Synthesis and evaluation of α-LNA

Article abstract of DOI:10.1081/NCN-100002438

Three different synthetic routes to the α-configured LNA thymine monomer starting from D-allose or D-arabinose were investigated. The introduction of one or four α-LNA monomers into α-DNA had a destabilizing effect on the duplexes. However, a fully modifi

Full text of DOI:10.1081/NCN-100002438

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SYNTHESIS AND EVALUATION OF α-LNA
Nanna K. Christensen a , Jakob K. Dalskov a & Paul Nielsen b
a Department of Chemistry , University of Southern Denmark , Odense M, 5230, Denmark
b Department of Chemistry , University of Southern Denmark , Odense M, 5230, Denmark
Published online: 07 Feb 2007.
To cite this article: Nanna K. Christensen , Jakob K. Dalskov & Paul Nielsen (2001) SYNTHESIS AND EVALUATION OF α-LNA,
Nucleosides, Nucleotides and Nucleic Acids, 20:4-7, 825-828, DOI: 10.1081/NCN-100002438
To link to this article: http://dx.doi.org/10.1081/NCN-100002438
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(4–7), 825–828 (2001)
SYNTHESIS AND EVALUATION OF α-LNA
Nanna K. Christensen, Jakob K. Dalskov, and Poul Nielsen∗
Department of Chemistry, University of Southern Denmark,
5230 Odense M, Denmark
ABSTRACT
Three different synthetic routes to the α-configured LNA thymine monomer
starting from D-allose or D-arabinose were investigated. The introduction of
one or four α-LNA monomers into α-DNA had a destabilizing effect on the
duplexes. However, a fully modified α-LNA sequence displayed strong recog-
nition of complementary RNA, but no transition with DNA.
LNA (locked nucleic acid), is defined as oligonucleotides containing the LNA
monomer, which is locked in a C-3ꢀ-endo conformation (1). Due to the unprece-
dented thermal affinities towards complementary RNA, LNA is a perfect candidate
for antisense therapeutics and diagnostic purposes. Oligodeoxynucleotides with
α-configuration (α-DNA) are known to hybridize to complementary nucleic acids
in a parallel manner (2). The α-anomer of LNA, α-LNA, would be the first analogue
of α-DNA (3) to be restricted in a C-3ꢀ-endo conformation. Therefore, it should po-
tentially display an unprecedented parallel recognition of complementary nucleic
acids (4).
For the synthesis of α-LNA several starting materials were considered. The
synthesis required an appropriate carbohydrate available in the furanose form, with
suitable protecting groups and with the configuration at C-3 defined in a D-glycero
configuration. From these requirements the D-allose derivative 1 was chosen as a
starting material. Thus, the same initial steps as in the original synthesis of β-LNA
(1) were used including a selective benzylation of the diol 2 (Scheme 1). Coupling
of thymine to the methyl furanoside 3 produced an anomeric mixture (α/β 1.3:1)
∗Corresponding author.
825
ꢁ
C
Copyright 2001 by Marcel Dekker, Inc.
www.dekker.com

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CHRISTENSEN, DALSKOV, AND NIELSEN
Scheme 1. a) ref. 5 (∼80%, four steps); b) ref. 6 (50%, three steps); c) i. Thymine, BSA, TMS-Cl,
CH3CN, then TMS-Tf, ii. TBAF, THF, iii. NaH, DMF (57%, three steps), iv. H2, Pd(OH)2-C, EtOH
(98%); d) i. DMTCl, AgNO3, Pyridine, THF (80%), ii. EtN(iPr)2, NC(CH2)2OP(Cl)N(iPr)2, CH2Cl2
(79%). T = thymin-1-yl.
in a reasonable yield using the best of several tested conditions. The two anomeric
LNA monomers 4a and 4b were tediously separated and the phosphoramidite 5
synthesized and used in automated DNA synthesis (4).
Fromthesamestartingmaterialasimplersyntheticroutehasbeenapproached.
Thus, coupling of thymine to the dimesylated methyl furanoside 6 gave the anomers
7a and 7b (α/β 1:1) which were easily separated but obtained in a very low yield
(Scheme 2).
In an earlier attempt, a bicyclic phenyl thioglycoside was synthesized from
3 affording a general bicyclic glycoside donor (6). However, a coupling reaction
afforded 4a in low yields and the strategy shown in Scheme 1 was superior to this
method.
Finally, a synthetic strategy starting from D-arabinose 8 was approached
(Scheme 3). Conversion to the protected analogue 10 was followed by a conve-
nient transformation to the diol 11. The transformation of the exact enantiomer of
11 to the α-L-LNA T-monomer in 8 steps has recently been reported (7), and since
no chiral reagents were used, the same reactions should apply to 11. The nucleobase
coupling was performed on a peracylated sugar revealing only the α-nucleoside in
a high yield (7).
Scheme 2. a) MsCl, pyridine (90%), ii. 20% HCl/MeOH (95%); b) i. Thymine, BSA, TMS-Cl,
CH3CN, then TMS-Tf, ii. TBAF, THF (24%); c) i. NaOH, EtOH, H2O, ii. H2, Pd(OH)2-C, EtOH
(42%, two steps).

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SYNTHESIS AND EVALUATION OF α-LNA
827
O
O
O
O
O
O
HO
HO
TBDPSO
HO
O
O
O
a
b
c
d
8
4a
BnO
HO
BnO
9
10
11
Scheme 3. a) ref. 8 (∼50%, three steps); b) ref. 9 (62%, two steps); c) i. (COCl)2, DMSO, Et3N,
CH2Cl2 (95%), ii. CH2O, NaOH, H2O, THF; d) ref. 7 (32% overall expected (7), eight steps).
The phosphoramidite 5 was used in combination with unmodified phospho-
ramidites in the synthesis of oligonucleotides 12–19. As expected, the introduction
of one α-LNA monomer into DNA (13) results in a very strong decrease in affinity.
In α-DNA, the exchange of one or four α-nucleotides with the α-LNA monomer (15
and 16) also results in strongly decreased affinity against both DNA and especially
RNA. This indicates that the α-LNA monomer do not influence the conformation
of the neighbouring α-nucleotides as suggested for the β-LNA monomers incor-
porated in DNA (10). The fully modified α-LNA sequence 19 shows no transition
with complementary DNA. However, a strong recognition of RNA is observed.
This duplex is confirmed by the fact that a clear melting transition with a decrease
in Tm of 8◦C is observed against the mismatched complementary RNA-sequence.
With the present sequence it is not possible to determine if the α-LNA prefers a
parallel recognition as expected.
In conclusion, three different synthetic pathways have been explored. The
first (Scheme 1) gives the best yields but includes an inconvenient separation of
Table 1. Hybridization Data of α-LNA Sequences
dA14 Complement
rA14 Complement
rA6CA7 Comp.
Sequence
Tm/◦Ca ꢀTm/◦Cb Tm/◦Ca
ꢀTm/◦Cb
Tm/◦Ca
12 5ꢀ-T14
33.0
21.5
32.0
25.5
26.0
22.0
18.0
no Tmc
30.0
22.0
43.0
35.0
24.5
20.0
33.5
45.0
13 5ꢀ-T6 αTLT7
14 5ꢀ-αT14
−11.5
−8.0
15 5ꢀ-αT7TLT6
16 5ꢀ-αT5TL4T5
17 5ꢀ-T10
−6.5
−1.5
−8.0
−4.6
18 5ꢀ-αT10
22.0
37.0
19 5ꢀ-αTL
+1.2d ; +2.5e
10
aMelting temperatures (Tm) obtained in a buffer containing 10 mM Na2HPO4, 100 mM
NaCl, 0.1 mM EDTA, pH 7.0 using 1.5 µM concentrations of each strand assuming identical
extinction coefficients for all thymine nucleotides.
bꢀTm/modification compared with the reference strands 12 and 14.
cNo clear cooperative transition was seen.
d Compared with 18.
eCompared with 17. TL = the α-LNA T-monomer.

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CHRISTENSEN, DALSKOV, AND NIELSEN
anomers. The last synthesis (Scheme 3) presents a longer route, but the target
compound is obtained as the only major product. In the present oligothymidylate
sequence, α-LNA displays the highest affinity towards RNA of any α-D-configured
oligonucleotide analogue. The preferred direction of recognition as well as the
RNA-selectivity indicated for 19 is under further investigation in our laboratory.
ACKNOWLEDGMENT
The Danish Natural Science Research Council is thanked for financial support.
REFERENCES
1. Koshkin A. A.; Singh S. K.; Nielsen P.; Rajwanshi V. K.; Kumar R.; Meldgaard
M.; Olsen C. E.; Wengel J. Tetrahedron, 1998, 3607–3630.
2. (a) Morvan F.; Rayner B.; Imbach J.-L.; Chang D.-K.; Lown J. Nucleic Acids Res.,
1986, 14, 5019–5035. (b) Lancelot G.; Guesnet J.-L.; Vovelle F. Biochemistry, 1989,
28, 7871–7878.
3. Other examples of analogues of α-DNA: (a) Bolli, M.; Lubini, P.; Leumann, C. Helv.
Chim. Acta, 1995, 78, 2077–2096; (b) Laurant, A.; Naval, M.; Debart, F.; Vasseur,
J.-J.; Rayner, B. Nucleic Acid Res., 1999, 27, 4151–4159.
4. Parts of the present work has been described in: Nielsen P.; Dalskov J. K. Chem.
Comm., 2000, 1179–1180.
5. Youssefyeh R. D.; Verheyden J. P. H.; Moffat J. G. J. Org. Chem., 1979, 44, 1301–1309.
6. Nielsen P.; Wengel J. Chem. Comm., 1998, 2645–2646.
7. Ha˚kansson A. E.; Koshkin A. A.; Sørensen M. D.; Wengel J. J. Org. Chem., 2000, 65,
5161– 5166.
8. Vasquez-Tato M. P.; Seijas J. A.; Fleet G. W. J.; Mathews C. J.; Hemmings P. R.;
Brown D. Tetrahedron, 1995, 959–974.
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10. Petersen M.; Nielsen C. B.; Nielsen K. E.; Jensen G. A.; Bondensgaard K.; Singh
S.K.; Rajwanshi V. K.; Koshkin A. A.; Dahl B. M.; Wengel J.; Jacobsen J. P.
J. Mol. Recognit., 2000, 13, 44–53.

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