2
S. Roy et al. / Journal of Molecular Structure 1036 (2013) 1–6
biological guests within their hydrophobic cavities to afford typical
host–guest complexes in aqueous solution [10–12]. A number of
inclusion complexes of cyclodextrins with drugs like paclitaxel
[13] and cinchona alkaloids [14] resulting in increasing their aque-
ous solubility have been reported.
prepared in methanol and 3.4
the buffer solution to maintain the final concentration of DIMs as
17.0 M in 2.0 ml. The -, b- and -CD (1.7 l) were added gradu-
ally to the cell to maintain the concentration of CD’s ranged from 0
to 18.7 ꢀ 10ꢁ3 mM. The solution was kept stirred continuously.
Fluorescence emission spectra were acquired on a Shimadzu RF-
5301PC fluorimeter (Shimadzu Corporation, Kyoto, Japan) by exci-
tation at the absorption maxima of each compound. Excitation and
emission bandwidths were set at 5 nm.
ll of this solution was added to
l
a
c
l
Herein, we report inclusion complexes of DIM derivatives 1–6
(Fig. 1), previously synthesized and characterized in our laboratory
[15], with
a-, b- and c-cyclodextrins, and characterization of the
mode of inclusion and stoichiometry of the complexes by means
of absorbance [16], fluorescence [17], circular dichroism and
phase–solubility studies.
2.4. Circular dichroism spectroscopy
Circular dichroism spectra of compound 2 (0.5 mM) in presence
a, b, c cyclodextrin (10.0 mM) were recorded on a JASCO J815
2. Experimental
of
spectropolarimeter (Jasco International Co., Hachioji, Japan) in
2.1. General procedures
rectangular quartz cuvettes of 1.0 cm path length at 20 0.5 °C.
Commercial a-, b- & c- CDs (Fluka) were dried in a desiccator in
vacuo over phosphorus pentoxide at 90 °C for at least 24 h and
stored in the same apparatus at 40 °C till use. Guests 5 and 6 were
prepared by the reaction of indole (Acros) and corresponding benz-
aldehyde (Acros) in presence of InCl3 (Aldrich). All other reagents
and chemicals were of analytical reagent grade. All solutions were
prepared using ultra pure water (MILLIQ). Sodium dihydrogen
phosphate and disodium hydrogen phosphate were dissolved in
double distilled, deionised water to make a 0.1 (M) buffer solution
of pH 7.2, which was used as solvent throughout the measurement.
All experiments were performed at 298.15 K.
2.5. Phase–solubility study
Phase–solubility studies were carried out according to the
method reported by Connors [18]. A fivefold molar excess of DIMs
were added to the aqueous solutions of a-, b- and c- cyclodextrins
with increasing concentrations of 0.1, 0.2, 0.3, 0.4, and 0.5 mM. The
resulting solutions, protected from light by wrapping with black
paper, were stirred for 72 h with similar stirring rates. After equil-
ibration, aliquots of the supernatant were filtered through a mem-
brane filter (0.45 lm). The filtrates were then analyzed using UV
spectrophotometer at kmax of each compound.
For the determination of stoichiometry of inclusion complexes
the total molar concentration of the different DIMs–CD solutions
(i.e. the combined concentrations of DIMs and CD) were kept
constant (3.0 mM), but the mole fraction of DIMs (i.e. [DIMs]/
[DIMs] + [CD]) were varied (0.2, 0.4, 0.6 0.8 mol fraction). The
solutions were stirred for 48 h. The absorbance of the resulting
solutions was measured at kmax of each compound by using spec-
trophotometer. The differences in absorbance in the presence of
2.2. Absorption spectral titrations
The inclusion complex formation phenomenon of CDs with
guest DIMs in aqueous phosphate buffer solutions was examined
at pH 7.2 by means of UV–visible spectral titration in a Shimadzu
1700 Spectrophotometer (Shimadzu Corporation, Kyoto, Japan). A
1.0 mM stock solution of DIMs (5.0 ml) was prepared in methanol,
and 6.0 ll of this stock solution was added to the phosphate buffer
CDs and in the absence of CDs (DA = A–Ao) were plotted against
molar ratio R; where R = [DIMs]/{[DIMs] + [CD]} for all solutions.
solution to maintain the final concentration of molecules as
2.5 ꢀ 10ꢁ7 mol dmꢁ3 in the cuvette. Then gradually
a-, b-, c-CD
were added in the cuvette so that the concentrations of CDs ranged
from 0 to 12.5 ꢀ 10ꢁ3 mol dmꢁ3. The absorption spectra were mea-
sured against an appropriate reagent blank.
3. Results and discussion
3.1. Absorption spectra
2.3. Fluorescence spectroscopy
The UV absorption spectrum of 2 in methanolic solution
(5 ꢀ 10ꢁ6 M) exhibited a kmax at 317 nm for
p ?
pꢂ transition with
Fluorescence eꢁm3ission spectra of DIM derivatives (1–4)
-CD (0–18.7 ꢀ 10ꢁ3 mM). 1.0 mM stock solutions of DIMs were
(17 ꢀ 10ꢁ6 mol dm ) were measured in presence of
a-, b- and
molar absorptivity of 55.272 ꢀ 103 dm3 molꢁ1 cmꢁ1. Qualitative
investigation of the inclusion complexation phenomenon of CDs
with guest DIMs was performed at various pH by means of UV–
visible spectral titration. The optimum results were obtained at
pH 7.2. Table 1 shows the UV spectral wavelength in terms of
the ratio of the initial absorbance (A1) to final absorbance (A2)
and the spectral wave length shift data for all the DIMs and
c
a-, b-, c- CDs complexes in aqueous phosphate buffer at pH 7.2.
Generally, in all cases, maximum absorption bands shifted in pres-
ence of CDs, while the absorption intensity gradually decreased
with the stepwise addition of CDs. The absorption intensity of com-
pound 2 gradually decreased with the increasing concentration of
CDs along with blue shifting of the absorption band. For compound
2 the absorption intensity varied from the initial 0.109 to 0.081
with
0.099 for
, b- and
ences harder environmental changes upon inclusion in the narrow-
est -CD cavity than in the b-CD and -CD cavity, respectively, in
agreement with the notion that the effectiveness of non-bonding
a
-CD and from 0.138 to 0.107 with b- and from 0.137 to
-CD. This corresponds to 26%, 23% and 28%, respectively,
-CD. This suggests that compound 2 in general experi-
c
c
a
a
c
Fig. 1. General structure of 3,30-diindolylmethane derivatives 1–6.