Mechanistic study on the enzymatic oxidation of flavonols

Article abstract of DOI:10.1016/j.tetlet.2007.11.147

Several flavonols have been transformed upon treatment by Trametes versicolor laccase. Most of the major oxidation products have been isolated by HPLC as pure compounds and their structures have been, when possible, investigated through spectral methods (HPLC-MS and NMR). The results are coherent with the predominance of a dismutation process, leading to cation formation, over direct radical-radical coupling.

Full text of DOI:10.1016/j.tetlet.2007.11.147

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Tetrahedron Letters 49 (2008) 619–623
Mechanistic study on the enzymatic oxidation of flavonols
Souhila Ghidouche a, Nour-Eddine Es-Safi a,b, Paul-Henri Ducrot a,*
a Unite´ de Chimie Biologique, UMR206 INRA-INAPG, INRA Route de Saint-Cyr, 78026 VERSAILLES Cedex, France
b
´
Laboratoire de Chimie Organique et d’Etudes Physico-chimiques, Ecole Normale Supe´rieure, B.P. 5118, Takaddoum Rabat, Morocco
´
Received 3 October 2007; revised 13 November 2007; accepted 23 November 2007
Abstract
Several flavonols have been transformed upon treatment by Trametes versicolor laccase. Most of the major oxidation products have
been isolated by HPLC as pure compounds and their structures have been, when possible, investigated through spectral methods
(HPLC–MS and NMR). The results are coherent with the predominance of a dismutation process, leading to cation formation, over
direct radical–radical coupling.
Ó 2007 Elsevier Ltd. All rights reserved.
Keywords: Polyphenols; Flavonoid; Enzymatic oxidation; Dismutation
The enzymatic oxidation of polyphenols1,2 is highly
implicated in numerous biological mechanisms in vegetal.
As the radicals, formed through these oxidation processes,
are relatively stable, they can undergo several coupling
reactions, reoxidation or dismutation reactions. The cou-
pling reactions of phenolic radicals are of major interest
in lignin, tanins and melanin biosynthesis.
Out of flavonoids, flavonols (yellow pigments with
chemical structure shown in Figure 1 and characterized
by a fully conjugated flavan backbone, through a 3-
hydroxy-pyranone C ring), often present in glycosidic
forms, are one of the main sub-classes.
In a programme dedicated to the study of the total syn-
thesis,3–5 and the chemical6–8 or enzymatic oxidation of
polyphenols, we focused our interest, in this latter field,
on the mechanistic features arising from the oxidation of
flavonols by Trametes versicolor laccase. Indeed, the main
transformations undergone by polyphenols in fruits and
plants during food processing are caused by oxidative reac-
tions catalyzed by phenol oxidases, such as laccases,9,10 cat-
echol oxidases, and tyrosinases, and by peroxidases.
Oxidation proceeds in the presence of molecular oxygen
for most polyphenol oxidases and with hydrogen peroxide
for peroxidases.11,12 Autoxidation and chemical oxidation
processes may also occur.13
An important specificity of our study, compared to
those already published, is also the use of a laccase as enzy-
matic oxidant. Indeed, other studies most often reported
the oxidation of flavonoids either with chemical radical ini-
tiators like AIBN and DPPH or enzymatic systems like
grape PPO (grape polyphenol oxidase; in this case, the
flavonoids are not the primary substrate of the enzyme,
the oxidation being mediated by the o-quinone from caf-
feoyl quinic acid, which is the true substrate of the
enzyme). Therefore, in these cases, oxidation of the flavo-
nols may occur through either a mono- or a di-electronic
transfer. The possibility of a di-electronic transfer may
indeed favour the oxidation of the B ring of flavonols
through the formation of quinones, reactivity of which
would thereafter favoured the formation of dimeric com-
pounds through nucleophilic addition reactions, due to
the high reactivity of aromatic rings derived from phloro-
glucinol. In the case of laccases, albeit the complete mech-
anism of the reaction, and mainly the pathway leading to
oxygen reduction, is not clearly determined,14 the mono-
electronic transfer is the privileged mechanism. Use of a
*
Corresponding author. Tel.: +33 1 308 833 641; fax: +33 1 308 833
119.
E-mail address: ducrot@versailles.inra.fr (P.-H. Ducrot).
0040-4039/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2007.11.147

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S. Ghidouche et al. / Tetrahedron Letters 49 (2008) 619–623
quercetin that this enzyme gave rise to the same oxidation
profiles than another laccase obtained from A. thaliana
seeds.18
To allow chemical mechanism determination for this
type of reaction, we decided to test our enzymatic system
on several commercially available flavonoids with various
substitution patterns either on their B or C rings. The
choice of the starting materials 1–6 (Fig. 1) was made on
account of several results published in the literature on
the structure–antioxidant activity relationships of flavo-
noids. It has indeed been clearly established that the B-ring
is the most important site for H-transfer and consequently
antioxidant capacity. In contrast, the A-ring seems to be
less important. On the other hand, the D2,3 double bond
should also contribute to the antioxidant activity, as it
ensures p-electron delocalization between the B- and C-
rings, which contributes to the stabilization of the ROÅ rad-
icals formed in the oxidation process after H-abstraction.19
Moreover, an important issue that is still under debate is
the role of the 3-OH group. In vitro studies demonstrated
that this hydroxy group contributes to the antioxidant
potential. Indeed, blocking the 3-OH group as in rutin or
removing it as in luteolin significantly decreases the
in vitro activity.20
Our starting compounds were therefore two flavones
with no hydroxy groups at C3, namely chrysine 1 and
luteolin 2, three flavonols with an hydroxy group at C3
exhibiting various hydroxylation patterns on their B ring,
namely galangin 3, kaempferol 4 and quercetin 5, and,
finally, quercetin 3-O-glucoside 6.
The first feature arising from our study21 was the non-
oxidation of both 1 and 2, thereby confirming the crucial
role of the 3-OH group in the oxidation process. Moreover,
this first result also showed that the presence of an highly
reactive catechol type B-ring is not sufficient to allow a
one-electron oxidative process to occur, also reflecting the
postulated important role (DFT study)22,23 played by the
hydroxy group at C3 in the first steps of oxidation of
flavonols.
Turning thereafter to the enzymatic oxidation of flavo-
nols 3–5, we were not surprised to observe that these com-
pounds, although not exhibiting the same hydroxylation
pattern on the B-ring, presented similar behaviours under
our oxidation conditions (although depsides24,25 are often
described as major compounds of flavonols oxidation,
resulting from a rapid degradation of the first formed oxi-
dation products, we were not able to detect them in our
study). Indeed, the reaction mixture was reflecting in all
cases the presence of only few compounds, MS analysis
of which showed that the major ones were, for compounds
4 and 5, isomeric products corresponding to the addition of
one oxygen atom (M+16) at C-2 with possible rearrange-
ment of the C-ring through trans acetalization of the highly
reactive tri-keto system and hydrate formation at C-3
(compounds 7–9, 11 and 12). Depending on the solvent
used for the reaction, the other products of the reactions
were, in these two cases, thereafter shown to be either
Fig. 1.
laccase as oxidizing enzyme would therefore allow us to
investigate the stability and the reactivity of the various
ROÅ potentially formed as primary products of the oxida-
tion. Moreover, laccases are highly implicated in enzymatic
evolution of flavonoids in Arabidopsis thaliana seeds.
Assuming that the course of the laccase oxidation of poly-
phenols should be more influenced by the chemical struc-
ture of the starting polyphenol than by the nature of the
enzyme used, we turned to the use of T. versicolor laccase,
a reliable, easily available fungus enzyme with a low sub-
strate specificity,15,16 to study flavonol oxidations. We
indeed preliminarily demonstrated17 with kaempferol and

S. Ghidouche et al. / Tetrahedron Letters 49 (2008) 619–623
621
dimeric compounds under aprotic conditions (acetonitrile,
compounds 14 and 1526) or products resulting from solvent
addition in protic media (MeOH or EtOH, 10 and 13). In
this case, turning from methanol to ethanol was the key
step allowing to unambiguously confirming this solvent
addition. Later on, NMR analysis confirmed the structure
of compounds 7–10 and 11–13 as oxidation products of
kaempferol and quercetin, respectively, structure of which
have already been more or less described in the
literature.24,25,27,28
As an example, 13a NMR spectra were used to deter-
mine its structure as follows. 1H spectrum of 13a exhibited
the same resonance pattern for the aromatic protons than
quercetin but also showed a supplementary resonance at
3.05 ppm, integrating for three protons in accordance with
the presence of a methyl group attached to an oxygen
atom. 13C spectrum was also quite similar to quercetin’s
with two quaternary sp2 carbon resonances less, which
were replaced by two resonances at 91.7 and 100.9 ppm.
HSQC and HMBC experiments were thereafter performed
and allowed the complete assignment of all carbon reso-
nances. Indeed, HMBC correlation (Fig. 2) were observed
for the resonance at 100.9 ppm with protons of the methyl
group as well as with protons H20 and H60. At the same
time no HMBC correlations were observed for carbon res-
onance at 91.7, which thereby was attributed to C3 when
the resonance at 100.9 ppm was assigned to C2, confirming
the introduction of a methoxy group at C2.
The same reaction, performed with galangin 3, however
yielded under all the conditions tested (solvent and concen-
tration changes) the exclusive formation of compound 16,
MS spectrum of which revealed its dimeric structure (m/z
556 [2Mꢀ2H+H2O]). This result clearly shows that
hydroxylation of ring B is not necessary for the oxidation
process to take place and thereby strengthens the hypothe-
sis of a kinetic oxidation of flavonols through the 3-OH
group. Moreover, obtention of 16 as the unique oxidation
product and the absence of adducts with solvent molecule
(MeOH or EtOH) clearly shows that the radical formed
at C-2 is not reactive enough to be directly quenched by
an ROH molecule (solvent or substrate molecule) but
should be first re-oxidized through the dismutation of
two radicals (Fig. 3), leading the corresponding cation,
which is thereafter trapped by a protic species; the proxim-
ity of the two radicals involved in the dismutation reaction,
favoured by p-staking interactions, led to the formation of
16 rather than quenching by a solvent molecule.
The structure of the dimeric compounds obtained
through oxidation of flavonols 4 and 5 in acetonitrile was
however more difficult to establish because of their low
abundance in the reaction mixture which did not allow us
to get them as pure compounds for NMR spectroscopy.
Kaempferol oxidation led to the formation of two dimeric
compounds (m/z 604 [2Mꢀ2H+O+H2O] and m/z 586
[2Mꢀ2H+O]) and quercetin allowed the formation of
two pseudo dimeric compounds at m/z 498 and 618
Fig. 2. 1H–13C long range correlations observed in 13a.
Fig. 3. Proposed mechanism for flavonols oxidation.

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S. Ghidouche et al. / Tetrahedron Letters 49 (2008) 619–623
[2Mꢀ2H+O]. Careful examination of the MS spectra of
compounds at m/z 604 (14) and 618 (15), obtained from
4 and 5, respectively, however reflected structural differ-
ences between these two compounds (we were not able to
isolate dimeric compound at m/z 586). Indeed, when the
spectrum of 15 seemed to be compatible with the formation
of either a carbon–carbon or a carbon–oxygen single bond
between the two monomeric subunits (and re-oxidation of
one of the subunit), formation of 14 is clearly only compat-
ible with the formation of an ether linkage between two
subunits. This assumption is supported by the observation
in the MS spectrum of 14 of a major fragment at m/z 302
also observed in compounds 10a,b, as a result of the easy
cleavage of the OR bond after the loss of an H2O molecule.
On the other hand, MS spectrum of 15 mainly revealed
classical fragmentations of the flavan skeleton.
Moreover, to allow incorporation of a H2O molecule
and a further oxygen atom, according to the molecular
weight of 14, the ‘upper’ subunit needs to be further oxi-
dized; the most satisfactory structure hypothesis for 14 is
therefore shown in Figure 1, involving the OH at C-40 in
the ether linkage between both subunits rather than the
OH at C-3 as in 16.
oxidation of phenolic species are of great importance and
will help in a better understanding of the biosynthesis of
phenolic oligomers and polymers in plants.
Acknowledgment
The authors thank L. Kerhoas (Inra) for LC–MS
measurements.
References and notes
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mutation process, due, in the case of 4, to the presence of
the hydroxy group at C-40, between p-stacking, dipole–
dipole and steric interactions leading a different spatial
arrangement of both subunits in the transition state than
that observed with 3. The hypothetical structure of 14,
however supports the hypothesis of a dismutation process
rather than radical–radical coupling, since we did not have
observed, in any cases, products resulting from a possible
phenoxy radical formation.
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A last interesting result is this obtained in the oxidation
of quercetin 3-O-glucoside 6. Indeed, even if the oxidation
seemed to proceed very slowly, this compound, albeit
exhibiting a protected hydroxy group at C-3, was partially
oxidized and the major oxidation product (m/z 908) did not
resulted from a loss of the glycosidic moiety. The fact that
under the same conditions, luteolin 2 remained untouched
may indicates that electro donating abilities of the oxygen
atom at C-3 is sufficient to promote oxidation at C-2.
In conclusion, we have demonstrated the enzymatic oxi-
dation of flavonols to be first promoted by the formation of
the radical on the oxygen at C-3 through H abstraction
even in the presence of phenolic hydroxy groups on the B
ring. We have also demonstrated that most of the sub-
sequent reactions either with solvent (water or alcohols)
or with another substrate molecule were initiated by a
dismutation reaction of two radicals, leading to the forma-
tion of a carbocation at C-2 that was thereafter quenched
by a protic species. Therefore, intermolecular interactions
are crucial factors for the reaction issue: increasing
unfavourable interactions would allow radical isomeriza-
tion and the formation of dimers with a diphenyl ether
link. These evidences for carbocation formation during
17. Ghidouche, S.; Ducrot, P.-H. unpublished results: incubating com-
pounds 1–6 in the presence of Arabidopsis thaliana seeds from a
mutant strain in which biosynthesis of flavonoids was silenced18
afforded qualitatively the same pattern of oxidation products as
described in this study. On the other hand, the use of Arabidopsis
thaliana seeds from mutant strains in which no laccase activity is
detected gave no oxidation products, allowing us to assume the
importance of the laccases in the oxidation of flavonoids.
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21. Typical procedure: A flavonol solution in methanol, ethanol or
acetonitrile (10 ml, 10 mg/ml) and 60 ml citrate/phosphate buffer
0.1 M was incubated with 30 ml of laccase (purchased from Fluka;
23.3 U/mg) solution (2 U/ml) at pH 4 and 30 °C for 2 h with stirring,
to allow oxygen dissolution in the reaction medium. Control reactions
were also made in the absence of enzyme to ensure the products
arising from enzymatic oxidations not being artefacts resulting from
autooxidation processes. The suspension was centrifuged at 6000 rpm
for 10 min; the supernatant was then filtered under vacuum through
an ultra filtration membrane, £ 44.5 mm (Millipore). The filtrate was
concentrated by evaporation. The oxidation products were separated

S. Ghidouche et al. / Tetrahedron Letters 49 (2008) 619–623
623
on a semi-preparative HPLC system including a Waters 600 pump, a
manual U6K injector, a Uvicord S II UV detector (Pharmacia LKB)
set at 254 nm, and a REC 102 recorder (Pharmacia Biotech). The
column was a C18 Kromasil 250 ꢁ 20 mm (10 lm packing). Elution
conditions: flow rate 18 ml/min, solvent A 0.5% acetic acid in H2O;
solvent B 0.5% in acetonitrile. Linear gradient from 15% to 50% B in
25 min followed by washing and reconditioning of the column. No
yields are given for these transformations, since we focused on
isolation of pure compounds out of a complex mixture. Products are
dissolved in DMSO-d6 for NMR spectroscopy.
23. Trouillas, P.; Marsal, P.; Siri, D.; Lazzaroni, R.; Duroux, J.-L. Food
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structure for compound 15 (see discussion in the text).
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1570–1581. and references cited therein.
`
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