A substituted triaza crown ether as a binding site in DNA conjugates

Article abstract of DOI:10.1039/b301100c

Synthesis of an asymmetrically substituted triaza crown ether, its incorporation into the 3′-end and 5′-end of nine-mer oligonucleotides, and the influence of various alkanediamine ligands on duplex thermostabilities are reported.

Full text of DOI:10.1039/b301100c

A substituted triaza crown ether as a binding site in DNA conjugates
Stefan Vogel,a Katja Rohr,a Otto Dahlb and Jesper Wengel*a
a Nucleic Acid Center†, Department of Chemistry, University of Southern Denmark, Campusvej 55, DK-5230
Odense M, Denmark. E-mail: jwe@chem.sdu.dk
b Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen,
Denmark
Received (in Cambridge, UK) 29th January 2003, Accepted 5th March 2003
First published as an Advance Article on the web 18th March 2003
Synthesis of an asymmetrically substituted triaza crown
ether, its incorporation into the 3A-end and 5A-end of nine-
mer oligonucleotides, and the influence of various alkane-
diamine ligands on duplex thermostabilities are reported.
coupling time), amidite 6 (92% coupling yield) and a universal
support. ON7 and ON9 were synthesized using standard DNA
amidites and LNA amidites ( > 98% coupling yields using 2 and
10 min coupling time, respectively), amidite 6 (for ON7, 98%
coupling yield) and standard supports. The coupling yields for
amidite 6 were estimated by trityl monitoring after having
performed an additional coupling reaction with a commercial
DNA phosphoramidite without a preceding capping step on a
sample of the support after coupling with amidite 6. Satisfactory
purities ( > 80%) of ON7–ON9 were verified by capillary gel
electrophoresis. Peaks, although only minor ones, correspond-
ing to the calculated masses of ON7 and ON8 were obtained by
MALDI-MS analysis supporting the composition of these
molecules.¶ Association of the crown ether moieties of ON7
and ON8 with the matrix is one possible explanation for the
problems experienced during MALDI-MS analysis. However,
the composition of ON8 was furthermore verified by ESI-MS
analysis ([M 2 3H]32 m/z 1099; calcd. 1099; major peak).
To study complexation of alkanediamine ligands with ON7
and ON8 and the importance of ligand chain length, a thermal
denaturation study was performed and the melting temperatures
(Tm values) were determined (Table 1). ON7 was hybridized
with its complementary strands ON8 or ON9 to give duplexes
bearing either two triaza crown ethers (ON7:ON8; Series A) or
a single triaza crown ether (ON7:ON9; Series B). In order to
distinguish effects of ligand complexation by the crown ether
unit X from nonspecific interactions of the ligand with the
duplex, we also studied duplex ON10:ON11∑ (Series C).
With no ligand present, duplex ON7:ON8 (Series A)
displayed a slightly higher thermal stability (Tm = 42.5 °C) than
Functionalized crown ethers are of special interest in terms of
host–guest chemistry, and their selectivity for complexation of
metal cations of different sizes and for organic molecules such
as amines and amino acids is a starting point for promising
applications.1 So far, only one example of incorporation of a
crown ether moiety, a monoaza crown ether, into an oligonu-
cleotide (ON) has been reported but no complexation studies
were performed.2
In searching for a model receptor acting as amine or amino
acid binding site in ONs we have focused on crown ethers.
Although 18-crown-6 and similar crown ethers are capable of
binding primary ammonium cations,3,4 binding to alkali metal
cations is appreciably stronger.5 A remarkable exception are
triaza crown ethers which show about ten times stronger
complexation of primary ammonium cations than sodium or
potassium cations.6 A protonated amine ligand is complexed by
a network of alternating O…H–N and N…H–N hydrogen bonds
with the arrangement of the three nitrogens in a triaza crown
ether matching the symmetry axis of a primary ammonium
cation enabling the formation of three strong N…H–N hydro-
gen bonds. We therefore decided to focus on conjugating a
triaza crown ether to ONs, and the phosphoramidite 6 designed
for automated end-conjugation of ONs was synthesized
(Scheme 1).
Commercially available amine 1 was first monobenzoylated
and then mesylated to give building block 2 which after
treatment with ethanolamine afforded a diamide. Subsequent
reduction led to triamine 4 as a suitable intermediate for a
macrocyclization reaction.7 Thus, treatment of triamine 4 with
diethyleneglycol ditosylate furnished the macrocycle 5 which
was eventually converted into the phosphoramidite 6‡ (Scheme
1).
The triaza crown ether unit X (Scheme 1 and Fig. 1) was
incorporated at the 5A-end and the 3A-end of 9-mer ONs (ON7
and ON8, see Fig. 1). In order to avoid low temperature
denaturations in these first studies, ON7 was synthesized with
three affinity-enhancing LNA§ monomers (Fig. 1).8–10 Synthe-
sis of ON7–ON9 was performed in 0.2 µmol scale on an
automated DNA synthesizer using the phosphoramidite ap-
proach.11 Standard procedures were used with modifications as
described previously12 for amidite 6 (including 10 min coupling
time using 1H-tetrazole as activator). Initially, coupling of
amidite 6 was not successful. Careful NMR monitoring of the
coupling reaction revealed water bound to the hygroscopic
crown as the major problem. Removal of residual water was
finally achieved by stirring amidite 6 in acetonitrile with 4 Å
molecular sieves for 24 h. The anhydrous solution obtained was
immediately used for coupling on the DNA synthesizer. ON8
was synthesized using commercial so-called “inverted ami-
dites” (3A-O-DMT amidites, > 98% coupling yields using 2 min
Scheme 1 Reagents and conditions a) (PhCO)2O, EtOH, reflux, 90%, b)
MsCl, pyridine, 80%, c) HO(CH2)2NH2, NEt3, MeCN, 70%, d) LiAlH4,
THF, 90%, e) TsO(CH2)2O(CH2)2OTs, Na2CO3, MeCN, 50%, f)
NC(CH2)2OP(Cl)N(i-Pr)2, (i-Pr)2NEt, CH2Cl2, 50%, g) DNA synthesizer.
† A research center funded by the Danish National Research Foundation for
studies on nucleic acid chemical biology.
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CHEM. COMMUN., 2003, 1006–1007
This journal is © The Royal Society of Chemistry 2003

duplex ON7:ON9 (Series B) bearing only one triaza crown
ether unit X (Tm = 41.0 °C). Different trends with respect to the
dependence of the thermal stabilities on ligand chain length and
ligand concentration for Series A, B and C were observed as
depicted in Table 1 (DTm values are calculated relative to the Tm
value recorded for each series in the absence of a ligand).
For duplex Series A involving duplexes with two juxtaposi-
tioned crown ether units X, optimal complexation of an
alkanediamine ligand should be for one equivalent of ligand of
the appropriate length allowing complexation-driven reversible
end-joining of the two strands of the duplex with concomitant
increased thermal stabilities. For duplex Series B, complexation
of the alkanediamine ligands was expected not to strongly
influence the thermal stability and no difference between the
different ligands and ligand concentrations was expected. In
Series A, all ligands induced an increase in Tm by 1.5–3.5 °C
except for the shortest ligand 12, and, in general, similar results
were obtained for Series B. Overall, no strong effect of the
equivalents of ligand present on the obtained DTm values was
observed
The results obtained in Series C are in strong contrast to those
obtained in Series A and B and therefore support specific
complexation of the alkanediamine ligands with the crown ether
unit(s) in Series A and B. Interstrand cross complexation
involving either the two juxtapositioned crown ether units in
Series A (e.g., with 1 eq. of ligands 13 and 14) or one crown
ether unit and the phosphate backbone (in Series A and B)
would explain the affinity enhancing effects observed. The
comparable results obtained with one and ten equivalents of
ligands render any conclusion regarding the actual mode of
complexation impossible at this stage.
Oligonucleotide crown ether conjugates have been in-
troduced herein as a novel class of potentially very useful
molecular building blocks for construction of self-assembling
molecular receptors. We are currently studying conformation-
ally restricted variants of the systems introduced herein and
synthesizing 15N-labelled crowns and ligands for NMR struc-
tural studies.
We thank the Nucleic Acid Center, The Danish Research
Agency and the Deutsche Forschungsgemeinschaft for financial
support and Ms Britta M. Dahl for oligonucleotide synthesis.
Fig. 1 Sequences synthesized, the structure of LNA nucleotide monomers
(TL and MeCL are LNA thymin-1-yl and LNA 5-methylcytosin-1-yl
monomers, respectively) and a representation of the putative mode of
complexation of a alkanediamine ligand by the two triaza crown ether units
in the oligonucleotide duplex ON7:ON8; only four base pairs are shown in
the structural model in which the dotted lines indicate probable hydrogen
bonds between the triaza crown ether units and ligand 14. Alkanediamine
ligands used: H2N(CH2)nNH2; n = 6 (12), n = 8 (13), n = 10 (14) and n
= 12 (15).
Notes and references
‡ Amidite 6: 31P NMR data: d (CH3CN) 148.5 ppm; MALDI-MS ([M +
Na]+ m/z 708.4224; calcd. 708.4229.
§ We have defined LNA as an oligonucleotide containing one or more
conformationally locked 2A-O,4A-C-methylene-b-
D-ribofuranosyl nucleo-
Table 1 Thermal denaturation experiments in the presence of alkanedia-
mine ligands 12–15a
tide monomer(s) (“LNA monomer(s)”).
¶ MALDI-MS m/z for ON7: [M 2 H]2 3327; calcd. 3325; [M 2 2H + Na]2
3349; calcd. 3347; MALDI-MS m/z for ON8: [M 2 H]2 3299; calcd. 3298;
[M 2 2H + Na]2 3322; calcd. 3320.
Duplex series
Eq.
Tm/°C
DTm/°C
∑ This duplex was available and chosen as reference because of close
resemblance to the ON7:ON8 and ON7:ON9 duplexes with respect to
sequence, composition and structure (see ref. 13 and ref. 14 for relevant
NMR structural work).
Series A (ON7:ON8)
Ligand 12
Ligand 13
Ligand 14
Ligand 15
Series B (ON7:ON9)
Ligand 12
Ligand 13
Ligand 14
Ligand 15
Series C (ON10:ON11)
Ligand 12
Ligand 13
Ligand 14
Ligand 15
42.5
1/10
1/10
1/10
1/10
42.0/42.0
45.0/44.0
46.0/45.0
44.5/45.0
41.0
40.0/44.5
43.5/43.5
43.5/42.0
44.5/43.5
43.5
20.5/20.5
+2.5/+1.5
+3.5/+2.5
+2.0/+2.5
1 H. J. Buschmann, L. Mutihac and K. Jansen, J. Incl. Phenom., 2001, 39,
1.
1/10
1/10
1/10
1/10
21.0/+3.5
+2.5/+2.5
+2.5/+1.0
+3.5/+2.5
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1/10
1/10
1/10
1/10
42.5/42.5
42.0/42.5
42.5/42.0
41.5/41.5
21.0/21.0
21.5/21.0
21.0/21.5
22.0/22.0
a Melting temperatures [Tm values (DTm values are calculated relative to the
Tm value recorded in the absence of a ligand)] measured as the maximum of
the first derivative of the melting curve (A260 vs. temperature; 10 °C to 80
°C with an increase of 1 °C min21) recorded in medium salt buffer (10 mM
sodium phosphate, 100 mM sodium chloride, 0.02 mM EDTA, pH 7.0)
using 1 µM concentrations of the two complementary strands. Hyper-
chromicity values and transition intervals were similar for all melting
curves. Each Tm value was determined in two independent experiments and
DTm values were within ±0.5 °C consistent for the two experiments. “Eq.”
denotes the molar equivalent(s) of ligand used relative to duplex. See
caption of Fig. 1 for structures of alkanediamine ligands 12–15.
CHEM. COMMUN., 2003, 1006–1007
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