Oligodeoxynucleotides containing amide-linked LNA-type dinucleotides: Synthesis and high-affinity nucleic acid hybridization

Article abstract of DOI:10.1039/b111000d

Synthesis of three different amide-linked LNA-type dinucleotides and their incorporation into 9-mer and 17-mer oligodeoxynucleotides is described; compared to the reference DNA-RNA duplex, incorporation of one of the three dimers (5′-DNA*LNA dimer) induce

Full text of DOI:10.1039/b111000d

Oligodeoxynucleotides containing amide-linked LNA-type dinucleotides:
synthesis and high-affinity nucleic acid hybridization
Anne Lauritsena and Jesper Wengel*b
a Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen,
Denmark
b Nucleic Acid Center†, Department of Chemistry, University of Southern Denmark, Campusvej 55, DK-5230
Odense M, Denmark. E-mail: jwe@chem.sdu.dk
Received (in Cambridge, UK) 6th December 2001, Accepted 29th January 2002
First published as an Advance Article on the web 12th February 2002
Synthesis of three different amide-linked LNA-type dinu-
cleotides and their incorporation into 9-mer and 17-mer
oligodeoxynucleotides is described; compared to the refer-
ence DNA–RNA duplex, incorporation of one of the three
dimers (5A-DNA*LNA dimer) induced significantly in-
creased duplex thermostabilities.
compared to the corresponding unmodified DNA–DNA duplex
(see Table 1, ON16 and ON17).3
In the chemical search for ideal antisense molecules, the
exchange of the phosphordiester moiety by various amide-
linked non-phosphorus internucleoside linkages has been
studied.1–7 Whereas amide linkages confer stability towards
nucleolytic degradation, only a few have shown increased
thermal stability of duplexes formed with RNA target
strands.1,2,6,7 Unfortunately, synthesis of the necessary building
blocks for most of the more promising of the known amide-
linked dinucleotides is rather troublesome requiring, e.g.,
stereoselective introduction of C3A-alkyl substituents and/or
C2A-substituents.2,6,7 Among the more easily accessible amide-
linked dinucleotides are several containing an additional atom
in the internucleoside linkage compared to the natural C3A-O-
P(NO)(OH)-O-CH2-C4A four-atom linkage,3,4 e.g., the T*T
dimer3 shown in Fig. 1 with an C3A-O-CH2-CH2-NH-C(NO)-
C4A linkage. This linkage was conveniently introduced by
condensing two easily available thymidine building blocks, i.e.
the deacetylated derivative of 1 and nucleoside 3 (Fig. 2).3 The
T*T dimer introduced once or twice into a 17-mer oligodeox-
ynucleotide sequence induced a small decrease in the Tm value
Fig. 2 Reagents and conditions: i), v), ix) DPPA, Et3N, DMF [i) 64%], [v)
60%], [ix) 46%]; ii), vi), x) sat. methanolic ammonia [ii) 81%], [vi) 74%],
[x) 75%]; iii), vii), xi) ClP(O(CH2)2CN)N(i-Pr)2, DIPEA, DCM [iii) 34%],
[viii) 31%], [xii) 50%]; iv), viii), xii) DNA synthesizer.
Table 1 Thermal denaturation experiments towards complementary single-
stranded DNA and complementary single-stranded RNAa
Tm/°C
RNA
Sequence (ON)b
Tm/°C DNA
5A-d(CACCAACTTCTTCCACA) (13) 57.5
57.5
5A-d(–CTLTCTTC–)b (14)
58.0 (+0.5)
61.0 (+3.5)
65.0 (+3.8)
n.d.
5A-d(–CTLTCTLTC–)b (15)
5A-d(–C[T*T]CTTC–)b (16)
5A-d(–C[T*T]C[T*T]C–)b (17)
5A-d(–C[TL*T]CTTC–)b (18)
5A-d(–C[TL*T]C[TL*T]C–)b (19)
5A-d(–C[TL*TL]CTTC–)b (20)
5A-d(–C[TL*TL]C[TL*TL]C–) (21)
5A-d(–C[T*TL]CTTC–)b (22)
5A-d(–C[T*TL]C[T*TL]C–)b (23)
60.0 (+1.3)
c(22.5)
c(21.5)
n.d.
49.5 (28.0)
43.0 (27.3)
51.5 (26.0)
47.5 (25.0)
59.0 (+1.5)
61.0 (+1.8)
53.5 (24.0)
50.5 (23.5)
57.5 ( 0)
58.0 (+0.3)
61.0 (+3.5)
64.5 (+3.5)
5A-d(GTGTTTTGC) (24)
31.0
30.5
5A-d(GTG[TL*T]TTGC) (25)
5A-d(GTG[TL*TL]TTGC) (26)
5A-d(GTG[T*TL]TTGC) (27)
18.0 (213.0)
17.5 (213.5)
35.0 (+4.0)
23.0 (27.5)
31.0 (+0.5)
39.5 (+9.0)
a Melting temperatures [Tm values (DTm values per modification)]
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 strands; A = adenine monomer, C = cytosine
monomer, G = guanine monomer, T = thymine monomer; See Fig. 1 for
structures of TL, T*T, TL*T, TL*TL and T*TL; ‘n.d.’ denotes ‘not
determined’. b For the sequences ON14–ON23, only the central fragment is
shown but their full base sequence is identical to the 17-mer unmodified
sequence ON13. c DTm values taken from ref. 3. The absolute Tm values of
ON16 and ON173 are not directly comparable to those recorded herein
because of the use of slightly different buffer conditions.
Fig. 1 Structures of a DNA*DNA phosphordiester dimer (TT), an
LNA*DNA phosphordiester dimer (TLT), a DNA*DNA amide-linked
dimer (T*T), an LNA*DNA amide-linked dimer (TL*T), an LNA*LNA
amide-linked dimer (TL*TL) and a DNA*LNA amide-linked dimer
(T*TL). T = thymin-1-yl.
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CHEM. COMMUN., 2002, 530–531
This journal is © The Royal Society of Chemistry 2002

Stimulated by the unprecedented binding affinity of LNA
(locked nucleic acids),‡ 8–11 we decided to evaluate the effect of
introducing locked furanose conformations in oligonucleotides
containing non-phosphorus linkages. Due to its relatively
straightforward synthesis we chose to study the T*T system
(Fig. 1) as the first amide-linked LNA-type oligonucleotide,
more precisely the three dimers TL*T, TL*TL and T*TL (Fig.
1). To synthesize the three amide-linked phosphoramidite
derivatives 8, 10 and 12, the four monomeric building blocks
1,§ 2,§ 33 and 4§ were prepared and condensed two by two
under mild neutral conditions using diphenyl phosphorazidate
(DPPA) as condensing agent following procedures described
previously3 furnishing the three amide-linked dinucleosides 7, 9
and 11. Subsequently, deacetylation and phosphitylation af-
forded the dimeric phosphoramidite building blocks 8, 10 and
12¶ suitable for incorporation of dimers TL*T, TL*TL and
T*TL, respectively, into oligonucleotides (Fig. 2).
Oligonucleotides ON13-ON15 and ON18-ON27 (Table 1)
were synthesized in 0.2 µmol scale on an automated DNA
synthesizer using the phosphoramidite approach.12 Standard
procedures were used but with modifications as described
previously13 when coupling the LNA-T amidite9 [leading to
incorporation of ‘TL’ (ON14 and ON15)], and amidites 8, 10
and 12. The coupling yields for all types of amidites were
498% with coupling times of 2 min (deoxynucleotide ami-
dites), 10 min (LNA-T amidite and amidites 8 and 12), and 20
min (amidite 10). Cleavage from the solid support and removal
of the protection groups was performed using concentrated
ammonia (55 °C, 12 h) to give ON13–ON15 and ON18–ON27
after precipitation from ethanol. Satisfactory purities ( > 80%)
were verified by capillary gel electrophoresis and the composi-
tions by MALDI-MS analysis.∑
despite the fact that the linker used herein contains an additional
atom compared to the natural phosphordiester linker, and that
incorporation of the LNA-type monomer in the 5A-end of the
dimer is strongly destabilizing. Previous studies on the effect on
introducing 2A-substituents (2A-fluoro and 2A-OMe substituents;
and thus conformational restriction) in the 5A-end positioned
monomer and/or the 3A-end positioned monomer of optimized
amide-linked dinucleosides likewise showed that the positive
effect of introducing 2’-substituents is by far larger at the 3A-end
of the dimer than at the 5A-end.6,7 However, introduction of
these 2A-substituted analogues at the 5A-end of the dimer was
reported to be neutral or even slightly stabilizing.6,7 The highly
variable effects observed for the three different dimers (TL*T,
TL*TL and T*TL) support our previous conclusion that
conformational tuning of neighboring unmodified deoxynu-
cleotide monomers by the presence of one (or several) LNA
monomers in an oligomer is much more pronounced in the 3A-
direction than in the 5A-direction.14 Thus, no favorable con-
formational tuning in the 5A-direction is apparently possible for
the LNA*DNA (TL*T) dimer or the LNA*LNA (TL*TL)
dimer. We are continuing our studies on the hybridization
properties and structural characteristics of oligomers containing
the three LNA-type amide-linked dimers introduced herein.
We thank The Danish Research Agency and The Danish
National Research Foundation for financial support, Ms Britta
M. Dahl for oligonucleotide synthesis, and Dr Michael
Meldgaard, Exiqon A/S, for MALDI-MS analysis.
Notes and references
† A research center funded by The Danish National Research Foundation
for studies on nucleic acid chemical biology.
The hybridization properties towards complementary single-
stranded DNA and RNA strands were evaluated by thermal
denaturation studies (Table 1). Relative to the reference DNA–
DNA and DNA–RNA duplexes, the data shown in Table 1 for
ON13–ON17 reveal a limited decrease in Tm value resulting
from incorporation of the DNA*DNA dimer (not evaluated
towards RNA), and increased Tm values towards DNA or RNA
resulting from incorporation of the LNA-T monomer TL. The
data obtained for ON18–ON23 demonstrate strikingly different
effects of the three amide-linked dimers TL*T (ON18 and
ON19), TL*TL (ON20 and ON21) and T*TL (ON22 and
ON23). Thus, with an LNA-type monomer at the 5’-end of the
dimer (TL*T), substantially decreased binding affinity towards
both DNA and RNA was obtained. The dimer with two LNA-
type monomers (TL*TL) likewise induced a significant de-
crease in Tm value towards DNA but had no effect when
hybridized towards RNA. Finally, a moderate affinity increase
towards DNA, and a significant affinity increase towards RNA
(DTm/mod = +3.5 °C), resulted from incorporation once or
twice of the dimer with one LNA-type monomer positioned at
the 3A-end of the dimer (T*TL). The results obtained for the
9-mer sequence (ON24–ON27) follow the same trends, but due
to the reduced sequence length the effects per modification were
more pronounced (e.g., DTm/mod = +4.0 °C and + 9.0 °C for
T*TL). For all oligomers containing one of the three LNA-type
amide-linked dimers, a weak (TL*T and T*TL) or moderate
(TL*TL) RNA-selectivity was observed.
‡ We have defined LNA as an oligonucleotide containing one or more
conformationally locked 2A-O,4A-C-methylene-b-D-ribofuranosyl nucleo-
tide monomer(s) (‘LNA monomer(s)’).
§ Synthesis of compounds 1, 2 and 4 will be reported elsewhere.
31P NMR data: d (CH3CN) 150.4, 148.8 (amidite 8); d (CHCl3) 149.7,
¶
149.4 (amidite 10); d (CHCl3) 149.5, 149.2 (amidite 12).
∑ MALDI-MS ([M 2 H]2 measured/[M 2 H]2 calculated): 5038/5038
(ON18), 5043/5043 (ON19), 5066/5066 (ON20), 5098/5099 (ON21),
5038/5042 (ON23), 2739/2740 (ON25), 2766/2768 (ON26), 2737/2740
(ON27).
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