LNA stereoisomers: Xylo-LNA (β-D-xylo configured locked nucleic acid) and α-L-LNA (α-L-ribo configured locked nucleic acid)

Article abstract of DOI:10.1039/a903189h

Synthesis of xylo-LNA containing one 2′-O,4′-C-methyleneβ-D-xylofuranosyl thymine nucleotide monomer and α-LLNAs containing one or four 2′-O,4′-C-methyIene-α-Lribofuranosyl thymine nucleotide monomer(s) has been accomplished using phosphoramidite chemistr

Full text of DOI:10.1039/a903189h

LNA stereoisomers: xylo-LNA (b-d-xylo configured locked nucleic acid) and
a-l-LNA (a-l-ribo configured locked nucleic acid)
Vivek K. Rajwanshi, Anders E. Håkansson, Britta M. Dahl and Jesper Wengel*
Center for Synthetic Bioorganic Chemistry, Department of Chemistry, University of Copenhagen, Universitetsparken
5, DK-2100 Copenhagen, Denmark. E-mail:wengel@kiku.dk
Received (in Cambridge, UK) 21st April 1999, Accepted 3rd June 1999
Synthesis of xylo-LNA containing one 2A-O,4A-C-methylene-
b-d-xylofuranosyl thymine nucleotide monomer and a-l-
LNAs containing one or four 2A-O,4A-C-methylene-a-l-
ribofuranosyl thymine nucleotide monomer(s) has been
accomplished using phosphoramidite chemistry with pyr-
idine hydrochloride as activator; oligothymidylate a-l-LNA
displays strongly enhanced affinity towards complementary
RNA.
nucleoside 2‡ in 50% yield after column chromatographic
separation from the 3A-O-DMT isomer (isolated in 29% yield).
Interactions (sterical interference and/or hydrogen bonding)
between the three substituents at the a-face of the furanose ring
are possible explanations for the unusually low reactivity, and
low selectivity, towards dimethoxytritylation of the primary
hydroxy group of nucleoside 1. Subsequent phosphitylation of
the 3A-hydroxy group afforded in 51% yield the phosphor-
amidite derivative 3.§ The 5A-O-DMT-protected derivative 512¶
was obtained in 80% yield by debenzylation of nucleoside 4 and
subsequently converted into the phosphoramidite 6 in low
yield.∑
In a series of papers, Seela et al. have synthesized and studied
xylo-DNA containing one or more 2A-deoxy-b-d-xylofuranosyl
nucleotide(s).1–4 Compared with the corresponding natural 2A-
deoxy-b-d-ribofuranosyl oligonucleotide reference (T, thymine
The oligomers were synthesized on an automated DNA
synthesizer by use of the phosphoramidite approach.13 Seela
et al. have reported that the use of 3A-O-phosphoramidites in
their syntheses of xylo-DNA required a ten-fold extension of the
standard coupling time. Therefore they applied phosphonate
chemistry for oligomerization of 5A-O-DMT protected 2A-
deoxy-b-d-xylofuranosyl monomers.3 Analogously, the cou-
pling yield of amidite 3 was only 15% after 10 min coupling
time using standard concentrations and 1H-tetrazole as activator
(sequence 5A-XLT6; coupling yields determined spectropho-
tometrically by the release of the 4,4A-dimethoxytrityl group
after each coupling step). This inefficient coupling of 3, which
strongly contrasts with the nearly quantitative coupling of the
parent diastereoisomeric LNA amidite,5–7 is probably caused by
steric hindrance during the reaction between the 5A-hydroxy
group of 5A-OH-T6 and the amidite 3, the latter having three
sterically demanding groups oriented towards the a-face of the
furanose ring. In an attempt to improve the coupling yield of
amidite 3, syntheses of 5A-XLT6 using different activators14–16
and coupling times were performed (Table 1). These experi-
ments showed that the use of pyridine hydrochloride as
activator may indeed allow efficient coupling of sterically very
hindered phosphoramidite building blocks. This is an important
result as research in this area moves towards bicyclic,9
tricyclic,17 functionalized9 and branched18 oligonucleotides.
We then turned to the synthesis of the xylo-LNA and a-l-
LNA sequences 8–10 shown in Table 2 using the optimized
conditions (pyridine hydrochloride; 10 min coupling time)
derivatives are shown for all the four different monomers), xylo-
DNA generally displays decreased thermal affinity towards
complementary single stranded DNA.1–4 We5–9 and others10
have recently reported unprecedented thermal stabilities of
duplexes involving LNA (Locked Nucleic Acid, TL)† and
complementary single stranded DNA or RNA. Here the first
stereoisomers of LNA are introduced, namely xylo-LNA (XL)
containing one or more 2A-O,4A-C-methylene-b-d-xylofurano-
syl nucleotide monomer(s), and a-l-LNA containing one or
more 2’-O,4’-C-methylene-a-l-ribofuranosyl nucleotide mon-
omer(s) (alTL).
O
O
NH
NH
O
N
O
R1O
N
O
O
OR2
O
OR2
R1O
O
1 R1 = R2 = H
Conversion of the thymine xylo-LNA nucleoside 111 and the
a-l-LNA nucleoside 4,12 which both were obtained from d-
glucose, into the phosphoramidite building blocks 3 and 6,
respectively, was performed as depicted in Scheme 1. Nucleo-
side 1 was reacted first with 1.5 equiv. of 4,4A-dimethoxytrityl
chloride (DMTCl) in anhydrous pyridine (25 h, room tem-
perature) and then with an additional 1.0 equiv. (21 h, room
temperature) to afford the 5A-O-DMT-protected xylo-LNA
4 R1 = DMT, R2 = Bn
5 R1 = DMT, R2 = H
6 R1 = DMT
i
iii
ii
2 R1 = DMT, R2 = H
3 R1 = DMT
ii
R2 = P(OCH2CH2CN)(NPri2)
R2 = P(OCH2CH2CN)(NPri2)
Scheme 1 Reagents and conditions: i, DMTCl, anhydrous pyridine (50%);
ii, Pri2NEt, 2-cyanoethyl N,N-diisopropylphosphoramidochloridite, anhy-
drous CH2Cl2 (3: 51%; 6: 7%∑); iii, ammonium formate, Pd/C, MeOH
(80%).
Chem. Commun., 1999, 1395–1396
1395

Table 1 Model syntheses of 5A-XLT6
ing one and four a-l-LNA monomers recognized com-
plementary RNA with remarkably increased affinity. Currently,
the binding properties of a variety of xylo-LNA and a-l-LNA
oligomers are being studied in full detail.
The Danish Natural Science Research Council, The Danish
Technical Research Council and Exiqon A/S are thanked for
financial support. Dr Carl Erik Olsen and Dr Thomas Kofoed
are thanked for recording MALDI-MS spectra.
Activator
t/min
Yielda,b (%)
1H-Tetrazole
1H-Tetrazole
10 min
30 min
30 min
10 min
b
15
31
71
4,5-Dicyanoimidazolec
Pyridine hydrochlorided
a
> 99
Refers to the coupling yield for amidite 3. The coupling yield of the
unmodified ß-cyanoethyl T-amidite was > 99%. c Ref. 14. d Ref. 15,16.
Notes and references
† We have defined LNA as an oligonucleotide containing one or more 2A-
O,4A-C-methylene-ß-d-ribofuranosyl nucleotide monomer(s).
Table 2 Xylo-LNA (8) and a-l-LNAs (9 and 10) synthesized; Tm values
measureda
‡ Selected data for 2: dH(C5D5N) 13.12 (1H, br s, NH), 8.07 (1H, d, J 1.2,
H-6), 7.84–7.00 (13H, m, DMT), 6.26 (1H, s, H-1A), 4.93 (1H, d, J 2.2, H-
2A), 4.61 (1H, m, H-3A), 4.38 (1H, d, J 8.0, H-5Ba), 4.30 (1H, d, J 8.0, H-5Bb),
4.20 (1H, d, J 10.0, H-5Aa), 3.87 (1H, d, J 10.0, H-5Ab), 3.72 (3H, s, OCH3),
3.71 (3H, s, OCH3), 1.97 (3H, s, CH3); m/z (FAB) 573 [M + H]+ (found: C,
66.6; H, 5.7; N, 4.7; C32H32N2O8.0.25H2O requires C, 66.6; H, 5.7; N,
4.9%). This compound was acetylated on an analytical scale (Ac2O,
anhydrous pyridine) yielding a mono-acetate for which the signal for the H-
3A proton was found (1H–1H COSY NMR analysis) at d 5.47 (compared
with d 4.61 for 2) proving compound 2 as being the 5A-O-DMT
derivative.
dA14
rA14
Complement Complement Mass
Tm/°C
(DTm/°C)
Mass
calc.
Tm/°C
(DTm/°C)
found
Sequence
[M 2 H]2 [M 2 H]2
7
8
5A-T14
5A-T7XLT6
32
19 (213)
32 (±0)
28
24 (24)
33 (+5)
46 (+4.5)
4221.2
4225.4
4309.0
4223.8
4223.8
4307.8
9 5A-T7(aLTL)T6
10 5A-T5(aLTL)4T5 36 (+1)
a
Melting temperatures (Tm values) obtained from the maxima of the first
derivatives of the melting curves (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 mM concentrations of the two complementary
strands (assuming identical extinction coefficients for all modified and
unmodified thymine nucleotides). Also shown are changes in Tm value per
modification (DTm) compared with the reference values obtained for 7.
§ Selected data for 3: dP(Me3CN) 154.0, 151.8.
¶ Selected data for 5: dH[(CD3)2SO] 11.39 (1H, br s, NH), 7.62 (1H, d, J 1.0,
H-6), 7.44–6.89 (13H, m, DMT), 5.97 (1H, s, H-1A), 5.94 (1H, d, J 4.3, HO-
3A), 4.44 (1H, d, J 4.3, H-3A), 4.23 (1H, s, H-2A), 4.13 (1H, d, J 8.4, H-5Ba),
3.92 (1H, d, J 8.4, H-5Bb), 3.74 (6H, s, OCH3), 3.31 (2H, m, H-5A), 1.86 (3H,
s, CH3); m/z (FAB) 573 [M + H]+.
∑ Selected data for 6: dP(MeCN) 149.9, 149.3; m/z (FAB) 773 [M + H]+.
This phosphitylation has not yet been optimized and the yield was only 7%
because of the need for repeated column chromatographic purification.
** All oligomers were prepared on a Biosearch 8750 DNA Synthesizer on
CPG solid supports using the standard conditions of the synthesizer.
However, the couplings of amidites 3 and 6 were performed with the
changes described in the text and after premixing the amidite with the
activator (1H-tetrazole 0.45 M; 4,5-dicyanoimidazole and pyridine hydro-
chloride 0.50 M) in anhydrous MeCN in a syringe followed by direct
injection of this mixture into the column reactor during the coupling time
applied. The 5A-O-DMT group was removed on the synthesizer immediately
after completion of the sequences. Subsequent treatment with concentrated
ammonia [32% (w/w), 12 h, 55 °C] and ethanol-precipitation afforded the
product oligomers.
affording coupling yields of ~ 99% for amidite 3 and 97–99%
for amidite 6.** Contrary to amidite 6, consecutive incorpora-
tion of amidite 3 resulted in reduced coupling yields (85–97%;
sequences not shown). The oligomers 8–10 were synthesized in
the DMT-off mode and directly ethanol-precipitated after
cleavage from the solid support yielding products with > 90%
purity as judged from capillary gel electrophoresis.
The results from preliminary binding studies in medium salt
buffer are shown in Table 2. The thermal stability of complexes
formed between xylo-LNA 8, containing a single XL monomer,
and complementary single stranded DNA (dA14) and RNA
(rA14) was significantly reduced (DTm/mod = 213 and 24 °C,
respectively) when compared with the unmodified T14 reference
7. Contrary to this, the binding affinity of a-l-LNAs 9 and 10
was unchanged or slightly improved towards complementary
DNA, and strongly increased towards complementary RNA
(DTm/mod = +4.5 and +5 °C). The latter results compare
closely with our results obtained earlier for the corresponding
LNA oligothymidylate sequences.5 Modeling studies on mono-
mers XL and aLTL clearly show their furanose conformations to
be very different. Thus, whereas XL is locked in a 3A-endo (N-
type) conformation, aLTL is locked in a 3A-exo (S-type)
conformation. The preliminary binding data indicate that the
inverted configuration at C-3A (compared with e.g. LNA and
DNA) in xylo-LNA monomer XL causes an unfavorable local
disruption of the regular duplex structures. However, the 2A-exo
conformation of monomer aLTL allows the formation of very
stable hetero duplexes despite the configuration being inverted
at both C-3A and C-4A. In fact, superimposition of models of the
LNA monomer TL and the a-l-LNA monomer aLTL reveals the
possibility of a very close three-dimensional positioning of the
thymine moieties and of the C-5A- and C-3A-oxygen atoms for
the two monomers existing in locked 3A-endo and 3A-exo
conformations, respectively.
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74, 2054.
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The first LNA stereoisomers, xylo-LNA and a-l-LNA, have
been synthesized using phosphoramidite chemistry on an
automated DNA synthesizer applying extended coupling times
and pyridine hydrochloride as activator. This synthetic method
should be generally applicable for coupling of sterically
hindered phosphoramidite building blocks. a-l-LNA contain-
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Communication 9/03189H
1396
Chem. Commun., 1999, 1395–1396