A "dual-function" photocage releasing nitric oxide and an anthrylmethyl cation with a single wavelength light

Article abstract of DOI:10.1002/chem.200901037

A dual function photocage able to associate the generation of an anthrylmethyl carbocation to the release of NO, under the control of a low energy single wavelength light, was reported. The O-alkylation of cupferron (CP) was accomplished by designing and

Full text of DOI:10.1002/chem.200901037

DOI: 10.1002/chem.200901037
A “Dual-Function” Photocage Releasing Nitric Oxide and an Anthrylmethyl
Cation with a ACTHUNRTGNEUNGSingle Wavelength Light
Elisa Vittorino, Enzo Cicciarella, and Salvatore Sortino*[a]
The design and development of new anticancer agents is
species in the desired bio-environment with accurate control
one of the most challenging enterprises of the modern scien-
tific research. Cancer chemotherapy mainly relies on the use
of drugs which interact with DNA, inducing modification
via different mechanisms including electron transfer, hydro-
gen-atom abstraction and generation of diffusible intermedi-
ates.[1]
in space and time.
On these grounds, the development of compounds able to
combine generation of NO with carbocation intermediates
under the control of light stimuli can be a significant step
forward in the development of smart “dual-function” anti-
cancer drugs. Such compounds aim to exploit either additive
or synergistic effects arising from the generation of two anti-
cancer species in the same region of space with the final
goal to maximize the efficiency of the treatment and, in
principle, might be activated in vivo by laser sources cou-
pled with fiber optics.
Motivated by our ongoing interest in developing photoac-
tivable NO-releasing systems,[9] we report herein an intrigu-
ing example of a dual function photocage able to associate
the generation of an anthrylmethyl carbocation to the re-
lease of NO, under the control of a low energy single wave-
length light. The design of this photocage was inspired by an
excellent work of Wang and co-workers[10] on a series of
substituted derivatives of cupferron (CP), a spontaneous
NO donor at room temperature[11] (Scheme 1a).[12] They
showed that O-alkylation of CP leads to thermostable O-
alkyl derivatives, which release NO only under light irradia-
tion (Scheme 1b). However, since all these derivatives ex-
hibit absorption in the extreme UV region, their practical
use is strongly limited by the very high excitation energy
(lexc =254 nm) required for the photolysis, which is not
suited for bio-applications because absorbed by the biologi-
cal environment. Furthermore, no mechanistic insights con-
cerning the photoactivated NO release were provided.
To accomplish our goals we sought to harness the O-alky-
lation of CP by designing and synthesizing compound 1
(Scheme 1c).
In recent years, nitric oxide (NO) has come to the lime-
light not only to be a pleiotropic bioregulator of key physio-
logical processes in living bodies,[2] but also for its promising
anticancer activity.[3] In this regard, DNA has been labeled
as the main target of NO, which can induce deamination of
the nucleobases producing abasic sites and, eventually, lead-
ing to strand breaks.[4] Besides, recent developments in drug
design witness an upsurge of interest in DNA-alkylating
agents as potential anticancer compounds.[5] In these cases,
the antineoplastic activity is mainly the result of the reaction
between carbocation intermediates with the 2-amino group
of guanine residues in DNA.[6] An important point to be
outlined is that both NO and carbocation species can act as
a double-edge sword either inhibiting or encouraging the
tumor proliferation depending on the dose.[7] Therefore, rap-
idity of delivery and precise spatiotemporal control of these
transient species are fundamental criteria that would be
meet in view of therapeutic applications. Using light as ex-
ternal trigger provides the great opportunity to satisfy both
the above requisites in the case of “caged molecules”,[8] with
the additional advantage of not affecting important physio-
logical parameters such as pH, temperature and ionic
strength. In fact, the fast response of the photochemical re-
actions associated to their instantaneous initiation/stopping
depending on the presence or not of the illumination, repre-
sent a powerful tool for the rapid introduction of bioactive
Due to the presence of the anthracene chromogenic
center, 1 offers the possibility to shift the excitation energy
well above 300 nm, indispensable prerequisite in the context
of bio-applications. We demonstrate that excitation of 1 with
[a] Dr. E. Vittorino, Dr. E. Cicciarella, Prof. S. Sortino
Dipartimento di Scienze Chimiche, Universitꢀ di Catania
Viale Andrea Doria 6, 95125 Catania (Italy)
E-mail: ssortino@unict.it
ꢀ
390 nm light induces heterolytic cleavage of C O bond lead-
ing to the formation of the 9-anthrylmethyl carbocation,
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COMMUNICATION
Scheme 1.
able to react with nucleosides, and to the release of NO as a
consequence of the CP decaging (Scheme 1c).
Compound 1 was easily synthesized in one step. Its irradi-
ation with low energy light excitation in a mixture phos-
phate buffer (10 mm, pH 7.4)/CH3OH (1:1 v/v) leads to a
satisfactory photodegradation with a quantum yield Fffi0.05.
Laser flash photolysis is a powerful tool to directly demon-
strate the involvement of the anthrylmethyl carbocation as
Figure 1. a) Transient absorption spectrum recorded 0.02 ms after 355 nm
laser excitation of a phosphate buffer (10 mm, pH 7.4)/MeOH (1:1 v/v)
solution of 1 (60 mm). The inset shows the top DA at 420 nmnm as a func-
tion of the laser intensity. b) Decay profile observed at 420 nm and relat-
ed first-order fitting. The inset shows the plot of kobs as a function of the
concentration of guanosine.
the key intermediate in the photodecomposition. ACTHNUGRTNEUNGFigure 1a
shows the spectrum observed upon 355 nm laser excitation
of a solution of 1. This transient is generated via a mono-
photonic mechanism (see inset Figure 1a) and exhibits a
maximum at 420 nm, a shoulder at shorter wavelengths and
an unstructured weaker absorption extending up to about
500 nm. Its decay was first-order with rate constant k0ffi1ꢂ
104 sꢀ1 (Figure 1b), unaffected by oxygen, and accelerated
by azide, an excellent scavenger of cations. According to ex-
tensive literature data on arylmethyl carbocation deriva-
tives,[13] these spectral and kinetic characteristics suggest the
assignment of this transient spectrum to the 9-anthrylmethyl
carbocation. It is worthy to note that the cation is complete-
ly formed within the 6 ns laser pulse, which means that its
formation rate is >1.6ꢂ108 sꢀ1. The potential of this transi-
ent species to react with the guanine moiety was tested by
quenching experiments carried out in the presence of in-
creasing amounts of guanosine. The slope of the straight line
of the plot kobs=k0 +kq[guanosine], afforded a bimolecular
quenching constant kqffi4ꢂ107 mꢀ1 sꢀ1 (inset Figure 1b), in
good agreement with values reported for similar carboca-
tions with amine derivatives.[13e]
toring of this radical species. To this end, we used an ultra-
sensitive NO electrode, which directly detects NO concen-
tration by an amperometric technique. The results illustrat-
ed in Figure 2 provide clear evidence that compound 1 is
stable to the release of NO in the dark. In contrast, light ir-
radiation induces constant generation of NO after a brief in-
itiation period, in according to the initial photodecaging of
CP which is the actual precursor of NO.
To get more insights into the mechanism responsible for
the photodecomposition of 1 we combined steady-state and
time-resolved experiments by using suitable model com-
pounds. The absorption spectrum of 1 in the 300–450 nm
does not show any significant difference with respect to that
of 9-anthracene methanol, chosen as a model compound,
suggesting negligible mutual interactions between the an-
thracene and the CP moieties in the ground state
(Figure 3a). In contrast, the fluorescence quantum yield of 1
is more than two orders of magnitude smaller than that of
the model compound (Figure 3b).
Photogeneration of the anthrylmethyl carbocation implies
the consequent release of the CP counterpart, which is ex-
pected to rapidly generate NO at room temperature. The
most convenient methodology to unambiguously demon-
strate the NO generation is the direct and in real-time moni-
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S. Sortino et al.
thracene derivatives.[15] This proposal is supported by the
large change in free energy calculated for this process,
DGCTffiꢀ1.1 eV[16], which is strongly corroborated by the in-
termolecular quenching experiments carried out by using
anthracene and the O-methyl derivative of CP (2) as model
compounds (Scheme 2).
Figure 2. NO release observed upon irradiation of a N2-saturated phos-
phate buffer (10 mm, pH 7.4)/MeOH (1:1 v/v) solution of 1 (60 mm) with
390 nm light excitation.
Scheme 2. Schematic illustration of the singlet-exciplex intermediate de-
rived from 1 (left) and the model compounds used for the intermolecular
quenching experiments (right).
As shown in Figure 4a, the fluorescence of anthracene is
effectively quenched by 2 according to the Stern–Volmer
equation, I0/I=1+kqt[2] where I0 and I are the fluorescence
intensities of anthracene in the absence and in the presence
of 2, respectively, kq is the bimolecular quenching constant
and t is the singlet lifetime of the anthracene. From the
slope of the linear plot and the value of t (ca. 4 ns), a value
for kq ffi6ꢂ109 mꢀ1 sꢀ1 was determined at room temperature.
Laser flash photolysis experiments provide a clear proof
that the quenching process occurs through an electron trans-
fer mechanism. In fact, as shown in Figure 4b, 355 nm laser
excitation of a solution of anthracene in the presence of 2
0.06m (>99% quenching) originates the typical transient
absorption of the anthracene radical cation characterized by
its diagnostic absorption with maxima at 710 and 420 nm.[17]
In summary, we have designed and synthesized a novel
dual function photocage that, to the best of our knowledge,
represents the first example of molecular system able to re-
lease NO and a carbocation species upon absorption of a
single wavelength light. We have demonstrated that the pho-
todecaging process is most likely triggered by a fast intramo-
lecular charge transfer and that the anthrylmethyl carbocat-
ion generated is able to react with guanosine, an essential
requisite in view of its anticancer properties. This photocage
satisfies a number of key criteria for potential biological ap-
plications including stability in the dark, satisfactory photo-
chemistry, adequate absorption at wavelengths longer than
300 nm and a high decaging rate constant. Finally, it is im-
portant to point out that since the antineoplastic activity of
both NO and carbocations comes from the direct reaction of
these species with DNA (see above), the presence a well-
known DNA intercalator such as the anthracene moiety into
the structure of the photocage offers, in principle, the addi-
tional advantage to generate these anticancer species in the
proximity of their main target. Study addressed to shed light
into the binding affinity of 1 with DNA and to evaluate the
Figure 3. a) Absorption and b) fluorescence emission spectra (lexc
365 nm) of 1 (a,c) and 9-anthracene methanol (b,d) in phosphate buffer
(10 mm, pH 7.4)/MeOH (1:1 v/v).
=
Since the lowest excited singlet state of the anthracene
chromophore (that is selectively excited) lies about 0.8 eV
below that of the CP unit,[14] a photoinduced energy transfer
between these two units is clearly endoergonic and thus,
cannot be responsible for the drastic fluorescence quenching
observed. This suggests the involvement of a non-emissive
intramolecular exciplex with some charge-transfer (CT)
ꢀ
character as a precursor for the heterolytic C O bond cleav-
age (Scheme 2) according to what observed for other an-
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Nitric Oxide
COMMUNICATION
liquid chromatography (HPLC) by using a LiChroCart RP-18 column
(5 mm packing, 4ꢂ250 mm; Hewlett–Packard) and eluting with CH3CN/
water (3:2 v/v). The quantum yield for the photodecomposition of 1 was
determined by using the following equation:
F ¼ ꢀD½1ŠV=DtI0F
where D[1]/Dt is the rate of disappearance of 1, V is the volume of the ir-
radiated solution, I0 is the intensity of the incident photons and F is the
fraction of the photons absorbed by 1 at the excitation wavelength.
Laser flash photolysis: The samples were excited with the third harmonic
of a Nd-YAG Continuum Surelite II-10 laser system (pulse width 6 ns
FWHM, at l=355 nm) and the excited solutions were analyzed at a right
angle geometry using a mini mLFP-111 apparatus developed by Luzchem
Research. Briefly, the monitoring beam was supplied by a ceramic xenon
lamp and delivered through quartz fiber optical cables. The laser pulse
was probed by fiber that synchronized the mLFP system with a Tektronix
TDS 3032 digitizer operating in the pre-trigger mode. The signals from a
compact Hamamatsu photomultiplier were initially captured by the digi-
tizer and then transferred to a personal computer that controlled the ex-
periment with Luzchem software developed in the LabView 5.1 environ-
ment from National Instruments. The energy of the laser pulse was mea-
sured at each laser shot by a SPHD25 Scientech pyroelectric energy mon-
itor. Oxygen was removed by vigorously bubbling the solutions with a
constant flux of argon previously passed through a water trap. The solu-
tion (in a flow cell of 1 cm pathlength) was renewed after each laser shot.
The sample temperature was 295Æ2 K.
NO detection: NO release was measured with a World Precision Instru-
ment, ISO-NO meter, equipped with a data acquisition system, and
based on direct amperometric detection of NO with short response time
(<5 s) and sensitivity range 1 nm–20 mm. The analog signal was digitalized
with a four-channel recording system and transferred to a PC. The sensor
was accurately calibrated by mixing standard solutions of NaNO2 with
0.1m H2SO4 and 0.1m KI according to the reaction:
Figure 4. a) Stern–Volmer plot for the fluorescence quenching of anthra-
cene by 2 at room temperature. b) Transient absorption spectrum record-
ed 0.05 ms after 355 nm laser excitation of a phosphate buffer (10 mm,
pH 7.4)/MeOH (1:1 v/v) solution of anthracene in the presence of 2
0.06m.
ꢀ
4 Hþ þ 2 Iꢀ þ NO2 ! 2 H2O þ 2 NO þ I2
NO measurements were carried out with the electrode positioned outside
the light path in order to avoid false NO signal due to photoelectric inter-
ference on the ISO-NO electrode.
extent of the photoinduced DNA photocleavage are cur-
rently underway in our laboratory.
Experimental Section
Acknowledgements
We express our sincere thank to Dr. Giuditta Leanza for her inestimable
technical assistance. We also thankful to the Universitꢀ di Catania (Pro-
getti di Ateneo) for the financial support.
Syntheses: Compounds 1 and 2 were synthesized in one step according to
the procedure reported in ref. [10]. Briefly, 9-chloromethyl anthracene
(in the case of 1) or methyl iodide (in the case of 2) were injected
through a septum to a DMF solution of CP cooled at 08C and stirred
overnight. The reaction mixtures were diluted with water, extracted with
CH2Cl2 and, after solvent evaporation, the products were purified by
silica gel chromatography using cyclohexane/ethyl acetate 80:20.
Keywords: antitumor agents · DNA · light · nitric oxide ·
photochemistry
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7.4 Hz), 7.50 (m, 2H), 7.45 (m, 1H), 7.40 (m, 2H), 6.45 ppm (s, 2H).
1
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Received: April 20, 2009
Published online: June 19, 2009
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