# Biomimetic Modeling of Copper Complexes: A Study of Enantioselective Catalytic Oxidation onD-(+)-Catechin and L-(−)-Epicatechin with Copper Complexes

Authors: Francesco G. Mutti; Roberta Pievo; Maila Sgobba; Michele Gullotti; Laura Santagostini
Journal: Bioinorganic Chemistry and Applications
 (2008)

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## Abstract

The biomimetic catalytic oxidations of the dinuclear and trinuclear copper(II) complexes versus two catechols, namely,D-(+)-catechin
and L-(−)-epicatechin to give the corresponding quinones are reported. The unstable quinones were trapped by the nucleophilic reagent, 3-methyl-2-benzothiazolinone hydrazone (MBTH), and have been calculated the molar
absorptivities of the different quinones. The catalytic efficiency is moderate, as inferred by kinetic constants, but the complexes exhibit significant enantio-differentiating ability towards the catechols, albeit for the dinuclear complexes, this enantio-differentiating ability is lower. In all cases, the preferred enantiomeric substrate is D-(+)-catechin to respect the other catechol, because of the spatial disposition of this substrate.


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## Body

## 1. Introduction

Reproducing complex biological  reactivity
within a simple synthetic molecule is a challenging endeavor with both
intellectual and aesthetic goals. The sequence of examining biological
reactivity, creating similar chemical architectures, and determining functional
reaction conditions for model systems is a process that allows the biological
code of reactivity to be deciphered. In the past years, the report on the
crystal structures of type 3 copper enzymes (e.g., catechol oxidase, hemocyanins,
and tyrosinase) [1–4], as too type 2-type 3 copper
enzymes (e.g., ascorbate oxidase, laccase, ceruloplasmin) [5–8] has taken a new
turn. The greater availability of such structural information now allows a
shift in the role of synthetic modeling from structural and spectroscopic
endeavors to the development of functional and catalytic models. Functional
models can provide an opportunity to examine a biological reactivity at a
small-molecule level of detail through systematic and comparative studies.
Although one goal of modeling is reproduction of reactivity, extension of this
reactivity beyond the scope of the inspiring system is perhaps an even more
important objective. Adequate synthetic models that have similar structural,
spectroscopic, and functional properties of active sites of copper proteins are
done [9–13]. These models provide many elegant examples of selective and
environmentally benign oxidants capable of performing interesting organic
transformations, and many of these are copper complexes that use dioxygen as
the ultimate oxidant above all in the catecholase activity [14–20]. The
interest of our group has mainly focused on dinuclear, and trinuclear [21–28]
copper complexes derived from octadentate nitrogen ligands which show catecholase
activities. Some of these compounds contain chiral centers [24–28], and we have
demonstrated the possibility to induce stereoselectivity in the catalytic
oxidation of chiral catechols, such as L- and D-Dopa and their methyl
esters. In the present paper we have extended this investigation on our chiral
complexes using as substrates other potential catechols, namely, D-(+)-catechin and L-(–)-epicatechin,
and we have found that the stereoselective catalytic oxidation of these
substrates depends on the chirality of the dinuclear or trinuclear copper
compounds, and on the spatial disposition of the catechols.


## 2. Experimental

### 2.1. General Remarks

The dinuclear and
trinuclear copper(II) complexes[Cu2-R-DABN-4Bz4][ClO4]4⋅2H2O(1); [Cu3-R-DABN-4Bz4][ClO4]6⋅2H2O(2); [Cu2-L-Lys-4Bz4]⋅[ClO4]4⋅6H2O(3); [Cu3-L-Lys-4Bz4][ClO4]6⋅6H2O(4); [Cu2-R-DABN-3Im4][ClO4]4⋅6H2O(5); [Cu3-R-DABN-3Im4][ClO4]6⋅2H2O(6) were prepared as described previously [27].
The trichiral complexes [Cu2-R-DABN-L-Ala-Bz4][ClO4]4(7) and [Cu3-R-DABN-L-Ala-Bz4][ClO4]6(8) were prepared with  standard procedures [29]. The
dinuclear complex [Cu2L-66][ClO4]4⋅6H2O(9) was prepared as described previously [30]. All
the compounds are shown in Figure 1.Figure 1
Dinuclear and
trinuclear copper complexes used in the biomimetic catalytic oxidations of
catechins.


### 2.2. Caution

Although
no problems were encountered during the preparation of perchlorate salts,
suitable care should be taken when handling such potentially hazardous
compounds.


### 2.3. Materials
and Physical Methods

Commercial
starting materials were used without purification and the solvents used for the
reactions were all spectrophotometric grade. Acetonitrile was distilled from
potassium permanganate and sodium carbonate; it was then stored over calcium
hydride and distilled before use under nitrogen. The pH of the solutions was
measured with an Amel instrument 338. Optical spectra were obtained with HP
8453 diode array spectrophotometer equipped with a thermostated cell holder at
the temperature of20±0.1°C. The data were treated with the commercial
program  FigSys (BioSoft, Cambridge,
UK). Formation kinetics was carried out under
pseudo-first-order conditions at 20±0.1°C.


### 2.4. Determination of Molar Absorptivities of the Quinones

It is well known  that
dinuclear and trinuclear model complexes, like  tyrosinase, oxidizeso-diphenols, triphenols, and flavonoids
to quinones, but, in all cases, the resulting quinones may undergo nonenzymatic
autopolymerization to produce colored compounds. To prevent further reactions
of  the quinones initially formed, a nucleophilic reagent,
3-methyl-2-benzothiazolinone hydrazone (MBTH), that traps the quinones and
generates chromophoric adducts, was used. Unfortunately, no molar absorptivities
of  these adducts for the substrates were available, so a spectrophotometric
method to determine the λmax and the molar absorptivities of  the
adducts was performed. In general, the method is based on the oxidation of the
substrate by an excess of sodium periodate, condition under which the reaction
was very fast [31, 32]. The unstable quinones were trapped by the nucleophilic
reagent (MBTH), and related λmax was detected. In all the
experiments, only one band developed in the range 300–900 nm, which
corresponds to the adducts with MBTH. Based on the recording of λmax,
an experimental design can be carried out to determine the molar absorptivities
of the different quinones, for example, performing spectra with different
substrate concentrations and fitting the data so obtained to a Lambert-Beer
equation by linear regression. Plots of the absorbance values obtained versus the different substrate
concentrations allow calculating
molar absorptivities. For D-(+)-catechin (CQ), the experimental
conditions were λmax=459nm; 50 mM phosphate buffer (pH 7.0)/MeOH (9:1, v:v)
at 20±0.1°C; 2 mM NaIO4;
1 mM MBTH; substrate concentrations (CQ) from
5 μM to 40 μM; quartz cell 1 cm path length; final volume
in cell 2 mL. The coefficient of determination (r2) was 0.998 and the molar absorptivity was 17230M−1cm−1. For
L-(−)-epicatechin (EQ), the experimental
conditions were λmax=463nm; 50 mM phosphate buffer (pH 7.0)/MeOH (9:1, v:v)
at 20±0.1°C; 2 mM NaIO4; 1 mM MBTH; substrate
concentrations (EQ) from 5 μM to 45 μM; quartz cell 1 cm path length; final volume
in cell 2 mL. The coefficient of determination (r2) was 0.994 and the molar absorptivity was 18950M−1cm−1.


### 2.5. Catecholase Activities

The kinetics of catalytic oxidation ofD-(+)-catechin and L-(–)-epicatechin
were studied by UV-Vis spectroscopy using a magnetically stirred and
thermostated 1-cm path length cell. The temperature during the measurements was
kept constant at 20±0.1°C.
A mixture of aqueous phosphate buffer (50 mM, pH 7.0)-methanol 9:1 (v:v) saturated
with atmospheric oxygen was used as solvent. All the kinetic experiments were
carried out in duplicate. The experiments performed over a substrate
concentration range were initiated by adding a few microlitres of the complexes
(final concentrations 0.2–1.4×10−5M) to the solution of the substrates; MBTH was
maintained 1.0×10−3M; the concentration of the substrate was varied
between 4.0×10−6 and 8.0×10−4M (final volume 2 mL). The formation of the
stable D-(+)-catechin-o-quinone-MBTH and
L-(–)-epicatechin-o-quinone-MBTH adducts was followed
through the development of the strong absorption band at 459 nm (ε=17230M−1cm−1)   for D-(+)-catechin-o-quinone-MBTH, and an absorption band
at 463 nm (ε=18950M−1cm−1) for L-(–)-epicatechin-o-quinone-MBTH, respectively. In all the
experiments, the noise was reduced by reading the absorbance difference between λmax and 1100 nm, where the absorption remains
negligible during the assay. The initial rates of oxidations were obtained by fitting the
absorbance versus time curves in the first seconds of the reactions.


## 2.1. General Remarks

The dinuclear and
trinuclear copper(II) complexes[Cu2-R-DABN-4Bz4][ClO4]4⋅2H2O(1); [Cu3-R-DABN-4Bz4][ClO4]6⋅2H2O(2); [Cu2-L-Lys-4Bz4]⋅[ClO4]4⋅6H2O(3); [Cu3-L-Lys-4Bz4][ClO4]6⋅6H2O(4); [Cu2-R-DABN-3Im4][ClO4]4⋅6H2O(5); [Cu3-R-DABN-3Im4][ClO4]6⋅2H2O(6) were prepared as described previously [27].
The trichiral complexes [Cu2-R-DABN-L-Ala-Bz4][ClO4]4(7) and [Cu3-R-DABN-L-Ala-Bz4][ClO4]6(8) were prepared with  standard procedures [29]. The
dinuclear complex [Cu2L-66][ClO4]4⋅6H2O(9) was prepared as described previously [30]. All
the compounds are shown in Figure 1.Figure 1
Dinuclear and
trinuclear copper complexes used in the biomimetic catalytic oxidations of
catechins.


## 2.2. Caution

Although
no problems were encountered during the preparation of perchlorate salts,
suitable care should be taken when handling such potentially hazardous
compounds.


## 2.3. Materials
and Physical Methods

Commercial
starting materials were used without purification and the solvents used for the
reactions were all spectrophotometric grade. Acetonitrile was distilled from
potassium permanganate and sodium carbonate; it was then stored over calcium
hydride and distilled before use under nitrogen. The pH of the solutions was
measured with an Amel instrument 338. Optical spectra were obtained with HP
8453 diode array spectrophotometer equipped with a thermostated cell holder at
the temperature of20±0.1°C. The data were treated with the commercial
program  FigSys (BioSoft, Cambridge,
UK). Formation kinetics was carried out under
pseudo-first-order conditions at 20±0.1°C.


## 2.4. Determination of Molar Absorptivities of the Quinones

It is well known  that
dinuclear and trinuclear model complexes, like  tyrosinase, oxidizeso-diphenols, triphenols, and flavonoids
to quinones, but, in all cases, the resulting quinones may undergo nonenzymatic
autopolymerization to produce colored compounds. To prevent further reactions
of  the quinones initially formed, a nucleophilic reagent,
3-methyl-2-benzothiazolinone hydrazone (MBTH), that traps the quinones and
generates chromophoric adducts, was used. Unfortunately, no molar absorptivities
of  these adducts for the substrates were available, so a spectrophotometric
method to determine the λmax and the molar absorptivities of  the
adducts was performed. In general, the method is based on the oxidation of the
substrate by an excess of sodium periodate, condition under which the reaction
was very fast [31, 32]. The unstable quinones were trapped by the nucleophilic
reagent (MBTH), and related λmax was detected. In all the
experiments, only one band developed in the range 300–900 nm, which
corresponds to the adducts with MBTH. Based on the recording of λmax,
an experimental design can be carried out to determine the molar absorptivities
of the different quinones, for example, performing spectra with different
substrate concentrations and fitting the data so obtained to a Lambert-Beer
equation by linear regression. Plots of the absorbance values obtained versus the different substrate
concentrations allow calculating
molar absorptivities. For D-(+)-catechin (CQ), the experimental
conditions were λmax=459nm; 50 mM phosphate buffer (pH 7.0)/MeOH (9:1, v:v)
at 20±0.1°C; 2 mM NaIO4;
1 mM MBTH; substrate concentrations (CQ) from
5 μM to 40 μM; quartz cell 1 cm path length; final volume
in cell 2 mL. The coefficient of determination (r2) was 0.998 and the molar absorptivity was 17230M−1cm−1. For
L-(−)-epicatechin (EQ), the experimental
conditions were λmax=463nm; 50 mM phosphate buffer (pH 7.0)/MeOH (9:1, v:v)
at 20±0.1°C; 2 mM NaIO4; 1 mM MBTH; substrate
concentrations (EQ) from 5 μM to 45 μM; quartz cell 1 cm path length; final volume
in cell 2 mL. The coefficient of determination (r2) was 0.994 and the molar absorptivity was 18950M−1cm−1.


## 2.5. Catecholase Activities

The kinetics of catalytic oxidation ofD-(+)-catechin and L-(–)-epicatechin
were studied by UV-Vis spectroscopy using a magnetically stirred and
thermostated 1-cm path length cell. The temperature during the measurements was
kept constant at 20±0.1°C.
A mixture of aqueous phosphate buffer (50 mM, pH 7.0)-methanol 9:1 (v:v) saturated
with atmospheric oxygen was used as solvent. All the kinetic experiments were
carried out in duplicate. The experiments performed over a substrate
concentration range were initiated by adding a few microlitres of the complexes
(final concentrations 0.2–1.4×10−5M) to the solution of the substrates; MBTH was
maintained 1.0×10−3M; the concentration of the substrate was varied
between 4.0×10−6 and 8.0×10−4M (final volume 2 mL). The formation of the
stable D-(+)-catechin-o-quinone-MBTH and
L-(–)-epicatechin-o-quinone-MBTH adducts was followed
through the development of the strong absorption band at 459 nm (ε=17230M−1cm−1)   for D-(+)-catechin-o-quinone-MBTH, and an absorption band
at 463 nm (ε=18950M−1cm−1) for L-(–)-epicatechin-o-quinone-MBTH, respectively. In all the
experiments, the noise was reduced by reading the absorbance difference between λmax and 1100 nm, where the absorption remains
negligible during the assay. The initial rates of oxidations were obtained by fitting the
absorbance versus time curves in the first seconds of the reactions.


## 3. Results and Discussion

### 3.1. Stereoselective Catalytic Oxidations

The
catalytic oxidations of catechols are the most widely employed test reaction to investigate the
behavior of tyrosinase and catechol oxidase model complexes. Previous studies
[24–27] have shown that chiral dinuclear and trinuclear copper complexes were
able to display stereo-discriminating ability towards optically active
catechols to give the corresponding o-quinones.
To confirm this behavior, new chiral catechols were employed in these catalytic
stereoselective  oxidations. D-(+)-catechin and L-(–)-epicatechin
(flavan-3-ols) (Figure 2) constitute a class of phenolic compounds ubiquitous
in plants and widely found in fruits, vegetables, and beverages [33–35]. In
particular, they are one of the major quality factors in grapes and then in the
resulting wine [36, 37].Absolute stereochemistry configuration of
the catechols.
(a)(b)The
catalytic oxidation of polyphenolic substrates, including catechins, was well
studied by many authors [38–41]. These reactions take place in the presence of
atmospheric oxygen when polyphenol oxidase (PPO) and the corresponding
substrates are mixed at the same time. The fundamental first step is the
transformation of o-diphenols
to the corresponding o-quinones.
The fate and stability of o-quinones
vary widely, depending both on the phenolic precursor and on environmental
factors. In particular, the o-quinones
of D-(+)-catechin and
L-(–)-epicatechin
were seen to be much less stable than those of other o-quinones. The prolonged autoxidation, either chemical or
enzymic, led to the formation of polymers resulting from repeated condensation
reactions between an aromatic ring of one molecule with an aromatic ring of
another (“head to tail” polymerization mechanism). Depending on how phenolic
compounds are oxidized, the condensation products formed from catechins may
differ. In fact, the pH of the solution influences considerably the obtained
products [42, 43], because at low pH values is favored the formation of
colorless condensation products, whereas yellow compounds tended to be formed
at higher pH values. To avoid any effect due to pH-dependence of oxidation products
and to stop the reaction at quinones formation, a nucleophilic reagent MBTH, that traps the
quinones and generates chromophoric adducts, was used (Scheme 1).Scheme 1
Nucleophilic attach of the
reagent MBTH to the catechols.We have then studied the pH
dependence of the reaction rates and have found that the better pH for the
catalytic oxidation of catechins in the presence of the chiral copper complexes
is pH 7.0. In order to make a comparison of the catalytic activity among the
various chiral complexes reported here, we also studied the catalytic oxidation
of the catechins in the presence of the achiral dinuclear complex[Cu2(L-66)]4+ and MBTH.Assuming that, for the present biomimetic catalytic reactions, a
two-step mechanism of catechol oxidation holds as in the case of our previous
studies with dinuclear [22], and trinuclear [23] copper(II) complexes, the
following simplified catalytic scheme can be hypothesized, where two molecules
of catechol (CatH2) per cycle are oxidized to quinone (Q):(1)Cu2II+CatH2↔[Cu2II/CatH2]→Cu2I+Q+2H+,Cu2I+CatH2+O2↔[Cu2/O2/CatH2]→Cu2II+Q+2H2O.  Since,
the kinetic experiments showed monophasic behavior and it was impossible to
separate the two steps. Thus, either the two steps have a similar rate or the
first one is slower. The dependence of the rates of the catalytic reactions as
a function of the substrate concentration exhibited a hyperbolic behavior in
all cases. However all the complexes exhibited substrate inhibition at high-substrate
concentrations, and therefore the kinetic parameters reported in
Table 1 were
estimated with the equation here reported:(2)v=Vmax[S]KM(1+Kc[S]2)+[S].Table 1
Kinetic
parameters for the stereoselective oxidations ofD-(+)-catechin and L-(–)-epicatechin in
methanol-aqueous phosphate buffer, pH 7.0 at 20±0.1°C.
ComplexesKM(M)kcat(s−1)kcat/KM(M−1s−1)R%Substrate[Cu2(R-DABN-4Bz4)]4+D-(+)-catechin(2.00±0.33)×10−5(1.21±0.75)×10−2604−32.0L-(–)-epicatechin(1.20±0.31)×10−5(1.41±0.96)×10−21169[Cu3(R-DABN-4Bz4)]6+D-(+)-catechin(1.33±0.32)×10−5(1.85±0.12)×10−2138732.8L-(–)-epicatechin(3.93±0.94)×10−5(2.75±0.31)×10−2701[Cu2(L-Lys-4Bz4)]4+D-(+)-catechin(1.14±0.38)×10−5(1.86±0.18)×10−2163246.0L-(–)-epicatechin(2.95±0.75)×10−5(1.78±0.17)×10−2604[Cu3(L-Lys-4Bz4)]6+D-(+)-catechin(1.37±0.33)×10−5(2.01±0.02)×10−2147060.4L-(–)-epicatechin(1.02±0.26)×10−4(3.71±0.58)×10−2363[Cu2(R-DABN-3Im4)]4+D-(+)-catechin(2.19±0.70)×10−5(1.09±0.15)×10−24987.2L-(–)-epicatechin(1.96±0.58)×10−5(8.44±0.69)×10−3431[Cu3(R-DABN-3Im4)]6+D-(+)-catechin(1.01±0.32)×10−5(5.10±0.39)×10−350742.6L-(–)-epicatechin(3.51±1.05)×10−5(7.15±0.80)×10−3204[Cu2(R-DABN-L-Ala-Bz4)]4+D-(+)-catechin(3.60±0.36)×10−5(2.77±0.11)×10−27698.8L-(–)-epicatechin(4.81±0.37)×10−5(3.10±0.11)×10−2644[Cu3(R-DABN-L-Ala-Bz4)]6+D-(+)-catechin(5.05±0.48)×10−5(6.46±0.15)×10−212805.3L-(–)-epicatechin(5.08±0.37)×10−5(5.84±0.10)×10−21150[Cu2(L66)]4+D-(+)-catechin(6.69±1.06)×10−5(7.57±0.60)×10−211311.9L-(–)-epicatechin(4.08±0.81)×10−5(4.44±0.13)×10−21088The copper complexes with chiral centers exhibited variable degree of
stereoselectivity (Table1) which has been evaluated using kinetic parameters
with the following equation:(3)R%=[(kcat/KM)D−(kcat/KM)L][(kcat/KM)D+(kcat/KM)L]×100, where D and  L are D-(+)-catechin and L-(–)-epicatechin.The catalytic  activity of all the
complexes, except[Cu2(R-DABN-4Bz4)]4+,
shows that the preferred coordination of the catechols is for D-(+)-catechin. This preference is probably dictated by the chirality of
the binaphthyl or lysine residues, as shown by our studies on related complexes
[24–26], and especially by the spatial disposition of the catechol substrates.
In fact, by simple calculation of molecular energy minimization, D-(+)-catechin shows a disposition almost planar with only the hydroxyl
group out of plane (Figure 3(a)).Three-dimensional structure of (a)D-(+)-catechin and
(b) L-(–)-epicatechin
with MM2 method.
(a)(b)On the contrary, theL-(–)-epicatechin
shows a more bulky spatial structure, because the two aromatic rings are
positioned on orthogonal planes, so the hindrance is very greater than in its
isomeric form (Figure 3(b)).Previous studies on the catalytic
oxidations of catechol derivatives demonstrated that the reaction needs the
cooperation of two close copper centers [22]  to enable
the binding of the catechol as a bridging ligand and allow a fast two-electron
transfer process. In the dinuclear copper complexes, the catechin substrate can
only form a productive complex  by binding the catechol residue to the two
copper ions in the A sites and that forces the resting part of the molecule to
approach the optically active residue so that chiral recognition is possible
(Scheme 2).Scheme 2
Proposed structures for
the putative intermediate adducts formed by the dinuclear copper(II) complexes
in the catalytic oxidations of the catechins.However,
the dinuclear complexes that contain as central core the 1,1-binaphthyl residue
show a rigid and bulky structure that reduces the possibility of effective
chiral recognition for the catechins. In this case, the coordination of the
catechols could occur on the outside of the complexes (Scheme2, Structure II).
The complex [Cu2(L-Lys-4Bz4]4+ has a different design. It contains a chiral L-Lysine residue as a central
unit, which is much more flexible than the 1,1-binaphthyl moiety and this is
connected with two arms carrying four benzimidazole donors through a pair of ortho-xylyl spacers. The high
flexibility of the spacer and the length of the two arms allow a better chiral
recognition of the substrates, as inferred by the kinetic constants and by the degree of stereoselectivity
(Scheme
2, Structure I).As
reported in the previous papers [26, 27], the trinuclear complexes display a
structure in which the Cu(II) center at B site and one of the two centers at A
site are mediated by a double hydroxide bridge (Scheme 3, Structure I). In this
case, the chiral recognition could depend not only on steric interactions but
also by coordination of the free aliphatic hydroxide to the other Cu(II): a site
(Scheme 3, Structure II) that allows a significant enantio-differentiating
behavior towards optically active substrates. In fact, considering the
three-dimensional structures of the two catechins reported before in
Figure 3,
one notices that the aliphatic hydroxide, in the D-(+)-catechin,
is opposite to the two catecholic groups, and therefore able to coordinate at
the Cu(II) center at A site. The L-(–)-epicatechin
shows the aliphatic hydroxide too far from the Cu(II) center at A site and, in
this case, the interaction needs a modification of the structure of the
complexes with a strong tension of the ligands.Scheme 3
Proposed structures
for the trinuclear copper(II) complexes (I) and for the putative intermediate
adducts (II) with the catechins.For[Cu2(L-66)]4+,
experimental data evidences a very low enantio-differentiation toward D-(+)-catechin
(see Table 1). This behavior could be due to the stacking interaction between
the aromatic ring of m-xylene residue and the aromatic ring far from the
catecholic one in the substrate; this interaction should be generated by a
parallel disposition of the xylene and the plane of the substrate molecule.


## 3.1. Stereoselective Catalytic Oxidations

The
catalytic oxidations of catechols are the most widely employed test reaction to investigate the
behavior of tyrosinase and catechol oxidase model complexes. Previous studies
[24–27] have shown that chiral dinuclear and trinuclear copper complexes were
able to display stereo-discriminating ability towards optically active
catechols to give the corresponding o-quinones.
To confirm this behavior, new chiral catechols were employed in these catalytic
stereoselective  oxidations. D-(+)-catechin and L-(–)-epicatechin
(flavan-3-ols) (Figure 2) constitute a class of phenolic compounds ubiquitous
in plants and widely found in fruits, vegetables, and beverages [33–35]. In
particular, they are one of the major quality factors in grapes and then in the
resulting wine [36, 37].Absolute stereochemistry configuration of
the catechols.
(a)(b)The
catalytic oxidation of polyphenolic substrates, including catechins, was well
studied by many authors [38–41]. These reactions take place in the presence of
atmospheric oxygen when polyphenol oxidase (PPO) and the corresponding
substrates are mixed at the same time. The fundamental first step is the
transformation of o-diphenols
to the corresponding o-quinones.
The fate and stability of o-quinones
vary widely, depending both on the phenolic precursor and on environmental
factors. In particular, the o-quinones
of D-(+)-catechin and
L-(–)-epicatechin
were seen to be much less stable than those of other o-quinones. The prolonged autoxidation, either chemical or
enzymic, led to the formation of polymers resulting from repeated condensation
reactions between an aromatic ring of one molecule with an aromatic ring of
another (“head to tail” polymerization mechanism). Depending on how phenolic
compounds are oxidized, the condensation products formed from catechins may
differ. In fact, the pH of the solution influences considerably the obtained
products [42, 43], because at low pH values is favored the formation of
colorless condensation products, whereas yellow compounds tended to be formed
at higher pH values. To avoid any effect due to pH-dependence of oxidation products
and to stop the reaction at quinones formation, a nucleophilic reagent MBTH, that traps the
quinones and generates chromophoric adducts, was used (Scheme 1).Scheme 1
Nucleophilic attach of the
reagent MBTH to the catechols.We have then studied the pH
dependence of the reaction rates and have found that the better pH for the
catalytic oxidation of catechins in the presence of the chiral copper complexes
is pH 7.0. In order to make a comparison of the catalytic activity among the
various chiral complexes reported here, we also studied the catalytic oxidation
of the catechins in the presence of the achiral dinuclear complex[Cu2(L-66)]4+ and MBTH.Assuming that, for the present biomimetic catalytic reactions, a
two-step mechanism of catechol oxidation holds as in the case of our previous
studies with dinuclear [22], and trinuclear [23] copper(II) complexes, the
following simplified catalytic scheme can be hypothesized, where two molecules
of catechol (CatH2) per cycle are oxidized to quinone (Q):(1)Cu2II+CatH2↔[Cu2II/CatH2]→Cu2I+Q+2H+,Cu2I+CatH2+O2↔[Cu2/O2/CatH2]→Cu2II+Q+2H2O.  Since,
the kinetic experiments showed monophasic behavior and it was impossible to
separate the two steps. Thus, either the two steps have a similar rate or the
first one is slower. The dependence of the rates of the catalytic reactions as
a function of the substrate concentration exhibited a hyperbolic behavior in
all cases. However all the complexes exhibited substrate inhibition at high-substrate
concentrations, and therefore the kinetic parameters reported in
Table 1 were
estimated with the equation here reported:(2)v=Vmax[S]KM(1+Kc[S]2)+[S].Table 1
Kinetic
parameters for the stereoselective oxidations ofD-(+)-catechin and L-(–)-epicatechin in
methanol-aqueous phosphate buffer, pH 7.0 at 20±0.1°C.
ComplexesKM(M)kcat(s−1)kcat/KM(M−1s−1)R%Substrate[Cu2(R-DABN-4Bz4)]4+D-(+)-catechin(2.00±0.33)×10−5(1.21±0.75)×10−2604−32.0L-(–)-epicatechin(1.20±0.31)×10−5(1.41±0.96)×10−21169[Cu3(R-DABN-4Bz4)]6+D-(+)-catechin(1.33±0.32)×10−5(1.85±0.12)×10−2138732.8L-(–)-epicatechin(3.93±0.94)×10−5(2.75±0.31)×10−2701[Cu2(L-Lys-4Bz4)]4+D-(+)-catechin(1.14±0.38)×10−5(1.86±0.18)×10−2163246.0L-(–)-epicatechin(2.95±0.75)×10−5(1.78±0.17)×10−2604[Cu3(L-Lys-4Bz4)]6+D-(+)-catechin(1.37±0.33)×10−5(2.01±0.02)×10−2147060.4L-(–)-epicatechin(1.02±0.26)×10−4(3.71±0.58)×10−2363[Cu2(R-DABN-3Im4)]4+D-(+)-catechin(2.19±0.70)×10−5(1.09±0.15)×10−24987.2L-(–)-epicatechin(1.96±0.58)×10−5(8.44±0.69)×10−3431[Cu3(R-DABN-3Im4)]6+D-(+)-catechin(1.01±0.32)×10−5(5.10±0.39)×10−350742.6L-(–)-epicatechin(3.51±1.05)×10−5(7.15±0.80)×10−3204[Cu2(R-DABN-L-Ala-Bz4)]4+D-(+)-catechin(3.60±0.36)×10−5(2.77±0.11)×10−27698.8L-(–)-epicatechin(4.81±0.37)×10−5(3.10±0.11)×10−2644[Cu3(R-DABN-L-Ala-Bz4)]6+D-(+)-catechin(5.05±0.48)×10−5(6.46±0.15)×10−212805.3L-(–)-epicatechin(5.08±0.37)×10−5(5.84±0.10)×10−21150[Cu2(L66)]4+D-(+)-catechin(6.69±1.06)×10−5(7.57±0.60)×10−211311.9L-(–)-epicatechin(4.08±0.81)×10−5(4.44±0.13)×10−21088The copper complexes with chiral centers exhibited variable degree of
stereoselectivity (Table1) which has been evaluated using kinetic parameters
with the following equation:(3)R%=[(kcat/KM)D−(kcat/KM)L][(kcat/KM)D+(kcat/KM)L]×100, where D and  L are D-(+)-catechin and L-(–)-epicatechin.The catalytic  activity of all the
complexes, except[Cu2(R-DABN-4Bz4)]4+,
shows that the preferred coordination of the catechols is for D-(+)-catechin. This preference is probably dictated by the chirality of
the binaphthyl or lysine residues, as shown by our studies on related complexes
[24–26], and especially by the spatial disposition of the catechol substrates.
In fact, by simple calculation of molecular energy minimization, D-(+)-catechin shows a disposition almost planar with only the hydroxyl
group out of plane (Figure 3(a)).Three-dimensional structure of (a)D-(+)-catechin and
(b) L-(–)-epicatechin
with MM2 method.
(a)(b)On the contrary, theL-(–)-epicatechin
shows a more bulky spatial structure, because the two aromatic rings are
positioned on orthogonal planes, so the hindrance is very greater than in its
isomeric form (Figure 3(b)).Previous studies on the catalytic
oxidations of catechol derivatives demonstrated that the reaction needs the
cooperation of two close copper centers [22]  to enable
the binding of the catechol as a bridging ligand and allow a fast two-electron
transfer process. In the dinuclear copper complexes, the catechin substrate can
only form a productive complex  by binding the catechol residue to the two
copper ions in the A sites and that forces the resting part of the molecule to
approach the optically active residue so that chiral recognition is possible
(Scheme 2).Scheme 2
Proposed structures for
the putative intermediate adducts formed by the dinuclear copper(II) complexes
in the catalytic oxidations of the catechins.However,
the dinuclear complexes that contain as central core the 1,1-binaphthyl residue
show a rigid and bulky structure that reduces the possibility of effective
chiral recognition for the catechins. In this case, the coordination of the
catechols could occur on the outside of the complexes (Scheme2, Structure II).
The complex [Cu2(L-Lys-4Bz4]4+ has a different design. It contains a chiral L-Lysine residue as a central
unit, which is much more flexible than the 1,1-binaphthyl moiety and this is
connected with two arms carrying four benzimidazole donors through a pair of ortho-xylyl spacers. The high
flexibility of the spacer and the length of the two arms allow a better chiral
recognition of the substrates, as inferred by the kinetic constants and by the degree of stereoselectivity
(Scheme
2, Structure I).As
reported in the previous papers [26, 27], the trinuclear complexes display a
structure in which the Cu(II) center at B site and one of the two centers at A
site are mediated by a double hydroxide bridge (Scheme 3, Structure I). In this
case, the chiral recognition could depend not only on steric interactions but
also by coordination of the free aliphatic hydroxide to the other Cu(II): a site
(Scheme 3, Structure II) that allows a significant enantio-differentiating
behavior towards optically active substrates. In fact, considering the
three-dimensional structures of the two catechins reported before in
Figure 3,
one notices that the aliphatic hydroxide, in the D-(+)-catechin,
is opposite to the two catecholic groups, and therefore able to coordinate at
the Cu(II) center at A site. The L-(–)-epicatechin
shows the aliphatic hydroxide too far from the Cu(II) center at A site and, in
this case, the interaction needs a modification of the structure of the
complexes with a strong tension of the ligands.Scheme 3
Proposed structures
for the trinuclear copper(II) complexes (I) and for the putative intermediate
adducts (II) with the catechins.For[Cu2(L-66)]4+,
experimental data evidences a very low enantio-differentiation toward D-(+)-catechin
(see Table 1). This behavior could be due to the stacking interaction between
the aromatic ring of m-xylene residue and the aromatic ring far from the
catecholic one in the substrate; this interaction should be generated by a
parallel disposition of the xylene and the plane of the substrate molecule.



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*Source: 762029-2008-09-23.xml*