Amplified nitric oxide photorelease in DNA proximity

Article abstract of DOI:10.1039/b800132d

A novel bichromophoric conjugate integrating a NO photodonor and a DNA intercalator in its molecular skeleton, allows the light-controlled NO delivery nearby DNA and amplifies NO release via effective photoinduced energy transfer mechanism. The Royal Society of Chemistry.

Full text of DOI:10.1039/b800132d

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Amplified nitric oxide photorelease in DNA proximityw
Fiorella L. Callari and Salvatore Sortino*
Received (in Cambridge, UK) 4th January 2008, Accepted 21st February 2008
First published as an Advance Article on the web 20th March 2008
DOI: 10.1039/b800132d
A novel bichromophoric conjugate integrating a NO photodonor
and a DNA intercalator in its molecular skeleton, allows the
light-controlled NO delivery nearby DNA and amplifies NO
release via effective photoinduced energy transfer mechanism.
when both the chromophores absorb the excitation light and
(2) the conjugate effectively binds to double strand DNA and
enables NO photodelivery nearby.
Compound 1 was easily synthesized in two steps (see ESIw)
and its spectroscopic features are illustrated in Fig. 2. The
absorption spectrum (a in Fig. 2A), shows bands that reflect
fairly well those of the equimolar mixture (d in Fig. 2A) of the
single components (b and c in Fig. 2A), accounting for only a
weak interaction between the two chromophoric units in the
ground state. On the other hand, the typical fluorescence emis-
sion of the anthracene moiety observed for the model compound
2 (b Fig. 2B) is totally quenched in the case of the conjugate 1 (a
Fig. 2B), suggesting a remarkable interaction in the excited state.
As far as the possible quenching mechanism is concerned, a
thermodynamically favored photoinduced energy transfer
from unit i (donor) to unit p (acceptor) seems to be the most
likely. The bichromophoric conjugate presents two indispen-
sable pre-requisites for this energy transfer to occur, that are:
(1) the lowest excited singlet state of i is ca. 0.5 eV higher than
that of p,9 (2) the emission spectrum of i considerably overlaps
the absorption of p.10 This hypothesis is well-supported by the
NO photorelease experiments reported below, which rules out
feasible photoinduced electron transfers involving the two
chromophoric units of the conjugate as responsible for the
fluorescence quenching.11
Exciting discoveries in biochemical research have demonstrated
that nitric oxide (NO), an ephemeral diatomic molecule, is much
more than a mere environmental pollutant. It is well established
that NO is a pleiotropic bioregulator of important physiological
processes encompassing neurotransmission, vasodilatation and
hormone secretion in living bodies.1 Besides, NO has proven to
be an excellent chain breaking antioxidant in the free radical-
induced lipid oxidation2 and an efficient anticancer agent that
inhibits key metabolic pathways to block growth or kills cells
outright.3 In this regard, DNA seems to be the main target of
NO, which can induce deamination of the nucleobases leading
to strand breaks.4
This scenario has stimulated fervent research activity de-
voted to developing NO delivery compounds for potential
therapeutic applications.5 In this perspective, light is an ap-
pealing external on/off trigger to accurately regulate the NO
dosage. This is crucial for biomedical purposes, in view of the
opposite effects NO may exert either inhibiting or encouraging
the tumor proliferation depending on the dose.6
On the basis of the above considerations, the achievement of
NO donors exhibiting binding affinity for DNA and, at the
same time, generating NO under appropriate light inputs,
would allow the NO delivery to be spatially and temporally
controlled, offering great advantages for bio-applications.
Prompted by our ongoing interest in developing functional
NO photodonors,7 we pursued this objective by designing and
synthesizing the conjugate 1 (Fig. 1). It integrates two chro-
mophoric units in its molecular skeleton: an anthracene
moiety, a typical DNA intercalator binder (unit i), and a
commercial nitroaniline derivative that we have recently dis-
covered to be a suitable NO photodonor under visible light
irradiation (unit p).8 Furthermore, it includes a secondary
amino group which is protonated at physiological pH. The
presence of this cationic charge is expected to improve the
water solubility of 1 and to further encourage its DNA binding
affinity by coulombic attraction with the opposite charged
phosphates backbone. Here, we demonstrate that (1) the NO
release from unit p of the conjugate can be significantly
amplified through a photoinduced energy transfer mechanism
The most convenient methodology to demonstrate the NO
generation is the direct and in real-time monitoring of this
radical species. To this end, we used an ultrasensitive NO
electrode, which directly detects NO concentration by an
amperometric technique (see ESIw). Fig. 3 shows the results
of two experiments carried out by exciting the conjugate 1 with
either 420 or 380 nm light, respectively. These wavelengths
were chosen such that the excitation light is absorbed
´
Dipartimento di Scienze Chimiche, Universita di Catania, Viale
Andrea Doria 8, I-95125 Catania, Italy. E-mail: ssortino@unict.it
w Electronic supplementary information (ESI) available: Synthetic
and experimental procedures. See DOI: 10.1039/b800132d
Fig. 1 The molecular structure of 1 (protonated form) and its
working principle.
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Fig. 2 (A) Absorption spectrum of 1 (a), the model compounds 2 (b)
and 3 (c) and their equimolar mixture 2 + 3 (d). (B) Fluorescence spectra
(lexc=365 nm) of 1 (a) and the model compound 2 (b). T = 25 1C,
phosphate buffer (pH 7.4, 1 mM): MeOH 2 : 1. The fluorescence
intensities have been corrected for the exact fraction of absorbed
photons by the anthracene moieties at the excitation wavelength.
selectively by the NO photodonor unit p in the former case and
by both the chromophoric units, p and i, in comparable
amounts, in the latter (see sketch in Fig. 3 and absorption
spectra in Fig. 2, for sake of clarity). In both experiments we
observed linear photogeneration of NO which promptly
stopped when the light was turned off and restarted as the
illumination was turned on again, obtaining unambiguous
evidences for the exclusively light-controlled generation of
NO from the conjugate 1.
Fig. 4 (A) Absorption spectra of 1 (6 mM) in 1 mM phosphate buffer
pH 7.4, in the presence in increasing amounts of ct-DNA in the range
0–150 mM. Cell path: 1 cm. The inset shows the half-reciprocal plot of
1 binding with ct-DNA determined at 388 nm. (B) Induced circular
dichroism of the 1-ct-DNA complex in 1 mM phosphate buffer pH 7.4;
[ct-DNA]bp = 10 mM; [1] = 6 mM. Cell path: 1 cm.
Despite the similar qualitative behavior, the rate of the NO
released in the two experiments was significantly different,
being ca. 0.3 nM sÀ1 and ca. 0.8 nM sÀ1 at 420 and 380 nm,
respectively. Such a difference cannot be ascribed to trivial
effects caused by a different number of either incident or
absorbed photon by the photodonor unit p at the two wave-
lengths. In fact, (1) care was taken to ensure the same photon
flux in the two experiments and (2) due to the larger extinction
coefficient of 1 at 380 nm, the fraction of absorbed photons by
the unit p at this wavelength (ca. 18%) is comparable to that
absorbed when the same unit is selectively excited at 420 nm
(ca. 20%). Furthermore, control experiments carried out by
irradiating solutions of the model compound 3 absorbing the
same fraction of photons at the two excitation wavelengths
(420 nm or 380 nm), did not show any significant difference in
the rate of the NO photogenerated, suggesting that the
primary photochemical process leading to NO by unit p does
not depend by the excitation energy in the range explored.
Therefore, on the basis of these findings, the higher NO release
observed upon 380 light irradiation can be unequivocally
interpreted on the basis of an amplification effect due to an
effective photoinduced energy transfer from unit i to p, in very
good agreement with the fluorescence quenching results
discussed before.
Interaction of compound 1 with double strand DNA was
proven by means of absorption and induced circular dichroism
(ICD) spectroscopy. Fig. 4A shows that the absorption bands
of 1 undergo both hypochromism and red-shift upon addition
of increasing amounts of calf-thymus DNA (ct-DNA). Be-
sides, a tight isosbestic point at 404 nm is observed, suggesting
homogeneity of the binding process. These spectral changes
are very similar to those typically observed for other anthra-
cene derivatives and are accepted as a confirmation of proxi-
mity of the polycyclic hydrocarbon to the DNA core.12
Hypochromism is in fact due to a strong interaction between
the electronic states of the intercalated chromophore and those
of the DNA bases.13
The binding constant, Kb, related to association complex
was calculated through the half-reciprocal plot of the absorp-
tion spectral changes, according to eqn (1):14
Fig. 3 NO released upon irradiation of 1 (10 mM) at 420 (a) and 380
nm (b). T = 25 1C, phosphate buffer (1 mM, pH 7.4): MeOH 99 : 1.
[ct-DNA]bp/(eAÀeF)=[ct-DNA]bp/(eBÀeF)+1/Kb(eBÀeF) (1)
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together with the ease of preparation, make the conjugate 1 an
appealing candidate to be tested in biological experiments.
Studies addressed to evaluate its capability to induce DNA
photocleavage are underway. We believe that the extension of
the present approach to the preparation of multicharged
conjugates integrating more intercalator and photodonor
units in their molecular skeleton may hopefully pave the way
for the development of novel classes of NO donors for
potential applications in biomedical research where NO re-
lease with precise spatiotemporal control is required.
This work was supported by University of Catania (Progetti
di Ateneo).
Fig. 5 NO released upon 420 nm light irradiation of the complex
Notes and references
1-ct-DNA phosphate buffer (1 mM, pH 7.4) at 25 1C. [ct-DNA]bp
10 mM; [1] = 6 mM.
=
1 (a) L. J. Ignarro, Nitric Oxide Biology and Pathobiology, 1st edn,
Academic Press, San Diego, CA, 2000, p. 41; (b) E. Culotta and D.
E. Koshland, Science, 1992, 258, 1862; (c) F. Murad, Angew.
Chem., Int. Ed., 1999, 38, 1857; (d) R. F. Furchgott, Angew. Chem.,
Int. Ed., 1999, 38, 1870.
where [ct-DNA]bp is the concentration of the polynucleotide in
base pairs, eA, eF and eB correspond to Aobs/[1], the extinction
coefficient for the free 1 and the extinction coefficient for the
totally bound form of 1, respectively.14 By the ratio of the
slope to intercept of the linear plot reported in the inset of
2 G. Hummel, A. J. Fisher, S. M. Martin, F. Q. Schafer and G. R.
Buettner, Free Radical Biol. Med., 2006, 40, 501.
3 (a) W. Xu, L. Z. Liu, M. Loizidou, M. Ahmed and I. G. Charles,
Cell. Res., 2002, 12, 311; (b) B. Mitrovic, L. J. Ignarro, H. V.
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Cantauria, S. Tarasia, L. Sharp, P. G. Braunschweiger and P. G.
Wang, Bioorg. Med. Chem. Lett., 1999, 9, 2255.
Fig. 4A, a Kb value of 1.1 Æ 0.3 Â 104
M
À1, in excellent
agreement with those reported for anthracene derivatives
including cationic functions,12 was obtained.
More insights into the binding mode were provided by ICD
spectroscopy. Due to the absence of chiral centers, compound
1 is not optically active by itself. As shown in Fig. 4B the
presence of ct-DNA induces optical activity as a consequence
of its close proximity with the asymmetric environment of the
biopolymer helix. In particular, it can be noted that the ICD
absorption maxima correspond fairly well to those of the
anthracene unit of 1 whereas no relevant ICD signals are
noted in the correspondence of the main absorption region of
the photodonor unit of 1 (dominant above 380 nm). Since ICD
signals are dependent on the cube of the distance of the two
interacting partners15 our findings seem to be consistent with a
binding fashion involving the intercalation of unit i of the
conjugate and the localization of the unit p mainly at the DNA
periphery, as pictorially sketched in Fig. 1.
4 (a) J. B. Hibbs Jr, R. R. Taintor and Z. Vavrin, Science, 1987, 235,
473; (b) T. deRojas-Walker, S. Tamir, H. Ji, J. S. Wishnok and S.
R. Tannenbaum, Chem. Res. Toxicol., 1995, 8, 473; (c) K. Fuku-
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5 P. G. Wang, M. Xian, X. Tang, X. Wu, Z. Wen, T. Cai and A. J.
Janczuk, Chem. Rev., 2002, 102, 1091.
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7 (a) S. Sortino, S. Petralia, G. Compagnini, S. Conoci and G.
Condorelli, Angew. Chem., Int. Ed., 2002, 41, 1914; (b) E. B.
Caruso, S. Petralia, S. Conoci, S. Giuffrida and S. Sortino, J.
Am. Chem. Soc., 2007, 129, 480; (c) E. B. Caruso, E. Cicciarella
and S. Sortino, Chem. Commun., 2007, 5028; (d) S. Conoci, S.
Petralia and S. Sortino, US Pat. Appl., pending.
8 The photochemical mechanism for NO photorelease involves a
nitro-to-nitrite rearrangement followed by the rupture of the O–N
bond to generate a phenoxyl radical and NO.
9 Estimated by the end of the absorption spectra of the model
compounds 2 and 3.
The suitability of 1 to generate NO when bound to ct-DNA
was demonstrated by NO photorelease experiments carried
out in the presence of 10 mM ct-DNA (1 complexation 4
90%). As shown in Fig. 5, NO photogeneration strictly
depending on the illumination conditions was observed. A
remarkable point of interest of this work is the comparable
efficiency of the NO photodelivery in the absence and in the
presence of DNA. This excludes potential photoinduced elec-
tron transfer between the excited 1 and the DNA bases as
competitive pathways to the NO generation.
10 The overlap integral J was numerically calculated to be 4.68 Â
10À11 MÀ1 cm3.
11 A potential photoinduced electron transfer involving the excited
anthracene and the adjacent secondary amino group as electron
donor is, of course, out of question in light of the protonation of
this latter under our experimental conditions (pKa 4 8).
12 See, for example: (a) C. V. Kumar and E. H. Asuncion, J. Am.
Chem. Soc., 1993, 115, 8547; (b) C. V. Kumar and E. H. Asuncion,
Chem. Commun., 1999, 1219.
13 C. V. Kumar, in Photochemistry in Organized and Constrained
Media, ed. V. Ramamurthy, VCH Publisher, New York, 1991, pp.
785–816.
14 A. M. Pyle, J. P. Rehmann, R. Meshoyer, C. V. Kumar, N. J.
Turro and J. K. Barton, J. Am. Chem. Soc., 1989, 111, 3051.
15 E. C. Long and J. K. Barton, Acc. Chem. Res., 1990, 23, 271.
16 An example of NO photodonor incorporating a potential DNA
intercalator in its molecular skeleton has been recently reported by
Miyata and colleagues.4c No investigations demonstrating affinity
for DNA and ability to generate NO nearby were reported,
however.
In summary, we report a photoactivated, multifunctional
conjugate that, to our knowledge, represents the first example
of an NO donor that effectively bind to DNA and enables
generation of this radical species in its close proximity, under
the exclusive control of visible light stimuli, with the additional
advantage of amplifying the NO delivery via an efficient
photoinduced energy transfer mechanism.16 These features,
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This journal is The Royal Society of Chemistry 2008
Chem. Commun., 2008, 1971–1973 | 1973