Incorporation of α- and β-LNA (Locked nucleic acid) monomers in oligodeoxynucleotides with polarity reversals

Article abstract of DOI:10.1016/S0960-894X(01)00298-0

The thymidine monomers of LNA with both α- and β-configuration are incorporated with polarity reversals (i.e., with 3′-3′ and 5′-5′ junctions) in oligodeoxynucleotides with β- and α-configuration, respectively. A 5′-O-phosphoramidite of the β-LNA monomer is synthesised. Large destabilisations of duplexes with both complementary DNA and RNA are observed for oligodeoxynucleotides containing the α-LNA monomer, whereas a duplex with complementary RNA of an α-oligodeoxynucleotide containing the β-LNA monomer is not destabilised.

Full text of DOI:10.1016/S0960-894X(01)00298-0

Bioorganic & Medicinal Chemistry Letters 11 (2001) 1765–1768
Incorporation of ꢀ- and ꢁ-LNA (Locked Nucleic Acid) Monomers
in Oligodeoxynucleotides with Polarity Reversals
Nanna K. Christensen,a Britta M. Dahlb and Poul Nielsena,*
aDepartment of Chemistry, University of Southern Denmark, Odense University, DK-5230 Odense M, Denmark
bDepartment of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen, Denmark
Received 5 March 2001; revised 21 March 2001; accepted 24 April 2001
Abstract—The thymidine monomers of LNA with both a- and b-configuration are incorporated with polarity reversals (i.e., with
30–30 and 50–50 junctions) in oligodeoxynucleotides with b- and a-configuration, respectively. A 50-O-phosphoramidite of the b-LNA
monomer is synthesised. Large destabilisations of duplexes with both complementary DNA and RNA are observed for oligo-
deoxynucleotides containing the a-LNA monomer, whereas a duplex with complementary RNA of an a-oligodeoxynucleotide
containing the b-LNA monomer is not destabilised. # 2001 Elsevier Science Ltd. All rights reserved.
Conformationally restricted nucleosides and corre-
sponding oligodeoxynucleotides (ODNs) have received
a lot of attention as potential candidates for antisense
and diagnostic purposes.1,2 For example, the use of
bicyclic nucleosides has significantly improved the
binding affinity of the corresponding ODNs towards
complementary nucleic acid sequences.2 As a prime
example, a bicyclic nucleoside containing a 20-O-40-C-
methylene bridge is known to be locked in a north (N)
conformation3 (Fig. 1) and ODNs containing this mod-
ification are known as LNA (locked nucleic acid).4ꢀ6
LNA displays an unprecedented affinity towards com-
plementary DNA and RNA (increases in thermal stabi-
sequence (T10), also a-LNA displayed a high affinity
towards complementary RNA but no recognition of
DNA, and mixed sequences containing a-LNA mono-
mers in combination with unmodified a-deoxynucleo-
sides displayed poor affinity towards both DNA and
RNA.10
Intensive investigation has been carried out on b-ODNs
containing one or more a-nucleosides incorporated with
reversed polarity allowing simultaneously for anti-
parallel recognition by the b-nucleosides and parallel
recognition by the a-nucleosides.11ꢀ16 Thus, chimeric a/
b ODNs with stretches of 4 to 12 a-nucleosides give
er only minor decreases in affinity compared to pure b-
ODNs.11,12 They show increased stability towards
nucleases11 as well as activation of RNaseH in a duplex
with complementary RNA.12 The incorporation of one
single a-configured thymidine with polarity reversals in
the middle of an otherwise b-configured mixed nonamer
ODN gives a decrease in affinity of 2.8 ꢁC towards
lity of duplexes ÁTm from +3 to +9 ꢁC
p
modification).4ꢀ6
ODNs containing exclusively a-configured nucleosides
(a-ODNs) recognise complementary nucleic acids in a
parallel manner.7ꢀ9 a-ODNs are resistant towards
degradation by nucleases and the affinity towards com-
plementary DNA and RNA is comparable to the anti-
parallel recognition by b-ODNs.7,8 However, the
binding does not induce RNaseH mediated degradation
of the RNA complement.9 We have recently introduced
a-LNA as a-ODNs containing a-d-configured bicyclic
nucleosides which are locked in an N-conformation (the
a-LNA monomers, Fig. 1).10 In a fully modified
*Corresponding author. Tel.: +45-6550-2565; fax: +45-6615-8780;
e-mail: pon@chem.sdu.dk
Figure 1. a- and b-LNA monomers and a nucleoside in a north con-
formation.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00298-0

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N. K. Christensen et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1765–1768
complementary RNA.13 With four single reverse incor-
porations of different a-deoxynucleosides each sepa-
rated by three or four b-nucleosides smaller decreases
(ÁTm=1.5 ꢁC p era-monomer) have been observed, and
furthermore, these sequences allowed RNaseH medi-
ated degradation of the RNA-strand and exhibited in
vivo antitumor activity.14 ODNs with alternating a- and
b-nucleosides (and hence, alternating 30–30 and 50–50
junctions) do not activate RNaseH,15 and the decreases
in affinity towards complementary RNA and DNA
strands are more pronounced (ÁTm from ꢀ1 to ꢀ5 ꢁC
per a-monomer).11,15 In self-complementary DNA dec-
amers, single incorporations of different a-deoxynucleo-
sides with polarity reversals afforded decreases in duplex
stability between 2.5 and 5.0 ꢁC per modification.16
was performed in the 50 to 30 direction using unmodified
50-O-phosphoramidites 3 as well as an a-LNA phos-
phoramidite 4 (Fig. 3). The a-thymidine and benzoyl
protected a-5-methylcytidine amidites 1 were both syn-
thesised from a-thymidine using published procedures18
and any required 50-O-phosphoramidites 3 were pur-
chased from Chemgenes. The synthesis of the a-LNA
phosphoramidite 4 has been reported earlier,10 whereas
a synthesis of the 30-O-dimethoxytrityl protected LNA
50-O-phosphoramidite 2 was developed (Scheme 1).
Thus, the LNA nucleoside monomer 54,5 was protected
on the primary hydroxyl group using a silyl ether to give
6 followed by tritylation of the 30-O position. The con-
ventional method using 4,40-dimethoxytrityl chloride in
pyridine failed in this case, and several combinations of
bases including DMAP as well as AgNO3 as an additive
did not bring the yield of 7 over 20%. However, the use
of 4,40-dimethoxytrityl triflate19 which has been used
before for tritylation of hindered secondary alcohols on
bicyclic nucleosides19 improved the yield of 7 to 66%.
Simple desilylation to give 820 followed by conventional
phosphorylation gave the phosphoramidite building
block 2 in 77% yield over the two steps.21
In order to perform a preliminary investigation on the
effects of conformational restriction in this context, we
decided to incorporate the a-LNA monomer with
polarity reversals into normal b-ODNs (Fig. 2A).
Hereby, the effect of an a-nucleoside with locked north
conformation concerning the affinity towards DNA and
RNA complements is investigated. For a further exam-
ination of strong conformational restriction in this type
of nucleic acid analogues, we also decided to incorpo-
rate the b-LNA monomer with polarity reversals into a-
ODNs (Fig. 2B).
All modified ODNs were synthesised in the 0.2 mmol
scale using phosphoramidite chemistry17 and the DMT-
ON mode on universal CPG-supports purchased from
Biogenex. All couplings were performed with tetrazole
activation. For 4, prolonged coupling time and, for 2
The building blocks needed for the automated synthesis
of ODNs using phosphoramidite chemistry17 are shown
in Figure 3. For synthetic simplicity, only the LNA
thymidine monomers and a-pyrimidine nucleosides
were used for this study. The synthesis of a-ODNs
containing reversely incorporated b-LNA monomers
demanded unmodified a-nucleoside phosphoramidites 1
in combination with the LNA 50-O-phosphoramidite 2
(Fig. 3) and was performed in the usual 30 to 50 direc-
tion. On the other hand, the synthesis of b-ODNs
containing reversely incorporated a-LNA monomers
Figure 3. Building blocks used for ODN synthesis. T=thymin-1-yl.
DMT=4,40-dimethoxy-
mCBz=3-N-benzoyl-5-methylcytosin-1-yl.
trityl.
Figure 2. (A) Segment of a b-ODN containing a-LNA with polarity
reversals; (B) segment of an a-ODN containing b-LNA with polarity
reversals.
Scheme 1. (a) TBDMS-Cl, pyridine, 91%; (b) DMT-triflate, pyridine,
66%; (c) TBAF, THF, 88%; (d) NC(CH2)2OP(Cl)N(iPr)2, CH2Cl2,
Et2NiPr, 88%. T=thymin-1-yl.

N. K. Christensen et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1765–1768
1767
and 3, prolonged detritylation times were used giving
>98% stepwise coupling yields. The modified oligomers
were cleaved from the solid support using LiCl in aqu-
eous ammonia and purified on disposable reverse phase
chromatography cartridges (Cruachem), yielding pro-
ducts with >90% purity according to capillary gel
electrophoresis.22 The composition of the sequences
(Table 1) were verified from MALDI-MS spectra.23
displayed a larger decrease in affinity towards DNA
(ꢀ7 ꢁC) but almost no decrease in affinity towards RNA
(ꢀ0.5 ꢁC).24 Thus, in the latter case the locked N-con-
formation of the b-monomer neutralises the destabilis-
ing effect of the polarity reversal incorporation.
The structures of duplexes containing ODNs with single
a-nucleosides incorporated with polarity reversals have
been studied by Germann and co-workers using NMR
techniques.13,16,25,26 It was demonstrated that the pre-
sence of these unnatural moieties leads to structural
variations in the section containing the modification
compared to an unmodified control. Thus, the a-
nucleoside is found to be in a south (S) conformation3
in both DNA–DNA and DNA–RNA hybrids,13,25
whereas the nucleotide downstream of the 50–50 junction
in a DNA–DNA duplex is shifted in conformational
equilibrium towards an S to N ratio of approximately
1:1 compared to higher than 80% S-conformation in the
unmodified control.16,25 In a DNA–RNA hybrid, where
the nucleotides of the DNA strand display a higher
propensity for adopting an N-conformation, the two
nucleotides upstream of the 30–30 junction turn out to be
shifted towards a higher percentage of S.13 In neither
case, however, does the overall structure of the duplex
alter decisively.13,25 Our results with reversely incorpo-
rated a-LNA are in line with these NMR analyses, as
the locked N-conformation of the a-nucleosides has
been found to be very unfavourable for duplex stabi-
lities. Hence, the high preference of a-nucleosides for
adopting S-conformations seems to be essential in
duplexes with polarity reversals.
The modified ODNs as well as their unmodified refer-
ence sequences were mixed with complementary single
stranded DNA or RNA sequences and the thermal sta-
bilities of the resulting duplexes were determined (Table
1). When normal a-thymidine is incorporated with
polarity reversals into b-ODNs (via 1 and 3), we observe
a decrease in thermal stability compared to unmodified
references that is comparable or slightly more pro-
nounced (ÁTm from ꢀ3.5 to ꢀ5.0 ꢁC) than what has
previously been reported13ꢀ16 (Table 1, ODNs 10 and 13
compared to 9 and 12). However, the decrease is much
more significant when the a-LNA nucleoside (via 4) is
similarly incorporated (ODNs 11 and 14). Thus, with
the mixed sequence 14 no stable duplexes are formed.
This indicates that the locked north conformation of the
a-monomer is very unfavourable for this type of duplex
formation. When b-thymidine was incorporated rever-
sely into a-ODNs (via 1 and 3) comparable destabilisa-
tions (ODNs 16 and 19 compared to 15 and 18) were
observed (ÁTm from ꢀ3 to ꢀ7 ꢁC) most significantly of
duplexes with complementary RNA. However, when
the b-LNA nucleoside (via 2) was incorporated, com-
pletely different results were obtained. Thus, for an oli-
gothymidylate sequence 17, a comparable decrease in
affinity towards DNA (ꢀ5 ꢁC) but a very large decrease
in affinity towards RNA (ꢀ13 ꢁC) was observed. On the
other hand, the mixed pyrimidine sequence (ODN 20)
On the other hand, the LNA monomer incorporated with
polarity reversals into a-ODNs improves the affinity
towards complementary RNA, at least in a mixed pyr-
Table 1. Hybridisation data of duplexes containing unmodified and modified ODNs with polarity reversals
Complementary ssDNA
Complementary ssRNA
Tm (ꢁC)a ÁTm (ꢁC)
ODN
Sequence
Tm (ꢁC)a
ÁTm (ꢁC)
b-ODNs containing a-nucleosidesb
50-b-T14
9
33.0
29.5
21.5
29.0
25.0
<10
ref.
ꢀ3.5d
ꢀ11.5d
ref.
30.0
25.0
22.0
26.5
22.5
ref.
0
0
0
0
10
11
12
13
14
50-b-T73 ꢀ3 aT5 ꢀ5 T6
ꢀ5.0d
ꢀ8.0d
ref.
0
0
0
0
50-b-T73 ꢀ3 aTL5 ꢀ5 T6
50-b-CTGATATGC
0
0
0
0
50-b-CTGA30 ꢀ3 aT5 ꢀ5 ATGC
ꢀ4.0e
ꢀ4.0e
0
0
0
50-b-CTGA3 ꢀ3 aTL5 ꢀ5 ATGC
<ꢀ19e
<10
<ꢀ16e
a-ODNs containing b-nucleosidesc
50-a-T14
15
16
17
18
19
20
31.5
28.5
26.5
42.5
39.5
35.5
ref.
43.0
36.0
29.0
32.0
28.5
31.5
ref.
0
0
0
0
50-a-T73 ꢀ3 bT5 ꢀ5 T6
ꢀ3.0f
ꢀ5.0f
ref.
ꢀ7.0f
ꢀ13.0f
ref.
0
0
0
0
50-a-T73 ꢀ3 bTL5 ꢀ5 T6
50-a-mCTmCmCTTmCTTT
0
0
0
0
50-a-mCTmCmCT30 ꢀ3 bT5 ꢀ5 mCTTT
ꢀ3.0g
ꢀ3.5g
0
0
0
50-a-mCTmCmCT3 ꢀ3 bTL5 ꢀ5 mCTTT
ꢀ7.0g
ꢀ0.5g
TL refers to LNA thymidine monomers.
aMelting temperatures (Tm) obtained from the maxima of the first derivatives of the melting curve (A260 vs temperature) recorded in a buffer con-
taining 10 mM Na2HPO4, 100 mM NaCl, 0.1 mM EDTA, pH 7.0 using 1.5 mM concentrations of the two complementary sequences, assuming
identical extinction coefficients for all thymine nucleotides. All values are means of double determinations.
bMixed with antiparallel complementary sequences dA14, rA14, 50-dGCATATCAG or 50-rGCAUAUCAG.
cMixed with parallel complementary sequences dA14, rA14, 50-dGAGGAAGAAA or 50-rGAGGAAGAAA.
dThe change in Tm value per modification compared with the reference strand 9.
eCompared with 12.
fCompared with 15.
gCompared with 18.

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N. K. Christensen et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1765–1768
imidine sequence, suggesting that the N-conformation is
preferable for the reverse b-nucleoside in this duplex
type. This confirms the potential of conformationally
locked nucleosides in ODNs also with polarity reversals
and indicates that more improved thermal stabilities
might be obtained in other sequences with chimeras of
conformationally restricted a- and b-nucleosides. Taken
together, our results and the NMR analyses (vide supra)
indicate that other conformational restrictions can
afford ODNs with higher binding affinities, for example
by incorporating restricted monomers up- or down-
stream of the polarity reversals. Furthermore, the fact
that a/b-chimeras with polarity reversals in some
duplexes with RNA induce RNaseH mediated cleavage
of the RNA strand14 and the fact that one incorpora-
tion of (b-)LNA does not significantly destabilise duplex
stability suggest, in combination, that chimeras of a/b-
LNA and other modified or unmodified nucleosides
might be useful in the construction of conformationally
restricted high-affinity ODNs with the ability of indu-
cing RNaseH mediated degradation of the target RNA.
5. Koshkin, A. A.; Singh, S. K.; Nielsen, P.; Rajwanshi, V. K.;
Kumar, R.; Meldgaard, M.; Olsen, C. E.; Wengel, J. Tetra-
hedron 1998, 54, 3607.
6. Wengel, J. Acc. Chem. Res. 1999, 32, 301.
7. Morvan, F.; Rayner, B.; Imbach, J.-L.; Thenet, S.; Ber-
trand, J.-R.; Paoletti, J.; Malvy, C.; Paoletti, C. Nucleic Acids
Res. 1987, 15, 3421.
8. Morvan, F.; Porumb, H.; Degols, G.; Lefebvre, I.; Pom-
pon, A.; Sproat, B. S.; Rayner, B.; Malvy, C.; Lebleu, B.;
Imbach, J.-L. J. Med. Chem. 1993, 36, 280.
9. Gagnor, C.; Rayner, B.; Leonetti, J.-P.; Imbach, J.-L.;
Lebleu, B. Nucleic Acids Res. 1989, 17, 5107.
10. Nielsen, P.; Dalskov, J. K. Chem. Commun. 2000, 1179.
11. Debart, F.; Tosquellas, G.; Rayner, B.; Imbach, J.-L.
Bioorg. Med. Chem. Lett. 1994, 4, 1041.
12. Boiziau, C.; Debart, F.; Rayner, B.; Imbach, J.-L.;
Toulme, J.-J. FEBS Lett. 1995, 361, 41.
13. Aramini, J. M.; Germann, M. W. Biochemistry 1999, 38,
15448.
14. Tan, T. M. C.; Kalisch, B. W.; van de Sande, J. H.; Ting,
R. C. Y.; Tan, Y. H. Antisense Nucleic Acid Drug Dev. 1998, 8,
95.
15. Koga, M.; Wilk, A.; Moore, M. F.; Scremin, C. L.; Zhou,
L.; Beaucage, S. L. J. Org. Chem. 1995, 60, 1520.
16. Aramini, J. M.; van de Sande, J. H.; Germann, M. W.
Biochemistry 1997, 36, 9715.
In conclusion, we have demonstrated the efficient
incorporation of conformationally locked a- and b-
LNA monomers into ODNs with polarity reversals. In
this context, we have efficiently synthesised a 50-O-
phosphoramidite of an LNA monomer. In accordance
with the NMR examination by Germann and co-work-
ers,13,25 our results suggest that the binding affinity of
ODNs containing a-nucleosides with polarity reversals
might instead be significantly improved by a-nucleoside
analogues that are conformationally restricted in S-type
conformations. The development of other con-
formationally restricted a/b-chimeric ODNs with
potential high-affinity nucleic acid recognition and,
therefore, also antisense purposes is now in progress.
17. Caruthers, M. H. Acc. Chem. Res. 1991, 24, 278.
18. Kurfurst, R.; Roig, V.; Chassignol, M.; Asseline, U.;
¨
Thuong, N. T. Tetrahedron 1993, 49, 6975.
19. Tarkoy, M.; Bolli, M.; Leumann, C. Helv. Chim. Acta
¨
1994, 77, 716.
20. Selected data for 8: 1H NMR; dH (CDCl3, 300 MHz) 7.43–
7.22 (9H, m, arom.), 7.18 (1H, s, H-6), 6.81–6.77 (4H, m,
arom.), 5.46 (1H, s, H-10), 4.30 (1H, d, J 7.6 Hz, H-100),
4.02 (2H, m, H-50), 3.89 (1H, d, 7.6 Hz, H-100), 3.77 (3H,
s, OCH3), 3.76 (3H, s, OCH3), 3.66 (1H, s, H-20 or 30),
3.34 (1H, s, H-20 or 30), 1.79 (3H, s, CH3). HR-MALDI
MS; m/z found 595.2062, calcd for C32H32N2O8+Na
595.2051.
21. Selected data for 2: 31P NMR; dP (CDCl3, 121.5 MHz with
85% H3PO4 as external standard) 150.68, 150.85.
22. ODN 13 was synthesised on a 30-O-DMT-dC CPG-sup-
port from Chemgenes and cleaved using concentrated aqueous
ammonia. After the usual chromatography (Cruachem), the
sequence was >70% pure according to capillary gel electro-
phoresis.
23. MALDI MS data for oligonucleotide sequences: m/z
(found/calcd); 10 (4195.3/4195.8 Da); 11 (4223.6/4223.8 Da);
13 (2712.2/2712.8 Da); 14 (2742.1/2740.8 Da); 16 (4194.8/
4195.8 Da); 17 (4220.8/4223.8 Da); 19 (2976.7/2975.0 Da); 20
(3003.3/3003.0 Da).
Acknowledgements
The Danish Natural Science Research Council is
thanked for financial support. Ms. Malene Larsson is
thanked for synthetic assistance.
References and Notes
1. Herdewijn, P. Biochim. Biophys. Acta 1999, 1489, 167.
2. Meldgaard, M.; Wengel, J. J. Chem. Soc., Perkin Trans. 1
2000, 3539.
3. For the conformational behaviour of nucleosides and
nucleotides see: Saenger, W. Principles of Nucleic Acid Struc-
ture; Springer: New York, 1984.
24. All thermal stabilities of duplexes containing 15–20 were
also determined in a high salt buffer (750 mM NaCl) and the
expected increases were observed independent of the mod-
ifications.
25. Aramini, J. M.; Mujeeb, A.; Germann, M. W. Nucleic
Acids Res. 1998, 26, 5644.
4. Singh, S. K.; Nielsen, P.; Koshkin, A. A.; Wengel, J. Chem.
Commun. 1998, 455.
26. Aramini, J. M.; Mujeeb, A.; Ulyanov, N. B.; Germann,
M. W. J. Biomol. NMR 2000, 18, 287.