Targeting abasic site-containing DNA with annelated quinolizinium derivatives: The influence of size, shape and substituents

Article abstract of DOI:10.1039/c3ob42140f

The interactions of regular DNA and abasic site-containing DNA (AP-DNA) with quinolizinium (1a), the linearly fused benzo[b]quinolizinium (2a), the angularly fused benzo[a]quinolizinium (3a), benzo[c]quinolizinium (4a), and dibenzo[a,f]quinolizinium (5a) as well as derivatives thereof were studied with photometric and viscosimetric titrations (regular DNA), fluorimetric titrations and thermal DNA denaturation experiments (regular DNA and AP-DNA). Whereas the parent quinolizinium ion (1a) and the benzo-annelated derivatives 2a, 3a and 4a exhibit no significant affinity to AP-DNA, additional benzo-annelation in 5a leads to an increased selective stabilization of AP-DNA by this ligand. Hence, the latter compound represents the first example of a ligand that does not require ancillary substituents for efficient AP-DNA stabilization. In addition, studies of derivatives with varied substitution patterns revealed an impact of substituents on the stabilization of the AP-DNA. We discovered that a chloro substituent affects the propensity of a ligand to bind to AP-DNA in a similar way as the methyl substituent and may be employed complementary to the known methyl effect to increase the binding affinity of a ligand.

Full text of DOI:10.1039/c3ob42140f

Organic &
Biomolecular Chemistry
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PAPER
Targeting abasic site-containing DNA with
annelated quinolizinium derivatives: the influence
of size, shape and substituents†
Cite this: Org. Biomol. Chem., 2014,
12, 1725
Katja Benner, Heiko Ihmels,* Sarah Kölsch and Phil M. Pithan
The interactions of regular DNA and abasic site-containing DNA (AP-DNA) with quinolizinium (1a), the lin-
early fused benzo[b]quinolizinium (2a), the angularly fused benzo[a]quinolizinium (3a), benzo[c]quinolizi-
nium (4a), and dibenzo[a,f]quinolizinium (5a) as well as derivatives thereof were studied with photometric
and viscosimetric titrations (regular DNA), fluorimetric titrations and thermal DNA denaturation experi-
ments (regular DNA and AP-DNA). Whereas the parent quinolizinium ion (1a) and the benzo-annelated
derivatives 2a, 3a and 4a exhibit no significant affinity to AP-DNA, additional benzo-annelation in 5a leads
to an increased selective stabilization of AP-DNA by this ligand. Hence, the latter compound represents
the first example of a ligand that does not require ancillary substituents for efficient AP-DNA stabilization.
In addition, studies of derivatives with varied substitution patterns revealed an impact of substituents on
the stabilization of the AP-DNA. We discovered that a chloro substituent affects the propensity of a ligand
to bind to AP-DNA in a similar way as the methyl substituent and may be employed complementary to
the known methyl effect to increase the binding affinity of a ligand.
Received 28th October 2013,
Accepted 23rd January 2014
DOI: 10.1039/c3ob42140f
www.rsc.org/obc
AP sites may be employed for analytical and therapeutical pur-
poses.3,7,8 Along these lines, structural features have been
Introduction
The damage of the nucleic bases of DNA is an essential identified that establish a selective association of a given
process within a cell that may lead to mutation or cell death.1 ligand with the abasic position in DNA. Thus, DNA intercala-
Specifically, oxidized, alkylated or protonated nucleic bases tors such as proflavine9 and berberine,10 or threading DNA
may be eliminated from the DNA either by hydrolysis of the intercalators,11 have been shown to bind to AP sites. High
N-glycosidic bonds of the nucleotides2 or by enzymatic repair affinities and selectivities of a ligand towards abasic site-con-
of damaged nucleobases by the base excision repair (BER) taining DNA (AP-DNA) are accomplished by hydrogen bonding
mechanism.3,4 In each case, abasic sites (apurinic or apyrimi- between the external ligand and the nucleic base that is oppo-
dinic sites; AP sites) are formed as mutagenic or carcinogenic site to the abasic site, thus mimicking the regular Watson–
DNA lesions.5 Notably, such AP sites add another type of recep- Crick base pairing. Typical examples of such ligands are
tor unit to DNA, which offers binding sites for a variety of naphthyridine derivatives or similar hetarenes that resemble
guest molecules, specifically DNA-intercalating ligands. Hence the structures and hydrogen-bond patterns of the nucleic
the AP site may readily accommodate a DNA intercalator as a bases.12,13 To increase the affinity of the ligand to the DNA, it
substitute for the missing nucleic base. However, other than may be connected through an appropriate linker with an inter-
intercalation into the regularly matched DNA, which is calator that provides an additional, non-specific binding inter-
accompanied by unwinding, lengthening, and kinking of the action with the DNA.14,15 In addition, it has been
double helix, the insertion of a ligand into an AP site does not demonstrated that aminoalkyl-substituted intercalators also
induce significant changes in the DNA structure.6 Further- bind selectively to abasic sites.16,17 In this case the intercalator
more, it has been proposed that ligands that bind selectively to is accommodated within the abasic site, presumably by π
stacking, while the protonated aminoalkyl substituent provides
an additional stabilization of the ligand–DNA complex by the
University of Siegen, Organic Chemistry II, Adolf-Reichwein-Str. 2, D-57068 Siegen,
association in the DNA groove. In a different approach, metal-
Germany. E-mail: ihmels@chemie.uni-siegen.de; Tel: +49 (0) 271 740 3440
loinsertors with sterically expansive ligands have been develo-
†Electronic supplementary information (ESI) available: Synthesis of quinolizi-
ped that exhibit a high selectivity to AP-DNA as compared with
nium derivatives; photometric and fluorimetric titration spectra, DNA melting
regular duplex DNA, because other than the latter the AP-DNA
curves. See DOI: 10.1039/c3ob42140f
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is able to compensate sufficiently the unfavorable energetic
contribution from the steric interactions with the ligand.6,18
Overall, the survey of AP-DNA ligands may lead to the con-
Results
Synthesis
clusion that in principle, any DNA intercalator is – with an The parent quinolizinium (1a)22 and the benzo-annelated
appropriate substitution pattern – a suitable ligand for AP- derivatives 3a,23 4a,24 and 5a25 were synthesized according to
sites. Nevertheless, to consider an AP-DNA ligand for thera- reported protocols.26 The aminoalkyl-substituted quinolizi-
peutic or analytical applications it has to bind with high nium derivatives 1e–1g were obtained by nucleophilic aromatic
affinity and selectivity in the abasic position. Therefore, the substitution reactions of the 2-bromoquinolizinium (1b) or
knowledge of the structural features of an intercalator that 4-chloroquinolizinium (1c).27 Thus, the reaction of N,N-
govern its interaction with abasic sites may be useful for the dimethyl-1,3-diaminopropane or N,N,N′-trimethyl-1,3-diamino-
rational development of AP-DNA targeting ligands. In this propane with 1b or 1c in isopropanol gave the quinolizinium
regard, however, the choice of intercalator structures seems derivatives 1e–1h, respectively, that were isolated by ion meta-
rather arbitrary, and to the best of our knowledge there are no thesis with hexafluorophosphate and subsequent crystalliza-
systematic studies reported on the influence of variations of tion. The yields were rather low (8–33%), as usually observed
size and shape of a given intercalator structure on their with nucleophilic substitution reactions at quinolizinium
AP-DNA binding properties. To gain more information about ions.27,28 The aminoalkyl-substituted quinolizinium-2-carboxa-
this particular aspect we were searching for a general structural mide 1i was obtained by amidation of quinolizinium-2-car-
basis for such systematic studies, with the long-term goal to boxylic acid (1d)29 with N,N-dimethyl-1,3-diaminopropane
assess the general design principles for efficient functional through a mixed-anhydride that is formed by treatment of the
AP-DNA ligands. For this purpose, we chose the quinolizinium acid with isobutyl chloroformate in acetonitrile in the presence
ion (1a) as the key structure, because it has been demonstrated of a mild base (N-methylmorpholine, NMM) (Scheme 1).
already that the form and dimension of annelated quinolizi-
9-Methylbenzo[a]quinolizinium (3b) and 9-methyldibenzo-
nium derivatives have a significant influence on the strength [a,f]quinolizinium (5b) were obtained by the cyclodehydration
and selectivity of their binding interaction with duplex, triplex method.30 For that purpose 2-(4-methylphenyl)pyridine (6a)
and quadruplex DNA.19 In addition, we have demonstrated and 2-(4-methylphenyl)quinoline (6b) were quaternized by the
that the aminoalkyl-substituted derivatives of the linearly reaction with chloroacetaldehyde oxime. Without further iso-
fused benzo[b]quinolizinium (2a) have the propensity to bind lation the resulting pyridinium derivatives 7a and 7b were con-
in abasic positions. Nevertheless, the interactions of the angu- verted to the cyclized products 3b and 5b in refluxing aq. HBr
larly fused derivatives benzo[a]quinolizinium (3a), benzo[c]qui- (48%) in very low yield (Scheme 2).
nolizinium (4a), and dibenzo[a,f]quinolizinium (5a) (Chart 1)
The benzo[c]quinolizinium derivatives 4b–h were syn-
with AP-DNA have not been reported so far. Hence, in an thesized according to established protocols (Scheme 3). Thus,
attempt to further identify the influence of the size and the the reaction of 2-methylpyridine with the corresponding
shape of quinolizinium ions on their association with AP-DNA dichlorobenzaldehyde derivatives 8b–f gave the trans-styrylpyri-
we examined the interactions of derivatives of the annelated dines 9b–f that were irradiated in acetone (λ > 250 nm) and
quinolizinium ions 3a–5a with regular DNA and with AP-DNA.
Furthermore, derivatives of the benzo[c]quinolizinium 4a–h
were studied to assess the influence of substituents. In particu-
lar, we were interested in the chloro substituent, because it is
well known that the chloro substituent may replace the methyl
group without changing the steric demand of a compound.20
Especially considering the known methyl effect on AP-DNA
ligands13d,e,21 we were interested in exploring the effect of the
chloro substituent, because in this particular case any differ-
ence or similarity is based exclusively on the electronic effect.
Scheme 1 Synthesis of aminopropylamino-substituted quinolizinium
derivatives 1e–1i.
For comparison, selected derivatives of the parent quinolizi-
nium (1a) were also included in this study.
Chart 1 Structures of the quinolizinium ion (1a) and its benzo- and
dibenzo-annelated derivatives 2a–5a.
Scheme 2 Synthesis of 9-methylbenzo[a]quinolizinium (3a) and 9-methyl-
dibenzo[a,f]quinolizinium (5b).
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Scheme 3 Synthesis of benzo[c]quinolizinium derivatives 4b–h (i:
SnCl2(H2O)2, EtOH, 90 °C, 3.5 h. ii: MeB(OH)2, Pd(dppf)Cl2–CH2Cl2, KF,
DME–H2O–MeOH (2/1/1), 110 °C, 70 h).
subsequently heated to 170 °C (except for the case of 9e) to
give the benzo[c]quinolizinium derivatives 4b–f in low to
moderate yields. The products were precipitated and isolated
as tetrafluoroborate salts. The 8-nitrobenzo[c]quinolizinium
(4e) was reduced to the 8-aminobenzo[c]quinolizinium (4g)
with SnCl2·2H2O. The 7-chlorobenzo[c]quinolizinium (4f) was
transformed in low yield to the 7-methyl-substituted derivative
4h by a Suzuki coupling with Pd(dppf)Cl2–CH2Cl2 (dppf = 1,1′-
bis(diphenylphosphino)ferrocene) as a catalyst.
All known compounds (1a–d, 2a, 3a, 4a, 5a, 9b–f) were
identified by comparison with literature data. All new com-
pounds (1e–i, 3b, 4b, 4d, 4f–h, 5b) were characterized by
1H-NMR and 13C-NMR spectroscopy, mass spectrometry and
elemental analysis.
Spectrometric titrations
To examine the general affinity of the quinolizinium deriva-
tives 3–5 to double-stranded DNA, the binding interactions of
these ligands with calf thymus DNA (ct DNA) were examined
by spectrophotometric titrations. As a general trend, the inten-
sity of the long-wavelength absorption bands decreased signifi-
cantly upon interaction with DNA, and a red-shifted band
developed with increasing DNA concentration, as exemplarily
shown for 1f, 1h, 3a, 4a, and 5b (Fig. 1). This effect is less pro-
nounced and in some cases even very small with quinolizi-
nium derivatives 1a–i (Fig. 1A). In most cases, isosbestic
points were maintained in the course of the titration. The
fitting of the resulting binding isotherms to the neighbor-
exclusion model31 gave the apparent binding constants
(Table 1). Among the tested compounds, the dibenzoquinolizi-
nium derivatives 5a and 5b have the largest binding constant
Kb (5a: 1.9 × 105 M−1 19d
of the monobenzo-annelated quinolizinium derivatives 3a,b, 4a–d,
and 4f–h are smaller, i.e. in the range of 2.4–6.7 × 104 M−1
;
5b: 1.5 × 105 M−1), whereas the ones
.
Fig. 1 Photometric titration of ct DNA to quinolizinium derivatives 1f
The fluorescence of the benzo- and dibenzo-annelated qui- (A), 1h (B), 3a (C), 4a (D), and 5b (E) in phosphate buffer (1f, 3a, 4a, 5b:
cLigand = 0.10 mM, 1h: cLigand = 0.05 mM; pH 7).
nolizinium derivatives was quenched upon addition of ct DNA
(cf. ESI†). The efficiency of the quenching process was deduced
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Table 1 Binding parameters of ligands 3–5 with DNA
ctDNA b
KcbtDNA a
−1 × 104 na
/
K
SV
/
Slope of plot
KTbX d
−1 × 104
/
M
M
−1 × 104 (η/η0)1/3 vs. LDR (r2)c
M
3a
3b 13
4.0
3
4
3
n.d.
2
4
3
3
1.1
2.4
0.5 (0.97)
0.5 (0.96)
0.3 (0.98)
0.2 (0.94)
0.7 (0.96)
0.3 (0.97)
0.5 (0.97)
0.4 (0.92)
0.5 (0.95)
n.d.
1.4
2.5
1.9
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
14
Chart 2 Structures of regular DNA TA and CG and of AP-DNA CX, TX,
AX, GX.
4a
2.7
1.0
0.3
1.6
1.0
1.2
1.3
2.8
4b n.d.e
4c
4d
4f
4g
4h
2.4
2.5
2.6
2.9
6.7
intensity of all tested compounds 3a, 3b, 4a, 5a and 5b (Fig. 3).
The data from the fluorimetric titrations were analyzed as
binding isotherms that were fitted to the theoretical data of a
1 : 1 stoichiometry to give the apparent binding constants, Kb,
according to established protocols (Table 1).13a These analyses
revealed that the binding constants of the ligands 5a and 5b
(1.4 × 105 M−1 and 1.2 × 105 M−1) are larger than the ones of 3a,
3b, and 4a that range from 1.4 × 104 M−1 (3a) to 2.5 × 104 M−1 (3b).
4
5a 19f
5b 15
3f
2
17 f
26
0.9 (0.99)
12
a Binding constant, KcbtDNA, and binding-site sizes, n (in base pairs),
obtained from photometric titrations with ct DNA (cLigand = 0.10 mM
in BPE buffer). b Stern–Volmer quenching constant, KSctVDNA, obtained
from fluorimetric titrations with ct DNA (cLigand = 10 µM in BPE
buffer). c Slope of the best-fit line of a plot of relative viscosity, (η/η0)1/3
,
vs. LDR (cDNA = 1.0 mM in BPE buffer); r2 of the linear fit is given in
brackets. d Binding constant, KTbX, obtained from fluorimetric titrations
with TX (c4a = 1.0 µM; c3a/3b/5a/5b = 10 µM in ODN buffer) considering a
1 : 1 stoichiometry. e n.d. = not determined. f Ref. 19d.
Viscometric titrations
For the determination of the binding mode with double-
stranded DNA, viscometric titrations of the ligands 1a, 3a,b,
4a–4d, 4f–4h, and 5a,b to ct DNA were performed and com-
pared with the data obtained with the known intercalator ethi-
dium bromide under identical conditions. Thus, the change of
the specific viscosity (η/η0)1/3 of the aqueous DNA solution
upon addition of the ligands was determined and plotted
versus the LDR as shown exemplarily for the ligands 4b, 4c and
4f (Fig. 4). The data were analyzed by linear regression, and
the slope of the best-fit line was determined as a measure of
the extent of DNA stiffening upon intercalation (Table 1). The
parent quinolizinium (1a) has essentially no influence on the
viscosity of the DNA solution (slope: −0.1), and the benzo[c]-
quinolizinium derivatives 4a, 4b, and 4d induce only a small
enhancement of the viscosity (slope: 0.2–0.3). In contrast, the
viscosity of the solution increases to a greater extent upon
addition of all other tested ligands (slope = 0.4–0.7). Neverthe-
less, in the presence of ethidium bromide a much stronger
effect was observed (slope = 0.8).
from the plot of the relative emission intensity, I0/I, versus the
DNA concentration (Fig. 2). According to the resulting Stern–
Volmer quenching constants, KSV, the quenching of the diben-
zoquinolizinium derivatives 5a and 5b by ct DNA (5a: 1.7 × 105 M−1;
5b: 2.6 × 105 M−1) is about 10 times more efficient than that
of the benzo-annelated quinolizinium derivatives 3 and 4
(0.3–2.8 × 104 M−1). Notably, the Stern–Volmer constants KSV of
the derivatives 3–5 are in the same range as the corresponding
binding constants with ct DNA, which indicates static quench-
ing, i.e. the radiationless deactivation of the excited quinolizi-
nium mainly takes place within the binding site.
The interactions of the quinolizinium derivatives with
AP-DNA were investigated exemplarily by fluorimetric titrations
with the double-stranded oligodeoxyribonucleotides TX that
contains an apurinic site opposite to thymine (Chart 2). To
maintain sufficient stability of the AP-DNA strand, the regular
deoxyribose residue at the AP site was substituted with tetra-
hydrofuran. The addition of TX led to a decrease of emission
Fig. 2 Relative fluorescence intensities of benzoquinolizinium deriva-
Fig. 3 Relative fluorescence intensities of benzoquinolizinium deriva-
■
●
○
□
●
□
■
△
◆
◇
▲
△
tives 3a ( ), 3b ( ), 4a ( ), 4b ( ), 4c ( ), 4d ( ), 4g ( ), 4h (☆), and 5b
(
tives 3a ( ), 3b ( ), 4a ( ), 5a ( ), and 5b ( ) upon addition of AP-DNA
TX in phosphate buffer (38 mM, pH = 7.0).
▲
) upon addition of ct DNA in phosphate buffer (16 mM, pH = 7.0).
1728 | Org. Biomol. Chem., 2014, 12, 1725–1734
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1 °C, resp.); and the same was shown exemplarily for the
chloro-substituted derivative 1c and AP-DNA TX. The
functionalization with 2- or 4-aminopropylamino substituents
resulted in different effects on the DNA melting temperature,
depending on the type and position of the substituent and on
the particular DNA form. Thus, the substituted derivatives 1e–i
induce no or only a very small shift of the DNA melting tempera-
ture, ΔTm, of regular DNA TA (−0.6 to 0.4 °C) or AP-DNA TX
(−1.4 to 0.3 °C), GX (0.3–2.9 °C), and AX (0.0–2.3 °C), whereas
the AP-site containing CX was significantly stabilized by the
ligands 1e and 1h, as indicated by higher melting temperatures
(1e: ΔTm = 6.2 °C; 1h: ΔTm = 6.0 °C). At the same time, the
derivatives 1f and 1g stabilize CX to a significantly lesser extent
(ΔTm = 3.5 and 1.0 °C).
The benzo[a]quinolizinium derivatives 3a and 3b have
essentially no effect on the melting temperature of regular
DNA (ΔTm < 1 °C, resp.), and also the AP-DNA is only slightly
stabilized in the presence of these ligands. Specifically, the
largest value of ΔTm induced by derivative 3a is just 3.6 °C for
TX, while the shifts of Tm of the other AP-DNA forms are even
smaller. Nevertheless, it should be noted that the introduction
Fig. 4 The relative specific viscosity of an aqueous solution of ct DNA
●
■
△
in the presence of the quinolizinium derivatives 4b ( ), 4c ( ) and 4f ( )
represented as a plot of relative viscosity, (η/η0)1/3, vs. ligand–DNA ratio,
LDR.
DNA denaturation studies
Because the induced shift of the DNA melting temperature
(ΔTm) in the presence of a ligand is directly related to the
stabilization or destabilization of the duplex DNA toward dis- of a methyl group in 3b induces a significantly larger increase
of the melting temperature of the AP-DNA as compared to 3a,
which is especially pronounced in the case of the apurinic
sociation, thermal DNA denaturation experiments represent a
powerful tool to assess ligand–DNA interactions.32 Thus, the
interaction of the quinolizinium derivatives 1–5 with the AP- DNA (3a: ΔTm = 2.2–3.6 °C; 3b: ΔTm = 6.2–6.5 °C). The benzo[b]-
quinolizinium ion (2a) does not have a stabilizing effect on
containing undecamers CX, TX, GX, AX (Chart 2) and of the
regular DNA TA and CG or apyrimidinic DNA AX and GX;
regular oligonucleotides TA and CG were determined by photo-
metric monitoring of the DNA melting at different LDR however, the association of 2a with the AP-DNA CX and TX
resulted in a stabilization (ΔTm = 4.4–5.1 °C; LDR = 2). The
parent benzo[c]quinolizinium 4a does not stabilize regular
(Table 2). The parent quinolizinium 1a neither stabilizes the
regular DNA TA or CG, nor the AP-DNA CX, TX, GX, AX (ΔTm
<
Table 2 Stabilization of regular and AP site-containing duplex DNA by quinolizinium derivatives 1–5 as determined from the thermal DNA dena-
turation studies
Induced ΔTm/°C at LDR = 0.5 and 2a
CX
0.5
TX
0.5
AX
0.5
GX
0.5
TA
CG
0.5
LDRb
2
2
2
2
0.5
2
2
1a
1c
1e
1f
1g
1h
1i
2a
3a
3b
4a
4b
4c
4d
4g
4f
−1.6
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
2.4
1.2
3.6
0.8
−0.5
2.6
2.8
3.5
0.5
−0.5
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
2.1
1.4
3.8
0.2
−0.3
3.1
2.1
4.1
0.0
0.4
−1.4
0.2
−0.2
0.3
−1.0
4.4
3.6
6.2
1.1
−0.1
5.4
5.7
7.0
0.0
−0.9
n.d.
2.3
0.5
0.0
2.0
0.3
−0.1
1.2
−0.5
1.5
0.2
0.1
1.1
2.8
−0.7
3.3
−0.3
n.d.
n.d.
n.d.
n.d.
n.d.
−0.5
0.6
0.6
1.4
0.4
−0.1
0.4
0.6
2.0
−0.1
n.d.
2.9
2.4
0.1
0.3
1.1
−0.9
1.8
3.1
1.0
1.5
1.1
3.1
4.1
0.8
4.8
7.9
9.6
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
−0.6
0.1
−0.3
0.0
−0.1
0.3
0.1
1.0
0.0
0.1
0.1
0.6
1.0
−0.9
0.4
0.3
0.4
0.3
−0.6
1.2
0.3
0.7
0.4
0.1
0.6
0.5
1.7
1.2
0.3
0.6
2.5
2.7
0.0
n.d.
n.d.
n.d.
n.d.
n.d.
1.4
0.2
0.0
0.3
0.1
0.1
0.0
0.1
0.2
0.3
0.0
0.9
0.9
−0.1
n.d.
n.d.
n.d.
n.d.
n.d.
1.9
0.4
0.1
1.8
0.7
0.2
0.2
0.4
0.6
1.4
0.6
2.4
2.8
n.d.
6.2
3.5
1.0
6.0
2.4
5.1
2.2
6.5
1.5
0.4
5.5
5.6
6.9
5.5
7.5
11.8
16.4
n.d.
n.d.
n.d.
n.d.
n.d.
−0.9
0.0
−0.7
−2.1
−0.3
0.3
0.6
−0.3
−0.9
−0.7
−0.3
3.9
2.7
4.2
6.2
10.3
2.6
3.1
8.2
13.1
5.1
6.9
11.3
15.6
0.7
1.6
3.7
4.3
4h
5a
5b
7.6
9.1
3.9
a ΔTm: shift of the DNA melting temperature in the presence of the ligand; LDR = ligand–DNA ratio; cDNA = 5.0 µM in aqueous phosphate buffer,
[Na+] = 38.1 mM; estimated error 0.5 °C, n.d. = not determined. b LDR values refer to the molar concentration of the oligonucleotide duplexes.
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DNA or AP-DNA (ΔTm < 1.6 °C). Whereas the introduction of a
Overall, the DNA-binding constants of the derivatives 3–5
chloro-substituent in 10-position in derivative 4b did not (Table 1) are comparable to those of known organic intercala-
change the DNA-stabilizing properties of the ligand, the tors with similar size and substitution pattern.36 Most notably,
addition of 7-, 8-, or 9-chlorobenzo[c]quinolizinium 4c, 4d or the dibenzo-annelated derivatives 5a and 5b bind significantly
4f to the AP-DNA CX or TX induced a small, but significant stronger to ct DNA than the monobenzo-annelated derivatives.
shift of the melting temperatures (ΔTm = 5.1–5.7 °C; LDR = 2).
This observation is in agreement with previous studies on
The melting temperatures of GX, AX, TA, and CG, however, ligand 5a19d and similar tetracyclic naphthoquinolizinium
remain essentially unchanged in the presence of these ligands. derivatives37 that have revealed a significant gain of DNA-
The ΔTm shifts induced by the 7-methyl-substituted benzo[c]- affinity of a given quinolizinium-type ligand with increasing
quinolizinium are just a bit larger than the ones of the 7-chloro- size of the π system of the ligand, as long as the shape of the
benzo[c]quinolizinium (4f). Also, the 8-aminoquinolizinium ligand fits the binding site appropriately.
(4g) induces only slightly larger shifts of the DNA melting
Interaction with abasic sites
temperatures than the 8-chloroquinolizinium (4d). As com-
pared to the benzo-annelated quinolizinium derivatives 2–4, The main objective of this work was to assess the propensity of
the addition of the dibenzo[a,f]quinolizinium ions 5a and 5b quinolizinium derivatives to bind to abasic sites and to iden-
to AP-DNA leads to a more effective stabilization of the duplex tify structural parameters that govern the strength and selecti-
structure. Hence, the melting temperatures, Tm, of TX and AX vity of this association.
increase by more than 10 °C, whereas the ones of the apyrimi-
Firstly, it is obvious from the collected data that the parent
dinic AP-DNA increase to a slightly lesser extent (e.g.: 5a: quinolizinium (1a) does not bind measurably to abasic sites as
ΔTCmX = 11.8 °C; ΔTGmX = 7.6 °C; LDR = 2). Again, the introduc- shown exemplarily with compounds 1a and 1c. This result was
tion of one additional methyl group into the ligand leads to a somewhat surprising because several bicyclic hetarenes are
more pronounced shift of the DNA melting temperature as known that bind to and stabilize abasic site-containing
shown with the methyl-substituted derivative 5b (ΔTCmX
=
DNA.13,21 Moreover, it has been proposed that the bicyclic
16.4 °C; ΔTGmX = 9.1 °C; LDR = 2). However, in the case of 5a indole fragment of the AP-DNA damaging tripeptide Lys-Trp-
and 5b even the regular duplex TA and CG is stabilized at Lys acts as a binding unit in the abasic site.38 However, in
LDR = 2 (ΔTm = 2.4–2.8 °C).
those cases additional binding interactions assist the accom-
modation of the ligand in the abasic position; namely hydro-
gen bonding of the ligand with the nucleic base opposite to
the abasic site, or groove binding by additional amino-alkyl
substituents. Therefore, we tested whether the affinity of the
quinolizinium to AP-DNA may be increased by functionali-
zation with amino-alkyl substituents as in 1e–i. Although a ten-
Discussion
Association with regularly paired DNA
It was demonstrated that the benzo-annelated quinolizinium dency for increasing stabilization of AP-DNA with aminoalkyl-
derivatives bind to double-stranded DNA, which is in agree- substitution of the quinolizinium ligand may be deduced from
ment with the known DNA-binding properties of quinolizi- the corresponding ΔTm values, it should be noted that only
nium derivatives.17 Specifically, the hypochromic effect and the AP-DNA CX is considerably stabilized by the derivatives 1e,
the red shift during photometric titrations are characteristic of 1f, 1h and 1i (ΔTm = 2.4–6.2 °C), and GX only to some extent
DNA-binding ligands.33 In addition, the isosbestic points indi- by 1e and 1f (ΔTm = 2.4–2.9 °C). At the same time, the other
cate that the complexation proceeds with one main binding AP-DNA is not significantly more stabilized as compared to 1a.
mode. At the same time, the lack of isosbestic points or the Apparently, the position of the aminoalkyl group at the quino-
slight fading thereof during titration reveals a marginal extent lizinium core as well as the substituents at the “internal”
of heterogeneous binding of the respective ligand in a few amino funtionality (secondary vs. tertiary) have an impact on
cases. Along these lines, it was shown by viscometric titrations the stabilizing effect of the ligand on the particular DNA form,
that most of the tested quinolizinium derivatives intercalate but the present data do not allow a structure-based explanation
into DNA as indicated by the increasing viscosity of the DNA of this observation.
solution upon ligand–DNA association (Fig. 4). Such a behav-
The fluorimetric titrations and thermal DNA-denaturation
ior usually indicates an intercalative binding mode, because experiments clearly indicate the association of the parent
this interaction leads to a stiffening and lengthening of the benzo-annelated quinolizinium derivatives 2a, 3a and 4a with
DNA biomacromolecule and in turn to an increased viscosity AP-DNA. In almost all cases the AP-DNA with apurinic sites is
of the solution;34 as supported by the direct comparison with more stabilized by the ligands 2a, 3a and 4a than the apyrimi-
the known intercalator ethidium bromide.
dinic DNA, presumably because the ligands experience more
The association of the quinolizinium derivatives with DNA steric repulsion in the smaller apyrimidinic binding site. The
was further demonstrated by fluorimetric titrations that exemplary binding constants are in a typical range (104 M−1) of
revealed a quenching of the DNA-bound ligand, most likely DNA intercalators whose association is mainly supported by
caused by a photoinduced electron-transfer reaction between dispersion interactions,39 i.e. with no further specific attractive
the excited quinolizinium and the nucleic bases.35
interactions with the DNA. For comparison, binding constants
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of AP-DNA ligands that bind also by hydrogen bonding are
usually two orders of magnitude larger.13
Table 3 Selectivity of DNA stabilization of the ligands 1–5
ΔΔTm a/°C at LDR = 0.5 and 2
As a general trend, the angularly annelated compound 2a
stabilizes the AP-DNA slightly more than the angular deriva-
tives 3a and 4a. This result may be explained by a better fit of
the linear ligand to the abasic site as opposed to the angular
ligands, which may interfere with the DNA backbone because
of their larger lateral steric demand. At the same time, the
introduction of an additional annelated benzene ring in the
S-shaped azoniachrysene 5a results in a higher affinity to
AP-DNA, as shown by larger binding constants (105 M−1) and
significantly larger induced shifts of the DNA melting tempera-
tures (ΔTm = 11–12 °C). These observations are in agreement
with preceding experimental and theoretical data, which have
provided evidence that the ligand 5a binds to regular DNA in
such a way that one naphthalene part of the ligand intercalates
between two base pairs, whereas the other naphthalene unit
points inside the groove to gain additional attractive dispersive
van der Waals interactions.19d Most likely, the ligands 5a and
5b bind to AP-DNA with a similar binding mode; however,
with a slightly different orientation of the ligand within the
abasic site, because the ligand occupies the place of the
missing base instead of being embedded between the two base
pairs in regular DNA. It should be noted, however, that the
ligands 5a and 5b should experience the same steric inter-
actions within the abasic position as the angular benzoquinoli-
zinium derivatives 3a and 4a. Presumably, the latter
unfavorable interaction is compensated by the additional
accommodation of one part of the ligand in the groove area.
In previous studies, it has been shown that the introduction
of a methyl substituent increases the binding affinity of a
ligand toward AP-DNA,21 which corresponds with the well-
established methyl effect of host–guest systems in pharma-
ceutical chemistry40 and medicinal chemistry.41 Upon associ-
ation of a ligand with AP-DNA the methyl group introduces a
favorable hydrophobic effect. Furthermore, the methyl effect
operates by a weak, but significant electron donation into the
π system that leads to increased stacking interactions. Notably,
our studies on quinolizinium derivatives confirmed the methyl
effect, because the methyl-substituted derivatives 3b, 4h, and
5b induce a larger shift of DNA melting temperature than the
parent compounds 3a, 4a, and 5a (Table 2). To be emphasized
is the observation that the combination of benzo-annelation
and methyl effect in derivative 5b induces ΔTm shifts for
AP-DNA of up to 16 °C, which are unusually large for otherwise
non-functionalized hetarene ligands.
CX
0.5
TX
0.5
AX
0.5
GX
0.5
LDR
2
2
2
2
1a
2a
3a
3b
4a
4b
4c
4d
4f
4g
4h
5a
5b
−1.6
2.2
1.2
3.3
0.7
−0.6
2.5
2.7
3.3
2.4
4.2
5.3
9.4
0.6
4.7
2.1
4.7
0.8
0.2
5.0
5.3
6.3
4.1
6.9
9.4
−0.3
2.0
1.7
3.8
0.3
−0.6
3.0
1.1
3.8
2.5
3.0
7.6
12.1
0.9
4.1
2.9
5.8
1.0
0.2
0.0
−0.3
0.4
0.6
1.1
0.3
−0.2
0.4
0.5
1.8
0.4
1.6
2.8
3.4
0.0
0.5
1.7
1.3
0.3
1.3
0.9
2.7
3.5
−0.1
−0.4
−2.1
−0.2
0.0
−0.2
0.5
−0.1
1.4
−0.4
−0.4
−0.6
1.6
−0.7
4.9
4.0
5.8
4.8
6.0
8.8
0.5
−1.3
−0.9
−0.8
−0.4
3.3
−1.0
−0.6
2.6
5.1
6.4
4.2
5.5
6.8
13.8
12.9
2.9
a ΔΔTm = ΔTm(NX) − ΔTm(TA or CG); N = C, T, A, G; conditions cf.
Table 2.
(ΔΔTm < 1.5 °C). From the parent compounds the linear benzo[b]-
quinolizinium 2a displays the most pronounced, but still
marginal differentiation between regular DNA and AP-DNA,
especially toward apurinic sites (ΔΔTCmX = 4.7 °C; ΔΔTmTX
=
4.1 °C, at LDR = 2). Thus, the latter has properties that are
similar to other cationic DNA-intercalators, such as ethidium
bromide,42 that also exhibit small, but significant selectivity
toward AP-DNA. It should be noted, however, that no
additional substituent effects contribute to the overall binding
of the parent compounds 2a, 3a and 4a. Thus, the data pre-
sented herein illustrate the intrinsic ligand properties that
govern the affinity and selectivity of a ligand toward AP-DNA.
The small series of substituted benzo[c]quinolizinium
derivatives was examined to explore a possible effect of the
chloro substituent. It has been observed already that the intro-
duction of a methyl group into a ligand increases its affinity to
AP-DNA (see above), and we demonstrated in this work that
this effect also operates in quinolizinium derivatives. Along
these lines, we reasoned that a chloro substituent may have a
similar effect. The chloro and methyl groups have essentially
the same steric demand,43 and like the methyl group, the
chloro substituent should increase the hydrophobic properties
of the ligand. In addition, the chloro atom leads to a slightly
changed dipole of the ligand, thus increasing dipole–dipole
interactions in the binding site, and it may even operate as a
hydrogen-bond acceptor. Indeed, the chloro-substituted
derivatives 4c, 4d and 4f stabilize especially the apurinic
As quinolizinium derivatives bind already to regularly
paired duplex DNA, it needs to be evaluated whether the
ligands exhibit selective interactions with either the regular
DNA or the AP-DNA. For that purpose, the selectivity of the
ligands for the abasic site may be expressed by their different
effects on the melting temperatures of the two DNA forms, as
AP-DNA more than the parent compound (e.g. 4a: ΔTmTX
=
1.1 °C; 4c: ΔTmTX = 5.4 °C; 4d: ΔTmTX = 5.7 °C; 4f: ΔTTmX = 5.1 °C;
LDR = 2), whereas this effect is smaller with the apyrimidinic
AP-DNA. As an exception, the 10-chlorobenzo[c]quinolizinium
(4b) exhibits basically no stabilizing effect toward AP-DNA,
most likely because this ligand is significantly twisted out of
planarity due to the repulsion between the chloro substituent
and the hydrogen atom at C1. Most notably, the 9-chlorobenzo-
quantified by the ΔΔTm values that are defined as ΔΔTm
=
ΔTm(AP-DNA) − ΔTm(regularly matched DNA)42 (Table 3).
These data revealed no or only a small selectivity of the parent
benzoquinolizinium derivatives 3a (ΔΔTm < 3.0 °C) and 4a
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[c]quinolizinium (4f) has only a slightly smaller effect on the choice of the size and shape of the intercalating part of the
melting temperatures of the employed AP-DNA strands as the ligand and the strategic selection of substituents that increase
9-methylbenzo[c]quinolizinium (4h). The same trend was the DNA-stabilizing properties.
observed for the selectivity of the binding as indicated by
similar ΔΔTm values (e.g. 4c: ΔΔTTmX = 4.9 °C; 4d: ΔΔTTmX
=
4.0 °C; 4f: ΔΔTmTX = 5.8 °C; 4h: ΔΔTTmX = 6.0 °C; LDR = 2;
Table 3). Overall, these results indicate that a chloro substitu-
ent affects the ability of a ligand to bind to AP-DNA in a
similar way as the methyl substituent.
Experimental
Equipment
Absorption spectroscopy: Varian Cary 100 Bio Spectrophoto-
meter. Emission spectroscopy: Varian Cary Eclipse. NMR spec-
troscopy: Bruker Avance 400, Varian VNMR-S 600.
To determine exemplarily the effect of a strong electron-
donating substituent we examined also the amino-substituted
derivative 4g. The electron-donating character of the amino
group in 4g is clearly indicated by the strong red shift of the
absorption (λAbs = 375 nm) and emission bands (λFl = 538 nm)
Materials
relative to the parent compound 4a (λAbs = 363 nm, λFl
388 nm). The induced shifts ΔTm as well as the ΔΔTm values
obtained with this ligand are just somewhat larger (e.g. ΔTmTX
=
All buffer solutions were prepared from purified water (resis-
tivity 18 MΩ cm−1) and biochemistry-grade chemicals. The
buffer solutions were filtered through a PVDF membrane filter
(pore size 0.45 µm) prior to use. BPE buffer: 6.0 mM Na2HPO4,
2.0 mM NaH2PO4, 1.0 mM Na2EDTA; total Na+ concentration
16.0 mM; pH 7.0. ODN buffer: 6.1 mM Na2HPO4, 3.9 mM
NaH2PO4, 1.0 mM Na2EDTA, 20 mM NaCl; total Na+ concen-
tration 38.1 mM; pH 7.0. Calf thymus DNA (type I; highly poly-
merized sodium salt; ε = 12 824 cm−1 M−1) was purchased
from Sigma (St. Louis, MO, USA). Oligodeoxyribonucleotides
5′-GCGTGNGTGCG-3′ (N = G, C, A, or T), 5′-CGCACNCACGC-3′
(N = G, A, or tetrahydrofuran spacer; purification: HPLC;
quality control: MALDI-TOF; synthesis scale: 1.0 µmol) were
purchased from Metabion International (Martinsried,
Germany). Melting temperatures of the corresponding duplex
=
7.0 °C; ΔΔTTmX = 4.8 °C; LDR = 2) than the ones recorded with
the chloro- or methyl-substituted ligands such as 4d or 4h.
These results demonstrate that the introduction of donating
substituents may also be employed to increase the affinity and
selectivity of an AP-DNA ligand. At the same time, it should be
noted that the resulting effect is not much stronger than the
one observed with chloro or methyl substituents, so all of
these substituents may be employed complementarily as
affinity-increasing structural elements.
Conclusions
DNA (cDNA = 5.0 µM) were determined in ODN buffer. CX: Tm
=
In summary, we have assessed the structural features that
determine the association of ligands with AP-DNA by means of
quinolizinium derivatives as the DNA-binding source. As has
been demonstrated with studies on triplex and quadruplex
DNA19 the quinolizinium ion serves as a useful reference
system to explore the effect of substituents and of the size and
shape of the ligand on the DNA-binding properties. As
expected the methyl effect and/or the annelation with an
additional benzene unit increase the ability of the ligand to
stabilize the DNA upon association, respectively, whereupon
the annelation effect depends on the shape of the polycyclic
system (angular vs. linear annelation). Most notably, we discov-
ered that the chloro-substituent may be employed complemen-
tary to the methyl group to increase the binding affinity.
Although we observed increased selectivity of a ligand towards
AP-DNA due to the above-given effect, it should be stressed
that as in the case of 5a these ligands are already very effective
binders to regular DNA and even exhibit selectivity towards
other DNA forms such as triplex DNA. Therefore, additional
variations of the substitution pattern, beyond the ones pre-
25.8 °C; TX: Tm = 26.4 °C; AX: Tm = 32.2 °C; GX: Tm = 32.9 °C;
TA: Tm = 55.1 °C; CG: Tm = 58.1 °C. The melting temperatures
of the double strands TA and TX are consistent with the
reported ones under identical conditions.16b
Methods
The spectrophotometric and spectrofluorimetric titrations
with DNA, and the thermal DNA denaturation studies were
performed according to reported protocols.16b,19c Binding con-
stants were determined from spectrofluorimetric titrations.
The binding constants of the ligands with ct DNA were deter-
mined by fitting the experimental data from the fluorimetric
DNA titration to the theoretical model according to the pub-
lished procedure.13a,31,44
Acknowledgements
sented herein, are necessary to accomplish higher selectivities. This paper is dedicated to Professor Berward Engelen, Univer-
For future studies in this direction the quinolizinium ion, sity of Siegen, on the occasion of his 70th birthday. Financial
especially considering the results obtained so far, may rep- support by the Deutsche Forschungsgemeinschaft is gratefully
resent a promising starting point. Overall these results may be acknowledged (Ih24/9-1). We thank Dr Anna Bergen for the
employed for the development of ligands that target abasic donation of a sample of the quinolizinium 1i and Ms. Sandra
site-containing DNA. Specifically, our findings may guide the Uebach for assistance during preparation of the manuscript.
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