Functionalized LNA (locked nucleic acid): High-affinity hybridization of oligonucleotides containing N-acylated and N-alkylated 2′-amino-LNA monomers

Article abstract of DOI:10.1039/b307026c

A molecular dynamics (MD) simulation, synthesis and very efficient hybridization for a series of N-acylated and N-alkylated derivatives of 2′-amino-LNA are reported.

Full text of DOI:10.1039/b307026c

Functionalized LNA (locked nucleic acid): high-affinity hybridization of
oligonucleotides containing N-acylated and N-alkylated 2A-amino-LNA
monomers†
Mads D. Sørensen,a Michael Petersenb and Jesper Wengel*b
a Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen,
Denmark
b Nucleic Acid Center‡, Department of Chemistry, University of Southern Denmark, Campusvej 55, DK-5230
Odense M, Denmark
Received (in Cambridge, UK) 19th June 2003, Accepted 14th July 2003
First published as an Advance Article on the web 28th July 2003
A molecular dynamics (MD) simulation, synthesis and very
efficient hybridization for a series of N-acylated and N-
alkylated derivatives of 2A-amino-LNA are reported.
functionalized 2A-amino-LNA monomers V–Z (Scheme 1; see
ESI for structure drawings). The bicyclic nucleoside 1 was
synthesized according to the published procedure12 and was
selectively N-benzoylated giving N-benzoyl derivative 2a and
N-alkylated by reductive alkylation furnishing the N-benzyl, N-
(pyren-1-yl)methyl and N-(2-phthalimido)ethyl derivatives 2b–
d. Subsequent protection of the 5A-hydroxyl groups afforded the
5A-O-4,4A-dimethoxytrityl (5A-O-DMT) derivatives 3a–d. DMT-
protection after acylation failed for the pyren-1-ylcarbonyl
derivative. Instead, the known 5A-O-DMT protected N-tri-
fluoroacetyl nucleoside 3e10 was converted into 3f by deacyla-
tion followed by reaction with pyrenyl-1-carbonyl chloride. The
phosphoramidite building blocks 4a–d and 4f were obtained by
standard phosphitylation of nucleosides 3a–d and 3f.
Studies on the attachment of hydrophobic and hydrophilic
moieties at the furanose ring of internally positioned ribonu-
cleotides of oligonucleotides (ONs) have been intensified over
recent years for the purpose of improved detection systems for
molecular diagnostics or enhanced antisense activity of ONs.1–6
Based on the high-affinity hybridization reported for LNA
(locked nucleic acid)7–9 and 2A-amino-LNA,10 § we decided to
evaluate N-acylated and N-alkylated derivatives of 2A-amino-
LNA monomers as novel building blocks for functionalized
ONs.
An MD simulation (1 ns duration) of a hybrid between a fully
modified nonamer N-benzoyl 2A-amino-LNA and its com-
plementary RNA was performed using standard methods.11 The
benzoyl moieties are located at the brim of the minor groove,
and they occupy roughly half of the space in the groove (Fig. 1).
The carbonyl oxygen is directed toward the solvent, and the
distance to one of the non-bridging phosphate oxygens is ~ 5.3
Å. The benzoyl moieties do not stack with one another, but
rather partake in van der Waals interactions with the sugar
moieties of the 3A-flanking sugars. A control simulation of the
corresponding LNA:RNA hybrid shows that the attachment of
the benzoyl moieties at the 2A-nitrogen does not introduce
significant changes in the duplex geometry.
Synthesis of the novel 2A-amino-LNAs (Tables 1 and 2)
containing monomers V–Z was performed in 0.2 mmol scale on
an automated DNA synthesizer. Standard procedures were used
with modifications13 for amidites 4. For synthesis of ONs that
contain V and X, tert-butylphenoxyacetyl protected DNA
amidites were applied to allow mild deprotection. The coupling
yields were ca. 99% for all modified (10 min coupling time) and
unmodified phosphoramidites. After deprotection and cleavage
from the solid support (32% aq. ammonia)¶ and desalting,
satisfactory purities ( > 80%) of all 2A-amino-LNAs were
verified by capillary gel electrophoresis∑ and their composition
by MALDI-MS analysis.
The influence on thermal denaturation of the functionalized
2A-amino-LNA monomers V–Z was studied toward DNA and
RNA complements (Table 1). All novel mixed sequence 9-mers
that contain two or three modified 2A-amino-LNA monomers
(ON4–ON8) hybridize efficiently and in general with very high
thermal stabilities comparable to those obtained for the LNA
(ON2 and ON107,8) and N-methyl 2A-amino-LNA (ON3)10
Based on this simulation we synthesized phosphoramidite
derivatives 4 as building blocks for ONs that contain the
Fig. 1 Average structure from molecular dynamics simulation. Stereo view
of a fully modified nonamer N-benzoyl 2A-amino-LNA [5A-(MeCTGA-
TATGMeC)L:RNA] hybrid viewed into the minor groove. For clarity, no
hydrogens are shown. The colouring scheme is: nucleobases, yellow; sugar–
phosphate backbone, red; benzoyl moieties, blue.
Scheme 1 Reagents and conditions: i) (2a): benzoyl chloride, Na2CO3,
MeOH, 0 °C, 93%; (2b): benzaldehyde, AcOH, NaCNBH3, MeOH, 85%;
(3c) pyrene-1-carbaldehyde, AcOH, NaCNBH3, MeOH, 94%; (3d) phthali-
midoacetaldehyde, AcOH, NaCNBH3, MeOH, 55%; ii) DMTCl, pyridine
(3a: 88%, 3b: 92%, 3c: 77%, 3d: 72%); iii) a) 3e,10 sat. NH3 in MeOH, 92%,
† Electronic supplementary information (ESI) available: structures of 2A-
amino-LNA monomers V–Z. See http://www.rsc.org/suppdata/cc/b3/
b307026c/
b) pyren-1-ylcarbonyl chloride, Na2CO3, MeOH,
0 °C, 67%; iv)
‡ A research center funded by the Danish National Research Foundation for
studies on nucleic acid chemical biology.
NC(CH2)2OP(Cl)N(i-Pr)2, (i-Pr)2NEt, CH2Cl2 (4a: 82%, 4b: 70%, 4c: 76%,
4d: 58%, 4f: 57%). T = thymin-1-yl.
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CHEM. COMMUN., 2003, 2130–2131
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containing monomers TL (purely conformational effect), X or Y
(once or twice) all displayed near to additive affinity increases
following the trend in Table 1. With ON19 [containing two N-
(pyren-1-yl)methyl 2A-amino-LNA monomers Y] as target
strand, significantly lower thermal affinities were obtained,
except for ON7, i.e. duplex ON7:ON19 that includes a “2 + 2
pyrene unit”. This indicates an interstrand stabilizing effect,
possibly stacking of the pyrenyl moieties, for duplexes that
contain the rather flexible N-(pyren-1-yl)methyl 2A-amino-LNA
monomer Y in the two strands. Furthermore, juxtapositioning
and interstrand interaction between the pyrene units of duplex
ON7:ON19 was indicated by steady-state fluorescence experi-
ments that show a strong pyrene–pyrene excimer band at
430–530 nm not observed for the duplex between ON7 and
complementary DNA or for single-stranded ON7.14
Table 1 Thermal denaturation studies toward DNA/RNA
Complementary
DNA
Complementary
RNA
Oligonucleotide (5A–3A)
Tm/°C
DTm/°C
Tm/°C
DTm/°C
ON1:d(GTG ATA TGC)
ON2:d(GTLG ATLA TLGC)
ON3:d(GMG AMA MGC)
ON4:d(GVG AVA VGC)
ON5:d(GWG AWA WGC)
ON6:d(GTG AXA XGC)
ON7:d(GTG AYA YGC)
ON8:d(GZG AZA ZGC)
27
Ref.
—
—
+6.7
+3.3
+5.5
20.5
+4.7
28
Ref.
—
—
+9.3
+7.3
+6.5
+3
447,8
3910
47
507,8
4910
56
37
38
26
41
50
41
34
49
+7
ON9:T10
18
Ref.
—
+6.3
—
+5.2
+7.8
18
Ref.
—
+6.1
—
—
+6.6
In conclusion, the 2A-nitrogen atom of 2A-amino-LNA
monomers is very suitable for functionalization of high-affinity
ONs, and we are currently expanding the structural diversity for
exploratory applications within biology and nanobiotechnol-
ogy.
ON10: (TL)9T
ON11: V9T
ON12: X9T
ON13: Y9T
ON14: Z9T
807
75
717
73
n.t.
65
86
n.t.
n.t.
77
Melting temperatures (Tm values) measured as the maximum of the first
derivative of the melting curve (A260 vs. temperature; 10 to 70 °C or 10 to
95 °C; increase 1 °C min21) 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. Each Tm value was
determined in two independent experiments, and DTm values, calculated per
monomer V–Z relative to the Tm value for ON1 and ON9, were within ±0.5
°C consistent for the two experiments. “n.t.”: No cooperative melting
transition. “TL” denotes an LNA thymine monomer.7,8
We thank the Danish National Research Foundation and the
Danish Research Agency for financial support, Ms. Britta M.
Dahl for oligonucleotide synthesis and Dr Michael Meldgaard,
Exiqon A/S, for MALDI-MS analysis.
Notes and references
§ We have defined a 2A-amino-LNA as an ON containing one or more 2A-
amino-2A-deoxy-2A-N,4A-C-methylene-b-D
-ribofuranosyl monomer(s).10
Table 2 Thermal denaturation studies toward N-(pyren-1-yl)carbonyl 2A-
amino-LNA (ON18) and N-(pyren-1-yl)methyl 2A-amino-LNA (ON19).
See the footnote in Table 1 for details and conditions
¶ Conditions: 2 h at room temperature for ON4, ON6, ON11, ON12, ON16
and ON18; 48 h at 55 °C for ON8; 48 h at 55 °C followed by 48 h at 70 °C
for ON14; 16 h at 55 °C for the remaining ONs.
∑ Analysis by standard capillary gel electrophoresis of the almost fully
modified ON12 and ON14 could not be performed.
ON18 d(GCA
XAX CAC)
ON19: d(GCA
YAY CAC)
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Tm/°C
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ON16: d(GTG AXA TGC)
ON17: d(GTG AYA TGC)
ON6: d(GTG AXA XGC)
ON7: d(GTG AYA YGC)
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compared to the methylene groups is indicated by comparison
of the influence on thermal stabilities of V vs. W and X vs. Y
[ON4–ON7 and the corresponding singly modified ONs (not
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(almost) fully modified N-benzoyl 2A-amino-LNA ON11 shows
remarkably efficient binding toward DNA and RNA comple-
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nyl 2A-amino-LNA ON12 no duplex is formed. Possibly,
unfavourable steric interactions impede duplex formation in this
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(2-amino)ethyl 2A-amino-LNA ON14 is able to efficiently target
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The possibility of interstrand stacking of the pyrenyl moieties
of monomers X and Y was evaluated (Table 2). With ON18
[containing two N-(pyren-1-yl)carbonyl 2A-amino-LNA mono-
mers X] as target strand, ON15–ON17, ON6 and ON7
14 M. D. Sørensen, N. H. Harrit and J. Wengel, manuscript in prepara-
tion.
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