Synthesis of 2′-amino-LNA: A novel conformationally restricted high-affinity oligonucleotide analogue with a handle

Full text of DOI:10.1021/jo9814445

J . Org. Chem. 1998, 63, 10035-10039
10035
duplex formation. However, as hydration is known to be
very important for the thermodynamic stability of du-
plexes,7 the thermodynamic parameters could be very
different in the case of, e.g., LNA/DNA and LNA/RNA
duplexes.8
Syn th esis of 2′-Am in o-LNA: A Novel
Con for m a tion a lly Restr icted High -Affin ity
Oligon u cleotid e An a logu e w ith a Ha n d le
Sanjay K. Singh, Ravindra Kumar, and J esper Wengel*
2′-Amino-LNA analogues appealed to us for several
reasons. First, the 2′-amino functionality should be a
structurally well-defined conjugation site9 in a confor-
mationally restricted LNA-type ON. Second, amine-
derivatized ONs have displayed increased thermal af-
finities toward complementary ONs possibly because of
the introduction of positively charged moieties (and thus
partial neutralization of the otherwise negatively charged
strands in a duplex) at physiological pH.10 In this report,
we describe the syntheses of the 2′-amino-LNA and 2′-
methylamino-LNA monomeric nucleosides 4 and 10, the
efficient oligomerization of the corresponding phosphor-
amidite building blocks 7 and 12, and the excellent
hybridization properties of 9-mer 2′-amino-LNAs (mono-
mers V and X).
Center for Synthetic Bioorganic Chemistry, Department of
Chemistry, University of Copenhagen, Universitetsparken 5,
DK-2100 Copenhagen, Denmark
Received J uly 23, 1998
In tr od u ction
The potential of chemically modified oligonucleotide
(ON) analogues as therapeutic agents or diagnostic
molecules has stimulated great interest in nucleic acid
mimics during recent years.1 Conformationally restricted
analogues containing bicyclic or tricyclic carbohydrate
moieties have been synthesized, and interesting proper-
ties, e.g., enhanced affinity toward complementary DNA
and/or RNA strands, have been obtained.2 We have
recently introduced LNA (locked nucleic acids) as a novel
class of conformationally restricted oligonucleotide ana-
logues containing 2′-O,4′-C-methylene ribonucleoside
LNA monomers.3-5 Among the intriguing properties of
LNA are unprecedented affinity toward complementary
DNA/RNA, excellent base-pairing selectivity obeying the
Watson-Crick hydrogen bonding rules, and the struc-
tural similarity with parent DNA/RNA allowing efficient
automated oligomerization without additional protecting
groups.3 The excellent hybridization characteristics of
LNA are expected to be related to the fixed 3′-endo (3E)
conformation of the monomeric LNA nucleosides6 (e.g.,
see monomer U) possibly resulting in entropically favored
Resu lts a n d Discu ssion
A key step in our synthesis of the parent LNA mono-
meric nucleosides was base-induced ring closure of 4′-C-
tosyloxymethyl derivatives by intramolecular nucleo-
philic attack from the 2′-OH group positioned at the
R-face of the pentofuranose ring.3a-c We envisioned that
synthesis of the 2′-benzylamino nucleoside 3 should be
possible via a double nucleophilic substitution reaction
on di-O-tosyl derivative 2.11 4′-C-Hydroxymethyl nucleo-
side diol 13b was transformed into nucleoside 2 in 80%
yield by treatment with p-toluenesulfonyl chloride and
4-N,N-(dimethylamino)pyridine (DMAP) in dichloro-
methane. Reaction of di-O-tosyl derivative 2 in neat
benzylamine at 130 °C afforded in 52% yield the 2′-
benzylamino-2′-deoxynucleoside 3 with the desired 2,5-
oxazabicyclo[2.2.1]heptane skeleton. In a small scale
(1) (a) Uhlmann, E.; Peyman, A. Chem. Rev. 1990, 90, 544. (b)
Beaucage, S. L.; Iyer, R. P. Tetrahedron 1993, 49, 6123. (c) Wagner,
R. W. Nature 1994, 372, 333. (d) De Mesmaeker, A.; Ha¨ner, R.; Martin,
P.; Moser, H. E. Acc. Chem. Res. 1995, 28, 366. (e) Herdewijn, P. Liebigs
Ann. Chem. 1996, 1337. (e) Akhtar, S.; Agrawal, S. Trends Pharmacol.
Sci. 1997, 18, 12. (f) Freier, S. M.; Altmann, K.-H. Nucleic Acids Res.
1997, 25, 4429. (g) Cool, E. T. Chem. Rev. 1997, 97, 1473.
(2) (a) Tarko¨y, M.; Leumann, C. Angew. Chem., Int. Ed. Engl. 1993,
32, 1432. (b) Altmann, K.-H.; Kesselring, R.; Francotte, E.; Rihs, G.
Tetrahedron Lett. 1994, 35, 2331. (c) Bolli, M.; Litten, J . C.; Schu¨tz,
R.; Leumann, C. J . Chem. Biol. 1996, 3, 197. (d) Marquez, V. E.;
Siddiqui, M. A.; Ezzitouni, A.; Russ, P.; Wang, J .; Wagner, R. W.;
Matteucci, M. D. J . Med. Chem. 1996, 39, 3739. (e) Steffens, R.;
Leumann, C. J . J . Am. Chem. Soc. 1997, 119, 11548. (f) Nielsen, P.;
Pfundheller, H. M.; Wengel, J . Chem. Commun. 1997, 825. (g) Nielsen,
P.; Pfundheller, H. M.; Olsen, C. E.; Wengel, J . J . Chem. Soc., Perkin
Trans. 1 1997, 3423. (h) Christensen, N. K.; Petersen, M.; Nielsen, P.;
J acobsen, J . P.; Olsen, C. E.; Wengel, J . J . Am. Chem. Soc. 1998, 120,
5458.
(6) Preorganization of LNA nucleosides into a 3′-endo type confor-
mation has been shown by X-ray crystallography (ref 5) and by NMR
studies (ref 3).
(7) The importance of hydration on duplex stability has been
reported for conformationally restricted N3′-P5′-phosphoramidate
DNA: Egli, M. Antisense Nucleic Acids Drug Dev. 1998, 8, 123.
(8) Preliminary thermodynamic analysis of binding between a fully
modified LNA and complementary DNA has indicated a significant
enthalpic advantage compared to the corresponding unmodified con-
trol: Koshkin, A. A.; Nielsen, P.; Meldgaard, M.; Rajwanshi, V. K.;
Singh, S. K.; Wengel, J . J . Am. Chem. Soc., in press. A favorable
entropy term has been reported for a duplex between a partly modified
LNA and RNA (ref 5).
(3) (a) Singh, S. K.; Nielsen, P.; Koshkin, A. A.; Wengel, J . Chem.
Commun. 1998, 455. (b) Koshkin, A. A.; Singh, S. K.; Nielsen, P.;
Rajwanshi, V. K.; Kumar, R.; Meldgaard, M.; Olsen, C. E.; Wengel, J .
Tetrahedron 1998, 54, 3607. (c) Koshkin, A. A.; Rajwanshi, V. K.;
Wengel, J . Tetrahedron Lett. 1998, 39, 4381. (d) Singh, S. K.; Wengel,
J . Chem. Commun. 1998, 1247.
(4) We have defined LNA as ONs containing one or more 2′-O,4′-
C-methylene bicyclic ribonucleoside LNA monomers. 2′-Amino-LNA is
defined as an ON containing one or more 2′-N,4′-C-methylene bicyclic
2′-amino-LNA monomers (possibly N-derivatized, e.g., N-methylated
to give 2′-methylamino-LNA).
(5) The LNA nucleosides containing uracil and cytosine nucleobases
have been synthesized independently by a linear approach: Obika,
S.; Nanbu, D.; Hari, Y.; Morio, K.; In, Y.; Ishida, T.; Imanishi, T.
Tetrahedron Lett. 1997, 38, 8735. Oligomers containing these pyrimi-
dine bases have recently been reported: Obika, S.; Nanbu, D.; Hari,
Y.; Andoh, J .; Morio, K.; Doi, T.; Imanishi, T. Tetrahedron Lett. 1998,
39, 5401.
(9) A 4′-C-alkylamino group has been used as a conjugation site:
Maag, H.; Schmidt, B.; Rose, S. J . Tetrahedron Lett. 1994, 35, 6449.
(10) (a) Fathi, R.; Huang, Q.; Coppola, G.; Delaney, W.; Teasdale,
R.; Krieg, A. M.; Cook, A. F. Nucleic Acids Res. 1994, 22, 5416. (b)
Ozaki, H.; Nakamura, A.; Arai, M.; Endo, M.; Sawai, H. Bull. Chem.
Soc. J pn. 1995, 68, 1981. (c) Nomura, Y.; Haginoya, N.; Ueno, Y.;
Matsuda, A. Bioorg. Med. Chem. Lett. 1996, 6, 2811. (d) Blasko´, A.;
Dempcy, R. O.; Minyat, E. E.; Bruice, T. C. J . Am. Chem. Soc. 1996,
118, 7892. (e) Horn, T.; Chaturvedi, S.; Balasubramaniam, T. N.;
Letsinger, R. L. Tetrahedron Lett. 1996, 37, 743. (f) Manoharan, M.;
Ramasamy, K. S.; Mohan, V.; Cook, P. D. Tetrahedron Lett. 1996, 37,
7675. (g) Linkletter, B. A.; Bruice, T. C. Bioorg. Med. Chem. Lett. 1998,
8, 1285. (h) Cuenoud, B.; Casset, F.; Hu¨sken, D.; Natt, F.; Wolf, R. M.;
Altmann, K.-H.; Martin, P.; Moser, H. E. Angew. Chem., Int. Ed. 1998,
37, 1288.
(11) In a communication, we have reported synthesis of the unpro-
tected 2′-amino-LNA and 2′-thio-LNA nucleosides: Singh, S. K.;
Kumar, R.; Wengel, J . J . Org. Chem. 1998, 63, 6078.
10.1021/jo9814445 CCC: $15.00 © 1998 American Chemical Society
Published on Web 11/26/1998

10036 J . Org. Chem., Vol. 63, No. 26, 1998
Notes
Sch em e 1. Syn th esis of 2′-Am in o-LNA Nu cleosid e
reaction conducted at only 80 °C, another product was
isolated in trace amounts, which on the basis of NMR
data (only one tosyl CH3 signal, changed chemical shift
values of the thymine moiety) and MS data was identified
as the 2,2′-anhydro nucleoside derivative 2a .12 Thus, in
analogy with earlier reports on double inversion at the
2′-position of nucleosides when introducing 2′-substitu-
ents,13 we postulate the intermediacy of 2,2′-anhydro
nucleoside 2a in the transformation of nucleoside 2 into
bicyclonucleoside 3. However, as intramolecular displace-
ment by an O-2 carbonyl is required for the suggested
mechanism, it can be anticipated that an alternative
synthetic strategy has to be developed for preparation of
the corresponding purine bicyclonucleosides. Debenzyl-
ation of 3 to give the unprotected 2′-amino-LNA nucleo-
side 4 was accomplished in 68% yield using ammonium
formate and 10% palladium on carbon (Scheme 1). This
method proved superior to catalytic hydrogenation (20%
Pd(OH)2/C, H2) furnishing a monobenzylated intermedi-
ate which eventually was transformed to product 4 using
ammonium formate and 10% palladium on carbon.11 For
automated oligonucleotide synthesis by the phosphor-
amidite approach,14 the 5′-O-dimethoxytrityl phosphor-
amidite 7 was synthesized. To avoid N-branching during
oligomerization, the N-trifluoroacetyl-protected deriva-
tive was prepared. Thus, using essentially the method
described by Ozaki et al.,10b nucleoside 5 was obtained
in 81% yield from 4 by reaction with ethyl trifluoroacetate
and DMAP. Subsequent 4,4′-dimethoxytritylation (to give
nucleoside 6 in 93% yield) and 3′-O-phosphitylation (57%
yield) afforded the nucleoside phosphoramidite 7 after
precipitation from cold petroleum ether (Scheme 1). It is
evident from the NMR data that the N-trifluoroacetyl
derivatives 5-7 exist as a mixture of rotamers.
4 a n d Am id ite Der iva tive 7a
As mentioned above, the 2′-amino group of a 2′-amino-
LNA nucleoside incorporated into an ON offers the
possibility of attachment of, e.g., reporter groups, lipo-
philic carriers, or an aminoalkyl chain. Although some
derivatizations of amino groups can be performed after
oligomerization,9 the use of N-alkylated 2′-amino-LNA
monomers is a more appealing alternative expected to
yield more well-defined oligomers. As the first step in this
direction, we have developed a synthetic route to the 2′-
methylamino-LNA nucleoside 10 and its phosphoramid-
ite derivative 12 (Scheme 2). Direct alkylation of the
unprotected 2′-amino-LNA nucleoside 4 using methyl
iodide and NaH in DMF afforded a number of products
according to analytical TLC. For simplicity, we therefore
decided to block the hydroxyl groups by reaction with the
bidentate reagent 1,3-dichloro-1,1,3,3-tetraisopropyldi-
siloxane in pyridine, affording 3,5-di-O-silyl tricyclo-
nucleoside 8 in 97% yield. By treatment with methyl
iodide and 1,8-diazabicyclo[5.4.0]undec-7-ene in a mix-
ture of THF and dichloromethane, alkylation proceeded
efficiently to give 2′-methylamino derivative 9 in 74%
yield and subsequent desilylation furnished 2′-methyl-
amino-LNA nucleoside 10 in 79% yield. One significant
advantage of introducing N-substituents before oligo-
merization is that, depending in the particular substitu-
a
Key: (a) TsCl, DMAP, anhydrous CH2Cl2; (b) BnNH2; (c) 10%
palladium on carbon, ammonium formate, CH3OH; (d) ethyl
trifluoroacetate, DMAP, CH3OH; (e) 4,4′-dimethoxytrityl chloride,
anhydrous pyridine, anhydrous dichloromethane; (f) N,N-diiso-
propylethylamine, 2-cyanoethyl N,N-diisopropylphosphoramido-
chloridite, anhydrous dichloromethane; (g) DNA synthesizer. DMT
) 4,4′-dimethoxytrityl.
ent, the need of introducing an additional N-protecting
group is obliterated. To allow evaluation of N-methylated
2′-amino-LNA (monomer X), the nucleoside phsophor-
amidite 12 (Scheme 2) was synthesized by 5′-O-4,4′-
dimethoxytritylation (to give nucleoside 11 in 72% yield)
and subsequent 3′-O-phosphitylation (52% yield after
precipitation from cold petroleum ether).
2′-Amino-LNAs (monomers V and X) were efficiently
synthesized using the phosphoramidite approach14 on an
automated DNA synthesizer. The stepwise coupling
efficiencies as evaluated spectrophotometrically by the
release of the 4,4′-dimethoxytrityl cation after each
coupling step were >99% for all amidites, with coupling
times of 12 min for amidites 7 and 12 and 2 min for
unmodified 2′-deoxynucleoside phosphoramidites. Due to
the satisfactory stepwise coupling yields rendering re-
versed-phase purification unnecessary, the 5′-O-4,4′-
(12) Derivative 2a : FAB-MS m/z 605 [M + H]+; 13C NMR (CDCl3,
62.9 MHz, selected signals) 172.0, 159.1, 21.7. No trace of bicyclo-
nucleoside 3 was detected.
(13) Verheyden, J . P. H.; Moffatt, J . G. J . Org. Chem. 1970, 35, 2868.
(14) Caruthers, M. H. Acc. Chem. Res. 1991, 24, 278.
(15) (a) Altona, C.; Sundaralingam, M. J . Am. Chem. Soc. 1973, 95,
2333. (b) Obika, S.; Morio, K.; Nanbu, D.; Imanishi, T. Chem. Commun.
1997, 1643.

Notes
J . Org. Chem., Vol. 63, No. 26, 1998 10037
Ta ble 1. Sequ en ces Syn th esized a n d Meltin g
Sch em e 2. Syn th esis of 2′-Meth yla m in o-LNA
Nu cleosid e 10 a n d Am id ite Der iva tive 12a
Tem p er a tu r es (Tm Va lu es) Mea su r ed
DNA
RNA
complement
complement
Tm,
°Cb
∆Tm, Tm, -∆Tm,
entry
sequencea
°Cc
°Cb
°Cc
Reference Sequences
1
2
3
4
5′-d(GTGATATGC)
30
32
44
52
28
30
50
67
5′-d(GTGTTTTGC)
5′-(GTLGATLATLGC)
5′-d(GTLGTLTLTLTLGC)
2′-Amino-LNA Sequences
5
6
5′-d(GTGAVATGC)
5′-d(GVGAVAVGC)
33
39
+3.0
+3.0
34
47
+6.0
+6.3
2′-Methylamino-LNA Sequences
7
8
9
5′-d(GTGAXATGC)
5′-d(GXGAXAXGC)
5′-(GXGVXVXGC)
33
39
47
+3.0
+3.0
+3.0
36
49
63
+8.0
+7.0
+6.6
a
A ) 2′-deoxyadenosine monomer, C ) 2′-deoxycytidine mono-
mer, G ) 2′-deoxyguanosine monomer, T ) thymidine monomer,
V ) 2′-amino-LNA monomer (see below), X ) 2′-methylamino-
LNA monomer (see below). b Tm ) melting temperature (measured
in medium salt buffer: 10 mM Na2HPO4, pH 7.0, 100 mM NaCl,
0.1 mM EDTA). c ∆Tm ) change in Tm per 2′-amino-LNA monomer
V and/or X.
a
Key: (a) 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane, anhy-
drous pyridine; (b) methyl iodide, 1,8-diazabicyclo[5.4.0]undec-7-
ene, anhydrous THF, anhydrous dichloromethane; (c) tetrabutyl-
ammonium fluoride, THF; (d) 4,4′-dimethoxytrityl chloride, an-
hydrous pyridine, anhydrous dichloromethane; (e) N,N-diisopro-
pylethylamine, 2-cyanoethyl N,N-diisopropylphosphoramidochlor-
idite, anhydrous dichloromethane; (f) DNA-synthesizer. [Si] )
1,1,3,3-tetraisopropyldisiloxane-1,3-diyl. DMT ) 4,4′-dimethoxy-
trityl.
toward DNA and ∆Tm ) +6.6 °C toward RNA) corre-
sponding to an approximately additive effect of the
monomers. The lack of coupling constants between H-1′
and H-2′ in the 1H NMR spectra of 4 and 10 indicate that
these 2′-amino-LNA nucleosides, like the parent LNA
nucleosides, exist in a 3′-endo type of conformation.15 This
is furthermore shown by the strong NOEs observed
between H-3′ and H-6 (6% NOE (for 4) and 8% NOE (for
10) in H-3′ by irradiation of H-6). Thus, the comparable
thermal stability of duplexes involving 2′-amino-LNA and
parent LNA3 probably reflects the similar monomeric
pentofuranose conformation and similar extent of hydra-
tion, and no indication of an additional stabilizing effect
by the introduction of the basic 2′-amino functionality
was observed. Therefore, the locked ribo-configured
bicyclo[2.2.1]pentofuranose skeleton appears in general
to be a convenient structural element in high-affinity
nucleic acid mimics designed to contain monomeric
nucleosides in a conformationally restricted 3′-endo type
conformation.
dimethoxytrityl group was removed after completion of
the oligomerizations. After deprotection and cleavage
from the solid support using concentrated ammonia,
evaporation, and precipitation from ethanol, the 2′-
amino-LNA 9-mer mixed-base sequences depicted in
Table 1 (entries 5-9) were obtained. The purity was in
all cases analyzed by capillary gel electrophoresis to be
>90%, and the composition was verified by MALDI-MS.
As expected, the N-trifluoroacetyl groups were efficiently
removed during treatment with concentrated ammonia.
The thermal stabilities (Tm values) of duplexes involv-
ing 2′-amino-LNAs were determined toward complemen-
tary DNA and RNA (Table 1, entries 5-9) and compared
to the thermal stabilities of the unmodified reference
duplexes and LNA reference duplexes (Table 1, entries
1-4, ∆Tm ) change in Tm per 2′-amino-LNA monomer
incorporated). Generally, as observed for LNA monomer
TL,3 significantly increased thermal affinities were ob-
tained toward both DNA and RNA, however, most
convincingly toward the latter (∆Tm ) +6.0 to +8.0 °C).
Whereas the effect toward DNA was identical for mono-
mers V and X (∆Tm ) +3.0 °C), it appears that toward
RNA the stabilizing effect of 2′-methylamino-LNA mono-
mer X is slightly more pronounced than that of 2′-amino-
LNA monomer V. It is noteworthy that the 2′-amino-LNA
shown in entry 9 containing both monomers V and X
displayed enhanced thermal stabilities (∆Tm ) +3.0 °C
Con clu sion
A synthetic route to unprotected 2′-amino-LNA nucleo-
sides has been developed embarking on double nucleo-
philic substitution of di-O-tosyl nucleoside 1 using ben-
zylamine. The overall retention of configuration at the
2′-position is suggested to be a result of the intermediacy
of 2,2′-anhydro nucleoside 2a , which apparently limits
the applicability of the described strategy to pyrimidine
nucleosides. Alkylation of the 2′-amino group to give 2′-
methylamino-LNA nucleoside 10 has been accomplished

10038 J . Org. Chem., Vol. 63, No. 26, 1998
Notes
[M + H]+; 1H NMR ((CD3)2SO, 250 MHz) δ 11.29 (bs, 1H, NH),
7.73 (d, 1H, J 1.1 Hz, 6-H), 5.31 (s, 1H, 1′-H), 5.29 (bd, 1H, J 3.7
Hz, 3′-OH), 5.13 (t, 1H, J 5.3 Hz, 5′-OH), 3.81 (s, 1H, 3′-H), 3.69
(d, 2H, J 5.1 Hz, 5′-H), 3.23 (s, 1H, 2′-H), 2.88 (d, 1H, J 9.8 Hz,
1′′-Ha), 2.55 (d, 1H, J 9.8 Hz, 1′′-Hb), 1.77 (d, 3H, J 0.8 Hz, CH3
); 13C NMR ((CD3)2SO, 62.9 MHz) δ 164.0, 150.1, 135.6, 107.8,
89.5, 87.9, 68.7, 61.9, 57.1, 49.4, 12.4. Anal. Calcd for C11H15N3O5‚
0.5H2O: C, 47.5; H, 5.8; N, 15.1. Found: C, 47.6; H, 5.3; N, 14.8.
1-(2-Am in o-2-N,4-C-m eth ylen e-2-N-tr iflu or oa cetyl-â-D-r i-
b ofu r a n osyl)t h ym in e (5). To a suspension of nucleoside 4
(0.050 g, 0.186 mmol) in methanol (2 mL) was added N,N-
(dimethylamino)pyridine (0.013 mg, 0.106 mmol) and ethyl
trifluoroacetate (0.029 mL, 0.242 mmol), and the mixture was
stirred at room temperature for 2.5 h. The solvent was removed
under reduced pressure, and the residue was subjected to column
chromatography on silica gel using methanol/dichloromethane
(2.5:97.5, v/v) as eluent to give nucleoside 5 as a white solid
material after evaporation of the solvents under reduced pres-
sure (0.055 g, 81%): FAB-MS m/z 366 [M + H]+; 13C NMR
efficiently, furnishing a convenient route for future
derivatizations of 2′-amino-LNA nucleosides. High-yield-
ing oligomerizations afforded the first 2′-amino-LNA
oligomers (containing monomers V and/or X), which
displayed significantly increased thermal stability toward
complementary DNA (∆Tm ) +3.0 °C) and RNA (∆Tm
)
+6.0 to +8.0 °C). The applicability of the 2′-amino group
as a handle for conjugation of high-affinity LNAs has
been indicated by the properties of 2′-methylamino-LNA.
Exp er im en ta l Section
Gen er a l. Reactions were conducted under an atmosphere of
nitrogen when anhydrous solvents were used. Column chroma-
tography was carried out on glass columns using Silica gel 60
(0.040-0.063 mm). Values for δ are in ppm relative to tetra-
methylsilane as internal standard (1H and 13C NMR) and
relative to 85% H3PO4 as external standard (31P NMR). 1H NOE
spectra were recorded for compounds 4 and 10. Fast-atom
bombardment mass spectra (FAB-MS) were recorded in positive
ion mode. Microanalyses were performed at The Microanalytical
Laboratory, Department of Chemistry, University of Copen-
hagen.
2
(CD3OD, 62.9 MHz) δ 166.5, 157.7 (q, J C,F 37.5 Hz, COCF3),
2
157.6 (q, J C,F 37.2 Hz, COCF3), 151.8, 136.8, 136.8, 117.6 (d,
1J C,F 287.5 Hz, CF3), 117.5 (d, 1J C,F 286.5 Hz, CF3), 110.8, 110.8,
90.7, 89.3, 87.7, 87.3, 70.1, 68.6, 66.2, 66.2, 64.5, 57.9, 53.3, 12.7.
Anal. Calcd for C13H14N3O6F3: C, 42.8; H, 3.9; N, 11.5. Found:
C, 42.5; H, 4.0; N, 11.2.
1-(3,5-Di-O-ben zyl-2-O-p-tolu en esu lfon yl-4-C-(p-tolu en e-
su lfon yloxym eth yl)-â-D-r ibofu r a n osyl)th ym in e (2). A solu-
tion of 1-(3,5-di-O-benzyl-4-C-(hydroxymethyl)-â-D-ribofuranosyl)-
thymine3b (1, 1.48 g, 3.16 mmol), N,N-(dimethylamino)pyridine
(1.34 g, 10.5 mmol), and p-toluenesulfonyl chloride (1.45 g, 7.6
mmol) in dichloromethane (20 mL) was stirred for 3 h at room
temperature. The reaction mixture was diluted with dichloro-
methane (30 mL), washed with saturated aqueous solutions of
sodium hydrogencarbonate (3 × 20 mL) and sodium chloride (2
× 25 mL), dried (Na2SO4), and filtered. After evaporation to
dryness under reduced pressure, the residue was subjected to
column chromatography on silica gel using methanol/dichloro-
methane (1:99, v/v) as eluent to give nucleoside 2 as a white
solid material after evaporation of the solvents under reduced
pressure (1.95 g, 80%): FAB-MS m/z 776 [M+]; 1H NMR (CDCl3,
250 MHz) δ 8.50 (bs, 1H), 7.70-6.79 (m, 19H), 6.03 (d, 1H, J
8.0 Hz), 5.00-4.93 (m, 2H), 4.59 (m, 2H), 4.48 (d, 1H, J 10.8
Hz), 4.37 (d, 1H, J 4.9 Hz), 4.10 (d, 1H, J 10.4 Hz), 4.02 (d, 1H,
J 10.4), 3.75 (d, 1H, J 10.2 Hz), 3.66 (d, 1H, J 10.2 Hz), 2.41 (s,
3H), 2.32 (s, 3H), 1.37 (s, 3H); 13C NMR (CDCl3, 62.9 MHz) δ
162.9, 149.8, 145.8, 145.2, 136.9, 136.8, 134.3, 132.1, 132.0, 130.0,
129.9, 129.0 128.9, 128.4, 128.3, 128.2, 128.0, 127.7, 111.2, 85.3,
84.0, 78.9, 78.3, 75.2, 74.3, 72.7, 69.1, 21.7, 11.9. Anal. Calcd
for C39H40N2S2O11: C, 60.3; H, 5.2; N, 3.6. Found: C, 60.0; H,
5.1; N 3.8.
1-(2-Am in o-2-N,4-C-m et h ylen e-2-N,3-O,5-O-t r ib en zyl-â-
D-r ibofu r a n osyl)th ym in e (3). A solution of nucleoside 2 (8.0
g, 10.0 mmol) in benzylamine (5 mL) was stirred at 130 °C for
20 h. The reaction mixture was subsequently directly subjected
to column chromatography on silica gel using methanol/dichloro-
methane (1:99, v/v) as eluent to give nucleoside 3 as a white
solid material after evaporation of the solvents under reduced
pressure (2.9 g, 52%): FAB-MS m/z 540 [M + H]+; 1H NMR
(CDCl3, 250 MHz) δ 7.58 (d, 1H, J 1.1 Hz), 7.40-7.23 (m, 15H),
5.79 (s, 1H), 4.68-4.48 (m, 4H), 4.08 (s, 2H), 3.95 (s, 1H), 3.78
(m, 2H), 3.66 (s, 1H, 2′-H), 3.04 (d, 1H, J 9.5 Hz), 2.73 (d, 1H, J
9.5 Hz), 1.57 (s, 3H, CH3); 13C NMR (CDCl3, 62.9 MHz) δ 163.9,
149.8, 139.2, 137.6, 137.3, 135.6, 128.5, 128.4, 128.3, 128.2, 128.0,
127.7, 127.0, 109.6, 88.2, 86.3, 76.7, 73.8, 72.0, 66.0, 63.8, 57.9,
57.8, 12.2. Anal. Calcd for C32H33N3O5‚0.5H2O: C, 70.1; H, 6.3;
N, 7.7. Found: C, 70.0; H, 6.1; N, 7.5.
1-(2-Am in o-5-O-4,4′-d im eth oxytr ityl-2-N,4-C-m eth ylen e-
2-N-tr iflu or oa cetyl-â-D-r ibofu r a n osyl)th ym in e (6). To a
solution of nucleoside 5 (0.030 g, 0.082 mmol) in anhydrous
pyridine (0.6 mL) at 0 °C was added dropwise (during 20 min)
4,4′-dimethoxytrityl chloride (0.054 g, 0.159 mmol) dissolved in
anhydrous pyridine/dichloromethane (0.6 mL, 1:1, v/v), and the
mixture was stirred for 10 h at room temperature. A mixture of
ice and water was added (5 mL), and the resulting mixture was
extracted with dichloromethane (3 × 5 mL). The combined
organic phase was washed with a saturated aqueous solution
of sodium hydrogencarbonate (3 × 2 mL), dried (Na2SO4), and
filtered. The filtrate was evaporated to dryness under reduced
pressure, and the residue was subjected to column chromatog-
raphy on silica gel using methanol/dichloromethane/pyridine
(1.5:98.0:0.5, v/v/v) as eluent to give nucleoside 6 as a white solid
material after evaporation of the solvents under reduced pres-
sure (0.051 g, 93%): FAB-MS m/z 667 [M]+, 668 [M + H]+; FAB-
HRMS calcd for C34H32N3O8F3+ 667.2142, found 667.2146; 13C
NMR (C5D5N, 100.6 MHz) δ 165.1, 165.0, 159.5, 159.5, 151.4,
145.7, 136.3, 136.1, 134.8, 134.6, 130.9, 130.9, 130.9, 128.9, 128.9,
128.7, 128.7, 128.4, 127.7, 123.2, 114.1, 114.1, 114.0, 110.4, 89.4,
87.9, 87.5, 87.4, 87.2, 70.8, 69.0, 66.0, 64.4, 60.5, 60.2, 55.5, 53.6,
53.4, 49.9, 13.2, 13.1.
1-(2-Am in o-3-O-(2-c ya n oe t h oxy(d iisop r op yla m in o)-
p h osp h in oxy)-5-O-4,4′-d im eth oxytr ityl-2-N,4-C-m eth ylen e-
2-N-tr iflu or oa cetyl-â-D-r ibofu r a n osyl)th ym in e (7). To a
solution of nucleoside 6 (0.121 g, 0.181 mmol) in anhydrous
dichloromethane (2 mL) were added N,N-diisopropylethylamine
(0.093 mL, 0.54 mmol) and 2-cyanoethyl N,N-diisopropylphos-
phoramidochloridite (0.057 mL, 0.26 mmol) at 0 °C, and the
mixture was stirred for 10 h at room temperature. The mixture
was diluted with dichloromethane (20 mL), extracted with a
saturated aqueous solution of sodium hydrogencarbonate (3 ×
10 mL), dried (Na2SO4), and filtered. The filtrate was evaporated
to dryness under reduced pressure, and the residue was sub-
jected to column chromatography on silica gel using methanol/
dichloromethane/pyridine (1.5:98.0:0.5, v/v/v) as eluent to give
crude 7 (0.107 g) after evaporation of the solvents under reduced
pressure. The residue was dissolved in anhydrous dichloro-
methane (1 mL), and by dropwise addition to vigorously stirred
petroleum ether (60-80 °C, 30 mL) at -30 °C, nucleotide 7
precipitated to give a white solid material after filtration (0.090
g, 57%): FAB-MS m/z 868 [M + H]+, 890 [M + Na]+; 31P NMR
(CD3CN, 121.5 MHz) δ 150.4, 150.2, 148.8, 149.1.
1-(2-Am in o-2-N,4-C-m et h ylen e-â-D-r ib ofu r a n osyl)t h y-
m in e (4). To a solution of nucleoside 3 (2.80 g, 5.20 mol) in
methanol (120 mL) was added ammonium formate (1.70 g, 0.027
mol) and 10% palladium on carbon (4 g), and the resulting
suspension was heated under reflux for 4 h. The catalyst was
filtered off (silica gel, washed with methanol, 15 mL), the filtrate
was concentrated to dryness under reduced pressure, and the
residue was subjected to column chromatography on silica gel
using methanol/dichloromethane (1:9, v/v) as eluent to give
nucleoside 4 as a white solid material after evaporation of the
solvents under reduced pressure (0.95 g, 68%): FAB-MS m/z 270
1-(2-Am in o-2-N,4-C-m eth ylen e-3,5-O-(1,1,3,3-tetr a isop r o-
p yld isiloxa n e-1,3-d iyl)-â-D-r ibofu r a n osyl)th ym in e (8). To a
solution of nucleoside 4 (0.20 g, 0.74 mmol) in anhydrous
pyridine (3 mL) at -15 °C was added dropwise (during 3 h) 1,3-
dichloro-1,1,3,3-tetraisopropyldisiloxane (0.305 mL, 0.0011 mol),
and the mixture was stirred for 10 h at room temperature.
MeOH (3 mL) was added, and the mixture was evaporated to
dryness under reduced pressure. The residue was subjected to

Notes
J . Org. Chem., Vol. 63, No. 26, 1998 10039
column chromatography on silica gel using methanol/dichloro-
methane (1:99, v/v) to give nucleoside 8 as a white solid material
after evaporation of the solvents under reduced pressure (0.370
mg, 97%): FAB-MS m/z 512 [M + H]+; 1H NMR ((CD3)2SO, 400
MHz) δ 11.37 (bs, 1H), 7.48 (s, 1H), 5.32 (s, 1H), 4.06 (d, 1H, J
13.5 Hz), 4.00 (s, 1H), 3.84 (d, 1H, J 13.5 Hz), 3.41 (s, 1H), 2.92
(d, 1H, J 10.2 Hz), 2.64 (d, 1H, J 10.2 Hz), 1.74 (s, 3H), 1.10-
0.92 (m, 28 H); 13C NMR ((CD)3SO2, 62.9 MHz) δ 163.8, 149.8,
134.1, 107.9, 89.5, 87.9, 70.1, 61.1, 57.9, 49.3, 17.2, 17.2, 17.0,
16.9, 16.8, 16.7, 12.6, 12.2, 11.7. Anal. Calcd for C23H41N3O6Si2:
C, 54.0; H, 8.1; N, 8.2. Found: C, 54.0; H, 8.3; N, 7.8.
1-(2-Me t h y la m in o -2-N ,4-C-m e t h y le n e -3,5-O -(1,1,3,3-
t et r a isop r op yld isiloxa n e-1,3-d iyl)-â-D-r ib ofu r a n osyl)t h y-
m in e (9). To a solution of nucleoside 8 (0.33 g, 0.64 mmol) in
anhydrous THF/dichloromethane (4:1, v/v) at -10 °C was added
dropwise (during 30 min) 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU, 0.125 mL, 0.836 mmol) and methyl iodide (0.05 mL, 0.79
mmol), and the mixture was stirred for 48 h at 10 °C. Additional
DBU (0.05 mL, 0.33 mmol) and methyl iodide (0.020 mL, 0.32
mmol) was added dropwise (during 15 min) to the reaction
mixture, and stirring at 10 °C was continued for 24 h. The
mixture was evaporated to dryness under reduced pressure, and
the residue was subjected to column chromatography on silica
gel using methanol/dichloromethane (1:99, v/v) as eluent to give
nucleoside 9 as a white solid material after evaporation of the
solvents (0.25 g, 74%): FAB-MS m/z 526 [M + H]+; 1H NMR
(CDCl3, 400 MHz) δ 8.19 (bs, 1H), 7.65 (d, 1H, J 1.3 Hz), 5.59
(s, 1H), 4.11 (s, 1H), 4.05 (d, 1H, J 13.2 Hz), 3.87 (d, 1H, J 13.2
Hz), 3.44 (s, 1H), 2.98 (d, 1H, J 9.5 Hz), 2.71 (d, 1H, J 9.5 Hz),
2.72 (s, 3H ), 1.91 (d, 1H, J 1.1 Hz), 1.12-0.96 (m, 28H); 13C
NMR (CDCl3, 62.9 MHz) δ 163.7, 149.6, 135.2, 109.7, 90.9, 85.7,
71.4, 67.3, 58.6, 58.2, 41.2, 17.5, 17.4, 17.3, 17.2, 17.1, 16.9, 13.3,
13.1, 13.0, 12.6, 12.1. Anal. Calcd for C24H44N3O6Si2‚0.25H2O:
C, 54.4; H, 8.3; N, 7.9. Found: C, 54.4; H, 8.1; N, 7.7.
organic phase was washed with a saturated aqueous solution
of sodium hydrogencarbonate (3 × 5 mL), dried (Na2SO4), and
filtered. The filtrate was evaporated to dryness under reduced
pressure, and the residue was subjected to column chromatog-
raphy on silica gel using methanol/dichloromethane/pyridine (1:
98:1, v/v/v) as eluent to give nucleoside 11 as a white solid
material after evaporation of the solvents under reduced pres-
sure (0.20 g, 72%): FAB-MS m/z 586 [M + H]+; 1H NMR (C5D5N
, 400 MHz) δ 13.2 (bs, 1H), 7.98 (d, 1H, J 1.3 Hz), 7.98-7.00 (m,
13H), 6.12 (s, 1H), 4.78 (d, 1H, J 3.7 Hz), 3.88-3.79 (m, 4H),
3.71 (s, 3H), 3.71 (s, 3H), 3.29 (d, 1H, J 9.3 Hz), 2.84 (d, 1H, J
9.3 Hz), 2.81 (s, 3H), 1.85 (d, 3H, J 0.9 Hz); 13C NMR (C5D5N,
62.9 MHz) δ 165.1, 159.2, 151.4, 145.9, 136.5, 136.4, 130.8, 130.7,
128.7, 128.4, 127.4, 113.8, 109.6, 89.8, 86.8, 85.1, 72.0, 68.7, 60.9,
59.4, 55.2, 40.1, 13.1. Anal. Calcd for C33H35N3O7‚0.25H2O: C,
67.2; H, 6.1; N, 7.1. Found: C, 67.2; H, 6.2; N, 6.9.
1-(3-O-(2-Cyan oeth oxy(diisopr opylam in o)ph osph in oxy)-
5-O-4,4′-dim eth oxytr ityl-2-m eth ylam in o-2-N,4-C-m eth ylen e-
â-D-r ibofu r a n osyl)th ym in e (12). To a solution of nucleoside
11 (0.130 g, 0.222 mmol) in anhydrous dichloromethane (2 mL)
at 0 °C were added N,N-diisopropylethylamine (0.088 mL, 0.514
mmol) and 2-cyanoethyl N,N-diisopropylphosphoramidochlori-
dite (0.065 mL, 0.291 mmol), and the mixture was stirred for
10 h at room temperature. Dichloromethane (30 mL) was added,
and the mixture was extracted with a saturated aqueous solution
of sodium hydrogencarbonate (3 × 10 mL), dried (Na2SO4), and
filtered. The filtrate was evaporated to dryness under reduced
pressure, and the residue was subjected to column chromatog-
raphy on silica gel using methanol/dichloromethane/pyridine
(0.5:98.5:1.0, v/v/v) as eluent to give crude 12 (0.120 g) after
evaporation of the solvents under reduced pressure. The residue
was dissolved in anhydrous dichloromethane (1 mL), and by
dropwise addition to vigorously stirred petroleum ether (60-80
°C, 30 mL) at -30 °C, nucleotide 12 precipitated to give a white
solid material after filtration (0.090 g, 52%): 31P NMR (CD3CN,
121.5 MHz) δ 147.7.
2′-Am in o-LNA Syn th esis a n d An a lysis. The 2′-amino-LNA
9-mers (Table 1, entries 3-7) were synthesized on an automated
DNA synthesizer using standard procedures and amidites 7 and
12 (12 min couplings) and commercial 2′-deoxynucleoside 2-cya-
noethyl N,N-diisopropylphosphoramidites (2 min couplings). The
stepwise coupling yield for all amidites was >99%. After
completion of the syntheses of the 5′-O-4,4′-dimethoxytrityl-OFF
2′-amino-LNAs, deprotection and cleavage from the solid support
was accomplished using concentrated ammonia (32% (w/w), 55
°C, 12 h). After evaporation under reduced pressure and
precipitation from ethanol, the purity was analyzed by capillary
gel electrophoresis to be >90% for all 2′-amino-LNAs. The
composition of the 2′-amino-LNAs was verified by MALDI-MS
[M - H]-: calcd for 5′-d(GTGAVATGC) 2779.9, found 2778.5;
calcd for 5′-d(GVGAVAVGC) 2833.9, found 2832.3; calcd for 5′-
d(GTGAXATGC) 2793.9, found 2793.5; calcd for 5′-d(GXGAX-
AXGC) 2876.0, found 2875.5; calcd for 5′-d(GXGVXVXGC)
2912.0, found 2910.8.
1-(2-Meth yla m in o-2-N,4-C-m eth ylen e-â-D-r ibofu r a n osyl)-
th ym in e (10). To a solution of nucleoside 9 (0.40 g, 0.76 mmol)
in THF at room temperature was added a solution of tetrabu-
tylammonium fluoride in THF (1.0 M, 2.2 mL, 2.2 mmol), and
the reaction mixture was stirred for 20 min whereupon pyridine/
water/methanol (6 mL, 3:1:1, v/v/v) was added. The mixture was
added to Dowex 50 × 200 resin (2.2 g, H+ (pyridinium) form,
100-200 mesh) suspended in pyridine/water/methanol (6 mL,
3:1:1, v/v/v), and the resulting mixture was stirred for 20 min.
After filtration, the residue was washed with pyridine/water/
methanol (3 × 3 mL, 3:1:1, v/v/v), and the combined filtrate was
evaporated to dryness under reduced pressure to give an oily
residue after coevaporation with methanol (2 × 5 mL). Column
chromatography on silica gel using methanol/dichloromethane
(1:49, v/v) as eluent gave nucleoside 10 as a white solid material
after evaporation of the solvents under reduced pressure (0.17
g, 79%): FAB-MS m/z 284 [M + H]+; FAB-HRMS calcd for
C
12H18N3O5+ 284.12465, found 284.12402; 1H NMR ((CD3)2SO,
400 MHz) δ 11.3 (bs, 1H, NH), 7.70 (d, 1H, J 1.1 Hz, 6-H), 5.50
(s, 1H, 1′-H), 5.26 (d, 1H, J 4.9 Hz, 3′-OH), 5.12 (t, 1H, J 5.7 Hz,
5′-OH), 3.87 (d, 1H, J 4.8 Hz, 3′-H), 3.67 (d, 2H, J 5.5 Hz, 5′-H),
3.12 (s, 1H, 2′-H), 2.87 (d, 1H, J 9.3 Hz, 5′′-Ha), 2.56 (s, 3H,
NCH3), 2.52-2.49 (1H, m, 5′′-Hb), 1.77 (s, 3H, CH3); 1H NMR
(CD3OD, 400 MHz) δ 7.80 (d, 1H, J 1.3 Hz, 6-H), 5.71 (s, 1H,
1′-H), 4.07 (s, 1H, 3′-H), 3.83 (s, 2H, 5′-H), 3.36 (s, 1H, 2′-H),
3.08 (d, 1H, J 9.9 Hz, 5′′-Ha), 2.68 (s, 3H, NCH3), 2.57 (d, 1H, J
9.9 Hz, 5′′-Hb), 1.88 (d, 3H, J 1.1 Hz, CH3); 13C NMR (CD3OD,
62.9 MHz) δ 166.6, 151.9, 137.4, 110.4, 91.3, 85.2, 71.4, 69.1,
59.4, 58.7, 40.2, 12.2.
Meltin g Tem p er a tu r e Mea su r em en ts. The thermal affin-
ity studies were performed as described earlier3b using 1.5 µM
concentrations of the two complementary strands, assuming
identical extinction coefficients for 2′-amino-LNA and the cor-
responding unmodified oligonucleotides.
Ack n ow led gm en t. The Danish Natural Science
Research Council, The Danish Technical Research
Council, The Danish International Development Agency,
and Exiqon A/S, Denmark are thanked for financial
support. Ms. Britta M. Dahl is thanked for oligonucle-
otide synthesis. Dr. Carl Erik Olsen (Department of
Chemistry, The Royal Veterinary and Agricultural
University,Denmark)isthankedfor MALDI-MSexperiments.
1-(5-O-4,4′-Dim e t h oxyt r it yl-2-m e t h yla m in o-2-N ,4-C-
m eth ylen e-â-D-r ibofu r a n osyl)th ym in e (11). To a solution
of nucleoside 10 (0.135 g, 0.477 mmol) in anhydrous pyridine
(1.5 mL) at 0 °C was added dropwise (during 20 min) a solution
of 4,4′-dimethoxytrityl chloride (0.238 g, 0.702 mmol) in anhy-
drous pyridine/dichloromethane (1.0 mL, 1:1, v/v), and the
resulting mixture was stirred for 10 h at room temperature. A
mixture of ice and water was added (5 mL), and the mixture
was extracted with dichloromethane (3 × 10 mL). The combined
J O9814445