Hydroxylated biphenyls as tyrosinase inhibitor: A spectrophotometric and electrochemical study

Article abstract of DOI:10.1016/j.ejmech.2016.12.028

A small collection of C₂-symmetry hydroxylated biphenyls was prepared by straightforward methods and the capability to act as inhibitors of tyrosinase has been evaluated by both spectrophotometric and electrochemical assays. Our attention was f

Full text of DOI:10.1016/j.ejmech.2016.12.028

Accepted Manuscript
Hydroxylated biphenyls as tyrosinase inhibitor: A spectrophotometric and
electrochemical study
Paolo Ruzza, Pier Andrea Serra, Davide Fabbri, Maria Antonietta Dettori, Gaia
Rocchitta, Giovanna Delogu
PII:
S0223-5234(16)31034-0
10.1016/j.ejmech.2016.12.028
EJMECH 9119
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 8 October 2016
Revised Date: 30 November 2016
Accepted Date: 12 December 2016
Please cite this article as: P. Ruzza, P.A. Serra, D. Fabbri, M.A. Dettori, G. Rocchitta, G. Delogu,
Hydroxylated biphenyls as tyrosinase inhibitor: A spectrophotometric and electrochemical study,
European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2016.12.028.
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Hydroxylated biphenyls as tyrosinase inhibitor: a spectrophotometric and electrochemical
study
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Paolo Ruzza , Pier Andrea Serra , Davide Fabbri , Maria Antonietta Dettori , Gaia Rocchitta ,
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Giovanna Delogu
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Istituto di Chimica Biomolecolare, Consiglio Nazionale Ricerche, Via Marzolo 1, I-35131 Padova, Italy
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Dipartimento di Medicina Clinica e Sperimentale, Università degli Studi, Viale S. Pietro 43/b, I-07100
Sassari, Italy
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Istituto di Chimica Biomolecolare, Consiglio Nazionale Ricerche, Traversa La Crucca 3, I – 07100 Sassari,
Italy
Abstract: A small collection of C -symmetry hydroxylated biphenyls was prepared by
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straightforward methods and the capability to act as inhibitors of tyrosinase has been evaluated by
both spectrophotometric and electrochemical assays. Our attention was focused on the
diphenolase activity of this enzyme characterized by the absence of the characteristic lag time of
enzymatic reaction of its monophenolase activity. To this purpose, we evaluated the capability of
tyrosinase to oxidize a natural o-diphenol substrate to o-quinone analyzing the changes in the UV-
Vis spectrum of a solution of caffeic acid and the reduction of the cathodic current in a tyrosinase-
biosensor, respectively. Results of both the methods were comparable. Most of the compounds
possessed higher inhibitory activity compared to compound 1, a known hydroxylated biphenyl
inhibitor of tyrosinase.
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Keywords: Tyrosinase, spectrophotometric assay, biosensor, hydroxylated biphenyls, synthesis
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These Authors contributed equally to this work
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*Corresponding author. Present address: Istituto di Chimica Biomolecolare, CNR, Traversa La Crucca 3, I –
07100 Sassari, Italy. Phone: +39 0792841220; Fax: +39 079 2841229
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E-mail address: giovanna.delogu@icb.cnr.it

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1.
Introduction
Tyrosinase [EC 1.14.18.1] is a copper-containing enzyme involved in melanin biosynthesis, in
unfavourable enzymatic browning of plant-derived foods, in pathological melanogenesis and in
insects moulting process [1]. It catalyses the oxidation of both monophenols (monophenolase or
cresolase activity) and o-diphenols (diphenolase or catecholase activity) to o-quinones (Figure 1A).
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Figure 1.
Three forms of this enzyme (oxy-, met-, and deoxy-tyrosinase) with different copper
structures of the active site are identified [2]. The monophenolase activity is catalysed by the oxy
form (less than 15%) that is transformed in the deoxy form in the successive oxidation to o-
quinone, while in the diphenolase cycle both the oxy and met forms are involved. The oxy form
oxidizes o-diphenol to o-quinone, yielding the met form, and this latter form transforms another o-
diphenol molecule into o-quinone and is reduced to the deoxy form, the only one capable of
reacting with molecular oxygen. For this reason, the monophenolase activity presents a
characteristic lag time that exists until a sufficient amount of catechol is produced [3]. The length of
the lag time is strictly connected to the enzyme source and concentration, the concentration of
monophenol and the presence of catalytic amounts of o-diphenol or transition metal ions, which
completely abolish the lag period [4].
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Investigation of tyrosinase inhibitors may lead to development of novel skin whitening
agents, medicinal products, antibrowning substances or insect control compounds [5]. Many
natural occurring compounds have been identified for those targets whose structure has inspired
the preparation of several collections of synthetic tyrosinase inhibitors [6]. The most studied
chemical scaffolds of tyrosinase inhibitors belong to the polyphenols group. In spite of a large
number of tyrosinase inhibitors only a few of these are generally used.
4,4’-Dihydroxy-biphenyl 1, a natural biphenol, was found to be a potent tyrosinase inhibitor,
more effective than kojic acid and arbutin [7]. Hydroxylated biphenyls are a class of polyphenols
widely present in nature [8], some of them manifest high biological activity like ellagitannins,
vancomicin, biphenomicins, others, structurally less sophisticated, are natural occurring dimers of
2-methoxy phenols and phenols. Compared to phenols, hydroxylated biphenyls manifest higher
antioxidant activity and generally they are less toxic than the corresponding phenolic monomer [9,
10]. There is an increasing interest in using the hydroxylated biphenyl unit as building block to
prepare bioactive molecules involved in human pathologies and disorders because most of them
manifest interesting pharmacological and biological activities such as neuroprotective and
antiproliferative properties [11].
In our continuing search on synthesis of hydroxylated biphenyls with interesting
stereochemical and biological features [12-15]. we have prepared a small collection of C2-

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symmetry biphenols and evaluated their capability to act as inhibitors of tyrosinase by both
spectrophotometric and electrochemical assays.
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Results and discussion
2.1.
Chemistry
Taking in account the high level of specificity of hydroxylated biphenyl structure in protein
binding in comparison to other aromatic compounds [16], we have considered C -symmetry
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isomer of biphenyl 1 (eg. compound 2) and its derivatives by introducing hydroxylated
functionalities in key positions (eg. compounds 3, 4) as depicted in Figure 2A.
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Figure 2
It is generally demonstrated that the presence of hydroxylated groups in a phenol structure
improves tyrosinase activity [17-19]. According to those considerations, a small collection of
biphenyls was selected. Different hydroxylated functionalities are present in compounds 6 and 7
whereas compounds 5 and 8 are lipophylic derivatives of biphenol 1. Compound 6 possesses a
benzodioxole moiety in a biphenylic structure that reminds sesamol structure, recently studied for
the effective activity as tyrosinase inhibitor [20].
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Except compound 7, all compounds studied in this work are known in literature, though
some of them were achieved in scarce yields or as side-products. Yields of compounds 5 and 8
were increased and new straightforward synthetic procedures improved the sustainability of the
process compared to those described in literature (Supplementary Data). Claisen rearrangement of
the corresponding 2-(prop-2-en-1-yl)phenol derivative gave compound 5 in high yield following two
microwave procedures carried out in aqueous solvents. Compounds 3, 4, 6-8 were obtained by
coupling reactions of the corresponding monomer with different reaction conditions according to
substituents in the phenolic ring. Synthesis and spectroscopic data of compounds 3 – 6 and 8
have been reported in the Supplementary Data. Compound 7 was obtained by oxidation of
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veratraldehyde C -symmetry dimer 9 in the presence of m-chloroperbenzoic acid (mCPBA) (Figure
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2B). Due to substitutions in ortho-ortho´ and ortho-meta positions, biphenyls 3 and 6 - 8 are
conformationally hindered. Biphenyls 6 - 8 were tested in racemic form.
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2.2
Biological evaluation

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With the aim to explore the biological properties of compounds 1-8, we evaluated the
capability of these molecules to inhibit the tyrosinase activity. In particular, our attention was
focused on the diphenolase activity of this enzyme [21]. To this purpose, we evaluated the
capability of tyrosinase to oxidize a natural o-diphenol substrate to o-quinone analyzing the
changes in the UV-Vis spectrum of a solution of caffeic acid. The spectrum recorded before mixing
the substrate and enzyme solutions, strongly immediately changes after the mixing and the
absorbance at 280 and 311 nm, characteristic of caffeic acid, decrease in time-dependent mode,
while a band at 413 nm attributable at the o-quinone appeared [22]. The time-course of caffeic acid
oxidation, obtaining monitoring the absorbance values at 311 nm at different time, has been
reported in Figure 3 and is characterized by the absence of the characteristic lag time of enzymatic
reaction catalyzed by tyrosinase due to its monophenolase activity.
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Figure 3
Any significant contribute of compounds 1-8 in the examined UV-Vis region of caffeic acid,
in presence or not of tyrosinase, in the experimental conditions was detected (data not shown).
With the exclusion of compounds 2 and 8, all tested hydroxylated biphenyl showed either a
comparable (compounds 3 and 5) or a superior efficiency to inhibit the tyrosinase activity respect to
biphenyl 1. It should be taken in account that compounds 5, structurally related to magnolol and
honokiol, showed no cytoxicity in normal human lymphocytes (IC50 < 100 µM) making this
compound a good candidate for further evaluation in vivo model [23].
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As show in Figure 4A, compound 4 is the most effective inhibitor of tyrosinase activity, while
compounds 6 and 7 have a lower ability to inhibit the caffeic acid oxidation by tyrosinase compared
to 4, even if it is significantly higher to that of biphenyl 1.
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Figure 4
Thereafter, tyrosinase inhibition activity of compounds 1-8 were determined by a tyrosinase-
biosensor as schematically represented in Figure 1B. A significant inhibition of tyrosinase enzyme
by all studied compounds was recorded in the range of 20 (compound 2) and 82 % (compound 6)
(Figure 4B). Taking as reference the inhibitor 1, only compound 2 showed a lower inhibition effect
while compounds 4, 7 and 8 had a similar inhibition power. A significant progressive increase of
tyrosinase inhibition was observed by exposing the biosensor to the compounds 3, 5 and 6,
respectively. Electrochemical detection of caffeic acid and inhibitor clearly demonstrate the
absence of the lag time of enzymatic reaction catalyzed by tyrosinase (Figure 4D). In a few
minutes, tyrosinase inhibition reached the steady-state. The diphenolase activity of tyrosinase have
been observed also in spectrophotometric assay where UV-Vis spectra of a solution of caffeic acid
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and inhibitor before and after addition of tyrosinase has been depicted in Figure 4C. After six
minutes a reduction of almost 60% of the curve has been observed.
Taking together, the results obtained with the two different analytical assays suggest that
compound 6 acts as inhibitor of the diphenolase activity of tyrosinase more efficiently than biphenyl
1. The main reason of this increased activity might be due to the high nucleophilic character of
phenolic-OH groups in virtue of the electron-donating effect of groups bonded in para-position.
Generally, the presence of hydroxyl groups in a phenol structure and the ability of substituents to
donate electrons increase the electron density of the aromatic ring through a resonance donating
effect that improves the potency of tyrosinase [17, 24, 25]. Nevertheless, in compound 8, steric
hindrance of methoxyl groups in ortho-position to phenolic-OH group would negatively influence
the interaction with the enzyme and, as results, a decrease of tyrosinase inhibition was evidenced
specially by spectrophotometric assay.
Significantly higher inhibitory activity was observed for biphenyl 4 with spectrophotometric
assay respect to biosensor detection, this seems due to the low lipophilicity of the compound
(LogP 2.15), more soluble in aqueous solution. Analogously to biphenyl 4, the difference in
inhibition activity of compounds 3, 5 and 7 observed in the two analytical assays might be due to
the environment of the experiment that could influence, albeit limited, tyrosinase activity as
documented in literature [26]. Infact, in spectrophotometric assay, tyrosinase forms an aqueous
solution whereas in biosensor probe, tyrosinase has been adsorbed in two lipophilic layers.
Compounds 2 showed a low efficiency of about 10% in both the experimental assays,
suggesting that the possible hydrogen bond between the two phenolic-OH groups would strongly
interfere with tyrosinase interaction. Surprisingly, compounds 6 possesses a high capability to
inhibit the oxidation of caffeic acid to o-quinone when detected by tyrosinase biosensor, while not
so marked activity was observed in spectrophotometric tyrosinase oxidation assay although it is
significantly different from 1. Compared to the other studied biphenyls, compound 6 possesses two
benzodioxole moieties that remind sesamol structure. It is acknowledged that sesamol is an
effective inhibitor that interact with tyrosinase by a mechanism influenced by the hindered position
of the benzodioxole ring [20]. Probably, the biphenyl structure enhances the conformational rigidity
of the benzodioxole moiety evidencing different response in the two analytical assays.
Recently, it has been observed that biphenyl 4 inhibited amyloid-β (Aβ) aggregation by 50%
in Congo red assays [27]. Although biphenyl 4 lacks in structural features generally required for an
efficient Aβ inhibitor [28], compound 4 appears to be a promising building block to develop a new
series of inhibitors of pathogenic Aβ peptide involved in neurodegenerative diseases. Taking in
account that amyloid formation has a critical role in melanin formation in humans [29] and different
form of metallo-Aβ peptide can damage neurotransmitters via reactive oxygen species (ROS) [30],
investigation of tyrosinase inhibitors having a biphenilic structure would be a useful start point to
identify efficient pathogenic Aβ peptide inhibitors. Our interest in this field is high [12] and our work

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on tyrosinase inhibitors would like to give insights also into targeting agents for neurodegenerative
disorders.
3.
Conclusions
A small collection of hydroxylated biphenyls was prepared by straightforward methods and
the inhibitory activity toward diphenolase tyrosinase oxidation was detected by spectrophotometry
and amperometric biosensor using caffeic acid as enzyme substrate. Results of both the methods
were comparable. Most of the compounds possessed higher inhibitory activity compared to
compound 1, a known hydroxylated biphenyl inhibitor of tyrosinase. Substituents in the aromatic
ring as well as steric hindrance of functional groups close to the phenolic-OH group influenced
conformational features of the biphenyl structure that affect tyrosinase interaction. Our results
suggest that hydroxylated biphenyls are suitable building block to develop effective tyrosinase
inhibitors, further studies are currently in progress.
4.
Experimental
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4.1.
Material and general remarks
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Compounds 1-2 and all starting materials were purchased by Sigma-Aldrich (Milan, Italy)
and used without purification. NMR spectra of compound 7 were recorded on Varian Mercury Plus
spectrometer at rt in acetone-d . Chemical shifts are given in ppm (δ) and coupling costants in
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Herz; multiplicities are indicated by s (singlet), bs (broad singlet), d (doublet). Elemental analyses
were performed using an elemental analyser Perkin-Elmer model 240 C.
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4.2. Synthesis of 5,5’,6,6’-tetramethoxy-[1,1’-biphenyl]-3,3´-diol 7.
To a solution of 5,5',6,6'-tetramethoxybiphenyl-3,3'-dicarbaldehyde (veratraldehyde dimer)
9 [13] (2 g, 6.11 mmol) in dry dichloromethane (100 mL) was added m-chloroperbenzoic acid 77%
(MCPBA) (2.98 g 13.32 mmol). The mixture was stirred at rt for 12h under N . The reaction mixture
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was quenched with a saturated sodium persulfate solution (10 mL) to remove mCPBA excess,
washed with brine (10 mL) and extracted with dichoromethane (3 x 30 mL). The organic phase was
dried over Na SO , filtered, and concentrated. The residue was dissolved in MeOH (30 mL), and
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then treated with 6N NaOH aqueous solution (62 mL). The mixture was stirred at room
temperature for 15 min. After removal of MeOH, the residue was dissolved in ethyl acetate (30
mL), washed with saturated NaHCO solution (20 mL) and brine (20 mL), and dried over Na SO .
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The organic phase was filtered and concentrated, to provide compound 7 as a white solid (1.12 g,
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60%): mp 212-213°C: H NMR (acetone-d ) δ 3.61 (s, 6H), 3.94 (s, 6H), 6.23 (d, J = 2.8 Hz, Ar,
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2H), 6.45 (d, J = 2.8 Hz, Ar, 2H), 8.21 (bs, 2 OH); C-NMR (acetone-d ) δ 55.05, 59.66, 99.90,
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108.44, 133.35, 139.92, 152.87, 153.45; Anal. Calcd for C H O : C, 62;74 H, 5.92; Found: C,
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62.77; H, 5.95.

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4.3. Spectrophotometric enzyme activity assay.
The capability of compounds 1-8 to inhibit oxidation of caffeic acid by tyrosinase/O2 oxidizing
system was monitored by means of a UV-VIS spectroscopy method, carried out in a Shimadzu UV-
Visible UV-2501 spectrophotometer using a Helma (Mülheim, Germany) duple chambers UV-cell
(2×4.375 mm). Briefly, 980 µL of a caffeic acid solution (180 µM) and 1000 µL of a mushroom
tyrosinase solution (166 U/mL, Sigma-Aldrich) both dissolved in 50 mM phosphate buffer (pH 6.8)
were separately introduced in the separated UV-cell chambers. 20 µL of an appropriate DMSO
stock solution of inhibitors (166 µM) were added to the chamber containing caffeic acid solution.
The tyrosinase activity in absence of inhibitors was detected adding 20 µL of DMSO. Ground UV-
Vis spectra were recorded in the 250-600 nm wavelength range. Thereafter, the solutions were
mixed and the spectra were registered again at fixed times (from 1 to 25 min). The inhibition
activity was determined as percentage of the caffeic acid consumption at 6 min.
The efficiency of compounds 1-8 to inhibit the diphenolase activity of tyrosinase was determined
analyzing the absorbance values at 311 nm using equation (1)[22, 31]:
% inhibition = (1-((D-C)/(B-A))*100
(1)
where C and D are the absorbance values at 311 nm in presence of inhibitor before the mixing and
after 6 min, respectively, while A and B are the corresponding values at 311 nm in absence of
inhibitor. The measurement was performed in triplicate and averaged before calculation.
4.4. Biosensor construction and immobilized tyrosinase inhibition assay.
Tyrosinase biosensors [32] were made using silver wires insulated with Teflon™ modifying
procedures previously described [33, 34]. Approximately 3 mm of the wire were exposed and
inserted into a glass capillary tube (20 mm in length; I.D. Ø = 1 mm) partly filled with graphite
loaded with epoxy resin (55% w/w) (Araldite-M®, Sigma-Aldrich, Milan, Italy) obtained by mixing
850 mg of graphite with 500 mg of Araldite-M and 200 mg of hardener. After 24 h at 40 °C, the
surface of the transducers where smoothed by using a high speed drill (Dremel® 300) equipped
with an aluminum oxide grinding wheel. Finally, the carbon transducer was immersed five times
into a solution of tyrosinase enzyme and let it dry for 5 min after each dip. A final net that
entrapped the enzyme was deposited on the top of enzyme layers by quick dipping of the
biosensors in a polyurethane solution (0.25%) as previously described [35].
Constant potential amperometry (CPA) was used for caffeic acid calibrations and in-vitro inhibition
experiments by applying a potential of -100 mV against an Ag/AgCl reference electrode (See
caffeic acid cyclic voltammetry in the Supplementary Data). Calibrations and experiments were
performed at room temperature and in a 10 ml electrochemical cell filled with fresh PBS (pH 7.40)
24 h after sensors’ fabrication. The percent inhibition of diphenolase activity of tyrosinase by
compounds 1 - 8 was assessed by recording the baseline-subtracted signal (Delta-I) of 500 µM of

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caffeic acid (100%) and the stabilized current after the introduction of the inhibitor (50 µM,
dissolved in 100 µL of DMSO) in the electrochemical cell. The measurement was performed in
triplicate and averaged before calculation. A control experiment was performed for each biosensor
by adding the same amount of DMSO alone.
Acknowledgements: Authors gratefully thank Sardinia Autonomous Region for providing financial
support, L.R. 7, 2007.
Supplementary data
Supplementary data associated with this article can be found, in the online version, at http://
These data include synthesis and characterization of the compounds described in this article and
supplementary electrochemical data.
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Caption of Figures
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Figure 1. (A) Schematic representation of the reactions catalyzed by tyrosinase. (B) Caffeic acid is
oxidized to the correspondinq quinone by the enzyme and reduced-back at carbon surface by
applying a reducing potential of -100 mV vs Ag/AgCl reference electrode. The first reaction,
inhibited by the compounds synthetized in this study, resulted in a significant reduction of the
cathodic current.
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Figure 2. (A) Hydroxylated biphenyl structures. (B) Preparation scheme of biphenyl 7.

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Figure 3. UV-Vis spectra of caffeic acid (88.2 µM) in 50 mM phosphate buffer, pH 6.8, before and
after addition of tyrosinase (83 U/mL). In the insert the time-course of caffeic acid oxidation
detected at three different wavelength.
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Figure 4. (A) Percentage of tyrosinase inhibition by caffeic acid oxidation with a unimmobilized
tyrosinase enzyme determined by UV-VIS spectroscopy. *, ** = p<0.05 and <0.01 respectively vs
compound 1.
(B) Percentage of tyrosinase inhibition by caffeic acid oxidation with a tyrosinase enzyme
immobilized on the surface of an amperometric biosensor monitored at a potential of -100 mV vs
Ag/AgCl reference electrode. *, **, *** = p<0.05, <0.01, <0.001 respectively vs compound 1.
(C) Spectrophotometric curve of tyrosinase activity decay in the presence of inhibitor 4.
(D) Amperometric plotting of tyrosinase activity decay in the presence of inhibitor 6; arrow a:
addition of caffeic acid; arrow b: biphenyl 6 inhibition.

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Highlights
•
•
•
A small collection of hydroxylated biphenyls was synthesized by straigtforward methods.
Significant tyrosinase inhibitor activity was detected in most of the compounds.
Selective diphenolase activity of tyrosinase enzyme have been detected by both
spectrophotometric and electrochemical assays.
•
Results of both the analytical methods were comparable.