Spectral and molecular modeling studies on hydroxybenzaldehydes with native and modified cyclodextrins

Article abstract of DOI:10.1007/s10895-013-1340-5

The inclusion complexation of 2-hydroxy-3-methoxybenzaldehyde (2HMB), 4-hydroxy-3-methoxybenzaldehyde (4HMB), 3,4-dimethoxybenzaldehyde (DMB) and 4-hydroxy-3,5-dimethoxybenzaldehyde (HDMB) with α-CD, β-CD, HP-α-CD and HP-β-CD were carried out by UV-Visibl

Full text of DOI:10.1007/s10895-013-1340-5

J Fluoresc
DOI 10.1007/s10895-013-1340-5
ORIGINAL PAPER
Spectral and Molecular Modeling Studies
on Hydroxybenzaldehydes with Native
and Modified Cyclodextrins
M. Jude Jenita & T. Mohandass & N. Rajendiran
Received: 16 September 2013 /Accepted: 27 November 2013
#
Springer Science+Business Media New York 2013
Abstract The inclusion complexation of 2-hydroxy-3-
methoxybenzaldehyde (2HMB), 4-hydroxy-3-
methoxybenzaldehyde (4HMB), 3,4-dimethoxybenzaldehyde
(DMB) and 4-hydroxy-3,5-dimethoxybenzaldehyde (HDMB)
with α-CD, β-CD, HP-α-CD and HP-β-CD were carried out
by UV-Visible, steady-state and time-resolved fluorescence
and PM3 methods. All the benzaldehydes shows dual fluores-
cence in aqueous and CD mediums and 1:1 inclusion com-
plexes were formed with CDs. PM3 geometry optimizations
results indicate that the HDMB/CD complex is significantly
more favorable than the other complexes. The negative en-
thalpy changes suggest that the inclusion complexation pro-
cesses are spontaneous. The geometry of the most stable
complex shows that methoxy/OH group of HMBs is
entrapped in the less polar CD cavities, while the aldehyde
group present in the upper part of the CDs cavities.
hydrophobic cavity of appropriate dimensions and hence can
form inclusion complexes with a variety of organic com-
pounds in the aqueous solution [2]. Model studies on CD
complexation offer valuable insights into molecular recogni-
tion and enzyme–substrate interactions [3]. In addition to
experimental approaches, theoretical calculations [4] can il-
lustrate the driving forces of the complexation [5] and the
inclusion regioselectivity in CD-catalyzed reactions [6]. Due
to the large size, most calculations on CD chose the molecular
mechanic (MM) method based on various empirical force
fields [7–10]. However, as MM method has difficulty in
modeling the molecules in their excited states, quantum me-
chanics (QM) methods must also be developed to study the
CD chemistry [11–13].
Investigation and application of cyclodextrins (CDs) and
their inclusion complexes in food, cosmetic and pharmaceu-
tical industries as well as in separation technologies has great-
ly increased in the last decades [14–16]. They are nontoxic
and safe to use as encapsulating materials for drugs and
biologically active compounds. Availability of a relatively
non-polar internal cavity in the toroidal structure of CD brings
about the inclusion complex formation of CDs with various
organic molecules. The properties (solubility, stability, test,
activity) of guest molecules placed inside the CD cavity can
be significantly changed. Thus, the practical importance of
CDs inclusion complexes is evident.
.
.
.
Keywords Aldehydes Cyclodextrin Inclusion complex
.
.
Intramolecular charge transfer Molecular modeling
Fluorescence
Introduction
α-, β-, γ-Cyclodextrins (CDs) are cyclic oligomers of six,
seven and eight α-d-glucose units connected through glyco-
sidic α-1,4 bonds [1]. These compounds, usually character-
ized as a doughnut or wreath-shaped truncated cones, have a
Our earlier studies on 4-hydroxy methoxybenzaldehydes
[17, 18], 4-hydroxy methoxybenzoic acids [19, 20] and hy-
droxy methoxybenzoic acids [21, 22] clearly shown that the
formation of ICT state in polar solvents and β-CD. In fact it
has been established that the barrier height for the ICT process
decreases with the increase in the solvent polarity. In this
paper, the effect of α-CD, β-CD, HP-α-CD and HP-β-CD
on the absorption and fluorescence spectra of 2-hydroxy-3-
methoxy benzaldehyde (2HMB, ortho vanillin), 4-hydroxy-
3-methoxy benzaldehyde (4HMB, vanillin), 3,4-dimethoxy
Electronic supplementary material The online version of this article
(doi:10.1007/s10895-013-1340-5) contains supplementary material,
which is available to authorized users.
:
:
M. J. Jenita T. Mohandass N. Rajendiran (*)
Department of Chemistry, Annamalai University, Annamalai
nagar 608 002, Tamilnadu, India
e-mail: drrajendiran@rediffmail.com

J Fluoresc
benzaldehyde (DMB, veratraldehyde) and 4-hydroxy-3,5-
dimethoxy benzaldehyde (HDMB, syringaldehyde) were
studied. The inclusion complexes of these benzaldehydes with
α-CD, β-CD, HP-α-CD and HP-β-CD were investigated by
UV-visible, steady state fluorescence, time resolved fluores-
cence and molecular modeling methods. The complexation
energies of the above mentioned molecules and CD during the
formation of inclusion complexes were studied using PM3
method in the aim to give some insights about the location of
hydroxyl group of the guest molecules toward primary or
secondary hydroxyl of CD. We also determine the driving
intermolecular interactions during the formation of such com-
plexes with the following points, (i) whether the encapsulation
of the hydroxyl groups same or different and (ii) the intramo-
lecular charge transfer effect of these molecules in CD media
to affect the complexation energy and intermolecular
interactions.
(Parametric method 3). Both CDs were fully optimized by PM3
without any symmetry constraint [25–27]. The glycosidic oxygen
atoms of CD were placed onto the XY plane and their centre was
defined as the centre of the coordination system. The primary
hydroxyl groups were placed pointing toward the positive Z axis.
The inclusion complexes were constructed from the PM3-
optimized the CD and the guest molecules. The longer dimension
of the guest molecule was initially placed onto the Z axis. The
position of the guest was determined by the Z coordinate of one
selected atom of the guest. The inclusion process was simulated
by putting the guest in one end of CD and then letting it pass
through the CD cavity. Since Density Functional Theory (DFT)
calculations are expensive (cost and takes long time) in treating
such large molecular systems, we used single point energy calcu-
lations to the PM3 optimized geometries using semiempirical
method as implemented in Gaussian 03 W [25–28].
Results and Discussion
Experimental
Effect of Cyclodextrins
Reagents and Methods
Figures 1, 2 and supporting Figures S1 and S2 depict the
absorption and emission spectra of 2HMB, 4HMB, DMB
and HDMB (2×10−5 M) in pH ~7 solutions containing differ-
ent concentrations of α-CD, β-CD HP-α-CD and HP-β-CD.
We provided the spectra of α-CD and β-CD only, because the
spectral shapes and shifts of the aldehydes with HP-α-CD and
HP-β-CD inclusion complexes were similar to the α-CD and
β-CD inclusion complexes respectively. The absorption max-
ima of all the above benzaldehydes in water (2HMB: λabs
~342, 310, 264, 217 nm, λflu ~338, 500 nm; 4HMB: λabs
~311, 279, 231 nm, λflu ~329, 425 nm; DMB: λabs ~307,
277, 230 nm, λflu ~335, 433 nm and HDMB: λabs ~304,
213 nm, λflu ~330, 434 nm) were red shifted than that of 4-
hydroxy benzaldehyde (HB: λabs ~294, 221 nm).
All the aldehydes and CDs were obtained from Sigma-Aldrich
and used as such. Solutions in the pH range 2.5–12.0 were
prepared by adding appropriate amount of phosphate buffer
(NaOH and H3PO4). A modified Hammett’s acidity scale (H0)
[23] for the solutions below pH ~2 (using a H2SO4-H2O
mixture) and Yagil’s basicity scale (H−) [24] for solutions
above pH ~12 (using a NaOH-H2O mixture) were employed.
Triply distilled water was used for the preparation of aqueous
solutions. The solutions were prepared just before taking
measurements. The concentration of the drug solutions were
in the order of 4×10−4 to 4×10−5 M. The concentrations of
CDs were varied from 1×10−3 to 10×10−3 M. The experi-
ments were carried out at room temperature 303 K.
With on increasing the four CDs concentrations, the above
benzaldehydes have not shown any significant spectral shifts
in the absorption spectra. However, the absorbance of all the
aldehyde molecules regularly increased along with the CDs
concentrations. The changes in the absorbance and fluores-
cence intensities of the above benzaldehydes measured as a
function of α-CD, β-CD, HP-α-CD and HP-β-CD were
plotted in the inset (Figs. 1, 2, 3 and 4). A similar trend was
also observed in pH ~1 solutions. In the absorption spectra,
the presence of isosbestic point in all the benzaldehydes
indicates the possibility of a single equilibrium involving 1:1
complexation between the guests and the CDs. The associa-
tion constants can therefore be evaluated from the Benesi–
Hildebrand equation [29, 30] for the 1:1 inclusion complexes
of all the CDs with the aldehydes. Figures 3 and 4 show a plot
of 1/A-A0 and 1/I-I0 as a function of 1/[CD] and a plot of 1/A-
A0 and 1/I-I0 as a function of 1/[CD]2. From the intercept and
Instruments
Absorption spectral measurements were carried out with a
Shimadzu UV-Visible spectrophotometer (model 1650 PC)
and fluorescence measurements were made by using a
Shimadzu spectrofluorimeter (model RF-5301). The pH
values in the range 2.5–12.0 were measured on an Elico pH
meter (model LI-120). The fluorescence lifetime measure-
ments were performed using a picoseconds laser and single
photon counting setup from Jobin-Vyon IBH.
Molecular Modeling
The theoretical calculations were performed with Gaussian 03 W
package. The initial geometry of the HBs, α-CD and β-CD were
constructed with Spartan 08 and then optimized by PM3

J Fluoresc
0.8
0.4
0
1.0
0.5
Fig. 1 Absorption spectra of a
2HMB, b 4HMB, c DMB and d
HDMB in different α-CD
concentrations (M) at pH ~7: (1)
0, (2) 0.002, (3) 0.004, (4) 0.006,
(5) 0.008 and (6) 0.01. Insert Fig.:
absorbance vs. α-CD
0.41
0.39
0.43
0.41
0.39
0.37
311 nm
(b)
342 nm
(a)
Ab
s
Abs
0.37
6
1
6
1
0.35
0
10
15
0
5
10
15
5
[α-CD] 10-3 M
[α-CD] 10-3 M
×
×
concentrations
0
280
200
350
400
200
320
360
250
300
240
Wavelength (nm)
Wavelength (nm)
1.5
1.0
0.90
0.59
(d)
304 nm
(c)
0.56
0.53
0.85
0.80
Abs
Abs
307 nm
10
0.50
0.75
6
1
15
0
5
10
15
0
5
6
1
[α-CD] 10-3 M
×
[α-CD] 10-3M
×
0.5
0.75
0
200
0
200
300
Wavelength (nm)
240
320
360
350
400
250
280
Wavelength (nm)
slope values of this plot, K values were determined. Good
linear correlations were obtained confirming the formation of
1:1 inclusion complexes.
The fluorescence spectra of all the benzaldehydes with α-
CD, β-CD, HP-α-CD and HP-β-CD are displayed in (Figs. 2
and S2). In the excited state, the above benzaldehydes reflect
remarkable changes in all the CD solutions. The above benz-
aldehydes exhibit a dual fluorescence of locally excited
(shorter wavelength, SW) and ICT (longer wavelength, LW)
states in water and CD solutions. Absorption and emission
maxima of 2HMB are red shifted than that of other aldehydes.
In aqueous and CD media, the LW band intensity of 2HMB,
4HMB and HDMB were greater than SW band intensity
whereas in DMB, the SW intensity was greater than LW band
intensity. With an addition of CDs both SWand LW intensities
increases in all the aldehydes. However, in DMB and HDMB
the rate of enhancement of the SWemission is greater than that
of LW band. In 2HMB and 4HMB, the LW fluorescence
intensity is greater than SW band intensity. In CD solutions,
the LW and SW band of 4HMB, DMB and HDMB increased
200
150
Fig. 2 Fluorescence spectra of a
2HMB, b 4HMB, c DMB and d
HDMB in different α-CD
concentrations (M) at pH ~7: (1)
0, (2) 0.002, (3) 0.004, (4) 0.006,
(5) 0.008 and (6) 0.01. Insert Fig.:
absorbance vs. α-CD
195
(b)
120
(a)
175
155
80
If
If
6
1
6
1
425 nm
10
50
500 nm
10
135
20
15
0
5
15
0
5
[α-CD] 10-3 M
100
×
75
[α-CD] 10-3 M
×
concentrations; Excitation
wavelength : a 2HMB – 342 nm,
b 4HMB - 310 nm, c DMB –
335 nm, and d HDMB – 330 nm
0
0
250
400
400
Wavelength (nm)
550
300
500
325
475
350
450
Wavelength (nm)
300
150
90
300
(c)
75
60
225
If
150
If
330 nm
10
336 nm
10
[α-CD] 10-3 M
6
45
75
0
15
0
5
[α-CD] 10-3 M
15
5
×
150
75
6
1
×
1
(d)
0
300
0
400
400
300
450
500
450
500
350
350
Wavelength (nm)
Wavelength (nm)

J Fluoresc
40
18.0
13.5
Fig. 3 Benesi-Hildebrand plot
for 1:1 inclusion complexation of
HMBs with α-CD and β-CD at
pH ~7: (a) and (b) plot of 1/A-Ao
vs. 1/[CD], (c) and (d) plot of 1/I-
Io vs. 1/[CD]
2HMB
4HMB
HDMB
DMB
(a)
(b)
2HMB
4HMB
HDMB
DMB
20
1/A-Ao
1/ A-Ao
9.0
0
4.5
-20
0
0
100
200
400
600
0
500
100
200
400
500
600
300
300
1/[α-CD] M
1/[β-CD] M
0.100
0.12
0.08
2HMB
4HMB
HDMB
DMB
2HMB
(c)
(d)
4HMB
HDMB
DMB
0.075
1/I-Io
1/ I-Io
0.050
0.04
0.025
0
0
0
100
200
400
600
0
100
200
400
600
500
500
300
300
1/[α-CD] M
1/[β-CD] M
at the same wavelength. Interestingly, in 2HMB, with increas-
ing the CDs concentrations, a large blue shift were observed in
the LW bands, whereas no significant spectral change were
seen in the SW bands. This can be seen more clearly from
looking at the (Figs. 2 and S2). The SW band intensities of
2HMB in 0.01 M CD solution is increased to 0.5 times more
than that of aqueous medium and LW band emission en-
hanced approximately 10 times. Further, the fluorescence
intensities of LW band increases with increase in the excita-
tion 270 nm to 320 nm. This may be the extended π-
conjugation would induce an excited state resonance contri-
bution of the carbonyl group to the benzene ring resulting in
the increased polarity to facilitate the interaction with water.
The SW fluorescence intensities increased from lower to
higher CD concentrations. Further LW band is largely red
shifted in more protic solvents than SW band. The above
results may assume ICT present in these molecules [22,
30–36]. If the LW band maximum was due to ICT, the
intensity of fluorescence may decrease in CD solutions. The
presence of LW emission maxima in DMB suggests ICT
present in these aldehydes. Further, appearance of LW band
in a hydrocarbon solvent indicates that ICT emission associ-
ated with H-bonding [17, 18] is present in these aldehydes.
The reason for the LW band is already explained in our earlier
40
18
Fig. 4 Benesi-Hildebrand plot
2HMB
4HMB
HDMB
DMB
(b)
for 1:2 inclusion complexation of
HMBs with α-CD and β-CD at
pH ~7: (a) and (b) plot of 1/A-Ao
vs. 1/[CD]2, (c) and (d) plot of 1/
I-Io vs. 1/[CD]2
2HMB
4HMB
HDMB
DMB
(a)
13.5
20
1/A-Ao
1/A-Ao
9
0
4.5
0
-20
300
250
300
200
1/[β-CD]2 × 103 M-1
250
0
50
100
150
200
0
50
100
150
1/[α-CD]2 × 103 M-1
0.08
0.1
2HMB
2HMB
4HMB
HDMB
DMB
(c)
(d)
4HMB
HDMB
DMB
0.06
1/I-Io
0.04
0.075
1/I-Io
0.050
0.025
0
0.02
0
50
100
150
200
250
300
100
200
250
300
0
0
50
150
1/[α-CD]2 × 103 M-1
1/[β-CD]2 × 103 M-1

J Fluoresc
Table 1 Absorption, fluorescence, lifetime, binding constant and ΔG
values of 2HMB, 4HMB, DMB and HDMB in water and α-CD and β-
CD environments
publications [19, 20, 31–36]. The LW band fluorescence
regularly red shifted from non-polar to polar solvents [19,
20, 35, 36]. The intensity of LW band also increased from
cyclohexane to water. Further LW band was largely red shifted
more in protic solvents than SW band. The appearance of the
LW band in a hydrocarbon solvent indicates that ICT is
present in all the aldehydes.
Compound Medium λabs log λflu <τ> Binding ΔG k cal
nm
ε
nm ns
constant mol−1
(K−1
)
Abs flu abs
flu
The full width at half the maximum height (FWHM) of the
ICT fluorescence band increased with increase in the CD
concentrations [17–22]. FWHM of the band ICT fluorescence
was different in water and CD solutions. This is because these
aldehydes have multi functional groups (OH, CHO, and
OCH3). Hence the emission of a state with a high ICT char-
acter is stabilized by H-bonding with the solvent. In such a
state, the donor group is the OH/methoxy/aromatic moiety of
the aldehydes molecules and the CHO group is the acceptor.
Further, the increase of SW fluorescence intensities in DMB
and LW fluorescence intensities in other HMBs confirm the
above prediction. These results again support that the ICT
emission present in all these molecules.
The CD dependent emission spectra show that the ICT
band is less sensitive to increase in all the CD concentrations,
whereas the SW band shows an enhancement. Inside the CD
cavities, HMB feels much less polar environment and the
main non-radiative path of the locally excited band (through
ICT) is restricted which also causes an enhancement of the
SW band. Further, the geometrical restriction of the cavity
would prevent the free rotation of the CHO or OCH3 groups in
the CDs cavities and thus hinder the formation of ICT state
causing an enhancement of normal SW band.
2HMB
Water
α-CD
β-CD
342 3.76 500 1.17
310 3.78 338
264 4.61
217 4.83
344 3.85 455 2.42 105 183 −2.80 −3.13
310 3.84 330
364 4.57
218 4.79
342 3.95 455 3.68 250 320 −3.32 −3.46
310 3.94 330
263 4.57
218 4.79
311 3.56 425 1.49
279 4.20 329
231 4.36
312 3.68 429 2.60 163 295 −3.06 −3.42
280 4.30 330
231 4.47
312 4.00 430 3.06 296 384 −3.42 −3.58
282 4.07 330
231 4.13
307 4.20 433 0.68
277 4.29 335
230 4.44
4HMB
Water
α-CD
β-CD
Water
α-CD
β-CD
DMB
307 4.33 435 1.06 98 124 −2.76 −2.90
277 4.41 337
230 4.54
306 4.43 436 1.42 157 243 −3.04 −3.30
279 4.36 336
The binding constant (K) values in α-CDs are greater than
β-CDs which indicate that the benzaldehyde molecules are
more tightly encapsulated in the α-CDs cavity than β-CDs.
The ‘K’ value of HDMB is less than other HMBs which
indicates that 2HMB, 4HMB and DMB are more deeply
included in the β-CDs cavities than HDMB. Since the band
distance between the two methoxy groups is greater than the
CDs cavities size; hence it is partially included in the CDs
cavities. Since the band distance of other aldehydes (2HMB/
4HMB/DMB) is less than CDs cavities size these molecules
may completely encapsulated in the CDs cavities. As can be
seen from Table 1, ΔG values are negative which suggest that
the inclusion processes are exothermic and spontaneous.
Table 1 show that the binding constants were apparently
varied with the properties of the substituents. The substituent
with a larger ‘K’ value fits more tightly into the CDs cavities.
Further, the higher polarizability of the substituent’s with
larger ‘K’ values favors binding through interaction between
permanent dipoles of CDs and induces dipoles of guests. The
strength of interaction is also dependent on the size of the CDs
cavities. As Figs. 3 and 4 shows the slope of the straight line
for α-CD is twice that of β-CD. This means that the interac-
tions are more sensitive to the size of substituents in the
232 4.44
304 4.40 434 2.22
213 4.52 330
306 4.52 433 3.71 269 373 −3.36 −3.56
215 4.65 330
304 4.70 433 4.25 372 493 −3.56 −3.73
217 4.80 330
HDMB
Water
α-CD
β-CD
complexation of α-CD than β-CD. Further the SW fluores-
cence intensity higher than LW supports this implication.
Several driving forces are possible for the inclusion com-
plexation of CD with guest compounds. However, in these
molecules, the hydrogen bonding interactions play a major
role in the inclusion complexation of CD with these alde-
hydes. In these cases, the inclusion process is determined by
the aldehyde and hydroxyl groups. The ‘K’ values are a
reasonable measure of hydrogen bonding and the change in
hydrogen bonding of aldehydes is caused only by the hydro-
gen ion concentration. Since the hydroxyl and methoxy sub-
stituent locate near the narrower rim of the CD cavity and
aldehyde group locates at wider rim of the CD cavity, the ‘K’
values are proportional to the hydrogen bonding interactions.
In DMB, the SW and in other HMBs the LW fluorescence

J Fluoresc
intensities are greatly enhanced with increasing CD concen-
trations which support this prediction. The difference in slopes
in Figs. 3 and 4 for the inclusion complexes indicates that the
interactions of hydrogen atoms, especially with β-CDs are
much stronger than α-CDs because HMB interactions are
approximate to the hydrogen bonding contact with α-CDs.
They are somewhat weak, since the hydroxyl groups are far
from the internal surface of the β-CD cavity in the inclusion
complexes. Thus for HMB, the binding constant with β-CDs
are greater than with α-CDs Table 1.
In order to substantiate this, the absorption and emission
maxima of all the aldehydes were measured in different
polorities of the solvents (Table 2). Compared to in any one
solvent, the absorption and emission maxima of 2HMB was
red shifted than that of 4HMB, DMB, HDMB and HB (cy-
clohexane: λabs ~288, 281, 274 and 265 nm, methanol: λabs
~294, 221 nm). The absorption spectra of 2HMB is different
from other molecules is due to the greater charge transfer
interaction of the aldehyde group with the aromatic moiety
and/or hydroxyl group. 2HMB show absorption maxima
around 350 nm in nonpolar solvents suggest that intramolec-
ular hydrogen bonding (IHB) present in this molecule. The
molar extinction coefficient is very high (~10−4 cm−1) and the
absorption maximum slightly blue shifted from 350 nm
to 340 nm as the solvent polarity increases imply that
this IHB band was affected by the polar solvents. The
absorption maxima of DMB and HDMB were red
shifted than 4HMB indicates, addition of methoxy groups in
HB molecules increasing the interaction between hydroxy and
aldehyde groups.
The LW band fluorescence were regularly red shifted from
non polar to polar solvents (Table 2). The intensity of LW
band was also increases from cyclohexane to water. It should
also be pointed out, the fluorescence intensity of the LW band
increases with increase in the λexc ~280 to 320 nm. Further
LW band was largely red shifted in more protic solvents (350
to 430 nm) than SW band (323 to 335 nm). The appearance of
the LW band in a hydrocarbon solvent indicates that intramo-
lecular charge transfer (ICT) present in all these aldehydes.
The SW band fluorescence intensities increased from lower
to higher CD concentrations. The above results may assume
different species present in these molecules. The presence of
LW emission maxima in DMB suggests ICT present in all
these aldehydes. The appearance of the LW band in a hydro-
carbon solvent indicates that ICT emission associated with H-
bonding [17, 18] present in these molecule. Supporting this
implication the excitation spectra exhibit the monitoring
wavelength dependence. The excitation spectrum for the
430 nm fluorescence was distinctly different from that for
the 330 nm fluorescence. Further, the fluorescence spectrum
in the water solution was significantly changed on addition of
dioxane showing a dual emission and an isoemissive point in
water-dioxane mixture. As expected [17, 18] the fluorescence
maximum of band LW red shifted with an increase of meth-
anol content. The ICT emission increases in protic solvents
suggesting that the hydrogen bonding interactions plays a
Table 2 Absorption and fluores-
Solvents
2HMB
4HMB
DMB
HDMB
cence spectral data (nm) observed
for 2HMB, 4HMB, DMB and
HDMB in different solvents
λabs
λflu
λabs
λflu
λabs
λflu
λabs
λflu
Cyclohexane
340
264
219
400 s
320
295
270
232
348
323
298
270
227
350
321
300
288
226
217
309
230
325
1,4-Dioxane
Acetonitrile
2-Propanol
Methanol
340
264
219
340
264
219
342
264
218
342
262
218
340
260
218
440
325
305
273
231
304
272
231
311
281
235
308
279
234
290
256
218
357
328
306
273
228
309
278
228
308
279
228
306
278
228
306
279
228
368
325
337
445
325
359
323
405
327
310
228
340
390 s
464
325
382
326
412
328
310
231
216
308
254
215
300
217
330 s
400
470
325
393
327
414
329
330
410
Water(pH ~6.5)
490
425
327
430
335
330
430
326 s

J Fluoresc
Table 3 Various prototropic maxima (absorption and fluorescence) of
2HMB, 4HMB, DMB and HDMB in aqueous, α-CD and β-CD medium
major role in the ICT emission. The dual emission observed in
DMB support the presence of ICT in these aldehydes, because
monocation formation is not possible in these aldehydes.
From the above results, it is clear that in CD solutions, the
surrounding polarity of aldehyde groups does not change very
much as there is no hypsochromic shift of the most polar (ICT)
state [37, 38] of aldehydes, but the relative intensity (IICT/ILE)
increases with the increase of CDs concentration. This may
only be possible if the orientation of these molecules is such
that the aromatic ring goes inside the CDs cavities and the
aldehyde group is present in the upper part of the CDs cavities.
Further, if aldehyde group is present in the inner CDs cavities,
a hypsochromic shift or absence of ICT emission could be
observed in the CD medium. However, a large hypsochromic
shifts observed in 2HMB indicates that this molecule is
completely encapsulated in the CDs cavities. This is because
the size of this molecule is smaller than CDs cavities. Further,
the emission enhancement in α-CDs solution is higher than β-
CDs, which reveals that, the smaller sized α-CDs cavities
restrict the free rotation of the benzaldehyde molecules.
Aqueous
α-CD
β-CD
λabs
λflu
λabs λflu
λabs λflu
2HMB
4HMB
DMB
Monocation 344
496
300
337
263
215
342
263
217
386
281
236
290
259
220
312
280
231
277
218
481
320
338
263
215
342
256
222
390
275
235
290
259
220
285
258
220
282
250
217
482
319
267
224
340
265
220
395
281
235
Neutral
468
324
455
300
468
332
Monoanion
550
353
517
316
459
319
Monocation 292
358
400
351
348
259
218
Neutral
289
256
218
299
216
426
325
429
330
425
330
Monoanion
359
415
358
Effect of Acid/Base Concentrations
Further, the effect of CD on the prototropic equilibrium be-
tween neutral, monoanion on the pH dependent changes in the
absorption and emission spectra of these molecules in CD
solution were measured. The spectral data of the prototropic
species of all the aldehyde molecules is complied in Table 3.
With an increase in pH from 7 to 10, a red shift is observed in
the absorption spectrum and this reflects the formation of the
monoanion. The proton can be removed from the hydroxyl
group because the pKa value for this reaction is 10, calculated
spectrophotometrically and it’s agreement with the value for
the deprotonation reaction of the OH group of all the aldehyde
molecules. A similar pKa value is observed in the case of all
the aldehyde molecules further confirmed deprotonation takes
place in the hydroxyl group. When the hydrogen ion concen-
tration is increased from pH ~7 to 1, a red shift is noted in the
absorption spectra indicates that, the monocation species
formed by protonating the carbonyl group. The above assign-
ments are consistent with the earlier finding shows that, the
red shift in the absorption when protonation takes place at the
carbonyl group or when deprotonation takes place from the
hydroxyl group [17–22].
Monocation 310
435
339
283
228
Neutral
308
280
226
430
336
307
277
230
432
335
306
277
227
430
355
HDMB Monocation 315.4
219.0
Neutral
300
217
420-
430
330
430
330
359
306
215
362
250
212
433
330
298
221
425
330
Monoanion
371
420
368
256
218
430
330
264 s
251
222
of all the aldehyde molecules were more deeply entrapped in
the CD cavities than aldehyde group.
The changes observed in the fluorescence spectra of all the
aldehyde molecules follow the same trend as that observed in
the corresponding reactions ground state. The absorption and
emission maxima were studied in 6×10−3 M CD solutions in
the pH range 0.1 to 11.0. When compared to aqueous medium,
a slight blue shift were observed in the absorption maxima of
neutral, whereas a large blue shift was observed in the
monoanion maxima of the benzaldehydes in CD medium than
aqueous medium. These results again supported OH group of
Excited State Singlet Lifetime
In order to confirm the interpretation of the steady-state fluo-
rescence, the time-resolved fluorescence decay times of all the
aldehydes in the CDs solutions were investigated. The average
fluorescence lifetimes are presented in Table 1. Analyzing the
fluorescence decay obtained at different observation, it is
found that for monitoring wavelength corresponding to the
maximum of SW (330 nm) fluorescence band, there are two

J Fluoresc
Fig. 5 CAChe – DFT optimized
structure of 2HMB, 4HMB,
DMB and HDMB
(a)
(b)
(c)
(d)
decay components, whereas for LW (430 nm) fluorescence
decays are very well fitted by a three-exponential function. On
the basis of the performed analyses of the experimental life-
time and pre-exponential factor, we suppose that in aqueous
solutions containing different concentrations of CDs, two or
three fluorescence emitting centers exist. The CD dependent
biexponential decay times of the SW emission lead us to
conclude that the fast decay component originates from the
emission of the hydrogen bonded complexes, whereas the
slow decay component results from 1:1 type inclusion com-
plexes. It is worth to note that the relative amplitude of the 1:1
type inclusion complex emission increases as the CD concen-
tration increases. In all the four CDs, the decay pattern mon-
itored at the LW emission differed considerably from that at
SW emission. For a monitoring wavelength corresponding to
the maximum of LW fluorescence band, an additional decay
component appears in the fluorescence decay.
FTIR Spectral Studies
(i) 2HMB/4HMB: The OH stretching vibrations in 2HMB/
4HMB produce a strong band in the 3159 cm−1 region
which is largely red shifted in the inclusion complex to
3397 cm−1. The OH bending at 1305 cm−1 and out of
plane deformation frequencies at 745 cm−1 also red
shifted in the inclusion complex to 1381 and 763 cm−1,
respectively. The C–O–CH3 symmetric stretching fre-
quency at 1095 cm−1 slightly red shifted in the complex.
Further C=C aromatic ring stretching at 1589 cm−1 also
red shifted in the complex to 1597 cm−1. However, the
C=O stretching vibration at 1673 cm−1 blue shifted in this
complex to 1682 cm−1. Further, the absorption intensity
of the inclusion complex is also significantly varied from
free guest molecules.
(ii) DMB: The OH stretching vibrations in DMB produce a
strong band in the 3077 cm−1 region which is largely red
shifted in the inclusion complex to 3345 cm−1 respec-
tively. The OH bending at 1379 cm−1 and out of plane
deformation at 735 cm−1 frequency also red shifted in the
inclusion complex to 1394 and 758 cm−1. The C–O–
CH3 symmetric stretching frequency at 1036 cm−1
Solid Inclusion Complex Studies
Further to confirm the HMB:CD interactions, we also pre-
pared the solid inclusion complexes and characterized by
FTIR and 1H NMR methods.

J Fluoresc
Fig. 6 The proposed inclusion
complex structure of 2HMB,
4HMB, DMB and HDMB
(a)
(b)
(c)
(d)
slightly red shifted in the complex. Further, C=C aromat-
ic ring stretching at 1598 cm−1 also red shifted in the
complex to 1608 cm−1. However, the C=O stretching
vibration at 1672 cm−1 blue shifted in this complex to
Table 4 Binding energies and
Properties
2HMB
4HMB
DMB
HDMB
α-CD
β-CD
HOMO, LUMO energy of
2HMB, 4HMB, DMB and
HDMB before inclusion com-
plexation by PM3 method
E
E
E
HOMO (eV)
−8.95
−0.68
−8.27
1.68
−9.15
−0.46
−8.69
1.24
−9.02
−0.42
−8.60
1.71
−9.05
−0.46
−8.59
2.16
−10.38
1.26
−10.35
1.23
LUMO (eV)
HOMO – ELUMO (eV)
−11.64
11.34
−11.58
12.29
Dipole Moment(D)
E*
−91.44
20.49
7.13
−92.48
23.44
5.55
−81.76
3.48
−125.25
38.16
6.24
−1247.52
−571.21
−676.73
−0.353
634.47
−1457.63
−667.55
−789.52
−0.409
740.56
G*
H*
33.29
0.099
108.00
S**
0.092
92.35
0.097
91.29
0.107
110.99
Unit * = Kcalmol-1 , ** = cal/mol-
Kelvin
Zero point energy

J Fluoresc
1681 cm−1. Further, the absorption intensity of the inclu-
sion complex is also significantly varied from free guest
molecules.
7.047 (7.264), 6H=7.428 (7.462); DMB: CHO=9.823
(9.523), OCH3=3.940 (3.911), OCH3=3.966 (3.879), 2H=
7.422 (7.458), 5H=6.993 (7.031), 6H=7.472 (7.645) and
HDMB: CHO=9.812 (9.654), OCH3, OCH3 =3.961
(3.887), OH=6.120 (6.457) and 2H and 6H=7.152 (7.207).
The Δδ values for host protons in the CDs indicate that all
the aldehydes are included in the CDs cavities. A significant
upfield shift of the CHO and OCH3 protons confirm that the
aldehydes were completely included in the CDs cavities.
Further, the well separated signals arising from these benzal-
dehyde molecules protons in the CD ring suggest that all the
benzaldehydes are tightly encapsulated within the CD cavity.
1HNMR Spectral Studies
Proton NMR (400 MHz in D2O) was used to compare the
environments of the pure and complexed molecules and in
particular to confirm interactions of the guest molecules.
Proton nuclear magnetic resonance (1H NMR) spectroscopy
has proved to be a powerful tool in the study of inclusion
complexes [30–33]. The resonance assignment of the protons
of CD are well established [13, 14] and consists of six types of
protons. The chemical shift of CD protons reported by
different authors are very close to those reported in this
work. The H-3 and H-5 protons are located in the
interior of the CDs cavities and it is, therefore likely
that the interaction of the host with the CD inside the
cavity will affect the chemical shifts of the H-3 and H-5
protons. A minor shift is observed for the resonance of
H-1, H-2 and H-4 located on the exterior of CD.
Significant chemical shifts changes Δδ were observed
with equimolar mixture of guest and host molecules.
Values of pure aldehyde and their (inclusion complexes)
are as follows:
Molecular Modeling of the Inclusion Complexes
A molecular modeling study was performed using PM3 method
to find out the most probable conformation of the HMB-CD
complex and to give a meaningful 3D visualisation of the
inclusion complex. The optimized structure of the aldehydes
are shown in Figs. 5, 6 and S3 present the best energy ranked
structures of HMB-CD inclusion complexes. The optimized
structure, bond distances, bond angles and most out of the
ordinary dihedral angles in these molecules before and after
complexation considered by PM3 method are presented in
Tables S1 and S2. Consistent with the fluorescence results, the
molecular modeling studies show that the methoxy/OH group
of HMBs is incorporated into the relatively less polar cavity of
CDs, while the aldehyde group is present in the upper part of the
CDs cavities. The studies also suggest that the probable mode of
inclusion of HMB is through its aldehyde group.
2HMB: CHO=10.310 (10.254), OCH3=3.997 (3.902),
OH=6.287 (6.567), 4H=7.990 (7.998), 5H=7.139 (7.312),
6H=8.073 (8.123); 4HMB: CHO=9.823 (9.578), OCH3=
3.960 (3.880), OH=6.390 (6.872), 2H=7.422 (7.458), 5H=
Table 5 Binding energies and HOMO, LUMO energy of 2HMB, 4HMB, DMB and HDMB after inclusion complexation by PM3 method
Properties
2HMB α-
2HMB - β-
4HMB - α-
4HMB - β-
DMB - α-
DMB - β-
HDMB - α-
HDMB - β-
CD
CD
CD
CD
CD
CD
CD
CD
EHOMO (eV)
−8.93
−0.70
−8.23
−9.09
−0.52
−8.57
−9.51
−0.61
−8.90
−8.90
−0.44
−8.46
−9.51
−0.68
−8.83
−9.13
−0.44
−8.69
−9.23
−0.67
−8.56
−9.52
−0.94
−8.58
ELUMO (eV)
EHOMO – ELUMO
(eV)
Dipole moment (D) 13.23
9.06
12.42
10.97
8.73
11.35
9.22
8.45
ΔD
0.21
−4.91
−0.16
−2.56
−4.32
−2.65
−4.28
−6.00
E*
−1336.80
−2.16
−559.91
−9.19
−679.95
−10.35
−0.392
−0.131
728.45
−1556.10
−7.03
−1344.83
−4.83
−1558.56
−8.45
−1329.68
−0.40
−535.28
32.45
−1539.12
−0.01
−638.78
25.29
−1378.40
−5.63
−1597.69
−14.81
−686.01
−56.64
−827.07
−43.79
−0.473
−0.171
853.69
ΔE*
H*
−665.80
−18.74
−800.97
−18.58
−0.453
−0.136
834.43
−567.47
−19.70
−686.42
−15.24
−0.398
−0.142
728.61
−667.95
−23.84
−803.41
−19.44
−0.454
−0.142
834.48
−579.87
−46.84
−701.29
−30.80
−0.407
−0.161
748.32
ΔH*
G*
−655.91
−12.47
−0.404
−0.150
744.96
−776.45
−20.22
−0.461
−0.151
850.83
ΔG*
S*
ΔS*
Zero point energy
Unit * = Kcalmol−1 , ** = cal/mol-Kelvin

J Fluoresc
Fig. 7 HOMO-LUMO structure
of the inclusion complex a
2HMB, b 4HMB, c DMB and d
HDMB with β-CD
HOMO
LUMO
(a)
(b)
(c)
(d)
Tables 4 and 5 shows the calculated binding energy of
HMBs complexed with α-CDs and β-CDs. The binding
energy change allowed us to evaluate the inclusion process
and to find the most stable inclusion complex between the
complexes studied. It was evaluated as
all the aldehydes with β-CD is more negative than that of α-
CD complexes. Also, the binding energy change of HDMB
with α-CD/β-CD is more negative than all the other com-
plexes. This indicates that HDMB:α-CD/β-CD forms more
stable inclusion complex. It is evidenced that the complexa-
tion process is energetically favorable.
The binding energy of the isolated molecules and com-
plexes suggested that stability of the complexes were high
compared to isolated molecule. From Tables 4 and 5, the
dipole moment of the 2HMB is 1.68 D, 4HMB is 1.24 D,
DMB is 1.71 and HDMB is 2.16 D. This was lower than that
of the complex, (2HMB:α-CD/β-CD complex ~13.23/9.06
D, 4HMB:α-CD/β-CD complex ~12.42/10.97 D, DMB:α-
CD/β-CD complex ~8.73/11.35 D and HDMB:α-CD/β-CD
ΔEcomplexaton ¼ Ecomplex – ðECD − EHMBs
Þ
ð1Þ
where Ecomplex, ECD and EHMBs represent the total energy of
the complex, the free optimized CDs and the free optimized
HMBs respectively. The negative binding energy changes
upon complexation clearly demonstrate that CDs can form
stable complexes with HMBs. The binding energy change of

J Fluoresc
complex ~9.22/8.45 D lower than the dipole moment for
resident molecules. The above quantum mechanical compu-
tation values demonstrated a strong relationship with the
complexation behavior.
negative than that for the other complexes. It means that all the
inclusion processes are enthalpically favorable in nature due
to the negative enthalpy changes. The ΔH and ΔS for all the
aldehydes with CDs are negative, suggesting that the forma-
tion of the inclusion complex is an enthalpy-driven process.
Further, a negative ΔG was obtained for all the benzaldehydes
imply that the inclusion complexation reaction between these
molecules and CDs proceeds spontaneously. Binding of all
molecules with CDs is enthalpy-entropy favourable showing
negative ΔH and ΔS values.
In Tables S1 and S2, we reported the selected bond
distances, bond angles and the most interesting angles
between the hydroxyl group and phenyl ring of these
aldehydes before and after complexation for the most
stable inclusion complex. From comparison of free
guest and the complex, it is clear that the geometrical
structures of the aldehydes after complexation are
completely altered. This alteration is achieved through
the variation of the dihedral angles between the phenyl ring
and the alkyl chain of these drugs which is subject to a
distortion to adopt a specific conformation leading to the
formation of a most stable complex. The non-bonded
interaction between the phenyl ring and CD might be
responsible for the difference in structure/stability of the
complex. Experimental thermodynamic values will be
helpful for numerical investigations in order to draw a
conclusion about the effect of solvent on the binding of
complexation.
From the optimized structures of the inclusion complexes,
one H-bond is formed between 4HMB/α-CD, HDMB with β-
CD. The H-bond lengths are shorter than 3.0 Ǻ which just falls
within the reported data [39]. This is justified the importance
of interaction energy between these molecules and CDs nec-
essary to ensure a better inclusion of the guest to the host. The
above values were supported by the fact that the flexibility of
the host molecule and substitutions of the guest molecules
may be one of the structural requirements for inclusion com-
plexes formation. Further Tables 4 and 5 confirmed that
hydrogen bonding interactions also played a major role in
the inclusion complexation process.
Frontier Molecular Orbitals
The highest occupied molecular orbitals (HOMOs) and the
lowest-lying unoccupied molecular orbitals (LUMOs) are
named as frontier molecular orbitals (FMOs). FMOs play in
important role in the optical and electric properties, as well as
in quantum chemistry and UV–vis spectra [40]. HOMO rep-
resents the ability to donate an electron. LUMO as an electron
acceptor represents the ability to obtain an electron. The
energy gap between HOMO and LUMO determines the ki-
netic stability, chemical reactivity and optical polarizability
and chemical hardness–softness of a molecule [41]. HOMO-
LUMO value of 4HMB:α-CD complex is more negative than
other inclusion complexes (Fig. 7). This suggests 4HMB:α-
CD inclusion complex is more stable than the other inclusion
complexes. However, the energy gap between HOMO and
LUMO of each complex suggests that these will be no signif-
icant change in the electronic spectrum of the guest molecules
driving molecular recognition and binding.
Conclusion
The inclusion complexes of α-CD, β-CD, HP-α-CD and
HP-β-CD with four aldehydes (2HMB, 4HMB, DMB and
HDMB) were investigated by UV–Vis, steady-state and time-
resolved fluorescence and molecular modeling techniques.
Dual fluorescence observed in solvents and the CD mediums
are responsible for intramolecular charge transfer effect in all
the aldehydess. The spectral data of all the benzaldehydes in
the CDs clearly demonstrate the formations of 1:1 inclusion
complexes were formed. The PM3 results suggest that the
complexation of HDMB/α-CD and β-CD are significantly
more favorable than the other complexes. The results con-
firmed aromatic ring for each compound is totally embedded
in CDs cavities. The statistical thermodynamic calculations
suggest that formation of the inclusion complexes is enthalpy
driven process.
Thermodynamics of Inclusion Process
The energetic features, thermodynamic characteristics and
electronic properties of these structures were summarized in
Tables 4 and 5. The statistical thermodynamic calculations
were performed using harmonic frequency analysis in PM3
method for the most stable structures, characterizing them as
true minima on the potential energy surface. The frequency
analyses were then used for the evaluation of the thermody-
namic parameters, such as enthalpy changes (ΔH), entropy
contribution (ΔS) and Gibbs free energy (ΔG), for the bind-
ing process of HMBs with α-CD and β-CD were summarized
in Tables 4 and 5. It can be observed that the inclusion
complexation of HMBs with α-CD and β-CD is exothermic
judged from the negative enthalpy changes. The enthalpy
change for all the HMBs:α-CD/β-CD complexes are more
Acknowledgements This work is supported by the CSIR [No.
01(2549)/12/EMR-II], and UGC [F.No. 41-351/2012 (SR)]. The authors
thank to Dr. P. Ramamurthy, Director, National centre for ultrafast pro-
cesses, Madras University for allowing the fluorescence lifetime mea-
surements available for this work.

J Fluoresc
21. Siva S, Sankaranarayanan RK, Prabhu AAM, Rajendiran N (2009)
Indian J Chemistry 48A:1515–1521
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