As it underlined in chapter 3, observing the chemical structure of OTA it is possible to note the presence of two acid groups: the carboxylic function on the phenylalaninic residue (pKa1 = 4.4) and the phenolic group on the isocoumarinic ring (pKa2 = 7.1), (Figure 5.3).
NH O
O
Cl O H OH
HOOC
pKa1 = 4.4
pKa2 = 7.1
Figure 5.3: Ka values for the two acidic groups present in the OTA structure.
The natural fluorescence of the mycotoxin is given by the presence of the isocoumarinic moiety, enhanced by the presence of a rigid system due to the formation of an intramolecular hydrogen bond between the phenolic moiety and the amidic carbonyl (α-form) or the esteric carbonyl group (β-form), as it is schematised in Figure 5.4.
NH O
O
Cl O H O
HOOC H
HN O
O
Cl O H O
HOOC H
Figure 5.4: Equilibrium between α- and β-forms for OTA at an acidic pH value.
Instead, in an alkaline environment, the presence of a phenate moiety allows a higher conjugation of the system, suggesting a more rigid structure where the amidic hydrogen is involved in a hydrogen bond with both the phenate and the carboxylate groups (Figure 5.5).
O O
Cl O N H O H
O O
Figure 5.5: Chemical structure of OTA in an alkaline environment.
Spectroscopic studies conducted by Dall’Asta et al.11 revealed for OTA a strong dependence of its spectroscopic properties (UV absorption and fluorescence emission) from the pH of the aqueous solution in which it is dissolved.
The UV spectra of aqueous solutions of ochratoxin A at different pH values is reported in Figure 5.6.
0 0.03 0.06
250 300 350 400 450 500
wavelenght (nm)
A
pH=4 pH=6 pH=7 pH=9
Figure 5.6: UV spectra of OTA at different pH values. Concentration of OTA: 1 µg/ml.
As it can be seen in Figure 5.6, at pH = 6 or lower, the predominant specie is the monoanionic
monoanionic and the dianionic species, are present since the absorption spectrum shows two different maxima, the former at 335 nm and the latter at 370 nm. At alkaline pH (pH = 9), only the dianionic specie is present, with an absorption maximum at 380 nm and a higher absorbance due to the increasing conjugation of the system.
Similarly, the fluorescence spectra of ochratoxin A performed at different pH values revealed greater emission properties for the dianionic form (Figure 5.7).
0 50 100 150 200 250 300 350 400
400 450 500 550 600
λ (nm)
F (u. a.)
pH = 7 pH 10
pH = 8 pH = 9
0 50 100 150 200 250 300 350 400
400 450 500 550 600
λ (nm)
F (u. a.)
pH = 7 pH 10
pH = 8 pH = 9
Figure 5.7: Fluorescence enhancements at different pH values.
As outlined in Figure 5.7, the fluorescence increases sharply from pH 7 to pH 9, consistently with the increasing concentration of the deprotonated form, while at higher pH values, a slight fluorescence decrease is observed, probably due to the hydrolysis of the lactone ring. Moreover, going from neutral to alkaline solutions a 9-fold fluorescence enhancement was obtained.
5.3.1 Spectroscopic measurements with neutral and negatively charged β-CDs.
First, we performed a screening of complexing properties of different commercially available cyclodextrins for OTA by fluorescence experiments (Table 5.1).
Table 5.1: Emission of OTA (10-7 M) in water in the presence of different commercially available CDs. pH of the solutions: 9.
Complex F/F0
water, pH = 9 1.0 ± 0.1
OTA-α-CD 1.0 ± 0.1
OTA-β-CD 1.2 ± 0.2
OTA-γ-CD 1.0 ± 0.1
DIMEB 0.99 ± 0.1
Hyp-β-CD 1.00 ± 0.2
Su-β-CD 0.13 ± 0.2
SBE-β-CD 0.99 ± 0.1
∆F = F/F0 where F is the OTA fluorescence intensity recorded in the presence of CD and F0 is the OTA fluorescence intensity recorded in water. Molar ratio OTA:CD = 1:105
As shown in Table 5.1, none of the commercially available cyclodextrins, neither neutral such as native α-, β- and γ-cyclodextrins, 2,6-di-O-methyl-β-cyclodextrin (DIMEB) and 2-hydroxypropyl-β-cyclodextrin (Hyp-β-CD), nor negatively charged such as succinyl-β-cyclodextrin (Su-β-CD) or tetra(6-O(sulfo-n-butyl))-β-cyclodextrin (SBE-β-CD) showed affinity for OTA. In particular, except for the case of the succinyl-β-cyclodextrin, where the number of the negative charges for molecule is high (from 4 to 6), the presence of substituents did not allow significant modifications of the interaction between the toxin and the hydrophobic cavity.
5.3.2 Spectroscopic measurements with positively charged β-CDs.
With the aim of evaluating the effect of electrostatic interactions on the OTA:cyclodextrins complex, we performed fluorescence measurements of aqueous solution of OTA, at pH 9, in the presence of β-cyclodextrin derivatives opportunely synthesized (see chapter 4) bearing one or more positively functional groups on the upper or on the lower rim.
5.3.2.1 Cyclodextrins bearing a single positive charge.
Although the mode of inclusion obtained by docking techniques suggested that the interaction of β-cyclodextrin with the mycotoxin occurred preferentially at the wider opening side of the host, we placed positive functional groups such as ammonium or guanidinium on both rims, in order to verify if the position of the charge could induce some differences in the host:guest interactions.
First of all, 2-amino-2-deoxy-β-cyclodextrin and 6-amino-6-deoxy-β-cyclodextrin were studied. In the former case, only a little fluorescence enhancement was obtained, as shown by the emission spectrum reported in Figure 5.8.
0 10 20 30 40
400 450 500 550 600
wavwlenght (nm)
F (a. u.)
OTA OTA + CD
pH = 8.0, OTA:CD 1:105 λex. 380 nm
Figure 5.8: OTA fluorescence in the presence and in the absence of 2-amino-2-deoxy-β-CD. [OTA] = 20 ng/ml; buffer solution: NH3/NH4Cl (20 mM).
In the case of the amino derivative at the 6-position no enhancement was observed since an F/F0 ratio of 1.0 (where F is the fluorescence of an aqueous solution of OTA at pH = 9 in the presence of the 6-amino-6-deoxy-β-cyclodextrin and F0 is the OTA fluorescence at pH = 9) was obtained.
Since the aim of these measures was to verify the capacity of cyclodextrin derivatives to promote significative enhancements of the emission of alkaline solutions of OTA (pH = 9), in both cases presented above it wasn’t possible to record the emission at pH grater than 8.0, since the pKa
of the ammonium group is ≈ 8.0. Therefore, the conditions used were not the best since at similar pH values, only 50 % of the guests added to the solutions were in the protonated form.
In order to overcome this problem, guanidinium derivatives of β-cyclodextrins were used, since the pKa of the guanidinium group is ≈ 13. The fluorescence spectra of OTA at pH = 9, in presence of 2-guanidinium-2-deoxy-2-(S)-β-cyclodextrin or 6-guanidinium-6-deoxy-β-cyclodextrin are shown in Figure 5.9 and Figure 5.10.
0 20 40 60
400 450 500 550 600
wavelenght (nm)
F (a. u.)
OTA OTA+CD pH = 9.0, OTA:CD 1:105 λex. 380 nm
O
NH O
H OH
H2N
NH2 O O
+
Figure 5.9: OTA fluorescence in the presence and in the absence of 2-guanidinium-2-(S)-β-CD. [OTA]= 20 ng/ml; buffer solution: NH3/NH4Cl (20 mM).
0 20 40 60
400 450 500 550 600
wavelenght (nm)
F (a. u.)
OTA OTA+CD
O
OH O
H NH
O O
H2N
NH2
+
pH = 9.0, OTA:CD 1:105 λex. 380 nm
Figure 5.10: OTA fluorescence in the presence and in the absence of 6-guanidinium-β-CD. [OTA]= 20 ng/ml; buffer solution: NH3/NH4Cl (20 mM).
In both cases no significative fluorescence enhancement was induced, suggesting that, electrostatic interactions due to the presence of a single positive charge placed on the upper or on the lower rim do not induce modifications of the mode of complexation of OTA.
5.3.2.2 Cyclodextrin derivatives bearing two positive charges on the upper rim.
All three 6A,6X-diamino-6A,6X-dideoxy-β-cyclodextrin isomers (Figure 5.11), synthesized as described in chapter 4, were tested as fluorescence enhancers for OTA emission.
HO O OH
OH O HO
OH OH
O HO
OH OH
O OH HO
HO O
OH HO HO
O HO HO O N+
HOHOO
O
O O O O
O N+
H HH H
H H
N+ O OH
OH O HO
OH OH
O HO
OH OH
O OH HO
HO O
OH HO HO
O HO HO O OH
HOHOO
O
O O O O
O N+
H H H H
H
H HO O OH
OH O N+
OH OH
O HO
OH OH
O OH HO
HO O
OH HO HO
O HO HO O OH
HOHOO
O
O O O O
O N+
H H H
H H H
6A,6B-diamino-β-CD 6A,6C-diamino-β-CD 6A,6D-diamino-β-CD
Figure 5.11: 6A,6X-diamino-β-CD derivatives.
The fluorescence spectra obtained adding the AB regioisomer to a solution of OTA, at pH 8.0 is reported in Figure 5.12.
0 10 20 30
400 450 500 550 600
wavelenght (nm)
F (a. u.)
OTA OTA + CD
pH = 8.0, OTA:CD 1:105 λex. 380 nm
Figure 5.12: OTA fluorescence in the presence and in the absence of 6A,6B-diamino-6A,6B-dideoxy- β-CD. [OTA] = 20 ng/ml; buffer solution: NH3/NH4Cl (20 mM).
Similarly, also in the case of the A,C and A,D diamino isomers, no fluorescence enhancements for OTA were obtained.
5.3.2.3 Application of per(6-guanidinium)-β-CD.
In order to check the performance of a perguanidilated β-cyclodextrin, rich of positive charges on the upper rim, the per-(6-guanidinium)-6-deoxy-β-cyclodextrn, synthesized as described in the chapter 4, was used as fluorescence enhancer. Below, the fluorescent spectrum recorded for a solution of OTA at pH = 9, in the absence and in the presence of the per-guanidilated derivative, is reported (Figure 5.13).
0 50 100 150 200
400 450 500 550 600
wavelenght (nm)
F (a. u.)
OTA OTA + CD
pH = 9.0, OTA:CD 1:105 λex. 380 nm
O O
NH
O
H OH
7 H2N
NH2
+
Figure 5.13: OTA fluorescence in the presence and in the absence of per(6-guanidinium)-6-deoxy-β-CD.
[OTA] = 20 ng/ml; buffer solution: NH3/NH4Cl (20 mM).
Although the experiment is preliminary, it is promising, but it deserves further attention.