cyclodextrin gave the best enhancement of fluorescence (Table 3.4). Unfortunately none of theme gave a significant fluorescence enhancement when added to a neutral aqueous solution of the toxin.
Table 3.4: Fluorescence enhancement recorded for OTA with α-, β- and γ-CD at neutral pH.
Complex F/F0
OTA-α-CD 1.0 ± 0.1
OTA-β-CD 1.2 ± 0.2
OTA-γ-CD 1.0 ± 0.1
The data are reported as F/F0, where F0 is the native fluorescence of the mycotoxin and F is the fluorescence obtained using cyclodextrin (molar ratio: mycotoxin/CD, 1:105).
Since fluorescence enhancement usually occurs when complexation inside the cyclodextrin protects the fluorophore from water quenching, the inclusion of the isocoumarinic portion, which is mostly responsible for OTA fluorescence, seems to be unlikely, based on spectroscopic data. For this reason, starting from these observations, the interaction between cyclodextrins (in particular β- and γ-cyclodextrin) and OTA was studied using molecular modelling.
3.5.2 Computational studies.
3.5.2.1 Predicted binding modes of OTA with β-CD and γ-CD.
The docking of OTA was carried out with the phenolic group in both the protonated and deprotonated forms, in agreement with its experimental pKa of 7.1. The two forms did not display differences regarding the inclusion mechanism, while different binding free energies were predicted (Table 3.5 reports a mean ∆G0 value).
Table 3.5: Hint scores and predicted binding free energies (kcal mol-1) calculated by ChemScore and AutoDock, relative to the best docking solutions selected for different OTA-CD complexes.
Best HINT/GOLD Best HINT/AutoDock Best ChemScore Best AutoDock
OTA β-CD 3881 2553 -9.05 -5.77
OTA-γ-CD 1403 2292 -6.77 -5.50
When investigating the docking of OTA into β.cyclodextrin, different docking scoring combinations produced different predictions. The two suggested alternative modes of OTA inclusion into β-cyclodextrin are shown in Figure 3.11 a and b, respectively.
a b
BestBestHINT/GOLDHINT/GOLD Best Best HINT/HINT/AutoDockAutoDock
Figure 3.11: Illustration showing the predicted inclusion modes of OTA into the β-CD. The Connolly surface of the host (CD), built using Sybyl MOLCAD tools, is coloured blue, while the guest molecule (mycotoxin) is presented in capped sticks. a) Best HINT/GOLD for OTA in β-CD. b) Best HINT/AutoDock for OTA in β-CD.
The best ChemScore and the best HINT/GOLD (Figure 3.11 a) solutions show the phenyl ring of L-phenylalanine inserted inside the cyclodextrin cavity, while the carboxylic function and the isocoumarinic ring protrude into the solvent towards the wider opening. In this way the carboxylate on the phenylalanine residue and the phenol and ester carbonyl groups of the isocoumarinic ring can form hydrogen bonds with the secondary hydroxyl groups placed on the cyclodextrin lower rim.
The best AutoDock and the best HINT/AutoDock solutions (Figure 3.11 b), on the contrary, show the whole phenylalanine portion inserted inside the cyclodextrin cavity, with the carboxylate group exposed to the solvent through the narrower edge of the hollow truncated cone. However, once again the isocoumarinic ring is not placed within the cyclodextrin cavity, but is more exposed, allowing hydrogen bond formations between its carbonyl group and cyclodextrin secondary hydroxyl groups.
In the presence of γ-cyclodextrin, instead, best ChemScore, best HINT/GOLD and best HINT/AutoDock solutions are in agreement regarding the inclusion of OTA in this larger cyclodextrin.
a b
BestBestHINT/GOLDHINT/GOLD Best Best HINT/HINT/AutoDockAutoDock
Figure 3.12: Illustration showing the predicted inclusion mechanism of OTA into γ-CD. The Connolly surface of the host (CD), built using Sybyl MOLCAD tools, is coloured blue, while guest molecule (mycotoxin) is presented with capped sticks. a) Best HINT/GOLD for OTA in γ-CD. b) Best HINT/AutoDock for OTA in γ-CD.
The predicted binding mode matches the one selected by AutoDock and HINT/AutoDock as best docking result for OTA inclusion in β-cyclodextrin. The phenylalanine portion is inserted inside the cyclodextrin cavity, with the exception of the carboxylate group, which is exposed to the solvent through the narrower opening of the γ-cyclodextrin. The isocoumarinic ring is exposed to the solvent, allowing for the hydrogen binding formation between its carbonyl group and the secondary hydroxyl groups located around the wider cyclodextrin opening (Figure 3.12 a). The best HINT/AutoDock solution shows, however, a very different behaviour (Figure 3.12 b). Only the phenylalanine phenyl group and, partially, the chlorine on the isocoumarinic ring are included inside the cyclodextrin cavity, while the entire molecule lies on the boundary of the cyclodextrin wider edge, allowing for hydrogen bond formation between OTA hydrophilic groups (carboxylate, carbonyl and phenol-OH) and the cyclodextrin hydroxyl groups.
3.5.2.2 Post-docking analysis.
Both of the two predicted mode of inclusion into β-cyclodextrin (Figure 3.11, a and b) can be explained at the light of the size and of the hydrophobic-polar balance of the OTA molecule (Figure 3.13).
aa
HINTHINT
Figure 3.13: a) Chemically structure of OTA. The distances (atom centre to atom centre) between different extremities of the molecule are highlighted. b) HINT hydrophobic-polar contour map of OTA. Hydrophobic regions are coloured green, while hydrophilic regions are coloured blue. The OTA molecule is presented by capped sticks.
According to the HINT hydrophobic-polar contour map of OTA, it is possible to see that the entire hydrophobic portion of the isocoumarinic ring cannot be included into β-cyclodextrin for steric reasons. Indeed, the measured distance, centre atom to centre atom, between the aromatic hydrogen atom and a hydrogen belonging to the methyl group is of 7.88 Å (Figure 3.13 a).
Therefore, the inclusion of the hydrophobic L-phenylalanine phenyl ring, which allows hydrogen bond formation between OTA hydrophilic groups and cyclodextrin secondary hydroxyl groups, seems to be realistic. Indeed, this predicted preferential inclusion mode exhibits a HINT score of 3881, against a score of 2553 assigned to the OTA conformation displaying the whole phenylalanine portion inserted inside the cyclodextrin cavity (corresponding to a ∆∆G0 of more than -2 kcal mol-1). The lower predicted affinity for the latter solution may be due to the inclusion of the hydrophilic amidic portion of L-phenylalanine inside the hydrophobic cyclodextrin cavity, but also to a more difficult hydrogen bond formation between the OTA carboxylate group and the primary hydroxyl groups located around the narrower opening of the β-cyclodextrin cavity. These hydroxyl groups are not orientated toward the interior of the hole. The predicted ∆∆G0 between HINT best solutions for the OTA-β-cyclodextrin complex (best HINT/GOLD) and the AFB1 -β-cyclodextrin complex (best HINT/GOLD) is about -4.5 kcal mol-1. This is consistent with a stronger binding of OTA than AFB1 with the β-cyclodextrin, that can be justified by the higher number of hydrogen bonds formed by OTA and particularly by its negatively charged carboxylate group (which should contribute to stronger interaction with the cyclodextrin hydroxyl groups, due to the Coulombic reinforcement). This finding suggests that the experimentally observed lack of fluorescence enhancement for OTA in the presence of β-cyclodextrin is probably due to the nature
of the inclusion mechanism and not to weak binding. Indeed, the isocoumarinic ring responsible for OTA fluorescence is probably not included in the hydrophobic cavity and, thus, not protected by the quenching effect exerted by water.
Similarly, in the presence of γ-cyclodextrin, the predicted inclusion mechanism of OTA into the host is also not supportive for a possible fluorescence enhancement. Neither of the two alternative predicted modes of binding (best ChemScore, best AutoDock and best HINT/GOLD, shown in Figure 3.12 a and best HINT/AutoDock, shown in Figure 3.12 b) display the isocoumarinic ring inserted within the cyclodextrin cavity. Furthermore, it should be noticed that the calculated HINT scores are lower than those for OTA inclusion into β-cyclodextrin (Table 3.5).
Therefore, the proposed model seems justified by the size of γ-cyclodextrin (diameter of 7.5-8.3 Å), which, although grater than β-cyclodextrin, is not wide enough to accommodate the isocoumarinic ring which bears a chlorine atom.
Also considering the water sites available inside the cavity by GRID analysis, no energetically favourable water sites within the β-cyclodextrin cavity were identified for the OTA-β-cyclodextrin complex, while the GRID water probe was able to detect different potential water sites within the cyclodextrin cavity of the OTA-γ-cyclodextrin complex. In a practical sense, herein, γ-cyclodextrin seems too small to include the isocoumarinic ring, but too large to produce a good fitting with the phenylalanine phenyl ring by displacement of all the water molecules.
3.5.2.3 Circular dichroism experiments.
As discussed above, molecular modelling simulations show that the complexation of OTA with β-cyclodextrin likely occurs through the inclusion of the phenylalanine residue and this model is in agreement with experimental fluorescence results: in the complex, the isocoumarinic nucleus is still exposed to solvent quenching, and thus, emission remains unchanged. Nonetheless, the inclusion of the phenyl ring should cause changes in the circular dichroism spectrum at 230-280 nm, which is the typical range of the phenylalanine UV absorption. For this reason, CD spectra could be an another test to verify the inclusion mechanism proposed by computational approach and fluorescence spectra.
Figure 3.14: Comparison between the circular dichroism spectra recorded for OTA and the inclusion complex OTA-β-CD.
As shown in Figure 3.14, no significant differences exist between the signals of OTA alone and of OTA in the presence of the β-cyclodextrin. In particular, no differences are found in the range of the isocoumarinic moiety (330-380 nm), while only a small variation can be observed at 230-250 nm, where phenyl ring absorption occurs.
3.5.2.4 Conclusions about OTA-CD complexes.
The “natural” force field HINT has been applied to investigate the inclusion of OTA into β- and γ-cyclodextrins. According to the fluorescence spectra recorded for aqueous solutions of the toxin in the presence of β- and γ-cyclodextrin, no enhancement of fluorescence was detected. By the molecular modelling approach presented here, it is suggested for the first time that a binding between the phenylalanine portion of OTA and the cyclodextrin cavity may occur. In particular, the model obtained seems to be feasible since the predicted exclusion from the cavity of the isocoumarinic ring, the most fluorescence portion of the OTA structure, results the crucial factor for the lack of fluorescent enhancement. Moreover, the models here obtained may allow to design more specific cyclodextrins, opportunely functionalized in order to increase the affinity to the guest and allowing their use as chemosensors.