Parallel RNA-strand recognition by 2′-amino-β-l-LNA

Article abstract of DOI:10.1016/j.bmcl.2009.03.079

A short synthetic route to the first β-l-ribo configured locked nucleic acid (LNA), that is, 2′-amino-β-l-LNA thymine phosphoramidite 6, has been developed from bicyclic nucleoside 1. Incorporation of 2′-amino-β-l-LNA thymine monomers into α-DNA strands r

Full text of DOI:10.1016/j.bmcl.2009.03.079

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2396–2399
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Parallel RNA-strand recognition by 20-amino-b-L-LNA
T. Santhosh Kumar a, Michael E. Østergaard b, Pawan K. Sharma c, Poul Nielsen a,
Jesper Wengel a, Patrick J. Hrdlicka b,
*
a Nucleic Acid Center, Department of Physics and Chemistry, University of Southern Denmark, DK-5230 Odense M, Denmark
b Department of Chemistry, University of Idaho, Moscow, ID 83844-2343, USA
c Department of Chemistry, Kurukshetra University, Kurukshetra 136119, India
a r t i c l e i n f o
a b s t r a c t
A short synthetic route to the first b-L L-LNA
-ribo configured locked nucleic acid (LNA), that is, 20-amino-b-
Article history:
thymine phosphoramidite 6, has been developed from bicyclic nucleoside 1. Incorporation of 20-amino-b-
Received 12 February 2009
Revised 18 March 2009
Accepted 20 March 2009
Available online 24 March 2009
L
-LNA thymine monomers into
a-DNA strands results in probes forming stable duplexes with comple-
mentary RNA in parallel orientation.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Antisense technology
Bridged nucleic acid (BNA)
Conformational restriction
Modified nucleotide
Modified oligonucleotide
Oligonucleotides (ONs) are widely explored as modulators of
gene expression within the antisense regime to develop powerful
research tools and therapeutics. Chemical modification of ONs is
necessary to protect them adequately from enzymatic degradation
by nucleases and to facilitate strong binding to complementary nu-
cleic acid targets.1 The use of ONs modified with conformationally
restricted nucleotide building blocks has been a particularly suc-
cessful approach toward this end.2 LNA3,4 (locked nucleic acid, b-
O
O
O
O
T
T
T
O
O
O
-DNA
-LNA
α
α
(αTL)
D-ribo configuration), arguably the most promising member from
O
O
O
O
O
O
this class of building blocks, exhibits greatly increased thermal
affinity toward complementary antiparallel (ap) DNA/RNA and
markedly improved stability toward nucleases.5
O
T
O
Similarly, a (a-D-ribo configuration, Fig. 1) has been ex-
-DNA6,7
α- -arabino bicyclic
D
plored as a potential building block within the antisense strategy
as it is highly resistant toward enzymatic degradation8–10 and
forms stable duplexes with complementary DNA and RNA. The
two strands are aligned in parallel orientation to form a right
handed helix held together by Watson–Crick base pairing.11,12
-L-LNA
β
nucleic acid
β
(
LTL)
(αTara
)
Figure 1. Building blocks utilized for parallel strand recognition.
More recently, analogs of
a-DNA with C5-propynyl pyrimi-
dines,13,14 modified backbones including cationic phosphorami-
date backbones14–18 or conformationally restricted sugar
moieties,19–21 have been explored in order to optimize recognition
of target oligonucleotide strands in parallel orientation.
As an example of the latter category, fully modified oligothy-
midylate,21a mixed pyrimidine,21b or mixed sequence21c
(a-D
a
-LNA
-ribo configuration, Fig. 1) exhibit markedly higher thermal
affinity toward complementary parallel (p) RNA than -DNA, while
not leading to duplex formation with complementary pDNA,
apDNA, or apRNA.21a–c However,
-LNA/ -DNA-mixmers, that is,
-DNA strands with interspersed incorporations of -LNA mono-
mers, exhibit lower affinity toward pDNA/pRNA than unmodified
-DNA.21b The functional incompatibility between these mono-
a
a
a
* Corresponding author. Tel.: +1 208 885 0108; fax: +1 208 885 6173.
E-mail address: hrdlicka@uidaho.edu (P.J. Hrdlicka).
Research center funded by the Danish National Research Foundation for studies
on nucleic acid chemical biology.
a
a
a
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.079

T. S. Kumar et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2396–2399
2397
mers has been attributed to the inability of
a
-DNA building blocks
yield) concluded this short sequence of protecting group manipu-
lations to provide the desired 20-amino-b-
-LNA phosporamidite
building block 6 (Scheme 1). The identity of all reported com-
pounds was fully ascertained by NMR (1H, 13C, COSY, and/or HET-
COR) and MALDI-HRMS, while purity was verified by 1D NMR.24
Several observations directly or indirectly support the sug-
to adopt N-type22 conformations imposed by
a
-LNA monomers.21b
L
In contrast, mixmers between [3.2.0]bicyclic monomer Tara (E-
a
type,22 Fig. 1) and
pRNA than unmodified
pDNA.21d Recent hybridization studies with fully modified ‘
LNA’ and ‘b- -LNA’ (b- -ribo configuration, Fig. 1) studied in the
mirror image world (i.e., -LNA and b- -LNA, respectively,
against -DNA/ -RNA targets), have suggested b- -LNA as a possible
structural and functional mimic of -LNA’ was
-DNA.21c Thus, ‘b-
found to form stable duplexes with both pRNA and pDNA but not
with apRNA and apDNA. While the furanose conformation of b-
LNA by definition is N-type due to its
-stereochemistry,22 the fura-
nose atoms overlay poorly with those of -LNA (N-type). Studies
evaluating b- -LNA/ -DNA mixmers as probes for parallel strand
recognition of nucleic acid targets have been precluded as the syn-
thesis of b- -LNA nucleotides has not been realized until now.
Herein, we have taken advantage of the recent synthetic avail-
ability of b- -ribo configured LNA nucleosides obtained as promi-
nent byproducts during our synthesis of 20-amino-
-LNA
monomers23 and report: (a) the first synthesis of a b-
-ribo config-
ured LNA phosphoramidite, (b) the automated solid-phase synthe-
sis of fully modified 20-amino-b-
-LNA and mixmers with DNA and
-DNA monomers, and (c) results from thermal denaturation
a
-DNA, display slightly higher affinity toward
-DNA, but decreased affinity toward
a
a
-
L
L
gested b-L-ribo configuration of bicyclic phosphoramidite 6: (a)
a
-L
D
key 1H NMR Nuclear Overhauser Enhancements (NOE) between
L
L
L
H6 and H20/H30, and between H10 and H500 in starting material 1
a
L
have previously been identified and discussed,23 (b) b-
L-ribo con-
figured nucleoside 2 exhibits specific rotation of identical magni-
L
-
tude but opposite sign relative to the known enantiomer25 (Table
L
S1),24 and (c) 1H/13C NMR data of b-
L
-nucleosides 2 and 4–6 are
identical to those of the corresponding enantiomers.24–26
Automated synthesis of ONs was performed on a 0.2 mol scale
using universal CPG solid supports. 20-Amino-b-
-LNA monomer X
was incorporated into DNA and -DNA strands, and was moreover
oligomerized to a fully modified ON. The corresponding phospho-
ramidite building blocks for the incorporation of -DNA thymine
a
L
a
l
L
L
a
L
a
a
-L
and 5-methylcytosine monomers were synthesized as previously
described.27,28 Standard procedures were applied for ON synthesis
except for extended coupling time (25 min) and the use of 1H-tet-
L
L
razole during incorporation of 20-amino-b-
L-LNA phosphoramidite
a
building block 6. The stepwise coupling yield was >95% for -DNA
a
experiments of complexes between these ONs and p/ap DNA/
phosphoramidites and ꢀ99% for the phosphoramidite building
block 6. Following standard workup and purification protocols,24
the composition and purity (>80%) of all modified ONs were veri-
fied by MALDI-MS (Table S4) and ion-exchange HPLC, respec-
tively.24 The hybridization properties of these ONs with DNA/
RNA targets in parallel/antiparallel orientation were studied by
thermal denaturation measurements (A260 vs T) using a neutral
medium salt phosphate buffer.
First, monomer X was incorporated once or thrice into mixed
sequence 9-mer DNA strands (ON1 and ON2). Singly modified
ON1 formed a significantly destabilized duplex with complemen-
tary apRNA, while transitions were not even observed with the
other mixtures, attesting detrimental effects of monomer X on du-
RNA targets.
The synthesis of 20-amino-b-
L
-LNA phosphoramidite building
block 6 initiates from b-
L-ribo configured bicyclic nucleoside 1
(Scheme 1).23 Nucleophilic displacement of the O50-mesylate
group of nucleoside 1 with a benzoate, and subsequent cleavage
thereof using saturated methanolic ammonia afforded amino alco-
hol 2 (53% yield, two steps). Protection of the secondary amino
group of 2 as a trifluoroacetamide furnished nucleoside 3 in 89%
yield, which upon debenzylation using hydrogen and 20%
Pd(OH)2/C in ethyl acetate gave diol 4 in 70% yield. Subsequent
O50-protection as the 4,40-dimethoxytrityl (DMTr) ether afforded
nucleoside 5 in 70% yield, which upon O30-phosphitylation (56%
O
O
ref 23
a
H
H
diacetone
OBn N
OBn N
glucose
MsO
O
HO
N
T
T
2
1
O
T
O
O
b
c
d
CF3
CF3
OBn N
OH
HO
HO
O
T
3
e
4
O
O
T
O
OH
O
f
H
T
CF3
CF3
N
O
N
O
N
DMTrO
DMTrO
NC
T
O
P
O
N
β
2'-amino- -L-LNA
monomer X
5
6
Scheme 1. Reagents and conditions: (a) (i) NaOBz, 15-crown-5, DMF,
D; (ii) satd NH3/MeOH, rt, 53% over two steps; (b) (CF3CO)2O, pyridine, CH2Cl2, 0 °C to rt, 89%; (c) H2,
Pd(OH)2/C, EtOAc, rt, 70%; (d) DMTrCl, DMAP, pyridine, rt, 70%; (e) NC(CH2)2OP(Cl)N(i-Pr)2, N,N-diisopropylethylamine, CH2Cl2, rt, 56%; (f) DNA synthesizer; T = thymin-1-yl;
DMTr = 4,40-dimethoxytrityl.

2398
T. S. Kumar et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2396–2399
20-Amino-b-
rated into a decameric non-selfcomplementary mixed pyrimidine
-DNA that has been used to study parallel recognition of DNA/
RNA targets by
-LNA21b and [3.2.0]bicyclic nucleic acid.21d As pre-
viously reported,21d unmodified
-DNA ON11 forms stable du-
L-LNA X monomers were subsequently incorpo-
Table 1
Thermal denaturation temperatures of duplexes between 20-amino-b-L-LNA and
complementary apDNA/apRNAa
a
ON
Sequence
Tm (°C)
a
a
+ apDNA
+ apRNA
plexes with complementary pDNA and pRNA but not with
ON1
ON2
ON3
50-d(GTG AXA TGC)
50-d(GXG AXA XGC)
50-d(GTG ATA TGC)
<10
<10
28.5
18.0
<10
26.5
complementary apDNA/apRNA (Table 3).34 Introduction of a single
20-amino-b-
L
-LNA monomer X into
a mild destabilization of the duplex with pRNA (
ble 3), while a substantial decrease in thermal affinity toward com-
plementary pDNA was observed
Tm = ꢁ7.5 °C, Table 3).
Incorporation of three or six X monomers into -DNA further sub-
stantiated these trends (see Tm values for ON13–ON14, Table 3).
Importantly, excellent discrimination of singly mismatched targets
a
-DNA (ON12) only resulted in
D
Tm = ꢁ1.0 °C; Ta-
a
Thermal denaturation temperatures [Tm values/°C] measured as the maximum
of the first derivative of the melting curve (A260 vs T) recorded in medium salt buffer
([Na+] = 110 mM, [Clꢁ] = 100 mM, pH 7.0 (NaH2PO4/Na2HPO4)), using 1.0
lM con-
(
D
centration of each strand. Tm-values are averages of at least two measurements. See
Scheme 1 for the structure of monomer X.
a
D
was observed for 20-amino-b- -LNA [Tm(ON12:50-d(GAG GCA GAA
L
Table 2
A)) = 28.0 °C; Tm(ON12:50-r(GAG GCA GAA A)) = 13.0 °C; no transi-
tions for ON14 with these mismatched pDNA/pRNA targets]. All
parallel duplexes with reported Tm-values exhibited smooth mon-
ophasic denaturation profiles (Fig. S3).24 Two experimental obser-
Hybridization data of fully modified 20-amino-b-L-LNA X10 and previously reported
oligonucleotidesa
ON
Sequence
Descriptor
Tm (°C)
Ref.
+ dA14
+ rA14
vations underline that mixmers between 20-amino-b-
L-LNA and a-
ON4
ON5
ON6
ON7
ON8
ON9
ON10b
50-X10
20-Amino-b-
L
-LNA
71.0
22.0
17.5
<10
<10
80.0
ndc
83.0
20.0
35.0
45.0
22.0
70.5
52.0
—
DNA (ON12–ON14) indeed form duplexes with pDNA/pRNA: (a)
identical Tm-values for ON12:pDNA were observed in pH 6 and
50-T10
DNA
21a
21d
21a
21d
3
50-
a
T10
a
a
-DNA
-LNA
pH
7
thermal denaturation medium salt buffers (i.e.,
50-( TL)10
a
50-( Tara
)
10
[3.2.0]bicyclic NA
LNA/DNA
b-
a
Tm = 35.5 °C), suggesting a lack of Hoogsteen base pairs typically
observed in triplexes, and (b) the Job plot between ON12 and pDNA
strongly suggests a 1:1 stoichiometry (Fig. S4).24 Duplexes with
antiparallel targets were generally not observed except for a weak
duplex between the heavily modified ON14 and apRNA. This is
more likely reflecting the formation of a parallel structure rather
than an antiparallel duplex, as five complementary Watson–Crick
base pairs between ON14 and apRNA can be formed if the se-
quences align in parallel orientation. Direct comparison of the
hybridization properties of ON12–ON14 with the corresponding
50-d(TL)9T
50-b- -d(bLTL)9T
L
L-LNA/b-
L
-DNA
32
a
For conditions see Table 1.
Studied in the mirror image world.
nd = not determined.
b
c
plex stability. This is in excellent agreement with previous obser-
vations where introduction of -nucleotides into DNA strands re-
L
mixmers between
a-DNA and a
-LNA21b or [3.2.0]bicyclic nucleic
sults in large decreases in thermal affinity toward p/ap DNA/
acid21d (Table S5),24 reveals that the thermal affinity toward pDNA
as well as pRNA decreases in the order [3.2.0]bicyclic nucleic
RNA.29–31
In stark contrast, fully modified decameric 20-amino-b-
L-LNA
acid > 20-amino-b-
L-LNA > a-LNA. This clearly suggests that the
ON4 exhibits very pronounced thermal affinity toward DNA and
RNA targets (Tm = 71.0 °C and 83.0 °C, respectively, Table 2) com-
pared with the previously reported unmodified DNA ON5,21a fully
conformationally restricted 20-amino-b-
L-LNA nucleotides are ade-
quately tolerated in combination with
a-DNA nucleotides in du-
modified
a a
-DNA ON6,21d -LNA ON7,21a or [3.2.0]bicyclic nucleic
plexes with pRNA, but less well than the E-type [3.2.0]bicyclic
nucleic acid nucleotides.
To conclude, significant improvement of parallel strand recogni-
tion by incorporation of conformationally restricted nucleotide
acid ON8,21d or the highly modified LNA ON93 or ‘b-
L
-LNA’
ON10.32 Interestingly, the UV-mixing curve (i.e., Job plots)33 be-
tween ON4 and complementary RNA strongly suggests the forma-
tion of triplexes with a 2:1 stoichiometry (Fig. S2).24 This is
noteworthy as the thermal denaturation curves between ON4
and complementary DNA/RNA only exhibit smooth monophasic
transitions (Fig. S1).24 Unfortunately, Job plots have not been
determined for the related ON5–ON10 preventing a direct compar-
ison with ON4. Importantly, triplexes between ON4 and DNA/RNA
strands with a central mismatch were significantly less stable
[Tm(ON4 + 50-d(A6CA7)) = no cooperative transition, Tm(ON4 + 50-
r(A6CA7)) = 74.0 °C].
building blocks into a-DNA strands, remains largely elusive despite
the development of numerous building blocks with differently
conformationally restricted sugar moieties.19–21 The 20-amino-b-
L
-LNA monomer reported herein, however, offers the prospect of
additional N20-functionalization in an equivalent manner as for
20-amino-LNA35 or 20-amino- -LNA.36 It is envisioned that the
a-L
hybridization properties thereby can be fine-tuned to facilitate
more efficient recognition of complementary parallel DNA/RNA
targets.
Table 3
Thermal denaturation temperatures of duplexes between
a
-DNA strands modified with monomer X and complementary parallel and antiparallel DNA/RNAa
Tm Tm/mod)
ON
Sequence
(D
pDNA
pRNA
32.0
31.0 (ꢁ1.0)
29.0 (ꢁ1.0)
23.5 (ꢁ1.4)
apDNA
apRNA
ON11b
ON12
ON13
ON14
50-
50-
50-
50-
a-
a-
a-
a-
D
D
D
D
-(MeCTMe
-(MeCTMe
C
C
MeCTT MeCTT T)
43.0
<10
<10
<10
<10
<10
<10
<10
17.5
MeCTX MeCTT T)
35.5 (ꢁ7.5)
22.5 (ꢁ7.2)
<10
-(MeCXMeC MeCTX MeCXT T)
-(MeCXMeC MeCXX MeCXX X)
a
For conditions see Table 1.
D
Tm/mod values for ON12–ON14 are given relative to ON11. For the structure of monomer X and a
-DNA see Figure 1 and Scheme 1; pDNA: 50-
d(GAG GAA GAA A); pRNA: 50-r(GAG GAA GAA A); apDNA: 30-d(GAG GAA GAA A); apRNA: 30-r(GAG GAA GAA A).
b
Tm-values previously reported in Ref. 21b.

T. S. Kumar et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2396–2399
2399
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Acknowledgments
We greatly appreciate funding from the Danish National Re-
search Foundation, Idaho NSF EPSCoR (Experimental Program to
Stimulate Competitive Research) and the Nucleic Acid Based Drug
Design Ph.D. school (NAC DRUG; supported by the Danish Agency
for Science Technology and Innovation). We thank Ms. B. M. Dahl
for ON-synthesis and the Reviewers for helpful comments.
17. Deglane, G.; Abes, S.; Michel, T.; Prevot, P.; Vives, E.; Debart, F.; Barvik, I.;
Lebleu, B.; Vasseur, J.-J. ChemBioChem 2006, 7, 684.
Supplementary data
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19. Keller, B. M.; Leumann, C. J. Synthesis 2002, 789.
General experimental section; preparation and characterization
of nucleosides 2–6; protocols for determination of specific rotation
and synthesis of ONs; representative RP-HPLC (Table S1) and ion-
exchange HPLC gradients (Table S2); MALDI-MS of synthesized
ONs (Table S3); protocol for thermal denaturation studies; repre-
sentative thermal denaturation profiles (Figs. S1 and S3); UV-mix-
20. Scheidegger, S. P.; Leumann, C. J. Chem. Eur. J. 2006, 12, 8014.
21. (a) Nielsen, P.; Dalskov, J. K. Chem. Commun. 2000, 1179; (b) Nielsen, P.;
Christensen, N. K.; Dalskov, J. K. Chem. Eur. J. 2002, 8, 712; (c) Christensen, N. K.;
Bryld, T.; Sørensen, M. D.; Arar, K.; Wengel, J.; Nielsen, P. Chem. Commun. 2004,
282; (d) Sharma, P. K.; Petersen, M.; Nielsen, P. J. Org. Chem. 2005, 70, 4918.
22. For further information regarding furanose conformations see: Saenger, W.
Principles of Nucleic Acid Structure; Springer: Berlin, 1984.
23. Kumar, T. S.; Madsen, A. S.; Wengel, J.; Hrdlicka, P. J. J. Org. Chem. 2006, 71,
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24. See Supplementary data.
25. Rosenbohm, C.; Christensen, S. M.; Sørensen, M. D.; Pedersen, D. S.; Larsen, L.
E.; Wengel, J.; Koch, T. Org. Biomol. Chem. 2003, 1, 655.
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ing curves (Figs. S2 and S4); comparison of Tm-values for
a-DNA
a
a
with incorporations of monomer X, TL and Tara (Table S5);
NMR spectra of nucleosides 2–6. Supplementary data associated
with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2009.03.079.
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