Extended ethidium bromide analogue as a triple helix intercalator: Synthesis, photophysical properties and nucleic acids binding

Article abstract of DOI:10.1039/b604281c

Ethidium bromide has been extended by fusing an additional aromatic ring resulting in a larger intercalator with increased affinity for poly r(A)·r(U), poly d(A)·d(T) and triple helices when compared to the parent heterocycle. The Royal Society of Chemist

Full text of DOI:10.1039/b604281c

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Extended ethidium bromide analogue as a triple helix intercalator:
synthesis, photophysical properties and nucleic acids binding{
Victor K. Tam, Qi Liu and Yitzhak Tor*
Received (in Cambridge, MA, USA) 23rd March 2006, Accepted 10th May 2006
First published as an Advance Article on the web 26th May 2006
DOI: 10.1039/b604281c
two key steps: (a) assembly of the phenyl-naphthalene core using a
Suzuki cross-coupling reaction of a suitably functionalized
naphthalene precursor with a highly functionalized benzene
precursor carrying a benzamide unit, and (b) a Morgan–Walls
ring-closure to assemble the bridging heterocyclic ring.
Ethidium bromide has been extended by fusing an additional
aromatic ring resulting in a larger intercalator with increased
affinity for poly r(A)?r(U), poly d(A)?d(T) and triple helices
when compared to the parent heterocycle.
Intercalation represents an important mode of nucleic acid small
molecule interactions.1 Polarizable polycyclic heteroaromatic
systems, such as ethidium and acridinium derivatives, represent
classical examples of nucleic acid intercalators.2 In general, such
aromatic molecules bind between adjacent base pairs and tend to
display limited selectivity between DNA and RNA as well as
inadequate discrimination between different sequences and folding
patterns.3 In recent years, structural modifications have been
employed to alter the selectivity of intercalators and improve their
nucleic acids recognition characteristics. In particular, attempts to
amplify the differentiation between DNA and RNA, between
double and triple stranded domains, as well as between matched
and mismatched or bulged sites have been reported.4 Here we
present a phenanthridinium analogue 2, where the aromatic
surface of the parent intercalator ethidium 1 is extended (Fig. 1).
The new large-surface intercalator, while displaying modestly
improved RNA over DNA selectivity, shows significantly
improved selectivity toward triple stranded oligonucleotides, when
compared to ethidium bromide.
The synthesis of the extended ethidium analogue 2 commenced
with triflation of commercially available 6-bromo-2-naphthol to
afford 3 in high yield (Scheme 1). Selective amidation at the
6-position was accomplished following a protocol developed by
Buchwald to cross-couple a benzamide group to the naphthalene
core 3 thereby installing one of the extranuclear amines.6
Palladium-mediated borylation between the triflate 4 and 4,4,5,5-
tetramethyl-[1,3,2]dioxaborolane afforded the boronic ester 5 in
modest yield.7 Completion of the aromatic scaffold was accom-
plished through a Miyaura–Suzuki coupling of 5 and 29-bromo-
59-nitrobenzanilide 6 to afford 7.8 Reduction of the aromatic nitro
functionality necessitated not only catalytic hydrogenation condi-
tions but the addition of hydrazine as well. To prevent any possible
oxidation or degradation of the free aromatic amine, 8 was
immediately reacted with benzoyl chloride under basic conditions
to form the tribenzamide 9. Treatment with phosphorus oxychlor-
ide to complete the extended phenanthridine core afforded 10 in
high yield. Alkylation of the phenanthridine nitrogen required
reaction with iodoethane in refluxing DMF over 20 h to afford 11
as a purple solid. Removal of the benzoyl groups in refluxing 48%
HBr over 12 h yielded the desired extended ethidium analogue 2.9
Similarly to ethidium bromide 1, the extended analogue 2
displays intense absorption in the UV range and a weaker charge
transfer band around 500 nm (Fig. 2A). Solvent polarity influences
both molecules in a similar fashion. Both chromophores experience
a hypsochromic shift with increasing solvent polarity that is mostly
apparent with the visible absorption bands (Fig. 2A). Excitation of
the major absorption bands leads to emission of both molecules at
ca. 600 nm. As with the ground state absorption bands, the
relatively broad emission band of both chromophores is sensitive to
its microenvironment. Increasing solvent polarity results in a
modest red shift of the emission maxima (Fig. 2B). Taken together,
these observations are consistent with stabilization of the charged
ground state upon increasing solvent polarity and concomitant
modest destabilization of the more delocalized excited state.
Both ethidium bromide and its extended analogue 2 are much
less emissive in water when compared to their emission in apolar
organic solvents. As a result, addition of nucleic acids to an
aqueous solution of the intercalators results in significant
enhancement of emission, which is accompanied by a slight
hypsochromic shift (Fig. 2C). While the extended analogue is
inherently less emissive than the parent intercalator,9 it is almost
In designing the extended ethidium analogue, we have
considered the electronic properties of the parent heterocycle.5
In particular, we have maintained the electronic communication
between the exocyclic amine at the 3 position and the quaternary
nitrogen by not perturbing this side of the molecule. In addition,
the exocyclic amine on the new aromatic ring electronically
communicates with the core structure in the same fashion the
8-amine does in ethidium. Synthetically, our strategy for the
formation of the extended phenanthridinium framework involved
Fig. 1 Structure of ethidium bromide 1 and its extended analogue 2.
Department of Chemistry and Biochemistry, University of California,
San Diego, La Jolla, California 92093. E-mail: ytor@ucsd.edu
{ Electronic supplementary information (ESI) available: synthetic proce-
dures & analytical data, absorption and fluorescence spectra, nucleic acid
binding isotherms and additional thermal melting profiles. See DOI:
10.1039/b604281c
2684 | Chem. Commun., 2006, 2684–2686
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Scheme 1 Synthesis of extended analogue 2.9 Reagents and conditions: (a) Tf2O, pyr., CH2Cl2, 0 uC to rt, 3 h, 98%; (b) N,N9-dimethylethylenediamine,
benzamide, CuI, K2CO3, toluene, 100 uC, 48 h, 96%; (c) 4,4,5,5-tetramethyl-[1,3,2]dioxaborolane, Pd(dppf)Cl2?CH2Cl2, Et3N, dioxane, 95 uC, 18 h, 65%;
(d) 6, Pd(dppf)Cl2?CH2Cl2, 1 M Na2CO3, DMF, 80 uC, 16 h, 90%; (e) 80% N2H4, Pd/C (10%), H2(1 atm), 65 uC, 4 h; (f) BzCl, Et3N, rt, 12 h, 85% over
2 steps; (g) POCl3, 100 uC, 18 h, 90%; (h) EtI, DMF, reflux, 20 h, 71%; (i) 48% HBr, reflux, 12 h, 80%.
completely quenched in water. This results in greater enhancement
of emission upon nucleic acids binding for 2, when compared to
the emission intensities of free and bound ethidium (Fig. 2C).
Nucleic acid affinity studies with calf thymus DNA (ctDNA)
revealed no discrimination between the extended ethidium
analogue or the parent compound (Table 1). When evaluating
the affinity to poly r(A)?r(U), however, 2 displayed a slightly
higher affinity for A-form RNA than ethidium bromide. Since
A-form RNA has a larger helical diameter compared to B-form
10
DNA (ca. 26 to 20 A, respectively), it provides a larger
˚
intercalating surface for the extended ligand, which may explain
the enhanced affinity compared to ethidium. To further explore
this hypothesis, the ability of 2 to bind triple helical oligonucleo-
tides was investigated. Fluorescence binding affinity studies reveal
that 2 has substantially higher affinity for triple helices compared
to ethidium bromide (Table 1). This is likely due to more favorable
stacking of the extended analogue 2 with the larger intercalating
surface provided by the base triplets, as has been observed with
other intercalating polyaromatic molecules.11
Thermal denaturation experiments with the homo-oligomers
dA19 and dT19 were employed to determine the amount of
stabilization imparted by 1 and 2 to double and triple helical DNA
oligonucleotides (Fig. 3).9 While minimal stabilization was
observed for the dT19?dA19 duplex with both intercalators,
dramatic stabilization of the dT19?dA19?dT19 triple helix was
observed with the extended ethidium analogue 2 (Table 2). As
shown in Fig. 3, under low salt conditions, the triple helical
dT19?dA19?dT19 begins to melt below 10 uC. While addition of
ethidium bromide slightly enhances the stability of the triple
stranded oligonucleotide (Tm = 24 uC),12 a dramatic stabilization is
Table 1 Nucleic acid affinities (Kd) of 1 and 2a
Nucleic acid polymers
1 (mM)
2 (mM)
calf thymus DNA
poly r(A)?r(U)
poly d(A)?d(T)
poly d(T)?d(A)?d(T)
poly r(U)?r(A)?r(U)b
a
15.1 ¡ 4.1
5.0 ¡ 0.9
30.8 ¡ 10.8
9.5 ¡ 5.2
16.1 ¡ 2.6
11.2 ¡ 1.6
1.6 ¡ 0.2
7.9 ¡ 1.3
1.0 ¡ 0.3
1.7 ¡ 0.3
Fig. 2 (A) UV-Vis absorbance spectra of 1 (left) and 2 (right). (B)
Excitation and emission spectra of 1 (left) and 2 (right). (C) 1 and 2 in the
presence and absence of ctDNA in pH 7.4 buffer.9 Solvents in (A) and (B):
H2O (blue), MeOH (yellow), CH3CN (green), CH2Cl2 (red).
Fluorescence titrations performed with 1.0 mM of 1 or 2 as small
volumes of increasing concentrations of nucleic acid were added.9
See ESI (Section S5) for additional information.
b
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Fig. 3 Melting profiles at 260 nm for (A) dT19?dA19?dT19 with no ligand
(bluecircles),ethidiumbromide1(greensquares),andtheextendedanalogue
2 (red triangles). (B) is an expansion of the triple helix melting profile.
a
Table 2 Thermal melting points of triple helix dT19?dA19?dT19
Tm(2A1)
Tm(3A2)
no ligand
49 ¡ 0.1 uC
53 ¡ 0.6 uC
54 ¡ 0.6 uC
,10 uC
24 ¡ 0.1 uC
37 ¡ 1.0 uC
1
2
a
Thermal melting experiments were performed in
a
buffer
containing 2.0 6 1022 M PIPES (pH 7.0), 2.0 6 1022 M NaCl,
1.0 6 1023 M EDTA. 10.0 mM of 1 or 2 were used for 1.0 mM of
triple helix concentration.9
apparent with the extended analogue (Tm = 37 uC). These results
correlate well with affinities determined by the fluorescence-based
titrations with polymeric oligonucleotides as listed in Table 1.
Triple helix DNA is of interest as a therapeutic target and
altering its stability could have potential biotechnological applica-
tions.13 Significant efforts in recent years have yielded a handful of
new heterocycles with diverse triple-helical selectivity traits, none,
however, were based on ethidium.11 Our observations suggest that
extending ethidium by fusing an additional aromatic ring yields an
analogue that can be viewed as a new triplex-selective motif.
Further structural modification can potentially fine tune the
nucleic acids affinity and selectivity of this extended heterocycle.
We thank the National Institutes of Health (AI 47673) for
support and the UCSD Mass Spectrometry Facility.
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2686 | Chem. Commun., 2006, 2684–2686
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