Synthesis and Studies of the Inhibitory Effect of Hydroxylated Phenylpropanoids and Biphenols Derivatives on Tyrosinase and Laccase Enzymes

The impaired activity of tyrosinase and laccase can provoke serious concerns in the life cycles of mammals, insects and microorganisms. Investigation of inhibitors of these two enzymes may lead to the discovery of whitening agents, medicinal products, anti-browning substances and compounds for controlling harmful insects and bacteria. A small collection of novel reversible tyrosinase and laccase inhibitors with a phenylpropanoid and hydroxylated biphenyl core was prepared using naturally occurring compounds and their activity was measured by spectrophotometric and electrochemical assays. Biosensors based on tyrosinase and laccase enzymes were constructed and used to detect the type of protein-ligand interaction and half maximal inhibitory concentration (IC50). Most of the inhibitors showed an IC50 in a range of 20–423 nM for tyrosinase and 23–2619 nM for laccase. Due to the safety concerns of conventional tyrosinase and laccase inhibitors, the viability of the new compounds was assayed on PC12 cells, four of which showed a viability of roughly 80% at 40 µM. In silico studies on the crystal structure of laccase enzyme identified a hydroxylated biphenyl bearing a prenylated chain as the lead structure, which activated strong and effective interactions at the active site of the enzyme. These data were confirmed by in vivo experiments performed on the insect model Tenebrio molitur.


General procedure for the synthesis of chalcones 15 and 16
To a stirred solution of KOH (90 eq) in water (4 mL) cooled to 0 °C in an ice bath was added dropwise a solution of OMe-dehydidrovanillin [8] (1 eq) and 4-hydroxy-3-methoxyacetophenone (for 15) or 2hydroxy-5-methoxyacetophenone (for 16) (3 eq) in methanol (20 mL). The reaction mixture was kept at 0 °C for 3 h, and then at room temperature for 96 h, under N2. The mixture was poured into ice-water (10 mL), adjusted to pH 3-4 with hydrochloridric acid (10% solution), and then extracted with ethyl acetate.
The organic layer was successively washed with water and saturated brine, dried over anhydrous sodium sulphate and purified by flash chromatography using a 2:1 mixture of ethyl acetate:petroleum as eluent to give 15 or 16. (

Preparation of Tyrosinase and Laccase biosensors
The biosensors were prepared starting from a Teflon™-insulated silver wire (30 mm in length; Ø = 125 μm), as previously described [11,12]. Briefly, 1 mm of the silver wire was exposed and then introduced into a silica capillary cylinder (10 mm in length; I.D. Ø = 180 μm, Polymicro Technologies, Phoenix, AZ, USA) partly packed with graphite-loaded (55% w/w) epoxy resin. By mixing 850 mg of graphite with 500 mg of Araldite-M and 200 mg of hardener, a preliminary disc electrode of carbon-composite was fabricated (Ø = 180 μm; area: 2.5 × 10 −4 cm 2 ) by filling the silica capillary tubing with the mixture. The silver wire assured a good electrical contact. After 24 h at 40 °C, the electrode surface was cleaned using a high-speed drill (Dremel® 300) equipped with an aluminum oxide grinding wheel. On top of the electrode surface a 1 µl of Tyrosinase or Laccase solution was deposited. After the complete evaporation of water at room temperature, the biosensor was quickly dipped in the polyurethane solution in order to entrap the enzyme on the electrode surface, and after the complete evaporation of the solvent biosensors were put to stabilize in PBS pH=6.0 overnight.

Biosensor Calibration protocol
The electrochemical measurements were performed at room temperature in a classical three-electrode electrochemical cell consisting of four biosensors as working electrodes, a reference electrode (Ag/AgCl in NaCl, 3.0 M) and the auxiliary electrode (a large surface steel wire), as previously described [13,14].
In order to characterize tyrosinase and laccase biosensors' performances, a preliminary cyclic voltammetry was performed in PBS pH=6.0, using dopamine as reference compound, so that to fix the working reduction potential of the corresponding quinone, obtained by the oxidation of the amine. In parallel, cyclic

Biosensor Inhibition protocols
Two different inhibition protocols have been used. The first has been set in order to assess the IC50 values of different inhibitors, as previously demonstrated. [15,16]. Briefly, a fixed concentration of dopamine (50 µM in 10 ml of PBS pH=6.0 at a fixed potential of -50 mV vs Ag/AgCl) has been injected in the electrochemical cell.
After having reached a stable baseline, known volumes of a stock solution of each inhibitor (10 mM) have been added in the cell up to the desired concentration. The second protocol has been explained in the manuscript S7 at 4.3. paragraph of the Materials and Methods section. For each inhibitor was built a group of biosensors (n=4) for both tyrosinase and laccase enzyme.

PC12 Cells Culture
PC12 cells, rat pheochromocytoma-derived cell line (ATCCCRL-1721) (passages 12-25) were maintained at 37 °C humidified air containing 5% CO2/95% atmospheric air and cultured in a 60 mm plastic culture plates with Dulbecco's modified Eagle's medium supplemented with 10% horse serum, 5% fetal bovine serum and 1% of penicillin/streptomycin. PC12 cells were treated for 24 h with the different compounds at different concentrations ranging from 1 to 40 μM, to evaluate their eventual toxicity. Then, in order to assess the eventual compounds' protection properties, PC12 cell treated with hydrogen peroxide (100μM) in association with increasing concentration of the above-mentioned molecules, from 1 up to 20 μM. For the compounds that had been shown to protect cells from the oxidative insult from H2O2, a co-treatment with MnCl2 1 mM and the compounds was performed, in order to evaluate whether the same compound could also able to protect against MnCl2 insult. For all treatments cell viability was measured by means of MTT assay, as explained in the following paragraph [17].

MTT Assay
At the end of the exposure time of each experiment, the cell viability was assessed by means of MTT (3-(4,5dimethyl-thiazol-2-yl)-2,5-diphenyltetrazoliumbromide) assay. For this purpose, 1 mg/mL of MTT was added to each sample and incubated for 4 h at 37 °C. Only viable cells are able to convert the soluble dye MTT into the insoluble formazan crystals. After the incubation the MTT supernatant solution was removed, the cells were washed in phosphate-buffered saline (PBS) and centrifuged while the pellet was dissolved in 2 mL of isopropanol. After centrifugation of the solution at 4,000 rpm for 5 min, the absorbance for each sample was assessed by means of a Bauty Diagnostic Microplate Reader at 578 nm. All experiments were performed in 24well plates (1 × 10 5 cells/mL/well) and repeated in triplicate.