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DNA Recognition with Large Calixarene Dimers.

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DNA Intercalation
DOI: 10.1002/ange.200502946
DNA Recognition with Large Calixarene
prove the existence of a regular cone conformation, whereas a
variable-temperature experiment revealed a fast dynamic
rotation of the calixarene around the bridge. A molecular
dynamics calculation confirmed the assumed high degree of
preorganization within the calixarene dimer 1 b carrying the
rigid aromatic spacer, whereas much more flexibility was
found for the aliphatic bridge in 1 a (Figure 1 a).
Reza Zadmard and Thomas Schrader*
DNA-binding molecules are of eminent medicinal significance because they constitute a large part of all anticancer
drugs.[1] In nature, DNA recognition mainly follows four
different paths: efficient binding agents either target the
phosphodiester backbone,[2] intercalate into the basepairs,[3]
or occupy the minor[4] or major groove.[5] Interestingly
enough, the zinc finger[6] and the leucine zipper motifs,[7]
two typical protein domains that fit snuggly into the major
groove of DNA, have similar dimensions (averaged a-helix
diameters of ca. 1.3 nm) and also a comparable overall
topology. Both motifs have a large basic cylinder with a
polar protic face that reads the nucleic bases on the groove.s
floor. Recently, Hannon et al. reported on the first artificial
noncovalent major groove binder, a tetracationic metallosupramolecular cylinder with a size of approximately 2 1
1 nm2.[8] This remarkable molecule binds tightly to the floor
of the major groove and induces DNA bending and intramolecular coiling. We have now found that a calixarene dimer
with six protonatable aniline NH2 groups at its upper rims,
connected by a suitable flexible linker, is also able to
recognize double-stranded (ds) DNA.[9] This molecule is
even larger than the triple-helix cylinder, with an average size
of about 3 1 1 nm2. Its dimensions therefore exclude a
potential intercalation or minor groove insertion.
Various dimeric cationic calixarenes are accessible by a
straightforward synthetic avenue. Triple protection of the
parent tetraaniline[10] with tert-butoxycarbonyl (Boc) groups
followed by amide coupling with diacid dichlorides leads to
dimeric protected precursors that are converted into the
cationic hosts by cleavage of the Boc groups under mild
conditions with trifluoroacetic acid. Reciprocal cross-peaks
between all adjacent aromatic calixarene protons (NOESY)
[*] R. Zadmard,[+] Prof. T. Schrader
Fachbereich Chemie
Universit5t Marburg
Hans-Meerwein-Strasse, 35032 Marburg (Germany)
Fax: (+ 49) 6421-28-25544
[+] Present address:
Chemistry and Chemical Engineering Research Center of Iran
P.O. Box 14335-186, Tehran (Iran)
[**] We are indebted to the Carell group (Munich) for a sample of
fluorescein-labeled triadenosine, as well as to the Marahiel group
(Marburg) for 45-bp DNA samples. Valuable advice came from Dr.
Mohamad Mofid. We also thank the Pingoud group (Giessen) for
the measurements of CD spectra. Funding by the Deutsche
Forschungsgemeinschaft is gratefully acknowledged.
Supporting information for this article is available on the WWW
under or from the author.
Angew. Chem. 2006, 118, 2769 –2772
Figure 1. a) Calix[4]arene dimers 1 a/1 b based on trifunctional anilinium building blocks. b) Molecular mechanics calculation for the rigid
hexaanilinium calixarene dimer 1 b. The syn conformer is energetically
almost identical to its anti counterpart (DH 1 kcal mol1).[11]
Preliminary binding experiments with negatively charged
substrates (see below) showed large upfield shifts for the
peripheral calixarene protons that are exposed to the
approaching guest. Such a shift was also seen for guest
protons that point towards the anisotropy cone of the
calixarene cavity. Subsequent 1H NMR spectroscopy and
fluorescence titrations[12] helped to characterize the binding
profile of compounds 1 a and 1 b: the sugars showed no
response, and the acidic peptides and the di- and oligophosphates were all weakly bound (except for oligonucleotides
such as fluorescein-labeled triadenosine).[13]
To investigate ds-DNA recognition, one of two complementary primer fragments containing 12 nucleic bases was
fluorescein-labeled at its 5’-terminus, and the resulting duplex
was incubated with increasing amounts of calixarene dimer. A
marked decrease in fluorescence intensity indicated a strong
interaction, producing a sharply kinked titration curve that
corresponds to an approximate 9:1 dimer/DNA stoichiometry
(Job plot)[14] with an association constant for each individual
dimer binding event of 1 1 105 m 1 (assuming no cooperativity). In buffer solutions that were tenfold more dilute, the
affinity increased to micromolar KD values, thus providing
experimental evidence for a certain contribution of polar
interactions. These, however, are most likely complemented
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
by strong hydrophobic forces[15] and possibly even reinforced
by the formation of aggregates.[16] The highest affinity,
however, was found for longer ds DNA with 45 nucleic
bases containing the whole primer (up to 6 1 107 m 1 in 2-[4-(2hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES)
buffer solution (0.1 mm)).[17] Interestingly, the stiff linear
dimer 1 b greatly laged behind the flexible 1 a by two orders of
magnitude; it simply could not adjust to the helical curvature
of the duplex. The relatively small dependence on ionic
strength and the high, variable stoichiometry factor could be
explained by major groove binding, with several dimers filling
up the groove where they engage in salt bridges with the
phosphodiester backbone. The DNA and RNA sequences
used herein are shown in Table 1.
To elucidate the binding mechanism, we measured UV as
well as fluorescence melting curves for various ds DNA
Figure 2. Melting curves of ss DNA and ds DNA before and after
complexation by a large excess of the dimer.
Table 1: DNA and RNA sequences used.[a]
tional hydrogen bond contacts. This
sequence selectivity was indeed established
with fluorescein-labeled (GC)12 and (AT)12
ds Fl-DNA
model DNA, which produced a pronounced
difference in the KD value of one order of
ds Fl-DNA
magnitude (Table 2). In sharp contrast,
(12 bp, 3 mm)
crystal structures for spermine–DNA com5’-[Fl]GGGGGGGGGGGG-3’/5’-CCCCCCCCCCCC-3’
ds Fl-DNA
plexes show that polyamines also bridge the
ds Fl-DNA
minor groove and thus lead to considerable
duplex stabilization.[19] Moreover, most
ds Fl-RNA
binders are known to distort the
from the structure of B6
ds DNA
A-DNA.[20] However,
in CD measurements before and after
[a] Fl = fluorescein.
complexation with a 50-fold excess of the
calixarene dimer, no major conformational
before and after addition of the dimer. In each case, we
changes were indicated in the critical region between 200 and
obtained almost the same melting points, ruling out extensive
270 nm (data not shown).[18]
cross-linking of the complementary DNA strands and interMolecular mechanics calculations of our 12-bp DNA
calation or minor groove insertion.[18] However, the UV
duplex complexed by the flexible ligand 1 a showed intriguing
minimum-energy structures in which the two ammonium
melting curves of ds DNA in the presence of the calixarene
groups of the calixarene units were deeply buried within the
dimers looked drastically different from those of pure DNA;
major groove. Depending on the base sequence, up to six
they were inverted and strongly enhanced with respect to
additional hydrogen bond contacts were established during
their absorption intensity (Figure 2). This is not an effect of
the minimization procedure; they all involve carbonyl oxygen
the calixarene dimer, which produced a straight horizontal
and imino nitrogen atoms of the nucleic bases and NH2 as well
line under identical conditions. Clearly, complexation affects
the nucleic base chromophores and strongly increases UV
as NH3+ groups of the calixarene (Figure 3). Subsequent
absorption for the duplex but
quenches it for the single-strand
Table 2: Association constants between various DNA strands and dimeric receptors 1.
(ss) DNA. This is in perfect agreeEntry Receptor Guest
Ka [m1][d]
ment with an insertion of the
ds Fl-DNA (12 bp)
HEPES (0.1 mm)
2 C 10 ( 20 %)
dimer corpus into the major
ds Fl-DNA (12 bp, 3 mm) HEPES (1 mm)
1 C 10 ( 16 %)
groove accompanied by hydrogen
ds Fl-DNA [(GC)12]
HEPES (1 mm)[b]
1 C 106 ( 7 %)
bonding to the nucleic bases
ds Fl-DNA [(AT)12]
HEPES (1 mm)[b]
1 C 105 ( 9 %)
(Hogsteen sites). As AT pairs
ds Fl-RNA [(AU)12]
HEPES (1 mm)[b]
6 C 106 ( 35 %) 30:1
present a methyl group into the
ds Fl-RNA [(AU)12]
HEPES (100 mm)[b] 8 C 105 ( 31 %) 35:1
ds DNA (45 bp)
HEPES (0.1 mm)[a]
6 C 107 ( 52 %) 30:1[c]
major groove and GC pairs do not,
ds Fl-DNA (12 bp)
HEPES (1 mm)
4 C 103 ( 45 %)
pure (GC)n DNA strands should be
preferred for insertion into the
[a] HEPES buffer in methanol/water (9:1). [b] HEPES buffer in methanol/water (1:1). [c] From best-curve
calixarene dimer owing to addifitting. [d] Statistical error from the curve fitting.
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. 2006, 118, 2769 –2772
According to recent reports, the
DNA recognition efficiency of major
groove binders can also be quantitatively characterized by ethidium bromide displacement assays;[22] hence,
the respective Ka values for 20-mer
ds DNA were calculated by the
competitive method and surpass
106 m 1, even at 100 mm NaCl.[23]
With the hexaaniline calixarene
dimer, we have introduced a large
amphiphilic molecule that selectively
binds ds DNA and RNA with submicromolar KD values in buffered
aqueous solution; contrary to most
other DNA binders, it most likely
Figure 3. a) B-DNA (12 bp, green and blue) with tightly bound anilinocalixarene dimer (red).
targets exclusively the major groove
b) View along the major groove, revealing three hydrogen bond contacts between the ligand NH2/
and causes no conformational
NHþ3 groups and various nucleic bases at the groove floor (MacroModel 7.0, MMFFs, water, 1000
change or (de)stabilization in the
DNA duplex.
We will, in the future, try to investigate in detail the
molecular dynamics calculations demonstrate the remarkable
postulated binding mode by employing linear dichroism,
stability of this arrangement: the overall structure is well
NOESY, and atomic force microscopy techniques. Most
maintained for 100 ps and even keeps five of six hydrogen
importantly, however, an element of sequence selectivity
bonds between DNA and the ligand.
can be introduced into the bridge, in the form of amide, ester,
Major grooves in RNA molecules are typically much more
and thioether groups or amino acid side chains that interact
confined, with a maximum width of about 10 G and a
with the base pairs on the floor of the major groove. Owing to
phosphatephosphate distance of even less. Their profile
(cross section) matches exactly the shape of our dimeric
calixarene binder. If its preferred nucleic acid binding mode is
truly insertion in the major groove, RNA should offer a
superior microenvironment for 1 a through maximizing the
van der Waals interactions. Remarkably, RNA affinities are
60-fold greater than that of DNA binding, and are only
slightly lowered in 100 mm buffer (from 170 nm to 1.3 mm),
indicating a perfect fit inside the major groove of the RNA
(Table 2).[21]
A standard test for efficient DNA binding is the replacement of already intercalated ethidium bromide. At 1 mm,
nonfluorescent 20-bp ds DNA with zero to three built-in
mismatches shows a steady increase in fluorescence emission,
which is in all cases almost completely quenched by an excess
(30 equivalents) of added dimer; its occupance of the major
groove consequently expels the ethidium bromide into the
Figure 4. Ethidium bromide displacement from unlabeled DNA
free solution. Although the values for the concentration at
duplexes (c = 1 mm) by dimer 1 a (H2O/MeOH 1:1, 9 mm or 100 mm
50 % dye displacement (C50) are modest (owing to the high
NaCl, 2 mm HEPES, pH 7.0). Frel = relative fluorescence emission
stoichiometric factor), the corresponding charge excess ratio
intensity; CE = charge excess ratio.
at 50 % dye displacement (CE50) are, even at high salt loads,
among the lowest ever reported for DNA binders, owing to
the relatively low total charge of the calixarene (Figure 4,
Table 3: Ethidium bromide displacement assay for different doublestranded DNAs at different concentrations of NaCl.
Table 3). Interestingly, the short 20-mer DNA strand bound
just as well as the calf thymus DNA (ca. 10 000 bp). Direct
ACHTUNGRE[NaCl] Nominal C50
comparison with spermine revealed again the striking differ[mm]
ence to polyamines: at physiological NaCl concentration, the
1 a + 20 bp (3 mm)
dimer–DNA interaction is hardly weakened, whereas C50 and
1 a + 20 bp (0 mm)
CE50 values for spermine drop by a factor of 300. We
1 a + 20 bp (0 mm)
1 a + calf thymus DNA
therefore conclude that the calixarene dimer does not attack
1 a + calf thymus DNA
the phosphodiester backbone by mere multiple salt bridges,
spermine + calf thymus DNA
but rather inserts into the spacious major groove where it can
spermine + calf thymus DNA 150
tolerate large amounts of competing ions.
Angew. Chem. 2006, 118, 2769 –2772
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
this new binding mode, we expect the dimeric calixarene to
find numerous applications as a sensitive analytical tool in
DNA chemistry with a promising potential to control gene
Received: August 18, 2005
Revised: January 20, 2006
Published online: March 20, 2006
Keywords: calixarenes · DNA · intercalation · major groove ·
supramolecular chemistry
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[9] For permanently charged aminocalixarenes that stabilize ionpairing interactions with ds DNA, see: Y. Shi, H.-J. Schneider, J.
Chem. Soc. Perkin Trans. 2 1999, 1797 – 1803; similar guanidinium calixarenes forming complexes with plasmid DNA: M.
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[13] Courtesy of the Carell group, LMU Munich, Germany.
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[15] A pKa titration of the monomeric parent tetraanilinium calixarene, although hampered by its low solubility, pointed to an
upper limit of pKa 7.0 for the last two deprotonation steps.
Thus, at neutral pH, it seems probable that each calixarene unit
carries no more than one positive charge. This value might be
slightly altered by the considerable methanol content in most
solvent mixtures.
[16] Dilution and variable temperature NMR experiments with 1 a
produced only small chemical shift changes; dynamic light
scattering (D. Joester, M. Losson, R. Pugin, H. Heinzelmann, E.
Walter, H. P. Merkle, F. Diederich, Angew. Chem. 2003, 115,
1524 – 1528; Angew. Chem. Int. Ed. 2003, 42, 1486 – 1490) or
GPC measurements would perhaps identify small oligomeric
aggregates. However, it is also conceivable, that these are only
formed after docking onto the DNA strand.
[17] In these cases, the calixarene dimer was precomplexed with
calcein, forming a quite efficient 2:1 complex (80 000 m 1). This
highly fluorescent reporter molecule was subsequently replaced
by the unlabeled native DNA in a competition experiment
(indicator displacement assay, IDA).
[18] M. H. Hou, S.-B. Lin, J.-M. P. Yuann, W.-C. Lin, A. H.-J. Wang,
L.-S. Kan, Nucleic Acids Res. 2001, 29, 5121 – 5128.
[19] L. W. Tari, A. S. Secco, Nucleic Acids Res. 1995, 23, 2065 – 2073.
[20] L. van Dam, N. Korolev, L. NordenskiLld, Nucleic Acids Res.
2002, 30, 419 – 428.
[21] The minor groove of RNA is much more shallow, but has the
same diameter as its major groove. It cannot, therefore, be ruled
out that the calixarene dimer also binds to this well accessible
cleft; such a recognition mode would also explain the high
observed stoichiometry factor of 30:1.
[22] Y.-H. Shim, P. B. Arimondo, A. Laigle, A. Garbesi, S. Lavielle,
Org. Biomol. Chem. 2004, 2, 915 – 921.
[23] We used the competitive method, with the binding constant for
ethidium bromide KEB = 107 m1 for calf thymus DNA, as
referenced in the literature. The noncompetitive method introduced by Boger et al. seems not to be applicable, because the
intercepts with the x-axis point to large binding site sizes of > 2
(compare the high stoichiometries found in fluorescence titrations): D. L. Boger, B. E. Fink, M. P. Hedrick, J. Am. Chem. Soc.
2000, 122, 6382 – 6394.
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. 2006, 118, 2769 –2772
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