Bovine serum albumin-cobalt(II) Schiff base complex hybrid: An efficient artificial metalloenzyme for enantioselective sulfoxidation using hydrogen peroxide

Article abstract of DOI:10.1039/c5dt04507j

An artificial metalloenzyme (BSA-CoL) based on the incorporation of a cobalt(ii) Schiff base complex {CoL, H₂L = 2,2′-[(1,2-ethanediyl)bis(nitrilopropylidyne)]bisphenol} with bovine serum albumin (BSA) has been synthesized and characterized. Attention is focused on the catalytic activity of this artificial metalloenzyme for enantioselective oxidation of a variety of sulfides with H₂O₂. The influences of parameters such as pH, temperature, and the concentration of catalyst and oxidant on thioanisole as a model are investigated. Under optimum conditions, BSA-CoL as a hybrid biocatalyst is efficient for the enantioselective oxidation of a series of sulfides, producing the corresponding sulfoxides with excellent conversion (up to 100%), chemoselectivity (up to 100%) and good enantiomeric purity (up to 87% ee) in certain cases.

Full text of DOI:10.1039/c5dt04507j

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Bovine serum albuminꢀcobalt(II) Schiff base complex hybrid:
an efficient artificial metalloenzyme for enantioselective
sulfoxidation using hydrogen peroxide
Jie Tang,ac Fuping Huang,a Yi Wei,a Hedong Bian,* ab Wei Zhanga and Hong Liang∗ a
An artificial metalloenzyme (BSAꢀCoL) based on the incorporation of cobalt(II)
Schiff base complex｛CoL, H2L = 2,2'ꢀ[(1,2ꢀethanediyl)bis(nitrilopropylidyne)]bisphꢀ
enol｝with bovine serum albumin (BSA) has been synthesized and characterized.
Attention is focused on the catalytic activity of this artificial metalloenzyme for
enantioselective oxidation of a variety of sulfides with H2O2. The influences of the
parameters such as pH, temperature, concentration of catalyst and oxidant on the
thioanisole as model are investigated. Under optimum conditions, BSAꢀCoL as hybrid
biocatalyst is efficient for the enantioselective oxidation of a series of sulfides,
producing corresponding sulfoxides in excellent conversion (up to 100%),
chemoselectivity (up to 100%) and good enantiomeric purity (up to 87% ee) in certain
cases.
Introduction
Optically active sulfoxides are useful building blocks as intermediates,1 auxiliaries2ꢀ3
*aState Key Laboratory for the Chemistry and Molecular Engineering of Medicinal
Resources (School of Chemistry and Pharmacy, Guangxi Normal University),
Guilin, 541004, P. R. China. Email: huangfp2010@163.com
*bSchool of Chemistry and Chemical Engineering, Guangxi University for
Nationalities, Key Laboratory of Chemistry and Engineering of Forest Products,
Nanning, 530008, P. R. China. Eꢀmail: gxunchem@163.com
cGuilin Normal College, Guilin 541001, P. R. China.

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and ligands4 in modern organic synthesis.5 They are also constituted important
biologically active compounds6 in the pharmaceutical industry.7ꢀ9 During the past few
decades, intense effort has been devoted to the development of various synthetic
methods toward enantioenriched sulfoxides.10 Among the available approaches
developed, it is widely appreciated that the asymmetric oxidation of corresponding
prochiral sulfide is the most powerful and reliable route.3,10
Many methods including different catalysts (natural enzymes,11ꢀ13 organocatalysts14
and metal complexes15) have found most applications in the asymmetric oxidation of
prochiral sulfides;16 high level enantioselectivity, satisfactory conversion and yield
have already been achieved.17ꢀ18 However, each system has obvious limitations that
include tedious separation process, relatively toxic reaction conditions or expensive
chiral metal complexes.10,19 Artificial metalloenzyme which is a promising new kind
of ecoꢀcompatible hybrid biocatalysts, represents an attractive alternative for
enantioselective catalysis.20ꢀ21 They are not only able, like enzymes, to work under
mild conditions with high activity, 22ꢀ24 but can also be stably used in a wide range of
substrates like homogeneous catalysts.25 For example, Viktor Mojr and coꢀworkers26
reported that smaller αꢀcyclodextrin conjugate was a suitable catalyst for the oxidation
of nꢀalkyl methyl sulfides while the larger βꢀcyclodextrin conjugates were optimal
catalysts for the oxidation of sulfides carrying bulkier substituents.
Artificial metalloenzymes are resulting from the introduction of a metalꢀcomplex
with catalytic activity into macromolecular hosts27ꢀ30 (such as a protein, DNA or an
antibody) with covalent31ꢀ33 or coordination bonds,7,20 as well as using supramolecular
anchoring.34ꢀ36 Therefore, various proteins (e.g., myoglobin, serum protein, avidin or
streptavidin) providing a chiral cavity21 as effective chiral hosts widely used for
synthesis of artificial metalloenzymes.34 Yoshihito Watanabe et al.37ꢀ40 employed
apoꢀMb and metal (Schiff base) complexes in preparing hybrid metalloproteins and
those were able to catalyze enantioselective sulfoxidation. Kyohei Watanabe and
coꢀworkers41 reported that the recombinant human serum albumin (HSA)
mutantꢀheme complex showed high peroxidase activity. In Valentina Oliveri’s
study,42 a novel artificial superoxide dismutase enzyme based on the nonꢀcovalent

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conjugation of bovine serum albumin (BSA) and the manganese(III) complex was
investigated.
In the context of supramolecular anchoring of metal cofactors within a host protein,
the serum albumin play a key role.43 Among them, BSA is attractive because of its
abundance, stability, low cost, ease of purification, excellent water solubility and
different substrate accessibility.38,44 However, the metalloenzymes using BSA as
protein scaffold generally display low activity in asymmetric sulfide oxidation. Atif
Mahammed et al.35 have reported that the BSAꢀconjugated manganese complex as the
catalyst for the enantioselective oxidation of prochiral sulfides obtaining good
enantioselectivity (up to 74% ee) but very low yield (16%). The incorporation of a
VOSO4 complex into BSA by Anca Pordea and coꢀworkers21 afforded a racemic
mixture in the asymmetric oxidation of thioanisole.
Schiff base complexes as cofactor for artificial metalloenzyme play an important
role in asymmetric catalysis due to their catalytic active properties10 and easy methods
of syntheses.45 In contrast to the remarkable success obtained of manganese46ꢀ48
,
iron49ꢀ50 vanadium22,51 metal complexes, few studies have demonstrated the catalytic
role of cobalt complexes in enantioselective sulfoxidation.
Previously, various strong oxidants were used for the oxidation of sulfides.52ꢀ55
However, these oxidants not only corroded the equipment but also generated large
amounts of wastes during the reaction process.56 As an alternative, hydrogen peroxide
(H2O2) is one of the most noteworthy “green oxidants” offering the advantages of
being abundant,8 cheap,21 relatively safe,50 atomꢀeconomical11 and environmentally
benign with the formation of water as the only byꢀproduct.57
Considering these remaining challenges and endeavoring to guide principles of
green chemistry, in this paper, we designed a BSAꢀcobalt(II) Schiff base complex
hybrid as biocatalyst for asymmetric sulfoxidation. The influence of parameters
through optimizing reaction conditions was studied using thioanisole as a model
substrate in the presence of “green oxidant” H2O2. Comparing with related
biomimetic systems,22ꢀ24,26 this artificial metalloenzyme provided efficient catalytic
activity for a broad panel of prochiral sulfides. In addition, it avoided a common

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problem which was overꢀoxidation of the sulfoxides to sulfones.58 Especially for
thioanisole, overꢀoxidation to sulfone was either entirely absent or very minimal in
most cases.
Experimental section
Materials
All starting chemicals were commercially available reagents of analytical grade and
were used without further purification. BSA was purchased from SigmaꢀAldrich. All
the sulfides were obtained from Energy Chemical. The preparation of
2,2'ꢀ[(1,2ꢀethanediyl)bis(nitrilopropylidyne)]bisphenol (H2L) was described in
previous publication.59 Cobalt(II) Schiff base complex (CoL) and BSA were dissolved
in dimethyl sulfoxide (DMSO) and 0.05 M Tris (trihydroxymethyl aminomethane)
buffer solution, pH 8.0, respectively, to prepare as standard stock solution
(concentration: 1 mM).
Physical measurements methods
UVꢀVis spectra were recorded on a Cary 100 UVꢀvisible spectrophotometer. The
FTꢀIR spectra were recorded on
a
PerkinꢀElmer Spectrum One FTꢀIR
spectrophotometer with a germanium attenuated total reflection (ATR) accessory, a
DTGS KBr detector and a KBr beam splitter ratio. ESIꢀMS (electrospray ionization
mass spectrum) spectra were recorded on a Bruker HCT Electrospray Ionization Mass
Spectrometer. HPLC experiments were carried out using UV2302II/P2302II High
1
performance liquid chromatograph. H and 13C NMR spectra were recorded on a
Bruker NMR. Optical rotations were measured with a WXGꢀ4 polarimeter.
Synthesis of CoL
A mixture of CoCl2ꢁ6H2O (0.5 mmol), H2L (0.5 mmol) and ethanol (15 ml) were
placed in a 15 ml Teflonꢀlined stainless steel vessel, heated at 80 °C for 72 h and then
cooled to room temperature at a rate of 5 °C per hour. The resulting crystalline
product was washed with ethanol and dried in air. Color: reddish brown. Yield: 60%
(based on Co(II)). Anal. Calc. for C40H44Cl2Co3N4O4: C, 53.83; H, 4.97; N, 6.28.

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Found: C, 53.63; H, 4.92; N, 6.15%. EISꢀMS (m/z): [C20H22CoN2O2]+, 381.10. FTꢀIR
(KBr phase, cmꢀ1): 3438s, 2977w, 1636s, 1540m, 1492w, 1441m, 1384m, 1329m,
1257w, 1218w, 1140w, 1101w, 1044w, 848m, 746m, 689w, 623w, 585w, 538m.
Crystal structure determination of CoL
Xꢀray structure analysis was performed at 293 K using Agilent Supernova
diffractometer (Mo, λ = 0.71073 Å). The structure was collected at 293(2) K and
solved by direct methods using SHELxsꢀ9760 and refined by fullꢀmatrix leastꢀsquares
techniques against F2 with SHELxsꢀ97.61 Anisotropic thermal parameters were
assigned to all nonꢀhydrogen atoms. The organic hydrogen atoms were generated
geometrically, the hydrogen atoms of the water molecules were located from
difference maps and refined with isotropic temperature factors. Analytical expressions
of neutralꢀatom scattering factors were employed, and anomalous dispersion
corrections were incorporated. The crystallographic data and structural refinement
details were summarized in Table 1.
Table 1 Crystal data and structure refinement for CoL
Formula
Formula weight
Crystal system
Space group
a (Å)
C20H22ClCo1.5N2O2
446.24
γ (°)
68.160 (4)
953.2 (4)
V (Å3 )
Triclinic
Z
2
Pꢀ1
Dc ( g mꢀ3)
1.555
1.087
8.327 (2)
9.764 (3)
13.148 (4)
75.049 (4)
Goodnessꢀofꢀfit on F2
b (Å)
θ range for data Collection (°) 2.3 to 26.4
c (Å)
Reflections collected/unique
10974/ 3881 [R(int) = 0.0246]
Final R indices [I > 2ó(I)]
R1 = 0.0277 ùR2 = 0.0821
R1 = 0.0360 ùR2 = 0.863
α (°)
β (°)
78.964 (4)
R indices (all data)
Synthesis of BSAꢀCoL
The design of artificial enzyme was performed by mixing BSA standard stock
solution (1 mM) with the CoL standard stock solution (1 mM) at equal concentrations

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and incubated overnight at 0 °C. The hybrid solution was dialyzed (3 cycles) in an ice
bath, leading to the separation of the proteinꢀcomplex hybrid from the nonꢀhybridised
complex solution. Then, the hybrid were purified by sephadex G75 gel
chromatography. The protein concentration was estimated by Coomassie Brilliant
Blue method62 after concentration.
Titration of CoL by a solution of BSA
Increasing amounts (from 0 to 1.5 molar equivalents, final concentration in the 0ꢀ6.75
ꢂM range) of a 1 mM solution of BSA (BSA was first solubilized in 0.05 M
phosphate buffered saline solution (PBS), pH 7.45) were added to a 4.5 ꢂM solution
of CoL in a 0.05 M PBS solution, pH 7.45. The absorbance of the solution As was
recorded between 250 and 500 nm, 5 min after each addition. The same experiment
was performed to measure the absorbance A0 of the complex CoL alone. After
correction of the dilution induced by the volume of cofactor added, the difference ꢃA
= A0 – As at 362 nm was plotted as a function of the number of equivalents of BSA
added. This allows to determine the stoichiometry of the complex CoL with BSA.50
Molecular docking of BSAꢀCoL
To understand further the right binding site and binding interaction forces of CoL on
BSA, the molecular docking of CoL with BSA was carried out using docking
software Autodock 4.2 (http://autodock.scripps.edu/, free software). The crystal
structure of BSA (PDB ID: 3V03) was obtained from the RCSB Protein Data Bank
(http://www.rcsb.org/pdb/home/home.do). Firstly, all the water molecules were
removed, and then hydrogen atoms were added to the protein structure.63 Secondly,
the partial atomic charges of BSA and CoL were calculated using Gasteigerꢀmarsili64
and Kollman methods,65 respectively. Thirdly, the different conformers of sorafenib
were generated by using Lamarkian genetic algorithm (LGA) with the same
parameters of each docking. Finally, according to the Autodock scoring function, the
dominating configuration of the BSAꢀCoL with minimum binding energy can be
obtained.66 After the molecular docking, the lowest Gibbs free energy of binding was

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illustrated by PyMol (http://www.pymol.org/).
Typical experimental procedure for enantioselective sulfoxidation reaction
A mixture of catalyst (2.7 ꢂmol) and sulfide (0.27 mmol) in 2 ml of phosphate buffer
(PB) solution (pH 5.1) was stirred in a test tube (5 ml capacity) for 5 hour at room
temperature. Then the reaction mixture was cooled to 0 °C and 30% H2O2 (1.5 equiv.)
as the oxidant was added under stirring. After another 20 hours, the solution was
quenched with sodium sulfite solution and extracted with dichloromethane (2 ml ×5).
Finally, the combined organic solutions were dried over by anhydrous sodium sulfate,
filtered, and evaporated under vacuum. The same procedure was followed with the
cofactor alone or without the cofactor. A sample of the crude reaction mixture
reꢀdissolved in a minimum amount of isopropanol before being analyzed by HPLC
with Chiralcel OBꢀH or Chiralpak ASꢀ3 (4.6Φ mm × 250 mm) column (Daicel
Chemical Industries, Tokyo) at room temperature. Further purification was achieved
by chromatography on silica gel then was taken for the NMR analysis. The
enantiomeric excess (ee) and chemoselectivity were calculated using the formulas:
ee % = [peak area (SꢀR)/(S+R)] × 100%; chemoselectivity % = [peak area
SO/(SO+SO2)] × 100%, SO = sulfoxide, SO2 = sulfone. Conversions were based on
sulfide substrate; yields were referred to isolated product after column
chromatography and were based on substrate; the absolute configurations of the
sulfoxides were assigned by comparison of HPLC elution orders and sign of optical
rotation reported in the literature.
Methyl phenyl sulfoxide. Yellow oil, purified by flash chromatography on silica
1
gel (ethyl acetate/petroleum ether, v/v = 3:1) (89%). H NMR (500 MHz, CDCl3) δ
(ppm): 7.59 (dd, J = 7.90, 1.4 Hz, 2H), 7.49ꢀ7.41 (m, 3H), 2.66 (s, 3H); 13C NMR
(126 MHz, CDCl3) δ (ppm): 145.6, 133.7, 131.0, 129.3, 127.3, 123.4, 43.9.
Determination of ee by HPLC analysis: Chiralcel OBꢀH, nꢀhexane/2ꢀPrOH (80:20),
0.8 ml/min, 254 nm, tr (S) = 14.0 min, tr (R) = 24.0 min, [α]D20 = +84.3° (c 1.0,
acetone), {ref. 67, [α]20D = +130° (c 1.7, acetone) for (R) 89% ee}.

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2ꢀChlorophenyl methyl sulfoxide. Colorless oil, purified by flash chromatography
1
on silica gel (ethyl acetate/petroleum ether, v/v = 2:3) (29%). H NMR (500 MHz,
CDCl3) δ (ppm): 7.99 (dd, J = 7.8, 1.6 Hz, 1H), 7.57 (td, J = 7.7, 1.2 Hz, 1H), 7.48 (td,
J = 7.6, 1.6 Hz, 1H), 7.43 (dd, J = 7.9, 1.1 Hz, 1H), 2.86 (s, 3H); 13C NMR (126 MHz,
CDCl3) δ (ppm): 143.6, 132.0, 129.8, 129.8, 128.2, 125.4, 41.7. Determination of ee
by HPLC analysis: Chiralcel OBꢀH, nꢀhexane/2ꢀPrOH (90:10), 1 ml/min, 254 nm, tr
20
D
20
(S) = 11.3 min, tr (R) = 22.0 min, [α]
+139.0° (c 1.0, THF) for (R) 80% ee}
= +151.2° (c 1.0, THF), {ref. 67, [α]D
=
3ꢀChlorophenyl methyl sulfoxide. Colorless oil, purified by flash chromatography
1
on silica gel (ethyl acetate/petroleum ether, v/v = 2:3) (40%). H NMR (500 MHz,
CDCl3) δ (ppm): 7.69 (t, J = 1.1 Hz, 1H), 7.53 (qd, J = 3.8, 1.3 Hz, 1H), 7.50 (td, J =
4.3, 2.0 Hz, 2H), 2.77 (s, 3H); 13C NMR (126 MHz, CDCl3) δ (ppm): 147.9, 135.8,
131.2, 130.6, 123.7, 121.6, 44.0. Determination of ee by HPLC analysis:
nꢀhexane/2ꢀPrOH (90:10), 1 ml/min, 254 nm, tr (S) = 15.0 min, tr (R) = 21.5 min,
20
20
[α]D = +93.5° (c 1.0, acetone), {ref. 67, [α] D = +128.8° (c 1.0, CHCl3) for (R) 99%
ee}.
3ꢀBromophenyl methyl sulfoxide. Colorless oil, purified by flash chromatography
1
on silica gel (ethyl acetate/petroleum ether, v/v = 2:3) (46%). H NMR (500 MHz,
CDCl3) δ (ppm): 7.84 (t, J = 1.7 Hz, 1H), 7.67ꢀ7.64 (m, 1H), 7.58 (dd, J = 7.8, 1.1 Hz,
1H), 7.43 (t, J = 7.9 Hz, 1H), 2.77 (s, 3H); 13C NMR (126 MHz, CDCl3) δ (ppm):
148.0, 134.2, 130.9, 126,5, 123.6, 122.0, 44.0. Determination of ee by HPLC analysis:
Chiralcel OBꢀH, nꢀhexane/2ꢀPrOH (80:20), 1 ml/min, 254 nm, tr (S) = 10.7 min, tr (R)
20
20
= 16.1 min, [α]D = +105.4° (c 1.0, acetone), {ref. 67, [α]D = +116.3° (c 1.2,
acetone) for (R) 97% ee}.
4ꢀChlorophenyl methyl sulfoxide. Colorless oil, purified by flash chromatography
1
on silica gel (ethyl acetate/petroleum ether, v/v = 2:3) (63%). H NMR (500 MHz,
13
CDCl3) δ (ppm): 7.63ꢀ7.60 (m, 2H), 7.55ꢀ7.52 (m, 2H), 2.74 (s, 3H); C NMR (126
MHz, CDCl3) δ (ppm): 144.2, 137.3, 131.1, 129.7, 128.9, 125.0, 44.0. Determination
of ee by HPLC analysis:
Chiralcel OBꢀH, nꢀhexane/2ꢀPrOH (90:10), 1 ml/min, 254
nm, tr (S) = 14.2 min, tr (R) = 23.7 min, [α]D20 = +76.6° (c 1.0, acetone), {ref. 67,

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[α]20D = +97° (c 2.0, acetone) for (R) 78% ee}.
4ꢀBromophenyl methyl sulfoxide. Colorless oil, purified by flash chromatography
1
on silica gel (ethyl acetate/petroleum ether, v/v = 2:3) (44%). H NMR (500 MHz,
CDCl3) δ (ppm): 7.72ꢀ7.68 (m, 2H), 7.57ꢀ7.53 (m, 2H), 2.75 (s, 3H); 13C NMR(126
MHz, CDCl3) δ (ppm): 144.9, 132.7, 132.6, 129.0, 125.5, 125,2, 44.0. Determination
of ee by HPLC analysis: Chiralcel OBꢀH, nꢀhexane/2ꢀPrOH (80:20), 0.8 ml/min, 254
nm, tr (S) = 12.2 min, tr (R) = 17.7 min, [α]D20 = +80.0° (c 1.0, acetone), {ref. 67,
[α]20D = +77° (c 1.8, acetone) for (R) 80% ee}.
4ꢀMethylphenyl methyl sulfoxide. Colorless oil, purified by flash chromatography
1
on silica gel (ethyl acetate/petroleum ether, v/v = 2:3) (91%). H NMR (500 MHz,
CDCl3) δ (ppm): 7.53ꢀ7.49 (m, 2H), 7.30 (d, J = 8.0 Hz, 2H), 2.68 (s, 3H), 2.38 (s,
3H); 13C NMR (126 MHz, CDCl3) δ (ppm): 142.3, 141.5, 130.0, 129.8, 123.5, 123.3,
43.9, 21.4. Determination of ee by HPLC analysis: Chiralcel OBꢀH, nꢀhexane/2ꢀPrOH
(50:50), 0.5 ml/min, 254 nm, tr (S) = 9.7 min, tr (R) = 16.5 min, [α]D20 = +112.3° (c 1.0,
acetone), {ref. 67, [α]20D = +150.4° (c 1.17, acetone) for (R) > 99% ee}.
4ꢀMethoxyphenyl methyl sulfoxide. Yellow oil, purified by flash chromatography
1
on silica gel (ethyl acetate/petroleum ether, v/v = 2:3) (98%). H NMR (500 MHz,
CDCl3) δ (ppm): 7.61ꢀ7.56 (m, 2H), 7.04ꢀ7.00 (m, 2H), 3.84 (s, 3H), 2.69 (s, 3H); 13C
NMR (126 MHz, CDCl3) δ (ppm): 162.0, 136.5, 129.5, 125.5, 114.9, 114.5, 55.5, 43.9.
Determination of ee by HPLC analysis: Chiralcel OBꢀH, nꢀhexane/2ꢀPrOH (80:20), 1
ml/min, 254 nm, tr (S) = 12.2 min, tr (R) = 24.6 min, [α]20D = +122.2° (c 1.0, CHCl3)
{ref. 67, [α]20D = ꢀ113.8° (c 3.09, CHCl3) for (S) 83% ee}.
Vinyl phenyl sulfoxide. Yellow oil, purified by flash chromatography on silica gel
(ethyl acetate/petroleum ether, v/v = 3:1) (23%). 1H NMR (500 MHz, CDCl3) δ (ppm):
7.57 (dt, J = 4.6, 2.7 Hz, 2H), 7.48ꢀ7.41 (m, 3H), 6.55 (m, J = 16.5, 9.6, 2.7 Hz, 1H),
6.17ꢀ5.81 (m, 2H); 13C NMR (126 MHz, CDCl3) δ (ppm): 143.3, 143.0, 132.1, 131.2,
129.4, 125.7, 124.6, 120.6. Determination of ee by HPLC analysis: Chiralcel OBꢀH,
nꢀhexane/2ꢀPrOH (90:10), 1 ml/min, 254 nm, tr (S) = 18.8 min, tr (R) = 30.1 min,
[α]20D = ꢀ115.1° (c 1.0, acetone), {ref. 67, [α]20D = ꢀ262.4° (c 1.7, acetone) for (S) 85%
ee}.

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Methyl benzyl sulfoxide. Colorless oil, purified by flash chromatography on silica
1
gel (ethyl acetate/petroleum ether, v/v = 1:1) (87%). H NMR (500 MHz, CDCl3) δ
(ppm): 7.43ꢀ7.36 (m, 3H), 7.33ꢀ7.30 (m, 2H), 4.02 (dd, J = 69.3, 12.8 Hz, 2H), 2.48 (s,
3H); 13C NMR (126 MHz, CDCl3) δ (ppm): 130.5, 130.0, 129.7, 129.2, 129.0, 128.5,
60.4, 37.3. Determination of ee by HPLC analysis: Chiralcel OBꢀH, nꢀhexane/2ꢀPrOH
(90:10), 1.5 ml/min, 210 nm, tr (S) = 13.7 min, tr (R) = 18.2 min, [α]20D = +21.7° (c 1.0,
acetone) {ref. 67, [α]20D = ꢀ33.6° (c 3.0, acetone) for (S) 58% ee}.
Methyl tertꢀButyl Sulfoxide. Colorless oil, purified by flash chromatography on
1
silica gel (ethyl acetate) (95%). H NMR (400 MHz, CDCl3) δ (ppm): (400 MHz,
CDCl3) 2.39, 2.37, 2.35, 2.34, 2.33, 2.33, 2.33, 2.31, 1.33, 1.25, 1.23, 1.23, 1.21, 1.20,
1.20, 1.19, 1.19, 1.17; 13C NMR (101 MHz, CDCl3) δ (ppm): 52.2, 31.4, 22.2.
Determination of ee by HPLC analysis: Chiralcel chiralpak ASꢀ3, nꢀhexane/2ꢀPrOH
(90:10), 1.0 ml/min, 210 nm, tr = 32.76 min, tr = 40.36 min.
Methyl nꢀOctyl Sulfoxide. White solide, purified by flash chromatography on
silica gel (ethyl acetate) (98%). 1H NMR (400 MHz, CDCl3) δ (ppm): 2.49 (1 H, td, J
7.5, 1.1), 2.09 (1 H, d, J 1.3), 1.66ꢀ1.53 (1 H, m), 1.43ꢀ1.26 (5 H, m), 0.88 (2 H, dd, J
7.2, 6.3); 13C NMR (101 MHz, CDCl3) δ (ppm): 53.6, 39.7, 37.4, 30.7, 28.1, 27.9,
27.8, 21.6, 13.1. Determination of ee by HPLC analysis: Chiralcel OBꢀH,
nꢀhexane/2ꢀPrOH (98:2), 0.6 ml/min, 210 nm, tr (S) = 26.4 min, tr (R) = 29.5 min.
[α]20D = ꢀ17.8° (c 1.0, acetone) {ref. 67, [α]25 = ꢀ71.3° (c 1, acetone) for (R) 85.1%
D
ee}.
Methyl nꢀDodecyl methyl Sulfoxide. White solid, purified by flash
1
chromatography on silica gel (10% methanol in dichloromethane) (56%). H NMR
(400 MHz, CDCl3) δ (ppm): 2.57, 2.43, 2.42, 2.40, 2.23, 2.02, 1.54, 1.52, 1.23, 1.19,
0.90, 0.88, 0.88, 0.87, 0.83, 0.81, 0.79; 13C NMR (101 MHz, CDCl3) δ (ppm): 54.5,
40.7, 34.3, 31.9, 29.6, 29.5, 29.3, 29.2, 28.8, 24.9, 22.7, 15.4, 14.1. Determination of
ee by HPLC analysis: Chiralcel OBꢀH, nꢀhexane/2ꢀPrOH (98:2), 0.5 ml/min, 220 nm,
tr (S) = 22.2 min, tr (R) = 24.4 min.

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Results and discussion
Description of the crystal structure
Complex CoL crystallized in the nonꢀcentrosymmetric space group Pꢀ1. Selected
bond lengths and bond angles were given in Table S1. Singleꢀcrystal Xꢀray
crystallographic analysis of CoL reveals a linear trinuclear Co(II) cluster (Fig. S1), in
which Co1 atom is located on a crystallographic inversion center and is coordinated
octahedrally by four O atoms from two Schiff base ligands (Co1ꢀO1 = 2.1401(15) Å,
Co1ꢀO2 = 2.1268(15) Å). Co1 is bridged to Co2 through two ꢁ2ꢀphenolatoꢀO atoms
from a Schiff base ligand and a ꢁ2ꢀCl atoms, with the Co1···Co2 distance of 2.8105(6)
Å. Co2 is coordinated to two iminoꢀN atoms as well as two ꢁ2ꢀphenolatoꢀO atoms
from a Schiff base ligand and one ꢁ2ꢀCl atom. The average CoꢀN and CoꢀO bond
lengths are 1.8795 and 1.8810 Å, respectively.
Spectroscopic characterization of the hybrid
The artificial metalloenzyme is dark brown, attesting to the presence of the Co
complex in the protein.68 Fig. S3 shows the UVꢀvisible spectra of CoL, BSA and
BSAꢀCoL hybrid at equal concentrations. The UVꢀvisible spectra of the BSAꢀCoL
hybrid is dominated by a single charge transfer (CT) band in the UV region. The
spectra show that attachment of the CoL to BSA results a shift in λmax from 279 to 274
nm accompanied by a substantial increase in its intensity. On the other hand, the
spectra of the complex in the presence of BSA depict one band in the UV region with
maxima at 366 instead of 362 nm, together with a slight increase in its intensity. In
addition, subtraction of BSAꢀCoL and BSA spectra leads to a difference spectrum that
is similar to the spectrum of CoL revealing the presence of interaction between the
CoL complex and the BSA.25,69
Insertion of the cofactor CoL in the BSA
The UV spectrum of a 4.5 ꢂM solution of CoL was recorded in 0.05 M PBS, pH 7.45,
alone or in the presence of BSA. The presence of BSA allows only a slight 4 nm shift

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of the band to be observed, from 362 to 366 nm, together with a decrease in its
intensity (Fig. S4). The slight shift of the band is in agreement with no change in the
coordination sphere of the metal42,70 Furthermore, it have been proven by several
authors that the decrease in absorbance might probably be the sign that the cofactor
inserts into a hydrophobic environment.50
In order to check the stoichiometry of the BSAꢀCoL, increasing amounts of BSA
were added to a 4.5 ꢂM solution of CoL in 0.05 M PBS, pH 7.45. The difference
between the absorbance value at 362 nm of the solution of BSAꢀCoL (As) and that of
CoL alone (A0), △A362nm, was plotted against the BSA/CoL ratio (Fig. S4 (insert)).
△A362nm clearly increases linearly with the BSA/CoL ratio until 1.0 equivalent of
BSA has been added to CoL, and then reaches a plateau. This indicates that the BSA
is able to bind one CoL.70 For a BSA/CoL ratio higher than 1.0, △A362nm slightly
decreases, which could be due to nonꢀspecific surface interaction of the CoL cofactor
with BSA in excess in the solution.50
Molecular docking of BSAꢀCoL
Molecular simulation is beneficial to further understand the interaction of CoL with
BSA. The globular protein BSA consists of three structurally similar domains and
each domain is also divided into subdomains A and B.71 The best ranked result, which
has the lowest free energy for BSAꢀCoL is shown in Fig. 1. As shown in Fig. 1(a),
CoL molecule is inserted into the hydrophobic cavity of subdomain IB (site I) of BSA.
The detailed docking results are presented in Fig. 1(b). The model shows that CoL is
surrounded by hydrophobic residues such as Pheꢀ133, Leuꢀ122, Leuꢀ115, Alaꢀ128 and
Trpꢀ134, polar residue Tyrꢀ160, as well as charge hydrophobic residues Lysꢀ136 and
Gluꢀ125. It is implied that CoL is bond to the site essentially via hydrophobic and Van
Der Waals forces.72 Similarly, P. Bourassa et al.73 also reported anticancer tamoxifen,
its metabolites 4ꢀhydroxytamoxifen or endoxifen could be bound within a
hydrophobic cavity in subdomain IB of BSA.

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Fig. 1(a) The each subdomain of BSA and the minimum energy docking conformation between
CoL with BSA. The BSA was presented by ribbon structure. CoL was presented by sphere model;
(b) The hydrophobic and hydrophilic amino acid residues surrounding the CoL within 10,000 Å;
Asymmetric sulfoxidation
In an effort to fulfill the demands of “economical and environmental” benefits,74 the
first attempt to use H2O2 as oxidant in asymmetric oxidation of sulfides was carried
out with methyl phenyl sulfide, one of the most frequently used model substrates for
initial sulfoxidation experiments (Scheme 1). As shown in Fig. 1, blank reaction with
thioanisole gave only 16% conversion with 52% chemoselectivity and the major
product was racemic. When only HSA was present, the reaction afforded 95%
conversion with 100% chemoselectivity and 14% ee of the sulfoxide, meanwhile 85%
conversion with 100% chemoselectivity and 8% ee were obtained in the presence of
BSA. This clearly demonstrates that the chiral environment provided by the protein
has a determinant influence on the stereoselective step.75 In contrast, the sulfoxide
formed with CoL alone was found to be almost a racemic mixture with an
enantiomeric excess of only 3% in favour of the R enantiomer due to the nonꢀachiral
nature of the catalyst.19 Clearly no other case studied here could match the
performance of the SAꢀCoL (BSAꢀCoL and HSAꢀCoL) in terms of enantioselectivity.
Interestingly, although BSA is homologous to HSA,76 BSA can catalyze the
sulfoxidation combined with CoL affording higher enantiomeric excess (40% ee for
the Rꢀenantiomer). These suggest that the host protein has an influence on the
enantioselection.35,46ꢀ50 This enantiomeric excess can be compared to those obtained

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for this type of reaction in the presence of some other artificial metalloenzymes. For
example, only 13% ee in favour of the S isomer was obtained in the oxidation of
thioanisole using a rather bulky ironꢀporphyrin complex associated with a
neocarzinostatin variant, NCSꢀ3.24, as catalyst. The lower enantioselectivity may be
due to the fact that the metal cofactor lay outside the binding site of the protein70. At
the opposite, the insertion of Feꢀcorrole into BSA led to 38% ee in favour of the R
isomer was reported by Atif Mahammed and Zeev Gross.35 In our case, the molecular
modelling experiments (Fig. 1(a) and Fig. 1(b)) show that the CoL molecule is
embedded in the hydrophobic environment of BSA which provides the asymmetric
environment around the catalyst. This could induce a enantioselectivity in the transfer
of the oxygen atom to the sulfur atom. It seems reasonable, therefore, to afford 40%
ee in favour of the R isomer. Additionally, the polar amino (Tyrꢀ160) acid surrounding
complex CoL (Fig. 1(b)) also enhances its reactivity and selectivity. 47
Scheme 1 Enantioselectivity of the thioanisole oxidation using aqueous 30% H2O2 as oxidant at
room temperature.
O
O
O
S
..
S
S
Catalyst
+
H2O2
+
100
100
100
100
100
90
92
95
85
90
52
16
40R)
25(R)
8(R)
3(R)
14(R)
Chemoselectivity %
Conversion %
ee %
t
s
y
l
L
o
a
t
A
a
C
S
c
ꢀ
B
o
n
Fig. 2 Screening of catalysts for oxidation of thioanisole at room temperature for 20 h. The mol
ratios of H2O2: thioanisole (0.27 mmol) were 150:100. The sulfoxide of thioanisole was in the R
configuration.

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The effect of oxidants such as tertꢀbutyl hydrogen peroxide (TBHP), sodium
hypochlorite (NaClO) and sodium periodate (NaIO4), were also tested along with 30%
aqueous H2O2, and respective data were shown in Fig. 3. All these oxidants gave fairly
good conversion (> 90%) and excellent product selectivity (100%). Remarkably, H2O2
effectively oxidized the sulfide affording the highest ee value (up to 40%). This
indicates the exclusive activation of BSAꢀCoL by H2O2 in this reaction.
S e l e c t i v i t y
%
C
o n v e r s i o n %
e e
%
1 2 0
1 0 0
8 0
6 0
4 0
2 0
0
1 0 0
1 0 0
1 0 0
1 0 0
9 5
9 3
9 2
9 2
4 0
P
O
O
l
I
O
H
C
a
B
T
a
H
N
N
Fig. 3 Screening of oxidants for oxidation of thioanisole catalyzed by BSAꢀCoL in PB (pH 5.1) at
room temperature after 20 h. The mol ratios of oxidant: thioanisole: BSAꢀCoL (2.7 ꢂmol) were
150:100:1. The sulfoxide of thioanisole was in the R configuration.
Influence of different pH
Following the results above, the reaction parameters such as pH value, substrate
concentration, oxidants or catalyst concentration, reaction temperature were
investigated in an attempt to optimize the enantioselective sulfoxidation and, in
particular, to improve the efficiency of the transformation. The experiments for
pHꢀdependent oxidation were carried out in the presence of H2O2 at room temperature
(Fig. 4). The chemoselectivities were observed quite similar in all cases (> 95 %). We
found that a maximum yield of 92% was achieved at pH 5.1 and the yield decreased
at pH 4.7 (87%). When the pH was over 5.1, the yield decreased drastically. Although
the best enantioselectivity (47% ee) was found at pH 6.0, pH of 5.1 was adopted as
the favorable pH in light of the optimum yield and chemoselectivity.

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1 2 0
1 0 0
8 0
1 0 0
9 2
8 7
5 5
6 0
4 7
4 0
S e le c tiv it y
Y ie ld
2 0
e e
4 .5
5 .0
5 .5
6 .0
6 .5
7 .0
7 .5
8 .0
8 .5
9 .0
9 .5
p H
Fig. 4 Effect of different pH on the oxidation of thioanisole in PB at room temperature for 20 h.
The mol ratios of H2O2: thioanisole: BSAꢀCoL (2.7 ꢂmol) were 150:100:1. The sulfoxide of
thioanisole was in the R configuration.
Influence of substrate mixing time
Due to the poor solubility of the substrate, the premixing time was considered in this
study. The results, which were summarized in Table 2, showed that both conversion
and yield increased steadily up to 5 h and attained a maximum value of 96% (entry 5)
while no change in the chemoselectivity (100%) was observed (in the range of 1ꢀ7 h).
A significant decrease in conversion (63%) was afforded when the mixing time was
increased from 5 to 8 h, while the ee’s slightly increased to 48% (entry 8). Therefore,
subsequent catalyst evaluation were conducted 5 h (taken as optimum) for thioanisole
premixing time to optimize other reaction parameters.
Table 2 Effect of substrate mixing time on the oxidation of thioanisolea
Entry Time (h)
Conversion (%)
Yield (%)
Chemoselectivity (%)
ee (%)
44
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
100
100
100
100
100
100
100
92
79
88
92
92
96
85
85
63
79
88
92
92
96
85
85
58
44
40
44
44
44
45
48
a Reactions were performed in PB (2 ml, pH 5.1) at room temperature for 20 h. The ratios of H2O2:
thioanisole: BSAꢀCoL (2.7 ꢂmol) were 150:100:1.

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Influence of substrate concentration
As shown in Table 3, 100% chemoselectivity in 96% yield and 44% ee was obtained
by oxidation of 0.135 mM thioanisole (entry 3) with 1 mol% BSAꢀCoL. Moreover,
0.203ꢀ0.338 mM substrate concentrations (entries 4ꢀ6) were also investigated. Yield
and enantioselectivity dropped to 78% and 34%, respectively, while the
chemoselectivity (100%) was not affected.
Table 3 Effect of substrate concentration on the oxidation of thioanisolea
Entry
Concentration (mM) Conversion (%)
Yield (%)
Chemoelectivity (%) ee (%)
1
2
3
4
5
6
0.034
0.068
0.135
0.203
0.270
0.338
80
85
96
75
78
75
68
75
96
75
78
75
85
44
46
44
38
38
34
88
100
100
100
100
a Reactions were performed in PB (2 ml, pH 5.1) at room temperature for 20 h. The ratios of H2O2:
thioanisole: BSAꢀCoL (2.7 ꢂmol) were 150:100:1. The sulfoxide of thioanisole was in the R
configuration.
Influence of oxidant and catalyst concentration
Using the conditions identified above, we next explored the effect of oxidant
concentration on the catalytic sulfoxidation at 0.135 mM substrate concentration.
Increasing the concentration of the oxidant from 0.135 to 0.203 mM, the oxidation of
thioanisole gave a marginal increase in the conversion from 99 to 100% along with an
increasing in the chemoselectivity from 88 to 96% (Fig. 5 (a)). However, the
conversion dropped quickly (down to 38%) when H2O2 concentration beyond 0.203
mM. It also showed a steady decrease in the sulfoxide selectivity from 100 to 76%,
whereas, the enantiomeric excess increased (from 44 to 63% ee for the Rꢀenantiomer).
The reason might be interpreted in terms of the oxidative destruction of the catalyst77
and the existence of a kinetic resolution process due to excess oxidant.78ꢀ79 Although
increasing the amount of oxidant was desirable from a enantioselectivity standpoint,
0.203 mM was adopted as the optimum choice in light of the high conversion and
chemoselectivity.
It is well known that the concentration of catalyst is an important factor in

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0
12
3
80
.
1.02
024
C
catalyzed oxidation. Clearly, the maximum of the curve corresponded to the optimal
catalyst concentration (Fig. 5(b)). In the absence of the catalyst, the oxidant reaction
gave only 16% conversion and racemic product. Phenyl methyl sulfoxide was
obtained when the catalyst concentration was 1.35 ꢂM (1 mol% of substrate) with
96% conversion in 44% ee. Higher catalyst concentration didn’t show positive effect
on catalytic activity but with somewhat less conversion (80ꢀ85%) and ee value
(38ꢀ41%). However, excellent chemoselectivities were obtained in all the cases except
blank reaction.
120
120
100
93
99
88
(b)
(a)
100
100
80
60
40
20
0
100
80
60
40
20
0
76
63
96
44
85
41
80
38
52
16
44
38
Conversion
Selectivity
ee
Conversion
Selectivity
ee
0.1
0.2
0.3
0.4
0.5
0
1
2
3
4
5
Concentration of H O (mM)
Concentration of catalyst (µM
)
2
2
Fig. 5 Effect of the concentration of (a) oxidant (H2O2) and (b) catalyst (BSAꢀCoL) on the
oxidation of thioanisole in PB (2 ml, pH 5.1) at room temperature for 20 h. The sulfoxide of
thioanisole was in the R configuration.
Influence of other factors
Low solubility of the sulfides in pure water could decrease the rate of the catalyzed
oxidation which is the reason for low enantioselectivity.81 Solubilization of the
substrate requires a small amount of organic solvents in some reactions.82ꢀ83 So the
effect of different organic coꢀsolvents was further investigated. Disappointingly, from
the results shown in Table 4, the use of methanol in concentration of 5% (v/v)
significantly decreased the conversion (entry 2), analogous to the results previously
found by Sergei V. Dzyuba and coꢀworkers.84 As a possible explanation, organic
solvent could results protein destruction although a higher solubility of substrate
could be obtained. Similar results also occurred when EtOH, acetone and THF as
coꢀsolvents (entries 3ꢀ5). Comparing with the polar coꢀsolvents (MeOH, EtOH and
acetone), the weak polar coꢀsolvent (THF) afforded the lowest conversion, yield and

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ee value (entry 5). In the absence of coꢀsolvent, the effect of reaction temperature was
investigated. The catalyst showed maximum activity at 25 °C (entry 1). A temperature
decreased to 0 °C resulted in a lower yield while the ee was not significantly affected
(entry 6). Meanwhile, a decrease both in yield (92%) and stereoselectivity (34% ee)
was observed when the temperature was increased to 50 °C (entry 7). It seems likely,
the higher temperature make denaturation of the artificial metalloenzyme85 or
temperature effect on activation energies for different enantiomers. Comparing to 20 h
at 0 °C, a higher ee (up to 50%) together with a higher yield (up to 98%) were
achieved by prolonging the reaction time (28 h) (entry 8). Decreasing the H2O2
dismutation would provide a very efficient oxygen transfer to sulfide86 because H2O2
dismutation could result catalyst destruction via hydroxyl radical generation.4 Thus we
also screened the adding H2O2 speed at 0 °C. Nevertheless, similar levels of activity
and enantiomeric purity (entries 9ꢀ11) were observed confirming no H2O2 dismutation
in this sulfoxidation system or the catalyst has a good stability.
Table 4 Effect of the coꢀsolvent, temperature and adding oxidant rate on the oxidation of
thioanisolea
ee
Entry
Temperature Coꢀsolvents
°C
Conversion
Yield
(%)
96
Chemoselectivity
(
)
(%)
96
82
81
75
55
85
92
98
87
92
93
(%)
100
100
100
100
100
100
100
100
100
100
100
(%)
44
42
41
35
34
44
34
50
50
49
50
1
25
25
25
25
25
0
None
MeOH
EtOH
Acetone
THF
2
82
3
81
4
75
5
55
6
None
None
None
None
None
None
85
7
50
0
92
8b
9b,c
10b,d
11b,e
98
0
87
0
92
0
93
a
Reactions were performed in phosphate buffer (2 ml, pH 5.1) for 20 h. The ratios of H2O2:
thioanisole: BSAꢀCoL (2.7 ꢂmol) were 150:100:1, aqueous 30% H2O2 was added all at once. b
Reaction was carried at 0 °C for 28 h. c H2O2 was added in two portions (20.5 ꢂl per hour ). d H2O2
e
was added in three portions (13.7 ꢂl per hour ). H2O2 was added in four portions (10.25 ꢂl per
hour ). The sulfoxide of thioanisole was in the R configuration.
To monitor the reaction process and investigate the existence of a kinetic resolution

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process during the course of the reaction, the time course curve of oxidation
thioanisole under the above optimized reaction condition was observed over a period
of hours. The data as given in Fig. 6(a) indicated that the chemoselectivity and
conversion increased steadily at 0 °C and attained a maximum value of 88% and
100% at 20 and 28 h, respectively, while enantioselectivity (46ꢀ49% ee) remained
nearly constant throughout the course of the reaction. Similar results were presented
in Fig. 6(b). The reaction was finished after 6 h at 25 °C affording a maximum value
of conversion (96%), whereas chemoselectivity (99ꢀ100%) and enantioselectivity
(43ꢀ45% ee) remained steady. This reaction profile, particularly with no change in
chemoselectivity, indicates that the oxidative kinetic resolution of the product is either
not occurring or negligible 87
.
120
100
80
60
40
20
0
120
100
80
60
40
20
0
100
(a)
(b)
98
96
Conversion
Selectivity
ee
Conversion
Selectivity
ee
ꢀ2
0
2
4
6
8
14
16
18
20
22
Time 1(0h)12
0
5
15
20
25
30
35
1T0 ime (h)
Fig. 6 The time profile diagram of oxidation of thioanisole at 0 °C (a) and 25 °C (b). Reactions
were performed in PB (2 ml, pH 5.1). The ratios of H2O2: thioanisole: BSAꢀCoL (2.7 ꢂmol) were
150:100:1. The sulfoxide of thioanisole was in the R configuration.
Oxidation of different sulfides
Having identified the optimized conditions, the scope of the asymmetric sulfoxidation
was investigated (Scheme 2). As shown in Table 5, all the products of aryl alkyl
sulfides obtained were of R configuration except for vinyl phenyl sulfoxide (entry 9).
The results indicated that the sulfides containing electronꢀwithdrawing groups on the
phenyl of the methyl phenyl sulfide (entries 2ꢀ6) had a stronger influence on the
reactivity and enantioselectivity of the oxidation than those containing
electronꢀdonating groups (entries 7ꢀ8). For example, the introduction of
electronꢀdonating Me (entry 7) and OMe (entry 8) groups at the para position resulted

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in similar activity and ee’s to unsubstituted methyl phenyl sulfide. In contrast, the
introduction of electronꢀwithdrawing Cl and Br groups at the para position (entries
5ꢀ6), meta position (entries 3ꢀ4) or ortho position (entry 2) of aromatic ring of the
sulfide gave (R)ꢀsulfoxides with higher enantioselectivity (ranging from 66 to 87%)
and much lower conversion (ranging from 28ꢀ67%) than those observed for
thioanisole. It was interesting to note that increasing the steric bulk of the aromatic
moiety of the sulfide led to an increase in enantioselectivity: 87% and 85% ee for the
sulfoxidation of oꢀchlorophenyl methyl sulfoxide and mꢀbromophenyl methyl
sulfoxide, respectively (entries 2, 4), in contrast with the results previously found by
Maria Annunziata M. Capozzi and coꢀworkers.88 A steric constraint also was detected
in the reaction of sterically bulky vinyl phenyl sulfide and benzyl phenyl sulfide. They
both caused a drastic reduction in stereoselectivity (entries 9ꢀ10) and afforded the
opposite enantiomers with only 35 and 10% ee, respectively. It was noteworthy that
good yields and excellent chemoselectivitives could be achieved even fort the sulfides
with branched or longer alkyl groups instead of aryl (entries 11ꢀ13), but disappointing
enantiomeric excesses were obtained. Oxidation of methyl nꢀoctyl sulfoxide produced
the corresponding sulfoxide with only 19% ee (R) while both methyl tertꢀbutyl
sulfoxide and methyl nꢀdodecyl methyl sulfoxide afforded racemic mixture.
Table 5 Enantioselective oxidation of various prochiral sulfides using BSAꢀCoLa
O
O
O
BSAꢀCoL, H2O2 (30%)
S
S
S
+
PB buffer (pH 5.1)
0 °C, 28 h
R1
R2
R1
R2
R1
R2
Conversion Yield Chemoselectivity
ee (%)
Entry
Substrate
(%)
(%)
(%)
100
68
(configuration)b
50 (R)
S
1
98
98
S
2
3
28
19
87 (R)
Cl
S
Cl
50
40
80
71 (R)

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Br
S
S
4
5
6
7
8
9
65
67
56
93
46
63
44
91
71
94
79
98
85 (R)
67 (R)
66 (R)
62 (R)
Cl
Br
S
S
S
100
44
98
23
98
51
69 (R)
35 (S)
O
S
S
10
11
12
13
98
95
99
57
87
95
98
56
89
100
99
10 (R)
S
_
19 (R)
_
tꢀBu
S
S
R
R = dodecyl
98
a
Reactions were performed in phosphate buffer (2 ml) at 0 °C for 28 h; The ratios of H2O2:
b
substrate: BSAꢀCoL (2.7 ꢂmol) were 150:100:1. Aqueous 30% H2O2 was added all at once.
Assigned by HPLC elution order with known literature data (see SI for details).
Conclusions
In summary, we have achieved efficient enantioselective sulfoxidation with wide
scope of application in water by using a BSAꢀCoL metalloenzyme/H2O2 system. This
procedure offers several major advantages: (1) apply manꢀmade catalyst under
biological catalytic mild conditions which carrying out in aqueous solution without
any additive; (2) the use of the most accessible and cheapest oxidant (H2O2) and
protein (BSA); (3) highly efficient for the selective oxidation of structurally diverse
sulfides in good to high activity and enantiomeric excess; and (4) the method conform
to several of the guiding principles of green chemistry. A further study of the reaction
mechanism and scope of this reaction is underway.

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Acknowledgements
We gratefully acknowledge the National Natural Science Foundation of China
(21361003, 21101035), the Scientific Research Foundation for the Returned Overseas
Chinese Scholars, State Education Ministry, the Talent’s Small Highland project of
Guangxi Medicinal Industry (1201), New Century Ten, Hundred, Thousand Talents
Project in Guangxi, and the Foundation of Key Laboratory for Chemistry and
Molecular Engineering of Medicinal Resources, IRT 1225.
References
1 M. Khorshidifard, H. A. Rudbari, B. Askari, M. Sahihi, M. R. Farsani, F. Jalilian, G.
Bruno, Polyhedron, 2015, 95, 1ꢀ13.
2 J.ꢀM. Brunel, P. Diter, M. Duetsch, H. B. Kagan, J. Org. Chem., 1995, 60,
8086ꢀ8088.
3 G. E. O’Mahony, P. Kelly, S. E. Lawrence, A. R. Maguireb, ARKIVOC, 2011, i,
1ꢀ110.
4 P. K. Bera, P. Kumari, S. H. R. Abdi, N. H. Khan,R. I. Kureshy, P. S. Subramaniana
and H. C. Bajaj, Rsc. Adv., 2014, 4, 61550ꢀ61556.
5 E. Wojaczyńska, J. Wojaczyński, Chem. Rev., 2010, 110, 4303ꢀ4356.
6 J.ꢀT. Sun, C.ꢀJ. Zhu, Z.ꢀY. Dai, M.ꢀH. Yang, Y. Pan, H.ꢀW. Hu, J. Org. Chem., 2004,
69, 8500ꢀ8503.
7 I. Fernández, N. Khiar, Chem. Rev., 2003, 103, 3651ꢀ3705.
8 K.Tanaka, K. Kubo, K. Iida, K. Otani, T. Murase, D. Yanamoto and M. Shiro, Asian
J. Org. Chem., 2013, 2, 1055ꢀ1060.
9 G. E. ÓMahony, A. Ford and A. R. Maguire, J. Org. Chem., 2012, 77, 3288ꢀ3296.
10 W. Dai, J. Li, B. Chen, G.ꢀS. Li, Y. Lv, L.ꢀY. Wang and S. Gao, Org. Lett., 2013, 15,
5658ꢀ5661.
11 K. NM, A. J. J. Olsthoorn, M. W. Fraaije, D. B. Janssen, Appl. Environ. Microbiol.,
2003, 69, 419ꢀ426.
12 S. Colonna, S. delꢀSordo, N. Gaggero, G. Carres, P. Pasta, Heteroatom Chem.,

Dalton Transactions
Page 24 of 28
View Article Online
DOI: 10.1039/C5DT04507J
2002,13, 467ꢀ473.
13 F. Zambianchi, M. W. Fraaije, G. Carrea, G. Gonzalo, C. Rodríguez, V. Gotor, Adv.
Synth. Catal., 2007, 349, 1327ꢀ1331.
14 S. Liao, I. Ǒorić, Q.ꢀG Wang and B. List, J. Am. Chem. Soc., 2012, 134,
10765ꢀ10768.
15 J. Fujisaki, K. Matsumoto, K. Matsumoto and T. Katsuki, J. Am. Chem. Soc., 2011,
133, 56ꢀ61.
16 A. Pordea and T. R.Ward, Chem. Commun., 2008, 4239ꢀ4249.
17 A. RiozꢀMartinez, G. de Gonzalo, D. E. T. Pazmino, M. W. Fraaije and V. Gotor,
Eur. J. Org. Chem., 2010, 6409ꢀ6416.
18 M. T. Reetz, F. Daligault, B. Brunner, H. Hinrichs and A. Deege, Angew. Chem.,
Int. Ed., 2004, 43, 4078ꢀ4081.
19 A.ꢀT. Li, J.ꢀD. Zhang, H.ꢀL. Yu, J. Pan, J.ꢀH. Xu, Process Biochem., 2011, 46,
689ꢀ694.
20 T. R. Ward, Chem. Eur. J., 2005, 11, 3798ꢀ3804.
21 A. Pordea, M. Creus, J. Panek, C. Duboc, D. Mathis, M. Novic, T. R. Ward, J. Am.
Chem. Soc., 2008, 130, 8085ꢀ8088.
22 E. SansiaumeꢀDagousset, A. Urvoas, K. Chelly, W. Ghattas, J.ꢀD. Maréchal, J.ꢀP.
Mahy, R. Ricoux, Dalton Trans., 2014, 43, 8344ꢀ8354.
23 Z.ꢀF. Ke, S. Abe, T. Ueno, K. Morokuma, Am. Chem. Soc., 2012, 134,
15418ꢀ15429.
24 R. Ricoux, Q. Raffy, J.ꢀP. Mahy, C. R. Chimie, 2007, 10, 684ꢀ702.
25 P. RousselotꢀPailley, C. Bochot, C. MarchiꢀDelapierre, A. JorgeꢀRobin, L. Martin,
J. C. FontecillaꢀCamps, C. Cavazza, S. Ménage, Chem. Biomol. Chem., 2009, 10,
545ꢀ552.
26 V. Mojr, M. Budĕšínský, R. Cibulka and T. Kraus, Org. Biomol. Chem., 2011, 9,
7318ꢀ7326.
27 F. Schwizer, V. Köhler, M. Dürrenberger, L. Knörr, T. R. Ward，ACS Catal., 2013,
3, 1752ꢀ1755.
28 J. M. Zimbron, T. Heinisch, M. Schmid, D. Hamels, E. S. Nogueira, T. Schirmer, T.

Page 25 of 28
Dalton Transactions
View Article Online
DOI: 10.1039/C5DT04507J
R. Ward, J. Am. Chem. Soc., 2013, 135, 5384ꢀ5388.
29 A. Chevalleyab, M. Salmain, Chem. Commun., 2012, 48, 11984ꢀ11986.
30 J. Bos and G. Roelfes, Curr. Opin. Chem. Biol., 2014, 19, 135ꢀ143.
31 L. Panella, J. Broos, J. Jin, M. W. Fraaije, D. B. Janssen, M. JeronimusꢀStratingh,
B. L. Feringa, A. J. Minnaard, Chem. Commun., 2005, 5656ꢀ5658.
32 J. R. Carey, S. K. Ma, T. D. Pfister, D. K. Garner, H. K. Kim, J. A. Abramite, Z.
Wang, Z. Guo, Y. Lu, J. Am. Chem. Soc., 2004, 126, 10812ꢀ10813.
33 C. A. Kruithof, M. A. Casado, G. Guillena, M. R. Egmond, A. van der Kerkꢀvan
Hoof, A. J. R. Heck, R. J. M. Klein Gebbink, G. van Koten, Chem. Eur. J., 2005,
11, 6869ꢀ6877.
34 K. Okrasa, R. J. Kazlauskas, Chem. Eur. J., 2006, 12, 1587ꢀ1596.
35 A. Mahammed, Z. Gross, J. Am. Chem. Soc., 2005, 127, 2883ꢀ2887.
36 M. T. Reetz, N. Jiao, Angew. Chem., Int. Ed., 2006, 45, 2416ꢀ2419.
37 T. Ueno, S. Abe, N. Yokoi, Y. Watanabe, Coord. Chem. Rev., 2007, 251,
2717ꢀ2731.
38 M. Ohashi, T. Koshiyama, T. Ueno, M.Yanase, H. Fujii, and Y. Watanabe, Angew.
Chem. Int. Ed., 2003, 42, 1005ꢀ1008.
39 T. Ueno, T. Koshiyama, M. Ohashi, K. Kondo, M. Kono, A. Suzuki, T. Yamane,
and Y. Watanabe, J. Am. Chem. Soc., 2005, 127, 6556ꢀ6562.
40 T. Ueno, T. Koshiyama, S. Abe, N. Yokoi, M. Ohashi, H. Nakajima, Y. Watanabe, J.
Organomet. Chem., 2007, 692, 142ꢀ147.
41 K. Watanabe, N. Ishikawa and T. Komatsu, Chem. Asian J., 2012, 7, 2534ꢀ2537.
42 V. Oliveri, G. Vecchio, Eur. J. Med. Chem., 2011, 46, 961ꢀ965.
43 J. Steinreiber, T. R. Ward, Coord. Chem. Rev., 2008, 252, 751ꢀ766.
44 Y.ꢀJ. Hu, Y. OuꢀYang, Y. Zhang, Y. Liu, Protein J., 2010, 29, 234ꢀ241.
45 A. M. I. Jayaseeli, A. Ramdass, S. Rajagopal, Polyhedron, 2015, 100, 59ꢀ66.
46 A. Pordea, D. Mathis, T. R. Ward, J. Organomet. Chem., 2009, 694, 930ꢀ936.
47 J.ꢀL. Zhang, D. K. Garner, L. Liang, Q. Chen, Y. Lu, Chem. Commun., 2008,
1665ꢀ1667.
48 R. Ricoux, M. Allard, R. Dubuc, C. Dupont, J.ꢀD. Maréchalc, J.ꢀP. Mahy, Org.

Dalton Transactions
Page 26 of 28
View Article Online
DOI: 10.1039/C5DT04507J
Biomol. Chem., 2009, 7, 3208ꢀ3211.
49 S. Gosiewska, M. Lutz, A. L. Spek, R. J.M. K. Gebbink, Inorg. Chim. Acta, 2007,
360, 405ꢀ417.
50 Q. Raffy, R. Ricoux, J.ꢀP. Mahy, Tetrahedron Lett., 2008, 49, 1865ꢀ1869.
51 F. v. d. Velde, I. W.C.E. Arends, R. A. Sheldon, J. Inorg. Biochem., 2000, 80,
81ꢀ89.
52 N. K. Jana, J. G. Verkade, Org. Lett., 2003, 5, 3787ꢀ3792.
53 B. Yu, A.ꢀH. Liu, L.ꢀN. He, B. Li, Z.ꢀF. Diao, Y.ꢀN. Li, Green Chem., 2012, 14,
957ꢀ962.
54 R. S. Varma, R. K. Saini, H. M. Meshram, Tetrahedron Lett., 1997, 38, 6525ꢀ6528.
55 N. Fukuda, T. Ikemoto, J. Org. Chem., 2010, 75, 4629ꢀ431.
56 K. Liu, Z.ꢀX. Yao and Y.ꢀF. Song, Ind. Eng. Chem. Res., 2015, 54, 9133ꢀ9141.
57 X.ꢀF. Wu, Tetrahedron Lett., 2012, 53, 4328ꢀ4331.
58 A. Bayat, M. ShakourianꢀFard, M. M. Hashemi, Catal. Commun., 2014, 52, 16ꢀ21.
59 A. Pui, I. Berdan, I. MorgensternꢀBadarau, A. Gref, M. PerréeꢀFauvet, Inorg. Chim.
Acta, 2001, 320, 167ꢀ171.
60 G. M. Sheldrick, Acta Crystallogr., 1990, A46, 467ꢀ473.
61 G. M. Sheldrick, SHELXSꢀ97, Program for Xꢀray Crystal Structure Solution,
University of Göttingen, Göttingen, Germany, 1997.
62 M. M. Bradford, Anal. Biochem., 1976, 72, 248ꢀ254.
63 G. M. Morris, D. S. Goodsell, R. S. Halliday, R. Huey, W. E. Hart, R. K. Belew, A.
J. Olson, J. Comput. Chem., 1998, 19, 1639ꢀ1662.
64 J. Gasteiger, M. Marsili, Tetrahedron, 1980, 36, 3219ꢀ3228.
65 R. Tiwari, K. Mahasenan, R. Pavlovicz, C. L. Li, W. Tjarks, J. Chem. Inf. Comput.
Sci., 2009, 49, 1581ꢀ1589.
66 J.ꢀH. Shi, J. Chen , J. Wang, Y.ꢀY. Zhu, Q. Wang, Spectrochim. Acta A., 2015, 149,
630ꢀ637.
67 (a) P. Pitchen, E. Duiiach, M. N. Deshmukh, and H. B. Kagan, J. Am. Chem. Soc.,
1984, 106, 8188ꢀ8193. (b) F. A. Davis, R. T. Reddy, W. Han, J. Am. Chem. Soc.,
1992, 114, 1428ꢀ1437. (c) J. Brunel, P. Diter, M. Duetsch, H. B. Kagan, J. Org.

Page 27 of 28
Dalton Transactions
View Article Online
DOI: 10.1039/C5DT04507J
Chem., 1995, 60, 8086ꢀ8088. (d) M. I. Donnoli, S. Superchi and C. Rosini, J.
Org. Chem., 1998, 63, 9392ꢀ9395. (e) M. A. M. Capozzi, C. Cardellicchio, F.
Naso, P. Tortorella, J. Org. Chem., 2000, 65, 2843ꢀ2846. (f) J.ꢀT. Sun, C.ꢀJ. Zhu,
Z.ꢀY. Dai, M.ꢀH. Yang, Y. Pan, H.ꢀW. Hu, J. Org. Chem., 2004, 69, 8500ꢀ8503.
(g) H. Egami, T. Katsuki, J. Am. Chem. Soc., 2007, 129, 8940ꢀ8941. (h) O. G.
Mancheno, J. Dallimore, A. Plant, C. Bolm, Org. Lett., 2009, 11, 2429ꢀ2432. (i)
K. Kubo, K. Iida, K. Otani, T. Murase, D. Yanamoto, M. Shiro, Asian J. Org.
Chem., 2013, 2, 1055ꢀ1060. (j) Y.ꢀZ. Chen, J.ꢀR. Zhuo, D.ꢀJ. Zheng, S. Tian, Z.
Li, J. Mol. Catal. B: Enzym., 2014, 106, 100ꢀ104.
68 Y. Daijima, T. Komatsu, Chem. Commun., 2014, 50, 14716ꢀ14719.
69 R. R. Davies, M. D. Distefano, J. Am. Chem. Soc., 1997, 119, 11643ꢀ11652.
70 E. Sansiaume, R. Ricou, D. Gori, J.ꢀP. Mahy, Tetrahedron: Asymmetry, 2010, 21,
1593ꢀ1600.
71 J.ꢀJ. Ao, L. Gao, T. Yuan, G.ꢀF. Jiang, Chemosphere, 2015, 119, 590ꢀ600.
72 J.ꢀH. Shi, Y.ꢀY. Zhu, J. Wang, J. Chen, Luminescence, 2015, 30, 44ꢀ52.
73 P. Bourassa, S. Dubeau, G. M. Maharvi, A. H. Fauq, T. J. Thomas, H. A.
TajmirꢀRiahi, Eur. J. Med. Chem., 2011, 46, 4344ꢀ4353.
74 S. M. G. Piresa, M. M. Q. Simõesa, I. C. M. S. Santosa, S. L. H. Rebelob, M. M.
Pereirac, M. G. P. M. S. Nevesa, J. A. S. Cavaleiroa, Appl. Catal. A: Gen., 2012,
439–440, 51ꢀ56.
75H. Egami, T. Katsuki, J. Am. Chem. Soc., 2007, 129, 8940ꢀ8941.
76 J. S. Mandeville, H. A. TajmirꢀRiahi, Biomacromolecules, 2010, 11, 465ꢀ472.
77 S. P. Das, J. J. Boruah, N. Sharma, N. S. Islam, J. Mol. Catal. A: Chem., 2012, 356,
36ꢀ45.
78 Z.ꢀW. Yang, C.ꢀF. Zhu, Z.ꢀJ. Li, Y. Liu, G.ꢀH. Liu, Y. Cui, Chem. Commun., 2014, 50,
8775ꢀ8778.
79 H. Srour, J. Jalkh, P. Maux, S. Chevance, M. Kobeissi, G. Simonneaux, J. Mol.
Catal. AꢀChem., 2013, 370, 75ꢀ79.
80 R. Rahimi, A. A. Tehrani, M. A. Fard, B. M. M. Sadegh, H. R. Khavasi, Catal.
Commun., 2009, 11, 232ꢀ235.

Dalton Transactions
Page 28 of 28
View Article Online
DOI: 10.1039/C5DT04507J
81 P. L. Maux and G. Simonneaux, Chem. Commun., 2011, 47, 6957ꢀ6959.
82 J.ꢀD. Zhang, A.ꢀT. Li, J.ꢀH. Xu, Bioproc. Biosyst. Eng., 2010, 33, 1043ꢀ1049.
83 Y.ꢀZ. Chen, J.ꢀR. Zhuo, D.ꢀJ. Zheng, S. Tian, Z. Li, J. Mol. Catal B: Enzym., 2014,
106, 100ꢀ104.
84 S. V. Dzyuba, A. M. Klibanov, Biotechnol. Lett., 2003, 25, 1961ꢀ1965.
85 S. J. Calvin, D. Mangan, I. Miskelly, T. S. Moody, P. J. Stevenson, Org. Process
Res. Dev., 2012, 16, 82ꢀ86.
86A. Marques, M. Marin, M.ꢀF. Ruasse, J. Org. Chem., 2001, 66, 7588ꢀ7595.
87 P. K. Bera, D. Ghosh, S. H. R. Abdi, N.ꢀu H. Khan, R. I. Kureshy, H. C. Bajaj, J.
Mol. Catal. A: Chem., 2012, 361ꢀ362, 36ꢀ44.
88 M. A. M. Capozzi, C. Cardellicchio, F. Naso, P. Tortorella, J. Org. Chem., 2000, 65,
2843ꢀ2846.