- Email: [email protected]
Fluorescence spectroscopic investigation of the interaction of citrinin with native and chemically modified cyclodextrins
Journal of Luminescence 172 (2016) 23–28
Contents lists available at ScienceDirect
Journal of Luminescence journal homepage: www.elsevier.com/locate/jlumin
Full Length Article
Fluorescence spectroscopic investigation of the interaction of citrinin with native and chemically modified cyclodextrins Miklós Poór a,n, Gergely Matisz b,c, Sándor Kunsági-Máté b,c, Diána Derdák b, Lajos Szente d, Beáta Lemli b,c a
Department of Pharmacology and Pharmacotherapy, Toxicology Section, University of Pécs, Szigeti út 12, Pécs H-7624, Hungary Department of General and Physical Chemistry, University of Pécs, Ifjúság útja 6, Pécs H-7624, Hungary c János Szentágothai Research Center, Ifjúság útja 20, Pécs H-7624, Hungary d CycloLab Cyclodextrin Research & Development Laboratory Ltd., Illatos út 7, Budapest H-1097, Hungary b
art ic l e i nf o
a b s t r a c t
Article history: Received 13 July 2015 Received in revised form 26 October 2015 Accepted 7 November 2015 Available online 30 November 2015
Citrinin (CIT) is a nephrotoxic mycotoxin produced by several Aspergillus, Penicillium and Monascus species. CIT is unavoidable contaminant of different foods and drinks due to its wide occurrence and high thermal stability. For this reason, development of new, more sensitive analytical methods and decontamination strategies has high importance. In our study, the complex formation of CIT with native and chemically modified cyclodextrins was investigated using fluorescence spectroscopy. Furthermore, thermodynamic and molecular modeling studies were also performed for the deeper understanding of these host-guest interactions. Our results show that among the tested compounds methylated βcyclodextrins form the most stable complexes with CIT and these derivatives cause the highest fluorescence enhancement of CIT as well. These observations recommend that some of the chemically modified derivatives show more favourable properties than the native cyclodextrin, and suggesting more promising analytical applicability and higher affinity as potential toxin binders. & 2015 Elsevier B.V. All rights reserved.
Keywords: Citrinin Cyclodextrin Fluorescence spectroscopy Host–guest interaction Fluorescence enhancement Toxin binder
1. Introduction Mycotoxins are toxic secondary metabolic products of various fungi. The foodborn mycotoxin citrinin (CIT; Fig. 1) is produced mainly by Aspergillus, Penicillium and Monascus species [1,2]. Due to the wide environmental occurrence of fungi, mycotoxins are unavoidable contaminants of foods, drinks and animal feeds. CIT appears principally in maize, rye, oats, barley, wheat and rice [1,2]. The high thermal stability of CIT makes also more difficult of its eradication from the food chain: It is decomposed at 175 °C by dry heating and at 140 °C in presence of water; however, the resulting decomposition products are as toxic as or more toxic than CIT itself [3]. CIT is a nephrotoxic mycotoxin with a complex mechanism of action (e.g. induction of cell cycle arrest, oxidative stress and apoptosis, etc.) [4–7]. Several analytical techniques are applied for the quantitative determination of CIT; HPLC-FLD (high-performance liquid chromatography with fluorescence detection) methods are commonly applied because of the strong fluorescence of CIT under acidic conditions [8–10].
n
Corresponding author. E-mail address: [email protected] (M. Poór).
http://dx.doi.org/10.1016/j.jlumin.2015.11.011 0022-2313/& 2015 Elsevier B.V. All rights reserved.
Cyclodextrins (CDs) are extensively studied host molecules built up from glucopyranose unites [11]. CDs have a conical structure with a hydrophobic interior as well as a hydrophilic exterior space [12]. The internal cavity can include different guest molecules and the stabilities of these host-guest complexes can be considerably increased by various chemical modifications [13]. Mycotoxins commonly form stable complexes with native or chemically modified cyclodextrins [14–17]. This complex formation is usually associated with the fluorescence enhancement of fluorescent mycotoxins (e.g., aflatoxin and zearalenone), resulting in the possibility to apply CDs in order to develop more sensitive fluorescent analytical methods [17–19]. Furthermore, recent studies also highlighted that mycotoxins can be effectively extracted from aqueous solutions by CD polymers [20,21], suggesting that CD technology may be suitable to decontaminate different toxinexposed drinks. The interaction of CIT with native β-cyclodextrin was also described [22]; on the other hand, we did not find any other references regarding CIT–CD interactions during our literature survey. In this study, the interaction of CIT with native α-, β- and γcyclodextrins as well as with chemically modified β-cyclodextrins was investigated using steady-state fluorescence spectroscopy. Because previously only the native β-cyclodextrin was studied, our main goal was to identify more appropriate CDs which form more
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2.3. Molecular modeling studies
Fig. 1. Chemical structure of the mycotoxin citrinin.
stable complexes with CIT and cause stronger fluorescence enhancement of CIT compared to β-cyclodextrin. Our results demonstrate that both of these requirements were fulfilled by some of the tested CD derivatives.
2. Materials and methods 2.1. Reagents All applied reagents were of analytical or spectroscopic grade. Citrinin (CIT) was purchased from Sigma-Aldrich; 5000 mM stock solution was prepared in ethanol (Reanal, spectroscopic grade) and stored at 4 °C protected from light. Cyclodextrins, namely αcyclodextrin (ACD), β-cyclodextrin (BCD), hydroxypropyl-βcyclodextrin (HPBCD), sulfobutylated-β-cyclodextrin (SBCD), randomly methylated-β-cyclodextrin (RAMEB), heptakis-2,6-di-Omethyl-β-cyclodextrin (DIMEB) and γ-cyclodextrin (GCD) were obtained from CycloLab Cyclodextrin Research & Development Laboratory, Ltd.
3. Results and discussion
2.2. Fluorescence spectroscopic measurements Fluorolog τ3 spectrofluorometric system (Jobin-Yvon/SPEX) was applied for fluorescence measurements. All analyses were performed at 25 °C except thermodynamic studies. During the measurements 1 mM CIT was applied in absence and presence of increasing CD concentrations (0–10 mM). Fluorescence spectra were recorded using 330 nm and 505 nm as excitation and emission wavelengths, respectively. Binding constants of CIT–CD complexes were determined by the Benesi–Hildebrand equation, assuming 1:1 stoichiometry: F0 1 1 ¼ þ ðF F 0 Þ A A K ½CDn
In the case of DIMEB, for each glucose units, two methyl groups were used to replace hydrogens of OH groups at the specified positions i.e. of the OH groups on the C-2 and C-6 carbon atoms. In the case of RAMEB, where the methyl substituents at the synthesis were placed randomly, all the OH groups were considered equivalent at the preparation of the model structure, thus at the lower rim four methyl groups were used to replace the hydrogen atoms of OH groups at C-6 , while at the upper rim eight at C-2 and/or C-3 positions. According to the carried out experimental studies, where aqueous solutions were used and pH ¼2.0 was applied (phosphate buffer), the citrinin molecule as having pKa ¼2.3 [22] was considered to have neutral charge. Its structure was optimized in vacuum by B3LYP/6-31G(d) density functional and basis set. The initial structures of the three CDs (BCD, RAMEB and DIMEB) were obtained from molecular dynamics simulation with explicit water molecules using the MMþ force field in the HyperChem 8.0 program. In the cases of CIT and the CDs, the atomic charges for the subsequent molecular docking studies were calculated using the B3LYP/6-31G(d) method and basis set by performing natural population analysis (NPA). The docking studies to determine the possible positions and orientations of CIT within the cavity of cyclodextrins and the associated binding affinities (thus approximating ΔG of the complexation – where van der Waals and electrostatic interactions, hydrogen-bond formations, desolvation and also entropy change at the complex formation are considered in the applied model) were carried out using the Vina 1.1.2 [23] program. The necessary . pdbqt input files were prepared using the OpenBabel program.
ð1Þ
3.1. Effect of pH on fluorescence emission spectrum of CIT In order to determine the optimal pH range for our measurements, 0.05 M phosphate buffers were prepared (pH ¼ 2.0, 2.5, 3.0 and 4.0). Under the applied conditions, CIT has excitation and emission wavelength maxima at 330 nm and 505 nm, respectively. In agreement with previous studies [22] we also observed that the decrease of pH results in the fluorescence enhancement of CIT (Fig. 2). Since the pKa value of CIT is 2.3 in water [22], this phenomenon can be attributed to the elevation of the highly fluorescent nonionic form of CIT, which predominates at pH 2.0. At
where K is the actual binding constant, F0 is the initial fluorescence intensity of CIT (in absence of CDs), F is the fluorescence intensity of CIT in presence of CDs, with concentration [CD], while A is a constant and n is the number of binding sites (n ¼1). To get insights into the formation details of CIT–BCD or CIT– DIMEB complexes the measurements were repeated and the binding constants were determined at five different temperatures: 20 °C, 25 °C, 30 °C, 35 °C and 40 °C. Thermodynamic parameters were determined for the formation of CIT–BCD and CIT–DIMEB complexes using the van't Hoff equation: ln K ¼
ΔG RT
¼
Δ H ΔS RT
þ
R
ð2Þ
where ΔG is the Gibbs free energy change, ΔS is the entropy change and ΔH is the enthalpy change associated with the complex formation.
Fig. 2. Fluorescence emission spectra of citrinin in 0.05 M phosphate buffer at pH 2.0, 2.5, 3.0, 4.0 [λexc ¼ 330 nm].
M. Poór et al. / Journal of Luminescence 172 (2016) 23–28
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Fig. 3. Fluorescence enhancement of citrinin by β- (BCD) and γ-cyclodextrin (GCD). Fluorescence emission spectra of CIT in absence and presence of increasing BCD (0–10 mM; left) and GCD (0-10 mM, right) concentrations in 0.05 M phosphate buffer (pH 2.0) [λexc ¼330 nm].
higher pH values (above pH 5), CIT does not exert fluorescence property. Furthermore, to test the suitability of the applied phosphate buffer, fluorescence emission spectra of CIT were also recorded in 0.05 M citrate buffer (pH 2.0) and in 0.01 M HCl solution. These spectra did not show differences compared to the fluorescence emission spectrum of CIT recorded in 0.05 M phosphate buffer (pH 2.0; data not shown). Therefore, similarly to the previous study of Zhou et al. [22], we also used phosphate buffer (pH 2.0) during our further experiments (see absorption spectrum of CIT in Fig. S1). 3.2. Effects of
Table 1 Log K values of CIT–CD complexes (where the unit of K is dm3/mol) and fluorescence enhancement of CIT in presence of 10 mM CD concentrations [λexc ¼330 nm, λem ¼ 505 nm].
GCD BCD HPBCD SBCD RAMEB DIMEB
log K (7 SD)
I/I0 ( 7SD)
1.84 7 0.10 2.34 7 0.14 2.34 7 0.13 2.487 0.16 2.59 7 0.03 2.93 7 0.09
1.28 7 0.03 1.677 0.02 1.65 7 0.03 1.977 0.02 1.95 7 0.01 2.067 0.01
α-, β- and γ-CD on fluorescence spectrum of CIT
To test the potential interaction of CIT with native CDs, increasing CD concentrations (0–10 mM) were added to standard amount of CIT (1 mM) in 0.05 M phosphate buffer (pH 2.0). The presence of ACD did not have any influence on the fluorescence spectrum of CIT suggesting the absence of complex formation of ACD with CIT (data not shown). On the other hand, both BCD and GCD resulted in the fluorescence enhancement of CIT (Fig. 3), causing approximately 1.7- and 1.3-fold increase compared to the fluorescence emission intensity of CIT in absence of CDs, respectively. This phenomenon can be explained by the cavity diameters of the CDs (ACD: 4.7 Å, BCD: 6.8 Å and GCD: 7.5 Å) [19], where the cavity of BCD with its 0.78 nm cavity diameter seems the most suitable for the inclusion of the CIT molecule [22]. 3.3. Fluorescence enhancement of CIT in presence of
β-CDs
BCD showed highly the most promising properties among the tested native CDs; therefore, during the following studies the interactions of CIT with chemically modified β-CDs were investigated. Increasing β-CD concentrations (0–10 mM) were added again to 1 mM CIT in 0.05 M phosphate buffer (pH 2.0). As Table 1 demonstrates the presence of each tested chemically modified βcyclodextrin increased the fluorescence signal of CIT in same or in higher extent compared to the native BCD. HPBCD induced similar while RAMEB and SBCD caused stronger fluorescence enhancement of CIT than BCD. DIMEB was the most effective in this respect causing approximately 2-fold increase of the fluorescence emission intensity of CIT (Table 1). These results demonstrate that considerably higher fluorescence enhancement of CIT can be induced with some of the chemically modified β-CDs compared to the native BCD.
Fig. 4. Benesi–Hildebrand plots of citrinin–cyclodextrin complexes (n ¼1) in 0.05 M phosphate buffer (pH 2.0) [λexc ¼ 330 nm, λem ¼ 505 nm].
3.4. Stabilities of CIT–CD complexes Thereafter, the stability constants (K) of CIT–CD complexes were determined using the Benesi–Hildebrand equation (see Section 2.2., Eq. (1)). As Fig. 4 demonstrates, Benesi–Hildebrand plots showed very good agreement with the assumed 1:1 stoichiometry. HPBCD bound CIT with similar stability than BCD (Table 1). Furthermore, SBCD, RAMEB and DIMEB complexes of CIT showed approximately 1.4-, 1.8- and 3.9-fold higher stability compared to the CIT–BCD complex, respectively. Therefore, DIMEB seems much more effective mycotoxin binder than the native BCD in the case of CIT. These results demonstrate that methylated βCDs form the most stable complexes with CIT among the tested
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Fig. 5. Benesi–Hildebrand plots of CIT–BCD and CIT–DIMEB complexes (n¼ 1) in 0.1 M hydrochloric acid and in 0.05 M phosphate buffer (pH 3.5) [λexc ¼ 330 nm, λem ¼505 nm].
compounds. One of the reasons of the higher binding constants of methylated β-CDs is that these derivatives show lowest aggregation in water than BCD, and their cavities are extended by methyl groups in both directions. Therefore, it provides a more complete and deeper penetration of CIT into the apolar cavities of DIMEB and RAMEB. 3.5. Effect of pH on the complex stabilities of CIT–BCD and CIT– DIMEB complexes Considering that the complex stabilities of CDs with nonionic and deprotonated forms of CIT can show differences, changes of the fluorescence signal of CIT can caused by the complex formation of CIT with the CD molecule or even partly resulted from the protonation/deprotonation of CIT. For this reason, we determined the complex stabilities of CIT–BCD and CIT–DIMEB complexes in 0.1 M hydrochloric acid and in 0.05 M phosphate buffer (pH 3.5), where the nonionic and the deprotonated form of CIT highly predominates, respectively. In 0.1 M hydrochloric acid CIT–BCD and CIT–DIMEB complexes showed log K values 2.31 (R2 ¼0.999) and 2.95 (R2 ¼ 0.999), respectively (see Benesi–Hildebrand plots on Fig. 5). These data show very good agreement with log K values previously determined in phosphate buffer at pH 2.0 (Table 1). On the other hand, considerably lower complex stabilities were observed at pH 3.5 in both cases, where the log K values 1.83 (R2 ¼ 0.999) and 2.49 (R2 ¼0.999) were determined for CIT–BCD and CIT–DIMEB complexes, respectively. These results suggest that BCD and DIMEB show preference towards nonionic CIT compared to its deprotonated form. Therefore, CDs seem more effective toxin binders at lower pH levels in the case of CIT. 3.6. Thermodynamic studies Fig. 6 and Table 2 summarize the results obtained from the temperature dependence of complex stabilities. Opposite tendencies of temperature dependence were obtained for CIT–BCD and CIT–DIMEB complexes: in the latter case higher stability is associated to the complexes at decreased temperatures while, surprisingly, in the case of CIT–BCD complex the stability increases at an elevated temperature. These properties reflect different formation thermodynamics of the CIT–BCD and CIT–DIMEB complexes. Since the free enthalpy changes are nearly the same around room-temperature, effect of enthalpy–entropy compensation can be assumed as follows. The attractive forces between the CIT and DIMEB molecules result in higher ordered structure of the
Fig. 6. van't Hoff's plot of CIT–BCD and CIT–DIMEB complexes.
Table 2 Thermodynamic parameters of complexation of citrinin with BCD and DIMEB (ΔG values were calculated at 25 °C).
CIT–DIMEB CIT–BCD
ΔG [kJ/mol] ( 7SD)
ΔH [kJ/mol] ( 7 SD)
ΔS [J/Kmol] ( 7 SD)
17.4 7 0.29 13.0 7 0.74
22.6 70.12 30.0 71.86
17.3 7 0.10 144.3 7 0.20
complexes included by its solvation shell. In contrast, the formation of the CIT–BCD complex preferably based on the destroying processes associated to the decreased order of the complex compared to the separated but solvated CIT and BCD molecules. In this case the positive enthalpy change reflects inhibited molecular interaction in condensed phase, however it is cannot be decided from the thermodynamic data if the interactions of the individual CIT and BCD molecules are repulsive or the heat associated to the decomposition of the solvation shells of molecules interacted shifts the overall enthalpy change to the positive region. Anyway, the huge entropy gain during formation of CIT–BCD complex overcompensates the positive enthalpy change and energetically favored formation of CIT–BCD complexes is observed around room temperature. The enthalpy–entropy compensation can also be explained in association with the solubilities of the host CD molecules. It is well-known that the solubility of the native BCD is much lower compared to the solubility of the DIMEB molecules which property originates from the highly ordered water molecules in the solvation shell of BCDs [24]. During complex formation the CIT molecules at least partly removes the water molecules from the CDs cavities and weakens the water–CD interactions in the cases of water molecules remained in the solvation shell. As a result, the freedom, i.e. the entropy of the small solvent molecules increases with higher content in the case when the BCD instead the DIMEB molecule form complexes with the CIT guest. Furthermore, considering the opposite dependence of the complex stabilities on the temperature, there is a characteristic temperature at 325.3 K where the free enthalpy changes associated to the formation of CIT–BCD and CIT–DIMEB complexes are the same. Below or above this temperature, the formation of CIT–BCD and CIT– DIMEB complexes is pronounced, respectively. 3.7. Molecular modeling studies The highest binding affinities were found in the cases of DIMEB ( 27.2 kJ/mol) and RAMEB ( 26.8 kJ/mol), while in the case of BCD, according to the measurements too, slightly lower affinity (by approximately 1.3 kJ/mol compared to DIMEB) has been found
M. Poór et al. / Journal of Luminescence 172 (2016) 23–28
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Fig. 7. The CIT–DIMEB complex from side (left) and top views (right) with the two preferred orientations of the CIT molecule (carboxyl group oriented up- or downward). The drawings were prepared with the Molekel 5.4 (Ugo Varetto, MOLEKEL 5.4, Swiss National Supercomputing Centre, Lugano, Switzerland) program. The electrostatic potential mapped to a constant electron density of 0.002 e/(a0)3 which gives adequately the molecular size and shape.
( 25.9 kJ/mol). In the cases of both methylated cyclodextrins, that complex was found to be preferred where the carboxyl groups were oriented downwards (and located close to the ether oxygen atoms at the lower rim). In the cases of CIT–BCD, CIT–RAMEB and CIT–DIMEB, 1.7 kJ/mol, 1.7 kJ/mol and 4.2 kJ/mol differences were obtained by the modeling study between the two orientations of CIT, respectively. The experimentally found highest fluorescence intensity in the case of DIMEB can be explained by the highest binding affinity obtained from modeling in that case, which is associated to reduced possibility of fluorescence quenching by the water molecules, and can be reasoned also by the placement of the – OCH3 groups (e.g. the lower rim is the most hydrophobic in this case, thus shielding the citrinin molecule the largest extent from the fluorescence quenching effect of the solvent water molecules). Another explanation of the higher fluorescence intensity of CIT– DIMEB compared to CIT–RAMEB complex can be the finding that in the latter case a preferred complex structure also exists with upwards position of the carboxyl group which can lead to less shielding of the aromatic part of CIT from the water molecules by resulting in the increased possibility of the up/down movement of citrinin in the host CD molecule which is caused by the water molecules located at the proximity of the upper rim. The obtained most preferred complex structures of two kinds are illustrated on Fig. 7 and Fig. S2 (see Cartesian coordinates of the CIT - CD complexes in Suppl. 3).
4. Conclusions In this study, the interactions of citrinin with various native and chemically modified cyclodextrins were investigated with fluorescence spectroscopy. Presence of β- and γ-CD results in the fluorescence enhancement of citrinin at lower pH levels (pH 1-4). Each tested chemically modified β-CD caused similar or higher
increase of the fluorescence of CIT than the native BCD. Furthermore, among the examined CDs the methylated β-CDs (DIMEB and RAMEB) formed the most stable complexes with CIT. Our results clearly demonstrate that some of the CD derivatives show more favorable properties compared to BCD during the complex formation with CIT, suggesting more promising analytical applicability and higher affinity as potential toxin binders.
Acknowledgements Financial support of the “Environmental industry related innovative trans- and interdisciplinary research team development in the University of Pécs knowledge base” (SROP-4.2.2.D-15/ 1/Konv-2015-0015) project is highly appreciated. The present scientific contribution is dedicated to the 650th anniversary of the foundation of the University of Pécs, Hungary.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jlumin.2015.11.011.
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Contents lists available at ScienceDirect
Journal of Luminescence journal homepage: www.elsevier.com/locate/jlumin
Full Length Article
Fluorescence spectroscopic investigation of the interaction of citrinin with native and chemically modified cyclodextrins Miklós Poór a,n, Gergely Matisz b,c, Sándor Kunsági-Máté b,c, Diána Derdák b, Lajos Szente d, Beáta Lemli b,c a
Department of Pharmacology and Pharmacotherapy, Toxicology Section, University of Pécs, Szigeti út 12, Pécs H-7624, Hungary Department of General and Physical Chemistry, University of Pécs, Ifjúság útja 6, Pécs H-7624, Hungary c János Szentágothai Research Center, Ifjúság útja 20, Pécs H-7624, Hungary d CycloLab Cyclodextrin Research & Development Laboratory Ltd., Illatos út 7, Budapest H-1097, Hungary b
art ic l e i nf o
a b s t r a c t
Article history: Received 13 July 2015 Received in revised form 26 October 2015 Accepted 7 November 2015 Available online 30 November 2015
Citrinin (CIT) is a nephrotoxic mycotoxin produced by several Aspergillus, Penicillium and Monascus species. CIT is unavoidable contaminant of different foods and drinks due to its wide occurrence and high thermal stability. For this reason, development of new, more sensitive analytical methods and decontamination strategies has high importance. In our study, the complex formation of CIT with native and chemically modified cyclodextrins was investigated using fluorescence spectroscopy. Furthermore, thermodynamic and molecular modeling studies were also performed for the deeper understanding of these host-guest interactions. Our results show that among the tested compounds methylated βcyclodextrins form the most stable complexes with CIT and these derivatives cause the highest fluorescence enhancement of CIT as well. These observations recommend that some of the chemically modified derivatives show more favourable properties than the native cyclodextrin, and suggesting more promising analytical applicability and higher affinity as potential toxin binders. & 2015 Elsevier B.V. All rights reserved.
Keywords: Citrinin Cyclodextrin Fluorescence spectroscopy Host–guest interaction Fluorescence enhancement Toxin binder
1. Introduction Mycotoxins are toxic secondary metabolic products of various fungi. The foodborn mycotoxin citrinin (CIT; Fig. 1) is produced mainly by Aspergillus, Penicillium and Monascus species [1,2]. Due to the wide environmental occurrence of fungi, mycotoxins are unavoidable contaminants of foods, drinks and animal feeds. CIT appears principally in maize, rye, oats, barley, wheat and rice [1,2]. The high thermal stability of CIT makes also more difficult of its eradication from the food chain: It is decomposed at 175 °C by dry heating and at 140 °C in presence of water; however, the resulting decomposition products are as toxic as or more toxic than CIT itself [3]. CIT is a nephrotoxic mycotoxin with a complex mechanism of action (e.g. induction of cell cycle arrest, oxidative stress and apoptosis, etc.) [4–7]. Several analytical techniques are applied for the quantitative determination of CIT; HPLC-FLD (high-performance liquid chromatography with fluorescence detection) methods are commonly applied because of the strong fluorescence of CIT under acidic conditions [8–10].
n
Corresponding author. E-mail address: [email protected] (M. Poór).
http://dx.doi.org/10.1016/j.jlumin.2015.11.011 0022-2313/& 2015 Elsevier B.V. All rights reserved.
Cyclodextrins (CDs) are extensively studied host molecules built up from glucopyranose unites [11]. CDs have a conical structure with a hydrophobic interior as well as a hydrophilic exterior space [12]. The internal cavity can include different guest molecules and the stabilities of these host-guest complexes can be considerably increased by various chemical modifications [13]. Mycotoxins commonly form stable complexes with native or chemically modified cyclodextrins [14–17]. This complex formation is usually associated with the fluorescence enhancement of fluorescent mycotoxins (e.g., aflatoxin and zearalenone), resulting in the possibility to apply CDs in order to develop more sensitive fluorescent analytical methods [17–19]. Furthermore, recent studies also highlighted that mycotoxins can be effectively extracted from aqueous solutions by CD polymers [20,21], suggesting that CD technology may be suitable to decontaminate different toxinexposed drinks. The interaction of CIT with native β-cyclodextrin was also described [22]; on the other hand, we did not find any other references regarding CIT–CD interactions during our literature survey. In this study, the interaction of CIT with native α-, β- and γcyclodextrins as well as with chemically modified β-cyclodextrins was investigated using steady-state fluorescence spectroscopy. Because previously only the native β-cyclodextrin was studied, our main goal was to identify more appropriate CDs which form more
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2.3. Molecular modeling studies
Fig. 1. Chemical structure of the mycotoxin citrinin.
stable complexes with CIT and cause stronger fluorescence enhancement of CIT compared to β-cyclodextrin. Our results demonstrate that both of these requirements were fulfilled by some of the tested CD derivatives.
2. Materials and methods 2.1. Reagents All applied reagents were of analytical or spectroscopic grade. Citrinin (CIT) was purchased from Sigma-Aldrich; 5000 mM stock solution was prepared in ethanol (Reanal, spectroscopic grade) and stored at 4 °C protected from light. Cyclodextrins, namely αcyclodextrin (ACD), β-cyclodextrin (BCD), hydroxypropyl-βcyclodextrin (HPBCD), sulfobutylated-β-cyclodextrin (SBCD), randomly methylated-β-cyclodextrin (RAMEB), heptakis-2,6-di-Omethyl-β-cyclodextrin (DIMEB) and γ-cyclodextrin (GCD) were obtained from CycloLab Cyclodextrin Research & Development Laboratory, Ltd.
3. Results and discussion
2.2. Fluorescence spectroscopic measurements Fluorolog τ3 spectrofluorometric system (Jobin-Yvon/SPEX) was applied for fluorescence measurements. All analyses were performed at 25 °C except thermodynamic studies. During the measurements 1 mM CIT was applied in absence and presence of increasing CD concentrations (0–10 mM). Fluorescence spectra were recorded using 330 nm and 505 nm as excitation and emission wavelengths, respectively. Binding constants of CIT–CD complexes were determined by the Benesi–Hildebrand equation, assuming 1:1 stoichiometry: F0 1 1 ¼ þ ðF F 0 Þ A A K ½CDn
In the case of DIMEB, for each glucose units, two methyl groups were used to replace hydrogens of OH groups at the specified positions i.e. of the OH groups on the C-2 and C-6 carbon atoms. In the case of RAMEB, where the methyl substituents at the synthesis were placed randomly, all the OH groups were considered equivalent at the preparation of the model structure, thus at the lower rim four methyl groups were used to replace the hydrogen atoms of OH groups at C-6 , while at the upper rim eight at C-2 and/or C-3 positions. According to the carried out experimental studies, where aqueous solutions were used and pH ¼2.0 was applied (phosphate buffer), the citrinin molecule as having pKa ¼2.3 [22] was considered to have neutral charge. Its structure was optimized in vacuum by B3LYP/6-31G(d) density functional and basis set. The initial structures of the three CDs (BCD, RAMEB and DIMEB) were obtained from molecular dynamics simulation with explicit water molecules using the MMþ force field in the HyperChem 8.0 program. In the cases of CIT and the CDs, the atomic charges for the subsequent molecular docking studies were calculated using the B3LYP/6-31G(d) method and basis set by performing natural population analysis (NPA). The docking studies to determine the possible positions and orientations of CIT within the cavity of cyclodextrins and the associated binding affinities (thus approximating ΔG of the complexation – where van der Waals and electrostatic interactions, hydrogen-bond formations, desolvation and also entropy change at the complex formation are considered in the applied model) were carried out using the Vina 1.1.2 [23] program. The necessary . pdbqt input files were prepared using the OpenBabel program.
ð1Þ
3.1. Effect of pH on fluorescence emission spectrum of CIT In order to determine the optimal pH range for our measurements, 0.05 M phosphate buffers were prepared (pH ¼ 2.0, 2.5, 3.0 and 4.0). Under the applied conditions, CIT has excitation and emission wavelength maxima at 330 nm and 505 nm, respectively. In agreement with previous studies [22] we also observed that the decrease of pH results in the fluorescence enhancement of CIT (Fig. 2). Since the pKa value of CIT is 2.3 in water [22], this phenomenon can be attributed to the elevation of the highly fluorescent nonionic form of CIT, which predominates at pH 2.0. At
where K is the actual binding constant, F0 is the initial fluorescence intensity of CIT (in absence of CDs), F is the fluorescence intensity of CIT in presence of CDs, with concentration [CD], while A is a constant and n is the number of binding sites (n ¼1). To get insights into the formation details of CIT–BCD or CIT– DIMEB complexes the measurements were repeated and the binding constants were determined at five different temperatures: 20 °C, 25 °C, 30 °C, 35 °C and 40 °C. Thermodynamic parameters were determined for the formation of CIT–BCD and CIT–DIMEB complexes using the van't Hoff equation: ln K ¼
ΔG RT
¼
Δ H ΔS RT
þ
R
ð2Þ
where ΔG is the Gibbs free energy change, ΔS is the entropy change and ΔH is the enthalpy change associated with the complex formation.
Fig. 2. Fluorescence emission spectra of citrinin in 0.05 M phosphate buffer at pH 2.0, 2.5, 3.0, 4.0 [λexc ¼ 330 nm].
M. Poór et al. / Journal of Luminescence 172 (2016) 23–28
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Fig. 3. Fluorescence enhancement of citrinin by β- (BCD) and γ-cyclodextrin (GCD). Fluorescence emission spectra of CIT in absence and presence of increasing BCD (0–10 mM; left) and GCD (0-10 mM, right) concentrations in 0.05 M phosphate buffer (pH 2.0) [λexc ¼330 nm].
higher pH values (above pH 5), CIT does not exert fluorescence property. Furthermore, to test the suitability of the applied phosphate buffer, fluorescence emission spectra of CIT were also recorded in 0.05 M citrate buffer (pH 2.0) and in 0.01 M HCl solution. These spectra did not show differences compared to the fluorescence emission spectrum of CIT recorded in 0.05 M phosphate buffer (pH 2.0; data not shown). Therefore, similarly to the previous study of Zhou et al. [22], we also used phosphate buffer (pH 2.0) during our further experiments (see absorption spectrum of CIT in Fig. S1). 3.2. Effects of
Table 1 Log K values of CIT–CD complexes (where the unit of K is dm3/mol) and fluorescence enhancement of CIT in presence of 10 mM CD concentrations [λexc ¼330 nm, λem ¼ 505 nm].
GCD BCD HPBCD SBCD RAMEB DIMEB
log K (7 SD)
I/I0 ( 7SD)
1.84 7 0.10 2.34 7 0.14 2.34 7 0.13 2.487 0.16 2.59 7 0.03 2.93 7 0.09
1.28 7 0.03 1.677 0.02 1.65 7 0.03 1.977 0.02 1.95 7 0.01 2.067 0.01
α-, β- and γ-CD on fluorescence spectrum of CIT
To test the potential interaction of CIT with native CDs, increasing CD concentrations (0–10 mM) were added to standard amount of CIT (1 mM) in 0.05 M phosphate buffer (pH 2.0). The presence of ACD did not have any influence on the fluorescence spectrum of CIT suggesting the absence of complex formation of ACD with CIT (data not shown). On the other hand, both BCD and GCD resulted in the fluorescence enhancement of CIT (Fig. 3), causing approximately 1.7- and 1.3-fold increase compared to the fluorescence emission intensity of CIT in absence of CDs, respectively. This phenomenon can be explained by the cavity diameters of the CDs (ACD: 4.7 Å, BCD: 6.8 Å and GCD: 7.5 Å) [19], where the cavity of BCD with its 0.78 nm cavity diameter seems the most suitable for the inclusion of the CIT molecule [22]. 3.3. Fluorescence enhancement of CIT in presence of
β-CDs
BCD showed highly the most promising properties among the tested native CDs; therefore, during the following studies the interactions of CIT with chemically modified β-CDs were investigated. Increasing β-CD concentrations (0–10 mM) were added again to 1 mM CIT in 0.05 M phosphate buffer (pH 2.0). As Table 1 demonstrates the presence of each tested chemically modified βcyclodextrin increased the fluorescence signal of CIT in same or in higher extent compared to the native BCD. HPBCD induced similar while RAMEB and SBCD caused stronger fluorescence enhancement of CIT than BCD. DIMEB was the most effective in this respect causing approximately 2-fold increase of the fluorescence emission intensity of CIT (Table 1). These results demonstrate that considerably higher fluorescence enhancement of CIT can be induced with some of the chemically modified β-CDs compared to the native BCD.
Fig. 4. Benesi–Hildebrand plots of citrinin–cyclodextrin complexes (n ¼1) in 0.05 M phosphate buffer (pH 2.0) [λexc ¼ 330 nm, λem ¼ 505 nm].
3.4. Stabilities of CIT–CD complexes Thereafter, the stability constants (K) of CIT–CD complexes were determined using the Benesi–Hildebrand equation (see Section 2.2., Eq. (1)). As Fig. 4 demonstrates, Benesi–Hildebrand plots showed very good agreement with the assumed 1:1 stoichiometry. HPBCD bound CIT with similar stability than BCD (Table 1). Furthermore, SBCD, RAMEB and DIMEB complexes of CIT showed approximately 1.4-, 1.8- and 3.9-fold higher stability compared to the CIT–BCD complex, respectively. Therefore, DIMEB seems much more effective mycotoxin binder than the native BCD in the case of CIT. These results demonstrate that methylated βCDs form the most stable complexes with CIT among the tested
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Fig. 5. Benesi–Hildebrand plots of CIT–BCD and CIT–DIMEB complexes (n¼ 1) in 0.1 M hydrochloric acid and in 0.05 M phosphate buffer (pH 3.5) [λexc ¼ 330 nm, λem ¼505 nm].
compounds. One of the reasons of the higher binding constants of methylated β-CDs is that these derivatives show lowest aggregation in water than BCD, and their cavities are extended by methyl groups in both directions. Therefore, it provides a more complete and deeper penetration of CIT into the apolar cavities of DIMEB and RAMEB. 3.5. Effect of pH on the complex stabilities of CIT–BCD and CIT– DIMEB complexes Considering that the complex stabilities of CDs with nonionic and deprotonated forms of CIT can show differences, changes of the fluorescence signal of CIT can caused by the complex formation of CIT with the CD molecule or even partly resulted from the protonation/deprotonation of CIT. For this reason, we determined the complex stabilities of CIT–BCD and CIT–DIMEB complexes in 0.1 M hydrochloric acid and in 0.05 M phosphate buffer (pH 3.5), where the nonionic and the deprotonated form of CIT highly predominates, respectively. In 0.1 M hydrochloric acid CIT–BCD and CIT–DIMEB complexes showed log K values 2.31 (R2 ¼0.999) and 2.95 (R2 ¼ 0.999), respectively (see Benesi–Hildebrand plots on Fig. 5). These data show very good agreement with log K values previously determined in phosphate buffer at pH 2.0 (Table 1). On the other hand, considerably lower complex stabilities were observed at pH 3.5 in both cases, where the log K values 1.83 (R2 ¼ 0.999) and 2.49 (R2 ¼0.999) were determined for CIT–BCD and CIT–DIMEB complexes, respectively. These results suggest that BCD and DIMEB show preference towards nonionic CIT compared to its deprotonated form. Therefore, CDs seem more effective toxin binders at lower pH levels in the case of CIT. 3.6. Thermodynamic studies Fig. 6 and Table 2 summarize the results obtained from the temperature dependence of complex stabilities. Opposite tendencies of temperature dependence were obtained for CIT–BCD and CIT–DIMEB complexes: in the latter case higher stability is associated to the complexes at decreased temperatures while, surprisingly, in the case of CIT–BCD complex the stability increases at an elevated temperature. These properties reflect different formation thermodynamics of the CIT–BCD and CIT–DIMEB complexes. Since the free enthalpy changes are nearly the same around room-temperature, effect of enthalpy–entropy compensation can be assumed as follows. The attractive forces between the CIT and DIMEB molecules result in higher ordered structure of the
Fig. 6. van't Hoff's plot of CIT–BCD and CIT–DIMEB complexes.
Table 2 Thermodynamic parameters of complexation of citrinin with BCD and DIMEB (ΔG values were calculated at 25 °C).
CIT–DIMEB CIT–BCD
ΔG [kJ/mol] ( 7SD)
ΔH [kJ/mol] ( 7 SD)
ΔS [J/Kmol] ( 7 SD)
17.4 7 0.29 13.0 7 0.74
22.6 70.12 30.0 71.86
17.3 7 0.10 144.3 7 0.20
complexes included by its solvation shell. In contrast, the formation of the CIT–BCD complex preferably based on the destroying processes associated to the decreased order of the complex compared to the separated but solvated CIT and BCD molecules. In this case the positive enthalpy change reflects inhibited molecular interaction in condensed phase, however it is cannot be decided from the thermodynamic data if the interactions of the individual CIT and BCD molecules are repulsive or the heat associated to the decomposition of the solvation shells of molecules interacted shifts the overall enthalpy change to the positive region. Anyway, the huge entropy gain during formation of CIT–BCD complex overcompensates the positive enthalpy change and energetically favored formation of CIT–BCD complexes is observed around room temperature. The enthalpy–entropy compensation can also be explained in association with the solubilities of the host CD molecules. It is well-known that the solubility of the native BCD is much lower compared to the solubility of the DIMEB molecules which property originates from the highly ordered water molecules in the solvation shell of BCDs [24]. During complex formation the CIT molecules at least partly removes the water molecules from the CDs cavities and weakens the water–CD interactions in the cases of water molecules remained in the solvation shell. As a result, the freedom, i.e. the entropy of the small solvent molecules increases with higher content in the case when the BCD instead the DIMEB molecule form complexes with the CIT guest. Furthermore, considering the opposite dependence of the complex stabilities on the temperature, there is a characteristic temperature at 325.3 K where the free enthalpy changes associated to the formation of CIT–BCD and CIT–DIMEB complexes are the same. Below or above this temperature, the formation of CIT–BCD and CIT– DIMEB complexes is pronounced, respectively. 3.7. Molecular modeling studies The highest binding affinities were found in the cases of DIMEB ( 27.2 kJ/mol) and RAMEB ( 26.8 kJ/mol), while in the case of BCD, according to the measurements too, slightly lower affinity (by approximately 1.3 kJ/mol compared to DIMEB) has been found
M. Poór et al. / Journal of Luminescence 172 (2016) 23–28
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Fig. 7. The CIT–DIMEB complex from side (left) and top views (right) with the two preferred orientations of the CIT molecule (carboxyl group oriented up- or downward). The drawings were prepared with the Molekel 5.4 (Ugo Varetto, MOLEKEL 5.4, Swiss National Supercomputing Centre, Lugano, Switzerland) program. The electrostatic potential mapped to a constant electron density of 0.002 e/(a0)3 which gives adequately the molecular size and shape.
( 25.9 kJ/mol). In the cases of both methylated cyclodextrins, that complex was found to be preferred where the carboxyl groups were oriented downwards (and located close to the ether oxygen atoms at the lower rim). In the cases of CIT–BCD, CIT–RAMEB and CIT–DIMEB, 1.7 kJ/mol, 1.7 kJ/mol and 4.2 kJ/mol differences were obtained by the modeling study between the two orientations of CIT, respectively. The experimentally found highest fluorescence intensity in the case of DIMEB can be explained by the highest binding affinity obtained from modeling in that case, which is associated to reduced possibility of fluorescence quenching by the water molecules, and can be reasoned also by the placement of the – OCH3 groups (e.g. the lower rim is the most hydrophobic in this case, thus shielding the citrinin molecule the largest extent from the fluorescence quenching effect of the solvent water molecules). Another explanation of the higher fluorescence intensity of CIT– DIMEB compared to CIT–RAMEB complex can be the finding that in the latter case a preferred complex structure also exists with upwards position of the carboxyl group which can lead to less shielding of the aromatic part of CIT from the water molecules by resulting in the increased possibility of the up/down movement of citrinin in the host CD molecule which is caused by the water molecules located at the proximity of the upper rim. The obtained most preferred complex structures of two kinds are illustrated on Fig. 7 and Fig. S2 (see Cartesian coordinates of the CIT - CD complexes in Suppl. 3).
4. Conclusions In this study, the interactions of citrinin with various native and chemically modified cyclodextrins were investigated with fluorescence spectroscopy. Presence of β- and γ-CD results in the fluorescence enhancement of citrinin at lower pH levels (pH 1-4). Each tested chemically modified β-CD caused similar or higher
increase of the fluorescence of CIT than the native BCD. Furthermore, among the examined CDs the methylated β-CDs (DIMEB and RAMEB) formed the most stable complexes with CIT. Our results clearly demonstrate that some of the CD derivatives show more favorable properties compared to BCD during the complex formation with CIT, suggesting more promising analytical applicability and higher affinity as potential toxin binders.
Acknowledgements Financial support of the “Environmental industry related innovative trans- and interdisciplinary research team development in the University of Pécs knowledge base” (SROP-4.2.2.D-15/ 1/Konv-2015-0015) project is highly appreciated. The present scientific contribution is dedicated to the 650th anniversary of the foundation of the University of Pécs, Hungary.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jlumin.2015.11.011.
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