Synthesis of abasic locked nucleic acid and two seco-LNA derivatives and evaluation of their hybridization properties compared with their more flexible DNA counterparts

Article abstract of DOI:10.1021/jo000275x

To investigate the structural basis of the unique hybridization properties of LNA (locked nucleic acid)¹ three novel LNA derivatives with modified carbohydrate parts were synthesized and evaluated with respect to duplex stabilities. The abasic LNA monomer² (X(L), Figure 1) with the rigid carbohydrate moiety of LNA but no nucleobase attached showed no enhanced duplex stabilities compared to its more flexible abasic DNA counterpart (X, Figure 1). These results suggest that the exceptional hybridization properties of LNA primarily originate from improved intrastrand nucleobase stacking and not backbone preorganization. Two monocyclic seco-LNA derivatives, obtained by cleavage of the C1' - O4' bond of an LNA monomer or complete removal of the O4' - furanose oxygen atom (Z(L) and dZ(L), respectively, Figure 1), were compared to their acyclic DNA counterpart³ (Z, Figure 1). Even though they are more constrained than Z, the seco-LNA derivatives Z(L) and dZ(L) destabilize duplex formation even more than the flexible seco-DNA monomer Z.

Full text of DOI:10.1021/jo000275x

J . Org. Chem. 2000, 65, 5167-5176
5167
Syn th esis of Aba sic Lock ed Nu cleic Acid a n d Tw o seco-LNA
Der iva tives a n d Eva lu a tion of Th eir Hybr id iza tion P r op er ties
Com p a r ed w ith Th eir Mor e F lexible DNA Cou n ter p a r ts
Lisbet Kværnø,† Ravindra Kumar,† Britta M. Dahl,† Carl Erik Olsen,‡ and J esper Wengel*,†,§
Center for Synthetic Bioorganic Chemistry, Department of Chemistry, University of Copenhagen,
Universitetsparken 5, DK-2100 Copenhagen, Denmark, and Department of Chemistry,
The Royal Veterinary and Agricultural University, DK-1871 Frederiksberg, Denmark
jwe@chem.sdu.dk
Received February 26, 2000
To investigate the structural basis of the unique hybridization properties of LNA (locked nucleic
acid)1 three novel LNA derivatives with modified carbohydrate parts were synthesized and evaluated
with respect to duplex stabilities. The abasic LNA monomer2 (XL, Figure 1) with the rigid
carbohydrate moiety of LNA but no nucleobase attached showed no enhanced duplex stabilities
compared to its more flexible abasic DNA counterpart (X, Figure 1). These results suggest that the
exceptional hybridization properties of LNA primarily originate from improved intrastrand
nucleobase stacking and not backbone preorganization. Two monocyclic seco-LNA derivatives,
obtained by cleavage of the C1′-O4′ bond of an LNA monomer or complete removal of the O4′-
furanose oxygen atom (ZL and d ZL, respectively, Figure 1), were compared to their acyclic DNA
counterpart3 (Z, Figure 1). Even though they are more constrained than Z, the seco-LNA derivatives
ZL and d ZL destabilize duplex formation even more than the flexible seco-DNA monomer Z.
In tr od u ction
stabilities toward complementary DNA and RNA (∆Tm/
mod ) 3-9 °C), stability toward 3′-exonucleolytic deg-
radation, efficient automated oligomerization, and good
aqueous solubility.15-20
In the search for new high-affinity antisense oligo-
ribonucleotide (ON) analogues, much of the recent re-
search has been focused on the synthesis of conforma-
tionally restricted derivatives.4,5 Several ONs contain-
ing bi- and tricyclic carbohydrate moieties have been
synthesized, their hybridization properties have been
studied,6-14 and a number of these rigid analogues have
displayed enhanced duplex stabilities compared with the
corresponding unmodified ONs. The analogue termed
LNA (locked nucleic acid, e.g., TL, Figure 1)1,15-20 display
most interesting properties: e.g., unprecedented thermal
The conformations of the furanose rings are decisive
for the type of duplex formed between two complemen-
tary nucleic acid strands. The pentofuranose moieties in
RNA normally exist in an N-type (north type) conforma-
tion leading to A-type duplexes, whereas an S-type (south
type) conformation as generally seen for DNA leads to
B-type duplexes. Studies on the interdependency between
the dihedral phosphate backbone angle C4′-C3′-O3′-P
and the furanose conformation likewise show that the
furanose conformation leads to preorganization in the
sugar-phosphate backbone.21-23 This preorganization is
also supported by recent NMR studies showing that
unmodified nucleotides flanked by two LNA monomers
(the rigid LNA monomer is restricted to an N-type (C3′-
endo/3E) conformation)16,24 mainly exist in an N-type
conformation, whereas all monomeric units of the un-
† University of Copenhagen.
‡ The Royal Veterinary and Agricultural University.
§ Present address: Department of Chemistry, University of South-
ern Denmark, DK-5230 Odense M, Denmark.
(1) We have defined LNA as an oligonucleotide containing one or
more 2′-O,4′-C-methylene-linked bicyclic ribonucleoside monomers.
(2) Kværnø, L.; Wengel, J . Chem. Commun. 1999, 657. This is a
preliminary report including the synthesis of the abasic LNA phos-
phoramidite building block and binding studies with oligonucleotides
containing the abasic LNA monomer XL.
(3) Vandendriessche, F.; Augustyns, K.; Van Aerschot, A.; Busson,
R.; Hoogmartens, J .; Herdewijn, P. Tetrahedron 1993, 49, 7223.
(4) Herdewijn, P. Liebigs Ann. Chem. 1996, 1337.
(5) Kool, E. T. Chem. Rev. 1997, 97, 1473.
(6) Tarkoy, M.; Bolli, M.; Leumann, C. Helv. Chim. Acta 1994, 77,
716.
(7) Altmann, K. H.; Kesselring, R.; Francotte, E.; Rihs, G. Tetrahe-
dron Lett. 1994, 35, 2331.
(8) Marquez, V. E.; Siddiqui, M. A.; Ezzitouni, A.; Russ, P.; Wang,
J . Y.; Wagner, R. W.; Matteucci, M. D. J . Med. Chem. 1996, 39, 3739.
(9) Steffens, R.; Leumann, C. J . J . Am. Chem. Soc. 1997, 119, 11548.
(10) Nielsen, P.; Pfundheller, H. M.; Olsen, C. E.; Wengel, J . J .
Chem. Soc., Perkin Trans. 1 1997, 3423.
(15) Singh, S. K.; Nielsen, P.; Koshkin, A. A.; Wengel, J . Chem.
Commun. 1998, 455.
(16) Koshkin, A. A.; Singh, S. K.; Nielsen, P.; Rajwanshi, V. K.;
Kumar, R.; Meldgaard, M.; Olsen, C. E.; Wengel, J . Tetrahedron 1998,
54, 3607.
(17) Obika, S.; Nanbu, D.; Hari, Y.; Andoh, J .; Morio, K.; Doi, T.;
Imanishi, T. Tetrahedron Lett. 1998, 39, 5401.
(18) Wengel, J .; Koshkin, A.; Singh, S. K.; Nielsen, P.; Meldgaard,
M.; Rajwanshi, V. K.; Kumar, R.; Skouv, J .; Nielsen, C. B.; J acobsen,
J . P.; J acobsen, N.; Olsen, C. E. Nucleosides Nucleotides 1999, 18, 1365.
(19) Koshkin, A. A.; Nielsen, P.; Meldgaard, M.; Rajwanshi, V. K.;
Singh, S. K.; Wengel, J . J . Am. Chem. Soc. 1998, 120, 13252.
(20) Wengel, J . Acc. Chem. Res. 1999, 32, 301.
(11) Christensen, N. K.; Petersen, M.; Nielsen, P.; J acobsen, J . P.;
Olsen, C. E.; Wengel, J . J . Am. Chem. Soc. 1998, 120, 5458.
(12) Raunkjær, M.; Wengel, J . J . Chem. Soc., Perkin Trans. 1 1999,
2543.
(13) Wang, G. Y.; Gunic, E.; Girardet, J . L.; Stoisavljevic, V. Bioorg.
Med. Chem. Lett. 1999, 9, 1147.
(21) Plavec, J .; Thibaudeau, C.; Viswanadham, G.; Sund, C.; Chat-
topadhyaya, J . Chem. Commun. 1994, 781.
(22) Plavec, J .; Thibaudeau, C.; Viswanadham, G.; Sund, C.; Sand-
strom, A.; Chattopadhyaya, J . Tetrahedron 1995, 51, 11775.
(23) Thibaudeau, C.; Chattopadhyaya, J . Nucleosides Nucleotides
1998, 17, 1589.
(14) Wang, G. Y.; Girardet, J . L.; Gunic, E. Tetrahedron 1999, 55,
7707.
(24) Obika, S.; Nanbu, D.; Hari, Y.; Morio, K.; In, Y.; Ishida, T.;
Imanishi, T. Tetrahedron Lett. 1997, 38, 8735.
10.1021/jo000275x CCC: $19.00 © 2000 American Chemical Society
Published on Web 07/26/2000

5168 J . Org. Chem., Vol. 65, No. 17, 2000
Kværnø et al.
F igu r e 1. Structures of DNA and LNA monomers. TL ) thymine LNA monomer; XL ) abasic LNA monomer; ZL ) monocyclic
thymine LNA monomer; d ZL ) deoxygenated monocyclic thymine LNA monomer; T ) thymine DNA monomer; X ) abasic DNA
monomer; Z ) acyclic thymine DNA monomer.
Sch em e 1a
modified reference oligodeoxynucleotides showed the
expected S-type conformation.25 As further investigation
of the importance of backbone preorganization on the
stability of nucleic acid duplexes, we have synthesized
and evaluated three novel LNA derivatives and compared
their hybridization properties with those of their less
conformationally restricted DNA counterparts. The aba-
sic LNA monomer2 XL (Figure 1), which is anticipated to
display the same restricted furanose and backbone
conformations as the parent LNA monomers (e.g., as the
monomer TL) with the exclusion of base-base stacking
interactions, has been compared to the abasic DNA
monomer X. Cleavage of the LNA C1′-O4′ bond or
complete removal of the O4′ furanose oxygen atom has
allowed the evaluation of the monocyclic seco-LNA mono-
mers ZL and d ZL, respectively. The synthesis of these was
stimulated by the possibility of inducing some conforma-
tional restriction in the backbone, thereby potentially
enhancing duplex stabilities compared to the totally
flexible acyclic DNA monomer Z.3 The rationale behind
the design of LNA and the analogues introduced herein
is the anticipation that more flexibility in a nucleic acid
single strand results in a larger loss of entropy during
duplex formation or less efficient nucleobase stacking
which are believed to be the major reasons for the
decreased duplex stabilities resulting from incorporation
of acyclic nucleotides.26,27 Furthermore, it is noteworthy
that earlier studies have indicated that it is rotational
restrictions in the sugar-phosphate backbone rather
than nucleobase stacking that induces the helical-type
structure reported for several single-stranded ONs.28
a
Reagents and conditions: (i) MsCl, pyridine; (ii) 15% HCl,
MeOH/H2O (7:1), 85% (2 steps); (iii) NaH, DMF; (iv) 80% AcOH,
(v) NaBH4, MeOH, 86% (3 steps); (vi) TsCl, DMAP, CH2Cl2, 83%;
(vii) NaH, DMF, 82%; (viii) H2, 10% Pd/C, 80%; (ix) DMTCl,
pyridine, 73%; (x) NC(CH2)2OP(Cl)N(i-Pr)2, EtN(i-Pr)2, CH2Cl2,
62%. DMT ) 4,4′-dimethoxytrityl.
Resu lts a n d Discu ssion
As previously reported, the 4-C-hydroxymethyl fura-
nose 129 (Scheme 1) was mesylated and transacetalized
to give the anomeric mixture of methyl furanosides 3 (via
2) which, by subsequent treatment with sodium hydride,
gave the cyclized anomeric mixture of methyl furanosides
4, and this, followed by hydrolysis (80% AcOH), afforded
the monocyclic aldehyde 5.30 Reduction of this aldehyde
(25) Bondesgaard, K.; Petersen, M.; Singh, S. K.; Rajwanshi, V. K.;
Kumar, R.; Wengel, J .; J acobsen, J . P. Chem. Eur. J ., in press.
(26) Nielsen, P.; Kirpekar, F.; Wengel, J . Nucleic Acids Res. 1994,
22, 703.
(27) Nielsen, P.; Dreiøe, L. H.; Wengel, J . Biorg. Med. Chem. 1995,
3, 19.
(28) Achter, E. K.; Felsenfield, G. Biopolymers 1971, 10, 1625.
(29) Waga, T.; Nishizaki, T.; Miyakawa, I.; Ohrui, H.; Meguro, H.
Biosci. Biotechnol. Biochem. 1993, 57, 1433.

Synthesis of Abasic LNA and seco-LNA Derivatives
J . Org. Chem., Vol. 65, No. 17, 2000 5169
Sch em e 2a
Sch em e 3a
a
Reagents and conditions: (i) Ac2O, DMAP, pyridine, 84%; (ii)
thymine, NaH, DMF, 52%; (iii) H2, 20% Pd(OH)2/C, EtOH, 87%;
(iv) NaOMe, MeOH, 82%; (v) DMTCl, pyridine, 87%; (vi) TBDM-
SCl, imidazole, CH2Cl2, 69%; (vii) Ac2O, DMAP, pyridine, 61%;
(viii) TBAF, THF; (ix) NC(CH2)2OP(Cl)N(i-Pr)2, EtN(i-Pr)2, CH2Cl2,
67% (2 steps). DMT ) 4,4′-dimethoxytrityl.
a
Reagents and conditions: (i) H2, 20% Pd(OH)2/C, EtOH, 86%;
(ii) TIPDSCl2, pyridine, 71%; (iii) methoxalyl chloride, DMAP,
CH2Cl2, 74%; (iv) Bu3SnH, AIBN, toluene, 84% (24:24a ) 5:1); (v)
thymine, NaH, DMF, 61%; (vi) TBAF, THF; (vii) DMTCl, 63% (2
steps); (viii) NC(CH2)2OP(Cl)N(i-Pr)2, EtN(i-Pr)2, CH2Cl2, 68%. [Si]
) TIPDS ) 1,1,3,3-tetraisopropyldisiloxane-1,3-diyl; DMT ) 4,4′-
dimethoxytrityl.
with NaBH4 proceeded smoothly in 86% yield (from 3)
to give the diol 6 which afterward was tosylated selec-
tively at the primary hydroxy group, affording the key
intermediate 7 in 83% yield.2 In the synthesis of the
abasic LNA monomer XL, intermediate 7 was cyclized
with NaH in DMF to give the bicyclic derivative 8 in 82%
yield, which was debenzylated by hydrogenolysis to afford
diol 9 in 80% yield. To prepare for automated oligonucleo-
tide synthesis using the phosphoramidite approach,31
derivative 9 was selectively DMT (4,4′-dimethoxytrityl)
-protected at the primary hydroxy group to give 10 in
73% yield by reaction with DMTCl in pyridine, where-
upon phosphitylation yielded the phosphoramidite build-
ing block 112 in 62% yield (Scheme 1).
As a key step during the synthesis of the monocyclic
seco-LNA thymine monomer ZL (Scheme 2), introduction
of the thymine moiety by nucleophilic substitution was
envisioned. To prevent cyclization into derivative 8,
protection of the tertiary hydroxy group of intermediate
7 was needed. Initial attempts of tert-butyldimethyl
silylation or benzoylation under various conditions failed,
probably because of sterical hindrance. On the contrary,
TMS-protection using TMSCl and imidazole in dichloro-
methane proceeded smoothly in 89% yield, but the O-Si
bond was not stable enough for the concomitant substitu-
tion step (NaH and thymine in DMF at 120 °C), and the
major product was in this case derivative 8. Alternatively,
after acetylation of 7 to give the fully protected derivative
12 in 84% yield using acetic anhydride and DMAP in
pyridine, introduction of the nucleobase was successfully
accomplished by reaction with NaH and thymine in DMF
at 120 °C, affording the thymine derivative 13 in 52%
yield. Unfortunately, debenzylation of 13 (H2, 20%
Pd(OH)2, EtOH) induced quantitative migration of the
acetyl group to the primary position, thereby furnishing
14 in 87% yield. This migration was indicated by changes
1
in the δ H of the neighboring methylene group and the
δ
13C of the quaternary carbon atom. It was furthermore
1
verified from a H NMR spectrum recorded in DMSO-d6
where OH couplings clearly showed the free hydroxy
groups to be secondary and tertiary. The migration was
also supported by the fact that 14 could not be DMT
protected, even with silver triflate as catalyst. To obtain
a convenient phosphoramidite derivative for automated
oligonucleotide synthesis, a number of protection group
transformations were needed. Thus, 14 was deacetylated
to give triol 15 in 82% yield using sodium methoxide in
MeOH followed by selective DMT protection to furnish
16 in 87% yield (DMTCl in pyridine), selective silylation
of the secondary hydroxy group in 69% yield (TBDMSCl
and imidazole in dichloromethane) to give 17, and
acetylation in 61% yield as described above, thereby
affording 18 with three orthogonal protecting groups.
Eventually, desilylation to give 19 (TBAF in THF) and
subsequent phosphitylation yielded the monocyclic seco-
LNA phosphoramidite building block 20 (67% for two
steps, Scheme 2).
In the first approach for synthesis of the O4′-deoxy-
genated (conventional nucleoside numbering used) mono-
cyclic seco-LNA thymine phosphoramidite building block
28 (Scheme 3), direct deoxygenation of the 4-O-methox-
alyl derivative of 7 using Bu3SnH and AIBN in toluene32
(30) Nielsen, P.; Wengel, J . Chem. Commun. 1998, 2645. No
experimental data were included in this communication.
(31) Caruthers, M. H. Acc. Chem. Res. 1991, 24, 278.

5170 J . Org. Chem., Vol. 65, No. 17, 2000
Kværnø et al.
Ta ble 1. Sequ en ces Syn th esized a n d Th er m a l
Den a tu r a tion Stu d ies tow a r d F u lly Com p lem en ta r y DNA
Sequ en cesa
entry
sequence
Y
Tm/oC
∆Tm
1
2
3
4
5
6
7
8
9
5′-d(CAGTGA-Y-ATGCGA)
T
47.6
52.8
27.2
26.7
32.6
33.3
58.6
b
TL
X
+5.2
-20.4
-20.9
-15.0
-14.3
+3.7
XL
ZL
d ZL
F igu r e 2. [Si] ) 1,1,3,3-tetraisopropyldisiloxane-1,3-diyl.
5′-d(CAG-Y-GA-Y-A-Y-GCGA) TL
afforded an inseparable mixture of the two diastereoiso-
mers resulting from reduction of the diastereotopic
tertiary radical. To induce the desired diastereoselectivity
via steric control, the 4-O-methoxalyl derivative 23 was
prepared. Thus, compound 7 was debenzylated (H2, 20%
Pd(OH)2, EtOH) to give triol 21 in 86% yield, which was
then selectively protected with the bidentate reagent 1,3-
dichloro-1,1,3,3-tetraisopropyldisiloxane (TIPDSCl2) in
pyridine, affording the TIPDS-protected derivative 22 in
71% yield. The 4-O-methoxalyl derivative 23 was pre-
pared by reaction of 22 with methoxalyl chloride and
DMAP in dichloromethane. In one experiment, column
chromatographic purification of the methoxalyl interme-
diate (74% yield) before the deoxygenation step (Bu3SnH
and AIBN in toluene) afforded a 5:1 mixture of O4-
deoxygenetated diastereoisomers 24 and 24a (Figure 2)
in 84% yield as a result of diastereoselective reduction
of the intermediary tertiary radical. Alternatively, direct
deoxygenation of the crude 4-O-methoxalyl intermediate
gave the same mixture in only 34% combined yield for
the two steps. When the synthesis of the 4-O-methoxalyl
derivative was repeated, this proved unstable during
column chromatography (∼50% decomposition to the
alcohol 22 using CH2Cl2/MeOH mixtures as eluents). This
could be minimized to ca. 10% decomposition (as judged
from analytical TLC) by the addition of 0.5% pyridine to
the eluent during column chromatography. Even though
this solvent system did not afford totally pure 23, the
deoxygenation step again yielded a 5:1 mixture of the
diastereomers 24 and 24a , but this time in satisfactory
66% combined yield for the two steps. Subsequently,
thymine was introduced to give derivative 25 in 61% yield
by a substitution reaction on 24 (thymine and NaH in
DMF), whereafter first desilylation (TBAF in THF to give
diol 26) and then selective DMT protection afforded 27
in 63% combined yield for two steps. Last, standard
phosphitylation furnished the desired phosphoramidite
derivative 28 in 68% yield (Scheme 3).
ZL
d ZL
b
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
5′-r(CAGUGA-Y-AUGCGA)
U
45.9
44.8
52.8
28.8
28.6
32.5
35.9
43.3
66.0
b
13.5
26.9
34.8
b
b
7.1
T
-1.1
+6.9
TL
X
-17.1
-17.3
-12.3
-8.9
XL
ZL
d ZL
T
5′-r(CAG-Y-GA-Y-A-Y-GCGA)
5′-d(GTGA-Y-ATGC)
-0.9
+6.7
TL
ZL
d ZL
T
-10.4
+7.9
TL
X
XL
ZL
d ZL
U
-19.8
-16.7
10.2
26.8
25.8
37.9
∼3
5′-r(GUGA-Y-AUGC)
T
-1.0
+11.1
-23.3
-17.6
-13.6
TL
XL
ZL
d ZL
T
9.2
13.2
32
20.1
19.3
28
5′-d(T7-Y-T6) toward 5′-d(A14
)
ZL
d ZL
T
-12.0
-13.0
5′-d(T7-Y-T6) toward 5′-r(A14
)
ZL
d ZL
20.5
20.5
-7.5
-7.5
a
Tm values measured as the maximum of the first derivative
of the melting curve (A260 vs temperature) recorded in medium
salt buffer (10 mM sodium phosphate, 100 mM sodium chloride,
0.1 mM EDTA, pH 7.0) using 1.5 µM concentrations of the two
complementary strands (assuming identical extinction coefficients
for T and all thymine-containing monomeric nucleotides and no
extinction for the abasic monomers). ∆Tm ) change in Tm value
calculated per modification. A ) adenosine monomer, C ) cytidine
monomer, G ) guanosine monomer, U ) uridine monomer, T )
thymidine monomer, TL, X, XL, Z, ZL, d ZL; see Figure 1. Oligo-
2′-deoxynucleotide sequences are depicted as d(sequence) and
oligoribonucleotide sequences as r(sequence). b No Tm was detected.
For conclusive structural verification, NOE difference
spectroscopy experiments on the individual compounds
of the two diastereoisomeric pairs 24/24a and 25/25a
were performed (see Figure 2 for numbering used in the
discussion below). Irradiation of H3 and H4 in 24 and
25 proved these two protons to be mutually close (NOEs
of all four irradiations were 4-10%). For 24a , irradiation
of H2 and H4 showed the proximity of these two protons
(NOEs of 2 and 4%, respectively), whereas irradiation of
one of the H5′ methylene protons gave an NOE of 4% on
H3. Irradiation of H4 in 25a likewise showed an NOE
on H2 of 4%. Altogether, these results unambiguously
verify the configuration of 24 and 25 and thus also of
26-28.
1 were synthesized, and their hybridization to fully
complementary ODN sequences 5′-d(TCGCATATCACTG)
and 5′-d(GCATATCAC) was studied. Prolonged coupling
times were used for amidites 11, 20, and 28 on an
automated DNA synthesizer as outlined in details in the
Experimental Section. The stepwise coupling yields for
the modified phosphoramidites were >99% (6 min cou-
pling time) for amidite 11, >98% (15 min coupling time)
for amidite 20, and 95-99% (20 min coupling time) for
amidite 28. The stepwise coupling yields for the unmodi-
fied amidites and the amidites leading to incorporation
of the TL and X monomers were >99%. Surprisingly, the
coupling with an unmodified RNA amidite (2′-O-TBDMS-
protected RNA amidites were used) following an incor-
poration of amidite 28 proceeded in only 92-98% yield
compared with yields of 95-99% following amidite 20 (20
min coupling time). Cleavage from the solid support and
removal of protecting groups was accomplished using
The modified 13- and 9-mer oligodeoxyribonucleotides
(ODNs) and oligoribonucleotides (ONs) depicted in Table
(32) Dolan, S. C.; Macmillan: J . J . Chem. Soc., Chem. Commun.
1985, 1588.

Synthesis of Abasic LNA and seco-LNA Derivatives
J . Org. Chem., Vol. 65, No. 17, 2000 5171
concentrated aqueous ammonia (55 °C for 16 h) except
for sequences containing monomer ZL or RNA monomers
where aq methylamine (40% (w/w); 10 min, 55 °C) was
used (for further details see the Experimental Section).
Capillary gel electrophoresis of all synthesized sequences
showed the purity as >90%, and the compositions were
verified by MALDI-MS analysis. When the standard
conditions (concentrated aq ammonia; 55 °C for 16 h)
were used during deprotection and cleavage from the
solid support for sequences containing monomer ZL, the
acetyl group at the 4′-O position of monomer ZL was
removed which could lead to intramolecular attack of the
resulting free tertiary hydroxy group on the phosphorus
atom and thereby to strand cleavage. This is a plausible
explanation for the generation of minor byproducts of the
predicted masses detected by MALDI-MS under these
conditions. As described above, all sequences containing
ZL were cleaved and deprotected using methylamine
instead which afforded complete deprotection without any
detectable strand cleavage. For each sequence, the melt-
ing temperature (Tm value) and the change in Tm value
per modification compared with the unmodified reference
(∆Tm) were determined33 (Table 1).
the exterior of the T13 strand.3 By the presence of one
additional tetrahydrofuran ring, the less flexible seco-
LNA counterparts ZL and d ZL could induce enhanced
binding of complementary strands if favorable conforma-
tions of the tetrahydrofuran rings are being adopted. In
parent LNA, this ring is locked in an envelope conforma-
tion with the C3 atom (numbering as in Figure 2) out of
the plane created by the remaining four ring atoms. As
judged by the variations in coupling constants between
the H2 and H3 atoms (numbering as in Figure 2) of 15-
18, 25, and 26 (J values of 2.7-8.1 Hz), it is clear that
there is no preference for the apparently ideal LNA-type
conformation of this ring. Thus, the possibility of in-
creased duplex stabilities (compared to the flexible DNA
monomer Z) for the monocyclic monomers ZL and d ZL
seems very much counteracted (see below) by overall
unfavorable conformations leading possibly to detrimen-
tal sterical interactions during duplex formation.35 Ex-
perimentally, very large decreases in melting tempera-
tures (∆Tm ) -9 to -17 °C for d ZL, entries 6, 9, 16, 20,
26, and 32; ∆Tm ) -12 to -17 °C for ZL, entries 5, 8, 15,
19, 25, and 31) were obtained. For the 14-mer oligo-
thymidylate sequence (entries 33-38), similar ∆Tm val-
ues were observed (toward both complementary DNA and
RNA). Summarizing, the tetrahydrofuran ring of mono-
mers ZL and d ZL seems to force these nucleotides into
conformations unfavorable for duplex formation possibly
by restricting the number of attainable conformations.
Incorporation of a single LNA monomer TL profoundly
increases the thermal stability (∆Tm values of 5.2-11.1
°C (entries 2, 12, 22, and 29 in Table 1). In accordance
with previous reports,34 we observed a detrimental effect
on the thermal stability upon incorporation of the abasic
DNA monomer X (entries 3, 13, and 23, ∆Tm values <
-17 °C). The Tm values obtained for the abasic LNA
monomer XL (entries 4, 14, 24, and 30) indicate that no
stabilization results from exchange of the abasic DNA
monomer X with the abasic LNA monomer XL. This is in
strong contrast to the results obtained by incorporation
of one LNA monomer TL instead of a thymidine monomer
and indicates that the direct effect of backbone pre-
organization is only a minor contributor to the increased
duplex stabilities of LNA. It is during these consider-
ations anticipated that the conformations of the fura-
nose-phosphate moieties of XL and TL are identical and
that the conformational effect of a nucleobase is negligible
in an LNA monomer. We believe that the strongly
constrained furanose moieties of XL and TL warrant these
assumptions. These results support the recent NMR
investigations leading to the conclusion that it is mainly
improved nucleobase stacking which confers preorgani-
zation to an LNA and thus the exceptional stability of
duplexes involving LNA.25 It should be noted that the
effect of this favorable nucleobase stacking could be of
both enthalpic as well as entropic origin and that we have
earlier reported the hybridization of a fully modified LNA
to be enthalpically driven and that of a partly modified
LNA to be entropically driven.19
Con clu sion
The results for the abasic monomers X and XL com-
pared with those of T and TL emphasize the importance
of the nucleobase as a mediator of conformational changes
leading to enhanced duplex stabilities. The structural
difference for both pairs examined (TL vs T and XL vs X)
is a methylene bridge between the O2′ and the C4′ atoms
thereby indicating that conformational restrictions of the
pentofuranose-phosphate backbone alone are not suf-
ficient to induce an effect on the thermal stability.
Despite the fact that the monocyclic seco-LNA derivatives
ZL and d ZL are more constrained than the completely
flexible acyclic DNA monomer Z,3 they destabilize du-
plexes even more than Z. Even though the results
obtained with the abasic monomers question the impor-
tance of backbone preorganization for entropically fa-
vored duplex formation, this destabilization is mainly
believed to be caused by the tetrahydrofuran ring forcing
the two monomers ZL and d ZL into conformations unfa-
vorable for duplex formation.
Exp er im en ta l Section
Gen er a l. All reagents were obtained from commercial
suppliers and were used without further purification. Petro-
leum ether of distillation range 60-80° was used. The silica
gel (0.040-0.063 mm) used for column chromatography was
purchased from Merck. After column chromatography, frac-
tions containing product were pooled, evaporated under re-
duced pressure, and dried under vacuum to give the product.
The monocyclic seco-LNA monomers ZL and d ZL were
incorporated in the same mixed sequences as XL as well
as in a homothymine sequence for direct comparison with
the previously reported results for the acyclic DNA
monomer Z.3 It was reported that monomer Z when
incorporated in the middle of an oligothymidylate 13-mer
induced a decrease in the Tm value of 8.1 °C toward
complementary DNA and a much less pronounced de-
stabilizing effect (∆Tm ) -0.3 °C) when incorporated in
1
All H NMR spectra were recorded at 400 MHz, all 13C NMR
spectra were recorded at 100.6 MHz (apart from two which
were recorded at 75.5 MHz), and the three 31P spectra were
recorded at 121.5 MHz. Chemical shifts are reported in ppm
(35) Reduced binding affinities have also been observed for other
ON analogues containing a flexible nucleobase on a rigified phosphor-
diester backbone. (a) Marangoni, M.; Van Aershot, A.; Augustyns, P.;
Rozenski, J .; Herdewijn, P. Nucleic Acids Res. 1997, 25, 3034. (b) Epple,
C.; Leumann, C. Chem. Biol. 1998, 5, 209.
(33) Tm values were obtained as reported earlier. See ref 16.
(34) Millican, T. A.; Chauncey, M. A.; Cutbush, S. D.; Eaton, M. A.
W.; Gunning, J .; Mann, J .; Mock, G. A.; Neidle, S.; Patel, T. P. Nucleic
Acids Res. 1984, 12, 7435.

5172 J . Org. Chem., Vol. 65, No. 17, 2000
Kværnø et al.
relative to tetramethylsilane as internal standard for 1H and
13C and relative to 85% H3PO4 as external standard for 31P.
Assignments of NMR spectra are based on 2D spectra when
given and follows standard carbohydrate/nucleoside nomen-
clature for easy comparison (i.e., the carbon atom next to the
nucleobase is assigned C1′ etc.) even though the systematic
compound names are given according to IUPAC nomenclature.
Fast-atom bombardment mass spectra (FAB-MS) were re-
corded in positive ion mode. Microanalyses were performed
at The Microanalytical Laboratory, Department of Chemistry,
University of Copenhagen.
3,5-Di-O-ben zyl-1,2-O-isopr opyliden e-4-C-m eth an esu lfo-
n yloxym eth yl-R-D-r ibofu r a n ose (2). Furanose 129 (12.357
g, 30.9 mmol) was dissolved in anhydrous pyridine (25 mL) at
0 °C under a N2 atmosphere, MsCl (5.00 mL, 64.3 mmol) was
added, and the mixture was stirred for 20 min at room
temperature. Half saturated aqueous NaHCO3 (200 mL) was
added, the mixture was extracted with EtOAc, and the
combined organic phase was successively washed with satu-
rated aqueous NaHCO3 and H2O. The combined organic phase
was evaporated to dryness under reduced pressure and co-
evaporated with toluene (20 mL) to give a crude product
(colorless oil, tentatively assigned as furanose 2) which was
used in the next step without purification. Rf (2% MeOH in
CH2Cl2, (v/v)) 0.70.
Meth yl 3,5-Di-O-ben zyl-4-C-m eth ylsu lfon yloxym eth yl-
R,â-D-r ibofu r a n osid e (3).30 Crude 2 was stirred in a mixture
of H2O (30 mL) and 15% HCl in MeOH (300 mL, w/w) for 10
h whereupon the mixture was neutralized with NaHCO3 (s).
H2O (200 mL) was added, the mixture was extracted with
EtOAc, and the combined organic phase was washed with H2O
and evaporated to dryness under reduced pressure. The
residue was coevaporated with acetonitrile (100 mL), and the
residue was subjected to silica gel column chromatography
(14.5 × 5.5 cm) eluting with a gradient of 30-40% EtOAc in
petroleum ether (v/v) to give furanosides 3 (11.896 g, 85.2%
from 1, anomeric ratio ) 1:4) as a colorless oil which was used
in the next step without further purification. Rf (EtOAc/
petroleum ether, 1:1 (v/v)) 0.37; 13C NMR (CDCl3) δ 137.48,
136.74, 128.43, 128.37, 128.32, 128.29, 128.08, 127.93, 127.89,
127.82, 127.70, 127.60, 107.35, 103.18, 84.49, 83.01, 81.76,
78.75, 74.89, 73.93, 73.59, 73.23, 72.43, 71.98, 70.73, 70.42,
70.08, 55.78, 54.84, 37.10, 37.00.
(1S,3RS,4R,7S)-7-Ben zyloxy-1-ben zyloxym eth yl-3-m eth -
oxy-2,5-d ioxa bicyclo[2.2.1]h ep ta n e (4).30 Furanosides 3
(9.188 g, 13.3 mmol) were dissolved in anhydrous DMF (50
mL) under a N2 atmosphere at 0 °C whereafter NaH (60% (w/
w), 1.576 g, 39.4 mmol) was added over 5 min. The mixture
was stirred at room temperature for 2 h, H2O (200 mL) was
added, and the suspension was extracted with EtOAc and
washed with saturated aqueous NaHCO3 and H2O. The
aqueous phase was extracted with EtOAc, and the combined
organic phase was evaporated to dryness under reduced
pressure to give a crude product (colorless oil, tentatively
assigned as 4) which was used without purification in the next
step. Rf (EtOAc/petroleum ether, 3:7 (v/v)) 0.37, 0.10 (two
epimers).
86.2% from 3) as a colorless oil. Rf (EtOAc/petroleum ether,
3:1 (v/v)) 0.31; 1H NMR (CDCl3) δ 7.34-7.25 (10H, m, Bn),
4.58 (4H, d, J 10.8 Hz, Bn), 4.05 (1H, m, H2), 3.87-3.74 (5H,
m), 3.60 (2H, m) (H3, H5, H3′, H5′), 2.4 (2H, bs, 2 × OH); 13
C
NMR (CDCl3) δ 137.54, 137.48, 128.32, 128.30, 127.74, 127.71,
127.67, 127.54 (Bn), 85.21, 84.83 (C2, C3), 81.50 (C4), 75.07
(C5), 73.68, 72.34 (Bn), 69.56 (C5′), 62.84 (C1).
(2S,3S,4S)-3-Ben zyloxy-4-b en zyloxym et h yl-2-(p -t olu -
en esu lfon yl)oxym et h yl-4-h yd r oxyt et r a h yd r ofu r a n (7).
Compound 6 (6.994 g, 20.3 mmol) was dissolved in anhydrous
CH2Cl2 (100 mL) at 0 °C under a N2 atmosphere, DMAP (6.262
g, 51.26 mmol) followed by TsCl (7.688 g, 40.33 mmol) were
added, and the mixture was stirred for 10 h at room temper-
ature. After evaporation to dryness under reduced pressure,
the residue was redissolved in EtOAc (250 mL), washed
successively with saturated aqueous NaHCO3 and H2O, and
the organic phase was evaporated to dryness under reduced
pressure. The residue was subjected to column chromatogra-
phy on silica gel (15 × 5.5 cm) using 1% MeOH in CH2Cl2 (v/
v) as the eluent to give furan 7 (8.387 g, 82.8%) as a white
solid material after coevaporation with n-hexane (100 mL). Rf
(EtOAc/petroleum ether, 1:1 (v/v)) 0.55; FAB-MS m/z 499 [M
1
+ H]+; H NMR (CDCl3) δ 7.75 (2H, d, J 8.2 Hz, Ar), 7.36-
7.24 (12H, m, Bn, Ar), 4.56 (1H, d, J 11.8 Hz, Bn), 4.55 (1H,
d, J 11.9 Hz, Bn), 4.53 (1H, d, J 11.8 Hz, Bn), 4.50 (1H, d, J
11.9 Hz, Bn), 4.14-4.09 (3H, m, H2, H1), 3.78 (1H, d, J 9.3
Hz, H5′), 3.76 (1H, s, H3), 3.76-3.74 (2H, m, H5), 3.49 (1H, d,
J 9.3 Hz, H5′), 2.41 (3H, s, ArCH3), 2.3 (1H, bs, OH); 13C NMR
(CDCl3) δ 144.74, (Ar), 137.32, 137.26 (Bn), 132.68 (Ar), 129.69
(Ar), 128.38, 128.33, 127.91, 127.86, 127.77, 127.69, 127.67 (Bn,
Ar), 84.28 (C3), 81.49 (C4), 81.33 (C2), 75.00 (C5), 73.65, 71.91
(Bn), 69.31, 69.19 (C5′, C1), 21.51 (ArCH3). Anal. Calcd for
(C27H30O7S): C, 65.0; H, 6.1; S, 6.4. Found: C, 64.9; H, 6.0; S,
6.5.
(1S,4S,7S)-7-Ben zyloxy-1-ben zyloxym eth yl-2,5-d ioxa -
bicyclo[2.2.1]h ep ta n e (8). Compound 7 (950 mg, 1.92 mmol)
was dissolved in anhydrous DMF (8 mL) at 0 °C under a N2
atmosphere, and NaH (134 mg, 60% (w/w), 3.35 mmol) was
added. The mixture was stirred at room temperature for 1.5
h, H2O (40 mL) was added, and the suspension was extracted
with EtOAc. The combined organic phase was washed succes-
sively with saturated aqueous NaHCO3 and H2O. The com-
bined aqueous phase was extracted with EtOAc, and the
combined organic phase was evaporated to dryness under
reduced pressure and coevaporated with anhydrous toluene
(6 mL). The residue was purified by silica gel column chro-
matography (8.5 × 4.8 cm) using EtOAc/petroleum ether (3:7,
v/v) as the eluent to give derivative 8 (516 mg, 82.0%) as a
colorless oil. Rf (EtOAc/petroleum ether, 1:1 (v/v)) 0.44; FAB-
MS m/z 327 [M + H]+; 1H NMR (CDCl3) δ 7.37-7.26 (10H, m,
Bn), 4.78 (1H, d, J 11.7 Hz, Bn), 4.63 (1H, d, J 12.9 Hz, Bn),
4.59 (1H, d, J 12.3 Hz, Bn), 4.58 (1H, d, J 12.0 Hz, Bn), 4.30
(1H, s, H3), 4.07-3.93 (5H, m), 3.73 (2H, s) (H1, H2, H5, H5′);
13C NMR (CDCl3) δ 137.79, 137.58, 128.40, 128.36, 127.83,
127.67, 127.58 (Bn), 85.74 (C4), 79.99, 76.45, 74.25, 73.75,
72.59, 71.94 (C1, C2, C3, C5′, Bn), 66.38 (C5).
(1S,4S,7S)-7-H yd r oxy-1-h yd r oxym et h yl-2,5-d ioxa b i-
cyclo[2.2.1]h ep ta n e (9). To a solution of 10% Pd/C (0.05 g)
in anhydrous EtOH (6 mL) was added 8 (516 mg, 1.58 mmol)
dissolved in anhydrous EtOH (6 mL) under a N2 atmosphere.
The solution was degassed with H2 and stirred under a H2
atmosphere for 3.5 h. The mixture was evaporated to dryness
under reduced pressure, and the residue was subjected to silica
gel column chromatography (6 × 2.8 cm) using 10% MeOH in
CH2Cl2 (v/v) as the eluent to give 9 (184 mg, 79.7%) as a white
solid material. Rf (10% MeOH in CH2Cl2 (v/v)) 0.22; FAB-MS
(2R,3S,4S)-3-Ben zyloxy-4-ben zyloxym eth yl-2-for m yl-4-
h yd r oxytetr a h yd r ofu r fu r a n (5).30 Crude 4 was stirred in
80% AcOH (100 mL) at 50 °C for 3.5 h whereupon the mixture
was evaporated to dryness under reduced pressure and co-
evaporated with anhydrous toluene (2 × 20 mL) to give a crude
product (colorless oil, tentatively assigned as 5) which was used
without purification in the next step. Rf (EtOAc/petroleum
ether, 1:1 (v/v)) 0.42.
(2S,3S,4S)-3-Ben zyloxy-4-ben zyloxym eth yl-2-h yd r oxy-
m eth yl-4-h yd r oxytetr a h yd r ofu r a n (6). Crude 5 was dis-
solved in MeOH (100 mL), and NaBH4 (2.138 g, 56.5 mmol)
was added over 5 min. After being stirred for 20 min at room
temperature, the reaction was quenched by the addition of 80%
AcOH (10 mL), and the mixture was evaporated to dryness
under reduced pressure and coevaporated with anhydrous
toluene (20 mL). The residue was purified by silica gel column
chromatography (15 × 5.8 cm) eluting with a gradient of 20-
80% EtOAc in petroleum ether (v/v) to give furan 5 (5.776 g,
1
m/z 147 [M + H]+; H NMR (CD3OD) δ 4.04 (1H, s, H2), 4.01
(1H, s, H3), 3.89 (1H, d, J 7.9 Hz, H5′), 3.85 (1H, dt, J 8.0, 0.5
Hz, H1), 3.75 (1H, d, J 7.9 Hz, H5′), 3.74 (1H, d, J 8.2 Hz,
H1), 3.70 (1H, d, J 15.2 Hz, H5), 3.67 (1H, d, J 15.2 Hz, H5);
13C NMR (CD3OD) δ 86.28 (C4), 78.72 (C2), 72.54 (C3), 72.38
(C5′), 71.21 (C1), 57.68 (C5).
(1R ,4S ,7S )-1-(4,4′-Dim e t h oxyt r it yl)oxym e t h yl-7-h y-
d r oxy-2,5-d ioxa bicyclo[2.2.1]h ep ta n e (10). Compound 9
(160 mg, 1.09 mmol) was dissolved in anhydrous pyridine (3

Synthesis of Abasic LNA and seco-LNA Derivatives
J . Org. Chem., Vol. 65, No. 17, 2000 5173
mL) at 0 °C under a N2 atmosphere, DMTCl (570 mg, 1.68
mmol) was added, and the mixture was stirred at room
temperature for 2.5 h. Sat. aq. NaHCO3 (25 mL) was added,
the mixture was extracted with EtOAc, and the combined
organic phase was washed with H2O. The organic phase was
evaporated to dryness under reduced pressure, and the residue
was coevaporated with anhydrous toluene (2 × 10 mL). The
residue was purified thrice by column chromatography using
(a) 97.5:2.0:0.5 CH2Cl2/MeOH/pyridine (v/v/v), (b) 98.5:1.0:0.5
CH2Cl2/MeOH/pyridine (v/v/v), and (c) 70:30:0.5 petroleum
ether/EtOAc/pyridine (v/v/v) as the eluents, thereby affording
10 (359 mg, 73.1%) as a white foam after coevaporation with
anhydrous acetonitrile (3 × 10 mL). Rf (5% MeOH in CH2Cl2
(v/v)) 0.41; FAB-MS m/z 448 [M]+; 1H NMR (CD3OD) δ 7.45-
7.43 (2H, m, DMT), 7.33-7.23 (6H, m, DMT), 7.19-7.17 (1H,
m, DMT), 6.84-6.81 (4H, m, DMT), 4.12 (1H, s, H2), 4.06 (1H,
s, H3), 4.05 (1H, d, J 8.1 Hz, H1/H5′), 3.97 (2H, d, J 7.0 Hz,
H1/H5′), 3.88 (1H, d, J 8.5 Hz, H1/H5′), 3.41 (1H, d, J 10.4
Hz, H5), 3.31 (1H, d, J 10.6 Hz, H5), 3.75 (6H, s, OCH3); 13C
NMR (CD3OD) δ 160.07, 146.34, 137.23, 137.06, 131.32,
131.28, 129.31, 128.70, 127.73, 114.05 (DMT), 87.34, 87.27
(CAr2Ph, C-1), 80.49 (C4), 75.13, 74.90 (C3/C6, C7), 73.21(C3/
C6), 62.37 (C2′), 55.70 (OCH3).
(1R,4S,7S)-1-(4,4′-Dim eth oxytr ityl)oxym eth yl-7-(2-cy-
a n oet h oxy(d iisop r op yla m in o)p h osp h in oxy)-2,5-d ioxa -
bicyclo[2.2.1]h ep ta n e (11). Derivative 10 (287 mg, 0.638
mmol) was dissolved in a mixture of anhydrous CH2Cl2 (2 mL)
and N,N-diisopropylethylamine (1 mL). 2-Cyanoethyl N,N-
diisopropylphosphoramidochloridite (0. 25 mL, 1.12 mmol) was
slowly added under a N2 atmosphere, and the resulting
mixture was stirred at room temperature for 1 h whereupon
MeOH (20 mL) and EtOAc (50 mL) were added. The mixture
was washed with saturated aqueous NaHCO3, and the organic
phase was evaporated to dryness under reduced pressure. The
residue was subjected to silica gel chromatography twice with
(a) 70:30:0.5 petroleum ether/EtOAc/pyridine (v/v/v) and (b)
75:25:0.5 petroleum ether/EtOAc/pyridine (v/v) as the eluents
to give 11 (258 mg, 62.3%) as a white foam after coevaporation
with anhydrous acetonitrile (6 × 10 mL). Rf (1:1 EtOAc/
petroleum ether (v/v)) 0.65, 0.57 (2 diastereomers); FAB-MS
m/z 649 [M + H]+; 31P NMR (CDCl3) δ 148.41, 148.32.
combined organic phase was washed successively with satu-
rated aqueous NaHCO3 and H2O, evaporated to dryness under
reduced pressure, and coevaporated with anhydrous toluene
(2 × 15 mL). The residue was purified by silica gel column
chromatography (9.8 × 5.5 cm) eluting with a gradient of 30%-
70% EtOAc in petroleum ether (v/v) to give 13 (1.484 g, 51.6%)
as a white foam after coevaporation with n-hexane (3 × 20
mL). Rf (1:1 EtOAc/petroleum ether (v/v)) 0.18; FAB-MS m/z
1
495 [M + H]+; H NMR (CDCl3) δ 8.81 (1H, bs, NH), 7.34-
7.26 (10H, m, Bn), 7.04 (1H, d, J 1.3 Hz, H6), 4.74 (1H, d, J
11.7 Hz, Bn), 4.62 (1H, d, J 11.5 Hz, Bn), 4.53 (1H, d, J 11.9
Hz, Bn), 4.49 (1H, d, J 11.9 Hz, Bn), 4.32 (1H, d, J 10.8 Hz,
H5′), 4.10 (1H, d, J 10.1 Hz, H5′′), 4.00-3.94 (4H, m, H1′, H2′,
H3′, H5′′), 3.84 (1H, d, J 10.8 Hz, H5′), 3.74 (1H, m, H1′), 2.04
(3H, s, CH3CO), 1.89 (3H, d, J 1.1 Hz, CH3);13C NMR (CDCl3)
δ 170.18 (CdO), 163.92 (C4), 150.82 (C2), 141.19 (C6), 137.65,
137.36, 128.33, 128.25, 127.89, 127.83, 127.62, 127.51 (Bn),
110.02 (C5), 89.26 (C4′), 84.23 (C2′), 82.07 (C3′), 73.71, 73.51,
73.29 (Bn, C5′), 66.55 (C5′′), 48.90 (C1′), 21.73 (CH3CO), 12.19
(CH3).
(2S,3S,4R)-4-Acetoxymethyl-3,4-dihydroxy-2-(thymin-1-yl-
m eth yl)tetr a h yd r ofu r a n (14). Compound 13 (1.897 g, 3.84
mmol) was dissolved in anhydrous EtOH (30 mL), 20% Pd-
(OH)2/C (0.08 g) was added, and the mixture was degassed
with H2 under reduced pressure. After being stirred at room
temperature under a H2 atmosphere for 36 h, the mixture was
evaporated to dryness under reduced pressure, and the residue
was subjected to column chromatography on silica gel (8.0 ×
5.5 cm) eluting with a gradient of 5-7% MeOH in CH2Cl2 (v/
v) to give 14 (1.054 g, 87.4%) as a white foam after coevapo-
ration with n-hexane (2 × 10 mL). Rf (10% MeOH in CH2Cl2
(v/v)) 0.24; FAB-MS m/z 315 [M + H]+; 1H NMR (CDCl3) δ
7.43 (1H, q, J 1.1 Hz, H6), 4.33 (1H, d, J 11.7 Hz, H5′′), 4.25
(1H, d, J 11.5 Hz, H5′′), 4.14 (1H, dd, J 3.8, 13.8 Hz, H1′),
4.05 (1H, m, H2′), 3.95-3.88 (4H, m, H1′, H3′, H5′), 2.12 (3H,
s, CH3CO), 1.90 (3H, d, J 1.3 Hz, CH3); 13C NMR (CDCl3) δ
170.97 (CH3CO), 165.00 (C4), 151.09 (C2), 142.32 (C6), 108.50
(C5), 84.36 (C2′), 79.93 (C4′), 78.44 (C3′), 74.11 (C5′), 64.78
(C5′′), 49.51 (C1′), 18.83 (CH3CO), 10.31 (CH3).
(2S,3S,4S)-3,4-Dih ydr oxy-4-h yd r oxym eth yl-2-((th ym in -
1-yl)m eth yl)tetr a h yd r ofu r a n (15). Compound 14 (1.037 g,
3.30 mmol) was dissolved in anhydrous MeOH (20 mL) at 0
°C under a N2 atmosphere, NaOMe (541 mg, 10.0 mmol) was
added, and the mixture was stirred for 50 min whereupon 80%
aq AcOH (2 mL) was added. The mixture was evaporated to
dryness under reduced pressure, coevaporated with anhydrous
toluene (10 mL), and the residue was purified by silica gel
column chromatography (5.5 × 5.5 cm) using 10% MeOH in
CH2Cl2 (v/v) as the eluent, affording 15 (716 mg, 81.6%) as a
white foam. Rf (20% MeOH in CH2Cl2 (v/v)) 0.32; FAB-MS m/z
273 [M + H]+; 1H NMR (CD3OD) δ 7.44 (1H, d, J 1.2 Hz, H6),
4.15 (1H, dd, J 3.7, 13.7 Hz, H1′), 4.03 (1H, dt, J 3.7, 8.1 Hz,
H2′), 3.94-3.84 (4H, m, H1′, H3′, H5′), 3.79 (1H, d, J 11.5 Hz,
H5′′), 3.71 (1H, d, J 11.5 Hz, H5′′), 1.90 (3H, d, J 1.3 Hz, CH3);
13C NMR (CD3OD) δ 165.03 (C4), 151.10 (C2), 142.34 (C6),
108.51 (C5), 84.19 (C2′), 81.37 (C4′), 78.72 (C3′), 74.18 (C5′),
62.14 (C5′′), 49.56 (C1′), 10.30 (CH3).
(2S,3S,4S)-4-Acetoxy-3-ben zyloxy-4-ben zyloxym eth yl-
2-((p-t olu en esu lfon yl)oxym et h yl)t et r a h yd r ofu r a n (12).
Compound 7 (3.442 g, 6.90 mmol) was dissolved in anhydrous
pyridine (20 mL) under a N2 atmosphere whereafter DMAP
(3.292 g, 26.9 mmol) and Ac2O (1.5 mL, 15.9 mmol) were added.
The reaction mixture was stirred at room temperature for 2
h, H2O (200 mL) was added, and the suspension was extracted
with EtOAc. The combined organic phase was washed succes-
sively with saturated aqueous NaHCO3 and H2O, evaporated
to dryness under reduced pressure, and coevaporated with
anhydrous toluene (3 × 10 mL). The residue was purified by
silica gel column chromatography (12.3 × 5.5 cm) with EtOAc/
petroleum ether (1:4, v/v) as the eluent yielding 12 (3.142 g,
84.2%) as a colorless oil after coevaporation with n-hexane (3
× 15 mL). Rf (1:1 EtOAc/petroleum ether (v/v)) 0.73; FAB-MS
1
m/z 541 [M + H]+; H NMR (CDCl3) δ 7.75 (2H, d, J 8.4 Hz,
Ar), 7.32-7.22 (14H, m, Ar, 2 × Bn), 4.67 (1H, d, J 11.5 Hz,
Bn), 4.51 (1H, d, J 11.5 Hz, Bn), 4.49 (2H, d, J 11.8 Hz, Bn),
4.25 (1H, d, J 10.6 Hz, H5), 4.10 (1H, d, J 10.1 Hz, H5′), 4.08
(1H, s, H3), 4.01 (2H, m, H1), 3.96 (1H, m, H2), 3.94 (1H, d, J
10.1 Hz, H5′), 3.85 (1H, d, J 10.6 Hz, H5), 2.42 (3H, s, ArCH3),
2.01 (3H, s, COCH3); 13C NMR (CDCl3) δ 170.18 (CdO), 144.78
(Ar), 137.71, 137.26 (Bn), 132.61 (Ar), 129.68, 128.31, 128.23,
127.87, 127.77, 127.58, 127.43 (Ar, Bn), 89.05 (C4), 83.17 (C3),
81.07 (C2), 73.70, 73.46, 73.07 (2 × Bn, C5), 68.52 (C1), 66.49
(C5′), 21.64, 21.51 (ArCH3, COCH3).
(2S,3S,4R)-3,4-Dih yd r oxy-4-(4,4′-d im eth oxytr ityl)oxy-
m eth yl-2-((th ym in -1-yl)m eth yl)tetr ah ydr ofu r an (16). Com-
pound 15 (706 mg, 2.59 mmol) dissolved in anhydrous pyridine
(20 mL) was concentrated under reduced pressure to half
volume and cooled to 0 °C under a N2 atmosphere. 4,4′-
Dimethoxytrityl chloride (1.345 g, 3.96 mmol) was added, and
the mixture was stirred at room temperature for 2 h. The
reaction was quenched with MeOH (5 mL), EtOAc (150 mL)
was added, and the mixture was washed successively with half
saturated aqueous NaHCO3 and H2O. The aqueous layer was
re-extracted with CH2Cl2 and EtOAc, the combined organic
phase was evaporated to dryness under reduced pressure, and
the residue was coevaporated with acetonitrile (2 × 30 mL).
The residue was purified by silica gel column chromatography
(2S,3S,4S)-4-Acetoxy-3-ben zyloxy-4-ben zyloxym eth yl-
2-(th ym in -1-ylm eth yl)tetr ah ydr ofu r an (13). Thymine (1.539
g, 12.2 mmol) was dissolved under
a N2 atmosphere in
anhydrous DMF (40 mL) at 120 °C. NaH (495 mg, 60% (w/w),
12.4 mmol) was added, and the suspension was stirred for 10
min whereupon 12 (3.142 g, 5.81 mmol) dissolved in anhydrous
DMF (25 mL) was slowly added. After 4 h, H2O (150 mL) was
added, and the mixture was extracted with EtOAc. The
(7.3 × 5.5 cm) with
a gradient of 0.5:2.0-5.0:97.5-94.5
pyridine/MeOH/CH2Cl2 (v/v/v) as eluent to give 16 (1297 mg,
87.0%) as a white foam after coevaporation with acetonitrile
(3 × 25 mL). Rf (10% MeOH in CH2Cl2 (v/v)) 0.56; FAB-MS

5174 J . Org. Chem., Vol. 65, No. 17, 2000
Kværnø et al.
1
m/z 574.1 [M]+; H NMR (CDCl3) δ 10.00 (1H, bs, NH), 7.41
Compound 18 (623 mg, 0.85 mmol) was dissolved in anhydrous
THF (20 mL), tetrabutylammonium fluoride in THF (5 mL, 1
M) was slowly added, and the mixture was stirred for 10 min.
After evaporation to dryness under reduced pressure, the
residue was purified by silica gel column chromatography (13.5
× 3.3 cm) eluting with a gradient of 0.5:25-35:74.5-64.5
pyridine/EtOAc/CH2Cl2 (v/v/v) to give 19 (533 mg) as a yel-
lowish foam after coevaporation with acetonitrile (3 × 15 mL).
Rf (1:1 EtOAc/CH2Cl2 (v/v)) 0.37; FAB-MS m/z 616 [M]+; 1H
NMR (CDCl3) δ 8.61 (1H, bs, NH), 7.38-7.36 (2H, m, DMT),
7.29-7.21 (7H, m, DMT), 7.09 (1H, d, J 1.1 Hz, H6), 6.84-
6.80 (4H, m, DMT), 4.20 (1H, d, J 10.8 Hz, H5′), 4.11 (1H, d,
J 11.4 Hz, H1′), 4.06 (1H, d, J 10.8 Hz, H5′), 4.01 (1H, m, H2′),
3.82-3.75 (2H, m, H1′, H3′), 3.79 (6H, s, OCH3), 3.64 (1H, d,
J 10.6 Hz, H5′′), 3.49 (1H, d, J 10.8 Hz, H5′′), 3.34 (1H, d, J
2.9 Hz, OH), 2.09 (3H, s, COCH3), 1.90 (3H, d, J 1.1 Hz, CH3);
13C NMR (CDCl3) δ 171.64 (COCH3), 163.86 (C4), 158.51
(DMT), 150.80 (C2), 144.25 (DMT), 141.04 (C6), 135.24, 135.17,
129.85, 127.84, 126.88, 113.12 (DMT), 110.18 (C5), 89.65 (C4′),
86.22 (CAr2Ph), 81.16 (C3′), 79.29 (C2′), 74.05 (C5′), 61.26
(C5′′), 55.10 (OCH3), 48.87 (C1′), 21.54 (COCH3), 12.20 (CH3).
This crude product contained impurities which could be
assigned as Bu4N+ and n-hexane: 1H NMR (CDCl3) 3.05 (m),
1.75 (m), 1.40 (sextet), 0.99 (t, J 7.4 Hz) (Bu4N+), 1.26 (m),
0.88 (t, J 6.9 Hz) (n-hexane); 13C NMR (CDCl3) 32, 23.14 (n-
hexane), 53.5, 25, 20, 13.5 (Bu4N+). The crude product was
used in the next step without further purification.
(2H, d, J 7.3 Hz, DMT), 7.31-7.16 (7H, m, DMT), 7.12 (1H, d,
J 0.9 Hz, H6), 6.79 (4H, d, J 8.8 Hz, DMT), 4.07-3.98 (3H, m,
H1′, H2′, H3′), 3.91-3.78 (3H, m, H1′, H5′), 3.74 (6H, s, OCH3),
3.44 (1H, bs, OH), 3.43 (1H, d, J 9.7 Hz, H5′′), 3.33 (1H, d, J
9.5 Hz, H5′′), 1.96 (1H, d, J 5.5 Hz, 3′OH), 1.83 (3H, s, CH3);
13C NMR (CDCl3) 164.60 (C4), 158.44 (DMT), 151.59 (C2),
144.41 (DMT), 142.00 (C6), 135.47, 129.96, 127.96, 127.81,
126.82, 113.14 (DMT), 110.06 (C5), 86.39 (CAr2Ph), 84.71 (C2′),
81.90 (C4′), 79.17 (C3′), 75.52 (C5′), 63.61 (C5′′), 55.11 (OCH3),
49.87 (C1′), 12.15 (CH3).
(2S,3S,4R)-3-(ter t-Bu tyldim eth ylsilyloxy)-4-(4,4′-dim eth -
oxytrityl)oxymethyl-4-hydroxy-2-((thymin-1-yl)methyl)tetra-
h yd r ofu r a n (17). Compound 16 (1266 mg, 1.84 mmol) was
dissolved in anhydrous CH2Cl2 (50 mL) under a N2 atmo-
sphere, imidazole (604 mg, 8.87 mmol) and TBDMSCl (909 mg,
5.35 mmol) were added, and the mixture was stirred at room
temperature. After 24 h, further TBDMSCl (380 mg) was
added, and after additional 48 h, the reaction mixture was
diluted with EtOAc (200 mL) and washed successively with
saturated aqueous NaHCO3 and H2O. The organic phase was
evaporated to dryness under reduced pressure and coevapo-
rated with acetonitrile (2 × 10 mL), and the residue was
purified by silica gel column chromatography (7.5 × 5.5 cm)
using 0.5:20:79.5 pyridine/EtOAc/CH2Cl2 (v/v/v) as the eluent
to give 17 (1042 mg, 68.6%) as a white foam after coevapora-
tion with acetonitrile (2 × 15 mL). Rf (1:1 EtOAc/CH2Cl2 (v/
v)) 0.56; FAB-MS m/z 689 [M + H]+; H NMR (CDCl3) δ 8.6
(2S,3S,4R)-4-Acet oxy-3-(2-cya n oet h oxy(d iisop r op yl-
am in o)ph osph in oxy)-4-(4,4′-dim eth oxytr ityl)oxym eth yl-2-
((th ym in -1-yl)m eth yl)tetr a h yd r ofu r a n (20). Nucleoside 19
(310 mg, 0.50 mmol) was dissolved in a mixture of anhydrous
CH2Cl2 (10 mL) and (i-Pr)2NEt (2.5 mL) under a N2 atmo-
sphere. 2-Cyanoethyl N,N-diisopropylphosphoramidochloridite
(0.6 mL, 2.7 mmol) was added, and the mixture was stirred
at room temperature for 4 h. The reaction was quenched by
the addition of H2O (5 mL), the mixture was diluted with
EtOAc (150 mL), and washing was performed using succes-
sively saturated NaHCO3 and H2O. The organic phase was
evaporated to dryness under reduced pressure, and the residue
was coevaporated with acetonitrile (2 × 20 mL). The residue
was purified by silica gel column chromatography (17.5 × 3.3
cm) using 0.5:24.5:75 pyridine/EtOAc/CH2Cl2 (v/v/v) as the
eluent. Impure fractions (all fractions were analyzed by 31P
NMR) were purified once more on a silica gel column (14 ×
3.3 cm) using a gradient of 0.5:15-25:74.5-64.5 pyridine/
EtOAc/CH2Cl2 (v/v/v) as eluent. Fractions containing pure
product were combined and evaporated to give amidite 20 (277
mg, 67.4% from 18) as a colorless foam after coevaporation
with acetonitrile (4 × 10 mL). Rf (1:1 EtOAc/CH2Cl2 (v/v)) 0.70;
FAB-MS m/z 817 [M + H]+; 31P NMR (DMSO-d6) δ 151.84,
151.74.
1
(1H, bs, NH), 7.38-7.21 (9H, m, DMT), 7.14 (1H, d, J 1.3 Hz,
H6), 6.85-6.81 (4H, m, DMT), 4.17 (1H, dd, J 3.1, 13.9 Hz,
H1′), 4.01 (1H, d, J 9.5 Hz, H5′), 3.95 (1H, dt, J 2.7, 8.4 Hz,
H2′), 3.85 (1H, d, J 9.5 Hz, H5′), 3.85 (1H, d, J 2.4 Hz, H3′),
3.79 (6H, s, OCH3), 3.70 (1H, d, J 8.4, 13.9 Hz, H1′), 3.36 (1H,
d, J 9.3 Hz, H5′′), 3.30 (1H, d, J 9.5 Hz, H5′′), 2.86 (1H, bs,
OH), 1.91 (3H, d, J 1.1 Hz, CH3), 0.71 (9H, s, C(CH3)3), 0.07,
0.02 (6H, 2s, Si(CH3)2); 13C NMR (CDCl3) δ 164.04 (C4), 158.48
(DMT), 150.68 (C2), 144.27 (DMT), 141.80 (C6), 135.39, 135.35,
129.87, 127.92, 127.77, 126.86, 113.10 (DMT), 109.60 (C5),
86.41 (CAr2Ph), 85.40 (C2′), 82.01 (C4′), 79.53 (C3′), 76.48 (C5′),
64.19 (C5′′), 55.09 (OCH3), 50.24 (C1′), 25.42 (C(CH3)3, 17.54
(C(CH3)3), 12.18 (CH3), -4.85, -5.07 (Si(CH3)2).
(2S,3S,4R)-4-Acetoxy-3-(ter t-bu tyld im eth ylsilyloxy)-4-
(4,4′-d im eth oxytr ityl)oxym eth yl-2-((th ym in -1-yl)m eth yl)-
tetr a h yd r ofu r a n (18). Compound 17 (1.006 g, 1.46 mmol)
was dissolved in anhydrous pyridine (20 mL) under a N2
atmosphere. DMAP (1.428 mg, 11.7 mmol) and Ac2O (1.0 mL,
10.6 mmol) were added, and the solution was stirred at room
temperature for 18 h. H2O (60 mL) was added, and the mixture
was extracted with EtOAc. The combined organic phase was
washed with saturated aqueous NaHCO3 and H2O and evapo-
rated to dryness under reduced pressure, and the residue was
coevaporated with acetonitrile (2 × 10 mL). The residue was
subjected to silica gel column chromatography (15.5 × 3.3 cm)
with 0.5:15-20:84.5-79.5 pyridine/EtOAc/CH2Cl2 (v/v/v) as the
gradient eluent to give 18 (650 mg, 60.9%) as a yellowish foam
after coevaporation with acetonitrile (3 × 15 mL). Rf (1:1
EtOAc/CH2Cl2 (v/v)) 0.73; 1H (CDCl3) δ 8.40 (1H, bs, NH),
7.38-7.35 (2H, m, DMT), 7.28-7.20 (7H, m, DMT), 7.16 (1H,
d, J 1.3 Hz, H6), 6.81-6.78 (4H, m, DMT), 4.36 (1H, d, J 10.4
Hz, H5′), 4.30 (1H, d, J 4.6 Hz, H3′), 4.13 (1H, dd, J 2.9, 14.1
Hz, H1′), 3.92 (1H, d, J 10.4 Hz, H5′), 3.89 (1H, d, J 10.4 Hz,
H5′′), 3.83 (1H, m, H2′), 3.79 (6H, d, J 0.4 Hz, OCH3), 3.57
(1H, d, J 7.8, 14.2 Hz, H1′), 3.24 (1H, d, J 10.4 Hz, H5′′), 2.07
(3H, s, COCH3), 1.92 (3H, d, J 1.3 Hz, CH3), 0.73 (9H, s,
C(CH3)3), 0.03, -0.09 (6H, 2s, Si(CH3)2); 13C NMR (CDCl3) δ
170.06 (COCH3), 163.78 (C4), 158.40 (DMT), 150.55 (C2),
144.61 (DMT), 141.38 (C6), 135.85, 135.55, 129.85, 129.81,
127.90, 127.66, 126.72, 112.99 (DMT), 109.90 (C5), 90.25 (C4′),
85.90 (CAr2Ph), 84.88 (C2′), 77.53 (C3′), 74.86 (C5′), 61.62
(C5′′), 55.06 (OCH3), 49.17 (C1′), 25.47 (C(CH3)3, 21.80 (COCH3)
17.61 (C(CH3)3), 12.20 (CH3), -5.00, -5.27 (Si(CH3)2). Anal.
Calcd for (C40H50N2O9Si) C, 65.7; H, 6.9; N, 3.8. Found: C,
65.8; H, 7.0; N, 4.1.
(2S,3S,4S)-3,4-Dih yd r oxy-4-h yd r oxym eth yl-2-((p-tolu -
en esu lfon yloxy)m eth yl)tetr a h yd r ofu r a n (21). Compound
7 (2.992 g, 6.00 mmol) was dissolved in anhydrous EtOH (20
mL), 20% Pd(OH)2/C (0.1 g) was added, and the mixture was
degassed with H2 under reduced pressure and stirred under
a H2 atmosphere. After 31 h, the mixture was evaporated to
dryness under reduced pressure, and the residue was subjected
to silica gel column chromatography (7.8 × 4.8 cm) using 5%
MeOH in CH2Cl2 (v/v) as eluent to give 21 (1.642 g, 86.0%) as
a white solid material. Rf (10% MeOH in CH2Cl2) 0.39; FAB-
1
MS m/z 319 [M + H]+; H NMR (CD3OD) δ 7.79 (2H, d, J 8.2
Hz, Ar), 7.43 (2H, d, J 8.0 Hz, Ar), 4.15 (2H, m, H1), 3.89 (1H,
m, H2), 3.82 (1H, m, H3), 3.82 (1H, d, J 9.2 Hz, H5), 3.71 (1H,
d, J 11.5 Hz, H5′), 3.70 (1H, d, J 9.2 Hz, H5), 3.62 (1H, d, J
11.5 Hz, H5′), 2.45 (3H, s, ArCH3); 13C NMR (CD3OD) δ 144.60,
132.35, 129.12, 127.12 (Ar), 83.75 (C2), 80.96 (C4), 77.71 (C1),
74.16 (C5), 69.74 (C1), 61.99 (C5′), 19.67 (ArCH3). Anal. Calcd
for (C13H18O7S) C, 48.7; H, 5.7. Found: C, 49.0; H, 5.7.
(2S,3S,4R)-3,4-Dih yd r oxy-4-h yd r oxym eth yl-3-O,O-(h y-
d r oxym eth yl)-(1,1,3,3-tetr a isop r op yld isiloxa n e-1,3-d iyl)-
2-((p-t olu en esu lfon yloxy)m et h yl)t et r a h yd r ofu r a n (22).
Compound 21 (2.401 g, 7.54 mmol) was dissolved in anhydrous
pyridine (25 mL) under a N2 atmosphere, and 1,3-dichloro-
1,1,3,3-tetraisopropyldisiloxane (2.8 mL, 9.0 mmol) was added.
(2S,3S,4R)-4-Acetoxy-3-h ydr oxy-4-(4,4′-dim eth oxytr ityl)-
oxym eth yl-2-((th ym in -1-yl)m eth yl)tetr a h yd r ofu r a n (19).

Synthesis of Abasic LNA and seco-LNA Derivatives
J . Org. Chem., Vol. 65, No. 17, 2000 5175
The mixture was stirred at room temperature for 35 h
whereupon H2O (150 mL) was added. The suspension was
extracted with EtOAc, and the combined organic phase was
washed successively with saturated aqueous NaHCO3 and
H2O. The organic phase was evaporated to dryness under
reduced pressure, and the residue was coevaporated with
anhydrous toluene (3 × 15 mL). The residue was subjected to
silica gel column chromatography (13.8 × 5.5 cm) eluting with
a gradient of 10-15% EtOAc in petroleum ether (v/v) to give
22 (3.015 g, 71.3%) as a white solid material. Rf (3:7 EtOAc/
petroleum ether (v/v)) 0.50; FAB-MS m/z 561 [M + H]+; 1H
NMR (CDCl3) 7.78 (2H, d, J 8.4 Hz, Ar), 7.32 (2H, d, J 8.1 Hz,
Ar), 4.25 (1H, m, H1), 4.12 (2H, m, H2, H1), 4.03 (1H, d, J
11.0 Hz, H5′), 3.98 (1H, s, H3), 3.74 (1H, d, J 9.3 Hz, H5),
3.65 (1H, d, J 9.3 Hz, H5), 3.54 (1H, d, J 11.2 Hz, H5′), 2.8
(1H, br s, OH), 2.44 (3H, s, ArCH3), 1.09-0.96 (28H, m,
TIPDS); 13C NMR (CDCl3) 144.64, 132.83, 129.65, 127.86 (Ar),
85.03 (C2), 83.61 (C4), 77.56 (C3), 72.94 (C5), 69.17 (C1), 60.68
(C5′), 21.53 (ArCH3), 17.14, 17.10, 17.06, 13.28, 12.51, 12.39,
12.21 (TIPDS). Anal. Calcd for (C25H44O8SSi2) C, 53.5; H, 7.9.
Found: C, 53.5; H, 7.8.
(2S,3S,4R)-3-H yd r oxy-4-h yd r oxym et h yl-4-O-m et h ox-
a lyloxy-3-O,O-(h yd r oxym et h yl)-(1,1,3,3-t et r a isop r op yl-
d isiloxa n e-1,3-d iyl)-2-((p-tolu en esu lfon yl)m eth yl)tetr a -
h yd r ofu r a n (23). Compound 22 (559 mg, 1.00 mmol) was
dissolved in anhydrous CH2Cl2 (10 mL) under a N2 atmosphere
at 0 °C. DMAP (1.047 g, 8.57 mmol) followed by methoxalyl
chloride (0.30 mL, 3.26 mmol) were added, and the mixture
was stirred at room temperature for 22 h. The reaction mixture
was evaporated to dryness under reduced pressure, and the
residue was purified by silica gel column chromatography (6.5
× 5.8 cm) using CH2Cl2 as the eluent to give 23 (480 mg,
74.4%) as a colorless oil. Rf (0.5% CH3OH in CH2Cl2 (v/v)) 0.70;
FAB-MS m/z 647 [M + H]+; 1H NMR (CDCl3) δ 7.80 (2H, d, J
8.2 Hz, Ar), 7.33 (2H, d, J 8.2 Hz, Ar), 4.54 (1H, d, J 12.3 Hz,
H5′), 4.40 (1H, d, J 11.0 Hz, H5), 4.27-4.22 (2H, m, H3, H1),
4.13-4.08 (2H, m, H1, H2), 4.01 (1H, d, J 12.3 Hz, H5′), 3.89
(3H, s, OCH3), 3.67 (1H, d, J 11.0 Hz, H5), 2.45 (3H, s, ArCH3),
1.07-0.91 (28H, m, TIPDS); 13C NMR (CDCl3) δ 157.34, 155.29
(CdO), 144.71, 132.82, 129.64, 127.83 (Ar), 94.67 (C4), 84.68
(C2), 75.82 (C3), 69.21 (C5), 68.53 (C1), 56.00 (C5′), 53.45
(OCH3), 21.52 (ArCH3), 17.09, 17.05, 17.00, 16.79, 13.22, 12.35,
12.34, 12.21 (TIPDS).
Hz, H5′), 3.82 (1H, ddd, J 2.4, 4.8, 9.9 Hz, H2), 3.66 (1H, dd,
J 7.8, 11.8 Hz, H5′), 3.48 (1H, t, J 8.5 Hz, H5), 2.44 (3H, s,
ArCH3), 2.43-2.37 (1H, m, H4), 1.08-0.87 (28H, m, TIPDS);
13C NMR (CDCl3) δ 144.58, 132.86, 129.59, 127.86 (Ar), 81.89
(C2), 73.68 (C3), 68.85 (C1), 68.47 (C5), 61.80 (C5′), 49.55 (C4),
21.51 (ArCH3), 17.40-16.91, 13.37-12.35 (TIPDS).
(2S ,3S ,4S )-3-H yd r oxy-4-h yd r oxym e t h yl-3-O,O-(h y-
d r oxym eth yl)-(1,1,3,3-tetr a isop r op yld isiloxa n e-1,3-d iyl)-
2-((th ym in -1-yl)m eth yl)tetr a h yd r ofu r a n (25). Thymine
(454 mg, 3.60 mmol) was dissolved in anhydrous DMF (20 mL)
at 120 °C under a N2 atmosphere, NaH (143 mg, 60% w/w,
3.58 mmol) was added, and the suspension was stirred for 5
min. Compound 24 (978 mg, 1.19 mmol) dissolved in anhy-
drous DMF (40 mL) was slowly added, and the mixture was
stirred at 120 °C for 2.5 h. After being cooled to room
temperature, H2O (100 mL) was added, and the mixture was
extracted with EtOAc. The combined organic phase was
washed successively with saturated aqueous NaHCO3 and
H2O, evaporated to dryness under reduced pressure, and the
residue was coevaporated with n-hexane (10 mL) and toluene
(10 mL). The residue was purified by silica gel column
chromatography (14.5 × 3.3 cm) eluting with a gradient of 30-
50% EtOAc in petroleum ether (v/v) to give 25 (548 mg, 61.2%)
as a white solid material after coevaporation with CH2Cl2 (3
× 15 mL). Rf (1:1 EtOAc/petroleum ether (v/v)) 0.31; FAB-MS
m/z 499 [M + H]+; 1H NMR (CDCl3) δ 8.94 (1H, bs, NH), 7.10
(1H, d, J 0.7 Hz, H6), 4.39 (1H, d, J 4.4 Hz, H3′), 4.19 (1H, dd,
J 4.3, 5.5 Hz, H2′), 3.92-3.82 (3H, m, H1′, H5′, H5′′), 3.76-
3.70 (2H, m, H5′′, H1′), 3.51 (1H, dd, J 8.1, 11.9 Hz, H5′), 2.30
(1H, m, H4′), 1.91 (3H, s, CH3), 1.07-0.87 (28H, m, TIPDS);
13C NMR (CDCl3) δ 163.65 (C4), 150.57 (C2), 140.97 (C6),
110.21 (C5), 86.38 (C2′), 72.93 (C3′), 67.88 (C5′), 57.13 (C5′′),
49.72 (C1′), 47.90 (C4′), 17.25, 17.21, 17.15, 16.98, 16.92
(TIPDS), 13.31 (CH3), 12.65, 12.40, 12.34, 12.19 (TIPDS).
(2S ,3S ,4R )-3-H yd r oxy-4-h yd r oxym e t h yl-3-O,(O-h y-
d r oxym eth yl)-(1,1,3,3-tetr a isop r op yld isiloxa n e-1,3-d iyl)-
2-((th ym in -1-yl)m eth yl)tetr a h yd r ofu r a n (25a ). In analyti-
cal scale using conditions similar to those described above for
the preparation of 25 but reacting with a mixture of 24 and
24a , 25a was obtained in a pure form after column chromato-
graphic purification eluting with a gradient of 20-30% EtOAc/
1
petroleum ether (v/v). FAB-MS m/z 499 [M + H]+; H NMR
(CDCl3) δ 8.64 (1H, bs, NH), 7.16 (1H, d, J 0.9 Hz, H6), 4.34
(1H, dd, J 2.2, 14.1 Hz, H1′), 4.05-3.93 (3H, m, H3′, H5′, H5′′),
3.88 (1H, ddd, J 2.2, 7.8, 8.5 Hz, H2′), 3.65 (1H, dd, J 8.1, 11.8
Hz, H5′′), 3.55 (1H, dd, J 7.0, 9.2 Hz, H5′), 3.50 (1H, dd, J 8.6,
14.1 Hz, H1′), 2.40 (1H, m, H4′), 1.90 (3H, d, J 0.7 Hz, CH3),
1.09-0.94 (28H, m, TIPDS); 13C NMR (CDCl3) 163.94 (C4),
150.59 (C2), 141.03 (C6), 109.96 (C5), 82.69 (C2′), 75.77 (C3′),
68.34 (C5′), 62.26 (C5′′), 49.53 (C1′, C4′), 17.43-16.94, 13.57,
13.31, 12.90, 12.51, 12.22 (TIPDS, CH3). Anal. Calcd for
(C23H42N2O6Si2) C, 55.4; H, 8.5; N, 5.6. Found: C, 55.4; H, 8.5;
N, 5.5.
(2S ,3S ,4S )-3-H yd r oxy-4-h yd r oxym e t h yl-3-O,O-(h y-
d r oxym eth yl)-(1,1,3,3-tetr a isop r op yld isiloxa n e-1,3-d iyl)-
2-((p -t olu en esu lfon yloxy)m et h yl)t et r a h yd r ofu r a n (24)
a n d (2S,3S,4R)-3-Hyd r oxy-4-h yd r oxym eth yl-3-O,O-(h y-
d r oxym eth yl)-(1,1,3,3-tetr a isop r op yld isiloxa n e-1,3-d iyl)-
2-((p-tolu en esu lfon yloxy)m eth yl)tetr a h yd r ofu r a n (24a ).
Compound 23 (406 mg, 0.628 mmol) was dissolved in anhy-
drous toluene (20 mL), the volume of the mixture was reduced
to 75% under reduced pressure, and the resulting mixture was
heated to 110 °C under a N2 atmosphere. Bu3SnH (0.5 mL,
1.86 mmol) and AIBN (37 mg, 0.22 mmol) were added, and
the mixture was heated under reflux for 1 h. After being cooled
to room temperature, the mixture was evaporated to dryness
under reduced pressure, and the residue was purified by silica
gel column chromatography (11.0 × 3.3 cm) using CH2Cl2 as
the eluent to give 24 (197 mg, 57.6%) and 24a (20 mg, 6%) as
colorless oils. In addition, a 2:1 mixture of 24 and 24a (68 mg,
20%) was obtained. Rf (3:7 EtOAc/petroleum ether (v/v)) 0.55.
(2S,3S,4R)-3-Hyd r oxy-4-h yd r oxym eth yl-2-((th ym in -1-
yl)m eth yl)tetr a h yd r ofu r a n (26). Compound 25 (502 mg,
1.01 mmol) was dissolved in anhydrous THF (20 mL) under a
N2 atmosphere at room temperature. A solution of tertra-
butylammonium fluoride in THF (1.0 M, 5 mL, 5.0 mmol) was
slowly added, and the mixture was stirred for 10 min. The
reaction mixture was evaporated to dryness under reduced
pressure, and the residue was purified by silica gel column
chromatography (13.5 × 3.3 cm) using 10% MeOH/CH2Cl2 (v/
v) as the eluent, affording 26 (397 mg) as a white foam after
coevaporation with n-hexane (3 × 10 mL). Rf (20% MeOH in
CH2Cl2 (v/v)) 0.51; FAB-MS m/z 257 [M + H]+; 1H NMR
(CD3OD) 7.45 (1H, q, J 0.9 Hz, H6), 4.21 (1H, dd, J 2.9, 6.0
Hz, H3′), 4.09 (1H, dd, J 7.7, 8.4 Hz, H5′), 4.05 (1H, ddd, J
3.3, 4.2, 10.6 Hz, H2′), 4.00 (1H, dd, J 4.2, 13.9 Hz, H1′), 3.88
(1H, dd, J 6.3, 10.9 Hz, H5′′), 3.78-3.69 (2H, m, H5′, H1′),
3.67 (1H, dd, J 7.4, 10.9 Hz, H5′′), 2.43 (1H, m, H4′), 1.90 (3H,
d, J 1.1 Hz, CH3); 13C NMR (CD3OD) 164.88 (C4), 151.08 (C2),
142.01 (C6), 108.67 (C5), 83.73 (C2′), 72.85 (C3′), 69.16 (C5′),
58.22 (C5′′), 49.01 (C1′), 45.04 (C4′), 10.30 (CH3). This product
contained impurities which could be assigned as Bu4N+ (1H
NMR (CD3OD) 3.30-3.26 (2H, m), 1.70 (2H, quintet), 1.46 (2H,
1
Data for 24: FAB-MS m/z 545 [M + H]+; H NMR (CDCl3) δ
7.77 (2H, d, J 8.2 Hz, Ar), 7.33 (2H, d, J 8.6 Hz, Ar), 4.33 (1H,
d, J 4.6 Hz, H3), 4.07 (1H, dd, J 4.6, 6.0 Hz, H2), 4.01 (1H, dd,
J 4.3, 10.3 Hz, H1), 3.91 (1H, dd, J 6.2, 10.3 Hz, H1), 3.84-
3.78 (2H, m, H5, H5′), 3.69 (1H, dd, J 4.2, 10.8 Hz, H5′), 3.44
(1H, dd, J 7.8, 11.9 Hz, H5), 2.45 (3H, s, ArCH3), 2.40 (1H, m,
H4), 1.08-0.93 (28H, m, TIPDS); 13C NMR (CDCl3) δ 144.76,
132.62, 129.68, 127.79 (Ar), 85.10 (C2), 72.82 (C3), 68.90 (C1),
67.99 (C5), 57.19 (C5′), 47.16 (C4), 21.51 (ArCH3), 17.22, 17.15,
17.04, 16.99, 13.25, 12.52, 12.46, 12.34 (TIPDS). 24a : FAB-
1
MS m/z 545 [M + H]+; H NMR (CDCl3) δ 7.79 (2H, d, J 8.4
Hz, Ar), 7.33 (2H, d, J 8.1 Hz, Ar), 4.30 (1H, dd, J 2.4, 10.6
Hz, H1), 4.18 (1H, dd, J 7.1 Hz, H3), 4.09 (1H, dd, J 4.8, 10.6
Hz, H1), 3.95 (1H, d, J 8.6 Hz, H5), 3.90 (1H, dd, J 4.3, 12.0

5176 J . Org. Chem., Vol. 65, No. 17, 2000
Kværnø et al.
sextet, J 7.3 Hz), 1.07 (3H, t, J 7.3 Hz); 13C NMR (CD3OD)
57.63-57.57, 22.89, 18.81, 12.03; FAB-MS m/z 242 (Bu4N+)).
This crude product was used in the next step without further
purification.
amidite 11, >98% stepwise yield (15 min coupling time) for
amidite 20, and 95-99% stepwise yield (20 min coupling time)
for amidite 28. The stepwise coupling yields for the unmodified
amidites and the amidites leading to incorporation of the TL
and X monomers were >99% (standard conditions were used
except for 6 min coupling time for the amidites leading to
incorporation of the TL and X monomers). However, the
coupling with an unmodified RNA amidite (2′-O-TBDMS-
protected RNA amidites were used) following an incorporation
of amidite 20 and amidite 28 proceeded in only 95-99% and
92-98% stepwise yield, respectively, even when using the
hand-coupling conditions described above (up to 20 min
coupling time). Cleavage from the solid support and removal
of protecting groups was in general accomplished using
concentrated aqueous ammonia (55 °C for 16 h). For all
sequences containing monomer ZL or RNA monomers, methyl-
amine (40% (w/w); 10 min, 55 °C) was used instead. 13-Mers
were purified by ethanol precipitation and 9-mers by desalting
(using a Pharmacia NAP column). All sequences containing
RNA monomers were desilylated and subsequently purified
by 1-butanol precipitation as described earlier.36 Capillary gel
electrophoresis of all synthesized sequences showed >90%
purity. The compositions were verified by MALDI-MS analysis
(m/z [M-H]-: found mass; calcd mass): 5′-d(GTGAXLATGC);
2658; 2656.7; 5′-d(GTGAXATGC); 2631; 2628.7; 5′-r(GUGAXL-
AUGC); 2760; 2756.3; 5′-d(CAGTGAXATGCGA); 3875; 3873.5;
5′-d(CAGXLGAXLAXLGCGA); 3710; 3709.1; 5′-r(CAGXL-
GAXLAXLGCGA); 3870; 3869.1; 5′-d(CAGTGAXLATGCGA);
3902; 3901.5; 5′-r(CAGUGAXLAUGCGA); 4069; 4065.1; 5′-
r(CAGUGAXAUGCGA); 4038; 4037.1; 5′-d(GTGAZLATGC);
2783.6; 2783.7; 5′-d(GTGAd ZLATGC); 26765.7; 2766.7; 5′-
d(GZLGAZLAZLGC); 2842.4; 2842.4; 5′-d(Gd ZLGAd ZLAd ZL-
GC); 2794.7; 2794.5; 5′d(GCATATCAC); 2682.0; 2681.8; 5′-
r(GUGAZLAUGC); 2883.1; 2882.3; 5′-r(GUGAd ZLAUGC);
2866.9; 2866.3; 5′-r(GZLGAZLAZLGC); 2939.1; 2938.4; 5′-
r(Gd ZLGAd ZLAd ZLGC); 2890.3; 2890.4; 5′-d(CAGTGAZLAT-
GCGA); 4030.4; 4027.5; 5′-d(CAGTGAd ZLATGCGA); 4013.2;
4011.5; 5′-d(CAGZLGAZLAZLGCGA); 4087.9; 4087.2; 5′-d(CAG-
d ZLGAd ZLAd ZLGCGA); 4039.2; 4039.3; 5′-d(T7ZLT6); 4222.1;
4225.6; 5′-d(T7dZLT6); 4206.5; 4209.6; 5′-d(TCGCATATCACTG);
3907.5; 3908.6; 5′-r(CAGUGAZLAUGCGA); 4191.2; 4191.1; 5′-
r(CAGUGAd ZLAUGCGA); 4176.3; 4175.1; 5′-r(CAGZLGAZLA-
ZLGCGA); 4247.2; 4247.2; 5′-r(CAGd ZLGAd ZLAd ZLGCGA);
4198.5; 4199.2.
(2S,3S,4S)-4-(4,4′-Dim eth oxytr ityl)oxym eth yl-3-h ydr oxy-
2-((th ym in -1-yl)m eth yl)tetr a h yd r ofu r a n (27). Crude prod-
uct 26 (371 mg) in anhydrous pyridine (20 mL) was concen-
trated under reduced pressure to ca. three-quarters volume,
and the resulting mixture was cooled to 0 °C under a N2
atmosphere. 4,4′-Dimethoxytrityl chloride (970 mg, 2.85 mmol)
was added, and after being stirred at room temperature for 2
h, MeOH (3 mL) was added. The mixture was evaporated to
dryness under reduced pressure, coevaporated with aceto-
nitrile (2 × 20 mL), and the residue was purified by silica gel
column chromatography (17.0 × 3.3 cm) using 0.5:25-60:74.5-
39.5 pyridine/EtOAc/CH2Cl2 (v/v/v) as the gradient eluent to
give 27 (333 mg, 63.4% from 25) as a white foam after
coevaporation with acetonitrile (2 × 10 mL) and n-hexane (2
× 10 mL). Rf (10% MeOH in CH2Cl2 (v/v)) 0.47; FAB-MS m/z
1
558.1 [M]+; H NMR (CDCl3) δ 9.27 (1H, bs, NH), 7.41-7.39
(2H, m, DMT), 7.31-7.18 (7H, m, DMT), 7.09 (1H, d, J 0.9
Hz, H6), 6.84-6.81 (4H, m, DMT), 4.16 (1H, m, H3′), 4.05-
3.92 (3H, m, H5′, H1′, H2′), 3.78 (6H, s, OCH3), 3.74-3.67 (2H,
m, H5′, H1′), 3.32 (2H, d, J 6.8 Hz, H5′′), 3.04 (1H, d, J 4.4
Hz, OH), 2.47 (1H, m, H4′), 1.89 (3H, d, J 0.7 Hz, CH3); 13C
NMR (CDCl3) δ 164.16 (C4), 158.35 (DMT), 151.27 (C2), 144.46
(DMT), 141.20 (C6), 135.65, 135.54, 129.75, 127.80, 127.74,
126.70, 113.06 (DMT), 110.23 (C5), 86.40 (CPhAr2), 83.67 (C2′),
73.84 (C3′), 69.78 (C5′), 60.37 (C5′′), 55.06 (OCH3), 49.24 (C1′),
43.17 (C4′), 12.17 (CH3).
(2S ,3S ,4S )-3-(2-C y a n o e t h o x y (d iis o p r o p y la m in o )-
p h osp h in oxy)-4-((4,4′-d im e t h oxyt r it yl)oxym e t h yl)-2-
((th ym in -1-yl)m eth yl)tetr a h yd r ofu r a n (28). Compound 27
(286 mg, 0.512 mmol) was dissolved in a mixture of anhydrous
CH2Cl2 (10 mL) and N,N-diisopropylethylamine (2.5 mL) under
a N2 atmosphere at 0 °C. 2-Cyanoethyl N,N-diisopropylphos-
phoramidochloridite (0.6 mL, 2.69 mmol) was added, and the
mixture was stirred at room temperature for 4 h. H2O (5 mL)
and EtOAc (150 mL) were added, and the solution was washed
successively with saturated aqueous NaHCO3 and H2O. The
combined organic phase was evaporated to dryness under
reduced pressure, and the residue was coevaporated with
acetonitrile (2 × 20 mL). The residue was purified by silica
gel column chromatography (17.0 × 3.3 cm) using 0.5:24.5:75
pyridine/EtOAc/CH2Cl2 (v/v/v) as the eluent, yielding amidite
29 (263 mg, 67.8%) as a white foam after coevaporation with
acetonitrile (3 × 20 mL). Rf (1:1 EtOAc/CH2Cl2 (v/v)) 0.52; FAB-
MS m/z 759 [M + H]+; 31P NMR (DMSO-d6) 150.62, 148.63.
Oligon u cleotid e Syn th esis. All oligomers were synthe-
sized on a DNA synthesizer using the phosphoramidite ap-
proach.31 The stepwise coupling yields were determined spec-
trophotometrically at 498 nm (quantifying the released 4,4′-
dimethoxytrityl group). The modified amidites 11, 20, and 28
were “hand-coupled” (premixing a ∼0.05 M solution of amidite
in anhydrous acetonitrile (0.2 mL, 10 µmol) and a ∼0.5 M
solution of tetrazole in anhydrous acetonitrile (0.3 mL, 150
µmol) in a syringe and via an adaptor slowly flushing this
mixture through a synthesis column for the coupling times
indicated below). The results for the modified amidites were
as follows: >99% stepwise yield (6 min coupling time) for
Ack n ow led gm en t. The Danish Natural Science
Research Council, The Danish Technical Research
Council, and Exiqon A/S are thanked for financial
support.
Su p p or tin g In for m a tion Ava ila ble: Copies of 13C NMR
spectra for 1, 3, 6-10, 12-19, 21-27, 24a , and 25a and copies
of the 31P NMR spectra for 11, 20, and 28. This material is
available free of charge via the Internet at http://pubs.acs.org.
J O000275X
(36) Wincott, F.; Direnzo, A.; Shaffer, C.; Grimm, S.; Tracz, D.;
Workman, C.; Sweedler, D.; Gonzalez, C.; Scaringe, S.; Usman, N.
Nucleic Acids Res. 1995, 23, 2677.