A simple protocol for the visual discrimination of natural cyclodextrins in aqueous solution using perichromic probes

Article abstract of DOI:10.1016/j.saa.2014.10.053

(Graph Presented) Three synthesized compounds, 4-(4-nitrostyryl)phenol, 2,6-dibromo-4-(2,4-dinitrobenzylideneamino) phenol and 2,6-dichloro-4-(2,4-dinitrobenzylideneamino)phenol, were deprotonated to generate the perichromic dyes 2b, 3b and 4b, respectively. These dyes were used as probes to investigate the micropolarity of natural cyclodextrins (CyDs) and it was observed that they interact differently with the CyDs according to the molecular structure of the dye and the diameter of the CyD. The solvatochromic bands of the dyes that interacted with the CyDs were bathochromically shifted, suggesting that the probes were transferred to the hydrophobic interior of the CyD in aqueous solution. Dyes 2b and 4b were found to be very selective for α-CyD and γ-CyD, respectively, while β- and γ-CyD changed the color of the solution of compound 3b. These dyes were then successfully used in a simple assay that allows the naked-eye discrimination of natural CyDs in aqueous solution, without the need for expensive techniques.

Full text of DOI:10.1016/j.saa.2014.10.053

Accepted Manuscript
A simple protocol for the visual discrimination of natural cyclodextrins in aque-
ous solution using perichromic probes
Leandro G. Nandi, Celso R. Nicoleti, Rafaela I. Stock, Tainá A. Barboza, Jürgen
Andreaus, Vanderlei G. Machado
PII:
S1386-1425(14)01550-9
http://dx.doi.org/10.1016/j.saa.2014.10.053
SAA 12868
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomo-
lecular Spectroscopy
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 June 2014
8 October 2014
15 October 2014
Please cite this article as: L.G. Nandi, C.R. Nicoleti, R.I. Stock, T.A. Barboza, J. Andreaus, V.G. Machado, A simple
protocol for the visual discrimination of natural cyclodextrins in aqueous solution using perichromic probes,
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2014), doi: http://dx.doi.org/10.1016/j.saa.
2014.10.053
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1
A simple protocol for the visual discrimination of natural cyclodextrins
in aqueous solution using perichromic probes
Leandro G. Nandi a, Celso R. Nicoleti a, Rafaela I. Stock a, Tainá A. Barboza a, Jürgen
Andreaus b, Vanderlei G. Machado a,∗
a Departamento de Química, Universidade Federal de Santa Catarina, UFSC, CP 476,
Florianópolis, Santa Catarina, 88040-900, Brazil
b Departamento de Química, Universidade Regional de Blumenau, FURB, Blumenau,
Santa Catarina, 89010-971, Brazil
∗ Corresponding author. Tel.: +55 48 3721 4542; fax: +55 48 3721 6850.
E-mail address: vanderlei.machado@ufsc.br (V. G. Machado)

2
ABSTRACT
Three synthesized
dinitrobenzylideneamino)phenol
compounds,
4-(4-nitrostyryl)phenol,
and
2,6-dibromo-4-(2,4-
2,6-dichloro-4-(2,4-
dinitrobenzylideneamino)phenol, were deprotonated to generate the perichromic dyes
2b, 3b and 4b, respectively. These dyes were used as probes to investigate the
micropolarity of natural cyclodextrins (CyDs) and it was observed that they interact
differently with the CyDs according to the molecular structure of the dye and the
diameter of the CyD. The solvatochromic bands of the dyes that interacted with the
CyDs were bathochromically shifted, suggesting that the probes were transferred to the
hydrophobic interior of the CyD in aqueous solution. Dyes 2b and 4b were found to be
very selective for α-CyD and γ-CyD, respectively, while β- and γ-CyD changed the
color of the solution of compound 3b. These dyes were then successfully used in a
simple assay that allows the naked-eye discrimination of natural CyDs in aqueous
solution, without the need for expensive techniques.
Keywords: Cyclodextrins; Perichromism; Solvatochromic probes; Visual detection;
Chromogenic chemosensors.

3
Introduction
Cyclodextrins [1, 2] (CyDs) are cyclic polysaccharides comprised of glucose units
(6, 7 or 8 for α-, β- or γ-CyD) connected at the 1 and 4 carbon atoms [3-5] and they are
formed during the degradation of starch through the action of CyD glucosyltransferases
[6-9]. These molecules have been used as receptors for a variety of organic compounds,
because they have lipophilic cavities with hydrophilic external walls, the diameter of the
cavities (470-830 pm) being determined by the number of glucose units [10]. The
ability of CyDs to form inclusion complexes in solution and in the solid state has led to
a great variety of research studies, not only related to their industrial applications [11-
13], but also to developments in other fields, such as bioorganic chemistry [14-16],
controlled drug-delivery systems [17-20], environmental chemistry [21-23] and
analytical techniques [24-27]. The hydrophobic character of the CyDs, which is crucial
for the formation of inclusion complexes, has been demonstrated by the use of
perichromic probes. The solvatochromic band of Reichardt’s pyridinium N-phenolate
dye [2,6-diphenyl-4-(2,4,6-triphenylpyridinium-1-yl)phenolate] is bathochromically
shifted from λmax = 453 nm in water to 503 nm in an aqueous solution of methyl-β-CyD
[28]. This shift of +50 nm suggests the formation of a 1:1 CyD:dye inclusion complex,
with the dye located at the less polar cavity of the CyD [28]. Similar results were
obtained
using
1-methyl-4-[(1-oxocyclohexa-2,5-dienylidiene)ethylidene]-1,4-
dihydropyridine (Brooker’s merocyanine) as the probe in the presence of several natural
and modified CyDs [29]. Although these dyes [5, 30, 31] and other probes [32-38] have
been used in studies on the micropolarity of CyDs in aqueous solution, no simple
colorimetric methods allowing the visual (naked-eye) discrimination of CyDs have been
published in the literature. The difficulties associated with developing such a method
are due to the small band shifts observed for the dyes in aqueous solution in the absence

4
and presence of the CyDs and also to the fact that, although the dye-CyD binding
constants can differ, no expressive band shifts are observed when different CyDs are
compared. Herein, we propose a simple protocol which allows the visual discrimination
between α-, β- and γ-CyD in water, by means of the use of perichromic probes 1b-4b.
((Scheme 1 here))
Experimental
Materials and methods
All chemicals used were high-purity commercial reagents. α-CyD (Cavitron
80000, batch number G8071, Mr 972.86 and water content 8.6 %) and β-CyD
=
(Cavitron® 82000 batch 1Q0011, Mr = 1135.02 and water content 10.7 %) were kindly
donated by Cerestar/Cargill while γ-CyD (Cavamax® W8 Pharma, batch number
80P200, Mr = 1297.15 and water content 5.1 %) was kindly donated by Wacker Chemie
GesmbH (Germany). All water contents were determined through Karl Fischer titrations
and the CyDs were used as received.
Deionized water was used in all measurements. This solvent was boiled and
bubbled with nitrogen and kept under nitrogen atmosphere to avoid the presence of
carbon dioxide. The experiments were performed in buffered medium at pH 11.5
(prepared by mixing 100 mL of an aqueous solution of Na2HPO4 1×10-3 mol L-1 with
some drops of NaOH 0.1 mol L-1 to adjust the pH value) in order to avoid the
protonation of the dye.
The melting points were uncorrected. The NMR spectra were recorded on a
Varian AS-400 spectrometer. Chemical shifts were recorded in ppm with the solvent
resonance as the internal standard. Data are reported as follows: chemical shift,
multiplicity (s = singlet, d = doublet, dd = double doublet), integration, and coupling

5
constants (Hz). IR spectra were obtained with KBr pellets. High-resolution mass spectra
were obtained with an electrospray ionization-quadrupole time-of-flight mass
spectrometer (HR ESI-MS QTOF), available at Laboratório Central de Biologia
Molecular Estrutural - CEBIME (UFSC). UV-vis measurements were performed with a
HP 8453 spectrophotometer at 25 °C, using a 1-cm quartz square cuvette. The maxima
on the UV-vis spectra (λmax) were calculated from the first derivative of the absorption
spectrum.
Syntheses of the compounds
2,6-Dibromo-4-nitrophenol
The synthesis followed the methodology described in the literature [39, 40]. In a
flask containing 5 mL of water at 40 °C, under magnetic stirring, potassium bromide
(7.14 g; 60 mmol), 4-toluenesulfonic acid monohydrate (7.61 g; 40 mmol), N-
bromosuccinimide (7.12 g; 40 mmol) and 4-nitrophenol (2.78 g; 20 mmol) were mixed.
In the course of reaction (2 h) the formation of a yellow solid was observed. The
reaction mixture was cooled to room temperature and the solid was filtered using a
Büchner funnel and washed with an aqueous solution of sodium metabisulfite 10 %
(3×10 mL). Thereafter, the solid was allowed to dry in a vacuum oven at 100 °C for 4 h.
The product was a light yellow solid with 80 % yield; m.p. obtained: 144-145 °C (m.p.
literature [39]: 145 °C); IR (KBr, ꢀ̅
max/cm-1): 3366 (O−H), 1688, 1574, 1464, (C=C),
1513, 1339 (N=O), 1232 (C−O); 1H NMR (DMSO-d6, 400 MHz) /ppm: 8.38 (s, 2H).
4-Amino-2,6-dibromophenol
The synthesis followed the methodology described in the literature [41]. In a
round-bottomed flask 2,6-dibromo-4-nitrophenol 3 (0.60 g; 2.0 mmol), tin chloride

6
(3.16 g; 14 mmol) and 60 mL of ethanol were added. The system was maintained under
magnetic stirring and reflux in an oil bath for 8 h. After the reflux, the reaction mixture
was removed from the heat and left to cool until room temperature. Sodium hydroxide
was then added until pH = 10. The organic layer was separated and the aqueous phase
was extracted with ethyl acetate (3×50 mL). The organic layer extracts were dried over
MgSO4, filtered and the solvent was evaporated under reduced pressure. The product
obtained was a pale brown solid with 70 % yield; m.p. obtained: 191-192 °C; m.p.
literature [42]: 191-193 °C; IR (KBr, ꢀ̅
max/cm-1): 3369, 3269 (N−H), 1607, 1481, (C=C),
1218 (C−O); 1H NMR (DMSO-d6, 400 MHz) /ppm: 7.43 (s, 2H).
4-(4-Nitrobenzylideneamino)phenol (1a)
This compound was synthesized by the reaction of 4-nitrobenzaldehyde with 4-
aminophenol in methanol at room temperature, according with procedures described in
the literature [43, 44]. yield obtained: 63 %; yield literature 89.8 % [43]. m.p. obtained:
175-176 °C; m.p. literature [43]: 176 °C; IR (KBr, ꢀ
̅
max/cm-1): 3439 (O−H), 3062
1
(C−H), 1623 (C=N), 1596, 1575, 1506 (C=C) and 1261 (C−O); H NMR (DMSO-d6,
400 MHz) /ppm: 9.70 (s, 1H), 8.79 (s, 1H), 8.34 (d, 2H, J = 8.6 Hz), 8.14 (d, 2H, J =
8.6 Hz), 7.32 (d, 2H, J = 8.2 Hz), 6.38 (d, 2H, J = 8.6 Hz).
4-(4-Nitrostyryl)phenol (2a)
The synthesis followed the methodology described in the literature [45]. 4-
Nitrotoluene (1.00 g; 7.3 mmol), 4-hydroxybenzaldehyde (0.89 g; 7.3 mmol) and
pyrrolidine (1.5 mL; 18.3 mmol) were mixed and the reaction was refluxed at 70-75 °C
under magnetic stirring for 38 h. The crude product was separated by silica gel column
chromatography using hexane/ethyl acetate (2/3 v/v), the solvent was rotary-evaporated

7
and the solid obtained was subsequently recrystallized from ethyl acetate. The product
was a light orange solid (19 % yield); m.p. obtained: 204-206 °C; m.p. literature [46]:
206 °C; IR (KBr, ꢀ
̅
max/cm-1): 3418 (O H), 1584, 1501, and 1437 (C=C), 1337 (N=O);
1H NMR (DMSO-d6, 400 MHz) /ppm: 9.77 (s, 1H), 8.18 (d, 2H, J = 8.0 Hz), 7.77 (d,
2H, J = 8.0 Hz), 7.50 (d, 2H, J = 8.0 Hz), 7.42 (d, 1H, J = 16.0 Hz), 7.16 (d, 1H, J =
16.0 Hz), 6.80 (d, 2H, J = 8.0 Hz).
2,6-Dibromo-4-(2,4-dinitrobenzylideneamino)phenol (3a)
2,4-Dinitrobenzaldehyde (0.20 g; 1.0 mmol), 4-amino-2,6-dibromophenol (0.13 g;
0.5 mmol) and methanol (5 mL) were placed in an Erlenmeyer flask. The reaction
mixture was stirred until complete dissolution of the reactants and one drop of acetic
acid was added. After magnetic stirring at room temperature for 4 h, the reaction
mixture was left to stand in a freezer overnight. The precipitate formed was filtered off
under vacuum, washed with iced methanol, and recrystallized three times from
ethanol/ethyl acetate 4/1 (v/v). The product, after drying, was an orange solid (0.10 g,
o
45 % yield), forming needle crystals; m.p. 212.1-214.3 C; IR (KBr, ꢀ
̅
max/cm-1): 3459
1
(O H), 1611 (C=N), 1476 (C=C), 1527, 1354 (N=O); H NMR (DMSO-d6, 400 MHz)
/ppm: 8.98 (s, 1H), 8.80 (d, 1H, J = 2.0 Hz), 8.60 (dd, 1H, J = 8.6 and 2.0 Hz), 8.36 (d,
1H, J = 8.6 Hz), 7.64 (s, 2H). 13C NMR (DMSO-d6, 100 MHz): /ppm: 155.26, 150.56,
148.99, 148.32, 143.41, 134.49, 131.12, 127.81, 125.75, 120.17, 112.34. HRMS (ESI,
TOF): m/z calcd. for C13H8Br2N3O5 [M + H]+ 445.8805, found 445.8798.
2,6-Dichloro-4-(2,4-dinitrobenzylideneamino)phenol (4a)
2,4-Dinitrobenzaldehyde (0.150 g; 0.7 mmol), 4-amino-2,6-dichlorophenol
(0.100 g; 0.55 mmol) and ethanol (5 mL) were added to an Erlenmeyer flask. The

8
mixture was stirred until complete dissolution of the reactants. One drop of acetic acid
was added and the reaction mixture was stirred for 4 h. The mixture was left to stand in
a freezer overnight. The precipitate formed was filtered off under vacuum, washed with
iced ethanol, and recrystallized three times from ethanol. The product after drying was
an orange solid (0.100 g; 50 % yield), forming needle crystals; m.p. obtained: 193.9-
o
195.2 C; IR (KBr, ꢀ
̅
max/cm-1): 3459 (O H), 1611 (C=N), 1476 (C=C), 1527, 1354
1
(N=O); H NMR (DMSO-d6, 400 MHz): /ppm: 10.49 (s, 1H), 8.98 (s, 1H), 8.80 (d,
1H, J = 2.0 Hz), 8.64 (dd, 1H, J = 8.6 and 2.0 Hz), 8.37 (d, 1H, J = 8.6 Hz), 7.48 (s,
2H). 13C NMR (DMSO-d6, 100 MHz) /ppm: 155.14, 148.98, 148.86, 148.29, 142.20,
134.50, 131.08, 127.81, 122.79, 122.21, 120.16. HRMS (ESI, TOF): m/z calculated for
C13H8Cl2N3O5 [M+H]+ 355.9836, found 355.9834.
UV-vis spectra of the dye in the solutions containing CyDs
A general procedure was used to make spectral measurements with the dye
solutions in the presence of the CyDs. Solutions of each dye (5.0×10-5 mol L-1) were
freshly prepared in buffered medium using a 50 mL volumetric flask. A part of this
solution was used in the preparation of a CyD stock solution in a 25 mL volumetric
flask. The concentration of CyDs was 1.0×10-2 mol L-1. The solution of the dye was
placed in a quartz cuvette hermetically closed with a rubber septum to avoid the
entrance of carbon dioxide. Small amounts of the CyD stock solution were then added
by means of a microsyringe, and the spectral readings were performed.
Measurements of binding constants
The spectral data collected in water with each dye were analyzed according to
the method described by Connors [47, 48], with Equation (1), where b is the path

9
length, c(dye) is the dye concentration, ∆ε11 is difference in molar absorptivities of the
complexed and free dye, K11 is the binding constant for a 1:1 complex formation and ∆A
is the change in absorbance at a fixed wavelength for the solution containing the dye
when the concentration of the CyD changes from zero to c(CyD). The binding constants
were calculated using the ORIGIN 7.0 program.
∆A/b= c(dye) K11 ∆ε11 c(CyD)/[1+K11 c(CyD)]
(1)
Results and discussion
The rationale for the present study considers that the use of carefully designed
dyes may allow a tight and selective space-filling accommodation of the dye in the CyD
cavity according to its internal diameter. Firstly, compounds 1a-4a were synthesized and
characterized. 4-(4-Nitrobenzylideneamino)phenol (1a) was prepared by mixing 4-
aminophenol with 4-nitrobenzaldehyde in ethanol. 4-(4-Nitrostyryl)phenol (2a) was
synthesized by means of the reaction of 4-nitrotoluene with 4-hydroxybenzaldehyde in
the presence of pyrrolidine as a base. Novel imines 3a [2,6-dibromo-4-(2,4-
dinitrobenzylideneamino)phenol]
and
4a
[2,6-dichloro-4-(2,4-
1
dinitrobenzylideneamino)phenol] were characterized by IR, H NMR, 13C NMR and
HRMS spectrometric techniques. Scheme 2 shows the route leading to the synthesis of
compound 3a. Firstly, 4-nitrophenol was brominated in the presence of KBr, 4-
toluenesulfonic acid and N-bromosuccinimide to form 2,6-dibromo-4-nitrophenol. This
intermediate was refluxed with tin chloride in ethanol to provide 4-amino-2,6-
dibromophenol, which in the last step was reacted with 2,4-dinitrobenzaldehyde in
methanol to form compound 3a. Imine 4a was synthesized by reacting 2,4-
dinitrobenzaldehyde with commercially available 4-amino-2,6-dichlorophenol.

10
((Scheme 2 here))
The pKa value for compound 1a, available in the literature [44], is 10.57 0.01
while the pKa values for compounds 2a-4a were found to be 10.79 0.08, 8.20 0.01 and
8.36 0.01, respectively. Thus, all experiments were performed using aqueous buffer
solutions at pH = 11.5 in order to ensure that all compounds were in the deprotonated
form (1b-4b).
Fig. 1 shows aqueous solutions of the dyes 1b-4b, where it can be observed that
dyes 1b and 2b are yellow and dyes 3b and 4b are orange. This figure also shows the
color of the aqueous solutions of the dyes in the presence of α-, β- and γ-CyD. For dye
1b, only α-CyD changed the color of the solution (from yellow to pale orange). For
stilbene 2b a change in the color of the solution also occurred only for α-CyD, but in
this case the change, from pale yellow to strong orange, was more perceptible. Since the
only alteration made in the molecular structure of the dye corresponded to the change in
the conjugated bridge, from C=N in 1b to C=C in 2b, these differences may be due to
an increase in the rigidity of the system caused by the C=C bond, leading to an
improvement in the space-filling accommodation of the dye in the CyD cavity. In
addition, an increase in the rigidity of the system may improve the molar absorptivity of
the dye. Indeed, the molar absorptivity values for 1b and 2b in buffered water (pH =
11.5) were found to be (1.19 0.12)×104 and (2.02 0.10)×104 L mol-1 cm-1, respectively.
Since the best result in the naked-eye detection assay was obtained for dye 2b, this dye
was used in the subsequent experiments.
((Fig. 1 here))

11
Fig. 1 also shows the behavior of dyes 3b and 4b in the absence and presence of
the CyDs. The solutions of these compounds under alkaline conditions are orange. In
the case of dye 3b, β- and γ-CyD changed the color of the solution to red and no change
was observed for α-CyD. Interestingly, only γ-CyD changed the color of the aqueous
solution of 4b (from orange to red). The molar absorptivity values for dyes 3b and 4b
were determined as (1.36 0.02)×104 and (9.59 0.02)×103 L mol-1 cm-1, respectively.
The spectral data show that the inclusion of the dyes in the CyDs in aqueous
solution could be visually observed due to a combination of changes in two of the
optical parameters of the dyes, the maximum wavelength (λmax) and the molar
absorptivity. The greatest differences were obtained for dye 4b in the presence of γ-
CyD, with ∆λmax = + 30 nm and ∆εmax = +1.04×104 L mol-1 cm-1. The data were
obtained for the dyes in aqueous solution (pH 11.5) in the absence and in an excess of
the CyD: for dye 2b, λmax = 420 nm and εmax = 1.93×104 L mol-1 cm-1 in the absence of
the CyD while λmax = 448 nm and εmax = 1.30×104 L mol-1 cm-1 with α-CyD; for dye 3b,
λ
max = 456 nm and εmax = 1.36×104 L mol-1 cm-1 in the absence of the CyD while λmax
=
=
470 nm and εmax = 9.38×103 L mol-1 cm-1 with β-CyD and λmax = 484 nm and λmax
1.00×104 L mol-1 cm-1 with γ-CyD; for dye 4b, λmax = 458 nm and εmax = 9.59×103 L
mol-1 cm-1 in the absence of the CyD while εmax = 488 nm and εmax = 2.00×104 L mol-1
cm-1 with γ-CyD.
Fig. 2(a) shows a sequence of UV-vis spectra of 2b as a function of the
concentration of α-CyD. In the absence of the CyD the dye exhibits a solvatochromic
band with λmax at 420 nm, which is bathochromically shifted with the addition of the
CyD, reaching λmax = 448 nm for c(CyD) = 3.94×10-3 mol L-1. The data reveal that the

12
dye was transferred to a less polar microenvironment with the addition of the CyD,
suggesting that inclusion of the dye in the hydrophobic cavity of the CyD occurred. Fig.
2(b) shows the plot of the variation in the absorbance at 420 nm for dye 2b as a function
of c(CyD), which shows that more than 95 % of the dye is included at a CyD
concentration of 3.94×10-3 mol L-1.
((Fig. 2 here))
In Fig. 3(a) the influence of the addition of β-CyD on the UV-vis spectra of the
buffered aqueous solution of dye 3b can be observed. The maximum of the
solvatochromic band of the dye was bathochromically shifted from 456 nm in the
absence of β-CyD to 470 nm in the presence of the CyD at a concentration of 1.33×10-3
mol L-1.
((Fig. 3 here))
A plot of the variation in the absorbance at 470 nm of the aqueous solution of 3b as a
function of the c(CyD) is shown in Fig. 3(b), which exhibits a shape typical for a 1:1
dye:CyD stoichiometry, with a maximum in absorbance being reached at ca. 1.00×10-3
mol L-1. A similar study was performed to evaluate the influence of γ-CyD on the UV-
vis spectra of dye 3b in aqueous solution. In this case, the solvatochromic band of the
dye showed a bathochromic shift of 28 nm, from λmax = 456 to 484 nm, with the
addition of 1.00×10-3 mol L-1 of the CyD [Fig. 4(a)]. In addition, an isosbestic point at
450 nm was observed, which suggests equilibrium between the free and included dye.
The increase in the absorbance at 484 nm with the increase in the concentration of γ-

13
CyD provided the titration curve shown in Fig. 4(b), which exhibited a pattern typical
for a 1:1 dye:CyD stoichiometry, with a plateau being reached at ca. 5.00×10-4 mol L-1.
((Figure 4 here))
The influence of the addition of increasing amounts of γ-CyD on the UV-vis
spectra of dye 4b in aqueous solution is shown in Fig. 5(a). On the addition of the CyD
the solvatochromic band of the dye, with λmax = 458 nm, is bathochromically shifted to
λmax = 488 nm, with the occurrence of an isosbestic point at 450 nm, suggesting an
equilibrium between free and complexed dye. Fig. 5(b) shows the corresponding
titration curve, obtained by collecting the absorbance values at the maximum of the
bathochromic band of the dye included in the CyD, at 488 nm. Again, the curve
obtained exhibits a 1:1 dye:CyD stoichiometric pattern, with a plateau occurring at ca.
5.00×10-4 mol L-1.
((Fig. 5 here))
All experimental data used to obtain the titration curves were fitted through non-
linear regression using Equation (1) [49], considering the dye and the CyD in a 1:1
stoichiometry [4, 47-49]. The results are displayed in Table 1 and show that the model
used fitted the experimental data very well (standard deviations below 1.53×10-6).
((Table 1 here))

14
Based on the observations made, compounds 2b-4b were used in a protocol
which allows the visual discrimination of the natural CyDs. In this simple procedure,
summarized in the Scheme 3, firstly the sample containing the unknown CyD is placed
in contact with dye 2b in aqueous solution. If the color of the solution changes from
pale yellow to orange, the sample contains α-CyD. If the test provides a negative result,
an aqueous solution of 4b is placed in contact with a new sample. The sample contains
γ-CyD if the color of the solution changes from orange to red. If the assay yields a
negative result, dye 3b is added to other aqueous solutions containing the sample and
the presence of β-CyD is suggested if the color of the solution changes from pale yellow
to dark orange.
((Scheme 3 here))
Conclusions
Compounds 2b-4b were used as perichromic probes for the investigation of the
micropolarity of natural CyDs in aqueous solution. Dye 2b is highly selective for α-
CyD and dye 4b for γ-CyD, while β- and γ-CyD changed the color of the aqueous
solution of compound 3b. Bathochromic shifts of the solvatochromic bands of the dyes
that interacted with the CyDs suggest that the dyes are transferred from a more polar
(water) to a less polar (the interior of the CyD) microenvironment, due to the space-
filling accommodation of the dye in the cavity of the CyD, according to its internal
diameter. Subsequently, these dyes were used in the development of an easy assay
allowing the visual discrimination of natural CyDs in aqueous solution, without the
need for expensive techniques. This assay is of interest as a simple analytical tool for
the fast characterization and quality control of natural CyDs.

15
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in the online
version, at http://dx.doi.org/10.1016/
Acknowledgements
The financial support of the Brazilian governamental agencies Conselho
Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES) as well as the Laboratório
Central de Biologia Molecular (CEBIME/UFSC) and UFSC is gratefully
acknowledged.
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19
Table 1
Binding constants for the inclusion complexes of dyes 2b-4b with CyDs at 25 °C in
aqueous solutions.a
CyD Dye K11 (L mol-1) b Standard deviation
(1.36 0.02)×103
(8.68 0.38)×103
(1.38 0.02)×104
(1.48 0.02)×104
4.22×10-6
4.40×10-9
4.90×10-7
1.53×10-6
2b
3b
3b
4b
α-CyD
β-CyD
γ-CyD
γ−CyD
a pH 11.5. b Calculated using Equation (1) [49].
Scheme 1. Molecular structures of the compounds used herein.

20
NH2
NO2
NO2
NO2
(i)
(ii)
(iii)
Br
Br
O2N
Br
Br
N
Br
Br
OH
3
a
OH
OH
OH
Scheme 2. The synthetic route to compound 3a. Reagents: (i) KBr, p-TsOH, N-
bromosuccinimide, H2O, 40 oC, (ii) SnCl2, EtOH, reflux, 8 h, (iii) 2,4-
dinitrobenzaldehyde, AcOH (one drop), methanol.
Scheme 3. Flowchart showing the protocol used for the visual identification of each
natural CyD. The vials show the solutions before (a) and after (b) the addition of the
dye.

21
Figure captions
Fig. 1. Solutions of dyes 1b-4b in the absence (a) and presence of α-CyD (b), β-CyD
(c) and γ−CyD (d) in water (pH 11.5). The concentration of each CyD was 1.0×10-2 mol
L-1 and of each dye was 5.0×10-5 mol L-1.
Fig. 2. (a) Influence of the addition of increasing amounts of α-CyD on the UV-vis
spectra of 2b (5.0×10-5 mol L-1) in water (pH 11.5) at 25 °C. (b) Curve for the variation
in the absorbance at 420 nm of 2b in water with increasing c(α-CyD).
Fig. 3. (a) Influence of the addition of increasing amounts of β-CyD on the UV-vis
spectra of 3b (5.0×10-5 mol L-1) in water (pH 11.5) at 25 °C. (b) Curve of the variation
in the absorbance at 470 nm of 3b in water with c(β-CyD).
Fig. 4. (a) Influence of the addition of increasing amounts of γ-CyD on the UV-vis
spectra of 3b (5.0×10-5 mol L-1) in water (pH 11.5) at 25 °C. (b) Curve of the variation
in the absorbance at 484 nm of 3b in water with c(γ-CyD).
Fig. 5. (a) Influence of the addition of increasing amounts of γ-CyD on the UV-vis
spectra of 4b (5.0×10-5 mol L-1) in water (pH 11.5) at 25 °C. (b) Curve of the variation
in the absorbance at 488 nm of 4b in water with c(γ-CyD).

22
(3b)
(4b)
(1b)
(2b)
(a) (b) (c) (d)
(a) (b) (c) (d)
Fig. 1
(a)
(b)
0,00
-0,04
-0,08
-0,12
-0,16
-0,20
0,8
0,6
0,4
0,2
0,0
350
400
450
500
550
600
0
1
2
3
4
103c(
α−
CyD) (mol L-1)
wavelength (nm)
Fig. 2

23
0.5
0.4
0.3
0.2
0.1
(b)
(a)
0.004
0.003
0.002
0.001
0.000
400
500
600
0.0
0.5
1.0
1.5
103c(β−CD) (mol L-1)
Wavelength (nm)
Fig. 3
0.5
0.4
0.3
0.2
0.1
0.0
0.10
(b)
(a)
0.08
0.06
0.04
0.02
0.00
400
500
600
0.0
0.2
0.4
0.6
103c(γ−CD) (mol L-1)
Wavelength (nm)
Fig. 4

24
1.0
0.8
0.6
0.4
0.2
0.25
0.20
0.15
0.10
0.05
0.00
(b)
(a)
400
500
600
0.0
0.2
0.4
0.6
103c(γ−CD) (mol L-1)
Wavelength (nm)
Fig. 5

25
Graphical abstract

26
Highlights
Probes 2b-4b were synthesized and used to investigate the micropolarity of CyDs
Dye 2b is highly selective for α-CyD and dye 4b for γ-CyD
β- and γ-CyD changed the color of the aqueous solution of compound 3b
A simple assay that allows the naked-eye discrimination of natural CyDs is described
This simple analytical tool can be applied for the quality control of natural CyDs