Spectroscopic studies of brilliant cresyl blue/water-soluble sulfonated calix[4]arene complex

Article abstract of DOI:10.1366/0003702981944760

Water-soluble sulfonated calix[4]arene (SCX4) was used as the host molecule for the guest dye(probe), Brilliant Cresyl Blue (BCB), which is commonly employed for studies of various biological systems. Absorption and fluorescence techniques were employed for the analysis of this system. The formation of a complex between the BCB dye and SCX4 results in a decrease in the BCB fluorescence intensity. A study of the changes in absorption and of temperature effects on the fluorescence of BCB/SCX4 was conducted. The binding constant for BCB/SCX4 at room temperature was estimated to be 8.49×10³ M^-1.

Full text of DOI:10.1366/0003702981944760

Spectroscopic Studies of Brilliant Cresyl Blue/Water-Soluble
Sulfonated Calix[4]arene Complex
Â
Ä
YULING ZHANG,* TUYET H. PHAM, MONTSERRAT SANCHEZ PENA,
REZIK A. AGBARIA,² and ISIAH M. WARNER*
Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803
Water-soluble sulfonated calix[4]arene (SCX4) was used as the host
highly soluble in water and can potentially be used as
mimics of enzyme-substrate complexes.25 The p-sulfo-
nated-calixarenes have been found to be a potent blocker
of outwardly rectifying chloride channels (ORCCs) with
subnanomolar inhibition constants26 resulting in the de-
velopment of chloride channel modulators. Further, an
electrode modi®ed by sulfonated calixarenes was report-
ed to be immobilized into a polypyrole ®lm to produce
a new ion-sensitive layer.27
molecule for the guest dye(probe), Brilliant Cresyl Blue (BCB),
which is commonly employed for studies of various biological sys-
tems. Absorption and ¯ uorescence techniques were employed for
the analysis of this system. The formation of a complex between the
BCB dye and SCX4 results in a decrease in the BCB ¯ uorescence
intensity. A study of the changes in absorption and of temperature
effects on the ¯ uorescence of BCB/SCX4 was conducted. The bind-
ing constant for BCB/SCX4 at room temperature was estimated to
1
be 8.49 3 103 M2
.
Along with the many reports on the applications of
calixarenes, many fundamental studies with these supra-
molecules have been noted. It is well established that the
cylindral-shaped calixarenes of varying cavity sizes can
form a variety of host±guest types of inclusion complexes
similar to cyclodextrins.1,28 However, calixarene host mol-
ecules have a unique composition that includes benzene
Index Headings: Water-soluble calixarene; Fluorescence; Absorp-
tion; Brilliant Cresyl Blue.
INTRODUCTION
Calixarenes1,2 are cyclic phenol oligomers linked by
ethylene groups. Although the usual forms of calixarenes
are insoluble in aqueous solution, a variety of calixarenes
with various solubilities can be obtained by synthetic
methods. Several water-soluble derivatives (e.g., sulfon-
ato,3±7 amino,8,9 nitro,10 carboxyl,11 and phosphonato-ca-
lixarenes12) have been synthesized. There have been
many reports about the application of calixarenes. One of
those involves the use calixarenes for extraction of acti-
nides,13±17 such as uranium and thorium, and lantha-
nides.14,16,17 Calixarenes are also used for transport studies
through supported liquid membranes with high ef®cien-
cy.18,19
Calixarenes have shown many applications not only in
the area of chemistry but also in biological systems. Re-
cently, several important articles have been published.
For example, we reported our synthesis of novel chiral
N-acylaminocalix[4]arene,9 and we employed this com-
pound as a pseudo-stationary phase for capillary electro-
kinetic electrophoresis to resolve an enantiomeric mix-
ture.20 One of the latest advances from another research
groups, which provide
groups for hydrogen bonding. Moreover, some calixa-
renes have polar functional groups and therefore dipole±
±
interaction, and hydroxyl
p p
dipole interactions. It has been clearly stated that
±
p p
interaction, hydrogen bonding, and dipole±dipole inter-
actions are extremely important for understanding bio-
logical interactions.29 The most attractive characteristic of
calixarenes is that they are comprised of phenol groups
that provide
±
p p
interactions.
The photophysics of calixarenes has been investigat-
ed3±6 because of their phenol chromophoroic units. It has
been shown that phenolate calixarenes have some dis-
advantages when polycyclic aromatic hydrocarbons are
used as ¯ uorescent probes because calixarenes can absorb
and emit electromagnetic radiation. To avoid absorption
problems with calixarenes, ¯ uorescent probes which ab-
sorb in the visible or near-infrared region are appropriate
for complexation studies. Examples of such ¯ uorescent
probes include Auramine O (AuO)30 and Brilliant Cresyl
Blue (BCB).
groups involves the synthesis of 1,1 -binaphthyl ca-
9
The BCB dye, which was ®rst synthesized in the early
1950s, is a water-soluble cationic and basic oxazine
dye.31±33 There have been many reports asserting the im-
portance of BCB as a stain marker in biological systems.
For example, this dye was initially used for staining liv-
ing cells.31±34 More recently, BCB has been applied as a
redox mediator for the study of protein±dye complexa-
tion,35 and has also been used to probe adsorption af®nity
to silica.36
lix[4]arene derivatives and their applications in the design
of molecular sensors to achieve visual discrimination be-
tween enantiomers.21 Calixarenes have also been reported
to show host-mediated potency in experimental tubercu-
losis.22,23 In another report, calixarene esters were used
for channel mimetic sensing membranes.24
Among the many different calixarene derivatives, the
p-sulfonated calixarenes provide not only a hydrophobic
environment (benzene rings) but also hydrophilic head
groups (SO32). The sulfonated calixarene derivatives are
By examining the structure of BCB, it is noted that
BCB provides electrostatic interaction in addition to
±
p p
interaction, dipole±dipole interaction, and hydrogen
bonding. Since our p-sulfonated calix[4]arene is nega-
tively charged and BCB is cationic, it should produce a
strong complex with BCB. In this paper, we examine the
Received 4 October 1997; accepted 6 March 1998.
* Authors to whom correspondence should be sent.
² Present address: Livermore Lawrence National Laboratory, Liver-
more, California 94550.
0003-7028 / 98 / 5207-0952$2.00 / 0
q 1998 Society for Applied Spectroscopy
952
Volume 52, Number 7, 1998
APPLIED SPECTROSCOPY

FIG. 1B. Three-dimensional structure of p-sulfonated calix[4]arene
(SCX4).
FIG. 1A. Chemical structures of p-sulfonated calix[4]arene (SCX4),
monomer (MH), and ¯ uorescent probe (BCB).
the pH increases, the absorbance decreases, producing a
peak at 620 nm, which gradually shifts about 16 nm to-
wards the red. Thus, the absorptivities are sensitive to the
pH of the environment. However, no single isosbestic
point was observed. It was visibly observed that the blue
color began to fade completely for the samples at pH
values of 10.5 and 11.6 and the blue color could not be
restored by readjusting the pH back to acidic condition.
Hence, it was concluded that the BCB dye must decom-
binding properties of p-sulfonated calix[4]arene as a host
molecule and BCB as a ¯ uorescence probe. The p-sul-
fonated calix[4]arene (SCX4), and the monomer of the
calixarene, p-sulfonated phenol (MH), have been used for
comparison of spectroscopic properties. The chemical
structures of these compounds and BCB are shown in
Fig. 1A. In addition, a three-dimensional structure of p-
sulfonated calix[4]arene is displayed in Fig. 1B. Our ab-
sorption and ¯ uorescence data are discussed in terms of
complex formation between the host calixarene and the
guest BCB molecules.
EXPERIMENTAL
Materials. The p-sulfonated-calix[4]arene was pur-
chased from Spectrum Chemical (MFG.CORP., NJ). Bril-
liant Cresyl Blue was purchased from Aldrich Chemical
Company (Milwaukee, WI) and used without further pu-
5
ri®cation. A 6.0
102 M stock solution of BCB was
prepared in deionized water. In all experiments, the BCB
3
6
concentration was held constant at 6.0
102 M, and the
3
pH of the samples are as stated in the text. Fluorescence
emission spectra were obtained with an excitation wave-
length of 548 nm. Buffers were prepared by following a
procedure described by Perrin and Dempsey.37
Apparatus. Steady-state ¯ uorescence measurements
were acquired with a SPEX-Fluorolog Model F2T21I
spectro¯ uorometer equipped with a cell compartment,
thermostated by use of a VWR Model-1160 constant tem-
perature circulator. The excitation and emission band-
widths were set at 5 and 3 nm, respectively. Absorption
spectra were recorded on a Shimadzu UV-3101PC scan-
ning spectrophotometer. All spectroscopic analyses were
conducted at room temperature unless otherwise noted.
RESULTS AND DISCUSSION
Spectroscopic Studies of BCB. Absorption of BCB
Dye. As shown in Fig. 1, BCB contains three amine
groups, which include a primary amine on the benzenoid
structure ( -NH ), a tertiary amine (R N), and a quater-
f
2
3
nary ammonium salt (R4N1 ). The absorption spectra of
BCB at various pH values from 2.0 to 11.6 were collected
and are shown in Fig. 2. The spectra in Fig. 2A were
acquired after the samples had equilibrated for 24 h. As
6
FIG. 2. (A) Absorption spectrum of BCB (6.0
102 M) at various
3
pH values measured after 24 h. (B) Absorption spectrum of BCB (6.0
102 M) at various pH values measured after 30 min.
6
3
APPLIED SPECTROSCOPY
953

6
6
FIG. 3. A plot of the absorption of BCB (6.0
(m); 8.0 (n); 9.0 (V); and 10.0 ( ) vs. time.
102 M) at pH
7.0
FIG. 4. Fluorescence of BCB (6.0 102 M) in the presence of various
3
5
3
concentrations of ethanol (EtOH) (%): (1) without EtOH; (2) 10%; (3)
20%; (4) 30%; (5) 40%; (6) 50%; (7) 60%; and (8) 70%.
1
pose at these basic pH values. Therefore, the absorption
spectra of BCB at various pH values were collected 30
min after sample preparation (Fig. 2B).
and the hydrophobicity of the microenvironment. In ad-
dition, the presence of EtOH will also weaken the hy-
drogen bonding between the nitrogen on the heterocyclic
ring of BCB and the bulk solvent molecules. A red shift
of the ¯ uorescence spectrum is observed.
By comparison of Figs. 2A and 2B, it is evident that
the decrease in the absorptivity in Fig. 2B is much less
than that in Fig. 2A. Again, it can be concluded that the
decrease in absorptivities is due to the decomposition of
the BCB dye, because there was about a 30 min interval
between the time of preparation until the time of mea-
surement of each sample. In order to investigate the cause
of this decrease, more sophisticated experiments were
conducted. These experiments employed the kinetic
mode of the Shimadzu UV spectrophotometer to monitor
the absorbance at a peak of 625 nm after sample prepa-
ration. The samples at pH values of 7.0, 8.0, 9.0, and
10.0 were monitored to generate Fig. 3, which demon-
strates that the absorption of BCB at pH
7.0 and 8.0
5
was evidently not altered with time. In contrast, the ab-
sorbance of BCB at pH values of 9.0 and 10.0 decreases
with time, indicating that the BCB dye is not stable at
pH values higher than 8.0. We have used a pH of 7.0 for
our probe studies. In general, the pK of a -NH group
a
f
2
is about 6.5. However, our experimental results do not
show a change in the absorption spectra over the range
of pH
2.0±8.0. This observation suggests that proton-
5
ation of BCB on the ±NH2 does not alter the absorption
of the BCB dye.
Fluorescence of BCB and the Effects of Alcohol. The
¯ uorescence spectra of BCB were acquired at various pH
values. Two peaks at 614 nm (peak I) and 642 nm (peak
II) are evident in the spectrum at pH 7.0, and the BCB
¯ uorescence does not change in terms of the relative vi-
brational intensities of the spectra at pH values between
2.0 and 8.0.
We have also examined the effects of EtOH on the
BCB ¯ uorescence (Fig. 4). The concentrations of EtOH
were varied from zero to 70% (v/v). Note that the ¯ uo-
rescence intensity around 642 nm increases dramatically
as the EtOH concentration increases. In addition, the peak
at 642 nm seems to gradually shift to the red by 7 nm
due to the larger increase of peak 642 nm. The increased
¯ uorescence and the decreased peak ratio (I614/I642) sug-
gest that the BCB ¯ uorescence is sensitive to the polarity
6
FIG. 5. (A) Fluorescence spectra of BCB (6.0
102 M) in the pres-
3
ence of SCX4 in the concentration range from 0.0 mM (1), 0.1 mM
(2), 0.2 mM (3), 0.4 mM (4), 0.6 mM (5), and 0.7 mM (6). (B) Fluo-
rescence spectra of BCB (6.0 102 M) in the presence of the monomer
(MH) at concentrations from 0.0 mM (1), 0.4 mM (2), 0.8 mM (3), 1.6
mM, (4), 2.4 mM (5), and 2.8 mM (6).
6
3
954
Volume 52, Number 7, 1998

FIG. 6. Absorption spectra of BCB/SCX4 in the presence of SCX4 at various concentrations from 0.0 mM (1), 0.2 mM (2), 0.4 mM (3), and 0.7
mM (4).
Complexation Studies of SCX4 with BCB. Fluores-
cence of BCB/SCX4. Steady-state ¯ uorescence spectra of
the BCB/SCX4 complex in aqueous solution are shown
in Fig. 5. The ¯ uorescence emission spectra of BCB at
various concentrations of SCX4 were acquired with an
excitation wavelength of 548 nm. It should be noted that
SCX4 does not absorb light at this wavelength. The ¯ uo-
rescence intensity of BCB was signi®cantly reduced by
the addition of SCX4 (Fig. 5A). However, little change
in the ¯ uorescence intensity was observed upon the ad-
dition of the monomer of equivalent concentrations (Fig.
5B). It is evident that the decrease is produced by the
presence of SCX4. As displayed in Fig. 1, calixarene pro-
due to a ground-state alteration as a result of complex
formation. The relatively slight decrease in the absor-
bance in the presence of SCX4 cannot be a reasonable
explanation for the large decrease in the ¯ uorescence in-
tensity.
Temperature Effects. In order to further characterize
the mechanism of the BCB ¯ uorescence reduction in the
presence of SCX4, temperature studies were also con-
ducted. It is well established that dynamic processes
heavily depend upon diffusion. Since higher temperatures
result in larger diffusion coef®cients, the bimolecular
complex formation constants are expected to increase
with increasing temperature. In contrast, increased tem-
perature is likely to result in a decrease in stability of
complexes. Thus, lower values of formation constants are
likely to be observed.
To estimate the binding constants, we measured the
¯ uorescence intensity in the presence and absence of
Ê
SCX4. Since calix[4]arene possesses a diameter of 3 A,
the stoichiometric ratio for this complex is expected to
be 1:1 for calix[4]arene and BCB. This assumption will
be tested by use of a Benesi±Hilderbrand plot.38 First, we
assume that the equilibrium for complex formation is as
follows:
vides rings, hydroxyl groups, and SO 2, which generate
p
interaction, hydrogen bonding, and electrostatic at-
3
±
p p
traction to cations. Such interactions exist in the ground
state (static) as well as in the excited state (dynamic).
This condition provides the possibility of proton transfer,
energy transfer, and charged transfer processes. There-
fore, it is important to determine whether the decrease in
BCB ¯ uorescence is static or dynamic. Additional studies
are necessary to provide this information. Speci®cally,
calixarene concentration effects on the absorbance spec-
tra and temperature effects on the association constants
will be used to investigate the nature of the BCB quench-
ing.38
SCX4
BCB
SCX4 BCB.
(1)
1
The equilibrium constant (K1) for a 1:1 association be-
*
>
Absorption of BCB/SCX4. The absorption spectrum of
BCB with 0.05 mM SCX4 slightly decreases and shifts
to the red region around 3 nm (Fig. 6). Further addition
of SCX4 causes a small decrease in the BCB absorbance
maximum. These absorption changes may be attributed
tween SCX4 and BCB is given by
[SCX4 BCB]
*
K1
(2)
5
[SCX4][BCB]
to
±
interactions, hydrogen bonding, and electrostatic
where [SCX4] and [BCB] are the respective concentra-
tions of SCX4 and BCB at equilibrium. The
[SCX4 BCB] is the equilibrium concentration of the in-
*
clusion complex for a given SCX4 concentration.
p p
interactions between BCB and SCX4. These interactions
result in a perturbation of the ground state of BCB, sug-
gesting that the decrease in BCB ¯ uorescence is likely
APPLIED SPECTROSCOPY
955

In general, the initial concentration of the host mole-
cule (SCX4) needs to be much larger than that of the
guest molecule (BCB). In our experiments, the concen-
tration of SCX4 is much larger than that of the complex,
tantly, the binding constants are much larger than those
of cyclodextrins with azo dyes, which are reported to be
1
41,42
approximately 300 M2
The binding constants of the complexes at various tem-
.
i.e., [SCX4]
[SCX4 BCB]. On the basis of experi-
peratures are calculated by using the Benesi±Hilderbrand
1
*
&
mental results, the ¯ uorescence of BCB is reduced upon
inclusion into the host. In other words, the reduced ¯ u-
orescence is caused by the formation of a complex be-
tween SCX4 and BCB. Therefore, the equilibrium con-
centrations of SCX4 and BCB are proportional to the
magnitude of the reductions in the ¯ uorescence intensi-
ties. A reasonable relationship between the equilibrium
concentrations and the ¯ uorescence peak areas is as fol-
lows:
equations. The association constants are 1.09
1
104 M2
,
3
8.49
103 M21, and 6.95
103 M2 for the temperature
at 15, 23, and 30 C, respectively. At higher temperatures,
3
3
8
the ¯ uorescence reduction is less evident than at lower
temperatures. This observation, again, indicates that a
static process is occurring within the system. Therefore,
the decrease of BCB ¯ uorescence in the presence of
SCX4 is likely due to complex formation (static mecha-
nism).
The relationship of the binding constants and the tem-
perature is thermodynamically given by
[BCB]0
(A0
(A0
A )
1
2
2
(3)
5
[SCX4 BCB]
A)
*
G
RT ln K .
1
(8)
(9)
D
5 2
where [BCB]0 is the initial analytical concentration of
BCB, and the parameters A0 and A1 denote the ¯ uores-
cence peak areas between 580 nm and 680 nm for BCB
dye in pure aqueous solution and in completely com-
plexed BCB, respectively; A is the peak area at a given
SCX4 concentration.
Rearrangement of Eq. 8 produces
G 1
H 1
S
D
R
D
D
ln K1
5 2
5 2
1
T
R T
R
1 2
the enthalpy change of formation; S refers to the entro-
where K1 represents the association constant, G refers
D
D
to the standard Gibbs free-energy change; H refers to
Rearrangement of Eqs. 2 and 3 produces the following:
D
1
1
1
(4)
5
1
py change of formation; R refers to the gas constant; and
T is the temperature. A plot of ln K1 against 1/T is linear
(not shown). Values of G, H, and S obtained from
A0
A
k1(A0
A )[SCX4]
1
A0
A
1
2
2
2
0
where [SCX4]0 is the initial analytical concentration of
A) vs. 1/[SCX4] should
0
D
D
D
SCX4, and a plot of 1/(A0
provide a straight line.
Similarly, if we assume a 2:1 complex, then
2
the linear line are 5.32 Kcal/mol, 5.20 Kcal/mol, and
2
0.40 mol deg/cal (e.u.), respectively.
2
CONCLUSION
2SCX4 BCB (SCX4) BCB.
5
(5)
1
*
2
On the basis of ¯ uorescence and absorption measure-
ments of the complexation of BCB and calixarene, sev-
eral important points are noted. First, the ¯ uorescence
and the absorption of the cationic dye BCB are stable
between pH values of 2.0 and 8.0, and virtually no
changes are observed at these pH values. In contrast, the
¯ uorescence is sensitive to the microenvironment, e.g.,
polarity and hydrophobicity. Second, the reduction of
BCB ¯ uorescence in the presence of SCX4 is a static
process resulting from guest±host inclusion, rather than
excited-state reactions. These conclusions are reached by
use of absorption and temperature studies.
The overall equilibrium constant (K2) is then expressed
as
[(SCX4)2 BCB]
*
K2
(6)
5
[BCB][SCX4]2
with the assumptions [SCX4]0
[SCX4 BCB]. Thus
[(SCX4)2 BCB]
*
&
&
*
1
1
1
(7)
5
1
2
A0
A
K2 (A0
A )[SCX4]
2
A0
A
2
2
2
2
0
2
and a plot of 1/(A0
straight line.
A) vs. 1/[SCX4] should give a
0
2
Use of this approach allows us to graphically deter-
mine the stoichiometry of the complexes used in our
studies. Benesi±Hildebrand plots38 (double-reciprocal
plots based on Eqs. 4 and 7) of the ¯ uorescence data are
not displayed in this article, but just explained in the text
ACKNOWLEDGMENTS
This work was supported by the Division of Chemical Sciences, Of-
®ce of Basic Energy Sciences, Of®ce of Energy Research, U.S. De-
partment of Energy (Grant DE-FG05-93ER14367). I.M.W. also ac-
knowledges the Philip W. West Endowment for partial support of this
research.
as follows. A linear relationship exists when 1/(A0
is plotted against 1/[SCX4], and a convex curvature is
A)
2
2
obtained when 1/(A0
A) is plotted against 1/[SCX4]
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Volume 52, Number 7, 1998

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