Action of tyrosinase on alpha and beta-arbutin: A kinetic study

<div><p>The known derivatives from hydroquinone, α and β-arbutin, are used as depigmenting agents. In this work, we demonstrate that the <i>oxy</i> form of tyrosinase (oxytyrosinase) hydroxylates α and β-arbutin in <i>ortho</i> position of the phenolic hydroxyl group, giving rise to a complex formed by <i>met</i>-tyrosinase with the hydroxylated α or β-arbutin. This complex could evolve in two ways: by oxidizing the originated <i>o</i>-diphenol to <i>o</i>-quinone and <i>deoxy</i>-tyrosinase, or by delivering the <i>o</i>-diphenol and <i>met</i>-tyrosinase to the medium, which would produce the self-activation of the system. Note that the quinones generated in both cases are unstable, so the catalysis cannot be studied quantitatively. However, if 3-methyl-2-benzothiazolinone hydrazone hydrochloride hydrate is used, the <i>o</i>-quinone is attacked, so that it becomes an adduct, which can be oxidized by another molecule of <i>o</i>-quinone, generating <i>o</i>-diphenol in the medium. In this way, the system reaches the steady state and originates a chromophore, which, in turn, has a high absorptivity in the visible spectrum. This reaction allowed us to characterize α and β-arbutin kinetically as substrates of tyrosinase for the first time, obtaining a Michaelis constant values of 6.5 ± 0.58 mM and 3 ± 0.19 mM, respectively. The data agree with those from docking studies that showed that the enzyme has a higher affinity for β-arbutin. Moreover, the catalytic constants obtained by the kinetic studies (catalytic constant = 4.43 ± 0.33 s^-1 and 3.7 ± 0.29 s^-1 for α and β-arbutin respectively) agree with our forecast based on 13 C NMR considerations. This kinetic characterization of α and β-arbutin as substrates of tyrosinase should be taken into account to explain possible adverse effects of these compounds.</p></div

Stereocontrolled Synthesis of β‑Lactams within [2]Rotaxanes: Showcasing the Chemical Consequences of the Mechanical Bond

The intramolecular cyclization of <i>N</i>-benzyl­fumar­amide [2]­rotaxanes is described. The mechanical bond of these substrates activates this transformation to proceed in high yields and in a regio- and diastereo­selective manner, giving interlocked 3,4-disubstituted <i>trans</i>-azetidin-2-ones. This activation effect markedly differs from the more common shielding protection of threaded functions by the macrocycle, in this case promoting an unusual and disfavored 4-<i>exo-trig</i> ring closure. Kinetic and synthetic studies allowed us to delineate an advantageous approach toward β-lactams based on a two-step, one-pot protocol: an intramolecular ring closure followed by a thermally induced dethreading step. The advantages of carrying out this cyclization in the confined space of a benzylic amide macrocycle are attributed to its anchimeric assistance

Computational docking of α-arbutin.

<p>Docking poses obtained with AutoDock of α-arbutin in the active site of the oxy form of mushroom tyrosinase are shown as sticks. The atom colors are as follows: red = oxygen, blue = nitrogen, brown = copper, green = carbon, and white = hydrogen. Polar interactions and hydrogen bonds are shown as black dotted lines. The distance from the <i>ortho</i> carbon of the phenolic ring to the oxygen atom of the peroxide ion is shown in blue lines.</p

Total oxygen consumption test (TBC).

<p>A total oxygen consumption test was carried out in the presence of <i>tert</i>-butylcatechol and different concentrations of α-arbutin (mM): a) 0, b) 5, c) 10 and d) 20. The rest of the experimental conditions were [<i>E</i>]₀ = 50 nM and [TBC]₀ = 1 mM. <b>Inset.</b> Total oxygen consumption test in the presence of <i>tert</i>-butylcatechol and different concentrations of β- arbutin (mM): a) 0, b) 5, c) 10 and d) 20. The rest of the experimental conditions were [<i>E</i>]₀ = 50 nM and [TBC]₀ = 1 mM.</p

Chemical structures of α-arbutin and β-arbutin.

<p>Chemical structures of α-arbutin and β-arbutin.</p

Action of tyrosinase on α-arbutin in the presence of MBTH.

<p>The experimental conditions were [<i>E</i>]₀ = 300 nM, [MBTH]₀ = 0.2 mM, [α-arbutin]₀ = 10 μM and DMF 2%. The spectrophotometric recordings were made every 60 seconds. <b>Inset. Determination of the MBTH saturation concentration.</b> The experimental conditions were [<i>E</i>]₀ = 100 nM, [α-arbutin]₀ = 20 mM and DMF 2%.</p

Diphenolase activity.

<p><b>A.</b> Representation of <i>i</i>_D (degree of inhibition of the diphenolase activity) <i>vs</i>. the concentration of α-arbutin. The experimental conditions were [<i>E</i>]₀ = 30 nM and [L-dopa]₀ = 0.5 mM. <b>Inset.</b> Spectrophotometric recordings of the effect of different concentrations of α-arbutin on the diphenolase activity of tyrosinase, using L-dopa as substrate. The experimental conditions were [<i>E</i>]₀ = 30 nM, [L-dopa]₀ = 0.5 mM and α-arbutin (mM): a) 0, b) 2.5, c) 5, d) 8, e) 13, f) 20 and g) 30. <b>B.</b> Representation of <i>i</i>_D (degree of inhibition of the diphenolase activity) <i>vs</i>. the concentration of β-arbutin. The experimental conditions were [<i>E</i>]₀ = 30 nM and [L-dopa]₀ = 0.5 mM. <b>Inset.</b> Spectrophotometric recordings of the effect of different concentrations of β-arbutin on the diphenolase activity of tyrosinase, using L-dopa as substrate. The experimental conditions were [<i>E</i>]₀ = 30 nM, [L-dopa]₀ = 0.5 mM and β-arbutin (mM): a) 0, b) 0.5, c) 2.5, d) 5, e) 12, f) 20 and g) 30.</p

Inhibition of monophenolase activity by arbutins.

<p>Graphical representation of the Lineweaver–Burk equation to show the inhibition of the monophenolase activity of tyrosinase in the presence of 3 mM α-arbutin. The experimental conditions were [<i>E</i>]₀ = 50 nM and R = [L-dopa]₀ / [L-tyrosine]₀ = 0.042. <b>Inset.</b> Graphical representation of the Lineweaver–Burk equation showing the inhibition of the monophenolase activity of tyrosinase in the presence of β-arbutin 3 mM. The experimental conditions were [<i>E</i>]₀ = 50 nM and R = [L-dopa]₀ / [L-tyrosine]₀ = 0.042.</p

Kinetic constants for the characterization of the activity of tyrosinase on α-arbutin and β-arbutin and chemical shift values of the carbon with the phenolic hydroxyl group.

<p>Kinetic constants for the characterization of the activity of tyrosinase on α-arbutin and β-arbutin and chemical shift values of the carbon with the phenolic hydroxyl group.</p

Kinetic characterization of the action of tyrosinase on arbutins.

<p>Representation of the initial rate values obtained for the action of tyrosinase on α-arbutin. The experimental conditions were [<i>E</i>]₀ = 100 nM, [MBTH]₀ = 0.2 mM and DMF 2%. <b>Inset.</b> Representation of the initial rate values obtained for the action of tyrosinase on β-arbutin. The experimental conditions were the same as Fig 7.</p