Optimization of and Mechanistic Considerations for the Enantioselective Dihydroxylation of Styrene Catalyzed by Osmate-Laccase-Poly(2-Methyloxazoline) in Organic Solvents

Article abstract of DOI:10.1002/cctc.201501083

The Sharpless dihydroxylation of styrene with the artificial metalloenzyme osmate-laccase-poly(2-methyloxazoline) was investigated to find reaction conditions that allow this unique catalyst to reveal its full potential. After changing the co-oxidizing agent to tert-butyl hydroperoxide and optimizing the osmate/enzyme ratio, the turnover frequency and the turnover number could be increased by an order of magnitude, showing that the catalyst can compete with classical organometallic catalysts. Varying the metal in the active center showed that osmate is by far the most active catalytic center, but the reaction can also be realized with permanganate and iron(II) salts.

Full text of DOI:10.1002/cctc.201501083

DOI: 10.1002/cctc.201501083
Full Papers
Optimization of and Mechanistic Considerations for the
Enantioselective Dihydroxylation of Styrene Catalyzed by
Osmate-Laccase-Poly(2-Methyloxazoline) in Organic
Solvents
Melanie Leurs, Pia S. Spiekermann, and Joerg C. Tiller*[a]
The Sharpless dihydroxylation of styrene with the artificial met-
alloenzyme osmate-laccase-poly(2-methyloxazoline) was inves-
tigated to find reaction conditions that allow this unique cata-
lyst to reveal its full potential. After changing the co-oxidizing
agent to tert-butyl hydroperoxide and optimizing the osmate/
enzyme ratio, the turnover frequency and the turnover
number could be increased by an order of magnitude, show-
ing that the catalyst can compete with classical organometallic
catalysts. Varying the metal in the active center showed that
osmate is by far the most active catalytic center, but the reac-
tion can also be realized with permanganate and iron(II) salts.
Introduction
Pure enantiomeric compounds are essential for the synthesis
of pharmaceuticals and other fine chemicals.[1] In many cases,
the use of an enantioselective catalyst, for example, an organo-
metallic one, is the most economical way to gain this. Enzy-
matic systems are being currently considered as alternatives to
organometallic catalysts.[2] In addition to their natural catalytic
activity, enzymes possess a defined chiral matrix, especially at
their active site, which offers the possibility to use them as
chiral ligands for catalytically active (transition) metal com-
plexes. This merging of proteins and metal catalysts results in
so called artificial metalloenzymes, which have the molecular
recognition ability of proteins and the broad reactivity scope
of small molecule catalysts.[3]
the metal ion in the active center is substituted by another.
Kaiser and co-workers were the first to generate such a hybrid
catalyst by replacing the natural zinc of carboxypeptidase A by
copper and they used the resulting artificial metalloenzyme in
oxidation reactions.[10]
Dihydroxylations of alkenes by using proteins as hosts are
the focus of much current research. For example, the use of
bovine serum albumin (BSA) (77%) and streptavidin (95%) as
ligands for osmate results in good enantiomeric excess (ee) for
the dihydroxylation of a-methylstyrene in aqueous media.[11]
Owing to the solubility and natural environment of proteins
and enzymes, most of the reactions catalyzed by artificial met-
alloenzymes are performed in aqueous media.[12] A few exam-
ples, which involve large amounts of organic co-solvents, have
been reported.[13]
Artificial metalloenzymes, generated by different anchoring
strategies, have been applied in various types of chemical reac-
tions, for example, sulfoxidation,[4] hydrogenation,[5] epoxida-
tion,[6] Diels–Alder reactions,[7] or diastereoselective coenzyme
reduction.[8]
In general, enzymes are used in organic solvents by immobi-
lizing them on designed carriers.[14] In our group, this approach
has been particularly successful for enzymes in amphiphilic
polymer co-networks (APCNs), which activate these biocata-
lysts by several orders of magnitude in membranes[15] and par-
ticles[16] in organic solvents and even in supercritical CO2.[17]
Chiral APCNs allow control of the enantioselectivity of enzymes
in organic solvents.[18]
There are three generally applied strategies for the incorpo-
ration of a metal complex within the biomolecule host; dative
anchoring, supramolecular anchoring, and covalent anchor-
ing.[9]
The dative anchoring strategy, which is used in this work, is
based on direct non-covalent interaction between the amino
acids of the enzyme and the metal ion or metal complex. One
way to achieve this, is to utilize natural metalloenzymes where
To overcome the limitations of artificial metalloenzymes in
aqueous systems, organo-soluble polymer enzyme conjugates
(PECs) have been investigated regarding their potential as arti-
ficial metalloenzymes for alkene dihydroxylation. Such PECs
based on conjugation of enzymes with poly(2-methyloxazo-
line) (PMOx) show enzymatic activity in aqueous and organic
solvents.[19] Their structures offer the possibility to apply them
as amphiphilic polymer nanocontainers[20] for dissolving inor-
ganic salts in organic solvents, forcing the salt to attach to the
protein, which represents the hydrophilic core of the nanocon-
tainer.
[a] M. Leurs, P. S. Spiekermann, Prof. Dr. J. C. Tiller
Chair of Biomaterials and Polymer Science
Department of Biochemical and Chemical Engineering, TU Dortmund
Emil-Figge-Strasse 66, 44227 Dortmund (Germany)
Fax: (+49)231-755-2480
E-mail: joerg.tiller@tu-dortmund.de
Supporting Information for this article is available on the WWW under
http://dx.doi.org/10.1002/cctc.201501083.
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An artificial enzyme composed of a laccase-PMOx conjugate
with an osmate at the active site acts as a chiral catalyst for
the asymmetric dihydroxylation of alkenes in chloroform, lead-
ing to highly enantioselective product formation (up to 99.4%
ee for 1-phenyl-1,2-ethanediol for styrene dihydroxylation) that
even exceeds the classical Sharpless catalysts (97% ee).[21,22]
Thus, we could transfer the concept of artificial metalloen-
zymes from water to organic solvents with even higher selec-
tivities.
acid (EDTA) solution to remove the copper of the enzyme and
subsequent addition of the potassium osmate in a molar
osmate/enzyme ratio (OER)=2 (Scheme 1).
This conjugate was found to convert styrene to 1-phenyl-
1,2-ethanediol in only 1%, which is calculated to a TOF of
0.25 hÀ1 and a TON of 42 (Table 1). This is about 300 times
lower TOF value compared with a commercial Sharpless cata-
lyst[24] with a somewhat lower selectivity (97% ee[21]).
The turnover might be limited as a result of the insolubility
of the co-oxidizing agent, K3[Fe(CN)]6, in organic solvents,
which restricts the accessibility of this reagent for the catalyst.
Another limiting factor might be the low solubility of the OHÀ
source (water) in chloroform (Scheme 2).
The drawback of all artificial metalloenzyme approaches,
and particularly for osmate-catalyzed dihydroxylation, is the
small turnover number (TON) and turnover frequency (TOF)
with regard to the amount of osmate catalyst used. Koehler
et al. reported a maximum TON of 27 when streptavidin is
used as host for osmate in an aqueous medium.[11] Laccase-
PMOx as the host for osmate in chloroform afforded a TON of
42.[22] In the present manuscript, the latter enzyme conjugates
were explored to find their full potential with respect to reac-
tion rate and turnover number.
Scheme 2. The asymmetric-osmate-catalyzed dihydroxylation of styrene.
Results and Discussion
To overcome these limitations, the co-oxidizing agent was
changed from the organo-insoluble K3[Fe(CN)]6/K2CO3 system
to tert-butyl hydroperoxide. As seen in Figure 1, this alteration
results in a homogeneous reaction solution, which affords
a six-fold increase in the TON without a significant change in
ee (Table 1). To further raise the turnover, an additional OHÀ
source (methanolic KOH) was added to the reaction mixture,
resulting in an increase in the TON from 258 to 328. Lowering
the OER to 1 gives a turnover number of 501 with an increased
ee value of 99.4. This value represents the detection limit of
the used HPLC analytics. In summary, a 12-fold increase in the
turnover number could be achieved by changing the co-oxidiz-
The basis of the organo-soluble osmate-laccase conjugates is
the modification of the enzyme with poly(2-methyloxazoline)
(PMOx)[23] and the subsequent exchange of copper with
osmate.[22] As shown previously, these conjugates show excel-
lent enantioselectivity in the dihydroxylation of styrene in
chloroform.
So far, the highest selectivity of ee (R)=99.4% could only be
obtained after relatively short reactions times of three days.
When running the reaction for seven days, the best result of
98.4% ee for the R enantiomer was obtained with laccase-
PMOx pretreated with aqueous ethylenediaminetetraacetic
Scheme 1. Strategy for enzyme modification with PMOx and pyromellitic acid dianhydride and the suggested copper type 1 exchange with osmate by treat-
ment with EDTA according to Ref. [22].
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Table 2. TON, TOF, yield, and ee values for the dihydroxylation[a] of sty-
rene by using different solvents and laccase-PMOx as ligand for osmate
with an OER of 1.
Solvent
TON[b]
TOF[c] [hÀ1
]
Yield [%]
ee (R) [%]
chloroform
CH2Cl2
MeOH
EtOH
DMF
DMSO
MeCN
501
600
315
266
38
2.98
3.57
1.87
1.58
0.23
0.06
0.29
6.6
7.8
4.1
3.5
0.5
0.1
0.6
99.4
94.1
0.0
0.0
0.0
10
49
75.7
0.0
Figure 1. Photographs of reaction mixtures for the dihydroxylation of sty-
rene with K3[Fe(CN)]6/K2CO3 as the co-oxidizing system (left) and tert-butyl
hydroperoxide/KOH as the co-oxidizing system (right).
[a] Reaction
performed
with
0.013 mmolmLÀ1
laccase-PMOx,
0.013 mmolmLÀ1 K2OsO2(OH)4, 110 mmolmLÀ1 tBuOOH, 10 mLmLÀ1 saturat-
ed methanolic KOH, and 100 mmolmLÀ1 styrene at 08C for seven days.
[b] The TON is defined as mmol product per mmol K2OsO2(OH)4. [c] The
TOF is defined as mmol product per mmol K2OsO2(OH)4 per h.
Table 1. Dependency of the ee, TON, TOF, and yield on the used co-oxi-
dizing system for the dihydroxylation of styrene in chloroform by using
osmate-laccase-PMOx. The reaction was run at 08C for seven days.
OER Co-oxidizing agent Base
TON[a] TOF[b] Yield ee (R)
[hÀ1
]
[%]
[%]
[c,d]
2
2
2
2
1
K3[Fe(CN)]6
tBuOOH[c]
tBuOOH[c,e]
tBuOOH[c,f]
tBuOOH[c,g]
K2CO3
–
K2CO3
KOH/MeOH 328
KOH/MeOH 501
42
258
232
0.25
1.54
1.38
1.95
2.98
1.0
6.5
6.0
8.5
6.6
98.4
97.2
97.8
98.6
99.4
[a] The TON is defined as mmol product per mmol K2OsO2(OH)4. [b] The
TOF is defined as mmol product per mmol K2OsO2(OH)4 per h. [c] Standard
reaction conditions: 0.013 mmolmLÀ1 laccase-PMOx, 0.026 mmolmLÀ1
K2OsO2(OH)4, 110 mmolmLÀ1 tBuOOH, and 100 mmolmLÀ1 styrene unless
otherwise stated. [d] 300 mmolmLÀ1 K3Fe(CN)6 used instead of tBuOOH,
plus 300 mmolmLÀ1 K2CO3. [e] Standard conditions plus 300 mmol K2CO3.
[f] Standard conditions plus 10 mLmLÀ1 saturated methanolic KOH.
[g] Standard conditions except 0.013 mmolmLÀ1 K2OsO2(OH)4 and plus
10 mLmLÀ1 saturated methanolic KOH.
Figure 2. Turnover number (TON) and enantiomeric excess (ee) of the asym-
metric dihydroxylation in different solvents catalyzed by osmate-laccase-
PMOx with an OER of 1. The reaction was performed with 0.013 mmolmLÀ1
laccase-PMOx, 0.013 mmolmLÀ1 K2OsO2(OH)4, 110 mmolmLÀ1 tBuOOH,
10 mLmLÀ1 saturated methanolic KOH, and 100 mmolmLÀ1 styrene at 08C for
seven days. The TON is defined as mmol product per mmol K2OsO2(OH)4.
ing reagent and providing methanolic KOH as an OHÀ source.
This shows the great potential of the artificial metalloenzyme.
It is known in the literature that the activity of enzymes and
the enantioselectivity of enzymatic reactions is influenced by
the solvent, an effect called medium engineering.[25] Following
this approach, a selection of solvents that are capable of dis-
solving the polymer-laccase conjugate were explored under
similar reaction conditions. The results clearly show the strong
dependence of the TON and enantioselectivity on the nature
of the solvent (Table 2, Figure 2).
of the laccase-PMOx to bind osmate by acting as an amphi-
philic polymer nanocontainer. Therefore, the specific binding
position within the active site of the laccase may not be fa-
vored anymore, which leads to a less selective product forma-
tion. Control experiments under the same conditions, but with-
out adding the PEC, showed significant conversion of styrene
in methanol and ethanol, also resulting in racemic product
mixtures. This indicates that the osmate/solvent complex is
catalytically active. The same control reaction in the other sol-
vents did not result in styrene conversion, indicating that the
complexation of osmate by laccase-PMOx is essential for the
catalytic activity in those solvents. Clearly, the complex forma-
tion in DMF, DMSO, and MeCN cannot be efficient judged by
the low activity. Further, only DMSO seems to direct the
osmate, at least in part, to the active site of the enzyme. Gen-
erally, TON, TOF, and selectivity can be controlled by the
choice of solvent.
The highest activity for the catalyst was found in dichloro-
methane, which exceeds that in chloroform by 20%. However,
the enantioselectivity of the reaction was 94.1% ee, which is
somewhat lower than that in chloroform (99.4% ee). In both
cases, significant conversion was obtained. The protic, polar
solvents methanol and ethanol also afforded a good TON, but
with complete loss of enantioselectivity in both cases. DMF
and MeCN afford a drastically lower activity and no enantiose-
lectivity at all. The lowest activity of osmate-laccase-PMOx was
found in DMSO, but the product formation is somewhat enan-
tioselective (75% ee).
Considering both the enantiomeric excess and the turnover
number, chloroform is the solvent of choice for further investi-
gations on the osmate-catalyzed dihydroxylation with laccase-
PMOx as a chiral ligand.
The loss of enantioselectivity of the diol formation in metha-
nol and ethanol might be due to the fact that potassium
osmate is soluble in these media. This lowers the driving force
So far, all reactions had been performed at 08C because it
was found previously that the dihydroxylation of alkenes with
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different PECs as ligand is subject to a dramatic temperature
dependency with regard to ee and TON.[22]
To explore if the TON can be increased at higher tempera-
ture without losing selectivity, the dihydroxylation of styrene
was performed at 22 and 48C by using different OERs. It was
expected that at some point the osmate only binds to the
active site, which should have the greatest complexation con-
stant according to previous experiments. It was further pre-
sumed that the selective substrate conversion at the osmate-
enzyme complex is faster than the unspecific conversion at the
free or non-specifically bound osmate owing to ligand acceler-
ation (and thus lower activation energy). Based on this, we ex-
pected that suppressing the formation of non-specifically
bound osmate might afford more enantioselective product for-
mation even at higher temperatures, and thus achieve greater
TONs. The results shown in Table 3 clearly prove that this con-
Figure 3. Enantiomeric access (ee (R) [%]) of the asymmetric dihydroxylation
with osmate-laccase-PMOx at different reaction temperatures. The reaction
was performed with different OERs, 110 mmolmLÀ1 tBuOOH, 10 mLmLÀ1 satu-
rated methanolic KOH, and 100 mmolmLÀ1 styrene for seven days.
bound osmate somewhere within the protein is catalytically
active, but not affording selective product formation. Thus,
considering the enantiomeric excess of the reaction, lower
temperatures are recommended for highly selective product
formation even at an OER of 1 or lower.
Table 3. Dependency of the ee, TON, and yield on the temperature of
the dihydroxylation of styrene[a] in chloroform by using osmate-laccase-
PMOx with different OERs.
The turnover number relative to the osmate after seven
days, and thus TOF, as expected, rises with increasing reaction
temperature (Table 3). However, the elevation (1.7-fold) in TON,
and thus TOF, when increasing the temperature by 22 K is not
as high as expected according to the Q10 temperature coeffi-
cient. According to the latter, it should be a four- to nine-fold
increase. This is typically an indication that the reaction is
somewhat influenced within its course.
OER
TON[b]
Yield
[%]
ee (R)
[%]
Reaction temperature: 228C
2
1
0.5
724
842
579
19.0
11.1
3.8
11.3
67.8
73.6
Reaction temperature: 48C
2
1
0.5
680
636
415
17.9
8.4
2.7
48.8
85.8
84.4
To investigate this, the reaction kinetics were followed for
seven days by taking a sample every 24 h. The data are pre-
sented in a normalized form in Figure 4. As can be seen, the
normalized TON increases linearly at 08C over the entire reac-
tion time. When increasing the reaction temperature to 48C,
the slope of the curve is steeper, but no further product is
formed after five days. The TON increases again at 228C, but
the product formation stops after three days. Comparing the
increase in absolute TON values in the linear region shows that
Reaction temperature: 08C
2
1
0.5
328
501
–
8.5
6.6
–
98.6
99.4
99.4
[a] Reaction performed with 110 mmolmLÀ1 tBuOOH, 10 mLmLÀ1 saturated
methanolic KOH, and 100 mmolmLÀ1 styrene for seven days. [b] The TON
is defined as mmol product per mmol K2OsO2(OH)4.
cept is valid. It was found that the ee (R) values increase dra-
matically when the osmate/enzyme ratio (OER) is reduced from
2 to 1 (for example, from 11.3 to 67.8% ee (R) at 228C,
Figure 3). Further lowering of the OER to 0.5 increased the
enantiomeric excess of the R enantiomer to 73.6%. A similar
picture is found for a reaction temperature of 48C. Although
the ee value increased from 48 to 84% when decreasing the
OER from 2 to 1, further decreasing the OER does not result in
higher ee values. The same relationship was found for a reac-
tion temperature of 08C. These results indicate that only one
osmate is bound to the enantioselectivity controlling region in
the active site of the laccase, because an OER of 2 affords dra-
matically reduced selectivity and an OER of 0.5 does not lead
to significantly higher selectivity. The TONs with respect to
osmate (protein concentration is kept constant) are not strong-
ly dependent on the OER, that is, nearly all osmate molecules
are catalytically active. This indicates that the non-specifically
Figure 4. Normalized turnover number (TON/TON(168 h)) versus time plot of
the asymmetric dihydroxylation with laccase-PMOx as ligand. The reaction
was performed at different temperatures and with 0.013 mmolmLÀ1 laccase-
PMOx, 0.013 mmolmLÀ1 K2OsO2(OH)4, 110 mmolmLÀ1 tBuOOH, 10 mLmLÀ1 sa-
turated methanolic KOH, and 100 mmolmLÀ1 styrene. The TON is defined as
mmol product per mmol K2OsO2(OH)4.
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the TOF increases from 2.98 hÀ1 at 08C to 3.74 hÀ1 at 48C and
8.60 hÀ1 at 228C. This means that the TOF, which is proportion-
al to the reaction rate in this case, increases three-fold when
raising the reaction temperature from 0 to 228C. This is closer
to the typical temperature/reaction rate relationship. Clearly,
there is a temperature-dependent loss of catalytic activity of
the osmate enzyme complex. This indicates that the active site
of the protein is somehow altered during the course of the re-
action, probably by the co-oxidizing reagent. Another possibili-
ty would be inactivation by the potassium osmate itself.
Another aspect of enzymatic reactions is their typical reac-
tion rate dependency on the substrate concentration accord-
ing to Michaelis–Menten (MM) kinetics.[26] According to this,
the substrate affinity to the active site is the controlling param-
eter for reaching the maximal reaction rate, that is, low sub-
strate affinity requires higher concentrations for maximal reac-
tion rates. To examine this, the reaction was performed at dif-
ferent styrene concentrations between 12.5 and 300 mmolmLÀ1
at two different temperatures (0 and 228C) for 24 h (Table S1
in the Supporting Information).
the substrate has a lower affinity to the active site of the
enzyme catalyst at 08C compared with at 228C. This might be
due to the fact that the protein folding at the active site
changes at this temperature. This is supported by the fact that
the enantioselectivity is much higher at the lower temperature,
indicating that the temperature-dependent enantioselectivity
of the reaction might not only be due to non-specific binding
of osmate, but also to refolding of the enzyme. The conse-
quence of this refolding is that the TOF can be increased by
up to 30% by increasing the styrene concentration. Thus, we
achieved another 1.4-fold increase in the turnover frequency
by optimizing the substrate concentration.
After optimizing the reaction conditions for the dihydroxyla-
tion of styrene with osmate-laccase-PMOx, we wondered if the
reaction can be influenced by variation of the metal in the
active site of the laccase. To explore this, potassium permanga-
nate and iron(II) chloride were added to the laccase-PMOx con-
jugate with a molar metal/enzyme ratio of 1 and tested for
their catalyzing potential for the dihydroxylation of styrene
(Table 4). In comparison with potassium osmate, potassium
A reaction time of 24 h was chosen to make sure that the
enzyme catalyst is not denatured during the course of the re-
action. It was found that the TOFs (proportional to reaction
rates) follow a Michaelis–Menten-like course and, thus, trend
towards a limit at high styrene concentrations.
Table 4. TON, TOF, yield, and ee values of the dihydroxylation[a] of styrene
by using different metal species and laccase-PMOx as ligand (ratio 1:1) at
08C.
It was observed that the TOF at 228C increases from 0.72 to
11 hÀ1 when increasing the styrene concentration from 12.5 to
150 mmolmLÀ1 and after that no significant increase in TOF at
higher substrate concentrations occurs (Figure 5). This means
that the TOF is near the maximal turnover frequency when
using a styrene concentration of 150 mmolmLÀ1. The apparent
Michaelis constant Km was calculated to be 35 mmolmLÀ1 by fit-
ting the data with the MM kinetics.
Metal species
TON[b]
TOF[c] [hÀ1
]
Yield [%]
ee [%]
K2OsO2(OH)4
KMnO4
FeCl2
501
51
20
2.98
0.30
0.07
6.6
0.6
0.2
99.4 R
25.6 R
11.0 R
[a] Reaction
performed
with
0.013 mmolmLÀ1
laccase-PMOx,
0.013 mmolmLÀ1 of the metal species, 110 mmolmLÀ1 tBuOOH, 10 mLmLÀ1
saturated methanolic KOH, and 100 mmolmLÀ1 styrene for seven days.
[b] The TON is defined as mmol product per mmol K2OsO2(OH)4. [c] The
TOF is defined as mmol product per mmol K2OsO2(OH)4 per h.
A different picture is found for the reaction temperature of
08C. Km was determined to be 115 mmolmLÀ1, indicating that
permanganate led to a dramatic decrease in ee (R) from 99.4
to 25.6% and a ten-fold lower TON (501 to 51) after seven
days. This might be due to the fact that KMnO4 is a stronger
oxidizing reagent than K2OsO2(OH)4, which might result in
faster protein denaturation. Another class of literature-known
reagents for dihydroxylation of alkenes are certain iron com-
plexes.[27] The FeII-laccase-PMOx conjugate catalyzes the dihy-
droxylation of styrene with an ee of only 11 % for the R enan-
tiomer and a turnover number of 20 after seven days. In con-
trast to K2OsO2(OH)4, this is a rather low product formation,
but the turnover still exceeds that of a literature-known system
(TON 9.4 for [FeII(4-MeO-C6H4-DPAH)2](OTf)2, no investigations
on enantiomeric excess[27b]). Nevertheless, the use of potassium
osmate gives the best results with respect to ee, TON, and TOF.
Conclusions
Figure 5. Turnover frequency (TOF) versus concentration of styrene (cstyrene
)
plot of the asymmetric dihydroxylation with osmate-laccase-PMOx. The reac-
tion was performed with 0.013 mmolmLÀ1 laccase-PMOx, 0.013 mmolmLÀ1
K2OsO2(OH)4, 110 mmolmLÀ1 tBuOOH, 10 mLmLÀ1 saturated methanolic KOH,
and different concentrations of styrene at different temperatures for 24 h.
The TOF is defined as mmol product per mmol K2OsO2(OH)4 per h.
The dihydroxylation of styrene by using an organo-soluble
osmate-laccase-PMOx artificial metalloenzyme was explored in
detail to find the limitations of this reaction. It was found that
exchanging the oxidizing agent from organo-insoluble
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150 mm NaCl, pH 7) as eluent followed by subsequent desalting
and lyophilization. The laccase activity of the resulting solid (pro-
tein content 30 wt%) was measured to 12370 UmgÀ1 with 2,6-di-
methoxyphenol as substrate (258C, pH 4.5, acetate buffer, 1 U is
defined as absorbance increase of 0.001 per min).
K3[Fe(CN)]6 to tert-butyl hydroperoxide results in a homogene-
ous reaction mixture that affords a 12-fold increase in reaction
rate. Investigations on the best-suited solvent showed that
chloroform is the most favorable solvent with respect to TOF,
TON, and ee. Changing the reaction temperature for optimized
molar metal/laccase ratios revealed that the enantioselectivity
is strongly temperature dependent. Investing the reaction ki-
netics showed that the affinity of the substrate styrene to the
active site of laccase is also temperature dependent. We hy-
pothesize that this is due to a temperature-dependent refold-
ing of the protein, which might also be the reason for the tem-
perature-dependent enantioselectivity. Unfortunately, the reac-
tion must be performed at 08C to avoid catalyst deactivation.
Exchanging the metal in the active site did not improve the re-
action rate or selectivity, but did reveal that, in principle, per-
manganate and iron(II) salts can also be used to create artificial
metalloenzymes with catalytic activity in dihydroxylation reac-
tions. Altogether, the increased reaction rate of TOF=12 hÀ1 at
228C shows that this catalyst is capable of performing the di-
hydroxylation of styrene in the range of the literature-known
Sharpless catalysts also with comparable ee values. The aspect
of using activating additives such as organic sulfonamides[21b]
to accelerate the reaction will be considered in future work.
Further, conformational studies on the artificial metalloenzyme
to get a deeper insight into the folding of the osmate-laccase
complex at different temperatures will be performed.
Sharpless dihydroxylation of styrene with poly(2-methyloxa-
zoline) laccase conjugates as ligand (general procedure)
The laccase-PMOx (10 mg, 0.13 mmol) was dissolved in 10 mL of
the respective solvent. Afterwards, K2OsO2(OH)4 (48 mg, 0.13 mmol
for an OER=1), KMnO4 (21 mg, 0.13 mmol) or FeCl2 (16 mg,
0.13 mmol) was added to the reaction mixture and stirred for
30 min. After that, styrene (115 mL, 1 mmol) and saturated metha-
nolic KOH (100 mL) were added. Finally, a tBuOOH solution in
decane (5.5m, 200 mL, 1100 mmol) was added and the reaction mix-
ture was vigorously stirred at different temperatures (0, 4, or 228C).
The reaction was stopped by extraction with an aqueous sodium
thiosulfate solution (1m, 2”10 mL). Afterwards, the phases were
separated and the aqueous phase was extracted five times with
chloroform (25 mL) and the organic phases were combined. After
removal of the solvent, the obtained solid was characterized by
HPLC.
The polymer synthesis and enzyme modification were performed
according to previously published work on dihydroxylation with
osmate-PECs.[22] Experimental details can be found in the Support-
ing Information.
Acknowledgements
Experimental Section
We thank B.Sc. Philipp Baumann for lab assistance. All polymers
were synthesized by using CEM Discover microwaves.
Materials
All chemicals were purchased from Sigma–Aldrich, Applichem,
Merck, ABCR, Acros, Armar Chemicals, or Carl Roth and were of an-
alytical grade or purer and, with exception of laccase, used without
further modification.
Keywords: asymmetric catalysis · asymmetric dihydroxylation ·
metalloenzymes
conjugates
·
organic solvents
·
polymer enzyme
Measurements
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Received: October 5, 2015
Revised: November 5, 2015
Published online on December 8, 2015
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