Inclusion complex formation of sanguinarine alkaloid with cucurbit[7]uril: Inhibition of nucleophilic attack and photooxidation

Article abstract of DOI:10.1039/c0ob00666a

The inclusion of sanguinarine, a biologically active natural benzophenanthridine alkaloid, in cucurbit[7]uril (CB7) was studied by NMR and ground-state absorption spectroscopy, as well as steady-state and time-resolved fluorescence measurements in aqueous solution. The iminium form of sanguinarine (SA⁺) produces very stable 1:1 inclusion complex with CB7 (K = 1.0 × 10⁶ M^-1), whereas the equilibrium constant for the binding of the second CB7 is about 3 orders of magnitude smaller. Marked fluorescence quantum yield and fluorescence lifetime enhancements are found upon encapsulation of SA⁺ due to the deceleration of the radiationless deactivation from the single-excited state, but the fluorescent properties of 1:1 and 1:2 complexes barely differ. The equilibrium between the iminium and alkanolamine forms is shifted 3.69 pK unit upon addition of CB7 as a consequence of the preferential encapsulation of the iminium form and the protection of the 6 position of sanguinarine against the nucleophilic attack by hydroxide anion. On the basis of thermodynamic cycle, about 225 M^-1 is estimated for the equilibrium constant of the complexation between the alkanolamine form of sanguinarine (SAOH) and CB7. The confinement in the CB7 macrocycle can be used to impede the nucleophilic addition of OH^- to SA⁺ and to hinder the photooxidation of SAOH.

Full text of DOI:10.1039/c0ob00666a

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PAPER
Inclusion complex formation of sanguinarine alkaloid with cucurbit[7]uril:
inhibition of nucleophilic attack and photooxidation†
Zsombor Miskolczy, M o´ nika Megyesi, G a´ bor T a´ rk a´ nyi, R e´ ka Mizsei and L a´ szl o´ Bicz o´ k*
Received 4th September 2010, Accepted 2nd November 2010
DOI: 10.1039/c0ob00666a
The inclusion of sanguinarine, a biologically active natural benzophenanthridine alkaloid, in
cucurbit[7]uril (CB7) was studied by NMR and ground-state absorption spectroscopy, as well as
steady-state and time-resolved fluorescence measurements in aqueous solution. The iminium form of
+
6
-1
sanguinarine (SA ) produces very stable 1 : 1 inclusion complex with CB7 (K = 1.0 ¥ 10 M ), whereas
the equilibrium constant for the binding of the second CB7 is about 3 orders of magnitude smaller.
Marked fluorescence quantum yield and fluorescence lifetime enhancements are found upon
+
encapsulation of SA due to the deceleration of the radiationless deactivation from the single-excited
state, but the fluorescent properties of 1 : 1 and 1 : 2 complexes barely differ. The equilibrium between
the iminium and alkanolamine forms is shifted 3.69 pK unit upon addition of CB7 as a consequence of
the preferential encapsulation of the iminium form and the protection of the 6 position of sanguinarine
against the nucleophilic attack by hydroxide anion. On the basis of thermodynamic cycle, about 225
-
1
M
is estimated for the equilibrium constant of the complexation between the alkanolamine form of
sanguinarine (SAOH) and CB7. The confinement in the CB7 macrocycle can be used to impede the
-
+
nucleophilic addition of OH to SA and to hinder the photooxidation of SAOH.
Introduction
the embedment of the benzimidazole moiety of the proton-pump
inhibitor lansoprazole and omeprazole drugs not only catalyzes
the transition into their physiologically active cyclic sulfenamide
form but also hinders the decomposition and dimerization, while
Encapsulation of species of biological interest in macrocyclic
compounds have been extensively studied because it can be utilized
in the delivery, stabilization, solubilization and controlled release
of drugs as well as in analyte sensing. Cyclodextrins are the
most widely used hosts in these applications. Recent reviews
provide good overview on the biopharmacological utilizations of
11
the biologically important reactivity is retained. The embedment
of antitumor platinum(II) complexes in CBn provides steric
hindrance to drug degradation by peptides and proteins, and
the use of different sized macrocycles allows of the tuning of
1
2,3
water-soluble calixarenes. Cucurbit[n]urils (CBn), the family of
relatively new, pumpkin-shaped cavitands composed of n glycoluril
12
drug release rates and toxicity. Antitumor metallocenes also
form inclusion complex with CBn. The molibdocene complex
catalysed the degradation of the cucurbituril framework in the
presence of oxygen, whereas encapsulation of titanocene dichlo-
ride by CB7 greatly slowed the hydrolysis of the cyclopentadienyl
4
units, are able to cross the cell membrane, which can be exploited
5
for drug delivery. The interaction of the lactone and carboxylate
forms of camptothecin, a cytotoxic quinoline alkaloid, with CBn
was examined and the effect of inclusion complex formation on
the solubility and anticancer activity was revealed. A significant
increase of thermal stability upon encapsulation in cucurbit[7]uril
13
ligands. For targeted drug delivery, stimuli-responsive polymer
6
14
nanocapsules, and reduction-sensitive, biocompatible vesicles
15
were self-assembled using cucurbit[6]uril (CB6) macrocycles.
(
CB7) was found for ranitidine, a histamine H
2
-receptor antagonist
Noninvasive remote-controlled release of drug molecules
was achieved from magnetic-core silica nanoparticles capped
7
employed for the treatment of excess stomach acid production.
The stability constants for the inclusion of local anaesthetics
in CB7 are 2–3 orders of magnitude greater than the values
reported for binding by the comparably sized b-cyclodextrin
16
with CB6.
Despite the pharmaceutical importance of natural alkaloids
very few information is available on their complexation with
CBn. The basicity of a pyridoindole type alkaloid, norharmane
8
host molecule. CBn complexation may provide a formulation
9
method to solubilize albendazole, an anticancer agent, and its
17
increased only 0.7 pK unit upon inclusion. The isoquinoline
10
derivatives for clinical application. Nau’s group have shown that
alkaloid, berberine proved to be a very sensitive fluorescent
18
probe, and 500-fold fluorescence quantum yield enhancement
Chemical Research Center, Hungarian Academy of Sciences, P.O. Box 17,
19
was observed upon binding in CB7. Sensitive analytical methods
1
1
525, Budapest, Hungary. E-mail: biczok@chemres.hu; Fax: +36-1-438-
143
have been developed on the basis of CBn complex formation for
20
21
the quantitative determination of coptisine and sanguinarine
in biological fluids.
†
1
Electronic supplementary information (ESI) available. See DOI:
0.1039/c0ob00666a
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Scheme 1 The two forms of sanguinarine.
1
Several studies have dealt with the effect of encapsulation in
resonances are shifted upfield into the 4–5 ppm region of the H-
NMR spectrum overlapping with the solvent and CB7 signals.
Therefore, the resonances of I and II are missing from the spectra.
Substantial upfield displacements are also observed for H-1 and
H-9 resonances upon complexation, which is in line with the fact
7,11,22
CBn on the protonation of guests,
but it is unknown how
the encapsulation influences the nucleophilic attack of organic
compounds. In the present work, we focus on sanguinarine,
a natural benzo[c]phenanthridine alkaloid (Scheme 1), which
23
+
exhibits anticancer, antimicrobial, antifungal properties and
that both ends of SA are embedded in a different CB7 macrocycle.
Scheme 2 illustrates the processes taking place in the solution
24
interacts with nucleic acids. A pH dependent reversible transition
+
+
occurs between its iminium (SA ) and alkanolamine (SAOH)
containing SA and CB7.
2
5,26
+
+
forms.
Since the carbon atom of SA at the position 6 has low p-
At 0.3 mM SA concentration, a second set of resonances
27
-
electron density, it is susceptible to nucleophilic addition of OH .
The biological activity is associated with the SA form, whereas the
emerges due to the partial dissociation of the 1 : 2 complex into
1 : 1 associate. In the spectrum of the latter species, the methylene
I peak moves to upfield into the domain of the solvent and
CB7 signals, which is accompanied by the significant upfield
displacement of the signal of H-9 and H-10. The geminal protons
of the methylene II show chemical non-equivalence possibly due
to the induced diastereotopic environment above the rim of the
CB7 in the 1 : 1 complex. Therefore, methylene II gives rise to two
broadened singlet resonances near ~6.1 ppm having a small (J<2
Hz) two-bond J-coupling but the location of the signals barely
alters compared to the corresponding resonance for uncomplexed
+
conversion to SAOH enhances the lipophilicity improving thereby
26
the cellular availability. The latter form undergoes an irreversible
28
oxidation in the singlet-excited state to produce oxysanguinarine.
The main goal of the present studies was to reveal how the inter-
action with CB7 affects the fluorescent properties, photostability
and the basicity of sanguinarine. Moreover, we demonstrate that
+
SA can associate with not only one but also two CB7 macrocycle.
Results and discussion
+
SA . These spectral changes suggest that the methylene I end of
+
Inclusion complex formation studied by NMR spectroscopy
SA is encapsulated in the 1 : 1 complex.
+
At large SA concentration, a third set of signals arises parallel
with the intensity enhancement of the peaks attributed to the 1 : 1
inclusion complex. Furthermore, the spectrum of the 1 : 2 inclusion
complex concomitantly vanishes when the SA :CB7 molar ratio
grows above 0.5. The third set of signals, coded with orange colour,
is probably due to weakly bound species formed by non-specific
Systematic NMR studies provide valuable information on the
structure and stoichiometry of inclusion complex formation.
Because of the inherently lower sensitivity of this technique,
much more concentrated sanguinarine solutions (typically 0.4–
mM) had to be used than in absorption and fluorescence
spectroscopic measurements. The high frequency region of the
+
1
+
-
binding. As shown in Scheme 1, sanguinarine aggregates (SA Cl )
n
1
H-NMR spectrum (6.0–10.0 ppm) proved to be appropriate to
+
and various loosely bound SA –CB7 complexes, in which the
alkaloid is not inserted into the CB7 cavity, may contribute to the
broad resonances coded with orange colour. Fast exchange occurs
among these species. In contrast, 1 : 2 and 1 : 1 confinements in
CB7 cavity lead to slow exchange among the solutes. Fig. 2 shows
the spectral change with increasing temperature. The three sets of
+
monitor the chemical shift changes of SA . Spectral assignment
+
1
of the SA protons was performed in the absence of CB7 by H-
1
3
C long-range J-correlation (gHMBC) and spatial correlation
1
(
1D-ROESY) experiments. For the complexes, homonuclear H-
1
1
1
H NOESY and H- H ROESY spectra were recorded.
Fig. 1 displays the effect of complexation on the spectrum of
1
H-NMR resonances reversibly coalesce into a single broad set,
+
SA at 283 K. The upper panel presents the spectrum of free
whose chemical shifts differ from those found in the absence of
CB7. According to the magnitude of the exchange cross-peaks
in the NOESY spectrum (Fig. 3), the extent of the population
transfer between the non-specific and 1 : 2 complexes is small,
however one can identify intense chemical exchange between non-
specific and 1 : 1 complexes and the correlations between 1 : 1 and
+
SA in neat D O, whereas the lower panel shows the spectral
2
+
alterations upon the gradual increase of SA concentration in 1
mM CB7 solution. The signal of H-6 is identified in all spectra
indicating that the iminium form (SA ) dominates over the SAOH
+
form under the condition of the NMR measurements (pH ª
+
6
). At 0.1–0.2 mM SA concentration range a single set of
1
: 2 inclusion complexes are also well observable in most cases.
resonances appears, where most aromatic protons have significant
+
chemical shift displacements with respect to the uncomplexed SA .
Energy-minimized structure of the 1 : 1 and 1 : 2 complexes
+
This spectrum is assigned to 1 : 2 SA :CB7 inclusion complex.
+
The methylenes I and II of this species experience a significant
shielding effect due to the encapsulation in CB7 cavity and their
To get more information on the molecular structure of SA –
CB7 complexes, quantum chemical calculations were carried out
1
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1
+
Fig. 1 Aromatic region of the H NMR spectra (599.9 MHz, D
2
O, 283 K) for SA –CB7 mixtures. The amount of CB7 (1.0 mM) was kept constant,
+
+
while the concentration of SA (indicated on the left) was increased. For comparison, the spectrum of SA recorded in the absence of CB7 is presented
on the top. The assignment of the color-coded resonances corresponds to Scheme 2.
with RM1 semiempirical method using HyperChem 8.0 program.
The energy-minimized structure of the ground-state complexes
are in accordance with the NMR spectra. As shown in the
this structure by electrostatic interactions. In addition to these
effects, the repulsion between the negatively charged oxygens of the
two hosts also renders the binding of two CB7 more difficult. The
lower panel of Fig. 4 displays that in the case of the 1 : 2 complex,
the positive charge of the nitrogen-heterocyclic ring interacts with
both hosts, and the benzodioxole part of the isoquinoline moiety
is less deeply incorporated in CB7 than in the 1 : 1 complex. The
latter phenomenon may explain the smaller upfield shift of the
H-6, H-10 and H-9 resonances for the 1 : 2 complex compared
to those observed for the 1 : 1 associate. The quantum chemical
+
upper panel of Fig. 4, SA can be only partially encapsulated
4
when 1 : 1 complex is formed because the length of CB7 is only
0
.91 nm. The benzodioxole part of the isoquinoline moiety is
encapsulated in the hydrophobic core of CB7 and the positive
charge of the nitrogen-heterocyclic ring interacts with the oxygens
+
of the macrocycle. The embedment of the other end of SA is
unfavourable because the penetration into the host is hindered
sterically by the N-methyl substituent, and the larger distance
of the positively charged nitrogen-heterocyclic ring from the
carbonyl-laced portal of CB7 does not allow the stabilization of
+
calculations show that SA is not planar. Consequently, the CB7
macrocycles do not have a parallel configuration in the 1 : 2
complex.
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+
Scheme 2 Binding modes of SA with CB7.
+
Effect of CB7 on the absorption and fluorescence properties of SA
[
Complex1: 2]
K =
(2)
at pH 4
2
[
Complex1:1][CB7]
The larger sensitivity of the absorption and fluorescence spec-
troscopy allowed the study of the complexation with CB7 in
much more diluted (16.4 mM) sanguinarine solution, where non-
specific binding plays negligible role. To avoid the formation of
the alkanolamine form (SAOH), the experiments were performed
at pH 4. As seen in Fig. 5, the absorption spectrum exhibits a
slight hypsochromic shift on addition of CB7 evidencing complex
formation. The inset displays the absorbance change at 490 nm.
A marked absorbance diminution is observed in the 0–0.04 mM
CB7 concentration range followed by a slight absorbance increase
at higher CB7 concentration. The two distinct domains suggest
that not only one but also two CB7 can form a complex with a
sanguinarine cation, in agreement with the results of NMR studies.
The equilibrium constants are defined as
The data of spectrophotometric titration were analyzed by
a home-made MATLAB 7.9 program as described in our pre-
29
vious paper. The line in the inset to Fig. 5 corresponds to
6
-1
the optimized parameters: K
1
= 1.0 ¥ 10 M , K
2
= 1000
-
1
-1
-1
M , e(Complex1:1) = 3400 M cm and e(Complex1:2) =
000 M cm , where e(Complex1:1) and e(Complex1:2) denote
-
1
-1
5
the molar absorption coefficients of 1 : 1 and 1 : 2 complexes,
respectively.
+
Binding to CB7 also alters the fluorescent behaviour of SA
+
(
Fig. 6). The fluorescence quantum yield of SA grows from 0.038
to 0.37 upon addition of 0.1 mM CB7. When the concentration
of CB7 is raised, the emission peak located initially at 604
nm moves gradually to 556 nm, and a concomitant intensity
enhancement is observed. As a representative example, the inset
shows the fluorescence intensity change at 558 nm and the
line represents the result of the nonlinear least-squares analysis.
[
Complex1:1]
K =
(1)
1
+
6
-1
[SA ][CB7]
The fitted equilibrium constants are K
1
= 1.4 ¥ 10 M and
1
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1
+
Fig. 2 Aromatic region of the H-NMR spectra for 0.43 mM SA and 1 mM CB7 mixture in D
2
O (599.9 MHz) as a function of the temperature. The
+
spectrum of free SA (4.2 mM) is shown in the absence of CB7 on the top.
-
1
+
+
K
2
= 1000 M , whereas I
F
(Complex1:1)/I
F
(SA ) = 17.7 and
concentration the amplitude of SA fluorescence vanishes, and
+
+
I
F
(Complex1:2)/I
F
(SA ) = 16.0 are obtained for the fluorescence
only the long-lived fluorescence of SA –CB7 1 : 1 complex is
intensity ratios at the detection wavelength (558 nm).
The fluorescence decay characteristics of SA also change
detected. Further increase of the amount of CB7 in the solution
brings about negligible change in the fluorescence decay indicating
that the difference between the fluorescence lifetimes of 1 : 2 and
1 : 1 complexes is less than the limit of experimental errors. The
similar enhancement of the apparent fluorescence yield and the
fluorescence lifetime implies that the confinement in CB7 affects
mainly the rate of nonradiative energy dissipation processes of the
+
considerably upon 1 : 1 binding to CB7. In accordance with
30
recently published results, exponential fluorescence intensity vs.
time profiles with t = 2.3 ns excited-state lifetime is measured
in water. Upon gradual addition of CB7, a new fluorescence
component with a lifetime of t = 17.5 ns emerges, whose amplitude
1
2
+
+
grows due to SA –CB7 complex formation. Around 13 mM CB7
singlet-excited SA .
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1
1
+
Fig. 3 Aromatic region of the H- H NOESY spectra of 0.43 mM SA and 1 mM CB7 mixture (599.9 MHz, D
2
O, 263 K, tmix = 0.1 s). The cross-peaks
+
stem from two types of correlations. The intense ones are exchange cross-peaks which appear between the same protons of SA that correspond to the
different binding states ( ). There are also NOE type cross-peaks encoding the spatial proximity between neighboring protons of the same species ( ).
Effect of pH variation
dependence of the sanguinarine spectra in CB7 solution (Fig. 7A)
is qualitatively similar to that found in water, but the alterations
25c
-
As Absol ´ı nov a´ and co-workers pointed out, there is a consider-
able discrepancy in the literature among the pK values measured
by various experimental techniques for the pH dependent equi-
appear at much higher pH. Increasing OH concentration leads to
+
the gradual disappearance of the SA absorption in the 380–520
nm range and the fluorescence band in the 470–740 nm domain
because the nucleophilic addition to the carbon atom of the
iminium bond eliminates the aromaticity of the heterocyclic ring.
The SAOH form absorbs only UV light, and its fluorescence band
+
librium between the iminium (SA ) and alkanolamine (SAOH)
forms of sanguinarine. The ionic strength, buffer, added organic
solvent, interaction with anions and the low solubility of SAOH
modify the pK values. To avoid these effects, acid–base titration
was performed in dilute neat aqueous solution. The results of the
absorption and fluorescence spectroscopic studies carried out in
the absence and presence of CB7 are compared in Fig. 7B. The pH
+
is strongly blue-shifted compared to that of SA . The fluorescence
maximum of SAOH is located at 415 nm in water, whereas it is
red-shifted to 419 nm in 1 mM CB7 solution indicating that SAOH
is able to produce inclusion complex with CB7.
1
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Fig. 5 Absorption spectrum of 16.4 mM sanguinarine at pH 4 in the
presence of 0, 5.5, 12 and 140 mM CB7. Inset presents the absorbance
change at 490 nm and the result of the nonlinear least-squares fit.
+
Fig. 4 Energy-minimized structure of the 1 : 1 and 1 : 2 SA –CB7
complexes in the ground state obtained by RM1 semiempirical calculations
+
with HyperChem 8.0 program. Color codes: CB7, green; SA , oxygen, red;
nitrogen, blue; carbon, light blue.
Fig. 6 Alteration of the fluorescence spectrum of 0.59 mM sanguinarine
on addition of 0, 0.15, 0.35, 0.59, 0.96, 1.7, 3.1, 29 and 160 mM CB7 at pH
4
. Inset displays the fluorescence intensity at 558 nm as a function of CB7
The open and filled squares in Fig. 7B show how the absorbance
at 460 nm and the fluorescence intensity at 600 nm vary with
increasing pH in the absence of CB7. The results of the two types of
measurements (M) fit to the same trend. The Boltzmann function
is widely used for the analysis of the sigmoid-shaped titration
concentration. Excitation is at 440 nm. The line corresponds to the fitted
function.
intensity at 560 nm and the absorbance at 460 nm follow the same
trend. A combined analysis of the two types of measurements gives
31
curves:
pK ¢ = 10.83 and the best fit of eqn (3) to the experimental data
is plotted in Fig. 7B. The inclusion complex formation with CB7
a
M −M
0
∞
M =
+ M∞
(3)
1
+ exp[(pH − pK )/ P]
a
leads to 3.69 pH unit displacement for the equilibrium between
+
where P denotes a fitting parameter, pK
logarithm of the equilibrium constant K
and M are the absorbances or fluorescence intensities at low
and high pH, respectively. The non-linear least-squares fit of eqn
3) to the experimental data gives pK = 7.14 ± 0.04 in water. This
result is in excellent agreement with that found by Absol ´ı nov a´ and
co-workers (pK = 7.21 ± 0.065) in the solution of sanguinarine
and 1 mM HNO
a
represents the negative
SA and SAOH. Similar basicity enhancements promoted by
+
+
a
= [SAOH][H ]/[SA ],
CB7-confinement have been reported for amines, but in those
cases, the effect originates from the preferential binding of the
M
0
•
7
,22a,22b,32
protonated guest.
The dissociation of the protonated
+
(
a
amines is impeded by the stabilization of the N –H bond through
hydrogen-bonding and ion–dipole interactions with the carbonyl
a
groups on the CB[7] portal. The apparent pK enhancement
a
25c
3
.
upon the confinement of sanguinarine in CB7 is due to an
-
As seen in Fig. 7B, the spectral changes occur at much higher
pH in the presence of 0.1 mM CB7, when the most of SA forms
entirely different effect. The macrocycle inhibits OH addition
+
+
by protecting the position 6 of SA against the nucleophilic
1
: 1 inclusion complex. The pH dependence of the fluorescence
attack. The NMR spectroscopic studies and quantum chemical
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6
-1
of these values and K
1
= 1.2 ¥ 10 M , the mean of the values
derived form absorption and fluorescence titrations, into eqn (4)
-
1
leads to KSAOH = 245 M as an estimate of the binding constant
of SAOH–CB7 complex. This quantity cannot be determined
directly by absorption or fluorescence measurements because the
photochemical instability of SAOH (vide infra) is accompanied
by a low solubility and small spectral changes upon confinement
in CB7. The small binding affinity of SAOH to CB7 is not
surprising because ion–dipole interactions cannot contribute to
the driving force of complexation in the case of this neutral guest.
Moreover, the addition of KOH brings about not only pH change
+
but also leads to the coordination of K to the carbonyl-fringed
portals of CB7, hindering thereby the ingression of the organic
3
3
-1
guest. Mezzini and co-workers reported 600 and 53 M for
+
the equilibrium constants of the first and second K binding to
3
4
+
CB7. The competitive complexation of K lessens the apparent
binding constant of SAOH–CB7 formation. In the presence of
0
.06 M KCl, which corresponds to the largest KOH concentration
4
-1
in the titration of CB7-bound sanguinarine, 3.9 ¥ 10 M was
obtained for the apparent equilibrium constant of SA inclusion
+
in CB7. Although this is about 28-fold smaller than the value
measured in neat water, it still ensures the complexation of about
+
9
6% of SA at 653 mM CB7 concentration, which is employed
in the fluorescence titration with KOH. Thus, we can conclude
Fig. 7 (A) Absorption spectra of 16 mM sanguinarine in 100 mM
CB7 solution at pH 7.16, 9.96, 10.83, 11.64 and 12.65. Inset shows the
fluorescence spectrum of 1.9 mM sanguinarine in 653 mM CB7 solutions at
pH 6.00, 9.60, 10.11, 10.59, 11.01, 11.36, 11.49 and 12.15. Excitation is at
+
+
that the competitive binding of K does not preclude SA –CB7
-
formation. At high pH, OH reacts probably with the small frac-
+
tion of unbound SA , which shifts the complexation equilibrium
-
1
3
09 nm. (B) Absorbance at 460 nm (ꢀ) and fluorescence intensity at 600
toward dissociation. Taking the above estimated KSAOH = 245 M
nm (ꢁ) for 10.5 mM sanguinarine aqueous solution as a function of pH.
Absorbance of 16 mM sanguinarine at 460 nm in the presence of 100 mM
CB7 (ꢀ) and fluorescence intensity of 1.9 mM sanguinarine at 560 nm (ꢀ)
in 653 mM CB7 solution at various pHs.
value, we can calculate that about 14% of SAOH is embedded
in CB7 at the end of the fluorescence titration displayed in
Fig. 7.
calculations demonstrated that position 6 is confined in the apolar
CB7 cavity, but located around the plane of the oxygens of
the macrocycle portal, whose high electron density hinders the
CB7-inhibited photooxidation
Maiti’s group demonstrated that in the presence of oxygen SAOH
undergoes an irreversible photooxidation in the excited singlet
state producing 6-oxysanguinarine. We studied the effect of
-
approach of the OH anion to the carbon atom of the iminium
28
+
bond of SA .
CB7 on this reaction in 6.1 mM sanguinarine solution at pH
On the basis of the thermodynamic cycle in Scheme 3, the
equilibrium constant of SAOH binding to CB7, KSAOH = [SAOH–
1
2.8 using excitation at 329 nm. The transformation of SAOH
was followed by the detection of its fluorescence intensity at 480
nm. Fig. 8 shows that the gradual increase of CB7 concentration
decreases the reaction conversion. This effect indicates that the
encapsulated excited guest is protected against interaction with
oxygen. Good linear correlations were obtained between the
reciprocal fluorescence intensity and irradiation time (inset in
Fig. 8) indicating that the disappearance of SAOH followed second
CB7]/{[SAOH][CB7]} can be estimated from the measured pK
a
=
1
4 - pK and pK ¢ = 14 - pK ¢ values and the equilibrium constant
b
a
b
+
of SA –CB7 1 : 1 complex formation (K
relationship:
1
) using the following
(4)
K
SAOH = K
1
K
b
/K ¢
b
-
1 -1
order kinetics. From the slopes, 840, 400 and 270 M s were
derived for the apparent rate constant of SAOH decomposition
in the presence of 0, 433 and 881 mM CB7. As photooxidation
of SAOH is probably a multistep reaction, CB7 may also affect
the rate constant of secondary reactions and the concentration
of reactive intermediates. Considering the above estimated low
fraction of the complexed SAOH under the experimental condi-
tions of photoirradiation, the inhibition caused by CB7 is efficient
since after 20 min reaction time, more than twice as large amount
of SAOH remains unreacted in the presence of CB7 as in water.
Unfortunately, the limited solubility of CB7 does not permit of
the complete complexation of SAOH.
Scheme 3 Thermodynamic cycle of OH- addition and 1 : 1 inclusion
complex formation of sanguinarine.
From the pK
a
and pK
a
¢ values determined above, K
b
= 1.38 ¥
-
7
-1
-4
-1
1
0 M and K
b
¢ = 6.76 ¥ 10 M can be derived. The substitution
1
068 | Org. Biomol. Chem., 2011, 9, 1061–1070
This journal is © The Royal Society of Chemistry 2011

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excited the samples, and the fluorescence decays were detected with
a Hamamatsu R3809U-51 microchannel plate photomultiplier,
which was connected to a Picoquant Timeharp 100 electronics (36
ps/channel time resolution). Time-resolved data were analyzed by
a nonlinear least-squares reconvolution method using Picoquant
FluoFit software. Molecular modeling calculations were carried
out with RM1 method using HyperChem 8.0 program (Hypercube
Inc., Gainesville, FL). NMR experiments were carried out on a
1
Varian NMR System (600 MHz for H) spectrometer. Samples
1
were placed into 5 mm NMR tubes. H chemical shifts were
DMSO
referenced to 7 volume% d
6
-DMSO as internal standard (d1H
=
2
.50 ppm). High purity deuterium oxide (99.9 atom%) solvent
was purchased from Sigma-Aldrich Inc. Solvent suppression was
performed by the standard PRESAT sequence. Spectra were
processed by the spectrometer software package: VnmrJ 2.2C.
Unless otherwise mentioned, NMR measurements were carried
out at 283 K.
Fig. 8 Diminution of SAOH concentration with reaction time detected
by the fluorescence intensity change upon irradiation at 329 nm. The initial
solutions contain 6.1 mM sanguinarine, 0.06 M KOH and 0 (᭹), 0.43 (ꢀ)
or 0.81 mM (ꢀ) CB7. The inset shows that the photooxidation of SAOH
follows second order kinetics with CB7 concentration dependent rate.
Acknowledgements
The authors very much appreciate the support of this work by the
Hungarian Scientific Research Fund (OTKA, Grant K75015). The
support of the Hungarian GVOP-3.2.1.-2004-04-0210/3.0 and
KMOP-1.1.2-07/1-2008-0002 grants are gratefully acknowledged.
Conclusions
+
The iminium form of sanguinarine (SA ) produces inclusion com-
plex with not only one but also two CB7. In the stable 1 : 1 complex,
the benzodioxole part of the isoquinoline moiety is encapsulated
in the hydrophobic core of CB7, and the positive charge of the
heterocyclic ring interacts with the oxygens of the macrocycle.
Although the examined natural alkaloid can be embedded only
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This journal is © The Royal Society of Chemistry 2011