevaporated with toluene (2 × 15 mL + 10 mL) and was
purified by dry column vacuum chromatography (EtOAc/
petroleum ether, 4:6-1:0 (v/v)) to give anomeric mixture 21
(2.05 g, 87%) as a white powder: Rf 0.37 (EtOAc/petroleum
ether, 7:3 (v/v)); 13C NMR (CDCl3) 169.2, 138.4, 137.4, 128.7,
128.6, 128.0, 127.8, 127.8, 127.2, 127.0, 97.7, 87.3, 84.6, 79.3,
73.2, 69.6, 69.5, 68.5, 66.5, 65.7, 37.9, 37.6, 26.8, 26.8, 24.0,
21.1, 20.9; MALDI-HRMS m/z calcd 681.16460 [M + Na]+, m/z
found 681.16090.
1
0.45 (methanol/dichloromethane, 1:9 (v/v)); H NMR (CDCl3)
9.12 (br s, 1H, NH), 7.49 (d, 1H, J ) 1.2 Hz, 6-H), 7.35-7.23
(m, 10H, Harom), 5.49 (s, 1H, 1′-H) 5.10 (s, 1H, 2′-H), 4.64 (d,
1H, J ) 10.4 Hz, CH2Ph), 4.53 (d, 1H, J ) 10.4 Hz, CH2Ph),
4.43 (s, 2H, CH2Ph), 4.38-3.95 (m, 4H, 5′-H and 5′′-H), 3.40-
3.25 (m, 2H, C3′-CH2CH2CH2OH), 2.18 (1H, 5′-OH), 1.85 (s,
3H, CH3), 1.79-1.56 (m, 4H, C3′-CH2CH2CH2OH); 13C NMR
(CDCl3) 164.0, 150.2, 138.2, 137.8, 134.7, 128.6, 128.5, 127.9,
127.8, 127.6, 127.6 (Carom), 110.5, 91.0 (C-1′), 89.2, 84.0, 79.6
(C-2′), 74.6, 73.2, 69.7, 68.0, 59.1, 25.3, 23.9, 12.6; MALDI-
HRMS m/z calcd 531.21017 [M + Na]+, m/z found 531.20890.
Anal. Calcd for C28H32N2O7: C, 65.20; H, 6.41; N, 5.43.
Found: C, 65.32; H, 6.46; N, 5.35.
(1R,3R,4R,7S)-7-Ben zyloxy-7-(3-b en zyloxy)p r op yl-1-
(4,4′-d im e t h oxyt r it yl)oxym e t h yl-3-(t h ym in -1-yl)-2,5-
d ioxa bicyclo[2.2.1]h ep ta n e (24). Nucleoside 23 (1.70 g, 3.3
mmol) was coevaporated with anhydrous pyridine (20 mL) and
dissolved in anhydrous pyridine (30 mL) whereupon DMTCl
(1.73 g, 5.1 mmol) was added. After the mixture was stirred
for 19 h at rt, methanol (20 mL) was added and the resulting
mixture was evaporated to dryness under reduced pressure.
The residue was dissolved in dichloromethane (150 mL) and
washed with satd aq NaHCO3 (3 × 100 mL). The organic phase
was evaporated to dryness under reduced pressure to give a
residue that was coevaporated with toluene (20 mL) and
purified by dry column vacuum chromatography (EtOAc/
petroleum ether, 1:1-7:3 (v/v)) to give the bicyclic nucleoside
24 (2.44 g, 90%) as a clear oil: Rf 0.47 (methanol/dichlo-
romethane, 1:19 (v/v)); 1H NMR 8.96 (br s, 1H, NH), 7.51-
7.14, 6.83-6.77 (2 × m, 24H, Harom), 5.52 (s, 1H, 1′-H), 5.05 (s,
1H, 2′-H), 4.58-4.15 (m, 8H, 5′-H, 5′′-H, CH2Ph), 3.76 (s, 6H,
2 × CH3), 3.56 (d, J ) 10.7 Hz, 1H, CH2Ph), 3.47 (d, J ) 10.7
Hz, 1H, CH2Ph), 3.22-3.15 (m, 2H, C3′-CH2CH2CH2OH), 1.86
(s, 3H, CH3), 1.72-1.34 (m, 4H, C3′-CH2CH2CH2OH); 13C NMR
(CDCl3) 163.9, 158.8, 158.7, 150.2, 144.6, 138.4, 138.1, 135.7,
135.6, 134.8, 130.3, 130.1, 128.5, 128.4, 128.2, 128.0, 127.7,
127.6, 127.6, 127.4, 127.1, 113.3, 110.4, 90.8 (C-1′), 89.0, 86.7,
84.0, 79.2 (C-2′), 74.9, 73.0, 69.7, 67.7, 60.0, 55.3, 25.2, 24.5,
12.7; MALDI-HRMS m/z calcd 833.34085 [M + Na]+, m/z found
833.34220. Anal. Calcd for C49H50N2O9‚0.5EtOAc: C, 71.64;
H, 6.37; N, 3.28. Found: C, 71.74; H, 6.37; N, 3.44.
(1R,3R,4R,7S)-1-(4,4′-Dim eth oxytr ityl)oxym eth yl-7-h y-
d r oxy-7-(3-h yd r oxy)p r op yl-3-(t h ym in -1-yl)-2,5-d ioxa b i-
cyclo[2.2.1]h ep ta n e (25). Nucleoside 24 (270 mg, 0.33 mmol)
and a catalytic amount of 10% Pd/C was dissolved in methanol
(20 mL), and HCOONH4 (547 mg, 8.70 mmol) was added. The
reaction mixture was heated under reflux for 6 h and subse-
quently filtered through silica gel, washing with absolute
ethanol (6 × 25 mL) to furnish nucleoside 25 (156 mg, 74%)
as a clear oil: Rf 0.12 (methanol/dichloromethane, 1:19 (v/v));
1H NMR (CDCl3) 9.67 (br s, 1H, NH), 7.50-7.18, 6.87-6.80 (2
× m, 14H, Harom), 5.45 (s, 1H, 1′-H), 4.78 (s, 1H, 2′-H), 4.31 (br
s, 1H, OH), 4.19 (s, 2H, 5′-H), 3.79 (s, 6H, 2 × CH3), 3.56-
3.34 (m, 4H, 5′′-H, C3′-CH2CH2CH2OH), 3.10 (br s, 1H, OH),
1.91 (s, 3H, CH3), 1.60-1.43, 1.34-1.23 (2 × m, 2 × 2H, C3′-
CH2CH2CH2OH); 13C NMR (CDCl3) 164.4, 158.8, 158.7, 150.6,
144.6, 135.8, 135.5, 134.6, 130.3, 130.1, 128.2, 128.0, 127.1,
113.4, 110.5, 90.0 (C-1′), 88.2, 86.6, 80.3, 80.0 (C-2′), 74.1, 62.4,
59.8, 55.4, 28.0, 27.3, 12.9; MALDI-HRMS m/z calcd 653.24695
[M + Na]+, m/z found 653.24470. Anal. Calcd for C35H38N2O9‚
1.25H2O: C, 64.36; H, 6.25; N, 4.29. Found: C, 64.49; H, 6.60;
N, 4.38.
1-(2-O-Acetyl-3-O-ben zyl-3-C-(3-ben zyloxy)p r op yl-5-O-
m esyl-4-C-m esyloxym eth yl-â-D-er yth r o-p en tofu r a n osyl)-
th ym in e (22). The anomeric mixture 21 (144.8 mg, 0.22 mmol)
was coevaporated with anhydrous acetonitrile (5 mL) and
dissolved in anhydrous acetonitrile (8 mL). Thymine (89.3 mg,
0.71 mmol) and N,O-bistrimethylsilyl acetamide (179 mg, 0.88
mmol) were added, and the stirred reaction mixture was
heated under reflux until the solution turned clear (ap-
proximately after 1 h). The reaction mixture was cooled to 0
°C, trimethylsilyl triflate (146.6 mg, 0.66 mmol) was added
dropwise, and the stirred reaction was heated under reflux
for 23 h whereupon a satd aq solution of NaHCO3 (50 mL)
was added. Extraction was performed using CH2Cl2 (70 mL).
The organic phase was washed with satd aq NaHCO3 (50 mL)
and evaporated to dryness under reduced pressure. The
residue was coevaporated with toluene (10 mL) and was
purified by dry column vacuum chromatography (EtOAc/
petroleum ether, 6:4-8:2 (v/v)) to give nucleoside 22 (148.2
mg, 93%) as a white solid material: Rf 0.31 (EtOAc/petroleum
1
ether, 7:3 (v/v)); H NMR (CDCl3) 8.61 (br s, 1H, NH), 7.41-
7.26 (m, 10H, Bn), 6.31 (d, 1H, J ) 8.3 Hz, 1′-H), 5.51 (d, 1H,
J ) 8.3 Hz, 2′-H), 5.05 (d, 1H, J ) 11.1 Hz, CH2Ph), 4.65 (d,
1H, J ) 11.1 Hz, CH2Ph), 4.55-4.37 (m, 6H, 5′-H, 5′′-H and
CH2Ph), 3.60-3.48 (m, 2H, C3′-CH2CH2CH2OH), 2.92, 2.90 (2
× s, 2 × 3H, 2 × CH3), 2.34-1.65 (m, 4H, C3′-CH2CH2CH2-
OH), 2.12, 1.94 (2 × s, 2 × 3H, 2 × CH3); 13C NMR (CDCl3)
169.6, 163.5, 150.9, 138.1, 137.7, 135.4, 128.8, 128.7, 128.6,
128.2, 128.0, 128.0, 127.4, 112.4, 86.0, 84.5, 84.2, 77.3, 73.5,
69.5, 68.6, 68.2, 67.1, 37.4, 37.4, 26.1, 24.2, 20.9, 12.3; MALDI-
HRMS m/z calcd 747.18640 [M + Na]+, m/z found 747.18940.
Anal. Calcd for C32H40N2O13S2: C, 53.03; H, 5.56; N, 3.87; S,
8.85. Found: C, 52.92; H, 5.65; N, 3.75; S, 8.73.
(1S,3R,4R,7S)-7-Ben zyloxy-7-(3-ben zyloxy)p r op yl-1-h y-
d r oxym e t h yl-3-(t h ym in -1-yl)-2,5-d ioxa b icyclo[2.2.1]-
h ep ta n e (23). Nucleoside 22 (2.04 g, 2.8 mmol) was dissolved
in a mixture of 1,4-dioxane (15 mL), H2O (15 mL), and an
aqueous solution of 6 M KOH (30 mL, 0.18 mol). The stirred
(1R,3R,4R,7S)-7-(3-Acet oxyp r op yl)-1-(4,4′-d im et h oxy-
tr ityl)oxym eth yl-7-h yd r oxy-3-(th ym in -1-yl)- 2,5-d ioxa bi-
J . Org. Chem, Vol. 69, No. 19, 2004 6319

Meldgaard et al.
cyclo[2.2.1]h ep ta n e (26). Nucleoside 25 (314 mg, 0.50 mmol)
was coevaporated with anhydrous pyridine (15 mL) and
dissolved in anhydrous pyridine (10 mL), and Ac2O (0.1 mL,
1.1 mmol) was added. After the mixture was stirred for 26 h
at rt, H2O (1 mL) was added, and the resulting mixture was
after 5 min evaporated to dryness under reduced pressure. The
residue was coevaporated with toluene (3 × 5 mL) and was
purified by dry column vacuum chromatography (EtOAc/
petroleum ether, 1:1-7:3 (v/v)) yielding nucleoside 26 as a
white foam (302 mg, 90%): Rf 0.64 (methanol/dichloromethane,
1:9 (v/v)); 1H NMR (CDCl3) 7.51-7.22, 6.88-6.82 (2 × m, 14H,
Harom), 5.45 (s, 1H, 1′-H), 4.72 (s, 1H, 2′-H), 4.20-4.09, 3.96-
3.82 (2 × m, 2 × 2H, 5′-H, 5′′-H), 3.80, 3.80 (2 × s, 2 × 3H, 2
× CH3), 3.54 (d, 1H, J ) 10.9 Hz, C3′-CH2CH2CH2OH), 3.36
(d, 1H, J ) 10.9 Hz, C3′-CH2CH2CH2OH), 1.96 (s, 3H, CH3),
1.50-1.42 (m, 7H, CH3, C3-CH2CH2CH2OH); 13C NMR (CDCl3)
171.1, 163.7, 158.8, 158.8, 150.2, 144.5, 135.6, 135.3, 134.4,
130.2, 130.1, 128.2, 128.1, 127.2, 113.4, 110.5, 89.7 (C-1′), 87.9,
86.8, 80.2 (C-2′), 80.0, 73.7, 64.4, 59.6, 55.4, 26.2, 24.4, 21.0,
12.9; MALDI-HRMS m/z calcd 695.25751 [M + Na]+, m/z found
695.25750.
(1R,3R,4R,7S)-7-(3-Acetoxyp r op yl)-7-(2-cya n oeth oxy)-
(N,N-(diisopr opyl)am in o)ph osph in oxy)-1-(4,4′-dim eth oxy-
tr ityl)oxym eth yl-3-(th ym in -1-yl)-2,5-d ioxa bicyclo[2.2.1]-
h ep ta n e (27). Nucleoside 26 (144 mg, 0.20 mmol) was
dissolved in anhydrous CH2Cl2 (5 mL) and N,N-(diisopropyl)-
ethylamine (0.15 mL, 0.8 mmol), and 2-cyanoethyl N,N-
(diisopropyl)phosphoramidochloridite (0.10 mL, 0.4 mmol) was
added. The reaction mixture was stirred for 26 h at rt,
whereupon methanol (2.5 mL) was added followed by dichlo-
romethane (5 mL). The resulting mixture was washed succes-
sively with satd aq NaHCO3 (3 × 10 mL), H2O (10 mL), and
brine (10 mL). The combined aqueous phase was extracted
with dichloromethane (10 mL), and the combined organic
phase was evaporated to dryness under reduced pressure. The
residue was coevaporated with toluene (10 mL) and purified
by dry column vacuum chromatography (EtOAc/petroleum
ether, 3:2-7:3 (v/v)) to give phosphoramidite 27 (120 mg, 64%)
as a clear oil: Rf 0.72 (methanol/dichloromethane, 1:9 (v/v));
31P NMR (CDCl3) 146.2, 145.8; MALDI-HRMS m/z calcd
895.36537 [M + Na]+, m/z found 895.36160.
was stirred for 3 h at rt whereupon ice-cold H2O (200 mL) was
added. The resulting mixture was extracted using CH2Cl2 (4
× 100 mL), and the combined organic phase was washed with
satd aq NaHCO3 (200 mL) and evaporated to dryness under
reduced pressure. The residue was coevaporated with toluene
(2 × 20 mL) and purified by dry column vacuum chromatog-
raphy (EtOAc/petroleum ether, 1:3-1:0 (v/v)) yielding fura-
noside 29 (3.54 g, 79%) as a clear oil: Rf 0.55 (EtOAc/petroleum
ether, 7:3 (v/v)); 1H NMR (CDCl3) 7.34-7.16 (m, 5H, Bn), 5.59
(d, 1H, J ) 3.7 Hz, 1-H), 4.77 (d, J ) 11.1 Hz, 1H, CH2Ph),
4.68 (d, J ) 11.1 Hz, 1H, CH2Ph), 4.38 (d, J ) 3.7 Hz, 1H,
2-H), 4.20-3.86 (m, 6H, 4-H, 5-H, 6-H, C3-CH2CH2CH2OH),
2.89 (s, 3H, CH3), 2.10-1.56 (m, 4H, C3-CH2CH2CH2OH), 1.53,
1.33, 1.29, 1.28 (4 × s, 4 × 3H, 4 × CH3); 13C NMR (CDCl3)
139.1, 128.3, 127.3, 127.2, 113.0, 109.9, 103.2 (C-1), 84.0, 83.9,
80.6, 73.1, 70.4, 68.7, 66.9, 37.5, 27.2, 27.1, 26.7, 26.7, 25.5,
23.4; MALDI-HRMS m/z calcd 509.18157 [M + Na]+, m/z found
509.18310.
3-C-(3-Azid op r op yl)-3-O-ben zyl-1,2:5,6-d i-O-isop r op yl-
id en e-R-D-a llofu r a n ose (30). To a stirred solution of furanose
29 (361 mg, 0.74 mmol) in anhydrous DMF (4 mL) was added
NaN3 (121.0 mg, 1.9 mmol). The reaction mixture was heated
to 100 °C and stirred for 24 h. H2O (25 mL) was added, and
extraction was performed using EtOAc (2 × 25 mL). The
combined organic phase was washed successively with brine
(20 mL), satd aq NaHCO3 (20 mL), and H2O (20 mL) and
evaporated to dryness under reduced pressure. The residue
was coevaporated with toluene (15 mL) and purified by dry
column vacuum chromatography (EtOAc/petroleum ether,
1:9-12-88 (v/v)) affording furanose 30 (271 mg, 84%) as a
clear oil: Rf 0.71 (EtOAc/petroleum ether, 7:3 (v/v)); 1H NMR
(CDCl3) 7.40-7.22 (m, 5H, Bn), 5.65 (d, 1H, J ) 3.6 Hz, 1-H),
4.83 (d, J ) 11.3 Hz, 1H, CH2Ph), 4.73 (d, J ) 11.3 Hz, 1H,
CH2Ph), 4.45 (d, J ) 3.6 Hz, 1H, 2-H), 4.21-3.91 (m, 4H, 4-H,
5-H, 6-H), 3.37-3.28 (m, 2H, C3-CH2CH2CH2OH), 1.95-1.62
(m, 4H, C3-CH2CH2CH2OH), 1.60, 1.39, 1.36, 1.35 (4 × s, 4 ×
3H, 4 × CH3); 13C NMR (CDCl3) 139.2, 128.2, 127.3, 127.1,
113.0, 109.8, 103.3 (C-1), 84.0 (C-2), 81.1, 73.2, 68.7, 66.9, 52.0,
28.2, 27.1, 26.7, 25.5, 23.0. MALDI-HRMS m/z calcd 456.21050
[M + Na]+, m/z found 456.21090; IR 2098 cm-1 (-N3). Anal.
Calcd for C22H31N3O6: C, 60.95; H, 7.21; N, 9.69. Found: C,
61.18; H, 7.22; N, 9.82.
3-O-Ben zyl-1,2:5,6-di-O-isopr opyliden e-3-C-(3-h ydr oxy)-
p r op yl-R-D-a llofu r a n ose (28). To a stirred solution of fura-
nose 6 (285 mg, 0.73 mmol) in anhydrous THF (5 mL) was
added dropwise a 2 M solution of BH3‚Me2S in diethyl ether
(1.1 mL, 2.2 mmol) at 0 °C. After being stirred for 4 h at rt,
the reaction mixture was cooled to 0 °C, and a mixture of 2 M
aq NaOH (1.4 mL, 2.8 mmol) and 35% aq H2O2 (0.3 mL) was
added dropwise. The reaction mixture was stirred for 67 h at
rt followed by extraction using EtOAc (25 mL). The organic
phase was washed with brine (2 × 25 mL) and the combined
aqueous phase extracted with EtOAc (25 mL). The combined
organic phase was evaporated to dryness under reduced
pressure. The residue was coevaporated with toluene (10 mL)
and purified by dry column vacuum chromatography (EtOAc/
petroleum ether, 1:1-3:2 (v/v)) to give furanose 28 (137 mg,
46%) as a white powder: Rf 0.22 (EtOAc/petroleum ether, 7:3
3-C-(3-Azid o)p r op yl-3-O-ben zyl-1,2-O-isop r op ylid en e-
R-D-a llofu r a n ose (31). Compound 30 (208 mg, 0.48 mmol)
was dissolved in a mixture of AcOH and H2O (8:2 (v/v), 5 mL)
and stirred for 41 h at rt. The reaction mixture was evaporated
and the residue coevaporated with toluene (2 × 10 mL) and
purified by dry column vacuum chromatography (EtOAc/
petroleum ether, 1:1-3:2 (v/v)) to give furanoside 31 (155 mg,
82%) as a clear oil: Rf 0.44 (EtOAc/petroleum ether, 7:3 (v/v);
1H NMR (CDCl3) 7.38-7.26 (m, 5H, Bn), 5.70 (d, J ) 3.6 Hz,
1H, 1-H), 4.74 (d, J ) 10.5 Hz, 1H, CH2Ph), 4.64 (d, J ) 10.5
Hz 1H, CH2Ph), 4.46 (d, J ) 3.6 Hz, 1H, 2-H), 4.11 (d, J ) 9.0
Hz, 1H, 4-H), 3.85-3.63 (m, 3H, 5-H, 6-H), 3.39-3.34 (m, 2H,
C3-CH2CH2CH2OH), 2.73 (br s, 1H, OH), 2.24 (br s, 1H, OH),
1.96-1.74 (m, 4H, C3-CH2CH2CH2OH), 1.61, 1.36 (2 × s, 2 ×
3H, 2 × CH3); 13C NMR (CDCl3) 137.9, 128.5, 127.9, 127.7,
113.3, 104.0 (C-1), 84.9, 82.7 (C-2), 78.8, 69.8, 67.7, 64.8, 52.0,
28.4, 26.9, 26.7, 23.3; MALDI-HRMS m/z calcd 416.17920 [M
+ Na]+, m/z found 416.18080; IR 2098 cm-1 (-N3). Anal. Calcd
for C19H27N3O6: C, 58.00; H, 6.92; N, 10.68. Found: C, 57.70;
H, 6.93; N, 10.44.
3-C-(3-Azid op r op yl)-3-O-b en zyl-4-C-h yd r oxym et h yl-
1,2-O-isop r op ylid en e-R-D-er yth r o-p en tofu r a n ose (32). To
a stirred ice-cold solution of NaIO4 (2.16 g, 10.1 mmol) in a
mixture of H2O and 1,4-dioxane (2:1 (v/v), 90 mL) was dropwise
added furanose 31 (2.21 g, 5.6 mmol) dissolved in THF (30
mL). Ethylene glycol (2.5 mL) was added after 5 h, the mixture
was filtered, and the filtering funnel was washed with EtOAc
(2 × 50 mL). The filtrate was extracted with EtOAc (100 mL),
and the combined organic phase was evaporated to dryness
under reduced pressure. The residue was dissolved in 1,4-
1
(v/v)); H NMR (CDCl3) 7.40-7.22 (m, 5H, Bn), 5.65 (d, J )
3.7, 1H, 1-H), 4.87 (d, J ) 11.2, 1H, CH2Ph), 4.73 (d, J ) 11.2,
1H, CH2Ph), 4.49 (d, J ) 3.7, 1H, 2-H), 4.30-3.92 (m, 4H, 4-H,
5-H, 6-H), 3.65 (t, J ) 5.9, 2H, C3-CH2CH2CH2OH), 1.99-
1.63 (m, 4H, C3-CH2CH2CH2OH), 1.60, 1.39, 1.35, 1.35 (4 ×
s, 4 × 3H, 4 × CH3); 13C NMR (CDCl3) 139.3, 128.2, 127.3,
112.9, 109.8, 103.3 (C-1), 84.0, 83.8 (C-2), 81.1, 73.1, 68.5, 66.9,
63.1, 27.5, 27.2, 26.7, 26.4, 25.5; MALDI-HRMS m/z calcd
431.20402 [M + Na]+, m/z found 431.20260. Anal. Calcd for
C
22H32O7‚0.2H2O: C, 63.84; H, 7.92. Found: C, 63.98; H, 7.90.
3-O-Ben zyl-1,2:5,6-di-O-isopr opyliden e-3-C-(3-m esyloxy)-
p r op yl-R-D-a llofu r a n ose (29). Compound 28 (3.77 g, 9.2
mmol) was coevaporated with anhydrous pyridine (20 mL) and
dissolved in ice-cold anhydrous pyridine (40 mL), and mesyl
chloride (1.0 mL, 12.9 mmol) was added dropwise. The solution
6320 J . Org. Chem., Vol. 69, No. 19, 2004

3′-C-Branched LNA-Type Nucleosides
dioxane (40 mL), and formaldehyde (2.0 mL, 37% aqueous
solution (w/w), 50.4 mmol) and 1 M aq NaOH (40 mL, 40 mmol)
were added. After the mixture was stirred for 20 h at rt, a
17.45 N aqueous solution of formic acid was added to neutral-
ize the mixture. H2O (50 mL) was added, and the resulting
mixture was extracted with EtOAc (3 × 100 mL). The
combined organic phase was evaporated to dryness, and the
residue was coevaporated with toluene (2 × 20 mL) and
purified by dry column vacuum chromatography (EtOAc/
petroleum ether, 2:3-3:2 (v/v)) to give furanose 32 (1.96 g,
89%) as a white foam: Rf 0.35 (EtOAc/petroleum ether, 7:3
CH2Ph), 3.51-3.37 (m, 2H, C3′-CH2CH2CH2OH), 3.15, 2.94 (2
× s, 2 × 3H, 2 × CH3), 2.33-1.67 (m, 4H, C3′-CH2CH2CH2-
OH), 2.14, 1.94 (2 × s, 2 × 3H, 2 × CH3); 13C NMR (CDCl3)
169.6, 163.6, 150.9, 137.4, 135.8, 128.9, 128.3, 127.4, 112.3,
85.9 (C-1′), 85.4, 83.9, 76.9 (C-2′), 68.1, 67.6, 67.1, 51.2, 37.8,
37.5, 26.3, 23.3, 20.9, 12.4; MALDI-HRMS m/z calcd 682.14593
[M + Na]+, m/z found 682.14400; IR 2101 cm-1 (-N3). Anal.
Calcd for C25H33N5O12S2: C, 45.52; H, 5.04; N, 10.62. Found:
C, 45.70; H, 5.10; N, 10.49.
(1S ,3R ,4R ,7S )-7-(3-Azid op r op yl)-7-b e n zyloxy-1-h y-
d r oxym e t h yl-3-(t h ym in -1-yl)-2,5-d ioxa b icyclo[2.2.1]-
h ep ta n e (35). Nucleoside 34 (825 mg, 1.3 mmol) was dissolved
in a mixture of 1,4-dioxane (6 mL), H2O (6 mL), and aq 6 M
KOH (12 mL, 72 mmol). The reaction mixture was stirred
under reflux for 41 h followed by the addition of brine (100
mL). Extraction was performed using EtOAc (3 × 80 mL), the
combined organic phase was evaporated to dryness under
reduced pressure, and the residue was coevaporated with
toluene (20 mL) and purified by dry column vacuum chroma-
tography (EtOAc/petroleum ether, 3:2-7:3 (v/v)) to give the
bicyclic nucleoside 35 (350 mg, 63%) as a white foam: Rf 0.69
1
(v/v)); H NMR (CDCl3) 7.39-7.26 (m, 5H, Bn), 5.79 (d, J )
4.3 Hz, 1H, 1-H), 4.71 (d, J ) 10.6 Hz, 1H, CH2Ph), 4.57 (d, J
) 10.6 Hz, 1H, CH2Ph), 4.50 (d, J ) 4.3 Hz, 1H, 2-H), 4.13 (d,
J ) 12.0 Hz, 1H, 5-H), 4.06 (d, J ) 12.0 Hz, 1H, 5-H), 3.85-
3.80 (m, 2H, 5-H′), 3.45-3.31 (m, 2H, C3-CH2CH2CH2OH), 2.56
(br s, 1H, OH), 2.47 (br s, 1H, OH), 2.08-1.74 (m, 4H, C3-
CH2CH2CH2OH), 1.66, 1.34 (2 × s, 2 × 3H, 2 × CH3); 13C NMR
(CDCl3) 138.0, 128.6, 128.0, 127.6, 113.0, 103.9, 87.7, 86.3, 84.4,
67.0, 63.7, 63.5, 52.0, 29.8, 26.3, 25.8, 23.0; MALDI-HRMS m/z
calcd 416.17920 [M + Na]+, m/z found 416.17900; IR 2098 cm-1
(-N3). Anal. Calcd for C19H27N3O6: C, 58.00; H, 6.92; N, 10.68.
Found: C, 57.76; H, 6.93; N, 10.20.
1
(methanol/dichloromethane, 1:1 (v/v)); H NMR (CDCl3) 9.28
(br s, 1H, NH), 7.45 (s, 1H, 6-H), 7.30-7.19 (m, 5H, Bn), 5.43
(s, 1H, 1-H′), 5.05 (s, 1H, 2-H′), 4.62-3.89 (m, 6H, 5-H′, 5-H′′,
CH2PH), 3.34-3.02 (m, 2H, C3′-CH2CH2CH2OH), 2.20 (br s,
1H, OH), 1.90 (s, 3H, CH3), 1.71-1.48 (m, 4H, C3′-CH2CH2-
CH2OH); 13C NMR (CDCl3) 164.1, 150.3, 137.5 (C-6), 134.5,
128.6, 128.6, 128.0, 127.7, 127.6, 110.9, 91.0, 89.2 (C-1′), 83.8,
79.6 (C-2′), 74.5, 68.1, 59.0, 51.7, 24.5, 24.4, 12.7; MALDI-
HRMS m/z calcd 466.16970 [M + Na]+, m/z found 466.1700;
IR 2099 cm-1 (-N3).
3-C-(3-Azidopr opyl)-1,2-di-O-acetyl-3-O-ben zyl-5-O-m es-
yl-4-C-m esyloxym eth yl-D-er yth r o-pen tofu r an ose (33). Com-
pound 32 (1.95 g, 5.0 mmol) was coevaporated with anhydrous
pyridine (20 mL) and dissolved in ice-cold anhydrous pyridine
(30 mL), and MsCl (0.96 mL, 12.4 mmol) was added dropwise
under stirring. The solution was stirred for 4.5 h at rt followed
by addition of satd aq NaHCO3 (50 mL) and brine (100 mL).
The resulting mixture was extracted with CH2Cl2 (3 × 50 mL),
and the combined organic phase was evaporated to dryness
under reduced pressure. The residue was coevaporated with
toluene (2 × 20 mL) and then dissolved in a mixture of TFA
and H2O (8:2 (v/v), 25 mL). After stirring for 3.5 h at rt, the
mixture was evaporated to dryness under reduced pressure,
and the residue was coevaporated successively with toluene
(2 × 30 mL) and anhydrous pyridine (20 mL). The resulting
residue was dissolved in anhydrous pyridine (30 mL) at rt
under stirring, and Ac2O (4.8 mL, 50.8 mmol) and DMAP (79
mg, 0.6 mmol) were added. The reaction mixture was stirred
for 15 h at rt followed by addition of ice (30 mL) and satd aq
NaHCO3 (50 mL). Extraction was performed using CH2Cl2 (3
× 100 mL), and the combined organic phase was first evapo-
rated to dryness under reduced pressure and then coevapo-
rated with toluene (2 × 20 mL). The residue was purified by
dry column vacuum chromatography (EtOAc/petroleum ether,
2:3 (v/v)) to give anomeric mixture 33 (1.98 g, 67%) as a white
foam: Rf 0.55 (EtOAc/petroleum ether, 7:3 (v/v)); 13C NMR
(CDCl3) 169.3, 169.1, 137.1, 128.8, 128.1, 127.3, 127.0, 97.6,
87.3, 84.5, 79.1, 68.4, 66.7, 65.6, 51.3, 38.0, 37.7, 37.6, 27.2,
23.2, 21.1, 20.8; MALDI-HRMS m/z calcd 616.12413 [M +
Na]+, m/z found 616.12170; IR 2099 cm-1 (-N3).
1-(2-O-Acetyl-3-C-(3-a zid op r op yl)-3-O-ben zyl-5-O-m es-
yl-4-C-m esyloxym eth yl-â-D-er yth r o-p en tofu r a n osyl)th y-
m in e (34). To a stirred solution of the anomeric mixture 33
(1.01 mg, 1.7 mmol) in anhydrous acetonitrile (20 mL) were
added thymine (644 mg, 5.1 mmol) and N,O-bistrimethylsilyl
acetamide (1.68 mL, 6.8 mmol). The mixture was stirred with
heating under reflux until the solution became clear (45 min)
and was then cooled to 0 °C followed by addition of trimeth-
ylsilyl triflate (0.92 mL, 5.1 mmol). The mixture was heated
under reflux for 43 h whereupon satd aq NaHCO3 (50 mL)
was added. Extraction was performed using CH2Cl2 (3 × 50
mL), the combined organic phase was evaporated to dryness
under reduced pressure, and the residue was coevaporated
with toluene (25 mL) and purified by dry column vacuum
chromatography (EtOAc/petroleum ether, 2:3-3:2 (v/v)) yield-
ing nucleoside 34 (841 mg, 75%) as a white foam: Rf 0.25
(EtOAc/petroleum ether, 7:3 (v/v); 1H NMR (CDCl3) 9.11 (s,
1H, NH), 7.43-7.26 (m, 6H, Bn), 6.20 (d, J ) 8.0 Hz, 1H, 1-H′),
5.61 (d, J ) 8.0 Hz, 1H, 2-H′), 5.01-4.41 (m, 6H, 5-H′, 5-H′′,
(1S,3R,4R,7S)-7-(3-(F lu or en -9-ylm eth oxyca r bon yl)a m i-
n op r op yl)-7-h yd r oxy-1-h yd r oxym et h yl-3-(t h ym in -1-yl)-
2,5-d ioxa bicyclo[2.2.1]h ep ta n e (36). Nucleoside 35 (293 mg,
0.7 mmol) was dissolved in methanol at rt under stirring (15
mL), and Pd(OH)2/C (105 mg, 0.1 mmol) was added. The
reaction flask was evacuated and filled with hydrogen gas
several times. The reaction mixture was stirred vigorously
under an atmosphere of hydrogen gas for 42 h at rt and was
then filtered through filter paper that was washed with
methanol (3 × 25 mL). The combined filtrate was evaporated
to dryness under reduced pressure, and the obtained residue
was coevaporated with anhydrous pyridine (5 mL) and then
dissolved in anhydrous pyridine (10 mL). Fluoren-9-ylmethoxy-
carbonyl chloride (175 mg, 0.7 mmol) was added, and the
reaction mixture was stirred for 19 h at rt followed by addition
of satd aq NaHCO3 (70 mL). Extraction was performed using
EtOAc (100 mL), the organic phase was evaporated to dryness
under reduced pressure, and the residue was coevaporated
with toluene (25 mL) and purified by dry column vacuum
chromatography (EtOAc/petroleum ether, 3:2 (v/v); methanol/
EtOAc, 3:50 (v/v)) affording nucleoside 36 (158 mg, 51%) as a
white powder: Rf 0.10 (EtOAc); 1H NMR (DMSO-d6) 7.76-
7.18 (m, 7H, Bn), 6.41-6.14 (m, 2H, Bn), 5.08 (s, 1H, 1-H′),
4.20 (s, 1H, 2-H′), 3.94-3.56 (m, 8H, 5-H′, 5-H′′, C3′-CH2-
CH2CH2OH and CHCH2), 2.60, 2.37 (2 × br s, 2 × 1H, 2 ×
OH), 1.68 (s, 3H, CH3), 1.44-0.68 (m, 4H, C3′-CH2CH2CH2-
OH), 1.04 (t, J ) 7.1 Hz, 1H, CH); 13C NMR (CDCl3) 162.8,
155.0, 149.3, 143.1, 142.9, 139.7, 133.4, 126.6, 126.1, 124.2,
119.1, 107.5, 89.6, 86.7, 78.8 (C-2′), 77.8, 72.0, 64.4, 58.8, 55.9,
45.7, 39.8, 26.4, 24.1, 13.1, 11.3; MALDI-HRMS m/z calcd
572.20033 [M + Na]+, m/z found 572.19960.
(1R,3R,4R,7S)-1-(4,4′-Dim eth oxytr ityl)oxym eth yl-7-(3-
(flu or en -9-yl-m eth oxyca r bon yl)a m in opr op yl)-7-h yd r oxy-
3-(th ym in -1-yl)-2,5-d ioxa bicyclo[2.2.1]h ep ta n e (37). Nu-
cleoside 36 (158 mg, 0.3 mmol) was coevaporated with
anhydrous pyridine (5 mL) and then dissolved in anhydrous
pyridine (10 mL). DMTCl (153 mg, 0.5 mmol) was added, and
the reaction mixture was stirred for 2 h at rt. Methanol (1.5
mL) was added, and the resulting mixture was extracted with
EtOAc (30 mL). The organic phase was washed with satd aq
NaHCO3 (2 × 20 mL), and the combined aqueous phase was
J . Org. Chem, Vol. 69, No. 19, 2004 6321

Meldgaard et al.
extracted with CH2Cl2 (20 mL). The combined organic phase
was evaporated to dryness under reduced pressure, and the
residue was coevaporated with toluene (15 mL) and purified
by dry column vacuum chromatography (EtOAc/petroleum
ether, 1:1-3:2 (v/v)) to give nucleoside 37 (166 mg, 68%,
rotamers observed in NMR) as a white powder: Rf 0.69
(EtOAc); 1H NMR (CDCl3) 9.33, 9.24 (2 × br s, 1H, NH), 7.71-
6.82 (m, 22H, Harom), 5.42, 5.34 (2 × s, 1H, 1-H′), 4.69 (s, 1H,
2-H′), 4.36-3.92 (m, 6H, 5-H′, 5-H′′, C3′-CH2CH2CH2OH), 3.79,
3.76 (2 × s, 2 × 3H, 2 × CH3), 3.51 (d, 1H, J ) 10.8 Hz, CH2-
CH), 3.35 (d, 1H, J ) 10.8 Hz, CH2CH), 3.25 (br s, 1H, OH),
1.92, 1.89 (2 × s, 3H, CH3), 1.81-1.11 (m, 5H, C3′-CH2CH2-
CH2OH, CH); 13C NMR (CDCl3) 163.8, 163.8, 158.8, 158.8,
158.8, 156.7, 150.3, 147.5, 144.6, 144.2, 144.1, 144.0, 141.4,
139.6, 135.6, 135.4, 134.2, 134.2, 130.2, 130.1, 129.3, 128.2,
128.1, 128.0, 127.9, 127.8, 127.2, 127.2, 125.2, 120.0, 113.4,
113.3, 110.7, 110.6, 89.9 (C-1′), 89.9, 88.1, 88.0, 86.8, 81.6, 80.4,
80.3 (C-2′), 80.0, 73.8, 73.3, 66.9, 59.5, 58.6, 55.4, 55.4, 47.3,
41.1, 29.8, 26.6, 26.4, 25.8, 12.9, 12.8; MALDI-HRMS m/z calcd
874.33101 [M + Na]+, m/z found 874.33480.
(1R,3R,4R,7S)-7-(2-Cyan oeth oxy)(N,N-(diisopr opylam i-
n o)p h osp h in oxy)-1-(4,4′-d im eth oxytr ityl)oxym eth yl-7-(3-
(flu or en -9-yl-m eth oxyca r bon yl)a m in o)p r op yl-3-(th ym in -
1-yl)-2,5-d ioxa bicyclo[2.2.1]h ep ta n e (38). Nucleoside 37
(140 mg, 0.2 mmol) was coevaporated with anhydrous aceto-
nitrile (2 mL) and then dissolved in anhydrous CH2Cl2 (2 mL).
N,N-(Diisopropyl)ethylamine (0.12 mL, 0.8 mmol) and 2-cya-
noethyl N,N-(diisopropyl)phosphoramidochloridite (0.06 mL,
0.3 mmol) were added, and the resulting mixture was stirred
for 24 h at rt. CH2Cl2 (20 mL) was added, and the resulting
mixture was washed with satd aq NaHCO3 (25 mL). The
organic phase was evaporated to dryness under reduced
pressure, and the resulting mixture was coevaporated with
acetonitrile (5 mL) and purified by dry column vacuum
chromatography using EtOAc/petroleum ether (9:11-1:1
(v/v)) to furnish phosphoramidite 38 (151 mg, 87%) as a
yellowish oil: Rf 0.52 (methanol/dichloromethane, 1:9 (v/v));
31P NMR (CDCl3) 145.8, 145.5; MALDI-HRMS m/z calcd
1074.43886 [M + Na]+, m/z 1074.43650.
Syn t h esis, Dep r ot ect ion , a n d P u r ifica t ion of LNA
ONs. LNA oligomers LNA-1, LNA-2, and LNA-3 were pre-
pared by the phosphoramidite approach as described earlier
on an automated DNA synthesizer on CPG solid support
(LNA-1 and LNA-2) or polystyrene solid support (LNA-3; and
also LNA-4 by on-column conjugation). The stepwise coupling
efficiencies for LNA phosphoramidites 27 and 38 were ∼85%
(15 min coupling time using pyridinium chloride as catalyst
for 27; 30 min coupling time using 1H-tetrazole as catalyst
for 38) and for unmodified deoxynucleoside phosphoramidites
(with 2 min standard coupling time) >99% using 1H-tetrazole
as activator. After standard deprotection and cleavage from
the solid support by 32% aqueous ammonia, LNA-1 and
LNA-2 were purified by DMT-on reversed-phase chromatog-
raphy on disposable purification cartridges, which included
detritylation. LNA-3 was purified by HPLC using an X-Terra
MS C18 column (7.8 × 300 mm) and 254 nm UV detection;
buffer A: MeCN:0.1 M NH4HCO3 (5:95 v/v); buffer B: MeCN:
0.1 M NH4HCO3 (75:25 v/v). Gradients: 5 min 100% A, linear
gradient to 70% B in 30 min, linear gradient to 100% B in 2
min (flow 1.5 mL/min), 8 min with 100% B (flow 1.5 mL/min),
linear gradient to 100% A in 1 min and 14 min with 100%.
The flow rate was 1.0 mL/min. The composition of the
synthesized ONs was confirmed by MALDI-MS analysis and
the purity by capillary gel electrophoresis/analytical RP-HPLC.
MALDI-MS: LNA-1 [M - H]- 3008.8, calcd 3012.1; LNA-2
[M - H]- 3009.9, calcd 3008.5; LNA-3 [M - H]- 2837.9, calcd
2838.7. ON LNA-3 (0.3 µmol), bound to a polystyrene solid
support following incorporation of the last nucleotide, was
suspended in a solution of 20% piperidine in anhydrous DMF
(1.0 mL). After the reaction mixture was vortexed for 20 min
at rt, the supernatant was removed and the support was
washed with anhydrous CH3CN (4 × 1.0 mL). To the resulting
dry support were then added HBTU (5.90 mg, 15.6 µmol),
Fmoc-glycine (4.68 mg, 18.8 µmol), DIPEA (0.01 mL), and
anhydrous DMF (1.0 mL). After the resulting mixture was
vortexed for 1 h, the supernatant was removed and the solid
support washed with first DMF (2 × 1.0 mL) and then MeOH
(2 × 1.0 mL). The solid support was subsequently suspended
in satd aq ammonia (1.0 mL) and left for 19 h at 55 °C
whereupon the liquid phase was removed and evaporated to
dryness. The resulting ON was purified by reversed-phase
HPLC (as described above) and evaporated to dryness. The
residue was treated with 80% aqueous AcOH (100 µL, w/w)
for 20 min, H2O (100 µL) and 3 M aqueous CH3COONa (50
µL) were added, the solution was transferred to an Eppendorf
tube, and absolute EtOH (500 µL) was added. The resulting
mixture was left at -20 °C for 22 h and centrifuged at 8000
rpm for 20 min. The supernatant was removed and the
precipitated LNA-4 isolated. MALDI-MS: LNA-4 [M - H]-
2894.5, calcd 2896.0.
Th er m a l Den a tu r a tion Stu d ies. The thermal denatur-
ation studies were performed in a low salt buffer (10 mM
sodium phosphate and 0.1 mM EDTA, pH 7.0) or in a medium
salt buffer (10 mM sodium phosphate, 100 mM sodium chloride
and 0.1 mM EDTA, pH 7.0). Concentrations of 1.0 µM of the
two complementary ON strands were used assuming identical
extinction coefficients for modified and unmodified ONs. The
absorbance was monitored at 260 nm while the temperature
was raised at a rate of 1 °C/min. The melting temperatures
(Tm values) of the duplexes were determined as the maximum
of the first derivatives of the melting curves obtained.
Ack n ow led gm en t. The Danish Natural Research
Foundation is gratefully acknowledged for financial
support. Ms. Britta M. Dahl, Department of Chemistry,
University of Copenhagen, is thanked for oligonucleotide
synthesis.
Su p p or tin g In for m a tion Ava ila ble: General methods.
Copies of 13C NMR spectra of compounds 26, 29, 32, 33, and
35-37. This material is available free of charge via the
Internet at http://pubs.acs.org.
J O049159A
6322 J . Org. Chem., Vol. 69, No. 19, 2004