Experimental and theoretical studies of the

photophysics of 7-amino-3-phenyl-2H-benzo[b]
[1,4]oxazin-2-one in homogeneous solvents
and b-cyclodextrin aqueous solutions
Silvana Valdebenito1, Gerald Zapata-Torres2, Else Lemp1 and Antonio L. Zanocco1*
Universidad de Chile, Facultad de Ciencias Químicas y Farmacéuticas, Departamento de Química Orgánica y
1

Fisicoquímica, Casilla 233, Santiago - 1, Santiago, Chile. 2Universidad de Chile, Facultad de Ciencias Químicas y
Farmacéuticas, Departamento de Química Inorgánica y Analítica, Casilla 233, Santiago - 1, Santiago, Chile

Estudios teóricos y experimentales de la fotofísica de la 7-amino-3-phenyl-2H-benzo[b][1,4]

oxazin-2-ona en disolventes homogéneos y soluciones acuosas de b-ciclodextrina

Estudis experimentals i teòrics de la fotofísica de la 7-amino-3-fenil-2H-benzo[b] [1,4]

oxazin-2-ona en solvents homogenis i solucions aquoses de b-ciclodextrina

Recibido: 6 de junio de 2014; aceptado: 17 de junio de 2014

RESUMEN Palabras clave: Ariloxazinonas; efecto solvente; sondas

El comportamiento fotofísico de la 7-amino-3-fenil-2H-
benzo[b][1,4]oxazin-2-ona en solventes orgánicos y en so-
luciones acuosas de b-ciclodextrinas, se estudió usando
métodos de fluorescencia estacionaria y de química com-
putacional.
En medio homogéneo, el espectro de fluorescencia mues-
tra un notable efecto solvatocrómico que produce gran-
des corrimientos de Stokes. El análisis de los corrimientos
de Stokes usando relaciones lineales de energía libre de
solvatación y la ecuación de Lippert-Mataga, indican que
se produce un aumento del momento dipolar en el esta-
do singulete excitado y la participación de un estado de
transferencia parcial de carga en el proceso de desacti-
vación. La incorporación de 7-amino-3-fenil-2H-benzo[b]
[1,4]oxazin-2-ona en la cavidad de b-ciclodextrina fue
monitoreada observando el incremento en la intensidad
de fluorescencia en función de la concentración de ci-
clodextrina. Los datos de fluorescencia analizados usan-
do gráficos de Job y la ecuación de Benesi-Hildebrand
son compatibles con la formación de un complejo 1:1. La
constante de asociación obtenida a partir de gráficos de
Benesi-Hildebrand fue igual a 597 M-1 a 298 K. Además,
los valores de los parámetros termodinámicos determina-
dos de la dependencia de la constante de asociación con
la temperatura, muestran que el proceso de inclusión es
controlado por la entalpía. Estudios de acoplamiento mo-
lecular sugieren que la estabilidad del complejo resulta de
interacciones de van der Waals favorables en el interior de
la cavidad y de una interacción de enlace de hidrógeno
entre el sustituyente amino de la oxazinona y un grupo
hidroxilo localizado en el borde más angosto de la cavidad
de la ciclodextrina. Las mismas conclusiones se obtuvie-
ron al utilizar la metodología de Mecánica Molecular-Area
Superficial de Poisson Boltzmann para determinar las
contribuciones energéticas a la energía libre total para el
proceso de inclusión.
fluorescentes; b-ciclodextrina; complejo de inclusión;
constantes de asociación.
SUMMARY
The photophysical behavior of 7-amino-3-phenyl-2H-
benzo[b][1,4]oxazin-2-one was studied in organic sol-
vents and in aqueous solutions of b-cyclodextrin using
steady-state fluorescence and computational chemistry
methods. In homogeneous media, fluorescence spectra
show a noteworthy solvatochromic effect leading to lar-
ge Stokes shifts. Linear solvation energy relationship and
Lippert-Mataga equation analysis of the Stokes shifts
indicate an increase of the dipolar moment in the singlet
excited state and the participation of a partial charge-
transfer state in the deactivation process. Incorporation of
7-amino-3-phenyl-2H-benzo[b][1,4]oxazin-2-one into the
b-cyclodextrin inner cavity was monitored by observing
the increase of fluorescence as a function of the cyclodex-
trin concentration. Analysis of fluorescence data in terms
of Job plots and the Benesi-Hildebrand equation are indi-
cate the formation of a 1:1 complex. The binding constant
obtained from Benesi-Hildebrand plots was 597 M-1 at 298
K. Also, the values of thermodynamics parameters deter-
mined from the dependence of the binding constant on the
temperature show that inclusion is an enthalpy-driven pro-
cess. Docking studies suggest that the complex stability
is due to favorable van der Waals interactions within the
cavity and a hydrogen bond interaction between the amino
substituent and hydroxyl groups located in the narrow rim
of the cavity. The same conclusion was achieved emplo-
ying the Molecular Mechanics Poisson-Boltzmann Surface
Area methodology to determine the energy contributions
to the total free energy for the inclusion process.
*Autor para la correspondencia: azanocco@ciq.uchile.cl

AFINIDAD LXXII, 569, Enero - Marzo 2015 53

Key words: Aryloxazinones; solvent effect, fluorescent
probes, b-cyclodextrin, inclusion complex, binding cons-
tants.
RESUM
El comportament fotofísic de la 7-amino-3-fe-
nil-2H-benzo [b] [1,4] oxazin-2-ona en solvents or-
gànics i en solucions aquoses de les b-ciclo-
dextrines, es va estudiar utilitzant mètodes de
fluorescència estacionària i de química computacional.
En un mitjà homogeni, l’espectre de fluorescència mos-
tra un notable efecte solvatocròmic que produeix grans
corriments de Stokes. L’anàlisi dels corriments de Stokes
utilitzant relacions lineals d’energia lliure de solvatació i
l’equació de Lippert-Mataga, indiquen que es produeix un
augment del moment dipolar en l’estat singulete excitat i la
participació d’un estat de transferència parcial de càrrega
en el procés de desactivació. La incorporació de 7-amino-
3-fenil-2H-benzo [b] [1,4] oxazin-2-ona a la cavitat de b-
ciclodextrina va ser supervisada observant l’increment en
la intensitat de fluorescència en funció de la concentració
de ciclodextrina. Les dades de fluorescència analitzades
mitjançant les gràficas de Job i l’equació de Benesi-Hilde-
brand són compatibles amb la formació d›un complex 1:
1. La constant d›associació obtinguda a partir de gràficas
de Benesi-Hildebrand va ser igual a 597 M-1 a 298 K. A
més, els valors dels paràmetres termodinàmics determi-
nats de la dependència de la constant d’associació amb la
temperatura, mostren que el procés de inclusió és contro-
lat per l’entalpia. Estudis d’acoblament molecular sugge-
reixen que l’estabilitat del complex resulta d’interaccions
de van der Waals favorables a l’interior de la cavitat i d’una
interacció d’enllaç d’hidrogen entre el substituent amino
de la oxazinona i un grup hidroxil localitzat en la part ex-
trema més estreta de la cavitat de la ciclodextrina. Les ma-
teixes conclusions es van obtenir en utilitzar la metodo-
logia de Mecànica Molecular-Àrea Superficial de Poisson
Boltzmann per determinar les contribucions energètiques
a l’energia lliure total pel procés d’inclusió.
Paraules clau: Ariloxazinones; efecte solvent; sondes
fluorescents; b-ciclodextrina; complex de inclusió; cons-
tants d’asociació.
INTRODUCTION
Cyclodextrins (CD) are cyclic oligosaccharides, the most
common consisting of six (a-CD), seven (b-CD) or eight
(g-CD) glucopyranose units linked by a-(1,4) bonds. These
toroidally-shaped capsules produced from starch have a
special structure characterized by a hydrophilic exterior
and an internal hydrophobic cavity. The size of the internal
cavity (5-8 Å) for the most common CDs, depends on the
number of glucopyranose units. These distinctive struc-
tural characteristics confer the CDs their unique ability
to form inclusion complexes in aqueous medium with a
wide variety of hydrophobic guest molecules [1-3]. Cova-
lent bonding is not involved in the complex formation, only
physical forces accounts for the complexes feasibility [4,5].
The stability of inclusion complexes in aqueous solutions
is mainly due to van der Waals forces and hydrophobic
interactions that help the hydrophobic moiety of the guest
54
molecule to be included within the hydrophobic CD cavity
[6,7]. Thus, the organic guest can enter entirely into the ca-
vity or in part depending on the polarity, molecular size and
geometrical factors. The complexation phenomenon often
involves remarkable variations in stability, degradation
paths, reactivity and bioavailability (in the case of drugs or
pro-drugs) of the guest molecule. Also, the photophysical
and photochemical properties of the included molecule
can change noticeably because of the non-polar microen-
vironment provided by the CD cavity. Formation of supra-
molecular complexes of fluorescent molecules with CDs
leads to a dramatic increase of the fluorescence quantum
yield of a probe [8,9]. Also, the rates of photochemical re-
actions decrease notably when CD inclusion complexes
are formed due to the geometric constraints imposed
on the guest molecule [10,11]. Moreover, the binding of
guest molecules within the host cyclodextrin is not fixed or
permanent but is rather a dynamic equilibrium, therefore
measurements of the inclusion constant allow determining
the thermodynamic parameters associated to the complex
formation.
Over the last years, our group has studied the photophy-
sical and photochemical behavior of a family of hydropho-
bic dyes, the aryloxazinones [12,13]. These compounds
behave in very similar way than coumarine derivatives.
Their fluorescence is characterized by intense and broad
emission bands, large Stokes shifts indicating a significant
sensitivity to the polarity of the environment, high fluores-
cence quantum yields and short fluorescence lifetimes [14,
15]. Also we found that these compounds are essentially
photostable in the absence of additives. Their photophy-
sical properties and large photostability in air-equilibrated
solutions suggest that aryloxazinone derivatives could be
valuable molecules to be used for several applications
such as laser dyes, fluoro-ionophores, fluorescence mar-
kers and probe molecules for the examination of electron
transfer processes and solvation effects as well as for
singlet oxygen photosensitization [12,13, 16-19]. Howe-
ver, the use of organic solvents (the simplest approach) is
incompatible with biologically- or environmentally-relevant
applications, and is also frequently discouraged by eco-
nomic and environmental considerations. The search for
fluorescent molecules with high photostability and bright-
ness in water is a topic of high interest since water is envi-
ronmentally benign and biologically relevant. Popular stra-
tegies for solubilizing hydrophobic fluorescence molecules
rely on the use of stabilizing, solubilizing, disaggregating
and enhancing additives. In this context we have studied
the formation of supramolecular systems between benzo-
and naphthoxazinones and b-cyclodextrin. As a first step
in this path we are interested in assessing the sensitivity of
their properties to the inclusion within a hydrophobic cavi-
ty in aqueous meda. We report in this paper an experimen-
tal and theoretical study of the inclusion complex formed
between 7-amino-3-phenyl-2H-benzo[b][1,4]oxazin-2-one
(7-ABZ), Figure 1, and b-cyclodextrin. The aims of this
study were (1) to determine the stoichiometry and binding
constants, K1, of the complex, (2) to determine the thermo-
dynamic parameters associated to the inclusion process,
and (3) to determine, through computational studies, the
optimum geometry of the complexes in order to estimate
the van der Waals and electrostatic contributions to the
binding energies of the host–guests complexes.
AFINIDAD LXXII, 569, Enero - Marzo 2015
 The method of continuous variations was employed to determine th

stoichiometry [20]. Briefly, the fluorescence intensity of the guest at the conc

F(M), was measured after equilibration of solutions prepared with different mo

ABZ:β-CD. From Jobs plots of F(M) against the molar fraction X of the
different molar ratios 7-ABZ:b-CD. From Jobs plots of F(M)
solution, the stoichiometry (n) was obtained from the X value correspon
against the molar fraction X of the guest in the solution, the
maximum value (n)
stoichiometry was obtained
of F(M) accordingfrom the X value
to equation (1): correspon-
N ding to the maximum value of F(M) according to equation (1):

X = ( n + 1)
−1
(1)
H2N O O
Figure 1. Structure of 7-amino-3-phenyl-2H-benzo[b][1;4]oxazin-2-one. Association
Association constants
constants determinations
determinations
The association constant for 7-ABZ …b-CD complex was
40000
Figure 1. Structure of 7-amino-3-phenyl- Thedetermined
associationusing thefor
constant 7-ABZ …β−CD complex
Benessi-Hildebrand method [21].
was In
determined using the
2H-benzo[b][1;4]oxazin-2-one. the case of a complex with 1:1 stoichiometry, equation (2),
30000 [β-CD]/mM the equilibrium
Hildebrand method constant is case
[21]. In the described by thewith
of a complex equation (3)
1:1 stoichiometry, equat
10
9 7-ABZ + β − CD ←  → [7-ABZ ⋅ ⋅ ⋅ β − CD ] (2)
β − CD  → by [7-ABZ ⋅ ⋅ ⋅]β (2)
⋅ β −(3)CD
7

EXPERIMENTAL 5
7-ABZ constant
equilibrium +7-ABZ β ← 
is+ described
− CD ⋅[⋅7-ABZ
the→equation − CD ] (2)
20000

←
IF/a.u.

3
2
1
0

K1 =
[7-ABZ ⋅ ⋅ ⋅ β − CD ] 7
Reagents 10000 [7-ABZ
K1 = K[7-ABZ [ ⋅ β][ −β CD
⋅ ⋅7-ABZ ⋅ ⋅]β]− CD ]
−⋅CD
(3)
All experiments were performed with analytical or spec- = (3) (3)
troscopic grade chemicals. b-CD (obtained from Merck, [7-ABZ
1 ][[7-ABZ
β − CD][]β − CD ]
99.9% HPLC) 450 was used 500 as
550 received.
600 The 700
650 fluorescent As inclusion
pro- of 7-ABZ in the β−CD cavity enhances the fluorescence quantum yield of the
wavelenght/nm
be 7-amino-3-phenyl-2H-benzo[b][1,4]oxazin-2-one As inclusion was of 7-ABZ
As inclusion Asofin the β−CD
inclusion
7-ABZ inof thecavity
7-ABZ
β−CD enhances
in the fluorescence
the enhances
cavity b-CD cavity quantumthe
enhances
the fluorescence yield of the
quantum yield of t
synthesized as described
Figure 2. Dependence of the fluorescenceearlier
intensity of [14] and
7-ABZ on guest
itsconcentration
the β-CD structure in in a fashionfluorescence
was dependent on quantum the β−CDyield concentration,
of the guest valuesin aoffashion
K1 can de-be obtained
confirmed by means of 1H NMR. The purity ofguest the in aguest
fashion
in in adependent on
onthe theβ−CD theconcentration, values ofvalues
K cancan
be obtained
aqueous solution (pH 6.0).
dye pendent
fashion dependent b-CD
on concentration,
β−CD concentration, values of1 K be
1 of K1 can be obtained
these samples has been established by thin layer fromchro-the analysis obtained
of the fluorescence
from the analysis intensitiesofinthe terms of the modified
fluorescence Benesi-Hildebrand
intensities
matography. Distilled water was purified trough fromathe analysis
MilliQ
from the of theterms
in
analysis fluorescence
of of
thethe intensities
modified
fluorescence in terms of
Benesi-Hildebrand
intensities in theterms modified
equation Benesi-Hildebrand
(4) for Benesi-Hildebr
of the modified
system. Citrate buffer solutions (pH 3 and pH 5, equation
0.1 M)(4) for 1:1 1:1stoichiometry
stoichiometry: :
21
were prepared according to the current procedures. equation equation(4)
All for 1:1(4)stoichiometry
for 1:1 stoichiometry: :
other solvents used were of the commercially available 1 1 1 1
1F − F 1( F = 1 − F ) 1+ K (1F − F 1) [1β CD ] 1 (4) (4)
spectroscopic grade.
=0 1
= 0+ 1
+ 1 0 (4)
Apparatus F − F0 F( −F1F− F0 () F K − 1F(0F)1 − K F0 () F[ β−CD F0]) [ βofCD ]
Absorption spectra were recorded in a Unicam UVwhere 4 spec- F and F0 are where F and F0 are
the fluorescence
0 the 1fluorescence
intensities of 7-ABZintensities
1 1
in the presence 7-ABZ
and absence of
trophotometer using 1 cm quartz cells. Data were where proces-
F and F0 Fareandin the
the presenceintensities
fluorescence and absence of 7-ABZ of b-CD, therespectively, and and
sed with Vision software. Steady-state fluorescence where
mea- F1Fis0 are the fluorescence intensities ofin7-ABZ presence
in theall absence
presence andofabsence of
β−CD, respectively, andtheF1 expected
is the expected fluorescence
fluorescence intensity
intensity when when all guest
guest molecules
surements were performed with a PC1 photon counting
β−CD, respectively, molecules are
and F1 is the expected included in a complex.
fluorescence intensity intensity
when all whenguest molecules
spectrofluorimeter from ISS, equipped with a thermostatic β−CD, respectively,
Molecularand F1 is theProcedures
Modeling expected fluorescence all guest molecu
are included in a complex.
rectangular cell holder. The experiments wereare carried
includedout in a complex.
Initial structure of 7-ABZ was built using Gaussian View
in the range 25–50 ◦0C, using a bath/circulation thermostat are includedO5X in aand
complex.
then energy minimized using DFT method with the
Molecular Modeling Procedures
Haake K. The fluorescence spectra of each set Molecular of solutions 6-31g**
ModelingModeling
Procedures orbital base in the Gaussian 09 environment [22].
were measured using identical experimental conditions.
Molecular Procedures
Initial structure ofThe 7-ABZrestrained
was built electrostatic
using Gaussian potential View (RESP)O5Xfitted charges,
and then energy minimized
According to the maximum in the absorptionInitial spectrum,structure employed in theusing molecular modeling procedure, were as-
Initial structure of 7-ABZ was built using Gaussian View O5X and thenminimized
of 7-ABZ was built Gaussian View O5X and then energy energy minimi
the excitation wavelength was set at 400 nm. using DFT method signed using
with the the RED-III-4
6-31g** orbital base program.
in the The Gaussianinitial 09
structure of
environment [22]. The
Solution preparation using DFT method with
b-CDthewas 6-31g**
obtained orbital from base theinCambridge
the Gaussian
using DFT method with the 6-31g** orbital base in the Gaussian 09 environment [22]. 09 environment
Structural Data- [22]. The
The dye stock solutions were prepared by dissolving restrained an electrostatic
base (code potential
BCDEXD10). (RESP) AutoDock4
fitted charges, [23, 24], employed
was used in tothe molecular
appropriate amount of benzoxazinone in methanol. restrained The electrostatic potential
accomplish the (RESP)
200 fitted
docking
restrained electrostatic potential (RESP) fitted charges, employedrunscharges,
by applyingemployed a in
Lamarckian the molecular
in the molec
standard solutions of b-CD in the corresponding modeling
buffer procedure, were Algorithm
Genetic assigned using (LGA)thetoRED-III-4
generate program. the population The initial
eva-structure of β-
were prepared with concentrations in the rangemodeling 1- 10 mM. procedure,
modeling were assigned
luated
procedure, usingwere using
theassigned theusing
following RED-III-4
parameters: program.
the RED-III-4 The initial
a population
program. ofThestructure
150 of β-
initial structure o
In order to study the influence of the b-CD on theCD was obtained individuals,
fluores- from the Cambridgea maximum Structural
of 2.5 xDatabase
106 energy (code BCDEXD10).
evaluations and AutoDock4
cence dye intensity at different pH’s, in all experiments CD was obtained CD from
the was obtained27000the from
Cambridge
generations
the Cambridge Structural
as an end Database
criterion.
Structural (code
Database TheBCDEXD10).
best
(codedocking AutoDock4
BCDEXD10). AutoDo
concentration of the fluorophore was kept constant. [23, 24], was used to accomplish
score was selected the for200further
docking runs by applying a Lamarckian Genetic
studies.
Inclusion complex preparation [23, 24], [23, was 24],
usedwas
toTheaccomplish
Amber
used 12thesoftware
to accomplish 200 docking 200 runs
the package docking by
was applying
used
runs byto a Lamarckian
carry out
applying Genetic Gen
a Lamarckian
Typically a set of 8 – 10 solutions was prepared containing Algorithm (LGA)molecular to generate the population
dynamics studies. evaluated
The complex using the following
obtained byparameters: a
b-CD in the range 0 – 10 mM and a constant concentration Algorithm (LGA) tomolecular
Algorithm generate
(LGA) the
docking
to generate population evaluatedin
waspopulation
the solvated using theusing
following
a pre-equilibrated
evaluated parameters:
oc-
the following a
parameter
of 7-ABZ. To prepare the final solution, 100 mL ofpopulation a stock of 150 individuals,
tahedral watera box maximum of 15 Å. of The 106 energy
2.5 xsolvated complexevaluations
was and 27000
6
solution of 7-ABZ 30 mM in methanol were added population 15of 150 individuals,
intopopulation first
of 150minimized a maximum
individuals, and then of 2.5 from
heated
a maximum xof10100 2.5 energy
xto 10
3006evaluations and 27000
K in equili-
energy evaluations and 27
ml PTFE test tube and the solvent was removed with a ni- generations as an bration runs of 1 ns each. Finally, a production runfor
end criterion. The best docking score was selected of further
10 ns studies.
trogen stream. 10 mL of a solution of b-CD in generations milliQ water as an endwas
generations ascriterion.
ancarried Theout.best
end criterion. TheThe dockingbest score
energetic docking wasscore
components selectedwas for
were furtherforstudies.
selected
analyzed further studies.
or citrate buffer at the appropriate concentration were then using the Molecular Mechanics Poisson-Boltzmann Surfa-
added to the dry 7-ABZ. The solutions were homogenized ce Area (MM-PBSA) module as implemented in Amber 12.
in an ultrasonic bath at room temperature by 2 min and fi- 8
nally shaken at controlled temperature in a Hangzhou mo- 8
del MSC-100 thermoshaker for 24 hrs. (650 rpm). RESULTS AND DISCUSSION 8
Determination of the Complex stoichiometry
The method of continuous variations was employed to de- Photophysical behavior in homogeneous solvents
termine the complex stoichiometry [20]. Briefly, the fluores- The absorption spectra of 7-ABZ are nearly independent
cence intensity of the guest at the concentration M, F(M), on solvent polarity with the maximum of the lowest-energy
was measured after equilibration of solutions prepared with absorption band, λmax(Abs), centered around 400 nm and

AFINIDAD LXXII, 569, Enero - Marzo 2015 55

 The absorption spectra of 7-ABZ are nearly independent on solvent polarity with the
N

maximum of the lowest-energy absorption band, λmax(Abs), centered around 400 nm and a

molar extinction coefficient around of 18000 M-1 cm-1. Spectra calculations employing H2N O O
Figure 1. Structure of 7-amino-3-phenyl-2H-benzo[b][1;4]oxazin-2-one.
DFT formalism (B3LYP-6311g+ for structure optimization and ZINDO-S to calculate the
a molar extinction coefficient around of 18000 M cm . -1 -1

Franck-Condon transitions)
Spectra overestimate
calculations λmax(Abs)
employing DFTbyformalism
about of 10(B3LYP- 40000
nm, however analysis of
6311g+ for structure optimization and ZINDO-S to calcu-
molecular orbitals indicate
late the a π-π* transition.
Franck-Condon Theseoverestimate
transitions) results are comparable
λmax(Abs) to those reported
by about of 10 nm, however analysis of molecular orbitals 30000 [β-CD]/mM
for other benzoxazinone derivatives
indicate a π-π* [25].
transition. The polarity
These of the
results are solvent has
comparable to a large effect on 10
9
those reported for other benzoxazinone derivatives [25]. 7
5
the fluorescence
Thespectrum of the
7-ABZ. Thehas
red ashift is quite
effectlarge in polar
fluo- solvents and the
20000

IF/a.u.
polarity of solvent large on the 3
2
rescence spectrum of 7-ABZ. The red shift is quite large 1
position of emission
in polar maximum, λmaxthe
solvents and (Em), shifts of
position from 451 nmmaximum,
emission in n-hexane to 524 nm in 0

λ (Em), shifts from 451 nm in n-hexane to 524 nm in 10000

methanol. These
max
shifts obtained
methanol. in a obtained
These shifts set of 16 insolvents,
a set ofwere analyzed in terms of the
16 solvents,
were analyzed in terms of the solvatochromic equation (5)
solvatochromic equation (5) introduced by Kamlet et al. [26, 27]:
introduced by Kamlet et al. [26, 27]:
450 500 550 600 650 700

ν = ν 0 + aπ * +bα + cβ + d ρ H2 (5) (5)

wavelenght/nm

where u is the cm-1, u0 Figure 2. Dependence of the fluorescence intensity of 7-ABZ on the β-CD concentration in
where υ is the position of position of themaxima
the emission emissioninmaxima
cm-1, υin
0 is a constant for a given aqueous solution (pH 6.0).
is a constant for a given fluorescent molecule, π* accounts Figure 2. Dependence of the fluorescence intensity of 7-ABZ
for dipolarities
fluorescent molecule, and polarizabilities
π* accounts of solvent,
for dipolarities and α is related
polarizabilities on the β-CD concentration in aqueous solution (pH 6.0).
of solvent, α is related
to the hydrogen bond donor solvent ability, β indicates the
to the hydrogensolvent
bondcapacity as hydrogen
donor solvent ability, bond acceptor,
β indicates and ρH2
the solvent is
capacity as Furthermore,
hydrogen the emission maximum of 7-ABZ shifts
2
ond acceptor, andthe
ρHsquare
is the of Hildebrand
square parameter,
of Hildebrand that accounts
parameter, for the
that accounts slightly towards the blue, from 538 nm in water at pH 6.8
for the solvent
solvent cohesive energy density and models the cavity ef- to about 529 nm as the host concentration
21 increases. The
fects [28,
ohesive energy density and29]. The result
models the cavity 9
of multilinear
effects regression
[28, 29]. Theanalysis increase in the fluorescence intensity and the shift of the
result of multilinear
by employing StatView 5.0 statistical software affords the emission maximum to shorter wavelength indicate that the
gression analysisequation
by employing
(6): StatView 5.0 statistical software affords the equationoxazinone
(6): derivative changes its environment upon inter-
action with the added b-CD. The inclusion of 7-ABZ in the
ν = 21941 − 1751π * −1005α − 1280 β (6) (6) cavity protects the fluorescent probe from the nor-
b-CD
mal quenching in aqueous solution and modifies the rate
The equation
he equation (6) (number of data(6)point
(number
N =16,of data point Ncoefficient
correlation =16, correlation
R=0.991 and Fisherof the competitive non-radiative processes that deactivate
coefficient R=0.991 and Fisher index F = 230), shows that the excited singlet state, explaining the threefold increase
dex F = 230), the
shows values
that of values of depend
theλmax(Em) λmax(Em)ondepend
the microscopic sol-
on the microscopic of the fluorescence intensity when compared with the fluo-
solvent
vent parameters π*, a and b, indicating that the emission rescence observed in pure water.
arameters π*, α red
and shifts in solvents
β, indicating that able to stabilize
the emission red charges
shifts in and dipoles
solvents The dependence of the fluorescence intensity enhance-
able to stabilize
as well as in solvents with high α and b values. This result ment on the b-CD concentration can be employed to de-
is strong evidence that supports a substantial charge-sep-
harges and dipoles as well as in solvents with high α and β values. This result is strong termine the complex stoichiometry using the method of
aration process occurring in the first excited singlet state. continuous variations. Figure 3 shows the Job plot ob-
The red
vidence that supports shift can be
a substantial interpreted in terms
charge-separation of an
process increased
occurring in di- tained for 7-ABZ in pure water, where r = [7-ABZ]/([7-ABZ]
the first excited
pole moment of 7-ABZ upon chromophore excitation and + [b-CD]) was varied from 0.1 to 0.9 and plotted against
nglet state. The subsequent
red shift can be solvent relaxation.
interpreted Indeed
in terms of anaincreased dipole moment the
Lippert-Mataga of 7-relative fluorescence intensity.
plot [30,31] of the Stokes shift vs. the solvent polarity func-
tion, yields
BZ upon chromophore the difference
excitation of dipolesolvent
and subsequent moments betweenIndeed
relaxation. the a Lippert-
excited- and ground states (μ* - μ0 ), equal to 6.8 Debye.
Mataga plot [30,31]
This of the was
value, Stokes shift vs.
obtained thean
using solvent
Onsagerpolarity
cavityfunction,
radius yields the
of 4.8 Å calculated from the minimized structure (B3LYP-
fference of dipole moments
6311g+) between
under Gaussianthe excited- and ground
09 environment. In states (μ* Mul-
addition, - μ0), equal to 6.8
liken charge analyses shows that the charge separation
ebye. This value, was obtained
mainly involvingusing atoms an in
Onsager cavity radius
the oxazinone of 4.8the
ring with Å di-
calculated from
pole in the molecular plane oriented close to the carbonyl
e minimized structure
bond axis. (B3LYP-6311g+)
These results are under Gaussianto09those
comparable environment.
obtained In addition,
for previously studied compounds of the family [13].
Mulliken charge analyses
Photophysicalshows behavior
that the charge separation
in cyclodextrin mainly involving atoms in the
solutions.
The complexation of a fluorescent molecule with CDs or
xazinone ring with
CDthe dipole in in
derivatives theaqueous
molecularmediaplane usually
orientedcauses
close toan thein-
carbonyl bond
crease of the fluorescence intensity and a blue shift of the
xis. These results are comparable to those obtained for previously studied compounds of
maximum of the fluorescence spectrum. This behavior
is largely due to the hydrophobic nature of the CD cav- The Job’s plot shows a maximum at r = 0.5, which indi-
e family [13].
ity, conduct similar to the observed when the media is cates the formation of 1:1 inclusion complex between
changed from polar solvents such as water or methanol to 7-ABZ and b-CD.
hotophysical behavior in cyclodextrin solutions.
non-polar solvents such as hexane or benzene. 7-ABZ has The enhancement of the fluorescence intensity measured as
he complexationaoflow fluorescence
a fluorescent quantum
molecule withyield
CDsinorwater that increases
CD derivatives a function of host concentration can be also used to obtain
in aqueous media
in a concentration dependent fashion when b-CD in added the binding constant by means of the Benesi-Hildebrand
sually causes an to the solution
increase of the (Figure 2). intensity and a blue shift of the maximum
fluorescence approach
of [32-34]. To apply the method, the fluorescence
intensity was monitored keeping the total concentration of
e fluorescence spectrum. This behavior is largely due to the hydrophobic nature of the

10
56 AFINIDAD LXXII, 569, Enero - Marzo 2015
 7-ABZ constant, whereas the concentration of b-CD was
increased. The 1:1 modified Benesi-Hildebrand equation (4)
was used to calculate the values of the equilibrium constant
K1 for the complex. Figure 4 shows the double reciprocal
plot obtained for 7-ABZ in water at 298 K.
5 water molecules off the b-CD internal cavity, and c) release
(F - F0)-1/(a.u.)-1x10-5
4
3 tropy, is concluded that the inclusion of 7-ABZ in b-CD is
2 ters, DG0, DH0 and DS0 increase as the pH diminishes and
0.0 0.2 0.4 0.6 0.8 1.0
[β-CD]-1/(10-3 M)-1
Figure 4. Benesi-Hildebrand plot for the 1:1 inclusion
Figure 4. Benesi-Hildebrand complex
plot for the formed between 7-ABZbecomes less spontaneous at lower pH’s because the in-
1:1 inclusion
and β-CD in aqueous water solution (pH 6.0).
complex formed between 7-ABZ and
b-CD in aqueous water solution (pH 6.0
The assumption of 1:1 association between 7-ABZ and
b-CD gives a linear relationship with good correlation coeffi-
cients (r = 0.99) for all pH values and temperatures employed.
A plot constructed assuming 1:2 association of 7-ABZ:b-CD
shows a downward curvature as the CD concentration in-
creases, which rules out the 1:2 stoichiometry. The values of
K1 were obtained from the ratio between the intercept and
the slope of the Benesi-Hildebrand plot (Table 1).
Table 1. Values of the binding constant and ther-
modynamic parameters for the inclusion of 7-ABZ
in b-CD at different pH values. T = 298 K.
K1/M-1
DH 0
/cal DS 0
/cal DG 0
/cal
Figure 5. The most stable structure obtained
mol-1 frommolthe-1docking
K-1 simulations
mol-1
inclusion of 7-ABZ in β-CD.
Water, pH = 6.8 597 -9500 -3780
-19.2 Figure 4. Benesi-Hildebrand plot forstructure
Citrate buffer, pH = 5 486 -7680 -3660 -13.5
Citrate buffer, pH = 3 156 -4730 -2990 -5.8
23
Data of Table 1 shows that binding constants are rela-
tively high at all pHs and decrease as pH diminishes. As
mentioned above, hydrophobic interactions are one of the
major factors that promote the inclusion of hydrophobic
guest into the cyclodextrin internal cavity and the inclu-
sion process is more favorable for uncharged molecules
than for charged ones. Consequently, lowering the pH
values increases the protonation of the guest -mainly of
the amino substituent in the benzene moiety- , improves
its water solubility, and diminishes the relative importance
of the hydrophobic effect leading to lower K1 values. In
addition, we measured the dependence of K1 on the tem-
perature in the range 25 to 45 ºC at all pH’s. Values of K1
diminish as the temperature increases, as expected for a
process driven by van der Waals interactions, electrostatic
interactions and hydrophobic effect. The enthalpy associ-
ated to the binding, DH0, was obtained from the slope of
the classical plot of ln K1 vs. 1/T that gives a straight line
in the temperature range employed, with correlation coef-
AFINIDAD LXXII, 569, Enero - Marzo 2015
ficients values 0.88 and 0.99. Thermodynamic parameters
DG0, DH0 and DS0 for the association of 7-ABZ with b-CD
are included in Table 1. The negative value of enthalpy at
all pH’s, corresponding to an exothermic host-guest inter-
action, implies that the complex dissociates as the tem-
perature increases and the enthalpy value could be a result
of a few factors partially compensating each other: a) a
diminution of hydrogen bonds in the system; b) release of
of water molecules around the organic molecule. The en-
tropy values are also negative indicating that the increase
of entropy resulting from the hydrophobic effect is lower
than the diminution caused for loss of translational and
rotational degrees of freedom due to complex formation.
As result of these negative changes in enthalpy and en-
an enthalpy-controlled process. Moreover, we determined
the dependence of the thermodynamic parameters for the
inclusion process with the pH of the medium. All parame-
the energetics of the incorporation of the guest into the
b-CD cavity is modified significantly. The inclusion process
crease of the fluorescent guest solubility decreases the
exothermicity due to the lower release of well-organized
water molecules surrounding the guest and the release of
enthalpy-rich water molecules from the cyclodextrin cav-
ity. Also, DS0 increases probably due to a greater com-
5
pensation between the negative entropy contribution due
to the loss of degrees of freedom and the entropy positive
(F - F0)-1/(a.u.)-1x10-5
contribution originated by the hydrophobic effect.
4
Docking and Molecular Mechanics Studies.
Computational chemistry has been used to study a diver-
sity of host–guest complexes involving CD [35-37]. Doc-
king 3is a method of molecular modeling appropriate to pre-
dict the preferred the relative orientation of one molecule
respect to a second when are bound to form a stable com-
plex.2To investigate the inclusion geometry of the complex
formed 0.0between
0.2 7-ABZ 0.4and b-CD,
0.6 molecular
0.8 docking
1.0 stu-
of thedies was carried out using -1the AutoDock4
-3 -1 program. The
[β-CD] /(10 M)
best docking score
is shown
the 1:1 inclusion in Figure
complex formed5.between 7-ABZ
and β-CD in aqueous water solution (pH 6.0).
Figure 5. Figure
The most5.stable structure
The most obtained
stable from the
structure dockingfrom
obtained simulations
the of the
docking simulations inclusion
of theofinclusion
7-ABZ inofβ-CD.
7-ABZ in β-CD.
The docking results shows that the favored relative orien-
tation of the complex is one in which 7-ABZ inserts dia-
gonally in the b-CD cavity
23 with the phenyl substituent in
57
 position 3 of the oxazinone ring oriented towards the wide tric solvent molecules from these groups during binding
edge of the truncated cone whereas the amino substituent diminishes
is exposed to the inner surface near the narrow edge of
b-CD. The hydrogen of the amino substituent is located at Table 3. Contributions to the total entropy for the binding
1.9 Å of the oxygen of a hydroxyl group oriented towards of 7-ABZ with b-CD calculated by the MM-PBSA method.
inside the narrow edge of the cavity. This result suggests
that two types of interactions contribute to the complex Contribution TDS/kcal mol-1 K-1
stability, van der Waals interactions in the inner of the ca-
DSTraslational -12.44
vity and a hydrogen bond between the amino and hydro-
DSRotational -9.80
xyl group. The presence of a hydrogen bond also allow to
explain the increase in the fluorescence intensity because DSVibrational 5.18
restrict the rotation of the amino group that is a well-known DSTotal -17.06
non-radiative deactivation path of the excited singlet of
amino substituted dyes [38]. the contribution of electrostatic interaction component to
the binding free energy. This suggestion is consistent with
restrict
Table 2. theContributions
rotation of thetoamino the totalgroupfreethat is a well-known
energy for the binding non-radiativethe deactivation path
loss in configurational entropy as consequence of the
restrict
ofthe
7-ABZrotation
withofb-CD the amino groupby
calculated thattheis MM-PBSA
a well-known non-radiative deactivation
method. binding process.path Table 3, includes the contributions to the
of the excited singlet of amino substituted dyes [38]. total entropy to the binding process, obtained using the
of the excited singlet of amino substituted dyes [38].
Contribution MM-PBSA/kcal mol-1 NMODE module of Amber 12 software. Data on Table 3
Table 2. Contributions to the total free energy for the binding of 7-ABZ with β-CD
Table 2. Contributions to the total free indicates
β-CD that, the loss in configurational entropy is due
DEvdW calculated byenergy
the MM-PBSA for the binding
-24.03 method. of 7-ABZ with
DEElec calculated by the MM-PBSA -13.36 method. to the decrease of the traslational and rotational degree
Contribution MM-PBSA/kcal mol -1 of freedom. The increase in vibrational movements is not
DEPB -59.65 -1
Contribution
∆EvdW MM-PBSA/kcal -24.03mol enough to compensate it. Considering the energy con-
DENPolar ∆EvdW -15.78-24.03 tributions to the free energy of the binding process, the
∆EElec -13.36
DEDisper ∆EElec∆EPB 27.47-13.36 -59.65 MM-PBSA calculations indicates that the spontaneous
∆E∆E -59.65 inclusion of 7-ABZ in b-CD is driven mainly by entalphic
-37.39 -15.78
PB NPolar
ΔGgas
∆ENPolar
∆EDisper -15.7827.47 factors.
ΔGsolv ∆EDisper -47.9627.47
ΔGgas -37.39 In summary, photophysical behavior of 7-amino-3-phen-
ΔGBinding ΔGΔG gas solv -85.34-37.39 -47.96 yl-2H-benzo[b][1,4]oxazin-2-one in organic media is de-
ΔGΔG solvBinding -47.96-85.34 pendent on the solvent micro-properties. Emission shows
The components to the ΔGtotal
Binding free energy are -85.34
showed in an important Stokes shift in polar solvents, compatible
kcal/mol: van der Waals interaction component (DEvdW), with an increase of the dipolar moment in the excited sin-
The components to the total free energy are showed in kcal/mol: van der Waals interaction component
electrostatic
The (∆E
components
), to interaction
the
electrostatic total free component
energy
interaction are
component showed (DEinElec),kcal/mol:
(∆E
), non-polar
non-polar van dersol-
Waals
solvation
glet(∆E
interaction
component
state
component and the participation of a partial charge-transfer
vdW Elec NPolar), polar
vation
(∆EvdW
solvation component
), electrostatic
component (∆EPB(DEN
interaction ),
component
), dispersion
Polar
polar
(∆E
componentsolvation
Elec ), non-polar
(∆E Disper
component
), solvation
interaction component
energy in state
the(∆E
gas in ),
phase
NPolar
the deactivation
polar
(ΔG gas),
process. In aqueous b-cyclodex-
(DEPBcomponent
solvation ), energy
solvation dispersion PB),component
(∆Esolv
(ΔG dispersion
binding (DE
component
energy ), Disper
(ΔGtotal).(∆E
Disper
interaction
), interaction energy
energy in the gas trin solutions,
phase (ΔGgas), 7-amino-3-phenyl-2H-benzo[b][1,4]oxazin-
solvation energy (ΔGsolv), binding energy (ΔGtotal).
in the gas phase (ΔGgas), solvation energy (ΔGsolv), binding 2-one incorporates spontaneously to the cyclodextrin
The bindingtotalenergies (complexation energies) were evaluated withinner
energy (ΔG ). cavity by means of an enthalpy driven process. All
the MM-PBSA
TheThe binding
binding energies
energies (complexation
(complexation energies)
energies) were were evaluated
evalua- with the MM-PBSA
thermodynamic parameters associated the inclusion pro-
ted with the[39,
methodology MM-PBSA
40]. Using methodology
this method, inclusion [39, 40].free Using cess increases
this were estimated
energies using theas the pH of the medium decreases and the
methodology [39, 40]. Using
method, inclusion this method,
free energies were inclusion
estimated free energies
using the were estimated bindingusing of the
7-ABZ to b-CD becomes less spontaneous.
trajectories
trajectoriesofofeach each species
speciesfromfrom the same
the same simulation run. The
simulation run.complexation energies were
trajectories of each species from the same simulation run. The complexation energies were
The complexation energies were evaluated using structu-
evaluated
res coming using from structures
simulation coming runsfrom simulation7-ABZ
on isolated runs on and isolated 7-ABZ and β-CD
ACKNOWLEDGEMENTS
evaluated using structures coming from simulation runs on isolated 7-ABZ and β-CD
b-CD monomers solvated with water molecules. Binding
monomers solvated with water molecules. Binding energies are thus computed as follows:
energies
monomers are thus
solvated withcomputed
water molecules.as follows:
Binding energies are thus computedFinancial as follows: support from FONDECYT (grant 1110636) is gra-
tefully acknowledged. S.V. acknowledges Ph.D. fellowship
7 − ANBisolated + β − CDisolated ←  → [7 − ABZ  β − CD ]
7 − ANBisolated + β − CDisolated ←  → [
 7 − ABZ  β − CD ]complex complex 21110237 from (7) CONICYT.
(7)
According to equation (7), the interaction (7) energy is calculated from free energies of three
According to equation (7), the interaction energy is calculated from free energies of three
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