Binding of dansylamide 1 derivatives to nucleotides and nucleic acids

Article abstract of DOI:10.1039/a801609g

Dansylamide ? has been modified by the introduction of several side chains into the sulfonamide substituent. Compounds with no, or with only one positive charge at the side chains were found not to be useful for association studies with nucleotides or nucleic acids. Relatively high binding constants with moderate base selectivities have been observed with a ligand obtained from dansyl chloride and N,N′-bis(3-aminopropyl)piperazine, which contains two dansyl units and two permanently charged peralkylammonium centers. Interactions of this ligand with double-stranded nucleic acids show a biphasic binding, the first at base pair concentrations below 10^-7 M being only detectable by a decrease of fluorescence intensity. The second phase is characterized by increased fluorescence and by wavelength changes similar to effects observed in lipophilic solvents and by affinities of up to 5 × 10⁵ M. At higher concentrations bathochromic shifts and extinction changes in UV spectra of the dye suggest an intercalation mechanism, in line with preliminary circular dichroism studies.

Full text of DOI:10.1039/a801609g

View Article Online / Journal Homepage / Table of Contents for this issue
Binding of dansylamide† derivatives to nucleotides and nucleic acids1
Xuemei Wang and Hans-Jörg Schneider*
FR Organische Chemie der Universität des Saarlandes, D 66041 Saarbrücken, Germany
Dansylamide† has been modified by the introduction of several side chains into the sulfonamide
substituent. Compounds with no, or with only one positive charge at the side chains were found not to
be useful for association studies with nucleotides or nucleic acids. Relatively high binding constants
with moderate base selectivities have been observed with a ligand obtained from dansyl chloride and
N,NЈ-bis(3-aminopropyl)piperazine, which contains two dansyl units and two permanently charged
peralkylammonium centers. Interactions of this ligand with double-stranded nucleic acids show a
biphasic binding, the first at base pair concentrations below 10؊7 M being only detectable by a decrease
of fluorescence intensity. The second phase is characterized by increased fluorescence and by wavelength
changes similar to effects observed in lipophilic solvents and by affinities of up to 5 × 105 M. At higher
concentrations bathochromic shifts and extinction changes in UV spectra of the dye suggest an
intercalation mechanism, in line with preliminary circular dichroism studies.
Introduction
R
(CH2)3
R
(CH2)3
N
N
(CH2)3
R
N
+
N
(CH2)3 R
+
The development of new ligands to recognize nucleotides
and nucleic acids is of analytical and potentially also of medi-
cinal interest.2 Considerable effort has been focused on the
development of new fluorescent dyes which might have specific
effects on nucleic acids. Little attention, however, has been paid
to dansylamide (DNSA) derivatives which are well-known
fluorescence probes for the study of proteins. We wanted to
explore the possibility of increasing the affinity of dansylamide
derivatives by the introduction of positive charges in side chains
of the dye, and/or to provide two dye residues in one ligand
molecule in order to increase stacking interactions with nucleo-
bases.
1
2
+
N
CH2 CH2 CH2
CH2 CH2 CH2
N
NH
R
NH
R
4
3
R
CH2
CH2
Results
R
CH2 CH2
N
CH2 CH2
R
Compounds such as 1 or 2 were considered to hold particular
promise both with respect to affinity constants and selectivity,
as they not only contain additional positive charges, but also
could allow two dansyl moieties to embrace an aromatic sub-
strate, leading to stacking interactions with potentially some
base selectivity. Ligand 2 bears two permanent charges, and
thus has the advantage of being usable at all desired pH values.
In contrast, the lower water solubility and/or the necessity to
use low pH values for complexation with ligand 1 severely limits
its application.
5
R
CH2
R
R
R
CH2
6
7
SO2NH
For comparison complexation studies were also carried out
with several other dansyl derivatives (3–7), which bear only one
positive charge. An additional charge can be introduced at the
sulfonamide function in the dansyl moieties by protonation at
pH < 4. With 2-naphthylsulfonic acid and 2-naphthylacetic acid
as test substrates, fluorescence titrations were carried out in
water with 0.5% DMSO, also under acidic conditions. In all
these cases the observed fluorescence emission intensity showed
a non-linear decrease with increasing substrate concentration.
Evaluation by least squares fitting to a 1:1 model was found to
be satisfactory (error values for association constants were
usually < 10%). The association lg K values for all these DNSA
derivatives as well as for DNSA itself, which bears one posi-
tive charge at the applied pH 2, and the naphthalene substrates
were all similar with lg K = 3.0 0.2. The dansyl derivatives
6 and 7, prepared by reaction of dansyl chloride with, e.g.
R =
N
α,α-diamino-p-xylene or p-phenylenediamine, had the dis-
advantage of very low water solubility of the complex, and
showed no regular association behaviour with aromatic sub-
strates or nucleotides. Table 1 summarizes the fluorescence
properties of ligands 1 to 7, and the few data which could be
obtained by addition of double-stranded calf thymus DNA to
ligands other than 1 and 2 at the accessible low concentrations.
The very low solubility of the derivatives 5 to 7 also limited UV
and melting point measurements to concentrations where no
significant effects could be found. The compounds with only
one dansyl unit also showed very low affinity to DNA, with a
slight increase of fluorescence intensity ∆I, i.e. with ligands 3
and 4, ∆I < 2% at 10Ϫ3  DNA. Also, there was no spectral shift
† Dansyl is 5-(dimethylamino)naphthalene-1-sulfonyl.
J. Chem. Soc., Perkin Trans. 2, 1998
1323

View Article Online
Table 1 Fluorescence data for dansyl derivatives 1 to 7a
Ligand excitation
Ligand emission
With DNA (∆λ/nm,d
blue shift)
With DNA
(∆I/I0 )
e
Liganda
wavelengthb (λ/nm)
wavelengthc (λ/nm)
1
2
3
4
5
6
7
326
330
330
330
350
350
350
520
547
545
545
490
493
496
Ϫ20
Ϫ29
Ϫ0.12
(I) Ϫ0.23; (II) 0.75
0.01
0.02
Ϫ0.74
Ϫ0.87
Ϫ0.39
0
0
0
0
0
a
Concentrations of ligands: 3 × 10Ϫ6  in SHE buffer, pH 7.0; ambient temperature. b Ligand excitation wavelength at fixed emission (at the λmax of
emission); c Ligand emission wavelength at fixed excitation (at the λmax of excitation); d ∆λ is the wavelength change at the highest DNA concentration
used. The concentration range of CT-DNA was varied usually from 10Ϫ8 to 10Ϫ5 , except for ligands 3 and 4 (from 10Ϫ6 to 10Ϫ3 ). e I0 is the
emission intensity of the ligand in the absence of CT-DNA, ∆I is the maximum emission intensity change upon addition of CT-DNA. For ligand 2,
(I) is the decrease of fluorescence at low ratio of [bp]/[2] (р0.01), (II) is the increase of fluorescence at higher DNA concentration (10Ϫ5 ).
Table 2 Binding constants (K, in Ϫ1) of host compound 2a
Benzenesulfonate
2-Naphthalenesulfonate
5Ј-AMP
5Ј-CMP
5Ј-GMP
5Ј-TMP
5Ј-UMP
5.2 × 102
1.3 × 103
5.6 × 103
2.0 × 103
2.9 × 103
1.5 × 103
7.3 × 102
a From fluorescence titrations; measurement conditions: 0.02

phosphate buffer, pH 7.0; the nucleotides at this pH bear two negative
charges. Error of binding constants р 10%.
observed in absorption and fluorescence spectra with these
compounds. With ligands such as 5, 6 and 7, having two or
three dansyl units with and/or without one positive charge, the
absorption and fluorescence intensities with added DNA were
found to decrease significantly and to reach a final saturation
value with increasing nucleic acid concentration, accompanied
by negligible spectral shifts. These spectral changes could not,
however, be fitted to binding isotherms. After these preliminary
experiments only ligand 2 was chosen for further investigation
of its interactions with nucleotides and nucleic acids.
Interaction of ligand 2 with nucleotides
Complexation of ligand 2 with aromatic substrates and in par-
ticular with nucleotides was studied by UV absorption and by
fluorescence spectroscopy. The absence of self-association of
the ligand at the concentrations used was ensured by the obser-
vation of linear dependencies (linear correlation coefficients
r > 0.9997) of intensity, or extinction, respectively, vs. ligand
concentration; these plots began to show curvature by fluor-
escence only at concentrations above 0.033 m of 2. Complex
formation of ligand 2 with all aromatic substrates was charac-
terized by the decrease of absorption or fluorescence intensity
with almost no shift of the peak positions. Affinities were
determined by fluorescence titrations, yielding a satisfactory fit
for a 1:1 association model (Fig. 1). The values (Table 2) show
relatively high equilibrium constants for simple nucleotides,
which compare favourably with more complicated synthetic
host compounds.2–5 It should be noted that the relatively high
buffer concentration used to maintain a constant pH weakens
the ion pairing and thus the affinities considerably. The lg
K values can be rationalized by salt bridges between the
ammonium centers and the phosphate units, and by moderate
stacking interactions between nucleobases and two naphthyl
units of the dye, which can be oriented in a cleft-like manner.
Computer aided molecular modeling indicates that such a
sandwich structure represents a local minimum in the calcul-
ated strain energies (Fig. 2). That stacking contributes to the
observed binding, although not to a very high degree, is borne
out by the smaller affinity observed with benzenesulfonate, as
Fig. 1 Fluorescent titration curves of ligand 2 with (a) 5Ј-TMP,
(b) 5Ј-GMP and (c) 5Ј-CMP. Excitation wavelength at 330 nm. For
Experimental conditions see Table 1.
well as by the observation of undetectable (estimated upper
value lg K < 3) complexation with the simple model compound
4, which bears only one stacking unit. Ligand 2 also exhibits a
1324
J. Chem. Soc., Perkin Trans. 2, 1998

View Article Online
Table 3 Binding constants (K, in Ϫ1) of ligand 2 with nucleic acidsa
K/Ϫ1 b
(Fluoresc.)
K/Ϫ1 c
(UV–range)
Single-stranded CT-DNA
Double-stranded CT-DNA
polyA
2.0 × 105 d
3.0 × 105 d
5.0 × 105 e
1.0 × 105 e
1.0 × 105 e
1.3 × 105 e
4.0 × 105 d
2.0 × 105 d
4.0 × 104 d
3.5 × 104 e
2.0 × 106 e
3.0 × 104 d
3.0 × 104 e
—
polyAؒpolyU
poly(dA)ؒpoly(dT)
poly(dG)ؒpoly(dC)
poly(dG-dC)
5.0 × 104 e
4.0 × 104 d
poly(dA-dT)
a Measurement conditions: MES buffer (0.01  MES, 0.001  EDTA),
pH 6.25. b Association constants K obtained from fluorescence titra-
tions: [2] = 0.010 m, base pair concentration [bp] was varied usually
from 0.0002 to 0.010 m; c Association constants K obtained from UV
absorption titrations: [2] = 0.020 m, usually [bp] from 0.003 to 0.030
m, except with CT-DNA from 0.005 to 0.080 m. d Association con-
stants K estimated from the concentration needed to reach 50% of ∆I,
where ∆I is the maximum emission intensity change upon addition of
nucleic acids. e Association constants K estimated from 1:1 curve fitting.
Fig. 2 CHARMm-simulated structure of the complex between ligand
2 and one nucleotide (5Ј-AMP)
Fig. 4 Fluorescent titration curve of ligand 2 with poly(dG)ؒpoly(dC).
Excitation wavelength at 330 nm. For Experimental conditions see
Table 2.
Fig. 3 Fluorescence spectra of ligand 2 (10 µ) with increasing
concentration of polyAؒpolyU. Concentrations of polyAؒpolyU were
as follows: 1, 0.0; 2, 2.1; 3, 4.2; 4, 6.3; 5, 8.4; 6, 10.5; 7, 12.6; 8, 14.6; 9,
17.8 µ. For Experimental conditions see Table 2. (a) Excitation spectra
determined with emission at 547 nm; (b) Emission spectra determined
with excitation at 330 nm.
systematic deviation from a fit to 1:1; in these cases the K value
was estimated from the concentration needed to reach 50% of
∆I, where ∆I is the maximum emission intensity change upon
addition of nucleic acids.
moderate binding selectivity to nucleotides, with a preference
for purines as in most other cases studied with artificial host
compounds.4
Dansylamine is also well known as a probe for the micro-
environment polarity, for instance in proteins. Therefore, to
further reveal the nature of the complexation sites, spectral
characteristics of ligand 2 in some organic solvents were
investigated. The results (Table 4) indicate that the fluorescence
intensity of 2 increases significantly, with a blue shift of
emission wavelength and a red shift of the excitation peak with
a change in solvent from water to hydrophobic organic solvent.
The fluorescence intensity of ligand 2 in alcohol is about ten
times stronger than that in aqueous solution. In comparison,
the spectral shifts on the second binding step of 2 with nucleic
acids show features similar to those in alcohol, which indicates
that the dansyl chromophore is involved in a distinctly more
hydrophobic environment when bound to nucleic acids.
UV Absorption spectra. Strong interactions of ligand 2 with
single- and double-stranded polynucleotides were also visible in
UV spectra. One advantage of UV–VIS spectroscopy is that
one can measure spectra at higher concentrations of base pairs,
until the system becomes insoluble. Typically, ligand 2 upon
addition of nucleic acids produced strong decreases of absorp-
tion, accompanied by a large red shift of the peak to a position
which is similar to that of 2 in organic solvents like methanol or
ethanol. In addition, one set of isosbestic points was observed
in each case (Table 5). Fig. 5 represents an example of an
absorption spectral change of 2 with poly(dA)ؒpoly(dT). The K
values (Table 3) obtained with the UV absorption measure-
Interaction of ligand 2 with nucleic acids
Fluorescence studies. The titrations of ligand 2 with nucleic
acids show a biphasic binding mode as a function of the
biopolymer concentration, as has also been found for other
fluorescent antibiotics, such as Hoechst 33 258.6 At base pair
(bp) concentrations of 0.0 < [bp] < 10Ϫ7 , with a low ratio of
r = [bp]/[2] (r р 0.01), one observes a decrease of fluorescence
intensity of 2, and no change of the peak wavelength with the
addition of nucleic acids. At higher ratios of [bp]/[2] the fluor-
escence shows a non-linear increase, with blue shifts of the
emission wavelength by up to ca. 30 nm, and red shifts of the
excitation wavelength by up to ca. 20 nm (Fig. 3). The first
phase could not be fitted to simple association equilibrium
models due to the overlap with the second binding mode; the
observed effects, however, indicate lg K1 values > 6.0. The
association at these low concentrations could involve a DNA-
induced self-aggregation of the ligand, as has been observed
with other groove binders.7 The association constants for the
second binding phase (Table 3) could be obtained in many cases
from curve fitting to a 1:1 model. Fig. 4 shows an example of
the curve fitting for fluorescence titrations of ligand 2 with
poly(dG)ؒpoly(dC). In other cases the titration curves showed
J. Chem. Soc., Perkin Trans. 2, 1998
1325

View Article Online
Table 4 Fluorescence wavelength changes of compound 2 bound either to nucleic acids or in organic solvents (compared to the absorption
wavelength in water)
Excitation
Emission
c
wavelength (λ/nm)a
wavelength (λ/nm)b
∆I/I0
Single-stranded CT-DNA
Double-stranded CT-DNA
polyA
350(ϩ20)
350(ϩ20)
350(ϩ20)
349(ϩ19)
349(ϩ19)
348(ϩ18)
345(ϩ15)
350(ϩ20)
347(ϩ17)
347(ϩ17)
349(ϩ19)
340(ϩ10)
515(Ϫ32)
518(Ϫ29)
515(Ϫ32)
515(Ϫ32)
515(Ϫ32)
516(Ϫ31)
525(Ϫ22)
515(Ϫ32)
518(Ϫ29)
515(Ϫ32)
509(Ϫ38)
490(Ϫ57)
(a) Ϫ0.26; (b) 2.52
(a) Ϫ0.23; (b) 0.75
(a) Ϫ0.20; (b) 0.81
(a) Ϫ0.04; (b) 1.40
(a) Ϫ0.15; (b) 1.44
(a) Ϫ0.04; (b) 1.15
(a) Ϫ0.13; (b) 0.36
(a) Ϫ0.27; (b) 1.56
—
polyAؒpolyU
poly(dA)ؒpoly(dT)
poly(dG)ؒpoly(dC)
poly(dG-dC)
poly(dA-dT)
In methanol
In ethanol
In acetone
In THF
—
—
—
a Excitation spectra with fixed emission wavelength at 547 nm. b Emission spectra with fixed excitation wavelength at 330 nm. c I0 is the emission
intensity of the ligand in the absence of nucleic acids, ∆I is the maximum emission intensity change upon addition of nucleic acids. (a) is the decrease
of fluorescence at low ratio of [bp]/[2] (р0.01), (b) is the increase of fluorescence at higher concentration of nucleic acids. For experimental
conditions see Table 3.
Table 5 Spectroscopic properties from UV titrations for ligand 2
Table 6 Effect of ligand 2 on DNA melting point Tm (ЊC) valuesa
bound to nucleic acidsa
CT-DNA
poly(dA)ؒpoly(dT)
(∆Tm)
∆λ/nm
Isosbestic
Rb
(∆Tm)c,d
Nucleic acids
H(%)b
(red shift)
points (λ/nm)
0.1
0.2
2.3
2.7
2.2
2.3
ss-CT-DNA
ds-CT-DNA
polyA
polyAؒpolyU
poly(dA)ؒpoly(dT)
poly(dG)ؒpoly(dC)
poly(dG-dC)
poly(dA-dT)
36
33
36
32
31
14
30
33
8.5
6.5
8.0
8.5
6.5
8.0
6.0
7.5
300, 304c
299, 307d
296e
295
298
a Measurement conditions: MES buffer (0.01  2-(N-morpholino)-
ethanesulfonic acid, 0.001  EDTA and pH 6.25, [NaCl] = 0.01 ). b R
is a ratio of the concentration of ligand 2 per mol of nucleic acid
phosphate of CT-DNA. c ∆Tm = Tm complex Ϫ Tm free nucleic acid.
The Tm values were obtained from first-derivative plots, dupulicate
runs. The error of Tm is 0.1 ЊC. d For ligand 1 (R = 0.1), ∆Tm with
CT-DNA is 0.6 ЊC; with all other ligands, ∆Tm < 0.1 ЊC.
308
300f
297
a Measurement conditions: MES buffer (0.01  MES, 0.001  EDTA),
pH 6.25. b The excitation change [H(%)] was obtained at a ratio of [2]/
[bp] = 1:1; for CT-DNA: [2]/[bp] = 1:2. c The isosbestic point shifts
from 300 to 304 nm with increasing ss-CT-DNA concentration. d The
isosbestic point shifts from 299 to 307 nm with increasing ds-CT-DNA
concentration. e Isosbestic point observed at 296 nm, but blurred at
higher concentration ([bp] > 15 µ) of polyA. f Isosbestic point
observed at 300 nm, but blurred at higher concentration ([bp] > 15 µ)
of poly(dG-dC).
acids, especially for polyA. One also observed a slight G:C
base pair preference. At the higher concentrations of nucleic
acids, accessible to UV–VIS measurements, the binding may
start to involve intercalation into the double-stranded poly-
mers. NMR analyses with related aminoalkylnaphthalenes
indicate intercalation only at such higher concentrations.8 The
magnitude of the excitation changes ∆ε and the bathochromic
shift is generally believed to indicate intercalation;9,10 the
observed ∆ε (usually by ca. 30% at the ratio [2]/[bp] = 11) and
absorption red shift suggest that the intercalation of the dansyl
chromophore leads to a close proximity of the dansyl chromo-
phore to the nucleobases. The changes are more pronounced
compared to other studies,9,10 where such large effects were only
reported for higher base pair/ligand ratios. The millimolar
affinities observed in this work indicate that simple ligand
structures may compete with the binding strength of more
complicated antibiotics.
Circular dichroism and melting studies. Further evidence for
the strong interaction of the ligands with nucleic acids comes
from preliminary circular dichroism (CD) spectra and melting
studies of nucleic acids. It is found that with the increase of
ligand concentration, CD spectra of the nucleic acids at 20 ЊC
changed with intensity decrease (Fig. 6), while the helix melting
temperature increased (Table 6).
Fig. 6 shows the CD spectra in the UV range upon addition
of ligand 2; they illustrate a considerable decrease of the peak
intensity indicating a distinct conformational change of the
DNA duplexes. Even the single-stranded polyA exhibited a
decrease of the peak intensity upon addition of ligand 2,
although this change is not as strong as that of the duplexes.
It is noteworthy that an isoelliptic point was observed at
about 260 nm at the concentrations of ligand 2 below 10 µ;
this was blurred above 10 µ of 2. These observations are
consistent with the absorbance changes mentioned above, indi-
cating similar affinities to those obtained from the quantitative
Fig. 5 Absorbance spectra of ligand 2 (20 µ) with increasing concen-
tration of poly(dA)ؒpoly(dT). Concentrations of poly(dA)ؒpoly(dT)
were as follows: 1, 0.0; 2, 6.1; 3, 12.2; 4, 18.3; 5, 30.8; 6, 42.9; 7, 49.0; 8,
53.2; 9, 58.0 µ.
ments show deviations compared to those of fluorometric
titrations, which indicate another binding mode at the higher
concentrations used in the UV measurement range. The K
values show an enhanced binding with single-stranded nucleic
1326
J. Chem. Soc., Perkin Trans. 2, 1998

View Article Online
preliminary circular dichroism data and with earlier results,8
where naphthyl-shaped moieties were shown to intercalate only
at such higher concentrations.
Experimental
Materials
Organic starting material was obtained from Fluka and
ACROS Chemical Co. Calf thymus DNA was purchased from
Aldrich, polyAؒpolyU, polyA, poly(dA-dT), poly(dA)ؒpoly(dT)
from Sigma Chemical Co., and poly(dG)ؒpoly(dC), poly(dG-
dC) from Pharmacia. The nucleic acid concentrations were
determined from the molar absorptivity: 9.8 mϪ1 cmϪ1 at 258
nm for polyA; 6.0 mϪ1 cmϪ1 at 260 nm for poly(dA)ؒpoly(dT);
7.4 mϪ1 cmϪ1 at 253 nm for poly(dG)ؒpoly(dC); 6.6 mϪ1 cmϪ1
at 262 nm for poly(dA-dT); 8.4 mϪ1 cmϪ1 at 254 nm for
poly(dG-dC); 6.0 mϪ1 cmϪ1 at 260 nm for polyAؒpolyU.
Nucleotide solutions were prepared in 0.02  phosphate buffer
(pH 7), while nucleic acids were dissolved in MES buffers [0.01
 2-(N-morpholino)ethanesulfonic acid, 0.001  EDTA and
pH 6.25, [NaCl] = 0.01 ].
N,NЈ-Bis(3-dansylaminopropyl)piperazine† (1). N,NЈ-Bis-
(3-aminopropyl)piperazine (13.6 mmol) was added to dansyl
chloride (34 mmol) in 200 ml dry toluene at 0 ЊC under stirring;
a solution of triethylamine (4 ml) was added dropwise; the mix-
ture was stirred at room temperature and the end of the reac-
tion was checked by TLC. The solid obtained was washed with
distilled water several times to obtain the pure compound
(45%). δH([2H6]DMSO) 1.37 (m, 4H), 2.00 (m, 12H), 2.80 (t, 4H),
2.84 (s, 12H), 7.28 (d, 2H), 7.63 (m, 4H), 7.91 (s, 2H), 8.11 (d,
2H), 8.30 (d, 2H), 8.48 (d, 2H); δC([2H6]DMSO) 25.90, 44.87,
52.20, 54.65, 114.89, 118.99, 123.17, 127.44, 128.05, 129.06,
136.07, 151.25. (Calc. for C34H46N6S2O4ؒH2O: C, 61.07; H, 7.06;
N, 12.27. Found: C, 60.80; H, 6.76; N, 12.17%).
Methylated N,NЈ-bis(3-dansylaminopropyl)piperazine (2).
Methyl iodide (1 mmol) was added to N,NЈ-bis(3-dansylamino-
propyl)piperazine (1) (0.5 mmol) in 10 ml dry DMF at 0 ЊC
under stirring, at 50 ЊC; the end of the reaction was checked by
TLC. After evaporation of the solvent and recrystallization
from methanol, the pure compound was obtained with a yield
of 32%. δH([2H6]DMSO) 1.88 (m, 4H), 2.83 (s, 12H), 2.87 (m,
4H), 3.15 (s, 6H), 3.56 (t, 4H), 3.80 (m, 8H), 7.32 (d, 2H), 7.65
(m, 4H), 8.06 (s, 2H), 8.12 (d, 2H), 8.30 (d, 2H), 8.48 (d, 2H);
δC([2H6]DMSO) 22.17, 45.30, 53.42, 115.64, 119.37, 123.90,
128.18, 128.59, 129.10, 129.62, 135.52, 151.00. (Calc. for
C36H52N6S2O4I2: C, 45.44; H, 5.47; N, 8.83. Found: C, 43.75; H,
5.20; N, 8.22%).
Fig. 6 (a) Circular dichroism (CD) spectra of 6.8 µ polyA in the
presence of different concentrations of ligand 2. Concentrations of 2
were (from top to bottom): 0.0, 3.4, 6.8, 10.2 µ; (b) CD spectra of 24
µ poly(dA)ؒpoly(dT) in the absence (top) and in the presence (bottom)
of 12 µ 2. All CD experiments in MES buffer at pH 7.0, 20 ЊC.
UV titrations. The lack of solubility of the complexes pre-
vented NMR or viscosimetric studies to further support the
intercalating mode.
N-Dansyl-3-(dimethylamino)propylamine (3). 3-(Dimethyl-
amino)propylamine (10.5 mmol) was added to dansyl chloride
(7 mmol) in 90 ml dry CH3CN at 0 ЊC under stirring. Then a
solution of triethylamine (2 ml) was added dropwise. The mix-
ture was stirred at room temperature overnight; after acidifi-
cation (pH 4), the mixture was extracted with diethyl ether. The
aqueous layer was made basic with aqueous NaOH, then
extracted with CHCl3 and dried over Na2SO4. A solid was
obtained after evaporation of solvent. Recrystallization from
ethanol yielded a pure compound (48%). δH([2H6]DMSO) 1.40
(m, 2H), 1.92 (s, 6H), 2.02 (t, 2H), 2.85 (m, 8H), 7.29 (d, 1H),
7.64 (m, 2H), 7.90 (s, 1H), 8.13 (d, 1H), 8.31 (d, 1H), 8.49 (d,
1H); δC([2H6]DMSO) 27.02, 40.10, 44.96, 45.13, 56.31, 115.18,
119.28, 123.62, 127.86, 128.44, 129.27, 129.42, 136.24, 151.49.
N-Dansyl-3-(trimethylamino)propylamine iodide (4). Methyl
iodide (4 mmol) was added to dansyl-3-(dimethylamino)-
propylamine (3) (2 mmol) in 40 ml dry methanol at 0 ЊC under
stirring. The mixture was stirred at room temperature for 18 h.
After evaporation, the pure compound was obtained by
recrystallization from methanol (55%). δH([2H6]DMSO) 1.79
(m, 2H), 2.85 (m, 8H), 2.97 (s, 9H), 3.23 (t, 2H), 7.29 (d, 1H),
7.64 (m, 2H), 8.07 (s, 1H), 8.13 (d, 1H), 8.29 (d, 1H), 8.49 (d,
1H); δC([2H6]DMSO) 23.01, 45.16, 52.40, 63.34, 115.26, 118.90,
Conclusions
The results show that a synthetically easily accessible fluor-
escent dye such as 2 can bind efficiently to aromatic substrates,
to nucleotides and to nucleic acids. With double-stranded DNA
the association starts at concentrations as low as 10Ϫ7 , prob-
ably involving binding in the major DNA groove which can
induce self-aggregation of the ligand (a similar decrease of
emission intensity I is observed in the first phase binding to
DNA as one finds in curves of I vs. ligand concentration, where
self-association and quenching start to lead to deviations from
linearity). At concentrations between 10Ϫ7 and 10Ϫ5 , access-
ible to fluorescence titrations with line fitting (second phase),
the binding with DNA is characterized by 1:1 association
isotherms with affinities as high as 5 × 105 . The observed
changes of fluorescence emission wavelength and intensities are
similar to those observed in alcoholic solvents; these data
would be in line with a deep groove association of the ligand.
At concentrations around 10Ϫ5 , accessed by UV titrations, the
observed extinction and wavelength changes point to inter-
calation as the dominant binding mode. This is in line with
J. Chem. Soc., Perkin Trans. 2, 1998
1327

View Article Online
123.69, 128.05, 128.48, 129.00, 129.61, 135.47, 151.49. (Calc.
for C18H28N3SO2I: C, 45.31; H, 5.92; N, 8.81. Found: C, 45.49;
H, 5.84; N, 8.71%).
Spectroscopic titrations were performed as described earlier,8
using non-linear least squares fit for association constants. With
the first part of the biphasic behavior of the fluorescence
titrations, no satisfactory fit could be obtained; in these cases lg
K values were approximated from the concentrations necessary
to reach 50% of the total observed change in fluorescence emis-
sion. The presence of 0.5% DMSO in the measured solution
does not lead to any significant lg K changes, as was established
in a test with compound 1 and 2-naphthylacetic acid with 15%
DMSO.
Tris(2-dansylethyl)amine (5). Tris(2-aminoethyl)amine (1
mmol) was added to dansyl chloride (3.5 mmol) in 30 ml dry
toluene at 0 ЊC under stirring. Then a solution of triethylamine
(1 ml) was added dropwise into the clear solution. The mixture
was allowed to stir at room temperature and the end of the
reaction was checked by TLC. The precipitate formed was
filtered and the solution was evaporated. The solid obtained
was washed several times with distilled water to obtain the pure
compound (38%). δH([2H6]DMSO) 2.11 (t, 6H), 2.64 (m, 6H),
2.84 (s, 18H), 7.18 (d, 3H), 7.54 (m, 6H), 7.76 (s, 3H), 8.08 (d,
3H), 8.28 (d, 3H), 8.44 (d, 3H); δC([2H6]DMSO) 45.13, 53.55,
115.09, 119.05, 123.52, 127.79, 128.11, 129.09, 129.35, 136.16,
151.38. (Calc. for C41H51N7S3O6ؒH2O: C, 58.38; H, 6.187; N,
11.35. Found: C, 58.25; H, 6.224; N, 11.31%).
Acknowledgements
An Alexander von Humboldt fellowship for X. W. and financial
support by the Deutsche Forschungsgemeinschaft and the
Fonds der Chemischen Industrie are gratefully acknowledged.
á,áЈ-Bis(dansylamino)-p-xylene (6). α,αЈ-Diamino-p-xylene (2
mmol) was added to dansyl chloride (5 mmol) in 20 ml dry tolu-
ene at 0 ЊC under stirring; a solution of triethylamine (2 ml) was
added dropwise; the mixture was stirred at room temperature
and the end of the reaction was checked by TLC. The precipi-
tate obtained was washed with distilled water several times to
obtain the pure compound (60%). δH([2H6]DMSO) 2.85 (s,
12H), 3.95 (s, 4H), 6.97 (s, 4H), 7.27 (d, 2H), 7.60 (m, 4H), 8.08
(d, 2H), 8.32 (d, 2H), 8.43 (s, 2H), 8.46 (d, 2H); δC([2H6]DMSO)
45.16, 45.74, 115.15, 119.28, 123.56, 127.20, 127.86, 128.30,
129.16, 129.42, 136.33, 136.64, 151.43. (Calc. for C32H34N4S2O4:
C, 63.76; H, 5.69; N, 9.29. Found: C, 63.16; H, 5.74; N, 9.11%).
1,4-Bis(dansylamino)phenylene (7). Phenylene-1,4-diamine (2
mmol) was added to dansyl chloride (5 mmol) in 20 ml dry
toluene at 0 ЊC under stirring; a solution of triethylamine (2 ml)
was added dropwise; the mixture was stirred at room temper-
ature and the end of the reaction was checked by TLC. The
precipitate obtained was washed with distilled water several
times to obtain the pure compound (48%). δH([2H6]DMSO)
2.82 (s, 12H), 6.80 (s, 4H), 7.21 (d, 2H), 7.52 (m, 4H), 8.07 (d,
2H), 8.24 (d, 2H), 8.42 (d, 2H), 10.40 (s, 2H); δC([2H6]DMSO)
45.24, 115.37, 118.87, 120.78, 123.61, 128.17, 129.15, 129.70,
130.19, 133.72, 135.10, 151.60. (Calc. for C30H30N4S2O4: C,
62.70; H, 5.26; N, 9.75. Found: C, 62.10; H, 5.10; N, 9.66%).
References
1 Supramolecular Chemistry, Part 80; part 79: V. P. Solov’ev, N. N.
Strakhova, V. P. Kazachenko, A. F. Solotnov, V. E. Baulin,
O. Raevsky, V. Rüdiger, F. Eblinger and H.-J. Schneider, Eur.
J. Org. Chem., 1998, in the press.
2 (a) M. J. Waring, in The Molecular Basis of Antibiotic Action,
ed. E. F. Gale, E. Cundliffe, P. E. Reynolds, M. H. Richmond and
M. J. Waring, Wiley, London, 2nd edn., 1981, p. 287; (b) D. W.
Wilson, Y. Li and J. Veal, in Advances in DNA Sequence Specific
Agents, ed. L. H. Hurley, JAI Press, Greenwich, vol. 1, 1992,
p. 89; (c) W. D. Wilson, in Nucleic Acids in Chemistry and Biology,
ed. M. Blackburn and M. Gait, IRL Press, Oxford, 1989, ch. 8;
(d) J. W. Lown, Anti-Cancer Drug. Des., 1988, 3, 25; (e) P. B. Dervan,
Science, 1986, 232, 464; ( f ) S. Neidle and Z. Abraham, CRC Crit.
Rev. Biochem., 1984, 17, 73; (g) S. Neidle and T. C. Jenkins, Mol.
Des. Model., Part B, 1991, 203, 433; B. H. Geierstanger and D. E.
Wemmer, Annu. Rev. Biophys. Biomol. Struct., 1995, 24, 463.
3 S. Claude, J.-M. Lehn, F. Schmidt and J.-P. Vigneron, J. Chem. Soc.,
Chem. Commun., 1991, 1182.
4 H.-J. Schneider, T. Blatter, B. Palm, U. Pfingstag, V. Rüdiger and
I. Theis, J. Am. Chem. Soc., 1992, 114, 7704.
5 A.-V. Eliseev and H.-J. Schneider, J. Am. Chem. Soc., 1994, 116,
6081.
6 G. Loontiens, P. Regenfuss, A. Zechel, L. Dumortier and R. M.
Clegg, Biochemistry, 1990, 29, 9029.
7 See e.g. J. Y. Fan, D. Sun, H. T. Yu, S. M. Kervin and L. H. Hurley,
J. Med. Chem., 1995, 38, 408; J. B. Chaires, N. Dattagupta and
D. Crothers, Biochemistry, 1982, 21, 3927.
Spectroscopic methods
8 J. Sartorius and H.-J. Schneider, J. Chem. Soc., Perkin Trans. 2,
1997, 2319.
9 A. M. Pyle, J. P. Rehmann, R. Mashoyrer, C. V. Kumar, N. J. Turro
and J. K. Barton, J. Am. Chem. Soc., 1989, 111, 3051.
10 W. D. Wilson and R. L. Jones, in Intercalation Chemistry, ed. M. S.
Whittingham and A. J. Jacobson, Academic Press, New York, 1992,
pp. 446–501.
Fluorescence spectra were obtained with a Hitachi Fluor-
escence Spectrophotometer F-2000 at 25 ЊC. The excitation and
emission slit width was 5 mm. UV spectra were recorded with a
Kontron Uvikon 860 instrument at 25 ЊC. Denaturation
experiments were performed in MES buffers with 6.0 × 10Ϫ5

poly(dA)ؒpoly(dT) or 7.6 × 10Ϫ5  CT-DNA phosphate. ∆Tm =
Tm complex Ϫ Tm free nucleic acid. The Tm values were
obtained from first-derivative plots. CD spectra were obtained
with a JASCO J-715 spectrophotometer interfaced to a PC. All
CD experiments were carried out at 20 ЊC in 1 cm path length
cuvettes. Curves presented are the average of three scans.
Paper 8/01609G
Received 25th February 1998
Accepted 16th March 1998
1328
J. Chem. Soc., Perkin Trans. 2, 1998