## 3 Results and Discussion  
### Design of the Imprinted Materials  
The interactions of the template with the functional monomers is the key step in formulating the MIP composition, based on weak non-covalent interactions such as hydrogen bonding, ionic interactions or van der Waals forces depending on the structure of the template. Both TY and HT have an aromatic ring that could be involved in \(\uppi\)-\(\uppi\) stacking with other aromatic functions or unsaturated systems, while the phenolic groups could act as donors or receptors for hydrogen bonds. In order to design imprinted polymers capable of both satisfactory rebinding and sensitive detection, we have chosen to explore the effect of the composition at three levels. First, we have selected non-fluorescent functional monomers capable of establishing significant interactions with the targets. To this end, compounds **5-9** reported in Figure 1 were chosen. Such monomers are widely used in the preparation of imprinted polymers and are in principle capable to establish the required interactions by hydrogen bonding. We have also chosen NIPAM **10** as a co-monomer. It is demonstrated that NIPAM usually does not interact with the template in polar solvents as DMSO and water, but acts as a stabilizer of the polymeric structure [20]. Second, we have considered two opposite approaches in the selection of the cross-linker. On one side we have chosen MBA **11**, a very common and minimal length cross-linker, capable in principle to contribute to ligand binding by hydrogen bonding. On the other side we have also considered a cross-linker fully capable of further hydrophobic interactions with the aromatic systems of the phenols, namely DVB **12**. Third, as to the fluorescent co-monomers, we have focused on fluorescein. The literature reports several examples of MIPs utilizing this fluorophore as signaling label. In most cases, quenching of its emission has been observed upon rebinding of the template [21; 22]. However, [PERSON] and colleagueshave described in 2014 a MIP for the recognition of cocaine with activation of fluorescein emission upon binding, using as the monomer a 4-vinylbenzamide derivative of fluorescein **12**[8]. The authors explain the increase of fluorescence on the basis of a proton transfer from the carboxylic group of fluorescein to the amino group of cocaine, that enhances the fraction of the more fluorescent fluorescein anion. However, in water fluorescein undergoes to a rather complex equilibrium between tautomers and protonated/deprotonated forms (Figure 2a) [23]. We reasoned that such equilibria could be shifted by the presence of the template even if it was not capable of proton transfer. The nonfluorescent lactonic tautomers H\({}_{2}\)Flat and HFlat\({}^{-}\) are in equilibrium with the fluorescent ones H\({}_{2}\)Fquin and HFquin\({}^{-}\) which are present in solution up to neutral pH. An aromatic ligand as our phenols could favorably shift the equilibrium towards the fluorescent tautomers. To evaluate this possibility, we have carried out DFT calculations at the B3 lyp-Df-6.31G**+ level in a pcm water model. The lactone tautomer H\({}_{2}\)Flat results more stable than the open acid H\({}_{2}\)Fquin by 10.44 KJ/mol, in agreement with the experimental observations [23]. A model of the \(\pi\)-stacked complexes of tyrosol and the two tautomers was in favor of the open acid complex (Figure 2b) by 13.2 KJ/mol thus giving an indication of a possible shift towards the fluorescent form in the presence of tyrosol. A similar behavior was obtained also by modelling the complexes of tyrosol with the monoanionic forms of fluorescein. We have therefore considered two fluorophore monomers, namely **12** as proposed by [PERSON] [8], and \(O\)-acryloyl-fluorescein **13**. The designed imprinted polymers are aimed at sensing phenols in aqueous extracts of olive leaves, where the most important derivative is oleuropein.