New activities of a catalytic antibody with a peroxidase activityFormation of FeII–RNO complexes and stereoselective oxidation of sulfides Re´my Ricoux, Edyta Lukowska, Fabio Pezzotti an
Trang 1New activities of a catalytic antibody with a peroxidase activity
Formation of Fe(II)–RNO complexes and stereoselective oxidation of sulfides
Re´my Ricoux, Edyta Lukowska, Fabio Pezzotti and Jean-Pierre Mahy
Laboratoire de Chimie Bioorganique et Bioinorganique, Institut de Chimie Mole´culaire et des Mate´riaux d’Orsay,
Universite´ Paris-Sud XI, Orsay, France
In order to estimate the size of the cavity remaining around
the heme of the 3A3–microperoxidase 8 (MP8)
hemo-abzyme,the formation of 3A3–MP8–Fe(II)-nitrosoalkane
complexes upon oxidation of N-monosubstituted
hydroxyl-amines was examined This constituted a new reaction for
hemoabzymes and is the first example of fully characterized
Fe(II)–metabolite complexes of antibody–porphyrin Also,
via a comparison of the reactions with N-substituted
hyd-roxylamines of various size and hydrophobicity,antibody
3A3 was confirmed to bring about a partial steric hindrance
on the distal face of MP8 Subsequently,the influence of
the antibody on the stereoselectivity of the S-oxidation of
sulfides was examined Our results showed that MP8 alone
and the antibody–MP8 complex catalyze the oxidation of thioanisole by H2O2and tert-butyl hydroperoxide,following
a peroxidase-like two-step oxygen-transfer mechanism involving a radical–cation intermediate The best system, associating H2O2as oxidant and 3A3–MP8 as a catalyst,in the presence of 5% tert-butyl alcohol,led to the stereo-selective S-oxidation of thioanisole with a 45% enantiomeric excess in favour of the R isomer This constitutes the highest enantiomeric excess reported to date for the oxidation of sulfides catalyzed by hemoabzymes
Keywords: artificial hemoproteins; abzymes; nitrosoalcanes; microperoxidase 8; S-oxidation
Catalytic antibodies with a metalloporphyrin cofactor,or
hemoabzymes,are not as efficient a category of catalysts as
their natural hemoprotein counterparts The hemoabzymes,
which display a peroxidase activity,are characterized by
kcat/Kmvalues that are three to four orders of magnitude
lower than those for natural peroxidases [1] The relatively
low efficiency of these porphyrin–antibody complexes is
probably the result,at least in part,of the fact that no
proximal ligand of the iron has been induced in these
antibodies To avoid this problem,we decided to use,as
a hapten,microperoxidase 8 (MP8),a heme octapeptide
where the imidazole side-chain of histidine 18 acts as a
proximal ligand of the iron atom A set of six monoclonal
antibodies was thus obtained: the best peroxidase activity – that found with the complex of MP8 and one of those antibodies,3A3 – was characterized by a kcat/Kmvalue of
2· 106M )1Æmin)1,the best ever reported for an antibody– porphyrin complex [2] Active-site topology studies sugges-ted that the binding of MP8 occurred through interactions
of its carboxylate substituents with amino acids of the antibody,and that the protein provided a partial steric hindrance of the distal face of the heme [2] In addition,
it was shown recently that 3A3–MP8 was a more efficient catalyst for the nitration of phenol by NO2/H2O2 than MP8 alone,and that the antibody protein not only protected MP8 against oxidative degradations but also induced a regioselectivity of the reaction in favor of the formation of 2-nitrophenol [3] Consequently,it was tempt-ing to examine whether the hemoabzyme 3A3–MP8 was able to catalyze the selective oxidation of other substrates
In the present study,compounds containing sulfur were chosen as substrates,as they play an important role in medicine and agriculture It has been reported that 10– 15% of medicinal and veterinary products and 33% of synthetic organic pesticides contain sulfur [4] The activity
of organosulfur compounds is often modified by oxidative metabolism Indeed,enzymatic oxidation produces sulf-oxide metabolites that are chemically more reactive than the starting substrate,and which are responsible for their direct toxicity [5] Numerous peroxidases catalyze the
in vitro S-oxygenation of alkyl-aryl-sulfides,with some-times a good enantioselectivity resulting from the inter-action of sulfides with a chiral environment in the heme’s active site [6] The prevailing sulfoxide has the R absolute configuration in the presence of chloroperoxidase (CPO) [6],lactoperoxidase (LPO) [7] and myeloperoxidase (MPO)
Correspondence to J.-P Mahy,Laboratoire de Chimie Bioorganique
et Bioinorganique,UMR 8124 CNRS,Institut de Chimie
Mole´culaire et des Mate´riaux d’Orsay,Baˆtiment 420,
Universite´ Paris-Sud XI,91405,Orsay cedex,France.
Fax: + 33 1 69 15 72 81,Tel.: + 33 1 69 15 74 21,
E-mail: jpmahy@icmo.u-psud.fr
Abbreviations: CcP,cytochrome c peroxidase; CH 3
COOEt,ethyl-acetate; CiP, Coprinus cinereus peroxydase; CPO,chloroperoxidase;
HRP,horseradish peroxidase; KLH,keyhole limpet hemocyanin;
LPO,lactoperoxidase; mCPBA,meta-chloroperbenzoic acid; MP8,
microperoxidase 8; MPO,myeloperoxidase; NOS,nitric acid
synthase; RNO,Fe(II)–nitrosoalkane complex; tBuOH,tert-butyl
alcohol; tBuOOH,tert-butyl hydroperoxide.
Enzymes: catalase (EC 1.11.1.6); horseradish
peroxidase,myelo-peroxidase,lactoperoxidase (EC 1.11.1.7); chloroperoxidase
(EC 1.11.1.10); cytochrome c peroxidase (EC 1.11.1.6).
(Received 8 December 2003,revised 21 January 2004,
accepted 6 February 2004)
Trang 2[8],as catalysts,and the S configuration in the presence
of horseradish peroxidase (HRP) [9], Coprinus cinereus
peroxydase (CiP) [7],cytochrome c peroxidase (CcP) [10]
and soybean peroxygenase [11] The S-oxidation of organic
sulfides by peroxidases could involve two types of
mech-anisms (Scheme 1) The first mechanism is a one-step
oxygen transfer mechanism,with an oxygen atom being
directely transferred from Compound I to the sulfur atom
of the organic sulfide; the second mechanism is the
two-step oxygen-transfer mechanism which involves a radical–
cation intermediate
In the present report,we first used coordination chemistry
to examine the topology of the binding site of the anti-MP8
Ig,3A3,especially to evaluate the size of the cavity
remaining around the iron atom For this purpose,we
studied the formation of Fe(II)-nitrosoalkane (RNO)
complexes upon the oxidation of N-substituted
hydroxyl-amines by the 3A3–MP8 complex The first was a rather
small and hydrophilic hydroxylamine bearing a branched
alkyl group,isopropylhydroxylamine 1 [R¼ (CH3)2-CH-],
whereas the second,
N-(1-p-chlorophenylpropyl)hydroxyl-amine-2 [R¼ (p-ClPh-CH2)(CH3)CH-],was more bulky
and hydrophobic A comparison of the results obtained
with both hydroxylamines confirmed that the antibody 3A3
brought a partial steric hindrance on the distal face of MP8
Consequently,the influence of the antibody on the
stereo-selectivity of the S-oxidation of sulfides was examined For
this study,the oxidation of thioanisole by different oxidants
was performed in the presence of either MP8 alone or with
the antibody–MP8 complex acting as a catalyst The results
described here show that,in the presence of 3A3 antibody,
the S-oxidation of thioanisole by H2O2occurs with a 45%
enantiomeric excess in favour of the R isomer This
constitutes the highest enantiomeric excess reported to date
for the oxidation of sulfides catalyzed by porphyrin–
antibody complexes
Materials and methods
Preparation of MP8 MP8 was prepared by sequential peptic and tryptic digestion
of horse-heart cytochrome c (Sigma),as described previously [12] The heme content was determined using the pyridine chromogen method [12] The purity of the sample was greater than 97%,based on MALDI-TOF mass spectrometry Preparation of monoclonal antibodies
MP8 was covalently attached to keyhole limpet hemo-cyanin (KLH) and to BSA,using glutaraldehyde as a coupling agent,in 1Mbicarbonate buffer,pH 9.5,according
to Tresca et al [13] The conjugates were then purified by column chromatography on Biogel P10 Hapten–protein ratios were determined spectrophotometrically using a molar absorption coefficient value (e) of 1.49· 105
M )1Æcm)1at
407 nm for MP8 In the case of BSA,6 mol of MP8 were bound per mol of protein,whereas in the case of KLH,
22 mol of MP8 were bound per 100 000 g of protein Two, 5-week-old,female BALB/c mice were immunized with the hapten–KLH conjugate,and the mouse showing the best immune response 12 days after the third immunization was killed Its splenocytes were fused with SP2O myeloma cells,as described by Ko¨hler & Milstein [14] The resulting hybrido-mas were screened by ELISA for binding to the hapten–BSA conjugate,using peroxidase-linked goat anti-mouse Ig [15] Positive hybridomas were cloned twice and produced in large quantities Antibodies were then purified from hybridomas supernatants on a column of protein A,and their homogen-eity and purity were checked by SDS gel electrophoresis All animal experimentation was carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC)
Hydroxylamines N-isopropylhydroxylamine (hydroxylamine 1) was pre-pared by reducing the corresponding 2-nitropropane with
Zn in the presence of ammonium chloride,according to a procedure described previously [16] The characteristics of the product were found to be identical to those reported
in the literature [17] N-(1-p-chlorophenylpropyl)hydroxyl-amine (hydroxylN-(1-p-chlorophenylpropyl)hydroxyl-amine 2) was prepared in two steps First, the condensation of p-chlorobenzaldehyde with nitroethane under acidic conditions generated the corresponding 1-p-chlorophenyl-2-nitropropene [18],which was then reduced into N-substituted hydroxylamine using a lithium-alumin-ium hydride,as described previously [19]
Reaction of MP8 and 3A3–MP8 with N-monosubstituted hydroxylamines
Forty microlitres of a 10)2M solution of N-monosubsti-tuted hydroxylamine in CH3OH was added to a cuvette containing 1 mL of 0.86 lM MP8–Fe(III) or 3A3–MP8 complex [obtained by preincubation of 0.86 lM MP8– Fe(III) with 2 lMantibody 3A3 for 1 h at room tempera-ture in 0.1M NaCl/Pi (PBS),pH 7.4] The evolution of the UV-visible spectrum of the solution was monitored,as
Scheme 1 Mechanisms of oxygen transfer reactions catalyzed by
per-oxidases.
Trang 3a function of time,between 350 and 550 nm Kinetic data
were then obtained by measuring the absorbance at the
maximum of absorption of the MP8–Fe(II)–RNO complex,
as a function of time
S-oxidation of thioanisole by various oxidants catalyzed
by MP8 or the antibody–MP8 complex
Optimization of reaction conditions Thioanisole,84 lM
in 0.1M Tris buffer,pH 7.5,was oxidized at room
temperature using MP8 (0.2 lM),as a catalyst,and various
oxidants (30 lM),such as H2O2, meta-chloroperbenzoic
acid (mCPBA) or tert-butyl hydroperoxide (t-BuOOH),in
the presence of 10% of various organic solvents [methanol,
CH3CN,tert-butyl alcohol (t-BuOH)] The reactions were
initiated by adding the oxidant,and the rate of S-oxidation
was observed by monitoring the decrease in the absorbance
at 254 nm for 10 min The concentrations of product
formed after 10 min were calculated using a mMabsorption
coefficient (e¼ 7.87 mM )1Æcm)1at 254 nm) in the
differ-ence spectrum between the sulfide and the corresponding
sulfoxide
Stereoselective oxidation of thioanisole Standard
incu-bations (total volume,0.5 mL) were performed at room
temperature in Tris buffer (0.1M,pH 7.5) containing
thioanisole (100 lM) and the catalyst,either 0.3 lMMP8
alone or 3A3–MP8 prepared by preincubation of 0.3 lM
MP8 with 0.87 lMantibody for 1 h at room temperature
An oxidant – H2O2,t-BuOOH or mCPBA (final
concentration,50 lM) – was then added dropwise to the
solution at a rate of 20· 5 lL drops over a period of
1.5 h The reaction was quenched by the addition of
excess of Na2SO3 The organic products were then
extracted with ethyl-acetate (CH3COOEt) and analyzed
by GC (to determine the degree of conversion of the
sulfide) and by HPLC on a Chiracel OD-H column
(iso-hexane/propan-2-ol; 95 : 5; v/v) to determine the
enantio-meric excess of the sulfoxide thus obtained
Results and discussion
Reaction of MP8 and 3A3–MP8 complexes with N-monosubstituted hydroxylamines
The addition of the N-monosubstitued hydroxylamines 1
or 2 (350 lM) to a solution of MP8–Fe(III) (0.86 lM), preincubated for 1 h at room temperature in 0.1MNaCl/Pi,
pH 7.4,with antibody 3A3 (2 lM), led to new complexes, 3a and 3b,respectively,characterized by an absorption spec-trum similar to those observed for the MP8–Fe(II)–RNO complexes,with aborption maxima at approximately 413 and 530 nm (Table 1) [20] The reactivity of these new complexes was very similar to that of the MP8–Fe(II)–RNO complexes and that of other already reported hemoprotein– Fe(II)–RNO complexes [20],in that (a) they were stable for 2 h in 0.1MNaCl/Pi,pH 7.4,in the presence of 1 mM sodium dithionite,and (b) conversely,they were rapidly destroyed upon the addition of 100 lM potassium ferri-cyanide [Fe(CN)6K3],with regeneration of the 3A3–MP8– Fe(III) complex This strongly suggested a 3A3–MP8– Fe(II)–RNO structure for these new complexes 3a and 3b Such a structure was confirmed by the following result,that the addition of 2 lM 3A3 to a solution of MP8–Fe(II)– (CH3)2NO,prepared previously by reaction of hydroxyl-amine 1 (350 lM) with 1 lMMP8 in 0.1MNaCl/Pi,pH 7.4, led to a spectrum that was almost identical to that of 3a (data not shown) Consequently,the above results show that the oxydation of N-monosubstituted hydroxylamines
in the presence of the hemoabzyme 3A3–MP8–Fe(III) leads
to the formation of 3A3–MP8–Fe(II)–RNO complexes This constitutes a new reaction of hemoabzymes,and is also the first example of an iron(II)–metabolite complex among the familly of porphyrin–antibody complexes Such com-plexes constitute good models for those formed not only
in vitro,but also in vivo,during the oxidative metabolism of drugs containing an amine function,such as amphetamine
or macrolids [20],and which lead to an inhibition of the catalytic functions of cytochrome P450 In addition,the
Table 1 UV-visible characteristics of microperoxidase 8 (MP8) and 3A3–MP8–RNO complexes and kinetic constants for their formation by reaction
of MP8 and 3A3–MP8 with N-substituted hydroxylamines.
Fe(II)–RNO complex R ¼
UV-visible
k max (nm), e (m M )1 Æcm)1)a k(min)1)d C 50 (l M )e k(min)1) C 50 (l M )
414 (96),532 413 (80),530 0.32 ± 0.03 285 ± 5 0.19 ± 0.02 565 ± 5
413 (77),530 413 (50),530 0.77 ± 0.04 300 ± 5 0.72 ± 0.02 285 ± 5
a
Calculated from the absorbance at 413 nm after reaction of 400 equivalents of RNHOH with 0.86 l M MP8–Fe(III) and 2 l M antibody 3A3 in 0.1 M NaCl/P i (PBS) buffer,pH 7.4.bRicoux et al 2000.cThis work.dk-values were calculated from the curves in Fig 1 which were fitted to pseudo first-order kinetics according to the equation: C ¼ C max (1-e –kt ),using Kaleidagraph e The RNHOH concentration which leads to 50% conversion of MP8 or 3A3–MP8 into the corresponding Fe(II)–RNO complex.
Trang 4above results validate the use of hemoabzymes as a
convenient model for hemoproteins used in toxicology
and pharmacology,such as cytochrome P450,peroxidases
and nitric oxide synthase (NOS) It is probable that the
mechanism of formation of these complexes is similar to
that described for the formation of the MP8–Fe(II)–RNO
complexes and for the Fe(II)–RNO complexes of
hemo-proteins (Scheme 2) It should involve,first,a one-electron
reduction of the Fe(III) into Fe(II) by the monosubstituted
hydroxylamine to give the RNHOH•+ radical cation A
second,one-electron oxidation could then be achieved using
O2 which,after losing two protons,should produce the
nitrosoalkane RNO that binds to MP8–Fe(II)
The values of the molar extinction coefficients at
413 nm for the 3A3–MP8–Fe(II)–RNO complexes have
been calculated according to Ricoux et al [20] (Table 1)
From Table 1,it is clear that (a) the e-values depend on
the nature of the R substituent of the hydroxylamine and
(b) for both hydroxylamines,the e-values are lower for the
3A3–MP8–Fe(II)–RNO complexes than for their MP8–
Fe(II)–RNO counterparts Indeed,when R¼ (CH3)2
CH-,calculated e-values are 77 mM )1Æcm)1 for the
MP8–Fe(II)–RNO complex and 50 mM )1Æcm)1 for the
3A3–MP8–Fe(II)–RNO complex Similarly,when R¼
(Cl-Ph-CH2)(CH3)CH-,a larger e-value is found for the
MP8–Fe(II)–RNO complex (96 mM )1Æcm)1) than for the
3A3–MP8–Fe(II)–RNO complex (80 mM )1Æcm)1) Overall,
the minor changes observed when comparing the spectral
characteristics of the 3A3–MP8–Fe(II)–RNO complexes
with those of the MP8–Fe(II)–RNO complexes (i.e
almost no shift and a slightly lower absorbance of the
soret band) have already been observed when inserting
MP8 into 3A3 [2] They are consistent with the insertion
of the MP8–Fe(II)–RNO complex into a hydrophobic
pocket with no change of the Fe(II) spin state and no
replacement of any of the two axial ligands of the iron,
His18 or RNO,by an amino acid side-chain of the
antibody protein
Binding site topology of antibody 3A3
Figure 1 shows the changes in the concentration of the
Fe(II)–RNO complex,formed upon addition of RNHOH
to MP8–Fe(III) or 3A3–MP8–Fe(III),as a function of time
From this figure,it appears that the formation of the Fe(II)–
RNO complexes follows pseudo first-order kinetics,and
that the formation rate of MP8–Fe(II) or 3A3–MP8–
Fe(II)–RNO complexes depends on the hydroxylamine
structure (Fig 1,Table 1) Interestingly,in both instances,
Fe(II)–RNO complexes derived from the smaller aliphatic hydroxylamine (1),formed more rapidly than those derived from the more bulky aromatic hydroxylamine (2) Indeed, rate constants of 0.77 ± 0.04 min)1 and 0.72 ± 0.02 min)1,and of 0.32 ± 0.03 min)1and 0.19 ± 0.02 min)1 could be calculated,respectively,for hydroxylamines 1 and
2 in the case of MP8 and 3A3–MP8 In addition,it is clear from these values that,for both hydroxylamines,the rate of complex formation is lower in the presence of the antibody,with a decrease in the rate constant of 7% being observed with hydroxylamine 1 and of > 40% with the more bulky hydroxylamine 2
The amount of Fe(II)–RNO complex formed after adding increasing concentrations of hydroxylamine 1 or 2
to a solution of either 0.86 lMMP8 or 0.86 lM3A3–MP8
in 0.1M NaCl/Pi buffer,pH 7.4,was determined using UV-visible spectroscopy With both hydroxylamines 1 and 2,the amount of Fe(II)–RNO complex increased with the RNHOH concentration,MP8 and 3A3–MP8 being totally converted into the corresponding Fe(II)–RNO complex at concentrations higher than 3 mM However,the concentra-tion necessary to convert 50% of MP8 or 3A3–MP8 (0.86 lM) into the Fe(II)–RNO complex (C50) also depen-ded on the hydroxylamine structure (Table 1) Indeed, whereas very similar concentrations of hydroxylamine 1 and 2 were needed to convert 50% of 0.86 lMMP8 into the corresponding Fe(II)–RNO complex (300 ± 5 lMand
285 ± 5 lM,respectively) (Table 1),a much higher con-centration of hydroxylamine 2 (C50¼ 565 ± 5 lM) than
of hydroxylamine 1 (C50¼ 360 ± 5 lM) was needed to convert 50% of 3A3–MP8 (0.86 lM) into the corresponding Fe(II)–(pCl-Ph)NO complex
From the results presented above,it first appears that the N-substituted hydroxylamine carrying a ramified donating
Fig 1 Time dependence of the formation of MP8- or 3A3–MP8–RNO complexes for the reaction of 0.86 l M MP8 or 0.86 l M MP8, associated with 2 l M antibody 3A3, with 333 l M RNHOH in 0.1 M NaCl/P i (PBS) buffer, pH 7.4 The concentration of Fe(II)–RNO complex,as a function of time,is shown Besides the points corresponding to experimental values,the indicated curves represent fit of the data to pseudo first-order kinetics,calculated from C ¼ C max (1-e–kt),using
KALEIDAGRAPH 3.0.2,where C is the concentration of Fe(II)–RNO complex formed at a given time and C max is the maximum concen-tration of Fe(II)–RNO complex formed MP8,microperoxidase 8; RNO,Fe(II)–nitrosoalkane complex.
Scheme 2 Mechanism of the formation of 3A3–MP8Fe(II)–RNO
complexes and oxidation of these complexes by potassium ferricyanide.
Trang 5alkyl group, N-isopropyl-hydroxylamine-1,is more reactive
than hydroxylamine 2, which is substituted by an
electro-attractive p-chlorophenyl group Indeed,it leads to the
highest rate constant and the lowest concentration necessary
to convert them into an Fe(II)–RNO complex (C50),with
either MP8 or 3A3-MP8 (Fig 1,Table 1) Second,with
both hydroxylamines,a decrease in the reaction rate,as well
as an increase in the C50value,are observed with the 3A3–
MP8 complex,when compared with MP8 alone These
phenomena are particularly important in the case of the
more bulky and hydrophobic N-substituted hydroxylamine
2,as the reaction rate decreases by a factor of 1.7 while the
C50value increases by a factor of 2 (Table 1) This suggests
that,although the antibody 3A3 does not prevent the
binding of a ligand,such as nitrosoalcane,to the iron of
MP8,it brings a partial steric hindrance on the distal face of
MP8 and thus controls access of the nitrosoalcane ligand
to the iron atom of MP8 Such a phenomenon has already
been observed in the case of hemoproteins having a narrow
active site,like hemoglobin,myoglobin and catalase,which
are unable to form Fe(II)–nitrosoamphetamine complexes,
and whose active site only enables the access of small
molecules,such as methyl and propylhydroxylamine,to the
heme iron [21] This is also consistent with our previously
reported results,which showed that the antibody protein
induced a regioselectivity of the nitration of phenol by
NO2/H2O2catalyzed by 3A3–MP8 in favor of the
forma-tion of 2-nitrophenol [3] Considering these findings,it was
reasonable to envision that the hemoabzyme,3A3–MP8,
could catalyze the selective oxidation of other substrates,
such as compounds containing sulfur,which are known to
play an important role in medicine and agriculture
Sulfoxidation of thioanisole
In a first experiment,H2O2(final concentration,30 lM) was
added to a solution of 84 lMthioanisole and 0.2 lMMP8 in
0.1M Tris buffer,pH 7.5,at room temperature The
concentration of product formed after 10 min was
calcula-ted as described in the Materials and methods The reaction
was quenched by the addition of excess Na2SO3,and the
organic products were then extracted with CH3COOEt and
analyzed by GC The only product formed,with a 2.5%
yield,was the corresponding sulfoxide that was identified by
comparison with an authentic sample (Fig 2,Table 2)
The involvement of the iron atom of MP8 in the catalysis
was indicated by the 100% inhibition of the reactions
performed in the presence of 100 mM CN– (data not
shown) Indeed,CN–anions are known to bind strongly to
the Fe(III) of MP8 [22],replacing the labile H2O ligand
in the sixth coordination position of the iron,to produce
a very stable and catalytically inactive hexacoordinate
MP8Fe–CN complex The effect of radical scavengers was also investigated The reaction was performed under the conditions described above,but in the presence of 200 lM ascorbic acid that quenches free radicals (data not shown) Under those conditions,a 100% inhibition of the sulfoxi-dation was observed,which means that,in this instance,a peroxidase-like mechanism was involved (Scheme 1) Optimization of reaction conditions
Before the 3A3–MP8 complex was assayed as a catalyst for the S-oxidation of thioanisole,the reaction conditions were optimized with MP8 alone acting as a catalyst For this purpose,thioanisole,84 lM in 0.1M Tris buffer,pH 7.5, was oxidized at room temperature with the use of MP8 (0.2 lM) as a catalyst,and various oxidants (30 lM),such
as H2O2,mCPBA or t-BuOOH,in the presence of 10% various organic solvents (methanol,CH3CN,t-BuOH) The reactions were initiated by adding the oxidant,and the concentrations of product formed after 10 min were calcu-lated as described in the Materials and methods The values thus obtained are compared in Table 2 It first appeared that H2O2 was the best oxidant for the sulfoxidation of thioanisole,as it produced the best yield in sulfoxide, regardless of the solvent used When tBuOOH was used in the buffer alone,no oxidation was observed However,in the presence of organic solvents,sulfoxide was produced, but in a lower yield than when using H2O2as an oxidant Finally,whatever the conditions,no sulfoxide was formed when mCPBA was used as an oxidant,which confirmed that the reaction occurred through a peroxidase two-step oxygen-transfer mechanism,involving a radical–cation intermediate,and not by a one-step oxygen-transfer mech-anism (Scheme 1) With both H2O2 and tBuOOH,the S-oxidation of thioanisole was more efficient in the presence
of an organic solvent,the best of which was t-BuOH As
it has been reported previously that (a) the addition of alcohols to the reaction buffer increased the rate of peroxidase-catalyzed asymmetric sulfoxidation of thioani-sole,owing to a better solubilization of the thioanisole substrate [23],and (b) the addition of 20–50% of organic solvent,such as methanol (v/v),to solutions of MP8 in water decreased the formation of MP8 dimers and aggre-gates [12],the increased concentration of sulfoxide could arise from the combination of two effects resulting from the addition of t-butyl alcohol to the reaction medium,namely,
a better solubilization of thioanisole and an increase in the catalytically active monomeric form of MP8
Fig 2 Activators and inhibitors of the sulfoxidation catalyzed by 3A3–
MP8 MP8,microperoxidase 8.
Table 2 Concentration of product for the S-oxidation of thioanisole by
H 2 O 2 , t-BuOOH or meta-chloroperbenzoic acid (mCPBA) in the pres-ence of various organic solvents, with 0.2 l M microperoxidase 8 (MP8)
as the catalyst.
Oxidant
PhSOCH 3 (%) Buffer alone
+ 10%
methanol
+ 10%
CH 3 CN
+ 10% t-BuOH
Trang 6Stereoselective S-oxidation of thioanisole
As the above results showed that the best system for the
S-oxidation of thioanisole associated H2O2as an oxidant
with MP8 as a catalyst in the presence of tBuOH as an
organic co-solvent (Table 2),the stereoselective S-oxidation
of thioanisole (100 lM) was performed at room
tempera-ture,in 0.1M Tris buffer,pH 7.5,containing 5% t-butyl
alcohol,in the presence of either 0.3 lM MP8 alone or
0.3 lMantibody–MP8 complex acting as a catalyst
More-over,H2O2 (final concentration of 50 lM) was added
dropwise to this solution,at a rate of 20· 5 lL drops over a
period of 1.5 h,in order to avoid a too high concentration
of oxidant in the reaction medium [6] This was
implemen-ted not only to limit the degradation of the catalyst,but also
to avoid a direct reaction of the sulfide with H2O2that could
lead to racemic sulfoxide The reaction was then quenched
by the addition of excess Na2SO3,and the organic products
were then extracted with CH3COOEt and analyzed by GC
and HPLC,as described in the Materials and methods The
results shown in Table 3 show that the antibody–MP8
complex is a more efficient catalyst than MP8 alone,either
with or without 5% tBuOH,and generates sulfoxide yields
of 30% and 49%,respectively,under these conditions,
whereas MP8 alone generates yields of 10% and 23%,
respectively,under the same conditions The yields did not
exceed 49%,even in the best case,because an oxidative
degradation of the catalyst occurred This was shown by a
progressive disappearance,in its absorption spectrum,of
the soret band at 396 nm that is characteristic of the heme
moiety This degradation was less important in the case of
the antibody–MP8 catalyst,which showed that the antibody
protected the heme against oxidative degradation and led to
higher yields in sulfoxide In addition,whereas almost no
enantiomeric excess is observed in the presence of MP8
alone,an important enantiomeric excess is observed with 3A3–MP8 used as a catalyst,with the best value of 45% obtained in favor of the R enantiomer in the presence of 5% tBuOH These results confirm the important role of the antibody,previously observed [2,3]: a protection of MP8 against oxidative degradation,which leads to a higher sulfoxide yield,and a steric hindrance on the distal face of the heme,which significantly increases the enantioselectivity
of thioanisole’s S-oxidation
Table 3 also compares the yields and enantiomeric excess obtained for S-oxidation of thioanisole with various hemo-proteins used as catalysts With the exception of CcP [10], which does not catalyze this reaction,all other peroxidases led to yields ranging from 80 to 100%,higher than that achieved with 3A3–MP8 CPO was the best catalyst and produced the (R)-sulfoxide with a 100% yield and a 98% enantiomeric excess [6] Most other fungal and plant peroxidases,such as HRP [9] and CiP [7],for which the crystal structures and the protein sequence are known
to be quite homologous,produced the (S)-sulfoxide with respective enantiomeric excess of 46 and 73% The mammalian peroxidases,MPO and LPO,which are also quite homologous in protein sequence [24],both produced the (R)-sulfoxide with respective enantiomeric excesses of 8 and 80%,like 3A3–MP8 Thus,although not as efficient as peroxidases themselves,3A3–MP8 constitutes an interesting model system for hemoproteins,especially for mammalian peroxidases,because it also leads to the oxidation of thioanisole into the (R)-sulfoxide,like these enzymes In addition,the enantiomeric excess (45%) represents the highest percentage reported,to date,for the oxidation of sulfides catalyzed by porphyrin–antibody complexes: the only other example is the stereoselective sulfoxidation of thioanisole by iodosylbenzene,catalyzed by a Ru(II)– porphyrin–antibody (SN 37.4),which produced the S-enantiomer sulfoxide with a 43% enantiomeric excess [25] Our results thus validate the use of the hemoabzyme 3A3–MP8 as a catalyst for the selective oxidation of interesting substrates such as alkanes and alkenes
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Catalyst
Yield
(%)
Enantiomeric excess (%) Configuration Reference
MP8 + 5%
t-BuOH
3A3–MP8 +
5% tBuOH
SN 37.4–
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