The adenine derivative of α-L-LNA (α-L-ribo configured locked nucleic acid): Synthesis and high-affinity hybridization towards DNA, RNA, LNA and α-L-LNA complementary sequences

Article abstract of DOI:10.1016/S0960-894X(01)00110-X

Synthesis of a 9-mer α-L-LNA (α-L-ribo configured locked nucleic acid) containing three 9-(2-O,4-C-methylene-α-L-ribofuranosyl)adenine nucleotide monomer(s) has been accomplished. The work involved synthesis of the bicyclic adenine nucleoside via a conden

Full text of DOI:10.1016/S0960-894X(01)00110-X

Bioorganic & Medicinal Chemistry Letters 11 (2001) 935–938
The Adenine Derivative of ꢀ-L-LNA (ꢀ-L-ribo Configured Locked
Nucleic Acid): Synthesis and High-Affinity Hybridization towards
DNA, RNA, LNA and ꢀ-L-LNA Complementary Sequences
Anders E. Hakanssona and Jesper Wengelb,*
aDepartment of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen, Denmark
bDepartment of Chemistry, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark
Received 11 December 2000; revised 5February 2001; accepted 14 February 2001
Abstract—Synthesis of a 9-mer a-l-LNA (a-l-ribo configured locked nucleic acid) containing three 9-(2-O,4-C-methylene-a-l-ribo-
furanosyl)adenine nucleotide monomer(s) has been accomplished. The work involved synthesis of the bicyclic adenine nucleoside via
a condensation reaction between l-threo-pentofuranose derivative 1 and 6-N-benzoyladenine followed by C20-epimerization.
Hybridization studies demonstrated very strong duplex formation with 9-mer complementary DNA, RNA, LNA and a-l-LNA
target sequences. # 2001 Elsevier Science Ltd. All rights reserved.
Synthesis of novel oligonucleotide analogues has been
conducted with the aim of developing nucleic acid
mimics with superior properties, for example, increased
stability towards nucleolytic degradation and increased
binding affinity and specificity towards complementary
nucleic acid targets.1 Following the discovery of LNA
(locked nucleic acid, b-d-ribo isomer, Fig. 1),2,3 we have
recently introduced the stereoisomeric analogue termed
a-l-LNA (a-l-ribo configured locked nucleic acid, a-l-
ribo isomer, Fig. 1).3,4 By virtue of their dioxabicy-
clo[2.2.1]heptane skeletons, the conformations of the
furanose rings of an LNA monomer and an a-l-LNA
monomer are efficiently locked in an N-type and an S-
type (or N-type, C30-endo)5 conformation, respectively
(Fig. 1). So far, only the properties of a-l-LNA con-
taining the thymine a-l-LNA monomer in homo-thymine
or in mixed sequence 9-mer contexts have been studied
indicating helical thermostability of a-l-LNA/DNA
and a-l-LNA/RNA duplexes approaching those of the
corresponding LNA/DNA and LNA/RNA duplexes.2,4
A similar reaction sequence is impossible for the corre-
sponding purine derivatives because of the inability of
these to form anhydro-nucleoside intermediates.
In this letter, a synthetic scheme for the purine a-l-LNA
monomers is disclosed, as exemplified by the synthesis
of the adenine derivative (Scheme 1). In addition, the
hybridization properties of a 9-mer a-l-LNA (ꢀ-L-LNA-2,
Table 1) containing three a-l-LNA adenine monomers
(
ꢀLAL, Scheme 1) towards complementary single-stranded
9-mer DNA, RNA, LNA and a-l-LNA target sequences
are evaluated.
The starting material for the synthesis of the target
bicyclic nucleoside 12 and the corresponding phosphor-
amidite derivative 15 was the known tetra-O-acyl-l-
threo-pentofuranose 1.7 Coupling between furanose 1
and 6-N-benzoyladenine using SnCl4 as Lewis acid
under thermodynamic control afforded the desired N9-
regioisomeric nucleoside 2 [9-(2-O-acetyl-5-O-benzoyl-
4-C-benzoyloxymethyl-3-O-benzoyl-a-l-threo-pentofur-
anosyl)-6-N-benzoyladenine] in 52% yield.8 As antici-
pated by the presence of the 20-acetoxy group suggesting
anchimeric assistance during the coupling reaction, the
b-anomeric nucleoside product was neither detected by
analytical TLC nor isolated. It should be noted that the
assignment of the synthesized nucleoside derivatives 2–
15 as the N9-regioisomers was confirmed by comparison
of the obtained 13C chemical shift values for derivatives
12 and 13 (vide infra) with the published values for
N7- and N9-regioisomeric b-d-ribofuranosyladenines.9 A
A key step in the synthesis of the thymine a-l-LNA
monomer was a cascade reaction on tri-O-mesyl
nucleoside A involving C20-epimerization, hydrolysis of
the resulting anhydro-nucleoside intermediate, cycliza-
tion, and introduction of a hydroxy group at C50
affording the bicyclic nucleoside B (Fig. 2).6
*Corresponding author. Tel.: +45-6550-2510; fax: +45-6615-8780;
e-mail: jwe@chem.sdu.dk
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00110-X

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936
Vorbruggen-type10 coupling reaction with TMS-triflate
as Lewis acid and N,O-bis(trimethylsilyl)acetamide as
silylating agent furnished nucleoside 2 in only 21%
yield. Next, epimerization at C20 via intermediates 3 and
4 was performed. Selective deacylation of the 20-O-ace-
tyl group to give intermediate 3 was accomplished using
half-saturated methanolic ammonia,11 whereupon reac-
tion with triflic anhydride in a mixture of dichloro-
methane and pyridine afforded intermediate 4 according
to analytical TLC. No purification of this intermediate
was necessary as direct reaction with potassium acetate
and 18-crown-6 in toluene at 80 ꢀC afforded the desired
C20-epimeric nucleoside 5 in excellent 84% yield from
2.12 Complete deacylation of 5 to give nucleoside 6 was
accomplished using a mixture of saturated methanolic
and aqueous ammonia, and via a transient protection
protocol (O-trimethylsilylation, 6-N-benzoylation and
desilylation) the corresponding 6-N-benzoyl derivative 7
was obtained in 91% from 5. To prepare for cyclization
by intramolecular attack from the 20-hydroxy group, the
di-O-mesyl derivative 8 was prepared in 79% yield by
reaction with 2.2 equiv of mesyl chloride in pyridine at
0 ꢀC. No mesylation of the secondary hydroxy group
was observed. Cyclization of nucleoside 8 with con-
comitant demesylation and debenzoylation was performed
in only 13% yield by reaction in a mixture of aqueous
sodium hydroxide and 1,4-dioxane (reflux, 72 h). Due to
the low yield and the need for repeated benzoylation of
the adenine moiety (required for automated oligonu-
cleotide synthesis), another approach was used. Thus,
nucleoside 8 was treated with sodium hydride in THF
which afforded derivative 9 with a bicyclic furanose
moiety which was subsequently converted into the
desired base-protected a-l-LNA adenine dihydroxy
nucleoside 12. Thus, substitution of the remaining
mesyloxy group of 9 with an acetate group to give 10,
selective deacetylation (half-saturated methanolic
ammonia) to give nucleoside 11 and subsequent deben-
zylation (Pd/C, ammonium formate) afforded the target
nucleoside 1213 in 32% yield from 8 (Scheme 1). In
addition, the fully deprotected nucleoside 13 was
obtained in 17% yield. The assigned a-l-ribo config-
uration of nucleoside 12 was confirmed by 1H NOE
experiments, that is, mutual NOE effects between H2/
H8 of the adenine moiety and the protons of the endo-
cyclic methylene group (10% and 3% when irradiating
H2/H8 and the protons of the endocyclic methylene group,
respectively) and between H10 and H20/H30 (5%/3% and
11% when irradiating H10 and H20/H30, respectively).14
Figure 1. The structures of nucleotide monomers of DNA, LNA and
a-l-LNA. The conformational equilibrium between N-type and S-type
conformers (e.g., the C30-endo/3E north(N)-type and C30-exo/3E
south(S)-type conformations shown) of an unmodified DNA monomer
is indicated. Also shown are the locked N-type (C30-endo/3E) and ‘S-
type’ (C30-exo/3E; N-type, C30-endo, 3E)5 furanose conformations of
an LNA and an a-l-LNA monomer, respectively.
Scheme 1. Synthesis of the a-l-LNA adenine nucleosides 12 and 13
and the phosphoramidite monomer 15 suitable for incorporation of
the a-l-LNA adenine monomer ꢀL L into oligonucleotides. Reagents,
A
conditions and yields: (i) 6-N-benzoyladenine (2 equiv), SnCl4 (3
equiv), acetonitrile, rt (52%); (ii) half-saturated NH3 in methanol,
0 ꢀC; (iii) Tf2O (3 equiv), pyridine, dichloromethane, ꢁ30 to 0 ꢀC;
(iv) KOAc (5equiv), 18-crown-6 (2 equiv), toluene, 80 ꢀC (84%, 3
steps); (v) saturated NH3 in methanol/32% aq NH3 (4:1), rt; (vi) (a)
TMSCl (15equiv), pyridine, rt; (b) BzCl (5equiv), pyridine, rt; (c)
32% aq NH3/H2O/methanol (6:3:4), rt (91%, two steps); (vii) MsCl
(2.2 equiv), pyridine, 0 ꢀC (79%); (viii) NaH (2 equiv), THF, rt; (ix)
KOAc (5equiv), 18-crown-6 (2 equiv), 1,4-dioxane, 100 ꢀC (87%, two
steps); (x) half-saturated NH3 in methanol, 0 ꢀC (83%); (xi)
HCOONH4 (3 equiv), 10% Pd/C, methanol, reflux (12: 44%, 13:
24%); (xii) DMTCl (2 equiv), pyridine, rt (82%); (xiii) 2-cyanoethyl-
N,N-diisopropylphosphoramidochloridite (2 equiv), N,N-diisopropy-
lethyamine (4 equiv), dichloromethane, rt (63%).
Figure 2. The key cascade reaction during the synthesis of the thymine
a-l-LNA monomer. The yield for the conversion shown was 58% and
the reagents used were 6 M NaOH and EtOH (1:1, reflux, 43 h; ref 6).

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937
Table 1. Melting temperatures (Tm values) obtained from the max-
ima of the first derivatives of the melting curves (A260 vs temperature)
recorded in a medium salt buffer (10 mM sodium phosphate, 100 mM
sodium chloride, 0.1 mM EDTA, pH 7.0) using 1.5mM concentra-
tions of the two complementary strands assuming identical extinction
coefficients for modified and unmodified oligonucleotides. The com-
plementary 9-mer DNA, RNA, LNA and a-l-LNA sequences are also
shown. ꢀ-L-LNA-2 represents the first a-l-LNA containing a-l-LNA
purine monomers. References are given in the table for published Tm
values. A, C, G, T and U denotes standard DNA/RNA monomers.
shown by Tm values of 60 and 56ꢀC, respectively. Only
the corresponding LNA:LNA duplex is thermally more
stable (Tm=74 ꢀC). As LNA has been established as an
RNA mimic2d,17 and as a-l-LNA, as shown herein,
hybridizes very efficiently with RNA targets, the for-
mation of very stable LNA:a-l-LNA duplexes was
expected.18
T
monomers
L and AL denotes LNA monomers. ꢀL L and ꢀL L denote a-l-LNA
T A
Conclusion
A viable synthetic route for the first a-l-LNA purine
monomer has been developed. The required C20-epi-
merization was efficiently accomplished by substituting
a 20-triflyloxy group with an acetate group. Subsequent
cyclization and preparation of the phosphoramidite
building block allowed synthesis of a 9-mer a-l-LNA
containing three a-l-LNA adenine monomers. In
hybridization studies towards complementary DNA and
RNA, high-affinity recognition was demonstrated thus
indicating the general affinity-enhancing character of
a-l-LNA compared with the corresponding unmodified
references (DNA-2:DNA-1 and DNA-2:RNA-1). It has
DNA-1
RNA-1
LNA-1
ꢀ-L-LNA-1
DNA-2
RNA-2
50-(GTGATATGC)
50-r(GUGAUAUGC)
50-(GTLGATLATLGC)
50-(G(ꢀLTL)GA(ꢀLTL)A(ꢀLTL)GC)
50-(GCATATCAC)
50-r(GCAUAUCAC)
LNA-2
ꢀ-L-LNA-2
50-(GCALTALTCALC)
50-(GC(ꢀLAL)T(ꢀLAL)TC(ꢀLAL)C)
Tm values/ꢀC
DNA-2
RNA-2
LNA-2
ꢀ-L-LNA-2
DNA-1
RNA-1
LNA-1
a-l-LNA-1
29
282b
382e
502b
454c
402d
46
742d
5
37
42
60
6
272e
442b
374c
5
6
furthermore been demonstrated that a-l-LNA:a-l-
LNA and a-l-LNA:LNA, like LNA:LNA, constitute
exceptionally stable duplex structures.
Following standard procedures, the phosphoramidite
building block 1515 was obtained from diol 12 by selec-
tive 4,40-dimethoxytritylation to give derivative 14 in
82% yield and subsequent phosphitylation (63% yield).
Phosphoramidite 15 was used on an automated synthe-
sizer to give a 9-mer a-l-LNA [ꢀ-L-LNA-2, 50-(GC
Acknowledgements
The Danish Natural Science Research Council, The
Danish Technical Research Council and Exiqon A/S are
acknowledged for financial support. Ms. Britta M. Dahl
is thanked for oligonucleotide synthesis and Drs. Carl
E. Olsen (The Royal Veterinary and Agricultural Uni-
versity) and Michael Meldgaard (Exiqon A/S) for
MALDI-MS analysis.
(
ꢀLAL)T(ꢀLAL)TC(ꢀLAL)C)]16 containing three a-l-
LNA adenine monomers (ꢀLAL, Scheme 1). The synth-
esis was performed as described earlier4 with a stepwise
coupling yield of >90% for amidite 15 (with coupling
times of 10, 15or 30 min) using 1 H-tetrazole as acti-
vator compared with >99% for unmodified 20-deoxy-
nucleoside phosphoramidites. It is noteworthy that
another sample of amidite 15 afforded stepwise coupling
yields of >99% using similar conditions8 which shows
that there is no significant sterical hindrance at the a-
face of the furanose ring of 15 during the coupling
reactions on the DNA synthesizer. Instead, variations in
purity in between the phosphoramidite samples may
explain the different stepwise coupling yields obtained.
References and Notes
1. (a) De Mesmaeker, A.; Haner, R.; Martin, P.; Moser, H. E.
Acc. Chem. Res. 1995, 28, 366. (b) Herdewijn, P. Liebigs Ann.
1996, 1337. (c) Freier, S. M.; Altmann, K.-H. Nucleic Acids
Res. 1997, 25, 4429. (d) Wengel, J. Acc. Chem. Res. 1999, 32,
301. (e) Herdewijn, P. Biochim. Biophys. Acta 1999, 1489, 167.
(f) Uhlmann, E. Curr. Opin. Drug Discovery Dev. 2000, 3, 203.
2. (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) Obika,
S.; Nanbu, D.; Hari, Y.; Andoh, J.; Morio, K.; Doi, T.; Ima-
nishi, T. Tetrahedron Lett. 1998, 39, 5401. (d) Koshkin, A. A.;
Nielsen, P.; Meldgaard, M.; Rajwanshi, V. K.; Singh, S. K.;
Wengel, J. J. Am. Chem. Soc. 1998, 120, 13252. (e) Singh,
S. K.; Wengel, J. Chem. Commun. 1998, 1247.
3. We have defined LNA as an oligonucleotide containing one
or more 20-O,40-C-methylene-b-d-ribofuranosyl nucleotide
monomer(s). The natural b-d-ribo configuration is assigned to
LNA (and LNA monomers) as the positioning of the N1, O20,
O30, and O50 atoms are equivalent to the one found in RNA.
Analogously, a-l-LNA has been defined as an oligonucleotide
containing one or more 20-O,40-C-methylene-a-l-ribofuranosyl
nucleotide monomer(s).
The hybridization properties of ꢀ-L-LNA-2 containing
three a-l-LNA adenine monomers and six DNA
monomers towards complementary 9-mer single-stran-
ded DNA, RNA, LNA and a-l-LNA targets were
evaluated and are shown in Table 1. In addition, a
number of already published Tm values are included for
comparison. The data obtained for ꢀ-L-LNA-2 show
that the affinity- enhancing effect of introducing a-l-
LNA thymine monomers4 can be extended to a-l-LNA
adenine monomers. Thus, the Tm values for ꢀ-L-LNA-2
are increased by 8 and 15 ꢀC towards complementary
DNA and RNA, respectively, compared with the corre-
sponding unmodified references (DNA:DNA and
DNA:RNA duplexes). It is furthermore revealed that ꢀ-
L-LNA-2 binds very strongly indeed towards the LNA
(LNA-1) and a-l-LNA (ꢀ-L-LNA-1) complements as

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A. E. Hakansson, J. Wengel / Bioorg. Med. Chem. Lett. 11 (2001) 935–938
938
4. (a) Rajwanshi, V. K.; Hakansson, A. E.; Dahl, B. M.;
Wengel, J. Chem. Commun. 1999, 1395. (b) Rajwanshi, V. K.;
Hakansson, A. E.; Kumar, R.; Wengel, J. Chem. Commun.
1999, 2073. (c) Rajwanshi, V. K.; Hakansson, A. E.; Sørensen,
M. D.; Pitsch, S.; Singh, S. K.; Kumar, R.; Nielsen, P.; Wen-
gel, J. Angew. Chem. Int. Ed. 2000, 39, 1656.
Hz, 500-H), 3.73 (2H, d, J=5.5 Hz, 50-H); 13C NMR
((CD3)SO) d 152.2, 151.6, 150.3, 133.4, 132.5, 128.5, 125.2 (Bz,
C-2, C-4, C-5, C-6), 142.5 (C-8), 90.7 (C-40), 84.4 (C-10), 79.4
(C-20), 72.9 (C-30), 72.6 (C-500), 57.5 (C-50).
14. Cyclization by intramolecular nucleophilic attack from the
50-hydroxy group [20-O-mesyl derivative of nucleoside 3,
NaOH (10 equiv) in H2O/dioxane (1:1), reflux, 94 h; 20-O-tri-
fluoromethanesulfonyl derivative of nucleoside 3, NaH (app. 6
equiv) in DMF, rt to 140 ꢀC, 140 h] was unsuccessfully
attempted. In contrast, Robins et al. have earlier reported
successful cyclization of the structurally similar nucleoside
9-(3-azido-3-deoxy-2-O-mesyl-b-d-xylofuranosyl)adenine to
the corresponding 20,50-anhydro nucleoside derivative: Robins,
M. J.; Hawrelak, S. D.; Kanai, T.; Siefert, J.-M.; Mengel, R. J.
Org. Chem. 1979, 44, 1317. It is therefore conceivable that the
cyclization described above will be possible using the right
conditions/reagents.
5. The furanose conformation of an a-l-LNA monomer could
alternatively be assigned as N-type (C30-endo, E) because of
3
its l-configuration. However, for a direct comparison with the
conformations of the natural DNA/RNA monomers and the
parent LNA monomers, the furanose conformation of an a-l-
LNA monomer is considered herein as equivalent to an S-type
conformation.
6. Hakansson, A. E.; Koshkin, A. A.; Sørensen, M. D.; Wen-
gel, J. J. Org. Chem. 2000, 65, 5161.
7. Rajwanshi, V. K.; Kumar, R.; Hansen, T. K.; Wengel, J. J.
Chem. Soc., Perkin Trans. 1 1999, 1407.
8. Hakansson, A. E.; Bryld, T.; Wengel, J. Manuscript in
preparation.
15. Data for (1S,3R,4S,7R)-3-(6-N-benzoyladenin-9-yl)-7-(2-
cyanoethoxy(diisopropylamino)phosphinoxy -1-(4,40 -dimeth-
oxytrityloxymethyl)-2,5-dioxabicyclo[2.2.1]heptane (15): Rf :
0.70, 0.77 (8% MeOH in CH2Cl2); 31P NMR (CH3CN) d
150.2, 150.4.
16. The 9-mer ꢀ-L-LNA-2 was purified (after standard depro-
tection and cleavage from the solid support using 32% aqueous
ammonia) by DMT-ON reversed phase chromatography on dis-
posable purification cartridges which includes detritylation. The
composition of the a-l-LNA was confirmed by MALDI-MS
analysis and the purity (>90%) by capillary gel electrophoresis.
17. (a) 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. (b) Bondensgaard, K.; Petersen,
M.; Singh, S. K.; Rajwanshi, V. K.; Kumar, R.; Wengel, J.;
Jacobsen, J. P. Chem. Eur. J. 2000, 6, 2687.
9. Chenon, M.; Pugmire, R. J.; Grant, D. M.; Panzica, R. P.;
Townsend, L. B. J. Am. Chem. Soc. 1975, 97, 4627.
10. (a) Vorbruggen, H.; Krolikiewicz, K.; Bennua, B. Chem.
Ber. 1981, 114, 1234. (b) Vorbruggen, H.; Hofle, G. Chem.
Ber. 1981, 114, 1256.
11. Neilson, T.; Werstiuk, E. S. Can. J. Chem. 1971, 49, 493.
12. This strategy for inversion of a secondary hydroxy group
has been applied earlier, for example, in a cyclopentane sys-
tem: Uchida, C.; Kimura, H.; Ogawa, S. Bioorg. Med. Chem.
Lett. 1997, 5, 921.
13. Data for (1R,3R,4S,7R)-3-(6-N-benzoyladenin-9-yl)-7-
hydroxy-1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptane (12):
Rf : 0.23 (13% MeOH in CH2Cl2); FAB-MS m/z 384
[M+H]+; 1H NMR ((CD3)SO) d ꢂ11.1 (1H, br s, 6-NH),
8.72 (1H, s, 2-H), 8.65(1H, s, 8-H), 8.04 (2H, d, J=8.2 Hz,
Bz), 7.65–7.51 (3H, m, Bz), 6.51 (1H, s, 10-H), 5.94 (1H, d,
J=4.4 Hz, 30-OH), 4.93 (1H, t, J=5.6 Hz, 50-OH), 4.44–4.42
(2H, m), 4.09 (1H, d, J=8.2 Hz, 500-H), 4.02 (1H, d, J=8.2
18. The formation of very stable LNA:a-l-LNA duplexes
is confirmed by the Tm value of 56 ꢀC obtained for the ꢀ-L-
LNA-1:LNA-2 duplex.