α-LNA, locked nucleic acid with α-D-configuration

Article abstract of DOI:10.1039/b003104f

The bicyclic thymine monomer of α-LNA (αT(L)) was efficiently synthesised and used in the synthesis of α-LNA sequences: incorporation of single αT(L)-monomers in α-configured oligothymidylates destabilises the affinity towards both complementary DNA and R

Full text of DOI:10.1039/b003104f

a-LNA, locked nucleic acid with a- -configuration
D
Poul Nielsen* and Jakob Kragh Dalskov
Department of Chemistry, University of Southern Denmark, Odense University, 5230 Odense M, Denmark.
E-mail: pon@chem.sdu.dk
Received (in Cambridge, UK) 18th April 2000, Accepted 16th May 2000
L
L
The bicyclic thymine monomer of a-LNA (aT ) was
efficiently synthesised and used in the synthesis of a-LNA
In order to synthesise the a-LNA thymine monomer (aT , 4,
Scheme 1) we first investigated the coupling of thymine to
appropriate bicyclic carbohydrate precursors. However, this
L
9
sequences: incorporation of single aT -monomers in a-
configured oligothymidylates destabilises the affinity to-
wards both complementary DNA and RNA, whereas a fully
modified a-LNA sequence displays a very efficient recogni-
tion of complementary RNA.
approach has never been optimised to give the target compound
in a satisfactory yield, and we hereby introduce an alternative
synthetic strategy. The starting methyl furanoside 1 was
9
obtained, as described previously, and used in a modified
Vorbrüggen nucleobase coupling reaction (Scheme 1). After
varying the reaction conditions, the best result in terms of yield
and ratio of products was obtained by using in situ TMS-
protection of both the 2A- hydroxy functionality in 1 and the
thymine, followed by coupling by using TMS-triflate as the
Lewis acid in refluxing acetonitrile for seven days. After
desilylation, the mixture of nucleosides was reacted with
sodium hydride to give the anomeric mixture 2 (a+b ≈ 1.3+1
Conformationally restricted oligonucleotide analogues have
been intensively investigated for their abilities in high affinity
1
nucleic acid recognition. As a prime example, LNA (locked
nucleic acid)† has recently been introduced as a nucleic acid
analogue displaying unprecedented affinity towards DNA and
2
RNA. The anomeric inverted analogue of DNA (a-DNA) has
been demonstrated to hybridise efficiently with complementary
DNA and RNA with a parallel strand orientation, and to be
1
according to H NMR) in 57% overall yield. After removal of
3
highly resistant towards degradation by nucleases. Chemically
the benzyl groups by hydrogenation, the b- and a-LNA
4
2
9
modified analogues of a-DNA have also been investigated,
monomers (3 and 4, respectively) were obtained in 98% yield
and separated. The configuration of 4 was confirmed by
including the introduction of a-configured bicyclic nucleoside
monomers.5
comparison of NMR data with its exact enantiomer and by
8d
The a-2A-deoxynucleoside monomers in a-DNA as well as
the b-2A-deoxynucleoside monomers in DNA exist in an
equilibrium between the two low-energy N- and S-type
NOE-difference spectra as mutual contacts were observed
between H-1A and H-3A and between H-5B and H-6, respectively.
L
The aT monomer 4 was prepared for incorporation into
6,7
conformational ranges. In none of the a-DNA analogues,
investigated so far,4 have the monomers been efficiently
restricted towards N-type conformations. Hoping to obtain an
unprecedented parallel nucleic acid recognition and, thereby, a
new tool in the development of diagnostic probes and antisense
therapeutics, we therefore decided to examine the incorporation
into oligonucleotides of an a-nucleoside analogue which is
conformationally locked in an N-type conformation, i.e. a-
LNA.‡
oligonucleotide sequences by protection, by using the dime-
thoxytrityl (DMT) group to give 5, followed by phosphitylation
to give the phosphoramidite synthon 6.§ This compound was
used in automated solid phase synthesis of oligonucleotides by
using the phosphoramidite approach.10 In connection with the
a-thymidine (aT) phosphoramidite, the a-LNA sequences were
obtained (Table 1) by using tetrazole activation and 10 min
coupling times giving > 98% stepwise coupling yields. The
modified oligomers 8–10 and 12, 13 were synthesised by using
the DMT-ON mode on universal CPG-support (Biogenex),
which allowed the synthesis of fully modified sequences after
cleavage from the solid support by using LiCl in aqueous
,5
Scheme 1 Reagents and conditions: i, (a) TMS-Cl, N,O-bistrimethylsilyl-
acetamide, thymine, MeCN, then TMS-OTf, (b) TBAF, THF; ii, NaH, DMF
(
57%, three steps); iii, H
Py, THF, DMF (80%); v, EtN(Pr )
79%). T = thymine-l-yl.
2 2 3
, Pd(OH) /C, EtOH (98%); iv, DMT-Cl, AgNO ,
i
i
2 2 2 2 2 2
, NC(CH ) OP(Cl)N(Pr ) , CH Cl
(
DOI: 10.1039/b003104f
Chem. Commun., 2000, 1179–1180
This journal is © The Royal Society of Chemistry 2000
1179

Table 1 Hybridisation data for a-LNA sequences and reference strands
In conclusion, a-LNA is able to form a duplex with
complementary RNA with a high thermal stability comparable
dA14
rA14
to other stereoisomers of LNA,8 even though the original
complement
complement
rA CA
6 7
(
b)LNA still displays the highest affinity towards com-
complement
plementary nucleic acids. Nevertheless, a-LNA in the present
/°Ca DT
/°Cb
T
m
/°Ca DT
/°Cb
T
m
/°C
a
Sequence
T
m
m
m
oligothymidylate sequence displays the highest affinity towards
c
RNA of any a- -configured oligonucleotide analogue. How-
D
7
8
9
0
1
2
3
5A-T14
33.0
32.0
25.5
26.0
22.0
18.0
no T
30.0
43.0
35.0
24.5
20.0
33.5
45.0
n.d.
n.d.
n.d.
n.d.
n.d.
c
c
c
c
ever, from the sequences presented here we are not able to
determine whether a-LNA prefers a parallel strand orientation
upon hybridisation. This subject is under investigation in our
laboratories via the synthesis of a-LNA sequences with mixed
nucleobase compositions, in addition to further examination of
the properties and applications of a-LNA.
5A-aT14
L
5A-aT
5A-aT
7
T T
6
26.5
21.5
28.0
24.6
L
1
1
1
1
5
4 5
T T
5A-T10
5A-aT 1L 0
22.0
37.0
d
e
5A-aT 10
M
+1.2 ;
f
+
2.5
The Danish Natural Science Research Council is thanked for
financial support. Ms. Britta M. Dahl, Department of Chem-
istry, University of Copenhagen, is thanked for synthesising
oligonucleotide sequences. Dr Henrik M. Pfundheller, Exiqon
A/S, is thanked for recording MALDI-MS spectra.
a
m
Melting temperatures (T ) obtained from the maxima of the first
derivatives of the melting curve (A260 vs. temperature) recorded in a buffer
containing 10 mM Na HPO , 100 mM NaCl, 0.1 mM EDTA, pH 7.0 using
.5 mM concentrations of the two complementary sequences, assuming
2
4
1
b
identical extinction coefficients for all thymine nucleotides. The change in
c
T
m
value per modification compared with the reference strand 8. Not
determined. No clear cooperative transition was seen. e Compared with
d
f
12. Compared with 11.
Notes and references
†
LNA is defined as an oligonucleotide containing one or more LNA
ammonia. The oligomers were purified by using disposable
reverse phase chromatography cartridges (Cruachem), which
yielded products with > 90% purity, as judged from capillary
gel electrophoresis. The compositions of a-LNA sequences
were verified from MALDI-MS spectra.¶
monomers which are bicyclic nucleosides preorganized in N-type con-
formations. a-LNA is therefore defined as an oligonucleotide containing
one or more monomeric a-
D-LNA nucleosides in connection to unmodified
a- -nucleosides.
D
8
‡ Three other stereoisomers of LNA have been recently introduced and
their affinities towards both complementary RNA and the enantiomeric L-
The a-LNA sequences 9, 10 and 13, as well as their a-DNA
counterparts 8 and 12, were mixed with their DNA and RNA
complements and the resulting hybridisation data are shown in
Table 1. Compared to the unmodified sequence 7, the affinity of
the unmodified a-sequence 8 towards dA14 is, as expected,3
similar. However, the introduction of one or four a-LNA
monomers (9 or 10, respectively) results in strongly decreased
affinities towards dA14. Towards complementary RNA, rA14, 8
has a higher affinity than 7 and the destabilising effect of one or
four a-LNA monomers is even more pronounced. However, in
both cases, the introduction of a block of a-LNA monomers
RNA have been investigated.8 In that sense, an a-LNA sequence has been
c
examined in form of the duplex between its enantiomer a-
L
-LNA and L-
RNA, but only as an (almost) fully modified sequence and only against
RNA.8
c
§
Selected data for 6: d
standard) 150.9, 151.1.
¶ MALDI-MS: m/z ([M 2 H]
(4309.2/4307.8); 13 (3261.6/3260.1).
P
(CDCl
3 3 4
, 121.5 MHz with 85% H PO as external
2
(found/calc.): 9 (4227.1/4223.8); 10
1
2
P. Herdewijn, Liebigs Ann. Chem., 1996, 1337; P. Herdewijn, Biochim.
Biophys. Acta, 1999, 1489, 167.
S. K. Singh, P. Nielsen, A. A. Koshkin and J. Wengel, Chem. Commun.,
diminishes the combined destabilising effect (comparing DT
m
for 9 and 10). This suggests that the N-type conformation a-
LNA monomers do not have the ability to alter neighbouring
nucleosides and, thereby, change the overall single strand
conformation towards a form which is more preferable for
duplex formation. As judged from NMR studies, this is an
1
998, 455; A. A. Koshkin, S. K. Singh, P. Nielsen, V. K. Rajwanshi, R.
Kumar, M. Meldgaard, C. E. Olsen and J. Wengel, Tetrahedron, 1998,
4, 3607; S. Obika, D. Nanbu, Y. Hari, J. Andoh, K. Morio, T. Doi and
5
T. Imanishi, Tetrahedron Lett., 1998, 39, 5401; A. A. Koshkin, P.
Nielsen, M. Meldgaard, V. K. Rajwanshi, S. K. Singh and J. Wengel,
J. Am. Chem. Soc., 1998, 120, 13 252; J. Wengel, Acc. Chem. Res.,
11
important feature of the original (b-)LNA.
The fully modified a-LNA sequence 13 displays strong
recognition of the complementary RNA-strand (DT = +1.2 °C
1
999, 32, 301.
3
4
F. Morvan, B. Rayner, J.-L. Imbach, S. Thenet, J.-R. Bertrand, J.
Paoletti, C. Malvy and C. Paoletti, Nucleic Acids Res., 1987, 15, 3421;
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Res., 1988, 16, 5039.
m
per monomer compared to a-DNA 12 and +2.5 °C compared to
the unmodified oligodeoxynucleotide sequence 11). On the
other hand, no clear cooperative transition was seen when 13
was mixed with dA14. This indicates either that a-LNA is
unable to recognise DNA and is thereby extraordinarily RNA-
selective or, alternatively, that the two sequences might form a
secondary structure not detectable by UV-spectroscopy at 260
nm. Thus, a broad non-cooperative transition at 40–45 °C is
seen in the mixture, but a similar transition is also observed for
C. H. Gotfredsen, J. P. Jacobsen and J. Wengel, Bioorg. Med. Chem.,
1
996, 4, 1217; K. Pongracz and S. M. Gryaznov, Nucleic Acids Res.,
1998, 26, 1099; F. Morvan, J. Zeidler and B. Rayner, Tetrahedron,
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5
3
049.
W. Saenger, Principles of Nucleic Acid Structure, Springer, New York,
984.
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6, 523.
1
3 alone. Even though the possibility of self-melting has been
6
7
12
described earlier for longer a-oligothymidylate sequences,
1
the broad transition and low hyperchromicity observed for 13
alone does not indicate the melting process of a duplex
structure, but rather a transition between secondary forms of
single strands. The presence of a duplex between 13 and rA14
was confirmed by the fact that a clear melting transition of a
duplex between 13 and a mis-matching complementary se-
1
8 (a) V. K. Rajwanshi, A. E. Håkansson, B. M. Dahl and J. Wengel, Chem.
Commun., 1999, 1395; (b) V. K. Rajwanshi, A. E. Håkansson, R. Kumar
and J. Wengel, Chem. Commun., 1999, 2073; (c) V. K. Rajwanshi, A. E.
Håkansson, M. D. Sørensen, S. Pitsch, S. K. Singh, R. Kumar, P.
Nielsen and J. Wengel, Angew. Chem., Int. Ed., 2000, 39, 1656; (d) A. E.
Håkansson and J. Wengel, personal communication.
m
quence was observed with T decreased by 8 °C (Table 1).
Furthermore, thermal stabilities measured at higher ionic
concentrations (data not shown) were increased, as expected,
for all the duplexes involving 11–13 and RNA-complements as
well as 11 and 12 with dA14, whereas no clear cooperative
transitions at increased temperature were observed with
9
P. Nielsen and J. Wengel, Chem. Commun., 1998, 2645.
1
1
0 M. H. Caruthers, Acc. Chem. Res., 1991, 24, 278.
1 M. Petersen, C. B. Nielsen, K. E. Nielsen, G. A. Jensen, K.
Bondensgaard, S. K. Singh, V. K. Rajwanshi, A. A. Koshkin, B. M.
Dahl, J. Wengel and J. P. Jacobsen, J. Mol. Recognit., 2000, 13, 44.
13+dA14 or with 13 alone.
12 U. Neidlein and C. Leumann, Tetrahedron Lett., 1992, 33, 8057.
1180
Chem. Commun., 2000, 1179–1180