Bis-4-aminobenzamidines: Versatile, fluorogenic A/T-selective dsDNA binders

Article abstract of DOI:10.1021/ol902501j

(Figure presented) N¹, N³-Bls(4-amldlnophenyl) propane-1, 3-dlamlne (BAPPA, R = H) Is a blsamlnobenzamldlne fluorogenlc derivative that displays a large Increase In Its emission fluorescence when bound to the minor groove of specific A/T DNA sites (K_d for an AATT site ～ 79 ± 6 nM). Moreover, the structural characteristics of BAPPA allow the easy Introduction of functional groups that protrude out of the DNA surface.

Full text of DOI:10.1021/ol902501j

ORGANIC
LETTERS
2010
Vol. 12, No. 2
216-219
Bis-4-aminobenzamidines: Versatile,
Fluorogenic A/T-Selective dsDNA
Binders§
Olalla Va´zquez,† Mateo I. Sa´nchez,† Jose´ Mart´ınez-Costas,‡
M. Eugenio Va´zquez,*,† and Jose´ L. Mascaren˜as*,†
Departamento de Qu´ımica Orga´nica and Departamento de Bioqu´ımica y
Biolog´ıa Molecular, UniVersidade de Santiago de Compostela,
15782 Santiago de Compostela, Spain
joseluis.mascarenas@usc.es
Received October 30, 2009
ABSTRACT
N1,N3-Bis(4-amidinophenyl)propane-1,3-diamine (BAPPA, R ) H) is a bisaminobenzamidine fluorogenic derivative that displays a large increase
in its emission fluorescence when bound to the minor groove of specific A/T DNA sites (Kd for an AATT site ∼ 79 ( 6 nM). Moreover, the
structural characteristics of BAPPA allow the easy introduction of functional groups that protrude out of the DNA surface.
Deciphering the human genome has opened new perspectives
in biomedical research, promising improved diagnostic
techniques and personalized therapies.1 However, to fully
exploit the knowledge about our genes, it is necessary to
develop probes capable of targeting and sensing specific
DNA sequences. While a number of sequence identification
methods that rely on single-stranded hybridization have been
successfully implemented, direct sensing of double-stranded
DNA (dsDNA) without the need for denaturation is clearly
underdeveloped.2 Of the few strategies so far described, those
based on distamycin-like hairpin polyamides are particularly
remarkable.3 However, adapting these systems for direct
dsDNA sensing is not trivial, owing to synthetic requirements
and cell internalization difficulties.4 Therefore, new sequence-
specific dsDNA probes based on simple, versatile, and readily
accessible structural scaffolds are highly desirable.
Benzamidines such as pentamidine (1) and propamidine
(2) (Figure 1) are very simple DNA binders that show good
(3) Polyamides with appended dyes: (a) Rucker, V. C.; Foister, S.;
Melander, C.; Dervan, P. B. J. Am. Chem. Soc. 2003, 125, 1195–1202. (b)
Fechter, E. J.; Olenyuk, B.; Dervan, P. B. J. Am. Chem. Soc. 2005, 127,
16685–16691. For a polyamide with an integrated fluorophore, see: (c)
Chenoweth, D. M.; Viger, A.; Dervan, P. B. J. Am. Chem. Soc. 2007, 129,
2216–2217.
§ Dedicated to Prof. Luis Castedo on the occasion of his 70th birthday.
† Departamento de Qu´ımica Orga´nica.
‡ Departamento de Bioqu´ımica y Biolog´ıa Molecular.
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Voelkerding, K. V.; Dames, S. A.; Durtschi, J. D. Clin. Chem. 2009, 55,
641–658.
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Natl. Acad. Sci. U.S.A. 2003, 100, 12063–12068. (b) Va´zquez, O.; Blanco-
Canosa, J. B.; Va´zquez, M. E.; Mart´ınez-Costas, J.; Castedo, L.; Mascaren˜as,
J. L. ChemBioChem 2008, 9, 2822–2829.
(2) (a) Ghosh, I.; Stains, C. I.; Ooi, A. T.; Segal, D. J. Mol. Biosyst.
2006, 2, 551–560. (b) Chan, E. Y.; Goncalvez, N. M.; Haeusler, R. A.;
Hatch, A. J.; Larson, J. W.; Maletta, A. M.; Yantz, G. R.; Carstea, E. D.;
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10.1021/ol902501j  2010 American Chemical Society
Published on Web 12/10/2009

tripyrrole component used in previously developed bivalent
minor-major groove DNA recognition systems.9 We focused
our attention on bisamidine systems owing to their structural
simplicity and potential for installing appropriate handles for
conjugation to the peptides.10
Although the electronic donor-acceptor configuration in
the bis-4-amidinophenoxy units in 2 might provide for the
observation of DNA-promoted fluorescence changes, the
overlap between the excitation wavelength of 2 (260 nm)
and the UV absorption of the DNA precludes its selective
excitation in the presence of DNA. Therefore, we decided
to prepare the aza-analogue of propamidine, namely, N1,N3-
bis(4-amidinophenyl)propane-1,3-diamine (BAPPA, 3), fea-
turing nitrogens instead of the two oxygen atoms present in
the parent propamidine (2). We anticipated that the increased
donating character of the amines would yield a push-pull
system with longer excitation and emission wavelengths than
2, together with a higher environment-sensitive fluoroge-
nicity.11 The synthesis of 2 and 3 was carried out in a
straightforward manner using a slightly modified version of
known procedures (Scheme 1).12
Figure 1. Pentamidine (1), propamidine (2), and BAPPA (3).
stability, A/T sequence selectivity, and excellent cell transport
properties in a variety of cell lines.5 Pentamidine and other
derivatives are either in use or in clinical trials against several
parasites like P. ViVax, P. carinii pneumonia, or P. falciparum
malaria,6 and there is great interest in developing new
analogues with improved efficacies and reduced toxicity.
Curiously, most of the DNA-binding studies of such ben-
zamidines are based on measurements of the thermal
denaturation of their DNA complexes, a technique that only
gives indirect and relatively coarse information of binding
properties and is difficult to implement in high-throughput
screenings.7 Therefore, the development of simple and direct
spectroscopic methods that allow a rapid monitoring and
quantification of the DNA binding affinity and selectivity
of these compounds would be highly desirable.8
Scheme 1. Synthesis of Propamidine (2) and BAPPA (3)
Herein, we demonstrate that replacing the bis-4-amidi-
nophenoxy unit found in classic benzamidine derivatives by
a bis-4-aminobenzamidine yields propamidine analogues that
display a large fluorescence enhancement upon sequence-
specific dsDNA binding. This provides for a simple, rapid,
and reliable method to monitor and quantify their DNA
affinity and selectivity, as well as to study the binding of
other nonfluorescent minor-groove binders.8 Importantly, in
contrast to classic nuclear stains such DAPI or Hoechst, these
analogues can be very easily assembled, and their structure
allows the straightforward introduction of functional groups
oriented toward the outer side of the DNA minor groove.
This research aroused in the context of our search for a
structurally simple and easily accessible substitute for the
As expected, propamidine analogue 3 (BAPPA) shows
longer maximum excitation (329 nm) and emission (387 nm)
wavelengths than propamidine (2). Moreover, the fluores-
cence emission intensity of BAPPA displays strong sensitiv-
ity to the solvent polarity, increasing from being almost
nonfluorescent in water to displaying strong emission in
MeOH or iPrOH. Interestingly, BAPPA displays weaker
fluorescence in nonpolar solvents, and in some of them like
(6) (a) Wilson, W. D.; Tanious, F. A.; Mathis, A.; Tevis, D.; Hall, J. E.;
Boykin, D. W. Biochimie 2008, 90, 999–1014. (b) Rodr´ıguez, F.; Rozas,
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(8) For examples of light-off fluorescence displacement assays for
analyzing minor groove binding, see: (a) Tse, W. C.; Boger, D. L. Acc.
Chem. Res. 2004, 37, 61–69. (b) Boger, D. L.; Fink, B. E.; Brunette, S. R.;
Tse, W. C.; Hedrick, M. P. J. Am. Chem. Soc. 2001, 123, 5878–5891. (c)
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Jenkins, T. C.; Neidle, S. Biochemistry 1993, 32, 13838–13843. (c) Edwards,
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(11) For spectroscopic effects in push-pull conjugated systems: (a)
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Mascaren˜as, J. L. Angew. Chem., Int. Ed. 2007, 46, 6886–6890. (b) Blanco,
J. B.; Dodero, V. I.; Va´zquez, M. E.; Mosquera, M.; Castedo, L.;
Mascaren˜as, J. L. Angew. Chem., Int. Ed. 2006, 45, 8210–8214. (c) Blanco,
J. B.; Va´zquez, O.; Mart´ınez-Costas, J.; Castedo, L.; Mascaren˜as, J. L.
Chem.sEur. J. 2005, 11, 4171–4178. (d) Blanco, J. B.; Va´zquez, M. E.;
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713–722. (e) Va´zquez, M. E.; Caaman˜o, A. M.; Mart´ınez-Costas, J.; Castedo,
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Org. Lett., Vol. 12, No. 2, 2010
217

THF or 1,4-dioxane we also observed a dual emission profile
(see the Supporting Information).13
More importantly, addition of a hairpin oligonucleotide
containing the consensus binding region AATTT promoted
a large fluorescence emission increase (>10-fold), probably
as a consequence of the change in hydration and viscosity
experienced by the probe when removed from the bulk water
into the DNA minor groove.14 In a titration experiment,
addition of successive aliquots of such a hairpin oligonucle-
otide to a 0.5 µM solution of BAPPA in buffer led to the
fluorescence profile represented in Figure 2. The increase in
Figure 3. Titrations of BAPPA with selected DNA hairpin
oligonucleotides containing the sequences: AATTT (b); AATT (O);
AAAA ((); AATG (]); AAGG (1); ATGG (∆); and GGCC (2).
Binding curves represent the best fit to a 1:1 binding mode
considering the contribution of the dsDNA to the fluorescence
emission. Emission monitored at 387 nm.
sites in the minor groove, although in such cases the target
sequences incorporate GC base pairs.17
Table 1. Sequence Selectivity of BAPPA from Titrations with
Selected dsDNAsa
Figure 2. Titration of BAPPA (0.5 µM in Tris-HCl buffer 20 mM,
pH 7.5, 100 mM NaCl) with the hairpin oligonucleotide 5′-GGCG
AATTT CGC TTTTT GCG AAATT CGCC-3′.
c
dsDNA siteb
KD
GGCC
ATGG
AAGG
AATG
AAAA
AATT
AATTT
∼16 µMd
6.6 ( 0.5 µM
5.7 ( 1.3 µM
1.1 ( 0.3 µM
85 ( 12 nM
79 ( 6 nM
the emission intensity at 387 nm could be fitted to a simple
1:1 binding mode.15 The observed KD of ∼70 nM is in
agreement with previously reported binding constants for
propamidine and related minor groove binders.16
BAPPA
70 ( 10 nM
a All titrations were performed by addition of successive aliquots of
the DNA stock solutions to a 0.5 µM solution of BAPPA (20 mM Tris-
HCl buffer, 100 mM NaCl, pH 7.5). b See Supporting Information for full
hairpin oligonucleotide sequences. c KD values are the average of at least
three independent titrations for each oligonucleotide DNA. d Saturation was
not reached with this DNA even at high concentrations.
Likewise, titrations of BAPPA with different hairpin
oligonucleotides with other sequences (Figure 3) allowed us
to establish the binding site profile (Table 1). As expected,
the preferred sequences were those containing at least four
A/T base pairs, with AATT being slightly better than AAAA.
Introduction of a single G/C mutation in the sequence led to
a 15-fold decrease in affinity (KD ∼ 1.1 ( 0.3 µM). Further
mutations of the preferred A/T-rich site drastically reduce
the binding affinity, with G/C-rich sequences showing only
residual binding to BAPPA.
Curiously, when using a dsDNA featuring a longer binding
site with 6 base pairs in length (AAATTT), the binding
isotherm was better represented assuming that the dsDNA
could accommodate two BAPPA molecules in the AAATTT
site. This result recalls a recent report that describes
simultaneous binding of other bisamidines in overlapping
The structural simplicity and synthetic accessibility of
BAPPA allows a straightforward preparation of analogues
featuring functional group handles in the propyl tether.
Therefore, the BAPPA derivative 4, which incorporates the
hydroxy group in the middle carbon, was very easily prepared
as shown in Scheme 2. Binding assays revealed a slight loss
in affinity with respect to BAPPA, for the consensus AATTT
site (KD ∼ 220 nM, see the Supporting Information).
The BAPPA dsDNA-induced fluorescence can also be
used to study the DNA binding profile of other A/T selective,
nonfluorescent, minor groove binders, by means of competi-
tive displacements. In a typical experiment, a solution of
BAPPA and the hairpin oligonucleotide containing the
(13) Da¨hne, S.; Freyer, W.; Teuchner, K.; Dobkowski, J.; Grabowski,
Z. R. J. Lumin. 1980, 22, 37–49.
(14) (a) Nguyen, B.; Neidle, S.; Wilson, W. D. Acc. Chem. Res. 2009,
42, 11–21. (b) Degtyareva, N. N.; Wallace, B. D.; Bryant, A. R.; Loo, K. M.;
Petti, J. T. Biophys. J. 2007, 92, 959–965.
(15) For data treatment, details, and derivation of the mathematical
expressions, see the Supporting Information.
(17) Munde, M.; Ismail, M. A.; Arafa, R.; Peixoto, P.; Collar, C. J.;
Liu, Y.; Hu, L.; David-Cordonnier, M.-H.; Lansiaux, A.; Bailly, C.; Boykin,
D. W.; Wilson, W. D. J. Am. Chem. Soc. 2007, 129, 13732–13743.
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218
Org. Lett., Vol. 12, No. 2, 2010

Scheme 2. Synthesis of Nucleophilic BAPPA 4
Figure 4. Displacement curves of 0.5 µM BAPPA and 1.4 µM of
selected DNA sequence was titrated with increased amounts
of the nonfluorescent minor groove binder. The fluorescence
emission spectra were recorded after each addition, and the
reduction in fluorescence resulting from the increased
displacement of the fluorescent BAPPA from the minor
groove was used to obtain the binding constant of the DNA
binder.18
We routinely used concentrations of 0.5 µM BAPPA and
1.4 µM dsDNA in these experiments and the concentration
of the external binder up to 150-500 µM, depending on its
relative binding affinity for the DNA. While propamidine
itself shows an affinity for the AATTT site similar to that of
BAPPA, introduction of an amino group in the middle of
the alkyl chain linker (analogue 5, Figure 4) led to a slight
decrease in DNA binding affinity. Interestingly, replacing
the amidinium group in BAPPA with a guanidinium group
also induced a near 10-fold decrease in affinity (analogue 6,
Figure 4).
hairpin oligonucleotide containing the AATTT site with: propamidine
2 (b, KD ) 161 ( 22 nM), aminopropamidine 5 (O, KD ) 452 (
7 nM), and guanidine analogue 6 (9, KD ) 479 ( 7 nM). Emission
monitored at 387 nm.
sites, displaying a remarkable fluorescence emission en-
hancement upon binding to the minor groove of such
sequences. This intrinsic DNA-dependent fluorogenicity,
together with the good cell translocation properties, suggests
that BAPPA and its derivatives might offer great potential
for the future development of dsDNA-dependent chemical,
optical, and biological applications.
Acknowledgment. This work is dedicated to Prof. Luis
Castedo on the occasion of his 70th birthday. We thank
the support given by the Spanish grants SAF2007-61015,
SAF2006-06868, CTQ2006-01339, Consolider Ingenio
2010 CSD2007-00006, and the Xunta de Galicia PGIDI-
T06PXIB209018PR, GRC2006/132, and PGIDIT08CSA-
047209PR. O.V. and M.I.S. thank the Spanish MICINN
for their PhD fellowships. M.E.V. also thanks the Human
Frontier Science Program (CDA0032/2005-C) and the
Spanish MICINN for his Ramon y Cajal contract.
We have also carried out preliminary tests of the in vivo
internalization and fluorescence emission properties of the
conjugation friendly analogue 4, using HeLa cells. We were
able to observe reasonable bright fluorescence emission
concentrated in the cell nuclei and clearly differentiated from
the autofluorescence background (see the Supporting Infor-
mation for details).
Supporting Information Available: Synthesis, charac-
terization, DNA binding, cell uptake experiments, and
spectroscopic data. This material is available free of charge
via the Internet at http://pubs.acs.org.
In summary, aza-propamidine BAPPA is a structurally
simple and efficient fluorescent probe for A/T-rich dsDNA
(18) For competitive displacement analysis, see: (a) Roehrl, M. H. A.;
Wang, J. Y.; Wagner, G. Biochemistry 2004, 43, 16056–16066. (b) Wang,
Z.-X. FEBS Lett. 1995, 360, 111–114.
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