Oxidation of organic molecules in homogeneous aqueous solution catalyzed by hybrid biocatalysts (based on the Trojan Horse strategy)

Article abstract of DOI:10.1016/j.tetasy.2010.03.050

New anionic metalloporphyrin-estradiol conjugates have been synthesized and fully characterized, and have been further associated to a monoclonal anti-estradiol antibody 7A3, to generate new artificial metalloenzymes following the so-called [Trojan Horse] strategy. The spectroscopic characteristics and dissociation constants of these complexes were similar to those obtained for the artificial metalloproteins obtained by association of cationic metalloporphyrin-estradiol conjugates to 7A3. This demonstrates that the nature of the porphyrin substituents, anionic or cationic, had little influence on the association with the antibody that is mainly driven by the tight association of the estradiol anchor with the binding pocket of the antibody. These new biocatalysts appeared to have an interesting catalytic activity in oxidation reactions. The iron(III)-anionic-porphyrin-estradiol-antibody complexes were found able to catalyze the chemoselective and slightly enantioselective (ee = 10%) sulfoxidation of sulfides by H₂O₂. The Mn(III)-porphyrin-estradiol-antibody complexes were found to catalyze the oxidation of styrene by KHSO₅, the Mn(III)-cationic-porphyrin- estradiol-antibody complexes even showing the highest yields so far reported for the oxidation of styrene catalyzed by artificial metalloproteins. However, a lack of chemoselectivity and enantioselectivity was observed, which was probably due to a weak interaction of the metalloporphyrin cofactor with the binding pocket of antibody 7A3, as suggested by the similar UV-visible characteristics and catalytic activities obtained with both anionic and cationic porphyrins.

Full text of DOI:10.1016/j.tetasy.2010.03.050

Tetrahedron: Asymmetry 21 (2010) 1593–1600
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Tetrahedron: Asymmetry
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Oxidation of organic molecules in homogeneous aqueous solution catalyzed
by hybrid biocatalysts (based on the Trojan Horse strategy)
Elodie Sansiaume a, Rémy Ricoux a, Didier Gori b, Jean-Pierre Mahy a,
*
a Equipe de Chimie Bioorganique et Bioinorganique, Institut de Chimie Moléculaire et des Matériaux d’Orsay, UMR CNRS 8182, Université Paris XI, 91405 Orsay Cedex, France
b Equipe de Synthèse Organique et Méthodologie et Equipe de Chimie des Procédés et Substances Naturelles, Institut de Chimie Moléculaire et des Matériaux d’Orsay,
UMR CNRS 8182, Université Paris XI, 91405 Orsay Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
New anionic metalloporphyrin–estradiol conjugates have been synthesized and fully characterized, and
have been further associated to a monoclonal anti-estradiol antibody 7A3, to generate new artificial
metalloenzymes following the so-called ‘Trojan Horse’ strategy. The spectroscopic characteristics and
dissociation constants of these complexes were similar to those obtained for the artificial metalloproteins
obtained by association of cationic metalloporphyrin–estradiol conjugates to 7A3. This demonstrates that
the nature of the porphyrin substituents, anionic or cationic, had little influence on the association with
the antibody that is mainly driven by the tight association of the estradiol anchor with the binding pocket
of the antibody.
Received 5 March 2010
Accepted 10 March 2010
Available online 18 May 2010
Dedicated to Professor Henri Kagan on the
occasion of his 80th birthday
These new biocatalysts appeared to have an interesting catalytic activity in oxidation reactions. The iro-
n(III)–anionic-porphyrin–estradiol-antibody complexes were found able to catalyze the chemoselective
and slightly enantioselective (ee = 10%) sulfoxidation of sulfides by H2O2. The Mn(III)–porphyrin–estra-
diol-antibody complexes were found to catalyze the oxidation of styrene by KHSO5, the Mn(III)–cat-
ionic-porphyrin–estradiol-antibody complexes even showing the highest yields so far reported for the
oxidation of styrene catalyzed by artificial metalloproteins. However, a lack of chemoselectivity and
enantioselectivity was observed, which was probably due to a weak interaction of the metalloporphyrin
cofactor with the binding pocket of antibody 7A3, as suggested by the similar UV–visible characteristics
and catalytic activities obtained with both anionic and cationic porphyrins.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
strategy’.1 They have obtained biocatalysts that are able to catalyze
the reduction of acetamido-acrylic acid by H2, with excellent enan-
Production of enantiopure compounds is a major issue in organ-
ic chemistry. Among the several strategies explored to induce ste-
reoselectivity into chemical reactions, the construction of artificial
metalloenzymes has appeared to be one of the most promising
ones. Indeed, such hybrid biocatalysts combine the efficiency and
wide scope of reactions of synthetic catalysts with the high selec-
tivity and ability to work under mild conditions of enzymes. More-
over, they can be optimized by directed evolution, a very powerful
way to enhance their performances.1
Artificial metalloenzymes can be obtained by insertion of a metal
cofactor into the cavity of a protein. This cofactor can be linked either
covalently2 or non-covalently3–5 to the protein. As an example of the
later case, Ward et al. have built artificial metalloenzymes by the
association of biotinylated rhodium complexes with (strept)avidin,
using the strong (strep)avidin/biotin affinity to anchor the cofactor
into the cavity of the protein, following the so-called ‘Trojan Horse
tiomeric excesses.6 In addition, by incorporation of achiral biotinyl-
ated manganese–salen complexes into streptavidin they got new
biocatalysts for aqueous sulfoxidation reactions using hydrogen
peroxide as an oxidant.7 Following the same strategy, we also previ-
ously reported the preparation of another artificial metalloenzyme
that was obtained by supramolecular association of a cationic metal-
loporphyrin–estradiol conjugate with an anti-estradiol antibody.8,9
This artificial metalloprotein possessed a peroxidase activity9 and
was able to catalyze the sulfoxidation of thioanisole by H2O2, with
a moderate enantiomeric excess.8 In an attempt to improve the cat-
alytic performances of such systems, we decided to design a new
metalloporphyrin cofactor, by covalent attachment of estradiol,
used as the ‘Trojan Horse’, to an anionic para-sulfonatophenyl-
substituted porphyrin metalated with iron(III) or manganese(III).
The choice of such porphyrins was guided by the observations in
previous literature reports that iron(III)- or manganese(III)-tetra-
para-sulfonatophenylporphyrin were excellent catalysts for oxygen
transfer reaction using KHSO5 or H2O2 as oxidants.10,11 New artificial
metalloenzymes were then obtained by supramolecular association
of the anionic metalloporphyrin–estradiol conjugates with the
* Corresponding author. Tel.: +33 169 157 421; fax: +33 169 157 281.
E-mail address: jean-pierre.mahy@u-psud.fr (J.-P. Mahy).
0957-4166/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2010.03.050

1594
E. Sansiaume et al. / Tetrahedron: Asymmetry 21 (2010) 1593–1600
anti-estradiol antibody 7A3 and we used them as catalysts for oxida-
tion reactions such as sulfoxidation of thioanisole by H2O2 and epox-
idation of styrene by oxone (KHSO5) in aqueous medium. Both
reactions are typically catalyzed by hemoproteins such as peroxi-
dase and cytochrome P450, respectively.
UV–visible spectroscopy (Fig. 1) and by HR-MS spectrometry that
were in agreement with the expected structure.
2.2. Insertion of the metalloporphyrin cofactors in the antibody
The UV–visible spectrum of 1-Fe, 1-Mn, and 2-Mn were re-
corded, alone and in the presence of a slight excess of antibody
7A317 (1.2 equiv per binding site in order to favor the binding of
the estradiol moiety of 1 inside the binding site of the antibody
7A3), respectively, in 50 mM phosphate-citrate buffer, pH 3, and
in 50 mM phosphate buffer, pH 7.4 (Fig. 1).
When comparing the spectra, it appears that in both cases, the
presence of the antibody allows only a slight shift of the Soret band
(4 nm) to be observed, together with a decrease in its intensity
(Table 1). The slight shift of the Soret band is in agreement with
no change in the coordination sphere of the metal, and thus sug-
gests that no amino-acid side chain of the antibody acts as a fifth
axial ligand of the metal. The decrease in absorbance may probably
be the sign that the porphyrinic part of the cofactors does not enter
deeply inside the hydrophobic binding pocket of 7A3. Indeed it has
been shown by several authors18–20 that, on the contrary, the inser-
tion of a metalloporphyrin in the hydrophobic environment pro-
vided by the binding pocket of anti-porphyrin antibodies led to a
significant increase of its Soret band.
2. Results and discussion
2.1. Synthesis of the new metalloporphyrin cofactors, iron(III)- and
manganese(III)-5,10,15-tris(p-sulfonatophenyl)-20-(4-phenyl
(3-O-amidoxymethylestradiol))porphyrin, 1-Fe and 1-Mn
The first step was the preparation of 5,10,15-tris(phenyl)-20-
(4-nitrophenyl)porphyrin (TP-p-NO2-PH2) upon nitration of
5,10,15,20-tetraphenylporphyrin (TPPH2) by NaNO2 as described
by Smith et al.12 TPPH2 was thus refluxed for 3 min in TFA at room
temperature with a stoichiometric amount of NaNO2 in order to
avoid polynitrations (Scheme 1). After TFA removal, the reduction
of the nitro group of p-NO2-TPPH2, to afford 5,10,15-tris(phenyl)-
20-(4-aminophenyl)porphyrin (TP-p-NH2-PH2), was then realized
by a classical reaction described by Collman et al.,13 that involved
the stirring of TP-p-NO2-PH2 with an excess of SnCl2ꢀ4H2O in a 4:1
mixture of 6 N HCl and CH2Cl2 for 24 h. at 60 °C. After neutraliza-
tion with solid NaOH at 0 °C, extraction with CH2Cl2 and purifica-
tion by chromatography on silica gel (eluent CH2Cl2/cyclohexane
95/5, Rf = 0.7) TP-p-NH2-PH2 was obtained in a 30% yield.
The sulfonation of (TP-p-NH2-PH2) was carried out according to
the method described by Fleischer et al.,14 by heating 94 mg of TP-
p-NH2-PH2 in concentrated H2SO4 at 80 °C under dry atmosphere
for 48 h. After cooling down to room temperature, protonated
5,10,15-tris(p-sulfonic acid-phenyl)-20-(p-aminophenyl)porphyrin
(T-p-SO3H-P-p-NH2-PH2) was precipitated into ice/water and ob-
tained as a green solid after centrifugation and washing with neu-
tral water until pH 3–4 as described in Section 4. Water-soluble
anionic 5,10,15-tris(p-sulfonatophenyl)-20-(p-aminophenyl)por-
phyrin (T-p-SP-p-NH2-PH2) was obtained by treatment with 30%
ammonium hydroxide and purified on a Na+-activated cation ex-
change column (AG-50W-X8). After lyophilization, T-p-SP-p-NH2-
PH2 was obtained as a brownish powder in a 36% yield.
In order to check the stoichiometry of the metal cofactor-7A3
complexes and to calculate their dissociation constant, increasing
amounts of 7A3 were added to a 5
lM solution of 1-Mn and 2-
Mn in 0.05 M phosphate buffer, pH 7.4, and to a 5
l
M solution of
1-Fe in phosphate-citrate buffer, pH 3. The titration was followed
by UV–visible spectroscopy as described in the Section 4, and the
difference between the absorbance values, respectively, at
420 nm (in the case of 1-Fe) and at 469 nm (in the case of 1-Mn
and 2-Mn) of the solution of antibody-cofactor and that of the
cofactor alone, respectively,
the metal cofactor/antibody ratio. Figure 2 shows the results ob-
tained in the typical case of the titration of 1-Mn by 7A3. A469
DA420 and DA469, was plotted against
D
clearly increases linearly with the 7A3/1-Mn ratio until 0.5 equiv
of 7A3 has been added to 1-Mn, and then reaches a plateau. This
indicates that the antibody is able to bind two cofactors, which is
in agreement with the binding of 1-Mn to the antibody, thanks
to the specific recognition of its estradiol anchor by the antibody
3-O-Carboxymethylestradiol was synthesized from estrone in
two steps as described previously.8 3-O-Carboxymethylestrone
was first prepared following the method described by Katzenel-
lenbogen,15 and the ketone function of estrone was then selectively
reduced by 5 equiv of NaBH4 (Scheme 1). The carboxy function
of 3-O-carboxymethylestradiol was activated by 1.2 equiv of
N-hydroxysuccinimide (NHS) in the presence of 1.2 equiv of carbo-
diimide in anhydrous THF at 0 °C. The activated ester was then iso-
lated as a white solid after THF removal. This step was necessary
because of the poor solubility of anionic T-p-SP-p-NH2-PH2 in
THF. Further condensation of 1.2 equiv of the activated ester with
porphyrin T-p-SP-p-NH2-PH2 was then carried out in DMF in the
presence of Na2CO3. After solvent removal and reprecipitation in
acetone/methanol (80:20) and after drying, the estradiol–porphy-
rin conjugate 1 was isolated as purple crystals (63 mg, 100% yield)
and characterized by UV–visible spectroscopy and Mass spectrom-
etry that were in agreement with the expected structure.
binding site. For a 7A3/1-Mn ratio higher than 0.7,
decreases, which could be due to non-specific surface interactions
DA469 slightly
of the 1-Mn cofactor with the antibodies in excess in the solution.
ꢀ
ꢁ
D
A
The function g
, relating the concentration of cofactor
ꢀn-M
ꢁꢀn-MꢁS
1 added with the absorbances measured (cf. Section 4), allows an
estimation of KD with a non-linear regression of the experimental
data (see Section 4). A dissociation constant KD = 1.9 ꢂ 10ꢁ6
M
could then be computed for the 7A3/1-Mn complex. When com-
pared to the dissociation constant of the antibody-antigen complex
(KD = 9.5 ꢂ 10ꢁ10 M), this value shows that the affinity of the anti-
body for the estradiol of the cofactor 1 has been lowered by only 3
orders of magnitude, despite the steric hindrance brought by the
porphyrin. This confirms that in the presence of an excess of anti-
body, and for concentrations above micromolar, almost every mol-
ecule of cofactor 1-Mn is accommodated in the antibody’s pocket.
Similar results were obtained for the titration of 1-Fe and 2-Mn
by antibody 7A3. In both cases a 1:2 antibody/metal cofactor stoi-
chiometry was found and respective KD values of 1.9 ꢂ 10ꢁ6 M and
9.0 ꢂ 10ꢁ6 M were calculated for the 7A3/1-Mn and 7A3/2-Mn
complexes.
1-Fe and 1-Mn were prepared following classical protocols de-
scribed, respectively, by Fleischer et al.14 and Adler et al.16 upon
reaction of 1 with, respectively, excess FeCl2ꢀ4H2O and excess
MnBr2 in DMF at 120–130 °C, under inert atmosphere to avoid
the formation of
l-oxo dimers. After solvent removal, the water-
Taken altogether the above-mentioned results are in agreement
with the anchoring of the metal cofactors 1-Fe, 1-Mn, and 2-Mn,
thanks to the specific recognition of the estradiol anchor by the
antibody 7A3, the metalloporphyrin being outside but in proximity
to the binding pocket.
soluble metalloporphyrins 1-Fe and 1-Mn were dissolved in water
and purified on an Na+-activated cation exchange column (AG-
50W-X8) and obtained, after lyophilization, as dark purple solids
in, respectively, 30% and 34% yield. Both were characterized by

E. Sansiaume et al. / Tetrahedron: Asymmetry 21 (2010) 1593–1600
1595
N
HN
N
NH
O
H
NaNO2,
TFA
H
H
HO
O
Br
N
HN
NO2
N
1. EtONa,
2. KOH
O
NH
O
SnCl2, HCl 6N
H
H
H
HO
O
N
HN
N
O
NH2
NH
NaBH4, MeOH, 0 °C
OH
H2SO4 cc
-
H
SO3
H
H
HO
N
O
HN
N
-O3S
NH2
NH
O
-O3S
OH
NHS, DCC
-
SO3
H
N
H
N
H
H
HN
N
-O3S
NH
O
O
1
-O3S
FeCl2.4H2O, or MnCl2,
DMF, 120°C
OH
-
SO3
H
N
H
N
O
N
H
H
-O3S
M
N
N
O
M = Fe: 1-Fe
M = Mn: 1-Mn
-O3S
Scheme 1.
2.3. Sulfoxidation of thioanisole by H2O2 catalyzed by the
antibody-(estradiol–metalloporphyrin) complexes
idases like horseradish peroxidase (HRP),21–23 which makes it a per-
fect candidate for a study of the enantioselectivity of the catalysis.
The oxidation of 2.5 mM thioanisole by 2.5 mM H2O2 was per-
formed in 0.05 M phosphate buffer, pH 3, as described in Section 4,
The influence of the antibody on the efficiency and the selectivity
of oxidation reactions catalyzed by the estradiol–iron(III)- and
-manganese(III)–porphyrin cofactors has been studied. For this,
the first reaction to be chosen was the oxidation of thioanisole by
H2O2. Indeed, thioanisole is well known as a typical substrate to be
oxidized into an asymmetric sulfoxide by naturally occurring perox-
using as catalyst 5
ously incubated with antibody 7A3 (7.5
presence of 500 M imidazole. After 1 h, the organic products were
l
M 1-Fe, 1-Mn, or 2-Mn either alone or previ-
l
M) for two hours, in the
l
extracted with ethyl-acetate and analyzed by GC using benzophe-
none as the internal standard to determine the degree of conver-

1596
E. Sansiaume et al. / Tetrahedron: Asymmetry 21 (2010) 1593–1600
Figure 1. (A) Superimposition of the spectra of 1-Fe, 10
lM in 0.05 M phosphate-citrate buffer, pH 3, alone (plain line), and in the presence of 5.85
lM 7A3 (dashed line). (B)
Superimposition of the spectra of 1-Mn, 10 lM in 0.05 M phosphate buffer, pH 7.4, alone (plain line), and in the presence of 5.85 lM 7A3 (dashed line).
No oxidation of thioanisole was observed, not only in the absence of
catalyst but also in the presence of Mn–porphyrin catalysts (data
not shown). This was not surprising, as manganese porphyrins have
been reported to be efficient catalysts for the catalase reaction [10]
(Eq. (2)).
Table 1
UV–visible characteristics of metalloporphyrin–estradiol cofactors 1-Fe, 1-Mn, and 2-
Mn and their complexes with 7A3
Complex
UV–visible characteristics:
kmax(nm) (
(Mꢁ1cmꢁ1))
Antibody/cofactor
stoichiometry
KD (Mꢁ1
)
e
1-Fe
1-Fe–7A3
1-Mn
1-Mn–7A3
2-Mn
2-Mn–7A3
415 (1 ꢂ 105), 544
—
1/2
—
1/2
—
1/2
2H2O2 ! 2H2O þ O2
ð2Þ
419 (93 ꢂ 103), 544
1.6 ꢂ 10ꢁ6
1.9 ꢂ 10ꢁ6
9 ꢂ 10ꢁ6
469 (96 ꢂ 103), 565, 604
469 (86 ꢂ 103), 565, 604
464 (96 ꢂ 103), 561, 602
464 (72 ꢂ 103), 564, 603
On the contrary, in the presence of both 1-Fe and 1-Fe–7A3 as
catalysts, the chemoselective formation of sulfoxide was observed
(Fig. 3). The quantity of sulfoxide formed with 7A3–1-Fe as catalyst
(44 lM) was comparable to that already obtained with 2-Fe as cat-
alyst8 and was about sixfold higher than that observed with 1-Fe
alone as catalyst. Since 7A3–2-Fe was reported to be only twice
more efficient as catalyst than 2-Fe alone, it is then clear that
7A3 better enhances the activity of 1-Fe than that of 2-Fe in the
sulfoxidation of thioanisole. This shows in both cases the protect-
ing effect of the antibody toward the oxidative degradation of
the porphyrin ring by H2O2.
Figure 3. Amounts of sulfoxide (lM) obtained for the oxidation of thioanisole by
H2O2 catalyzed by 1-Fe and 2-Fe and their complexes with antibody 7A3.
Figure 2. UV–visible titration of 1-Mn by antibody 7A3. (A) Evolution of the UV–
visible spectrum of 5 M solution of 1-Mn in 0.05 M phosphate buffer, pH 7.4, upon
addition of increasing amounts of antibody 7A3 (0–7.5 M). (B) Difference between
the absorbance value at 469 nm of the antibody-cofactor solution and that of the
cofactor alone, A469 plotted against the metal cofactor/antibody ratio.
l
l
Concerning the enantioselectivity of the catalysis, whereas the
sulfoxide formed with 1-Fe alone was found to be a racemic mix-
ture, an enantiomeric excess of 10% in favor of the S enantiomer
was measured for the reaction in the presence of 7A3. This moder-
ate excess was only slightly higher than that reported in the case of
7A3–2-Fe8 (Table 2). It was also in the range of the enantiomeric
excesses obtained for the sulfoxidation of thioanisole catalyzed
by artificial metalloproteins obtained by the ‘Trojan Horse’ strategy
or by dative insertion of metal–salen cofactors into S112D
streptavidine mutant7 and apomyoglobin and mutants.24 It was
however lower than those obtained with artificial metalloproteins
obtained by covalent25 or supramolecular26 insertion of Mn–salen
complexes into apomyoglobin mutants, supramolecular insertion
D
sion of the sulfide, and by HPLC on a Chiracel OD-H column to
determine the enantiomeric excess of the sulfoxide thus obtained.
O
S
O
S
S
CH3
CH
3 + H2O2
Catalyst
CH3
(S)
ð1Þ
(R)
Catalyst = 1-Fe, 1-Mn, 2-Mn,
7A3-1-Fe, 7A3-1-Mn, 7A3-2-Mn

E. Sansiaume et al. / Tetrahedron: Asymmetry 21 (2010) 1593–1600
1597
Table 2
UV–visible characteristics of metalloporphyrin–estradiol cofactors 1-Fe, 1-Mn, and 2-Mn and their complexes with 7A3
Protein
Metal complex
Strategy
ee (%)
Configuration
Ref.
Anti-estradiol antibody 7A3
Anti-estradiol antibody 7A3
Streptavidine
Apomyoglobine mutants
Apomyoglobine mutants
Apomyoglobine mutants
Anti-microperoxidase 8 (MP8)-antibody 3A3
Bovine serum albumin
Streptavidin
1-Fe
2-Fe
‘Trojan Horse’
‘Trojan Horse’
‘Trojan Horse’
Dative
10
8
11
5–33
12–51
60
46
38
(S)
(S)
(R)
(R) or (S)
(S)
(S)
(R)
(S)
(S)
This work
8
Mn–salen–biotin
Cr–salen
Mn–salen
Mn–salen
MP8
25
26
27
28
29
30
31
Covalent
Supramolecular
Supramolecular
Supramolecular
Dative
Fe–corrole
VOSO4
93
of microperoxidase8 into monoclonal antibody 3A327, and of Fe–
corrole into Bovine Serum Albumin.28 Finally it was also far from
the enantiomeric excess observed in the presence of the artificial
metalloprotein obtained by dative incorporation of VOSO4 into
streptavidin (93%).29
the double bond, followed either by the epoxide ring closure
and/or by the formation of phenylacetaldehyde 4 by the so-called
‘NIH shift’ mechanism.34 It is then likely that the formation of 3
and 4 as major products should arise from a similar mechanism
in the reactions of KHSO5 with styrene catalyzed by 1-Mn and 2-
Mn and their complexes with antibody 7A3.
2.4. Epoxidation of styrene by KHSO5 catalyzed by the antibody-
(estradiol–metalloporphyrin) complexes
When comparing the results shown in Table 3, it first appears
that, in the presence of 10 equiv of imidazole as co-catalyst, the
anionic porphyrin-based catalysts 1-Mn and 7A3–1-Mn are less
efficient than their cationic counterparts 2-Mn and 7A3–2-Mn, as
shown by total yields of 20% for both 1-Mn and 7A3–1-Mn and
90% and 85%, respectively, for 2-Mn and 7A3–2-Mn. Consequently,
it also appears that the anchoring into the antibody 7A3 of both
the anionic porphyrin-based catalyst 1-Mn and the cationic porphy-
rin-based catalyst 2-Mn does not cause any significant change in the
total oxidation yield. However, concerning the epoxide/phenylacet-
aldehyde (3/4) ratio, the anchoring of the estradiol–Mn–porphyrin
catalysts to the antibody 7A3 had a noticeable effect. Indeed,
whereas with the anionic porphyrin-based catalysts, 1-Mn, the
epoxide 3 was obtained as the major product with a 3/4 ratio of 2,
3 and 4 were obtained in comparable amounts with 7A3–1-Mn, as
catalyst (3/4 = 1). In this case, then, the antibody protein induced a
chemoselectivity of the oxidation of styrene in favor of phenylacet-
aldehyde 4. On the contrary, no change in the chemoselectivity was
observed when the cationic porphyrin-based catalyst 2-Mn was in-
serted into 7A3 as both 2-Mn and 7A3–2-Mn catalysts appeared to
favor the productionof thephenylacetaldehyde 4 versus the epoxide
3 with a 4/3 ratio of about 3. This discrepancy in the influence of the
antibody protein on the chemoselectivity of the oxidation of styrene
catalyzed by either 1-Mn or 2-Mn could be explained by the fact that
1-Mn is more tightly associated, through its estradiol anchor, to 7A3
than 2-Mn, as suggested by a fivefold larger KD value for 7A3–2-Mn
than for 7A3–2-Mn (Table 1). Finally, no enantiomeric excess could
be observed with any of the four catalysts used, showing that no
asymmetric induction was produced by the anchoring of the estra-
diol–Mn–porphyrin catalysts to antibody 7A3.
The second reaction to be chosen to test the influence of the
antibody on the efficiency and on the selectivity of oxidation reac-
tions catalyzed by the estradiol–iron(III)- and -manganese(III)–
porphyrin cofactors was the oxidation of styrene. This reaction
was chosen for several reasons: (i) It constitutes a reaction of
interest in organic synthesis, (ii) the epoxidation of alkenes is an
important reaction in the metabolism of alkene derivatives by
cytochrome P450-dependent monooxygenases,30 and finally (iii)
very few artificial metalloenzymes have been shown to be able
to catalyze this reaction.31–33
O
*
O
KHSO5
Catalyst
+
+
O
Phenyloxirane
Phenylacetaldehyde
Benzaldehyde
3
4
5
Catalyst = 1-Fe, 1-Mn, 2-Mn
7A3-1-Fe, 7A3-1-Mn, 7A3-2-Mn
ð3Þ
The oxidation of styrene by H2O2 appeared to be totally ineffi-
cient, whatever the catalyst used (data not shown), we thus
decided to use as oxidant oxone (KHSO5) since it was previously re-
ported that metallo-tetra-para-sulfonatophenylporphyrin were
excellent catalysts for oxygen transfer reaction using KHSO5 as oxi-
dant.10 Accordingly, the oxidation of 10 mM styrene by 2.5 mM
KHSO5 was carried out in 0.05 M phosphate buffer, pH 7.4, in the
presence of 5
lM 1-Fe, 1-Mn, or 2-Mn in the presence or not of
7.5 M 7A3, and in the presence of 10 equiv of imidazole as co-cat-
l
Experiments were also performed in the presence of 100 equiv
of imidazole as co-catalyst. No change in the 3/4 ratios was ob-
served in any of the reactions performed when compared to those
obtained in the presence of 10 equiv of imidazole (data not shown).
alyst, as described in Section 4. After 1 h, the organic products were
extracted with ethyl-acetate and analyzed by GC using benzophe-
none as the internal standard to determine the degree of conver-
sion of styrene and the nature of the products obtained, and by
HPLC on a Chiralpak AD-H column to determine the enantiomeric
excess of the epoxide thus obtained.
Table 3
Under these conditions, no oxidation was observed with both 1-
Fe and 7A3–1-Fe as catalysts, whereas three products were
obtained with the manganese catalysts: phenyloxirane 3 and phe-
nylacetaldehyde 4 as major products and benzaldehyde 5 as a very
minor product with yields lower than 2%, whatever the catalyst
(Table 3). As already shown for the oxidation of styrene by oxygen
atom donors, such as iodosobenzene, in the presence of metallo-
porphyrins as catalysts, it is likely that both products 3 and 4
derive from the addition of a high-valent metal-oxo species on
Epoxidation of styrene by oxone in phosphate buffer, pH 7.4, in the presence of 1-Mn
and 2-Mn as catalysts and 10 equiv of imidazole as co-catalyst
Catalyst
Yield ( 1%)
3
3/4
5
4
Total
1-Mn
1-Mn–7A3
2-Mn
2
2
2
1
6
9
66
63
12
9
22
21
20
20
90
85
2
1
0.3
0.3
2-Mn–7A3

1598
E. Sansiaume et al. / Tetrahedron: Asymmetry 21 (2010) 1593–1600
No change in the total yield of oxidation products was observed in
the presence of either 2-Mn or 7A3–2-Mn, the only change ob-
served was a twofold increase in the total yield of oxidation prod-
ucts in the presence of 1-Mn or 7A3–1-Mn (38%).
protein, and then to reduce the distance between metal complex
and the estradiol anchor. This could be achieved either by directly
substituting the estradiol moiety on one meso-carbon of the porphy-
rin ring or alternatively by substituting the porphyrin ligand with an
about twice as small salen ligand.
Until now only a few results have been reported for the epoxida-
tion of alkenes catalyzed by artificial metalloproteins. In a first pa-
per, Reetz et al.31 briefly mention preliminary results suggesting
that an artificial metalloenzyme, obtained by covalent linkage of
Mn- and Rh-salen complexes to papain, could be able to catalyze
the epoxidation of styrene with less than 10% ee. Two other papers
have been published more recently that are dealing with the epoxi-
dation of styrene derivatives by H2O2, catalyzed by bovine and hu-
man carbonic anhydrase, in which the catalytic Zn2+ ion has been
4. Materials and methods
4.1. Physical measurements
UV–visible spectroscopy studies were performed on double
beam UVIKON 860 XL and CARY 300 BIO VARIAN spectrophotom-
eters. 1H NMR spectra were recorded on BRUKER AM 250, 300, 360
spectrometers.
replaced by Mn2+ 32,33 One of them by Soumillon et al.32 reports
.
yields between 9% and 57% with enantiomeric excesses between
18% and 52%, whereas the other by Okrasa and Kazlauskas33 reports
conversions between 1% and 12%, and enantiomeric excesses be-
tween 50% and 66%. Therefore, it is clear that our new hybrid estra-
diol–Mn–porphyrin catalysts 7A3–1-Mn and 7A3–2-Mn lead to a
better conversion of styrene (38–85%), which is linked to the
remarkable catalytic efficiency of the metalloporphyrin cofactor.
However, contrary to other authors,31–33 the chemoselectivity was
in favor of the formation of phenylacetaldehyde, and we could not
observe any stereoselectivity, which could be due to the fact that
in both the Mn2+-carbonic anhydrase and Mn– and Rh–salen–pa-
pain complexes, the catalytic metal cofactor is deeply inserted into
the protein pocket, whereas in our case the metalloporphyrin cofac-
tor most probably stays outside, though in proximity to the binding
pocket of the anti-estradiol antibody 7A3.
4.2. Synthesis of 5,10,15-tris(p-sulfonatophenyl)-20-(4-phenyl
(3-O-amidoxymethylestradiol)) 1 and of its iron(III)- and
manganese(III) complexes 1-Fe and 1-Mn
4.2.1. Synthesis of free base 5,10,15-tris(p-sulfonatophenyl)-20-
(p-aminophenyl)porphyrin
The 5,10,15-trisulfonato-20-p-aminophenylporphyrin was first
prepared in two steps by nitration and further sulfonation of tetra-
phenylporphyrin (TPPH2). The nitration of tetraphenylporphyrin
was first carried out according to the method described by Smith
et al.,12 by reacting 315 mg of TPPH2 (0.5 mmol) with exactly
1.4 equiv of NaNO2 in 12 mL TFA at room temperature during
3 min to avoid polynitrations. After TFA removal under reduced
pressure, the crude product was then reduced with 660 mg SnCl2
(5 mmol) in a mixture of 40 mL of concentrated HCl (37%) and
10 mL of CH2Cl2 under argon, during 24 h under stirring at 60 °C.13
The reaction mixture was then neutralized with KOH pellets and ex-
tracted with CH2Cl2. The crude product was further purified by col-
umn chromatography on silicagel, using increasing amounts of
CH2Cl2 in cyclohexane as eluant. The pure 5,10,15-phenyl-20-p-
aminophenylporphyrin (TPP-p-NH2-PH2) was isolated as purple
crystals (75 mg, yield: 24%). Rf = 0.7 in 95:5 CH2Cl2/cyclohexane.
UV–visible in CH2Cl2: kmax (nm) = 420 (Soret), 518, 555, 592, 649
1H NMR (CDCl3, 360 MHz, d (ppm)): 8.91 (d, J = 4.8 Hz, 2H); 8.78
(m, 6H); 8.28 (m, 6H); 8.4 (d, J = 8.3 Hz, 2H); 7.79 (m, 9H); 8.61 (d,
J = 8.3 Hz, 2H).
The sulfonation of the 5,10,15-phenyl-20-p-aminophenylporph-
yrin was carried out according to the method described by Fleischer
et al.14 94 mg (0.15 mmol) of TPP-p-NH2-PH2 in 10 mL of concen-
trated H2SO4 were heated at 80 °C under dry atmosphere for 48 h.
After cooling down to room temperature, the mixture was poured
into ice/water (50 mL) giving a suspension that was centrifuged four
times (10 min at 6000 rpm). The supernatant was repeatedly re-
moved and replaced with neutral water until pH 3–4. The residual
solid was then dissolved in 30% ammonium hydroxide (20 mL)
and the solvent was removed by rotary evaporation. The solid was
dissolved in a minimum amount of water and applied to a Na+-acti-
vated cation exchange column (AG-50W-X8). The product was
lyophilized. UV–visible, kmax (nm) in H2O = 415 (Soret), 516, 558,
587, 638. 1H NMR (MeOD, 250 MHz, d (ppm)): 8.8 (m, 16H); 7.9
(d, 6H, J = 8.5 Hz); 7.1 (d, 6H, J = 8.5 Hz). MALDI: m/z: 870.12
(M+H+) (exact mass = 869.12 g molꢁ1).
3. Conclusion
Our aforementioned results describe the synthesis and charac-
terization of new anionic metalloporphyrin–estradiol conjugates
that were further associated to a monoclonal anti-estradiol anti-
body 7A3 to generate new artificial metalloenzymes following
the so-called ‘Trojan Horse’ strategy. The spectroscopic characteris-
tics and dissociation constants of these complexes were rather
similar to those obtained for the artificial metalloproteins obtained
by association of cationic metalloporphyrin–estradiol conjugates
to 7A3. This demonstrated that the nature of the porphyrin substit-
uents, anionic or cationic, had little influence on the association
with the antibody that was, as expected, mainly driven by the tight
association of the estradiol anchor with the binding pocket of the
antibody.
Our new biocatalysts appeared to have an interesting catalytic
activity in oxidation reactions. The iron(III)–porphyrin–estradiol-
antibody complexes were found able to catalyze the chemoselective
and slightly enantioselective sulfoxidation of sulfides by H2O2. The
10% enantiomeric excess observed with the anionic iron(III)-
5,10,15-tris(p-sulfonatophenyl)-20-(4-phenyl(3-O-amidoxyme-
thylestradiol))porphyrin–7A3 (1-Fe–7A3) catalyst was however
about the same as that already reported in the case of the cationic ir-
on(III)- 5,10,15-tris(4-N-methylpyridiniumyl)-20-(4-phenyl(3-O-
amidoxymethylestradiol))porphyrin–7A3 (2-Fe–7A3).8 The Mn
(III)–porphyrin–estradiol-antibody complexes were found to cata-
lyze the epoxidation of styrene by KHSO5 with the highest yields
ever reported for the epoxidation of styrene catalyzed by artificial
metalloproteins. However, a lack of chemoselectivity and enantiose-
lectivity was observed, which was probably due to a weak interac-
tion of the metalloporphyrin cofactor with the binding pocket of
antibody 7A3, in agreement with the UV–visible characteristics
and the catalytic activities that are similar with both anionic and cat-
ionic porphyrins. In the light of the literature results,31–33 it is then
likely that to get better enantioselectivities, it will be necessary to in-
crease the interaction of the metal cofactor with the antibody
4.2.2. Synthesis of free base 5,10,15-tris(p-sulfonatophenyl)-20-
(4-phenyl(3-O-amidoxymethylestradiol)) 1
3-O-Carboxymethyloestrone was synthesized from estrone fol-
lowing the method described by Katzenellenbogen.15 The ketone
function (634 mg, 1.93 mmol, 1 equiv) was then selectively re-
duced by 8 equiv of NaBH4 (600 mg, 15.8 mmol) in methanol at
0 °C to obtain 3-O-carboxymethylestradiol (574 mg, yield = 90%).
The carboxylic acid function was activated by N-hydroxy-

E. Sansiaume et al. / Tetrahedron: Asymmetry 21 (2010) 1593–1600
1599
succinimide (9.7 equiv) and dicyclohexylcarbodiimide (DCC)
(9.7 equiv) in freshly distilled THF at 0 °C. The solvent was evapo-
rated and the product was filtered after precipitation of dicyclo-
hexylurea (DCU). The activated ester (0.08 mmol, 1.5 equiv) was
condensed with porphyrin (0.054 mmol, 1 equiv) in 5 mL of DMF
in the presence of 0.54 mmol. of Na2CO3. The solvent was evapo-
rated and the estradiol–porphyrin conjugate 1 was precipitated
in a mixture of MeOH/acetone and isolated as purple crystals
(63 mg, yield = 100%). UV–visible: kmax (nm) in H2O = 415 (Soret),
517, 560, 653. 1H NMR (MeOD, 360 MHz, d (ppm)): 8.8 (s, 8H);
8.3 (m, 12H); 7.1 (d, 2H, J = 8.5 Hz); 6.6 (d, 2H, J = 8.5 Hz); 2.8 (m,
2H); 2.2 (d, 2H, J = 16 Hz); 1.9–1.7 (m, 7H), 0.725 (s, 3H). HR-MS
(MALDI-MS) m/z: 1182.33 (M+H+) (exact mass = 1181.34 g molꢁ1).
antigen obtained by covalent linkage of estradiol in 3-position to
BSA.17
4.4. Characterization of the metalloporphyrin–antibody 7A3
complexes
4.4.1. Comparison of the UV–visible spectra of the
metalloporphyrin cofactors, 1-Fe, 1-Mn, and 2-Mn, with those
of the corresponding metalloporphyrin–7A3 complexes
A 5
1-Mn and 2-Mn in phosphate buffer of pH 7.4 was prepared by
mixing 25 L of a 200 M solution of 1-Fe and 475 L of 0.05 M
phosphate-citrate buffer, pH 3, and 25 L of a 200 M solution of
1-Mn, 2-Mn, and 475 L of 0.05 M phosphate buffer, pH 7.4.
M solution of 1-Fe in phosphate-citrate buffer, pH 3, and 1-
Mn–, 2-Mn–7A3, and in phosphate buffer, pH 7.4, was obtained
in a similar way by replacing 268 L of 0.05 M phosphate-citrate
buffer, pH 3, by 268 L of a 14 M solution of antibody 7A3 in
lM solution of 1-Fe in phosphate-citrate buffer of pH 3, or
l
l
l
l
l
l
4.2.3. Insertion of iron into free base 1
5
l
The insertion of iron into 1 was achieved by heating the free base
porphyrin (20 mg, 0.017 mmol) and FeCl2ꢀ4H2O (33 mg, 10 equiv) at
120 °C in DMF under inert atmosphere for one night.14 Quantitative
insertion of iron into the starting material was confirmed by UV–vis-
ible monitoring. When the reaction was over, the solvent was evap-
orated under reduced pressure and the obtained solid was dissolved
in water. The aqueous solution was then applied first to a dialysis
membrane (Float-a-Lyzer pore size = 500) for four days and, second,
to a Na+-activated cation exchange column (AG-50W-X8). Finally
freeze-drying afforded the complex 1-Fe as dark purple solid
(6.3 mg, yield = 30%). UV–visible: kmax (nm) in (0.01 M HCl): 393
l
l
l
50 mM phosphate buffer, pH 7.4. The UV–visible spectra of both
solutions were then recorded between 300 and 750 nm.
4.4.2. Titration of antibody 7A3 by the metalloporphyrin
cofactors, 1-Fe, 1-Mn, and 2-Mn
Increasing amounts (from 0 to 1.5 M equiv) of a 14
of antibody 7A3 in 50 mM phosphate buffer, pH 7.4, were added to
a sample cuvette that contained a mixture of 25 L of a 200
solution of cofactor 1-Fe, 1-Mn, or 2-Mn in 0.1 M phosphate-cit-
rate buffer, pH 3, and 475 L of 0.1 M phosphate-citrate buffer,
lM solution
(Soret,
e
= 1 ꢂ 105 molꢁ1 L cmꢁ1), 528, 680. HR-MS (MALDI-MS):
l
lM
m/z: [M]+ = 1235.39 (exact mass = 1235.12 g molꢁ1).
l
4.2.4. Insertion of manganese into free base 1 and into 5,10,15-
tris(4-N-methylpyridiniumyl)-20-(4-phenyl(3-O-
pH 3. The absorbance of the solution AS was recorded between
250 and 750 nm, 5 min after each addition. The same experiment
amidoxymethylestradiol)) porphyrin 2
was performed with 268 lL of 0.05 M phosphate-citrate buffer,
Manganese insertion into 1 was achieved by heating the free
base porphyrin (23.5 mg, 0.019 mmol) and MnCl2 (25 mg,
10 equiv) at 120 °C in DMF under inert atmosphere for three
days.16 Quantitative insertion of manganese into the starting mate-
rial was confirmed by UV–visible monitoring. When the reaction
was over, the solvent was evaporated under reduced pressure
and the obtained solid was dissolved in water. The aqueous solu-
tion was then applied first to a dialysis membrane (Float-a-Lyzer
pore size = 500) for four days and, second, to a Na+-activated cation
exchange column (AG-50W-X8). Finally freeze-drying afforded the
complex 1-Mn as dark purple solid (7.9 mg, yield = 34%). UV–visi-
pH 3, instead of the 7A3 solution, to measure the absorbance of
the cofactor alone A1. After correction of the dilution induced by
the volume of cofactor added, the difference
D
A = As ꢁ A1 at
420 nm (1-Fe) or 470 nm (1-Mn or 2-Mn) was plotted as a function
of the number of equivalents of 7A3 added. This allowed the deter-
mination of the stoichiometry of the complex formed by 1-Fe, 1-
Mn, or 2-Mn with the antibody 7A3.
4.4.3. Determination of the dissociation constants of the 1-Fe–,
1-Mn–, and 2-Mn–7A3 complexes
To calculate the dissociation constant KD of the 1-Fe–, 1-Mn–,
or 2-Mn–7A3 complexes, the two estradiol-binding sites (S) of
the antibody were considered as being independent from each
other.
ble: kmax (nm) in H2O: 469 (Soret,
e
= 0.96 ꢂ 105 molꢁ1 L cmꢁ1),
517, 566, 604. HR-MS (MALDI-MS): m/z: [M]+ = 1234.24 (exact
mass = 1234.47 g molꢁ1).
5,10,15-Tris(4-N-methylpyridiniumyl)-20-(4-phenyl(3-O-amid-
oxymethylestradiol)) porphyrin 2 was prepared by coupling of
5,10,15-tris(4-pyridyl)-20-(4-aminophenyl) porphyrin with 3-O-car-
boxymethylestradiol as described previously.8 2-Mn was obtained by
reaction of MnCl2ꢀ2H2O (259 mg, 55 equiv) and the free base porphy-
rin (32 mg, 0.029 mmol) and NaOH (1 M) under argon at 50 °C during
72 h. The mixture was acidified and the porphyrin was precipitated
with NH4PF6 (96 mg, 20 equiv). Then the precipitate was centri-
fuged and redissolved in acetone. 2 mL of a concentrated solution of
tetra-butylammonium chloride (TBAC) (100 g/L) were added and
the precipitate was centrifuged, washed with acetone, dissolved in
methanol, and then dried. 2-Mn was obtained as dark purple solid
(29.8 mg, yield = 95%). UV–visible: kmax (nm) in H2O: 378, 401, 428,
The dissociation constant KD can then be written as follows:
½n-Mꢃ ꢀ ½Sꢃ
KD
¼
ð4Þ
½n-M ꢁ Sꢃ
where n-M = 1-Fe, 1-Mn, or 2-Mn, [S] stands for the concentration
of the free recognition sites of the antibody, and [n-M-S] for the
concentration of recognition sites engaged in a complex with the
cofactor n-M.
For each concentration of cofactor added to the antibody, the
following two equations can be written:
465 (Soret,
e
= 96 ꢂ 103 molꢁ1 L cmꢁ1), 557, 766 HR-MS (MALDI-
½n-Mꢃ0 ¼ ½n-Mꢃ þ ½n-M-Sꢃ
½Sꢃ0 ¼ ½Sꢃ þ ½n-M-Sꢃ ¼ 2½7A3ꢃ0
ð5Þ
ð6Þ
MS) m/z: [M]+ = 1073.43 (exact mass = 1073.45 g molꢁ1).
4.3. Preparation of the anti-estradiol antibody 7A3
where [n-M]0 and [S]0 stand for the initial cofactor and recognition
site concentrations, respectively.
The antibody that was used as apoprotein was a monoclonal
IgG, 7A3, that was generated by immunization of mice with an
The dissociation constant KD can then be written as a function of
[n-M-S]:

1600
E. Sansiaume et al. / Tetrahedron: Asymmetry 21 (2010) 1593–1600
ð½n-Mꢃ0 ꢁ ½n-M-SꢃÞ ꢀ ð½Sꢃ0 ꢁ ½n-M-SꢃÞ
½n-M- Sꢃ
tration: 0.01 M) were then added followed by 25 lL of a 0.01 M
solution imidazole (final concentration = 0.5 mM). The oxidant, ox-
one (final concentration, 2.5 mM), was then added to the solution
KD
¼
ð7Þ
Which gives:
at a rate of 5 ꢂ 5
lL drops of 0.5 mM oxone over a period of 1 h.
ꢂ
ꢃ
KD
The same procedure was followed with the cofactor alone, and
without cofactor. The organic products were extracted with
ethyl-acetate and analyzed by GC using benzophenone as the
internal standard to determine the degree of conversion of styrene
and the nature of the products obtained. The enantiomeric excess
of the epoxide obtained was analyzed using a Dionex HPLC system
(U3000) with a peltier effect oven, and a diode array detector. The
separation was accomplished with a Chiralpak AD-H column
½Sꢃ0 ¼ ½n-M-Sꢃ ꢀ 1 þ
ð8Þ
½n-Mꢃ0 ꢁ ½n-M-Sꢃ
On the other hand, the absorbance values AS and A1 A1 mea-
sured for each of the two solutions follow the Beer–Lambert law:
AS ¼ en-M-S ꢀ ½n-M-Sꢃ ꢀ ‘ þ en-M ꢀ ½n-Mꢃ ꢀ ‘
A1 ¼ en-M ꢀ ½n-Mꢃ0 ꢀ ‘
As a result,
A ¼ AS ꢁ A1 ¼ en-M ꢀ ð½n-Mꢃ0 ꢁ ½n-MꢃÞ ꢀ ‘ ꢁ en-M-S ꢀ ½n-M-Sꢃ ꢀ ‘
en-M ꢀ ½n-M-Sꢃ ꢀ ‘ ꢁ en-M-S ꢀ ½n-M-Sꢃ ꢀ ‘
¼ ðen-M en-M-SÞ ꢀ ½n-M-Sꢃ ꢀ ‘
which gives (with ‘ = 1 cm):
DA can be linked to [n-M-S] by the following equation:
(5
l
M, 4.6 mm id ꢂ 250 mm L). The column temperature was
25 °C. The eluent was n-hexane and ethanol (98:02) with a flow
rate of 1 mL/min, and with a injection volume of 2
2 mg/mL).
D
lL (around
¼
ꢁ
References
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ꢁ
A
en-M-S
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½n-M-Sꢃ ¼
ð9Þ
ð
en-M
Þ
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!
D
ꢁ
A
KD
½n-Mꢃ0 ꢁ
½Sꢃ0 ¼
ꢀ
en-M-S
1 þ
D
ꢁ
A
en-M
en-M
en-M-S
6. Collot, J.; Gradinaru, J.; Humbert, N.; Skander, M.; Zocchi, A.; Ward, T. R. J. Am.
Chem. Soc. 2003, 125, 9030.
ꢂ
ꢃ
D
ꢁ
A
en-M-S
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¼ g
ð10Þ
en-M
e
n-M-S and en-M have been calculated with AS and A1, considering
that the addition of 0.5 equiv of 7A3/n-M cofactor, the absorption
of the antibody-cofactor solution measured is that of the 7A3–n-
M complex.
As a result, KD can be estimated with a non-linear regression of
[S]0 (2[7A3]0) with the function g
ꢀ
ꢁ
D
ꢁ
A
.
en-M
en-M-S
4.5. Catalytic activity of the metalloporphyrin–antibody 7A3
complexes
4.5.1. Sulfoxidation of thioanisole catalyzed by the metallo
porphyrin–7A3 complexes
The antibody 7A3 (7.5
Fe-, 1-Mn-, or 2-Mn (5
pH 3. 12.5 L of a 0.1 M solution of thioanisole in acetonitrile (final
concentration: 2.5 mM) and 25 L of a 0.01 M solution of imidazole
in bi-distilled water (final concentration: 500 M) were then
added. The oxidant, H2O2 (final concentration, 2.5 mM), was then
added to the solution at a rate of 5 ꢂ 5 L drops of 0.5 mM H2O2
l
M) was incubated for two hours with 1-
M) in 412.5 mL 0.05 M phosphate buffer,
l
l
l
l
l
over a period of 1 h. The same procedure was followed with the
cofactor alone, and without cofactor. The organic products were
extracted with ethyl-acetate and analyzed by GC using benzophe-
none as the internal standard to determine the degree of conver-
sion of the sulfide, and by HPLC on a Chiracel OD-H column
(iso65 hexane/propan-2-ol, 95:5; v/v) to determine the enantio-
meric excess of the sulfoxide thus obtained.
30. Hirao, H.; Kumar, D.; Thiel, W.; Shaik, S. J. Am. Chem. Soc. 2005, 127, 13007.
31. Reetz, M. T.; Rentzsch, M.; Pletsch, A.; Maywald, M. Chimia 2002, 56, 721.
32. Fernandez-Gacio, A.; Codina, A.; Fastrez, J.; Riant, O.; Soumillon, P.
ChemBioChem 2006, 7, 1013.
4.5.2. Epoxidation of styrene catalyzed by the metallo
porphyrin–7A3 complexes
The antibody 7A3 (7.5
Fe-, 1-Mn-, or 2-Mn (5
100 L of a 0.05 M solution of styrene in acetonitrile (final concen-
l
M) was incubated for two hours with 1-
33. Okrasa, K.; Kazlauskas, R. J. Chem. Eur. J. 2006, 12, 1587.
34. Collman, J. P.; Kodadek, T.; Raybuck, S. A.; Brauman, J. I.; Papazian, L. M. J. Am.
Chem. Soc. 1985, 107, 4343.
l
M) in 0.05 M phosphate buffer, pH 7.4.
l