Exploring the effect of supramolecular structures of micelles and cyclodextrins on fluorescence emission of local anesthetics

Article abstract of DOI:10.1039/c0pp00286k

Benzocaine (ethyl 4-aminobenzoate, 4) and its derivatives ethyl 2-aminobenzoate, 2, and ethyl 3-aminobenzoate, 3, were found to form association complexes with supramolecular structures of micelles and cyclodextrins (CDs). The fluorescence emission of 2, 3 or 4 dissolved in the pseudo-micellar phase or included into α-, β-, or γ-CD cavity increases dramatically with respect to that observed in only water. High percentages of organic solvents like dioxane, acetonitrile, DMSO in the aqueous solution lead to a similar effect. The stability constants of the complexes formed between these drugs and cyclodextrins have been determined. In neutral or acid medium, a 1:1 stoichiometry for drug: CD complexes have been found, whereas in alkaline medium 1:2 stoichiometry was also detected in some cases. Kinetic studies of both the nitrosation of the amine group and the alkaline hydrolysis of the ester function was employed to infer the conformation of the complexes as well as to evaluate their stability constants. Theoretical calculations to optimize the molecular structure of 2, 3 and 4 allow us to propose possible geometries of the complexes that are in agreement with the experimental data. The Royal Society of Chemistry and Owner Societies 2011.

Full text of DOI:10.1039/c0pp00286k

View Article Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links
Photochemical &
Photobiological Sciences
Cite this: Photochem. Photobiol. Sci., 2011, 10, 531
www.rsc.org/pps
PAPER
Exploring the effect of supramolecular structures of micelles and
cyclodextrins on fluorescence emission of local anesthetics†
Emilia Iglesias*
Received 22nd September 2010, Accepted 25th November 2010
DOI: 10.1039/c0pp00286k
Benzocaine (ethyl 4-aminobenzoate, 4) and its derivatives ethyl 2-aminobenzoate, 2, and ethyl
3-aminobenzoate, 3, were found to form association complexes with supramolecular structures of
micelles and cyclodextrins (CDs). The fluorescence emission of 2, 3 or 4 dissolved in the
pseudo-micellar phase or included into a-, b-, or g-CD cavity increases dramatically with respect to that
observed in only water. High percentages of organic solvents like dioxane, acetonitrile, DMSO in the
aqueous solution lead to a similar effect. The stability constants of the complexes formed between these
drugs and cyclodextrins have been determined. In neutral or acid medium, a 1 : 1 stoichiometry for drug
: CD complexes have been found, whereas in alkaline medium 1 : 2 stoichiometry was also detected in
some cases. Kinetic studies of both the nitrosation of the amine group and the alkaline hydrolysis of the
ester function was employed to infer the conformation of the complexes as well as to evaluate their
stability constants. Theoretical calculations to optimize the molecular structure of 2, 3 and 4 allow us to
propose possible geometries of the complexes that are in agreement with the experimental data.
microstructures, such as micelles, that can be used as drug carriers
Introduction
and in the study of the physicochemical properties of membrane-
associated drugs.6–8 In the same sense, the hydrophobic cavities of
cyclodextrins, in combination with their different size, are capable
of selectively incorporating organic guest or drug molecules
to form inclusion complexes in aqueous solution, which show
chemical and spectral properties greatly modified in comparison
to that observed in the bulk water phase.9–13
In previous works we have studied the interactions of some local
anesthetics, including novocaine, tetracaine and procainamide,
with closed environments of micelles and cylodextrins under
different experimental conditions.14–16 The common characteristic
of these LA is that the amine group is in the para-position with
respect to the ester (or amide) group. We have found that the
most appropriate experimental conditions that yield the highest
interaction between the cyclodextrin and the LA correspond to
both neutral LA and cyclodextrin, that is, when the van der Waals
interactions and the hydrophobic effect that excludes the LA from
the bulk water phase reach the maximum level.14
The aim of this work is the analysis of the effect of both the
local anaesthetic geometry and intra- or inter-molecular hydrogen-
bonding interactions in the association of drug molecules to closed
environments. For that, we have performed a comparative study
of the three compounds shown in Scheme 1 in the membrane-
mimetic environment existing in micelles or in the cavity of
cyclodextrins. The interaction with micellar interfaces of both
cationic and anionic micelles has been analysed by fluorescence
emission, since it is known that the medium plays an important
role in determining the primary photoprocesses of fluorophores in
solution. The characterization of the inclusion complexes formed
Benzocaine, ethyl 4-aminobenzoate (4), and its derivatives ethyl 2-
aminobenzoate (2) and ethyl 3-aminobenzoate (3) are interesting
ester-type local anesthetics (LA) used in pharmaceutical formula-
tions for external and mucous applications, with unique features
such as the lack of an amine group ionisable near physiological pH,
thus being uncharged substrates. Nevertheless, their parenteral
administration is restricted by their limited water solubility.1,2
Besides, it is generally believed that interaction of the anesthetic
molecules with membrane lipids or membrane proteins leads to
the inactivation of neuronal nerve transmission, a key feature
on anesthesia mechanism. Therefore, research with anesthetic
molecules has two main objectives: first, it is interesting to know
their behavior in the presence of membranes and membrane-
like environments, and, second, to look for new formulations
that control drug delivery in biological systems, prolonging the
anesthetic effect and reducing their toxicity or side effects.3
Many biological processes that occur at the surface of biological
membranes or within their hydrophobic moiety can be imitated
in vitro by using simple systems of supramolecular structures
(like micelles, cyclodextrins, polymer, DNA, etc.). Association
of LA to these structures provides a way to increase solubility,
stability and bioavailability of drugs.4,5 In water above the critical
micelle concentration, cmc, self-aggregating surfactants form
Departamento de Qu´ımica F´ısica e E. Q. I. Facultad de Ciencias. Universidad
de La Corun˜a., 15008-La Corun˜a, Spain. E-mail: qfemilia@udc.es
† Electronic supplementary information (ESI) available: Absorption and
emission spectra of compounds 2, 3, and 4 dissolved in water, micelles, and
CDs; reaction spectra of2, 3, and4 in water. See DOI: 10.1039/c0pp00286k
This journal is
The Royal Society of Chemistry and Owner Societies 2011
Photochem. Photobiol. Sci., 2011, 10, 531–542 | 531
©

View Article Online
Scheme 1 Molecular structures of the local anesthetics.
between 2, 3, or 4 and a-, b-, or g-CD has been performed
from either fluorescence or reactivity studies. In acid medium,
i.e. in conditions of neutral CD host, the nitrosation of the amine
group was also investigated; whereas, in alkaline medium, i.e. in
conditions of anionic CD host, the hydrolysis of the ester group
was analysed, too. Different behaviour was observed in acid or
in alkaline medium. The results were interpreted on the basis of
different guest–host binding modes and supported by theoretical
guest-structure optimization
nm, respectively. Data acquisition and analysis of fluorescence
spectra were performed with the Fluorescence Data Manager
Software supported by Aminco.
Time-resolved fluorescence measurements were performed us-
ing the time-correlated single photon counting technique with an
Edinburgh Instruments as described in the literature.17
Results and discussion
1. Studies in homogeneous and microheterogeneous media
Experimental
Absorption and emission spectra of the three anesthetics have been
studied in solvents of various polarities and hydrogen bonding
capability. The absorption spectrum of 2, 3, or 4 in all solvents, or
in water under different acidity conditions, is characterized by two
bands; a larger wavelength band (LW) with a maximum around
300 nm and a shorter wavelength band (SW) at 220–240 nm.
Both SW and LW bands are ascribed to p→p* transition of the
benzenoid system and suffer small solvent shift. The protonation
of H2N group in, for instance, compound 2 (pKa ~ 2.19)18 causes
a strong decrease in the absorption intensity of the LW, as well as
a pronounced blue shift of the SW with respect to the neutral
form; Fig. 1a shows representative results. The effect of acids
observed with the other two isomers is much smaller; both the
absorption and emission wavelength maxima of 2 are largely red
shifted in every medium when compared to 3 or 4 because of the
intramolecular hydrogen bonding, Fig. 1b. The red shift increases
according to the sequence 4 < 3 < 2, which indicates that the
position of the substituent in the phenyl ring is the key factor
for the absorption and emission behavior, because of the different
electronic densities of the HOMO on each atom. The relevant data
are listed in Table 1.
All three compounds give only one broad structureless emission
band that shows normal Stokes shift in all solvents. The emitting
chromophore is the benzene ring, and emission occurs from the
(p,p*)S1 state. This fact indicates that the excited and emitting
species are the same. Nevertheless, the Stokes shifts observed for
2 are higher than that of 4 (Fig. 1b), due to the intramolecular
hydrogen bonding in the former, which stabilizes the excited state.
On the other hand, the larger dipole moment of the excited
state than that of the ground state provokes a larger Stokes
shift in polar and/or protic solvents, such as water or methanol,
than in apolar and/or aprotic solvents such as dioxane. Both
excitation and emission spectra of 2 recorded in various solvents
are displayed in Fig. 2a. While the fluorescence emission yield
in water of both 3 and 4 is practically negligible, the 2 isomer
shows a reasonable good fluorescence yield; however, it is strongly
Materials
The local anesthetics investigated in this work were of the
highest purity available (>99%) and were used without further
purification. Solvents of spectrophotometric grade were used as
received. Cyclodextrins and surfactants were purchased from
Sigma of the maximum purity and used as received. The other
reagents, acetic acid, sodium acetate, sodium nitrite, or sodium
hydroxide, were commercial products of the maximum purity. The
concentration of the aqueous solution of NaOH was determined
by titration against standards, whereas the acidity of aqueous
buffered solutions was obtained from pH measurements. The
reported [buffer] refers to the total buffer concentration.
Methods
The UV-vis spectra and kinetic experiments were recorded with a
double beam UV-vis spectrophotometer fitted with thermostatted
cell holder at 25 ◦C. Data acquisition of both UV-vis spectra
and kinetics were performed with software supported by the
manufacturer and converted to ASCII format for their analysis
with common packet programs. Kinetic measurements were
performed under pseudo-first order conditions with the local
anaesthetic as the limiting reagent; we applied the integration
method to record the absorbance–time (A–t) data during at least
2.5 half-lives and fit the data to the first-order integrated rate
equation, At = A• + (Ao - A•) ¥ exp(-kot) by non-linear regression
analysis, to obtain the pseudo-first order rate constant, ko, and
A• (the absorbance at infinite time) and (Ao–A•) as optimizable
parameters. In every experiment perfectly first-order behaviour
was observed (r > 0.9999).
Steady-state fluorescence measurements were performed with
an Aminco-Bowman Series 2 spectrofluorometer at 25 ◦C. Emis-
sion intensity was detected at right angles by exciting optically thin
solutions (A £ 0.15 for a 10 mm path length) in the region 270–
350 nm. Excitation and emission slits were fixed at 4 and 2 (or 4)
532 | Photochem. Photobiol. Sci., 2011, 10, 531–542
This journal is
The Royal Society of Chemistry and Owner Societies 2011
©

View Article Online
Table 1 Spectroscopic properties of local anesthetics ethyl 2-aminobenzoate, 2; ethyl 3-aminobenzoate, 3, and ethyl 4-aminobenzoate or benzocaine, 4,
in different solvents
Absorption spectrum
Aqueous medium
Emission spectrum
LA
l1/nm (log e)
l2/nm (log e)
Solvent
era
lex/nm
l
em/nm
Stokes shift/cm-1
2
neutralb
244 (3.89)
243 (3.90)
226 (4.06)
326 (3.62)
326 (3.63)
273 (3.07)
dioxane
MeCN
DMSO
MeOH
water
2.21
345
341
349
345
339
393
395
402
407
422
3540
4010
3780
4420
5800
mild acidc
strong acidd
35.94
46.45
32.66
78.30
3
4
neutralb
242
shoulder
226 (4.05)
312 (3.36)
312 (3.33)
312 (3.01)
dioxanee
MeCNe
DMSOe
water
—
—
—
—
320
320
343
320
406
409
418
460
6620
6800
5230
9510
mild acidc
strong acidd
neutralb
219 ((4.09)
shoulder
226 (4.18)
284 (4.24)
284 (4.24)
275 (3.48)
dioxane
MeCN
DMSO
MeOH
water
2.21
295
295
305
300
305
328
331
339
346
356
3410
3690
3290
4430
4700
mild acidc
strong acidd
35.94
46.45
32.66
78.30
a Relative dielectric constant. b Water. c Buffer solution of acetic acid–acetate pH 4.65. d HCl 0.04 M. e >95% v/v.
Fig. 1 (a) Absorption spectra of 2 0.19 mM in ( ◊ ◊ ◊ ) neutral medium; (---) mild acid medium of acetic acid-acetate buffer, and in (—) strong acid medium
of HCl; (b) normalized fluorescence excitation (ex) and emission (em) spectra of ( ) benzocaine and of (—) 2 in methanol. See Table 1 for excitation and
᭺
emission wavelength values.
enhanced in apolar solvents. Fig. 2b shows comparative results of
the fluorescence intensity variation against the solvent percentage
for compounds 2, 3, and 4 in dioxane, as a representative solvent.
These emitting molecules are sensitive to the hydrogen-bonding
character of the solvent. The fluorescence intensity of all them
increases in going from protic (e.g. water) to polar aprotic media
(e.g. dioxane) as clearly manifest the results of this figure, with
drastic effects in benzocaine and in the meta-amine isomer. Two
different ground-state conformers (I and II of Scheme 1) can
be possible for ethyl 2-aminobenzoate, whereas only conformer
I is possible in compounds 3 and 4. However, 2 displays only
one emission band and normal Stokes shift, which excluded
intramolecular proton transfer in the excited state. The contrary
was observed in, for instance, methyl salicylate fluorophore19 due
to the stronger O ◊ ◊ ◊ H ◊ ◊ ◊ O intramolecular hydrogen bond. The
existence of isomer II in ethyl 2-aminobenzoate contributes to the
high fluorescence yield observed in water in comparison to the
meta- and para-amine isomer.
The time-resolved fluorescence of 2 was examined in neutral
aqueous medium. Analysis of the fluorescence decay17 shows good
fit to biexponential function affording the lifetimes t1 = 1.800
0.004 ns and t2 = 6.5 0.1 ns with preexponential factors all
positive and the relative amplitude as a1 = 33 ¥ 10-3 (93%) and
a2 = 1 ¥ 10-3 (7%). The two emitting species were attributed to I
and II conformers of 2. The form I predominates, but conformer
II is more stable. Attempts to measure the lifetimes of 3 and 4
gave no reliable data due to the low quantum yield observed in
water.
The properties of the aqueous medium can also been drastically
modified in the presence of micelles that are generated by self-
aggregating surfactants to produce microheterogeneous media.
For the sake of simplicity, we used both anionic micelles of sodium
dodecyl sulfate (SDS) and cationic micelles of tetradecyltrimethy-
lammonium bromide (TTABr) in neutral aqueous medium (no
acid or base have been added). Addition of 0.025 M SDS or
0.030 M TTABr very slightly affects the absorption spectrum of
This journal is
The Royal Society of Chemistry and Owner Societies 2011
Photochem. Photobiol. Sci., 2011, 10, 531–542 | 533
©

View Article Online
Fig. 2 Excitation and emission spectra of ethyl 2-aminobenzoate 42 mM in different solvents: (a) non-normalized spectra in (1) water, (2) dioxane, (3)
acetonitrile, (4) MeOH, (5) DMSO; (b) fluorescence emission intensities as a function of the percentage (v/v) of dioxane in the aqueous solution of
compounds 2, ethyl 2-aminobenzoate; 3, ethyl 3-aminobenzoate, and 4, ethyl 4-aminobenzoate.
Fig. 3 Plot of fluorescence emission intensities as a function of (a) TTABr concentration and (b) SDS concentration for (᭢) ethyl 2-aminobenzoate, 2;
(
) benzocaine, 4, and (᭡) ethyl 3-aminobenzoate, 3. The inset shows the lowest two curves at full y-axis scale.
᭹
2, shifting both SW and LW to the red – 4 and 14 nm, respectively,
in TTABr, but only 2 and 4 nm, in SDS (Fig. S1 to S3, ESI†);
nevertheless, the fluorescence intensity increases strongly and the
wavelength emission maxima (lem) shift to shorter wavelength.
Whatever the surfactant, the emission intensity increases with
the surfactant concentration above the cmc (critical micelle
concentration). Representative data are displayed in Fig. 3.
The comparison between micellar media and homogeneous
media of water-solvent mixtures indicates that, while in micelles
the position of the emission maxima (i.e., lemmax)remains invariable
as the surfactant concentration increases just above the cmc20
the probe microenvironment is changing parallel to the solvent
composition.
Incorporation of LA into micelles can be treated as the
partitioning between two phases: the micelles, being visualized
as the micellar pseudo-phase, and the bulk water phase, ac-
cording to the equilibrium process LAw + Dn ꢀ LAm with Km
representing the binding constant of LA to micelles (Dn refers
to the micellized surfactant). The LA dissolved in the aqueous
micellar pseudo-phase displays similar absorption spectrum as
that observed in water; hence, the excitation wavelength neither
changes significantly; however, both excitation and emission
spectra were recorded in every case to find the optimal conditions.
By contrast, the fluorescence emission intensities in both water
and micellar phases, that is Iw and Im respectively, would differ
significantly because, on fluorescence time scales, micelles can
be considered as rigid host systems carrying the solubilized
fluorophore molecules, where the lifetimes (t) and/or fluorescence
yields (f) are different, mainly due to the strong reduction of
solvent quenching. As benzocaine and its 2 and 3 isomers are freely
moving non-covalently-bound fluorophores, it is envisaged that
both parameters increase in the micellar pseudo-phase. Therefore,
(e.g. from 5 mM to 0.29 M in TTABr or from 9 mM to
max
0.27 M in SDS), a gradual blue shift of lem
of more than
435 ¥ 103 cm-1 was observed in water–solvent mixtures (Table 2).
This fact reflects the drastic difference between homogeneous
and microheterogeneous media. Thus, while micellar systems can
be considered as composed of particles, usually much smaller
than the wavelength of light, that can be seen as hydrophobic
pockets with restricted or no access to water molecules, where the
fluorophore incorporated and senses a unique microenvironment,
in homogeneous media of water–solvent mixtures the nature of
534 | Photochem. Photobiol. Sci., 2011, 10, 531–542
This journal is
The Royal Society of Chemistry and Owner Societies 2011
©

View Article Online
Table 2 Maximum wavelength of fluorescence emission and the corresponding intensities measured as a function of the dioxane percentage in the binary
aqueous mixtures and as a function of the surfactant concentration in the aqueous micellar medium, showing the progressive blue shift in homogeneous
media, but the sharp shift in micelles
Homogeneous solvent of dioxane/water
mixtures (ethyl 3-aminobenzoate, 3)
Microheterogeneous solvent: aqueous micellar
medium (ethyl 2-aminobenzoate, 2)
% Dioxane (v/v)
lemmax/nm
IF/a.u.
[SDS]/M
lemmax/nm
IF/a.u.
0
457
453
445
441
437
431
421
406
0.038
0.142
0.530
1.059
1.758
3.933
6.877
8.527
0
421
421
414
414
414
414
0.446
0.541
1.416
2.692
3.378
4.187
16.7
33.3
42.0
50.0
63.0
83.3
96.7
7.47 ¥ 10-3
9.33 ¥ 10-3
0.014
0.028
0.240
[TTABr]/M
0.240
lemmax/nm
409
IF/a.u.
6.728
Table 3 Experimental conditions and fluorescence intensities measured in water, Iw, and optimized values in cationic micelles of TTABr and anionic
micelles of SDS, Im, along with the association constant of the local anesthetic (LA) to micelles, Km, eqn (1)
LA (c/mM)
lex/lem (nm)
Iw/a.u.
Im/a.u.
Km/dm3 mol-1
fm/fw
TTABr (tetradecyltrimethylammonium bromide, cmc 3.5 mM)20
2 (13.5)
3 (13.6)
4 (15.7)
350/409
340/434
300/343
0.406
0.0195
0.1124
6.85 0.10
4.41 0.07
6.00 0.10
250 19
17
23
53
79
6
9
103
SDS (sodium dodecylsulfate, cmc 7.5 mM)20
2 (13.5)
350/413
350/413
326/443
300/347
0.420
0.470
0.028
0.143
4.31 0.07
3.73 0.07
0.622 0.017
0.797 0.010
140 10
145 14
10
8
22
6
2 (13.5) buffera
3 (13.6)
43
75
5
5
4 (15.7)
a Aqueous solution of acetic acid acetate 0.033 M pH 4.55
the enhancement of emission intensity as a function of [surfactant]
is due to different lifetimes of the fluorophore in water (Iw
different Im values obtained at 100% incorporation of the drug
should reflect different locations inside the micelle, i.e. for the
same micelle hydrophobicity, the experimental differences must be
attributed to the fluorescence probe. In this respect, the 2 isomer
is more hydrophobic than the others two due to intramolecular
H-bonding. Therefore, this compound resides deeper inside the
micellar interface where it senses a less polar environment. The
higher value of Km measured for isomer 2 corroborates this
statement and is also supported by larger wavelength shift of the
LW absorption than that observed with 3 or 4 (Fig. S4, ESI†).
The peculiar geometry of 4 allows it to enter lengthwise among
the surfactant monomers in the micelle with a significant degree
of protrusion; which makes the aromatic ring sense an apolar
microenvironment. However, the isomer 3 must reside outside in
the micellar interface in order to avoid strong disruption of the
micelles due to the bulky geometry of this molecule; in fact, the
corresponding association constant takes the smallest value of the
three LA. Even though, in the case of 4 its molecular geometry
facilitates good association to micelles, the interactions (H-
bonding) with water molecules reduce incorporation to micelles;
the observed results are the balance of all these driving forces.
The situation observed with anionic micelles of SDS is quite
different for compound 2 in comparison to 3 and 4. The possibility
of H-bonding formation between the sulfate head groups (–O–
μ
fw[LA]w), at 100% incorporation in micelles (Im μ fm[LA]m), and
at intermediate micelle concentration, (IF μ fw[LA]w + fm[LA]m).
The experimental values of IF measured at each [surfactant]
and depicted in Fig. 3, show a sharp increase at low surfactant
concentration just above the cmc and levels off at high surfactant
concentration. Solid lines were drawn by applying eqn (1), where
[Dn] = [surfactant] - cmc represents the micellized surfactant
concentration. Using non-linear regression analysis, the best
fitting values of Im and Km were determined and are listed in
Table 3, along with the experimental conditions. The ratio Im/Iw
equals the ratio of the fluorescence quantum yield of the bound
(fm) and free molecule (fw); the corresponding enhancement ratio
is listed also in Table 3.
Iw + ImKm[Dn]
1+ Km[Dn]
IF =
(1)
Even though the fluorescence emission measured in water, Iw,
is quite different for 2, 3, and 4 compound (being Iw (3) ꢀ Iw (4)
ꢀ Iw (2) following the ratio of 1 : 6 : 21 approximately), the values
measured in the micellar pseudophase, Im, follows the same trend
but, by contrast, reach quite close levels (the ratio is 1 : 1.4 : 1.6);
in other words, micelles enhance the fluorescence quantum yield
and the enhancement is higher in TTABr than in SDS micelles.
As neither compound can specifically interact with TTABr
micelles (for instance, by electrostatic of H-bonding forces), the
-
SO3 ) of the surfactant monomers and the amino group of the
fluorophore, forces compounds 3 and 4 to reside probably in
the micellar interface, outside the Stern-layer; on the contrary,
This journal is
The Royal Society of Chemistry and Owner Societies 2011
Photochem. Photobiol. Sci., 2011, 10, 531–542 | 535
©

View Article Online
Fig. 4 (a) Excitation and emission spectra of 4 as a function of a-CD concentration. Scans a and b were recorded in the absence of CD and the intensity
was amplified by a factor of 2; the next scans were recorded at [CD] equal to: (c,d)0.75; (e,f )2.25; (g,h)6.0, and (i,j)30 mM. (b) Variation of the emission
intensity as a function of a-CD for (᭡) 2, 10.6 mM; ( ) 3, 46.7 mM; and (᭢) 4, 9.2 mM. Conditions in Table 4.
᭹
the intramolecular H-bonding that is possible in compound 2,
allows this compound to incorporate more inside the micelle.
Nevertheless, if one observes the data in Table 2, the comparison
of the results measured in 0.24 M or either [SDS] or [TTABr],
indicates that, firstly, the emission intensity in SDS (Im = 4.2) is
quite lower than that measured at the same [TTABr] (Im = 6.7), and
secondly, the emission maxima in SDS (lem = 414 nm) is red shifted
with respect to TTABr (lem = 409 nm). Both observations reflect
the more hydrophobic microenvironment sensed by 2 in TTABr
micelles than in SDS micelles. These results evidence the drastic
differences in homogeneous and microheterogeneous media, as
well as the importance of the molecular structure of the LA and
the nature of the interface in the magnitude of mutual interaction.
The time-resolved fluorescence of 2 was examined in aqueous
micellar medium of 0.025 M SDS and 0.030 M TTABr, that is,
according to Fig. 3, in a region where the IF reaches almost the
maximum value. Global deconvolution analysis of the two time
microenvironment of the CD cavity enhances the fluorescence22–24
and decreases the reactivity of the included guest,25 except when
the reaction was CD mediated.26
The absorption spectrum of 2, 3 or 4, recorded in the presence
of cyclodextrins (Fig. S1 to S3, ESI†), shows small red shift in
lmax (<4 nm) and no significant absorbance changes for a reliable
evaluation of their interaction with the CD cavity. By contrast,
the emission spectra show strong variation in the presence of
cyclodextrins; in the same manner, the reactivity of the amine
group towards nitrosation or the hydrolysis of the ester function
is notably affected in aqueous cyclodextrin solutions.
2.1 Fluorescence measurements. The fluorescence intensity
in water of the local anesthetics of Scheme 1 increases gradually
upon addition of either a-, b-, or g-CD, due to the enhanced
dissolution of the guest into the hydrophobic CD cavity, which
indicates the formation of inclusion complexes. The excitation
and emission spectra of 4 recorded as a function of a-CD
concentration are shown in Fig. 4a. Similar spectra were obtained
in any other possible combination of 2, 3, and 4 and a-, b-, or
g-CD. Fig. 4b and 5 display representative plots (of nine possible
combinations) of emission intensity, IF, against [CD].
Under experimental conditions of neutral CD, these plots
describe saturation curves at high CD concentration. This fact
suggests the formation of 1 : 1 inclusion complexes. In this sense,
the stoichiometry of the benzocaine, 4, and b-CD complex was
determined by Job-plot analysis27 as being 1 : 1.28 However, the
most general means for assessing stoichiometry is the constancy
of stability constants as the host concentration is varied, that is, the
success of an assumed stoichiometry model in accounting for the
data. This procedure is followed in the quantitative treatment of
the following experimental data. Then, assuming the formation of
1 : 1 inclusion complex between the LA and CD according to eqn
(2), and taking into account that [CD]o ꢁ [LA]o (i.e. the total CD
concentration is much higher than that of the LA), along with the
mass balance on host [CD]o ª [CD]free and guest [LA]o = [LAw] +
[LA·CD], and the stability constant K11, it can be easy to arrive
2
profiles afforded a good fit (c < 1.1) on the basis of a biexponential
function with lifetimes t1 = 2.28 0.08 ns (5.7%) and t2 = 9.01
0.015 ns (94.3%) in SDS micelles and t1 = 1.4 0.1 ns (1.3%) and
t2 = 10.20 0.01 ns (98.7%) in TTABr micelles. The comparison
with the results obtained in water indicates that the longer-lived
component in the emission, which is the most affected by the
presence of micelles and attributed to the II-form of 2, is the main
species in micellar media.
2. Studies in aqueous cyclodextrin solutions
A third common way to alter the structure of water solvent is
the addition of cyclodextrins. In aqueous solutions, the non-
polar cyclodextrin cavity is occupied by water molecules, which
can be readily replaced by appropriate guest molecules of lower
polarity than water. The driving forces leading to the inclusion
complexation of cyclodextrins were thought to include electro-
static, van der Waals, hydrophobic effect and hydrogen-bonding
interactions.21 Upon inclusion into the CD cavity, the chemical and
spectral properties of the guest can be affected. The hydrophobic
536 | Photochem. Photobiol. Sci., 2011, 10, 531–542
This journal is
The Royal Society of Chemistry and Owner Societies 2011
©

View Article Online
Table 4 Experimental conditions and fluorescence intensities measured in water, Iw, and optimized values in cyclodextins, Ic, along with the stability
constant of the 1 : 1 inclusion complex formed between local anesthetic (LA) and cyclodextrins, K11 (eqn (2) and (3))
LA ([LA])
Cyclodextrin
lex/lem (nm)
Iw/a.u.
Ic/a.u.
K
11/dm3 mol-1
fc/fw
2 (10.6 mM)
a-CD
b-CD
g-CD
342/410
343/410
343/417
0.604 0.030
0.723 0.050
0.580 0.035
4.2 0.1
6.6 0.1
5.3 0.5
50
184
73 15
3
7
7
9
9
3 (46 mM)
4 (9.2 mM)
a-CD
b-CD
g-CD
322/418
325/427
326/439
0.024
0.021
0.036
1.96 0.08
1.22 0.03
0.770 0.015
48
185
21.0 0.07
5
9
82
58
21
a-CD
b-CD
g-CD
b-CD
b-CD
295/341
300/345
300/345
302/346
0.043
0.048
0.048
0.113
3.09 0.04
194
780 30
60.0
740 30
549 42
9
72
11
5.3
13
0.529 0.005
0.257 0.004
1.46 0.02
1
4 (27 mM)
4 (5 to 14 mM)
Phase solubility measurements 28
Fig. 5 (a) Variation of emission of fluorescence intensity as a function of b-CD concentration for 4 (᭢) 9.2 mM and (᭜) 27 mM; ( ) 3, 46.7 mM and (᭡)
᭹
2, 10.6 mM. The excitation and emission wavelength are reported in Table 4; (b) Increase of the emission of fluorescence at 439 nm (lex 326 nm) of 3, 46
mM, as a function of g-CD concentration.
at eqn (3) to express the fluorescence intensity of the guest as a
function of cyclodextrin concentration. In this equation, Iw and
Ic refer to the emission intensity in water and at 100% complexed
with CD, respectively.
The guest 2 complexed with whatever CD is very protected
from water molecules; in fact, Ic values are comparable to those
obtained in solvents or in cationic micelles of TTABr. On the other
hand, the complexes formed with b-CD are more stable than that
formed with a-, or g-CD, probably due to both the tight fitting of
2 to the CD cavity and to the gain in energy when high energetic
water molecules inside the b-CD are replaced by the hydrophobic
guest.
Benzocaine and the guest 3 hardly show fluorescence in water
as a consequence of the very fast radiationless decay, thus no
intramolecular hydrogen-bonding is possible; but, the presence
of CDs enhances strongly the fluorescence emission. The highest
effect was observed in a-CD solutions and following the trend:
Ic (a-CD) > Ic (b-CD) > Ic (g-CD) with increasing factors of 80 : 50 : 18 for
the case of 3 and of 70 : 13 : 5 for the case of 4. These results reflect
that the fluorophore moiety of 3 and 4 remains very exposed to
water molecules; the effect is very marked in benzocaine, which, in
spite of forming the most stable complexes with whatever CD, the
Ic values are very low in comparison to that obtained in solvents
or, for instance, in cationic micelles.
K11
(2)
ꢀꢀꢀꢀꢁ
LAw + CD
LA·CD
ꢂꢀꢀꢀꢀ
Fluorescence measurements applied to the determination of
stability constants are based on the proportionality of fluorescence
intensity to fluorophore concentration, where the proportionality
constant includes the quantum yield and the molar absorptivity,
eab. Both parameters, but mainly the quantum yield, take different
values for the free (fw) and included (fc) guest, i.e., Iw = dw[LA]o
and Ic = dC[LA·CD]t, with di = eabfi.
Iw + IcK11[CD]
1+ K11[CD]
IF =
(3)
The non-linear regression analysis of the experimental data by
means of eqn (3), affords the optimized values of the unknown
parameters Ic and K11 that are collected in Table 4, along with the
ratio fc/fw that shows the effect of CD on the emission quantum
yield.
For the sake of comparison, Table 4 also includes the stability
constant of the inclusion complex formed between 4 and b-
CD determined from solubility measurements.28 The datum
This journal is
The Royal Society of Chemistry and Owner Societies 2011
Photochem. Photobiol. Sci., 2011, 10, 531–542 | 537
©

View Article Online
Fig. 6 Plot of ko against cyclodextrin concentration obtained in the nitrosation of (a) ethyl 2-aminobenzoate, 2, in the presence of a-CD followed the
increase in absorbance at (
, ) 270 nm or the decrease in absorbance at (D,᭡) 336 nm: open points represent total cyclodextrin concentration; solid
᭹
᭺
points represent free cyclodextrin concentration; (b) benzocaine in the presence of b-CD at [nitrite] = 2.9 mM, [buffer] = 0.078 M, pH 4.50, (᭡) ko against
total [b-CD]; ( ) k against free [b-CD]; dashed line, fitting to eqn (4) with the assumption k = 0.
᭹
o
c
(Kc = 549 M-1) is significantly lower than that obtained in this
work. The sensibility of fluorescence determinations is notably
higher and the required amount of guest is considerably lower,
which leads to more accurate results.
The time-resolved fluorescence of 2 was measured in 30 mM
a-CD and 7.0 mM b-CD. In both cases, a bi-exponential decay
with lifetimes t1 = 2.29 0.03 ns (18.6%) and t2 = 8.43 0.02 ns
(81.4%) in a-CD and t1 = 2.02 0.04 ns (8.9%) and t2 = 9.26
0.01 ns (90.1%) in b-CD were observed. Against, the longer
lifetime, which corresponds to the emitting conformer II, is the
main species in the presence of cyclodextrins and that which is
included into the CD cavity.
The reaction spectra of 4, recorded also between 240 and 400 nm
at [4] = 9 ¥ 10-5 M and [nitrite] = 2.9 ¥ 10-3 M, show a broad
band centered at 283 nm that decreases with time and shifts the
absorption maximum to 263 nm, by drawing also two well-defined
isosbestic points at 318 and 271 nm (Fig. S5, ESI†). We have chose
l = 290 nm (De ~ 14 000 mol-1 dm3 cm-1) to follow the reaction.
The observed rate constant, ko, obtained for either 2 or 4 de-
creases as the cyclodextrin concentration increases. Representative
plots of ko versus [CD] are given in Fig. 6. Taking into account that
the nitrosating agents XNO (formed from the HNO2 according
to the equilibrium reactions HNO2 + X- + H+◦ ꢀ X–NO, K1,
or 2HNO2 ꢀ N2O3, K2 = 3 ¥ 10-3 M-1 at 25 C29) are highly
hydrophilic species, the complexation process leads to the reactant
separation; in other words, the LA included into the CD cavity
is protected from the attack of XNO in water, with XNO being
H2NO+, AcNO or N2O3. The degree of encapsulation of H2N
group will determine the possible attack of the nitrosating agent.
The overall reaction mechanism is that of Scheme 2, in which
we consider different reactivities for free (kw) and complexed (kc)
guest, the concentration of both are related by the equilibrium
constant, Kc, with LA representing to either 2 or 4. The mass
balance of [LA]t = [LA] + [LA·CD] ([LA] ꢀ [CD]) leads to eqn
(4), where kw = k1[XNO] and kc = k2[XNO].
2.2 Reactivity measurements. In order to postulate a more
exact picture of the conformation of inclusion complexes, we
studied the effect of cyclodextrins on the reactivity of the guest
towards the nitrosation of the amine group in mild acid medium
and the alkaline hydrolysis of the ester group. The results could
inform us of the position of these reactive groups in the CD
complexes.
Nitrosation in acid medium. The influence of CDs on the
nitrosation of the primary amine group of benzocaine, 4, and of
ethyl 2-aminobenzoate, 2, has been analyzed in aqueous buffered
medium of acetic acid–acetate ([buffer] = 0.075 M) at pH ~ 4.50.
The nitrosation of ethyl 3-aminobenzoate, 3, resulted complicated
by products phase separation (strong turbidity).
kw + kcKc[CD]
ko =
(4)
1+ Kc[CD]
The reaction spectra of 2, recorded from 240 to 400 nm at
[2] = 1.5 ¥ 10-4 M and [nitrite] = 2.9 ¥ 10-3 M, shows two broad
bands centered at 327 and 270 nm. The former decreases with time
meanwhile the absorption maximum shifts to 311 nm, whereas the
later increases with time; the spectral changes describe two well-
defined isosbestic points at 311 and 251 nm (Fig. S5, ESI†). The
reaction was followed at both 336 (decrease in absorbance, De ~
3300 mol-1 dm3 cm-1) and 270 nm (increase in absorbance, De ~
7300 mol-1 dm3 cm-1). Under the same experimental conditions,
the observed rate constant takes the same value independent of
the wavelength used to follow the reaction, which indicates both
changes correspond to the same reaction process.
Scheme 2 Equilibrium and reaction steps in nitrosation.
The ko vs. [CD] profiles obtained in the nitrosation of 2 fit
perfectly to eqn (4) if kc = 0, i.e. the complex formed between 2
538 | Photochem. Photobiol. Sci., 2011, 10, 531–542
This journal is
The Royal Society of Chemistry and Owner Societies 2011
©

View Article Online
Table 5 Experimental conditions and results obtained in the nitrosation of 2 and 4 in aqueous buffered solutions of acetic acid–acetate, [buffer] =
0.075 M of pH 4.50, in the presence of cyclodextrins. See Scheme 2 and eqn (4) for the interpretation of rate constants
LA (c/M)
Cyclodextin
[nitrite]/M
l/nm (De/M-1 cm-1)
kw/10-3 s-1
kc/10-3 s-1
Kc/M-1
Kcap/M-1
2 (1.5 ¥ 10-4)
a-CD
a-CD
b-CD
2.9 ¥ 10-3
2.9 ¥ 10-3
3.5 ¥ 10-3
270 (7300)
336 (3200)
270 (7300)
1.06
1.05
1.57
~0
~0
~0
40.80 0.95a
42.5 1.0a
182 2b
21.0 0.5
22.9 0.7
131
2
4 (0.9 ¥ 10-4)
a-CD
b-CD
g-CD
2.9 ¥ 10-3
3.5 ¥ 10-3
3.5 ¥ 10-3
290 (14000)
290 (14000)
290 (14000)
1.42
1.78
1.85
(0.35 0.03)
(0.41 0.02)
(0.34 0.05)
160 10a
684 20b
65 6c
61
370 20
45.5 3.0
3
a The value of KI used to calculate the free cyclodextrin concentration was 15 M-1. b KI = 5 M-1. c KI = 4 M-1.
and a- or b-CD is unreactive. The amine group must be completely
protected from the attack of the nitrosating agent. By contrast, the
ko vs. [CD] profiles obtained in the nitrosation of 4 fit to eqn (4)
with significant values of kc, the rate constant for the nitrosation
of the complex. The dashed line in Fig. 6 was drawn by assuming
kc = 0. The determined values of both kc and Kc, along with the
experimental conditions, are collected in Table 5.
12.33; 12.20, and 12.08 respectively to a-, b-, and g-cyclodextrin
at 25 ◦C)31 and also to obtain adequate reaction rates. Therefore,
under these experimental conditions, the cylodextrins are nega-
tively charged.
The LW absorption band displayed in the spectra of 2, 3, or
4 (with maxima at 327, 311, and 285 nm, respectively; Table 1)
decreases with time in alkaline medium meanwhile shifts to shorter
wavelength and fixed, at the end of the reaction, at 309, 302 and
265 nm, respectively, by describing, in each case, two isosbestic
points centered, for instance, at 270 and 232 nm in the benzocaine
hydrolysis (Fig. S6 and S7, ESI†). Therefore, a clean reaction
occurs to give, as the reaction products, 2-, 3-, or 4-aminobenzoate
and ethanol, respectively to the hydrolysis of 2, 3, or 4.
The reaction kinetic was studied by recording the decrease in
absorbance as a function of time at 350 (log e = 3.30), 330 (log e =
3.0), and 300 nm (log e = 4.0), respectively to 2, 3, or 4. The
compound 3 hydrolyses nearly 10-fold faster than the other two,
due to the inductive effect of the amine group in either ortho or para
positions of the benzoic moiety reduces the electrophilic character
of the carbonyl group by resonance effects.
Addition of cyclodextrins to the reaction medium results in
a progressive reduction of pseudo-first order rate constant. The
ko vs. [CD] profiles describe saturation curves. Fig. 7 shows
representative results. Considering the same CD, the inhibition
effect is stronger for benzocaine than for the other two LA,
which show similar reduction effect; considering the same LA and
different molecular receptor, a concentration of b-CD near 4-fold
lower than that of a- or g-CD yields similar inhibition effect.
The ko vs. [CD] profiles can be fitted to the overall eqn (6),
derived from Scheme 3, with some modifications depending on
the LA being considered. In this equation, kw and kc represent
the pseudo-first order rate constants for the hydrolysis of free
and complexed LA; K¢11 and K12 are the stability constants of
the inclusion complexes for 1 : 1 and 1 : 2 stoichiometries, formed
between the LA and ionized cyclodextrin, and [CD-] represent the
stoichiometric ionized CD. The non-linear regression analysis of
the experimental data by means of eqn (6) leads to the results of
Table 6.
The optimized values of Kc are significantly lower than that
obtained from fluorescence measurements in pure water (compare
data in Tables 4 and column 8 of Table 5). Obviously, Kc values
must be independent of the method used to evaluate them.
Nevertheless, there is an important difference in the experimental
conditions of nitrosation experiments that refer to the presence of
acetic acid and acetate ions ([buffer] = 0.075 M). Both components
of the buffer form inclusion complexes with CDs;21,30 then, the Kc
values obtained in the fitting of ko against total CD concentration
ap
are, in fact, Kc (apparent values modified by the competition of
the buffer for the CD cavity). By representing HAc or Ac- by I,
then I + CD ꢀ I·CD, KI, the mass balance on [I]o = [I] + [I·CD]
and on [CD]o = [CD] + [I·CD] leads to eqn (5) to determine the
free cyclodextrin concentration, [CD].
⎛
⎜
⎜
⎝
⎞
⎟
⎟
⎟
⎟
⎠
1
[CD]o
2
⎜
[CD] +[CD] [I] +
−[CD]
−
= 0
⎜
(5)
o
o
KI
KI
Solving this quadratic equation for each initial [CD]o, one
determines the corresponding free cyclodextrin concentration. For
that, we assumed the KI reported in Table 5.30 The plot of ko vs.
free [CD] is also shown in Fig. 6. The corresponding fit to eqn
(4) yields the values of Kc reported in Table 5. As can be seen,
the comparison with those obtained from fluorescence (Table 4) is
now in very good agreement.
However, the most important observation of nitrosation results
is that the inclusion complex of 4 reacts with the nitrosating agent
at reaction rates 4-fold lower than that of uncomplexed compound,
whereas those of 2 do not. This fact indicates that in the inclusion
complexes of benzocaine, the amine group remains exposed to
water, a result that confirms the fluorescence Ic values, which are
much lower than those read in solvents or in cationic micelles. By
contrast, in the inclusion compounds of 2 the amine group is not
exposed to water molecules.
−
′
kw + kcK11[CD ]
(6)
ko =
−
− 2
′
′
1+ K11[CD ]+ K11K12[CD ]
Hydrolysis in alkaline medium. The alkaline hydrolysis of the
ester function of 2, 3, or 4 compound was studied in aqueous
solutions of cyclodextrins at high and fixed [OH-], to ensure
Inclusion complexes of 2 appear unproductive, irrespective of
the cyclodextrin being considered, i.e. kc ~ 0 in every case, and
with a- and g-CD the best fit was obtained if both 1 : 1 and 1 : 2
the ionization of secondary OH-group of cyclodextrins (pKa
=
This journal is The Royal Society of Chemistry and Owner Societies 2011
Photochem. Photobiol. Sci., 2011, 10, 531–542 | 539
©

View Article Online
Scheme 3 Equilibrium and reaction steps in alkaline hydrolysis.
(kc = 0) or productive (kc π 0) complexes; as [2] = 0.5 mM is much
lower than that of a- and g-CD, we ruled out the possibility of two
molecules of 2 inside the CD cavity. As can be seen in Fig. 7(c), the
best fit of the model to the experimental points was obtained when
unproductive complexes of both 1 : 1 and 1 : 2 stoichiometries are
considered.
The apparently different behaviour observed with b-CD is the
consequence of the smaller concentration interval used, i.e. the
maximum [b-CD] is nearly 5-fold lower than that used with a-
or g-CD, due to the lower solubility of b-CD. On the contrary,
good agreement between the measured ko at each [CD] and the
calculated values from eqn (6) is obtained in the case of the
hydrolysis of 3 and 4 if one considers the formation of only
1 : 1 inclusion complexes, that is, K12 is negligible. The complexes
formed between 4 and g-CD showed to undergo hydrolysis at
approximately half-the rate measured in water.
In alkaline conditions, the CDs have the secondary –OH group
ionized, i.e., they are anionic hosts. Then, the hydration of the
wider rim is more important in alkaline medium than in neutral
or acid medium (neutral host); this fact difficult the inclusion
of hydrophobic guests. In spite of that, the complex stability
constants reported in Table 6 reach values close to that measured
in neutral (Table 4) or acid medium (Table 5). These results can
be understood if the local anesthetic inclusion mode is oriented
tail-first (T-F) in alkaline medium, but head-first (H-F) in neutral
or acid media, Scheme 4. The T-F inclusion mode, stabilized by
strong H-bonding between the –NH2 group of LA and the alkoxide
group, –O-, of CD, leaves the ester function inside the CD cavity,
completely protected from the nucleophilic attack of HO-. In
addition, if this conformation is assumed for the binding mode of
compound 2, the hydrophobic ethyl group would remain exposed
to water; therefore, a second CD molecule protecting this moiety
would explain the formation of complex of 1 : 2 stoichiometry,
which would be specially stable in the case of a-CD, whose narrow
cavity difficult the guest protection. On the other hand, the H-
F inclusion mode, stabilized by H-bonding between the ester-
group of the LA and the –OH group of CD, leaves the amine
group protected from the nitrosating agent, XNO. The molecular
geometry of benzocaine gives ‘loose fit’ inclusion complexes
with different degree of encapsulation, mainly in the case of
g-CD host, which can be exposed to the reagents in the bulk
Fig. 7 (a) Influence of b-CD on the basic hydrolysis of (᭡) 2, [OH-] =
0.078 M, ( ) 3 [OH-] = 0.048 M, right y-axis, and (᭢) 4, [OH-] = 0.083 M,
᭹
and variation of ko against [g-CD] obtained in the alkaline hydrolysis of
(b) 4 at [OH-] = 0.075 M; the dashed line shows the calculated points if
kc = 0 (unproductive complex), and (c) 2 at [OH-] = 0.095 M; lines show
the fits to eqn (6) when kc = 0 (—); when K12 = 0 but kc is negative, kc =
(-1.4 0.2)¥10-4 s-1, which has no sense ( ◊ ◊ ◊ ); and when kc and K12 are
zero (---). The results reported in Table 6 correspond to the solid lines.
complexes are considered, in other words both K¢11 and K12 are
significant. However, different models have been essayed for this
particular case, including the formation of only 1 : 1 unproductive
540 | Photochem. Photobiol. Sci., 2011, 10, 531–542
This journal is
The Royal Society of Chemistry and Owner Societies 2011
©

View Article Online
Table 6 Experimental conditions and results obtained in the alkaline hydrolysis of 2, 3, and 4 in the presence of cyclodextrins; kw and kc are the
¢
pseudo-first order rate constants for the hydrolysis of the free and complexed LA, respectively, and K11 and K12 represent the stability constants on
inclusion complexes of 1 : 1 and 1 : 2 (LA : CD) stoichiometry, see eqn (6)
LA (c/M)
Cyclodextrina
[OH-]/M
kw/10-4 s-1
kc/10-4 s-1
K¢11/M-1
K
12/M-1
2(5.3 ¥ 10-4)
a-CD (0–40) mM
b-CD (0–9) mM
g-CD (0–40) mM
0.075
0.075
0.095
3.43
3.50
4.16
—
—
—
49
114
34
2
2
78
—
13
3
3
2
3(5.6 ¥ 10-4)
4(9 ¥ 10-5)
a-CD (0–40) mM
b-CD (0–9) mM
g-CD (0–40) mM
0.040
0.045
0.040
12.8
14.1
12.7
—
—
—
34.0 0.8
123
41.8 0.8
—
—
—
2
a-CD (0–40) mM
b-CD (0–9) mM
g-CD (0–15) mM
0.075
0.075
0.075
2.23
2.23
2.23
—
—
86
420
3
—
—
—
8
0.94 0.05
140 10
a Cyclodextrin type and concentration interval used.
Scheme 4 Dimensions and sizes of the optimized structures of host and guest and the proposed geometries of inclusion complexes: head-first in neutral
medium and tail-first in alkaline medium.
aqueous phase. The HyperChem software package was used for
the geometrical optimization of the molecular structures of local
anesthetics that are displayed in Scheme 4 and compared to that of
cyclodextrins.
micelles – formed by either cationic or anionic surfactants – and
to a-, b-, and g-cyclodextrin showing different location sites or
geometries of the inclusion complexes.
The hydrophobic local anesthetic molecules, such as ethyl 2-
aminobenzoate, together with the absence of specific interactions
with interfaces (e.g. electrostatic or hydrogen bonding), allow
local anesthetics to penetrate more efficiently across hydrophobic
interfaces and, by extension, across biological membranes, which
redound in high anesthetic effect.
Conclusions
The results presented here indicate that ethyl 2-aminobenzoate,
ethyl 3-aminobenzoate, and ethyl 4-aminobenzoate bind to
This journal is
The Royal Society of Chemistry and Owner Societies 2011
Photochem. Photobiol. Sci., 2011, 10, 531–542 | 541
©

View Article Online
The dominant interactions – van der Waals interactions and
the hydrophobic effect – observed in the complexation of local
anesthetics bearing a tertiary amine moiety, such as novocaine or
tetracaine, are drastically changed when specific interactions with
CD are allowed. The strong hydrogen bonding between the H2N
group of LA and the alkoxide –O- group of CD not only give high
stable inclusion complexes, but also a change in the binding mode
of the LA and even in the stoichiometry of the complex. Therefore,
the complexation with cyclodextrins enhances the stability of LA,
which are protected from reagents in the bulk water phase.
13 N. Takisawa, K. Shirahama and I. Tanaka, Interactions of amphiphilic
drugs with a-, b-, and g-cyclodextrins, Colloid Polym. Sci., 1993, 271,
499.
14 E. Iglesias, Inclusion complexation of novocaine by b-cyclodextrin in
aqueous solutions, J. Org. Chem., 2006, 71, 4383.
15 E. Iglesias, Investigation of physico-chemical behaviour of local
anesthetics in aqueous SDS solutions, New J. Chem., 2008, 32,
517.
16 I. Iglesias-Garc´ıa, I. Brandariz and E. Iglesias, Fluorescence study of
tetracaine-cyclodextrin inclusion complexes, Supramol. Chem., 2010,
22, 228.
17 B. Reija, W. Al-Soufi, M. Novo and J. Va´zquez-Tato, Specific In-
teraction in the inclusion complexes of pyronines Y and B with b-
cyclodextrin, J. Phys. Chem. B, 2005, 109, 1364.
18 R. Stewart, The Proton Applications to Organic Chemistry, Academic
Press, Inc., 1985, chap. 3.
Acknowledgements
19 G. S. Cox and N. J. Turro, Methyl salicylate fluorescence as a probe of
the geometry of complexation to cyclodextrins, Photochem. Photobiol.,
1984, 40, 185.
20 A. Dom´ınguez, A. Ferna´ndez, N. Gonza´lez, E. Iglesias and L.
Montenegro, Determination of critical micelle concentration of some
surfactants by three techniques., J. Chem. Educ., 1997, 74, 1227.
21 M. V. Rekharsky and Y. Inoue, Complexation thermodynamics of
cyclodextrins, Chem. Rev., 1998, 98, 1875.
We are indebted to Dr Mercedes Novo of the Facultad de Ciencias,
Campus de Lugo, University of Santiago de Compostela, for the
time-resolved fluorescence measurements. Financial support from
the Direccio´n General de Investigacio´n (Ministerio de Educacio´n
y Ciencia) of Spain and FEDER (Project CTQ2005-07428/BQU)
and from Direccio´n General de Programas y Transferencia de
Conocimiento (Ministerio de Ciencia e Innovacio´n) of Spain
(Project CTQ2008-04429/BQU) is gratefully acknowledged.
22 A. Ueno and T. Osa, Host–guest photochemistry in solution, in
Photochemistry in Organized and Constrained Media, V. Ramamurthy,
ed., VCH Publishers, Inc., New York, 1991, chap. 16.
23 G. Krishnamoorthy and S. K. Dogra, TICT of 2-(4¢-N,n-
dimethylamino phenyl)pyrido[3,4-d]imidazole in cyclodextrins: Effect
of pH, J. Phys. Chem. A, 2000, 104, 2542.
References
24 (a) S. Monti, L. Flamigni, A. Martelli and P. Bortolus, Photochemisty
of benzophenone-CD inclusion complexes., J. Phys. Chem., 1988,
92, 4447; (b) S. Monti, G. Ko¨hler and G. Grabner, Photophysics
and photochemistry of methylated phenols in b-cyclodextrin inclusion
complexes, J. Phys. Chem., 1993, 97, 13011; (c) S. Monti, G. Marconi,
F. Manoli, P. Bortolus, B. Mayer, G. Grabner, G. Ko¨hler, W. Boszczyk
and K. Rotkiewicz, Aspectroscopic and structural characterization
of the inclusion complexes of p-dimethylaminobenzonitrile with cy-
clodextrins., Phys. Chem. Chem. Phys., 2003, 5, 1019; (d) S. Monti, P.
Bartolus, F. Manoli, G. Marconi, G. Grabner, G. Ko¨hler, B. Mayer, W.
Boszczyk and K. Rotkiewicz, Microenvironmental effects in the excited
state properties of p-dimethylaminobenzonitrile complexed to a- and
b-cyclodextrin, Photochem. Photobiol. Sci., 2003, 2, 203.
25 K. Takahashi, Organic reactions mediated by cyclodextrins, Chem.
Rev., 1998, 98, 2013.
26 E. Iglesias, Cyclodextrins as enzyme models in nitrosation and in acid–
base hydrolysis reactions of alkyl nitrites., J. Am. Chem. Soc., 1998,
120, 13057.
27 K. A. Connors, Binding Constants. The Measurements of Molecular
Complex Stability, Wiley, New York, 1987, chap. 8.
28 L. M. A. Pinto, L. F. Fraceto, M. H. A. Santana, T. A. Pertinhez, S. Jr.
Oyama and E. Paula, Physicochemical characterization of benzocaine-
b-cyclodextrin inclusion complexes, J. Pharm. Biomed. Anal., 2005, 39,
956.
1 H. Matsuki, H. Satake, S. Kaneshina, P. R. Krishna and I. Ueda,
Surface and colloid properties of local anesthetic solutions, Curr. Topics
Coll. Interface Sci., 1997, 2, 69.
2 P. T. Frangopol and D. Mihailescu, Interactions of some local
anesthetics and alcohols with membranes, Colloids Surf., B, 2001, 22,
3.
3 W. C. Bowman and M. J. Rand, Textbook of Pharmacology, Blackwell
Sci. Publs., University Press, Cambridge, 1990.
4 Local Anesthetics, Handbook of Experimental Pharmacology, ed.
J. M. Ritchie and G. R. Strichartz, Springer-Verlag, Berlin, 1987, vol.
81, chap. 2.
5 J. Szejtli, Cyclodextrin complexed generic drugs are generally not-bio-
equivalent with the reference products: therefore the increase in number
of marketed drug/cyclodextrin formulations is so slow, J. Inclusion
Phenom. Macrocyclic Chem., 2005, 52, 1.
6 G. Savelli, R. Germani and L. Brinchi, in Reaction and Synthesis in
Surfactant Systems, T. Texter, ed., Marcel Dekker, Inc.: New York,
2001, vol. 100, chap. 8.
7 C. A. Bunton, F. Nome, F. H. Quina and L. S. Romsted, Ion binding
and reactivity at charged aqueous interfaces, Acc. Chem. Res., 1991, 24,
357.
8 P. Speicer, Biological and technological relevance of amphiphilic
structures in apolar medium, in Reverse Micelles, P. L. Luise and
B. E. Straub, ed., Plenum Press: New York, 1984, pp. 339–346.
9 K. A. Connors, Measurement of cyclodextrin complex stability con-
stants, in Comprehensive Supramolecular Chemistry, J. Szejtli, T. Osa,
ed. Pergamon, 1996, vol. 3, pp. 205–241.
10 J. Szejtli, Cyclodextrin inclusion complexes, Cyclodextrin Technology,
Kluwer Acad. Publishers, Netherlands, 1988, chap. 2.
11 O. S. Tee, The stabilization of transition states by cyclodextrins and
other catalysts., Adv. Phys. Org. Chem., 1994, 29, 1, and references
therein.
29 D. L. H. Williams, Nitrosation Reactions and the Chemistry of Nitric
Oxide, Elsevier, 2004, chap. 1.
30 (a) D. M. Davies and M. E. Deary, Stability of 1 : 1 and 2 : 1a-CD-p-
nitrophenyl acetate complexes and the effect of a-CD on acyl transfer
to peroxide anion nucleophiles, J. Chem. Soc., Perkin Trans. 2, 1999,
1027; (b) E. Iglesias, Ester hydrolysis and enol nitrosation reactions of
ethyl cyclohexanone-2-carboxylate inhibited byb-cyclodextrin., J. Org.
Chem., 2000, 65, 6583.
31 J. Szejtli, Introduction and general overview of cyclodextrin chemistry,
Chem. Rev., 1998, 98, 1743.
12 W. Saenger, Cyclodextrin inclusion compounds in research and indus-
try, Angew. Chem., Int. Ed. Engl., 1980, 19, 344.
542 | Photochem. Photobiol. Sci., 2011, 10, 531–542
This journal is
The Royal Society of Chemistry and Owner Societies 2011
©