Visible light-activatable cyclodextrin-conjugates for the efficient delivery of nitric oxide with fluorescent reporter and their inclusion complexes with betaxolol

Article abstract of DOI:10.1039/d1nj00039j

This contribution reports the design, synthesis, photochemical properties and drug inclusion capability of two novel β-cyclodextrin (βCD) conjugates, βCD-NBFNO1 and βCD-NBFNO2, covalently integrating an N-nitroso amino-nitro-benzofurazan in the primary and secondary hydroxyl rims of the βCD scaffold, respectively through flexible spacers of different length. Both βCD conjugates are water-soluble and release nitric oxide (NO) under the input of either blue or green light, with quantum yields ΦNO (blue) = 0.13, 0.31 and ΦNO (green) = 0.007, 0.013 respectively, the former representing the largest values ever reported for nonmetal-containing NO donors activatable by visible light. The good contrast between the fluorescence green emission of the chromogenic moiety after and before the NO release permits the easy and in real-time quantification of the amount of NO generated, without the addition of external fluorescent agents. Despite the presence of the appendages, these βCD derivatives are also able to complex betaxolol, a β-blocker drug widely used for the reduction of the intraocular pressure, with binding constants Kb = 500 ± 50 and 1100 ± 100 M-1, respectively, without affecting the photochemical performances. In view of the well-known vasodilator properties of NO, the present βCD derivatives represent intriguing candidates for biopharmaceutical research studies addressed to combined therapeutic ocular applications.

Full text of DOI:10.1039/d1nj00039j

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Visible light-activatable cyclodextrin-conjugates
for the efficient delivery of nitric oxide with
fluorescent reporter and their inclusion
complexes with betaxolol†
Cite this: DOI: 10.1039/d1nj00039j
Mimimorena Seggio,a Sara Payamifar,‡ab Aurore Fraix,a Eszter Kalydi,cd Petr Kasal,e
f
g
Ovidio Catanzano,
Salvatore Sortino
Claudia Conte,
Fabiana Quaglia *g and
a
*
This contribution reports the design, synthesis, photochemical properties and drug inclusion capability of
two novel b-cyclodextrin (bCD) conjugates, bCD-NBFNO1 and bCD-NBFNO2, covalently integrating an
N-nitroso amino-nitro-benzofurazan in the primary and secondary hydroxyl rims of the bCD scaffold,
respectively through flexible spacers of different length. Both bCD conjugates are water-soluble and
release nitric oxide (NO) under the input of either blue or green light, with quantum yields FNO (blue) = 0.13,
0.31 and FNO (green) = 0.007, 0.013 respectively, the former representing the largest values ever reported
for nonmetal-containing NO donors activatable by visible light. The good contrast between the fluorescence
green emission of the chromogenic moiety after and before the NO release permits the easy and in real-
time quantification of the amount of NO generated, without the addition of external fluorescent agents.
Despite the presence of the appendages, these bCD derivatives are also able to complex betaxolol, a
b-blocker drug widely used for the reduction of the intraocular pressure, with binding constants Kb = 500 ꢀ 50
and 1100 ꢀ 100 Mꢁ1, respectively, without affecting the photochemical performances. In view of the well-
known vasodilator properties of NO, the present bCD derivatives represent intriguing candidates for
biopharmaceutical research studies addressed to combined therapeutic ocular applications.
Received 4th January 2021,
Accepted 25th March 2021
DOI: 10.1039/d1nj00039j
rsc.li/njc
and hormone secretion,3 and its superb antioxidant, anticancer and
antibacterial activity.4 NO has shown vasodilator effects on eye
Introduction
The design of molecules able to generate nitric oxide (NO) has vasculature5–7 and wound healing effect on injured corneal surface
received increasing attention, especially over the last decade.1,2 opening new therapeutic perspectives in ophthalmology.8–10 This
This is due to the multifaceted role NO plays in a number of scenario has given a boost to the development of compounds able to
physiological processes including neurotransmission, vasodilatation release NO under physiological conditions as potential therapeuti-
cals to fight a variety of diseases.1,2,11,12
The strict dependence of the biological effects of NO by its
a PhotoChemLab, Department of Drug and Health Sciences, University of Catania,
dosage and location13 has made NO-photodonors (NOPD) more
Catania, Italy. E-mail: ssortino@unict.it
b Organic Chemistry Institute for Advanced Studies in Basic Sciences (IASBS),
appealing than spontaneous NO releasers due to the superb
spatiotemporal accuracy that light triggering offers.14–18 In
particular, NOPD activation by environmental light can be very
suited for supplying NO for therapeutic purposes.19,20 Unfortu-
nately, most NOPD have poor water solubility requiring specific
formulations for their delivery to the anterior segment of
the eye.
Zanjan, Iran
c
´
CycloLab Ltd., Illatos ut 7, H-1097 Budapest, Hungary
d
¨
+
´
Department of Pharmacognosy, Semmelweis University, H-1085 Ulloi ut 26,
Budapest, Hungary
e Department of Organic Chemistry, Charles University in Prague,
Hlavova 8/8 128 43, Prague 2, Czech Republic
f Institute for Polymers, Composites and Biomaterials, CNR, Via Campi Flegrei 34,
80078 Pozzuoli, Napoli, Italy
Among the strategies available to tackle this issue, the use of
cyclodextrins (CDs) used for decades to increase the water-
solubility of lipophilic drugs still offers room to innovate. CDs
are cyclic oligosaccharides, well-known for their capability to
complex, stabilize and solubilize guest compounds.21–25 CD
solubilizing activity is strictly related to their aptitude to form
g Drug Delivery Laboratory, Department of Pharmacy, University of Napoli Federico
II, Via Domenico Montesano 49, 80131, Napoli, Italy. E-mail: quaglia@unict.it
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d1nj00039j
‡ S. P. carried out the work at the University of Catania during her stage from the
IASBS.
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without further purification. All solvents used (from Carlo Erba)
were spectrophotometric grade.
Synthetic procedures
All bCD conjugates were synthesized according to the proce-
dures reported in ESI.†
Instrumentation
1H-NMR spectra were recorded on a Varian VXR-600 at 600
MHz. UV-Vis spectra absorption and fluorescence emission
spectra were recorded with a JascoV-560 spectrophotometer
and a Spex Fluorolog-2 (mod. F-111) spectrofluorimeter, respec-
tively, in air-equilibrated solutions, using quartz cells with a
path length of 1 cm. Fluorescence lifetimes were recorded with
the above fluorimeter equipped with a TCSPC Triple Illumina-
tor. The samples were excited with a pulsed diode excitation
source (Nanoled) at 455 nm, the decays were monitored at
550 nm, and ethanol solution itself was used to register the
prompt at 455 nm. The system allowed a time-resulution 4200 ps.
The multiexponential fit of the fluorescence decay was obtained by
the following equation:
Scheme 1 Structures of bCD-NBFNO1, bCD-NBFNO2, their respective
fluorescent photoproducts bCD-NBF1, bCD-NBF2 formed after NO
photorelease, and BTX used as guest molecule.
supramolecular complexes with a guest molecule while covalent
modification of the CDs ring through functionalization of the
primary and/or secondary hydroxyl groups with suitable appen-
dages has been much less explored. The integration of photo-
responsive units into the CDs scaffold is of great interest as
recently proven by a variety of photoresponsive CD-based nano-
constructs with potential phototherapeutic applications.26,27
Several NOPD have been supramolecularly combined with CDs
derivatives.28 In contrast, only limited examples of NO photo-
donor covalent conjugates with CDs are reported to date.29,30 In
the context of ocular diseases, the covalent modification of the CD
with NOPD could bring the benefit of obtaining a functional
NO-releasing scaffold whilst, at the same time, maintaining the
macrocycle capacity for encapsulation of small therapeutics in a
combinatory therapeutic approach.29–32
In this contribution, we provide a proof of concept of this
approach by synthesising two novel bCD conjugates, bCD-NBFNO1
and bCD-NBFNO2, integrating an N-nitroso amino-nitro-benzo-
furazan (NBF-NO) within the primary and secondary rim of the
bCD scaffold, respectively through flexible spacers of different
length (Scheme 1). The N-nitroso amino-nitro-benzofurazan photo-
responsive unit was selected since it represents the non-metal based
NO releaser generating NO with the highest quantum yield upon
excitation with Vis light developed so far.33 Concomitantly to NO
release, this compound forms the highly fluorescent non-nitrosated
derivative as the sole stable photoproduct, representing an optical
reporter useful for the NO detection even at the naked eye. Despite
the excellent photochemical performances, NBF-NO has poor solu-
bility in an aqueous medium, which severely limits its ocular
application in the absence of any carrier system. After an in depth
characterization of the photophysical properties of the bCD con-
jugates, we explore their host–guest complexation properties with
betaxolol (BTX), a well-known b-blocker drug used against glaucoma
for the reduction of intraocular pressure.34,35
I(t) = Saiexp(ꢁt/t
)
i
Absorption spectral changes were monitored by irradiating
the sample in a thermostated quartz cell (1 cm path length,
3 mL capacity) under gentle stirring, using a continuum laser
with lexc = 405 nm, 20 mW, and lexc = 532 nm, ca. 100 mW, and
having a beam diameter of ca. 1.5 mm.
Direct monitoring of NO release in solution was performed
by amperometric detection (World Precision Instruments), with
an ISO-NO meter, equipped with a data acquisition system, and
based on direct amperometric detection of NO with short
response time (o5 s) and sensitivity range 1 nM–20 mM. The
analogue 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 0.1 M
H2SO4 and 0.1 M KI according to the reaction:
ꢁ
4H+ + 2Iꢁ + 2NO2 - 2H2O + 2NO + I2
Irradiation was performed in a thermostated quartz cell
(1 cm path length, 3 mL capacity) using the continuum laser
with lexc = 405 nm or 532 nm. NO measurements were carried
out under stirring with the electrode positioned outside the
light path in order to avoid NO signal artefacts due to photo-
electric interference on the ISO-NO electrode.
Fluorescence and photodecomposition quantum yields
Fluorescence quantum yields (Ff) were determined using
optically-matched solutions at the excitation wavelength of
conjugates and Fluorescein NaOH 0.1
M as reference
(Fref = 0.95)36 through the following equation:
Experimental section
Materials
Ff = Ff(s) (I/I(s)
)
All reagents (Sigma-Aldrich, Alfa Aesar, Molar Chemicals Kft where Ff(s) is the fluorescence quantum yield of the standard; I
and Cyclolab) were of high commercial grade and were used and I(s) are the areas of the fluorescence spectra of compounds
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and standard, respectively; absorbance at the excitation wave-
length was less than 0.1 in all cases.
Photodecomposition quantum yield (FNO) was determined
at lexc = 405 nm and 532 nm within the 20% transformation of
the conjugates by using the following equation
FNO = [C]V/t(1–10ꢁA)I
where [C] is the concentration of phototransformed bCD-
NBFNO1 or bCD-NBFNO2, V is the volume of the irradiated
sample, t is the irradiation time, A is the absorbance of the
sample at the excitation wavelength and I the intensity of the
excitation light. The concentration of the phototransformed
conjugates was determined spectrophotometrically, by taking
into account the absorption changes at 385 nm and the related
De at the same wavelength. I was calculated by potassium
ferrioxalate actinometry.
Dark stability tests
The stability of both conjugates in the dark was evaluated by
recording the absorption spectra of water solution of each
compound in thermostated baths at different temperatures.
The percent of decomposition was evaluated by the changing of
the absorption spectra, taking into account the molar extinc-
tion coefficients of the starting compounds and their respective
non-nitrosate derivatives.
Fig. 1 (A) Absorption and fluorescence emission spectra (lexc = 450 nm)
of aqueous solutions of bCD-NBFNO1 (a and b) and bCD-NBFNO2 (c and
d). (B) Absorption and fluorescence emission spectra (lexc = 450 nm) of
aqueous solutions of denitrosated bCD-NBF1 (a and b) and bCD-NBF2
(a and d). Fluorescence emission spectra were carried out with optically
matched solutions of all compounds at the excitation wavelength.
T = 25 1C.
Host–guest complexation experiments with BTX
To 2 ml of an aqueous solution of BTX (1.4 ꢂ 10ꢁ3 M), different
volumes of either bCD-NBFNO1 or bCD-NBFNO2 stock solu-
tions (6 ꢂ 10ꢁ3 M) were added. After the addition of each
aliquot, the solutions were stirred for 5 min, and the absorption
spectra were recorded. The spectral changes were evaluated by
subtracting the absorption of the same concentration of con-
jugates in neat water. The absorbance changes were then
plotted as a function of the concentration of the conjugates
according to the Benesi–Hildebrand equation (vide infra).
non-nitrosate derivative. However, chromatographic analysis
(see ESI†) and the photolysis carried out upon irradiation of
this band with green light (vide infra) ruled out this hypothesis.
In contrast, such a shoulder can be due to either intra
or intermolecular non-covalent interaction between the N-nitroso
appendages and the bCD moiety. The presence of this absorption,
even in very dilute solutions, make the former hypothesis the more
likely.37
Analogously to what already observed for NBF-NO, the
presence of the nitroso group has a significant effect on the
emission properties. bCD-NBFNO1 and bCD-NBFNO2 exhibited
Results and discussion
bCD-NBFNO1 and bCD-NBFNO2 show excellent solubility in fluorescence emission in the green region (Fig. 1A) but with
water up to ca. 10ꢁ2 M. Fig. 1 shows the UV-Vis spectroscopic quite low quantum yields, being Ff = 0.026 and 0.018, respec-
properties of aqueous solutions of the two derivatives and, for tively, that are values ca. 4 and 7-fold smaller than those of the
the sake of comparison, those of the non-nitrosated bCD analogues non-nitrosated derivatives (Ff = 0. 10 and 0.13)
conjugates bCD-NBF1 and bCD-NBF2.
(Fig. 1B).
The broad absorption band and the quite large extinction
The absorption spectra of bCD-NBFNO1 and bCD-NBFNO2
(Fig. 1A) exhibit a dominant absorption at ca. 380 nm and a coefficient of the conjugates make possible their excitation in
shoulder at 475 nm. The main absorption is blue-shifted by the visible region, even in a reduced concentration range.
more than 90 nm as compared to that of the non-nitrosated Fig. 2A shows the absorption and fluorescence emission spectral
analogues (see Fig. 1B) as a consequence of the loss of the changes observed upon blue light excitation of an air-equilibrated
‘‘charge transfer’’ character due to the presence of the electron- solution of bCD-NBFNO1. They show the bleaching of the main
withdrawing NO group at the amino functionality. Note that the absorption at 377 nm and the formation of a new, intense
shoulder in the visible region is much more intense if com- absorption band at 475 nm. The spectral evolution is also
pared with that observed for the same chromogenic unit non- characterized by the presence of 3 isosbestic points, accounting
linked to the bCD scaffold.33 In principle one may think that for a quite clean photochemical process. Note that, the spectrum
such absorption can be due to the presence of impurities of observed at the end of the photolytic process is identical to that of
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value among those reported for any organic NOPD activatable
in the Vis range.
Interestingly, the restoring of the emission of the optical
reporter is visible even at naked eye (Fig. 3A) and gives easily
readable information about the NO generated. The NO photo-
release measured by amperometric monitoring was then
related to the increase of the fluorescence emission for both
compounds. As shown in Fig. 3B, we found a very good
correlation between the concentration of NO liberated by both
compounds upon light stimuli and the increase of the fluores-
cence intensity of the related optical reporters.
It needs to be stressed that the remarkable values of FNO
found for both conjugates permit the generation of a consider-
able amount of NO without the need of long irradiation times,
which in some cases can be deleterious to cells. According to
the literature, anilinyl radical derivatives formed after the
homolytic N–NO photocleavage evolves to stable photoproducts
by H-transfer from the solvent medium. Since in neat water,
like in our case, this process is thermodynamically not feasible,
Fig. 2 (A) Absorption (solid lines) and fluorescence emission, (lexc = 424 nm,
isosbestic point) (dotted lines) spectral changes observed upon exposure of an
aqueous solution of bCD-NBFNO1 (35 mM) at lexc = 405 nm (ca. 20 mW cmꢁ2
)
for time intervals from 0 to 300 s. The arrows indicate the course of the spectral
profile with the illumination time. The inset shows the different absorbance
changes at l = 377 nm (’) and fluorescence changes at l = 550 nm (K),
respectively. (B) NO release profile observed for an aqueous solution of bCD-
NBFNO1 (35 mM) upon alternate cycles of irradiation (lexc = 405 nm, ca.
20 mW cmꢁ2) and dark. T = 25 1C.
the non-nitrosated conjugate bCD-NBF1 (compare spectra a in
Fig. 1A and B) showing the typical charge transfer band at 475 nm
of the amino-nitro-benzofurazan chromogenic unit.38
Besides, the evolution of the fluorescence emission spectra
observed upon irradiation shows a significant restoring of the
green emission with lmax = 550 nm, typical of the amino-nitro-
benzofurazan fluorophore (Fig. 2A).38 These findings clearly
account for the NO photorelease from bCD-NBFNO1 and the
concomitant formation of the non-nitrosated fluorophore that
act as suitable fluorescent reporter to follow the NO uncaging in
real time. The inset of Fig. 2A shows a very good agreement
between the evolution of the absorption and fluorescence
changes as a function of the irradiation time and indicates
that the photolysis was complete within ca. 2 min of irradiation.
This accounts for a very effective photochemical reaction as
confirmed by the high of the quantum yield for the NO
photorelease, FNO = 0.13, a value very close to that observed
for the non-water-soluble NBF-NO (FNO = 0.15).33 NO release
was demonstrated by the direct detection of this radical species
using an ultrasensitive NO electrode. Fig. 2B shows that NO is
Fig. 3 Fig. 3 (A) Absorption (solid lines) and fluorescence emission,
(lexc = 427 nm, isosbestic point) (dotted lines) spectra changes observed
before (a and b) and after the complete photolysis (c and d) at lexc = 405 nm
(ca. 20 mW cmꢁ2) of aqueous solution of bCD-NBFNO2 (16 mM) and actual
images of the solutions before (bottom) and after (top) the photolysis
promptly released upon illuminator, of the aqueous solution of acquired upon excitation at l = 350 nm. The inset shows the NO release
profile observed for an aqueous solution of bCD-NBFNO2 (16 mM) upon
bCD-NBFNO1, stops in the dark and restarts once the light
source is switched on again.
alternate cycles of irradiation (lexc = 405 nm, ca. 20 mW cmꢁ2) and dark.
(B) Correlation of the fluorescence increase observed upon photolysis of
Fig. 3A and related inset show that bCD-NBFNO2 exhibited a
bCD-NBFNO1 (’) and bCD-NBFNO2 (K) and the concentration of NO
similar photobehavior with an even higher quantum yield for
the NO photorelease, FNO = 0.31, which represents the largest emission after and before irradiation, respectively. T = 25 1C.
photoreleased. I and I0 represent the fluorescence intensities at the lmax of
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the high values observed for FNO indicate a key role of the bCD
scaffold as a reactant, providing a source of 14 easily abstract-
able H atoms and very close to the anilinyl radical intermediate.
Moreover, it should be noted that the absorption and emission
spectral and time evolution observed upon irradiation were
identical in the case of an N2-saturated solution (data not
shown), suggesting that both the efficiency and nature of the
photochemical reaction are not dependent by the presence of
oxygen. This observation rules out the participation of a long-
lived excited triplet state in the photodecomposition, suggest-
ing a NO photodetachment occurring more likely from the
short-lived excited singlet state. This hypothesis is supported
well by (i) the negligible population of the excited triplet state
reported for amino-nitro benzofurazan derivatives in polar
solvents,39 and (ii) the very short singlet lifetimes found for
bCD-NBFNO1 and bCD-NBFNO2. As shown in Fig. 4, both
conjugates exhibited a biexponential behavior with dominant
components (ca. 80%) with t = 2.30 and 0.84 ns, respectively.
As discussed above, the absorption spectrum of both bCD
conjugates shows a pronounced shoulder extending up to the
green region, which is negligible in the case of NBF-NO.33
These spectral features prompted us to investigate the photo-
reactivity of the compounds upon excitation with green light.
Therefore, aqueous solution of either bCD-NBFNO1 or bCD-NBFNO2
were irradiated at lexc = 532 nm. We observed changes in the
absorption and fluorescence emission spectral profiles basically
identical to those already observed under blue light stimuli (data
not shown) although with lower photochemical efficiency. In
fact, the values calculated for FNO were = 0.007 and 0.013 for
bCD-NBFNO1 and bCD-NBFNO2, respectively. Fig. 5 shows unam-
biguous evidence that NO release for both compounds can also be
triggered by green light.
Fig. 5 NO release profiles observed for an aqueous solution of
bCD-NBFNO1 (35 mM, a) and bCD-NBFNO2 (16 mM, b) upon alternate
cycles of irradiation with green light (lexc = 532 nm, ca. 100 mW cmꢁ2) and
dark.
Fig. 6 (A) Dark stability of the aqueous solution of bCD-NBFNO1 (square)
and bCD-NBFNO2 (circles) incubated in the dark at 25 1C (filled symbols)
or 4 1C (open symbols) at different times. (B) Dark stability of the aqueous
solution of bCD-NBFNO1 (square) and bCD-NBFNO2 (circles) incubated
for 15 min at different temperatures.
The photoreactivity dependence by the excitation wave-
length is not surprising and may be due to the participation
of upper excited singlet states populated with blue light as
mediators of the photodecomposition route.
The stability of the conjugates was also evaluated in the dark
at different temperatures and times. Fig. 6A shows moderate
decomposition for both compounds (ca. 20%) at 25 1C, which is
almost totally inhibited if samples are incubated for the same
time at 4 1C. Moreover, incubation of the solution in a thermo-
stated bath at a different temperature for 15 min each showed
satisfactory stability up to 40 1C (Fig. 6B).
the reasons motivated in the introductory part. Titration of an
aqueous solution of BTX was then carried out using increasing
amounts of either bCD-NBFNO1 or bCD-NBFNO2. BTX offers
good spectroscopic requisite to follow the titration by UV-Vis
absorption spectroscopy since its absorption maximum falls at
275 nm, a region in which both conjugates display small
absorption. Fig. 7A and B show the absorption spectral changes
in the BTX region observed upon the addition of the host
molecules and after subtracting the same amount of hosts
added to the same volume of water. We observed a hypo-
chromic shift of the absorption band of BTX after addition of
the bCD conjugates, according to typical host–guest encapsula-
tion processes. The reciprocal of the absorbance changes at the
absorption maximum was then plotted as a function of the
reciprocal concentration of the host molecules, according to
the Benesi–Hildebrand equation:40
Finally, we investigated the host–guest complexation ability
of the bCD conjugates. As a prototype guest, we chose BTX for
1
1
1
¼
þ
DA Kass ꢃ De ꢃ ½hostꢄ De
Fig. 4 Fluorescence decay and the related biexponential fitting of the
aqueous solution of bCD-NBFNO1 (40 mM) (A) and bCD-NBFNO2 (B)
recorded at lexc = 455 nm and lem = 550 nm.
where Kass represents the association constants for the supra-
molecular host–guest process, De is the difference of the molar
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triggered by visible light. However, although with lower effi-
ciency, both conjugates release NO even under stimuli of green
light. The good fluorescence contrast between the highly fluo-
rescent stable photoproducts and the poorly fluorescent start-
ing compounds permits the formers to be excellent optical
reporters for the easy detection of the NO generated, with the
release process being followed even by the naked eye. Moreover,
the reporter can be excited by using the same excitation
wavelength used for NO uncaging, facilitating the real-time
monitoring of NO, which is crucial given the transient nature of
this diatomic radical, without requiring a double excitation
source. It is also important to stress that, as extensively demon-
strated in our recent studies on other nitroso-derivatives,41
these photoactivatable compounds do not show significant
cytotoxicity in the dark in the concentration range used. Inter-
estingly, functionalization of the CD scaffold with the photo-
activatable moieties does not preclude the encapsulation guest
as demonstrated by the association of both conjugates with the
b-blocker BTX. In this regard, in view of the well-known
vasodilator properties of NO, the present work may open
intriguing prospects for biological studies on formulations for
ocular application against glaucoma, addressed to explore the
combinatory effect of BTX and NO, with this latter slowly
released at physiological temperature but rapidly released upon
environmental light. These studies are currently underway in
our laboratories.
Fig. 7 Absorption spectral changes observed upon addition of different
amounts of bCD-NBFNO1 from 28 mM to 210 mM (A) and bCD-NBFNO2
from 62 mM to 500 mM (B) to aqueous solutions of BTX (1.4 mM). The
related double-reciprocal plots and the linear fitting of the data, according
to the Benesi–Hildebrand equation, are reported for bCD-NBFNO1 (C) and
bCD-NBFNO2 (D). T = 25 1C.
extinction coefficient between the free and complexed guest
and [host] is the concentration of the bCD conjugates. As
shown in Fig. 7C and D, we obtained very good linear plots in
both cases and values of Kass of 500 ꢀ 50 Mꢁ1 and 1100 ꢀ 100 Mꢁ1
for bCD-NBFNO1 and bCD-NBFNO2, respectively, were obtained
from the intercept/slope ratio. The higher value observed for
bCD-NBFNO2 is probably attributable to the longer flexible spacer
between the CD scaffold and the chromogenic unit, which allows a
better accommodation of the host within the hydrophobic cavity.
In order to be used in combination, one of the indispensable
requisites for this host–guest system is that the encapsulation
of BTX in the CD cavity does not affect the photochemical
performances of the photoactivatable conjugates. Therefore,
photolysis experiments on bCD-NBFNO1 and bCD-NBFNO2
were carried out in the presence of BTX. We observed that
the presence of the guest molecule changes neither the nature
nor the efficiency of the photoreactivity of both compounds,
ruling out any intermolecular communication between the host
and the guest through competitive photoinduced processes.
This is not a trivial result for host–guest supramolecular com-
plexes. In fact, it is known that the photoreactivity of the host
and guest components can be remarkably influenced in effi-
ciency nature or both upon complexation as result of their close
proximity and the presence of specific interactions, steric
constrains and reduced polarity.27
Conflicts of interest
We have no confict of interests to declare.
Acknowledgements
We thank the Programma Operativo Nazionale Ricerca e Inno-
vazione 2014–2020 (CCI 2014IT16M2OP005), Fondo Sociale
Europeo, Azione I.1 ‘‘Dottorati Innovativi con caratterizzazione
Industriale’’.
Notes and references
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for Pharmaceutical and Biological Applications. Wiley-VCH
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2 P. G. Wang, M. Xian, X. Tang, X. Wu, Z. Wen, T. Cai and
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3 L. J. Ignarro, Nitric Oxide: Biology and Pathobiology. Elsevier
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4 L. J. Ignarro, Nitric Oxide Chemistry and Biology,
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5 L. Schmetterera and K. Polak, Prog. Retinal Eye Res., 2001,
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Conclusions
We have designed, synthesized and characterized two novel
photoactivatable bCD conjugates. These compounds show
excellent water solubility, fairly good stability in the dark within
24 h at room temperature, and release of the biologically
relevant NO under visible light stimuli. In particular, excitation
with blue light leads to NO release with the highest quantum
yield never observed for non-metal based NO photoreleaser
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