Multicore Artificial Metalloenzymes Derived from Acylated Proteins as Catalysts for the Enantioselective Dihydroxylation and Epoxidation of Styrene Derivatives

Article abstract of DOI:10.1002/chem.201802185

Artificial metalloenzymes (AME′s) are an interesting class of selective catalysts, where the chiral environment of proteins is used as chiral ligand for a catalytic metal. Commonly, the active site of an enzyme is modified with a catalytically active metal. Here we present an approach, where the commercial proteins lysozyme (LYS) and bovine serum albumin (BSA) can be converted into highly active and enantioselective AME′s. This is achieved by acylation of the proteins primary amino groups, which affords the metal salts in the core of the protein. A series of differently acylated LYS and BSA were reacted with K₂OsO₂(OH)₄, RuCl₃, and Ti(OMe)₄, respectively, and the conjugates were tested for their catalytic activity in dihydroxylation and epoxidation of styrene and its derivatives. The best suited system for dihydroxylation is fully acetylated LYS conjugated with K₂OsO₂(OH)₄, which converts styrene to 1,2-phenylethanediol with an enantioselectivity of 95 % ee (S). BSA fully acylated with hexanoic acid and conjugated with three moles RuCl₃ per mole protein shows the highest ee values for the conversion of styrene to the respective epoxide with enenatioselectivities of over 80 % ee (R), a TON of more than 2500 and a yield of up to 78 % within 24 h at 40 °C. LYS has two favored selective binding sites for the metal catalyst and BSA has even three. The AME′s with titanate in the active center invert the enantioselectivity of styrene epoxidation.

Full text of DOI:10.1002/chem.201802185

A Journal of
Accepted Article
Title: Multicore Artificial Metalloenzymes derived from acylated
proteins as catalysts for the enantioselective dihydroxylation and
epoxidation of styrene derivatives.
Authors: Melanie Leurs, Bjoern Dorn, Sascha Wilhelm, Magiliny
Manisegaran, and Joerg Tiller
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
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content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201802185
Link to VoR: http://dx.doi.org/10.1002/chem.201802185
Supported by

Chemistry - A European Journal
10.1002/chem.201802185
FULL PAPER
Multicore Artificial Metalloenzymes derived from acylated
proteins as catalysts for the enantioselective dihydroxylation and
epoxidation of styrene derivatives.
[
a]
[a]
[a]
[a]
[a]
Melanie Leurs , Bjoern Dorn , Sascha Wilhelm , Magiliny Manisegaran and Joerg. C. Tiller*
4
g
Abstract: Artificial metalloenzymes (AME’s) are an interesting class
of selective catalysts, where the chiral environment of proteins is
used as chiral ligand for a catalytic metal. Commonly, the active site
of an enzyme is modified with a catalytically active metal. Here we
present an approach, where the commercial proteins lysozyme
nanofibers as enzyme activating matrices.
In order to widen the diversity of enzyme catalyzed reactions the
active sites of enzymes were modified with alternative metals.
This led to so-called artificial metalloenzymes (AME’s), which
combine the molecular recognition ability of proteins with the
7
(LYS) and bovine serum albumin (BSA) can be converted into highly
broad reactivity scope of small molecule catalysts. Kaiser and
active and enantioselective AME’s. This is achieved by acylation of
the proteins primary amino groups, which affords the metal salts in
the core of the protein. A series of differently acylated LYS and BSA
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 AME in oxidation reactions. AME’s,
8
were reacted with K
2
OsO
2
(OH)
4
, RuCl
3
, and Ti(OMe)
4
, respectively,
generated by different anchoring strategies, have been applied
7
k
and the conjugates were tested for their catalytic activity in
dihydroxylation and epoxidation of styrene and its derivatives. The
best suited system for dihydroxylation is fully acetylated LYS
in various types of chemical reactions , for example, imine
7
g
9
10
reduction ,
sulfoxidation ,
hydrogenation ,
Diels-Alder
reduction ,
11
12
reactions ,
diastereoselective
coenzyme
1
3
13d, 14
conjugated with
phenylethanediol with an enantioselectivity of 95 %ee (S). BSA fully
acylated with hexanoic acid and conjugated with three mol RuCl per
K
2
OsO
2
(OH)
4
,
which converts styrene to 1,2-
dihydroxylation or epoxidation.
Most of the reactions catalyzed by AME’s are carried out in
3
aqueous media, but there are also some examples, which
7
c, 7d, 14b, 15
mol protein shows the highest ee values for the conversion of
styrene to the respective epoxide with enenatioselectivities of over
involve organic co-solvents.
A multitude of these reactions can be performed with natural
enzymes, too, which are in many cases much more active but
often less stable as artificial enzymes particularly in
aqueous/organic co-solvent systems.
80 %ee (R), a TON of more than 2500 and a yield of up to 78 %
within 24 h at 40 °C. LYS has two favored selective binding sites for
the metal catalyst and BSA has even three. The AME’s with titanate
in the active center invert the enantioselectivity of styrene
epoxidation.
Recently, we have shown that the conjugation of enzymes with
polymers, such as poly(2-methyl-2-oxazoline) (PMOx) renders
the biocatalysts soluble in organic media with improved activity
1
6
compared to the native enzyme powder. The organo-soluble
conjugates were also used as core-shell nanocontainers to drive
organo-insoluble salts into the enzyme structure that forces the
heavy metal ions to bind in the chiral environment of the enzyme,
possibly in the active site. This leads to the generation of
organo-soluble AME’s. Those are active in the dihydroxylation of
styrene in chloroform, which affords in the case of laccase as
enzyme enantioselectivities of the product formation of more
Introduction
Enzymes have been established as highly selective and active
catalysts in water, but also in organic solvents. The biocatalysts
are used in fields such as biosensors and production of fine
1
chemicals. In general, the naturally limited substrate spectrum
and activity of enzymes in organic solvents has been improved
17
than 99 %ee ((R)-enantiomer). However, the method of
2
by several techniques including protein engineering , medium
preparing these conjugates is rather tedious and the used
enzymes are expensive. Besides making the protein organo-
soluble, the major role of the conjugation might be blocking the
primary, easily accessible amino groups in the protein scaffold.
This way, the heavy metals are forced to bind in less accessible
sites of the protein, which are more likely forming a chiral
environment than binding positions rather at the surface of the
biomacromolecule.
3
4
engineering , and binding on suitable supports. The latter has
been explored in our group, by using amphiphilic conetworks as
4
e, 4f,
5
4h,
6
films
and particles
or by applying electrospun
[
a] Dipl.-Chem. Melanie Leurs, M.Sc. Bjoern Dorn, M.Sc. Sascha Wilhelm,
B.Sc. Magiliny Manisegaran, Prof. Dr. rer nat. Joerg C. Tiller*
Chair of Biomaterials and Polymer Science, Department of Biochemical
and Chemical Engineering, TU Dortmund, Emil-Figge-Str. 66, 44227
Dortmund, Germany
In the present study, we explore this concept of blocking the
amino groups of readily available proteins by acylation and
rendering them into AME’s active in oxidation reactions.
*
2
To whom correspondence should be addressed. Phone: +49 (0)231 755
479, Fax: +49 (0)231 755 2480, Email: joerg.tiller@tu-dortmund.de
Supporting information for this article is given via a link at the end of
the document.
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Chemistry - A European Journal
10.1002/chem.201802185
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Results and Discussion
The objective of this work was to render inexpensive proteins
into AME’s for the enantioselective synthesis in organic solvents.
The rational is that blocking the primary amino groups by
acylation might block the outer non-chiral binding sites, mostly
the lysine-based primary amino groups, for heavy metal
catalysts and force those into less accessible, but likely better
chirality controlling sites of the protein. (see Figure 1).
Figure 2. Degree of functionalization vs. molar equivalents of anhydride per
primary amino group of enzyme plot for the functionalization of lysozyme
(
LYS). The degree of functionalization was measured spectrophotometrically
20
by an adjusted TNBS-Assay according to literature known protocols (see
experimental section).
The resulting acylated LYS’s are not soluble in organic solvents
in contrast to the respective formerly reported poly(2-oxazoline)
1
6c, 17a
(
POx)-conjugates
, but form stable suspensions in various
organic solvents such as toluene and chloroform (Figure 3).
Figure 1. Concept of acylated artificial metallo enzymes for asymmetric
dihydroxylation.
Lysozyme (LYS) and bovine serum albumin (BSA) were chosen
as readily available inexpensive and stable proteins. They were
modified with different acyl anhydrides according to established
1
8
literature protocols for N-acylation. First the results of LYS
acylation are discussed. The conversion was followed by
photometric titration of the remaining primary amino groups. As
seen in Figure 2, the conversion of the amino groups strongly
depends on the excess of acyl anhydride, providing the
opportunity to control the degree of modification. In general, the
acetylation (AA) seems to be less efficient than propionylation
Figure 3. Pictures of suspensions of 4.2 mg fully acetylated LYS (LYS-AA) in
10 mL chloroform (left, top) and toluene (left, bottom) and DLS measurements
of the fully acylated LYS in toluene (right).
(
PA) and hexanoylation (HA). To verify the selectivity of the
DLS measurements of the acylated LYS in toluene revealed that
the size of the formed aggregates decreases in the order AA (4-
acylation reaction for primary amine groups, unmodified LYS
and LYS-AA were pH-titrated between pH 3.3 and 11.6 (see
6
µm)>PA (1-2 µm)>HA (0.5-0.8 µm) (Figure 3, right). This is in
1
9
Figure S1) according to a literature protocol. The given plot
displays the number of titratable groups in the chosen pH range.
Comparison of the final values at pH 11.6 shows 28 titratable
groups for LYS und 22 for LYS-AA, which is in good agreement
with the expectations for the acetylation of the 6 primary amine
groups of the lysine units. Furthermore this experiment strongly
indicates that no titratable functional group other than the
primary amine groups was modified during the acylation.
good compliance with the expectation that nonpolar solvents
such as toluene should afford finer dispersions of the conjugates
because the modification inserts an increasingly less polar
carbon chain to the protein. No particles could be detected by
DLS in the chloroform suspensions, indicating particle sizes
larger than 10 µm.
First, the established dihydroxylation of styrene was carried out
with
a 1:1 (mol/mol) osmate complex with the modified
lysozymes (Os-LYS, metal to protein ratio (MPR) = 1). It is
known to literature, that enzyme activity in organic solvents
3
b
strongly depends on the protonation state of the protein. The
pH can be adjusted in aqueous environment and the resulting
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Chemistry - A European Journal
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protonation state remains frozen in organic media (pH-
3
b
memory). This way, the acetylated LYS with varying degrees of
modification were adjusted to three different pH values (pH 6.3,
7.0, and 8.4) prior to lyophilization. Then, the acetylated as well
as the unmodified LYS’s were suspended in chloroform,
potassium osmate was added, and after 30 min of stirring
styrene and peroxide were given to the suspension. The product
was isolated after 7 d at 0 °C and analyzed by HPLC. No
product formation was found for the suspended pristine enzyme
powder. In contrast, Os-LYS-AA affords a significant formation
of 1,2-phenylethanediol. All derivatives favor synthesis of the
(
S)-enantiomer of the product (Fig. 4).
As seen in Figure 4 a), the highest activity and selectivity was
achieved at pH 7. The higher the number of modified amino
groups, the higher was the selectivity and also the activity. This
proves the concept of this work to be effective, i.e. the blocking
of the amino groups of proteins forces the catalytic metal into
regions of the protein that better control the enantioselectivity
and also the activity.
Figure 4: a): Turnover number (TON) and enantiomeric excess (ee, (S)-
enantiomer) vs. pH plot of the asymmetric dihydroxylation catalyzed by Os-
LYS-AA with an MPR of 1. The reaction was carried out with 0.03 µmol/mL
LYS-AA, 0.03 µmol/mL
2 2 4
K OsO (OH) , 110 µmol/mL tBuOOH and 100
µmol/mL styrene at 0°C for 7 d in chloroform. The TON is defined as µmol
product per µmol protein. The degree of modification is the percentage of
primary amines of the protein blocked by acylation.
b): Comparison of turnover frequency (TOF, coloured bars) and enantiomeric
excess (ee, (S)-enantiomer, black bars) of the asymmetric dihydroxylation
catalyzed by osmate based AME’s (pH 7, fully acylated) with an MPR of 1 at
different temperatures. The reaction was carried out with 0.03 µmol/mL protein
conjugate, 0.03 µmol/mL
2 2 4
K OsO (OH) , 110 µmol/mL tBuOOH and 100
µmol/mL styrene. The reaction times were adjusted to previously determined
surviving times of a respective Os-laccase-PMOx under the used conditions
17a
and temperatures, being 7 d at 0 °C, 72 h at 20 °C, and 24 h at 40 °C. The
TOF is defined as µmol product per µmol protein per hour.
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All further investigations were carried out with fully acylated
proteins adjusted to pH 7. The different acylated LYS’s were
conjugated with osmate and the catalytic activity for the
dihydroxylation of styrene was explored at different
temperatures.
Taking a look at Figure 4 b), top, shows that the best
enantioselectivity in the dihydroxylation of styrene with lysozyme
based AME’s was achieved with some 98 %ee (S) by using Os-
LYS-HA at 0 °C. The respective Os-LYS-PA also affords a high
enantioselectivity with 95 %ee (S) under the same reaction
conditions but about 4-fold lower activity. In comparison to the
latter Os-LYS-AA shows the lowest enantioselectivity with
and also the enantioselectivity is with 50 %ee (S) significantly
lower. The substrates α-methylstyrene and 4-methylstyrene are
converted with lower rates and the product formation cannot be
considered enantioselective. 1H-Indene and allylphenylether are
not converted at all. Investing the conversion of these substrates
with Os-BSA-AA leads to a similar picture, with exception of 4-
methylstyrene, which is converted with a 5-fold higher TON and
the (S)-enantiomer is favored with an enantioselectivity of
90 %ee (S). The Os-BSA-AA is also not active in the conversion
of 1H-Indene and allylphenylether. The high substrate selectivity
of the acylated protein complexes might be usable for separation
of product mixtures of styrene derivatives. The previously
5
7 %ee (S) and also a lower activity. Increasing the temperature
reported Os-laccase-PMOx shows
selectivity, e.g., catalyzing the dihydroxylation of 1H Indene
(TON 5.2 after at °C, 71 %ee (1R,2S)) and
allylphenylether (TON = 1.2 after 7 d at 0 °C, 87 %ee (R)) but no
conversion was found for 4-methylstyrene and -methylstyrene.
This underlines the strong role of the nature of protein for the
catalytic process of AME’s.
a
different substrate
to 20 °C expectedly increases the activity by a factor of 2-5 and
lowers the enenatioselectivity. Os-LYS-HA and OS-LYS-PA
retain enenatioselectivities of 88 and 85 %ee (S), respectively.
Further increasing the reaction temperature to 40 °C leads to
greatly enhanced activities, but the enantioselectivity drops to
below 40 %ee (S) in both cases.
=
7
d
0
The same experiments were carried out with fully acylated BSA
(
Figure 4 b), bottom). The optimal pH for high activity and
Table 1: Comparison of turnover frequency (TOF), yield, turnover number
selectivity was found to be 7.0 as well. Os-BSA does catalyze
styrene dihydroxylation. Os-BSA-AA, however, shows the
(TON) and enantiomeric excess (ee) of the asymmetric dihydroxylation
catalyzed by Osmate-based AME’s (pH 7, fully acylated) with an MPR of 1
with different alkenes at 20 °C for 3 d. The reaction was carried out with 0.03
-1
highest activity (TOF 0.9 h , TON of 275 after 7 d at 0 °C) and
also a high enantioselectivity (73 %ee (S)), favoring formation of
the (S)-enantiomer. In case of Os-BSA-HA the product formation
is hardly selective (25 %ee (S) at 0 °C), while Os-BSA-PA
affords an enantioselectivity of 94 %ee (S) at 0 °C. Increasing
the temperature to 20 °C also leads to increased TOF values
and in the cases of Os-BSA-PA and Os-BSA-AA, the
enantioselectivity is still high in a range of 76 – 80 %ee (S) at
µmol/mL protein conjugate, 0.03 µmol/mL
2 2 4
K OsO (OH) , 110 µmol/mL
tBuOOH and 100 µmol/mL alkene for 3 d in chloroform. The TON is defined as
µmol product per µmol protein. The TOF is defined as µmol product per µmol
1
protein per hour. H-Indene and allylphenylether are not converted by both
AME’s.
-
1
AME
alkene
ee [%] TON TOF [h ] Yield [%]
2
0 °C. Remarkably, Os-BSA-AA shows a selectivity which is
Os-LYS-HA 4-chlorostyrene 50
Os-LYS-HA 4-methylstyrene 43
Os-LYS-HA α-methylstyrene 31
Os-BSA-AA 4-chlorostyrene 51
Os-BSA-AA 4-methylstyrene 90
Os.BSA-AA α-methylstyrene 26
112 1.5
3.4
0.6
0.09
1.7
3.3
0.1
higher than 60 %ee (S) at 40 °C.
Altogether, the measured selectivities of the 1:1 complexes of
acylated LYS and BSA with osmate are with up to 98 %ee (S)
20
3
0.3
1
3a, 17b
comparable to that of other designed AME’s.
This shows
0.04
0.8
that simple acylation of readily available proteins and
complexation with osmate renders those into enantioselective
metalloenzymes for the dihydroxylation of styrene in organic
solvents. The enantioselectivity depends on the nature of the
protein and the protein specific choice of the carbonic anhydride
used for modification. BSA is more stable with respect of
retaining enantioselectivity at higher temperatures.
55
108 1.5
5
0.06
As shown previously, the solvent plays an important role in the
1
7a
selectivity of enzymes.
the experiments above at 0 °C with acylated LYS’s in toluene
results provided in Table S2 in the supplements). The activity of
This was addressed by carrying out
In order to broaden the concept of using AME’s prepared by
complexation of acylated proteins with metals, LYS and BSA
were conjugated with catalysts suitable for epoxidation.
(
all three conjugates is similar to that of the most active AME Os-
LYS-AA in chloroform, but the enantioselectivity is below
4 2 3
Ti(OMe) , Mn(Ac) , and RuCl , respectively, were considered as
2
1
potential candidates as suggested in the literature. Fully
acylated LYS and BSA, respectively, adjusted to pH 7.0 were
reacted in 1:1 molar ratio with the respective metal compound.
The reaction was carried out in dichloromethane (DCM).
Preliminary investigations showed that the by far highest
conversion of styrene was achieved when using RuCl
catalytic metal salt. The results of ruthenium-based AME’s
derived from acylated LYS will be discussed first.
30 %ee favoring the (S)-enantiomer in all cases. This confirms
the great influence of solvents on stereoselective reactions of
even artificial metalloenzymes.
A number of alkenes were investigated as substrates for the
most active and selective AME Os-LYS-HA in chloroform at
3
as
20 °C. The results, summarized in Table 1, show that this
complex catalyzes the dihydroxylation of 4-chlorostyrene, 4-
methylstyrene and α-methylstyrene. In case of 4-chlorostyrene
the achieved TON is about 2 times lower than that for styrene
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The kinetics of the styrene epoxidation catalyzed by Ru-LYS-AA
was measured at 0 °C, 20 °C, and 40 °C, respectively. As seen
in Figure 5, the TON increases linearly at 0 °C during the entire
reaction time of 7 d leading to a TON of 500 which calculates to
to 40 °C leads to a 6-fold increase in TOF and thus an epoxide
yield of 46 % after 24 h with a selectivity of 50 %ee (R). When
raising the temperature of this reaction to 50 °C no increase in
activity was observed, but the enatioselectivity was found to be
only 30 %ee (R).
16 % conversion of styrene to styrene oxide. Only small
amounts of aldehyde byproducts were found indicating product
selectivities higher than 90 %. The enantioselectivity of the
product formation favoring the (R)-enantiomer is with 95 %ee
exceptionally high. When increasing the reaction temperature to
2
0 °C the slope of the curve is steeper, but no further product is
formed after three days. The achieved TON of 749 calculates to
2 % yield, but the enantioselectivity drops to 63 %ee (R). The
2
activity further increases at 40 °C. However, no significant
conversion occurs after 24 h. The yield is again increased to
47 % with a drop of enantioselectivity to 50 %ee (R). This
indicates that the generated active site for epoxidation in the
protein is somehow altered during the course of the reaction like
it has been found for dihydroxylation of styrene with Os-laccase-
17a
PMOx in a previous study.
It is worth noting that the
enantioselectivity does not significantly change during the
course of the reaction (see Table S3 in the supplements). All
following reactions were stopped after 24 h at 40 °C, after 72 h
at 20 °C, and after 7d at 0 °C, respectively.
Figure 6: Comparison of turnover frequency (TOF, coloured bars) and
enantiomeric excess (ee, (R)-enantiomer, black bars) for the asymmetric
epoxidation catalyzed by Ru-based AME’s (pH 7, MPR = 1) in DCM at different
temperatures. The reaction was carried out with 0.06 µmol/mL protein, 0.06
Figure 5: Turnover number (TON) vs. time plot for the asymmetric epoxidation
catalyzed by Ru-LYS-AA (pH 7, MPR = 1) in DCM. The reaction was carried
3
µmol/mL RuCl , 220 µmol/mL tBuOOH and 200 µmol/mL styrene for 7 d at
out with 0.06 µmol/mL LYS-AA, 0.06 µmol/mL RuCl
3
, 220 µmol/mL tBuOOH
0 °C, 72 h at 20 °C, and 24 h at 40 and 50 °C. The TOF is defined as µmol
product per µmol protein per hour.
and 200 µmol/mL styrene at 0 °C, 20 °C, and 40 °C, respectively. The TON is
defined as µmol product per µmol protein. The black lines illustrate the trend of
the data points. The enantioselectivity does not change in the curse of the
reaction and the ee values are presented in Figure 6.
Carrying out the epoxidation of styrene catalyzed by acylated
Ru-BSA AME’s at 20°C reveals that the hexanoated Ru-BSA is
the best suited epoxidation catalyst of styrene regarding ee
The styrene epoxidation catalyzed by the fully acylated Ru-
LYS’s was investigated at 20 °C. The activity of the AME’s
increases in the order LYS<LYS-HA< LYS-PA<LYS-AA (see
Figure 6). The (R)-conformation of the formed epoxide is favored
in all cases. The pristine Ru-LYS shows a high activity in
-1
(
(
88 %ee (R)) and activity (TOF = 6 h , TON = 452 after 3 d)
Figure 6). Ru-BSA-PA still affords an enantioselectivity of
72 %ee (R), but the activity is less than half of that of Ru-BSA-
-1
HA. Epoxidation catalyzed by Ru-BSA-AA leads to product
formation with only 30 %ee (R) and rather low activity. The
pristine Ru-BSA also catalyzes epoxidation of styrene, but the
product formation is hardly enantioselective resulting in an ee
value of only 20 % for the (R)-enantiomer.
The TOF of Ru-BSA-HA increases nearly 6-fold when increasing
the temperature from 20 °C to 40 °C. More remarkably, the
selectivity is with 80%ee (R) still high. Further increasing the
temperature to 50 °C allows 47 % conversion of styrene within
epoxidizing styrene (TON = 361 after 3 d, TOF = 5.0 h ), but the
product formation is hardly enantioselective (10 %ee (R)). The
Ru-LYS-HA affords higher enantioselectivity of 56 %ee (R) with
-1
slightly increased activity (TOF = 6.2 h , TON = 449 after 3 d)
-1
compared to Ru-LYS. Ru-LYS-PA (TON = 561, TOF = 7.7 h )
-1
and Ru-LYS-AA (TON = 749, TOF = 10.4 h ) show a product
selectivity of 74 and 63 %ee (R), respectively, and even higher
activities than Ru-LYS-HA. In the case of the styrene
epoxidation catalyzed by Ru-LYS-AA an increase in temperature
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Chemistry - A European Journal
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FULL PAPER
24 h, but the selectivity was found to be only 56 %ee (R).
3
three selective binding sites for ruthenium. This Ru -BSA-HA is
Regarding the course of selectivity loss over temperature it can
be concluded that the selectivity loss occurs at lower
temperatures for the acylated Ru-LYS AME’s than for respective
Ru-BSA and thus the latter are more robust at higher
temperatures.
the most active and still highly enantioselective AME for the
epoxidation of styrene. The reaction at 40 °C with only 0.18
µmol/L ruthenium chloride and 0.06 µmol/L BSA-HA leads to an
-1
epoxide yield of 78 % (TON = 2613, TOF = 109 h ) after 24 h
and a high enantiomeric excess of 82 %ee (R). This AME can
compete with a recently reported ruthenium aqua complex
supported on silica particles where an ee value of 79 % and a
conversion of 92 % was found after 6 h for styrene at 25 °C
2
Acylated LYS was also treated with Mn(Ac) . The resulting
AME’s were investigated as catalysts for the epoxidation of
styrene at 20 °C for 3 d. The results are summarized in Table S4
in the supplements. The acylated Mn-LYS’s show low TOFs of
21c
using 1 mol% Ru. By increasing the MPR to 4 even a yield of
88 % can be achieved but the enantioselectivity decreases to
44 %ee (R).
-1
some 0.1
enantioselectivities similar to RuCl
are in the range of a literature known Mn-Carbonic Anhydrase
–
0.3
h
(TON
=
8-24 after
3
d) and
3
based system. The activities
(
7
TON between 2.2 and 21 at 30 °C using BES buffer (0.1 M, pH
.2) as reaction medium).
1
4c
4
Alternatively, acetylated LYS’s were treated with Ti(OMe) ,
which binds supposedly to the enzyme as titanate species, and
investigated as catalysts for epoxidation of styrene at 20 °C for 3
-1
d. The found TOF values are between 0.2 and 0.3 h (TON = 14
22 after 3 d) for Ti-LYS-PA and Ti-LYS-AA, respectively, and no
conversion was found in the presence of the Ti-LYS-HA.
Interestingly, the Ti(OMe) based AME’s favors the (S)-
conformation of the product with remarkably high ee values of
6 and 90 %ee (S) for Ti-LYS-PA and Ti-LYS-AA, respectively.
-
4
8
This inversion of stereoselectivity might be due to different
mechanisms of the titanium and ruthenium-based catalysts, e.g.
formation of metallacyclo intermediates of the acylated Ti-LYS
22
and electrophilic oxidation in case of the acylated Ru-LYS.
Also the used tert-butylperoxide might play a role in this,
because the titanium peroxide complexes form intermediates,
which would then carry an additional ligand (tert-butyl), what
might slow the reaction rate down and change the
enantioselectivity. The Ti-BSA-HA also catalyzed the
epoxidation of styrene with a slow rate but a fairly high
enantioselectivity (75 %ee (S)). The design of the AME’s was
based on previous work, where the best enantioselectivities
were achieved by 1:1 (molar ratio) complexes of protein
Figure 7: Metal to protein ratio (MPR) vs turnover number (TON) and
enantiomeric excess (ee) plot for the asymmetric epoxidation of styrene
catalyzed by Ru-LYS-AA and Ru-BSA-HA (top, (R)-enantiomer) and Ti-LYS-
AA and Ru-BSA-HA (bottom, (S)-enantiomer) (PH 7) in DCM at 20 °C for 3 d
for LYS-AA and at 40 °C for 24 h for BSA-HA. The reaction was carried out
with 0.06 µmol/mL protein, 0.06 to 0.24 µmol/mL metal species, 220 µmol/mL
tBuOOH and 200 µmol/mL styrene. The TON is defined as µmol product per
µmol protein.
(
laccase) and metal compound (osmate). In order to explore, if
The same scenario was found for the Ti-LYS-AA, indicating that
the titanate binds at similar positions as the ruthenium (Figure 7,
bottom). The only difference is that the third binding site affords
a larger drop in enantioselectivity than found for the respective
the investigated proteins have more than one binding site that
controls the enantioselectivity of the product formation, the metal
salt RuCl
reaction was carried out at 20 °C for 3 d. As seen in Figure 7,
top, Ru -LYS-AA with two ruthenium centers is nearly doubling
its activity, while slightly losing its enantioselectivity (65 %ee to
6 %ee (R)). The addition of one more ruthenium to the protein
3
was added in varying ratios to LYS-AA and the
Ru -LYS-HA. Comparison of these two AME’s shows that the
3
2
titanate based systems are more selective and less active than
the respective ruthenium systems. Besides this difference, the
binding sites of titanate and ruthenium seem to be similar in both
acylated proteins.
Having established that the proteins might have more than one
metal binding site, we investigated, if the thermally inactivated
catalyst with one metal center can be re-activated by
subsequent adding of another portion of metal species. If only
the surrounding of the metal ion is oxidized, the additional metal
ions might bind in other regions of the protein renewing the
activity. Thus, the epoxidation of styrene was carried out with
Ru-LYS-AA and Ru-BSA-HA, respectively, at 40 °C and another
5
leads again to an increase in activity accompanied by a
significant loss in enantioselectivity (35 %ee (R)). This means
that the acetylated lysozyme contains two binding sites for
ruthenium that enantioselectively catalyze the epoxidation of
styrene to the (R)-enantiomer of the respective epoxide, i.e. it
represents an AME with two active sites. There is also a third
binding site that actively catalyzes this reaction but with no
selectivity. Alternatively, it is thinkable that the excess of salt is
only changing the equilibrium between metal salt and one active
center that complexes the metal species. However, this seems
less likely, because the salt is not soluble in the organic solvent.
The same experiment was carried out with BSA-HA at 40 °C for
molar equivalent RuCl was added to the reaction mixture after
3
24 h reaction time. The activity of both AME’s could be renewed
this way. Also subsequent lowering of the reaction temperature
24 h. As seen in Figure 7, top, BSA-HA seems to contain even
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Chemistry - A European Journal
10.1002/chem.201802185
FULL PAPER
has no re-activating effect on the AMEs. Thus we propose that
the deactivation of the enzyme might be due to irreversible
oxidation of the complexing groups in the protein or reaction with
the formed styrene oxide with these groups.
Tab. 2: Comparison of turnover frequency (TOF), yield, turnover number
(
TON) and enantiomeric excess (ee) of the asymmetric epoxidation catalyzed
by ruthenium based AME’s (pH 7) at 40 °C for 24 h for Ru -BSA-HA and at
20 °C for 3 d for Ru -LYS-AA with different alkenes. The reaction was carried
out with 0.06 protein, 0.18 and 0.12 µmol/mL µmol/mL K OsO (OH) , 220
3
2
2
2
4
µmol/mL tBuOOH and 200 µmol/mL alkene. The TON is defined as µmol
product per µmol protein. The TOF is defined as µmol product per µmol
protein per hour.
To identify possible metal binding sites we took a deeper look at
the protein structure (see Figures S5 and S6 in the
supplements). The reaction with unmodified protein resulting in
low ee values indicates that the easily accessible primary amino
groups of lysine on the surface of the protein are possible
binding sites of the metal ions. Blocking these groups by
acylation leads to greatly increased enantioselectivities.
Comparison with successful epoxidation catalyst ligands
suggests that the secondary amines of tryptophan and histidine
plus the tyrosine residues as potential docking sites. Lysozyme
-1
AME
alkene
ee [%] TON TOF [h ] Yield [%]
Ru
3
Ru
3
Ru
3
Ru
2
Ru
2
Ru
2
-BSA-HA 4-chlorostyrene 79
-BSA-HA 4-methylstyrene 36
-BSA-HA α-methylstyrene 91
-LYS-AA 4-chlorostyrene 97
-LYS-AA 4-methylstyrene 80
-LYS-AA α-methylstyrene 67
1381 58
42
21
24
10
9
703
789
341
318
28
29
23
contains at least 3 tyrosines, 6 tryptophanes and 1 histidine.
33
Due to the high achieved ee values and thus the necessity of a
stable binding in a chiral environment, it was assumed that the
formed metal-protein complex leading to enantioselective
product formation is at least bidental. For such a binding
scenario we recognized three possible binding sites in lysozyme.
Two of them (via Trp28, Tyr23 and Trp 108, Trp 111) are located
at helical structures whereas the third one (Trp 62, Trp 63) is
situated within a random coiled area of the protein (Figure S5).
The fact that only two binding sites direct the stereoselectivity
suggests that the best binding sites are in the region of the α-
helices. BSA shows a more complicated structure containing 20
4.7
4.4
0.4
0.8
Conclusions
2
4
tyrosines, 2 tryptophanes and 17 histidines and thus much
more possible binding sites for metal compounds. Looking for
opportunities to form bi- or multi-dental complexes metal binding
also different opportunities are possible like it is, exemplarily,
shown in Figure S6.
The concept idea of the present study was to render common
proteins into highly selective AME’s by blocking the primary
amino groups by acylation. The chosen enzymes lysozyme and
bovine serum albumin were acylated with different carboxylic
2 2 4 4 3
acids and treated with K OsO (OH) , Ti(OMe) , and RuCl ,
To broaden the reaction scope a number of styrene derivatives
respectively, in different organic solvents. It was found that
acylation is required to obtain enantioselective dihydroxylation
and epoxidation of styrene derivatives with AME’s. This proves
the concept to be valid and opens a great opportunity to readily
achieve AME’s as highly active and selective catalysts from
natural sources. With respect to activity and selectivity, the
acylated protein conjugates outperform literature known artificial
metalloenzymes. For example, a xylanase equipped with a
manganese porphyrin leads to a styrene oxide yield of 17 % and
with an enantiomeric excess of 8.5 %ee (S) after 1 h at RT in
3 2
were investigated as substrates for Ru -BSA-HA and Ru -LYS-
AA catalyzed epoxidation at 40 °C and 20 °C, respectively. The
results, shown in Table 2, reveal that the reaction can also be
carried out with 4-chlorostyrene, 4-methylstyrene and α-
methylstyrene with significant conversion. 1H-Indene and
allylphenylether are not converted. For example, 4-chlorostyrene
is epoxidized by Ru
high ee with 78 %ee (R). The epoxidation of this substrate
catalyzed by Ru -LYS-AA leads to an increase in
enantioselectivity to 97 %ee (R) with 10 yield. 4-
methylstyrene is epoxidized to 21 % with a low enantioselectivity
of 32 %ee of the (R)-enantiomer catalyzed by Ru -BSA-HA. In
contrast, a high enantiomeric excess of 80 %ee (R) but a lower
yield of 9 % is achieved by catalysis with Ru -LYS-AA of the
3
-BSA-HA catalysis with 42 % yield and a
2
%
14b
phosphate buffer (50 mM, pH 7). Two other reports describe
the epoxidation of different styrene derivatives by H O as
2 2
3
oxidant, catalyzed by bovine and human carbonic anhydrases,
2+
in which the original catalytic Zn ion was replaced by a
2
manganese species. The one published by Okrasa and
Kazlauskas reports conversions between 1% (TON = 2.2 after 2
h) and 12 % (TON = 21 after 16 h), and enantiomeric excesses
epoxidation of the latter substrate. -methylstyrene is converted
to 24 % with a very high ee value for the (R)-enantiomer of
91 %ee (R) for when catalyzed by Ru
3
-BSA-HA. In the case of
between 50 %ee and 66 %ee at 30 °C using BES buffer (0.1 M,
Ru -LYS-AA a dramatic drop in both, conversion and ee was
2
14c
pH 7.2) as reaction medium.
Soumillion et al. used a
observed for this substrate. Thus, the efficiency of catalysis is
strongly dependent on the nature of the protein and the styrene
derivative. Also the spectrum for asymmetric epoxidations is
broader than that of dihydroxylation catalyzed by osmate-based
systems, but still limited to structures close to styrene.
phosphate buffer (50 mM, pH 7)/DMF mixture and reported
yields between 9 % and 57 % with 18 % to 52 % ee overnight at
14a
4
°C.
Epoxidation of styrene with an artificial manganese
chloroperoxidase from Caldariomyces fumago in BES buffer (0.1
M, pH 7.2) yields to 40 % after 5 h (TON 1500) with an
14c
enantiomeric excess of 49 %ee (R).
Also natural
monooxygenases can be used for epoxidation reactions. For
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Chemistry - A European Journal
10.1002/chem.201802185
FULL PAPER
example the usage of
pseudomonas sp. leads to very high specific epoxidation
a
fused StyA/StyB system from
Functionalization with hexanoic anhydride was carried out at room
temperature because of the better suspension of hexanoic acid in water
at this temperature. Afterwards the respective molar amount of anhydride
for the desired degree of functionalization (see figure 2) was added and
the mixture was allowed to react under vigorous stirring for 30 min at the
preset temperature followed by subsequent lyophilisation to obtain a
white solid. Then the solid was suspended in water and the pH was
adjusted with NaOH and HCl solutions (0.01 M) prior to lyophilisation.
-
1
activities for styrene of 95 min (99 %ee (S) within the first 5 min
2
5
(
TON = 475). Hollmann et al. reached a productivity of
maximum 6.4 mM/h with 6.5 µM StyA after 15 min, what means
-1
a TOF of around 66 min , and a total turnover number after 2 h
2
6
of 662 with more than 98 %ee (S) for styrene epoxidation.
Thus the natural enzymes are much more active, but less stable
than the artificial metalloenzymes used in this study.
It is particularly remarkable that the proteins used in this study
might have more than one enantioselectivity controlling binding
site. Lysozyme could contain two selective sites, and at least a
third non-selective binding site. The BSA might have three
selective sites and minimum a fourth that might direct to the
opposite enantiomer. This shows that the protein structures are
indeed rich of different chiral binding sites and there might be
more opportunities to direct the catalytic metal to those, e.g. by
blocking other functional groups or even introducing new and
more efficient ones by chemical modification. Introducing metal
complexing functional groups that are less susceptible for
oxidation or alkylation might make the AMEs more stable and
possible more active.
Determination of degree of acylation
The degree of functionalization of acetylated proteins was determined by
an adjusted TNBS-assay according to literature known protocols. . To
20
-
1
1
0
00 µL of protein mixture (1.4 mg mL in water) were added 400 µL of a
.1 M NaHCO buffer at pH 8.5 and 250 µL of 0.01 % TNBS solution
3
prepared in the same buffer. The solution was allowed to react at 40 °C
for 2 h. Then, 250 µL 10 % (w/v) SDS was added followed by addition of
125 µL aqueous 1 M HCI. The absorbance (E) of the mixture was
measured spectrophotometrically in a quartz glass cuvette (d = 1 cm) at
346 nm at 20 °C against a blank treated as above but with 500 µL of
buffer instead of the protein solution. The number of free primary amino
groups of the modified and unmodified protein were calculated according
to Lambert-Beers law with a molar extinction coefficient of ε = 14600
-
1
-1
mM cm .
Experimental Section
Materials
All chemicals were purchased from Sigma-Aldrich, Applichem, Merck,
ABCR, Acros, Armar Chemicals, or Carl Roth and were of analytical
grade or purer. Lysozyme from henn egg white (EC 3.2.17, ≥20000 U/g
Dihydroxylation of styrene (general procedure).
(
pH 6.2)) was purchased from AppliChem and bovine serum albumin
The acetylated protein (0.3 µmol) was dispersed in 10 mL of chloroform
(BSA, ≥ 98 % content) from Roth. All chemicals were used without further
or toluene. Afterwards, 111 µL of K
2 2 4
OsO (OH) dissolved in methanol
purification.
-
1
(1mg mL in MeOH, 0.3 µmol for an MPR = 1) was given to the reaction
mixture and is was stirred for 30 min. Then styrene (1mmol) was added.
Finally, a 200 µL of a 5.5 M tBuOOH solution in decane (1100 µmol) was
added and the reaction mixture was vigorously stirred at different
temperatures (0 °C, 20 °C or 40 °C).
The reaction was stopped by filtration through a 0.2 µm PTFE-filter
followed by the extraction with a 1 M aqueous sodium thiosulfate solution
Measurements
The turnover number (TON) and enantiomeric excess (ee) of the
dihydroxylation and epoxidation of alkenes was determined by high
performance liquid chromatography (HPLC) by using a HPLC system
(LaChrom Elite, Hitachi) equipped with a chiral column (Chiralpack IC,
(2 x 10 mL). Afterwards, the phases were separated and the aqueous
Daicel Chemical Industries). As eluent, a mixture of n-heptane and
isopropanol in a volume ratio of 9:1 was used. A flow rate of 0.5 mL min
phase was extracted 5 times with 25 mL chloroform and the organic
phases were combined. After removal of the solvent the obtained solid
was characterized by HPLC.
-
1
and a diode array detector at 254 nm at 8 °C was applied. Calibration
curves were performed with enantiopure samples of the respective diols
and epoxides purchased from Sigma-Aldrich.
The DLS measurements were performed on a Malvern Zetasizer nano S
at 20 °C. All samples were measured in toluene or chloroform. The
equilibration time before measurement was 2 min.
Epoxidation of styrene with acetylated proteins as ligand (general
procedure).
The acetylated protein (0.3 µmol) was dispersed in 5 mL of DCM.
Afterwards, RuCl , Ti(OMe) or Mn(Ac) (1 mg/mL in MeOH, 0.3 µmol for
3 4 2
an MPR = 1) was given to the reaction mixture and it was stirred for 30
min in a sealed vessel. After that the alkene (1 mmol) was added. Finally,
a 5.5 M tBuOOH solution in decane (200 µl, 1100 µmol) was added and
the reaction mixture was vigorously stirred at different temperatures (0 °C,
All pH-values were determined using
International) calibrated by three point calibration (buffer solutions: pH
.00/7.00/10.00 (HACH)).
a Qph70 pH-meter (VWR
4
Acylation of proteins (general procedure)
2
0 °C or 40 °C) in a sealed vessel.
The protein modification with different acyl anhydrides was performed
The reaction was stopped by filtration through a 0.2 µm PTFE-filter
followed by removal of the solvent. After that the obtained fluid was
characterized by HPLC.
18
according to established literature protocols for N-acetylation. First 0.3
µmol of the protein (4.2 mg for lysozyme, 19.9 mg for BSA) was
dissolved in 3 mL of bidistilled water and cooled to 4 °C for the
functionalization with acetic anhydride or propionic anhydride.
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Chemistry - A European Journal
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Entry for the Table of Contents
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[
a]
The commercially available
proteins lysozyme (LYS) and
bovine serum albumin (BSA) were
converted into highly active and
enantioselective artificial
Melanie Leurs , Bjoern
[
a]
[a]
Dorn ,Sascha Wilhelm , Magiliny
[
a]
[a]
Manisegaran and Joerg. C. Tiller*
Page No. – Page No.
metalloenzymes for dihydroxylation
and epoxidation by acylation of the
primary amino groups, which
affords the proteins to take up
metal salts in the core of the
protein.
Multicore Artificial
Metalloenzymes derived from
acylated proteins as catalysts for
the enantioselective
dihydroxylation and epoxidation of
styrene derivatives.
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