Combined heterogeneous bio- and chemo-
catalysis for dynamic kinetic resolution of (rac)-
benzoin†
R. Nieguth,
a
J. ten Dam,
b
A. Petrenz,
c
A. Ramanathan,
bd
U. Hanefeld
b
and M. B. Ansorge-Schumacher*
ac
Dynamic kinetic resolution (DKR) of racemic starting material is a promising route to optically pure (S)-
benzoin (2-hydroxy-1,2-di(phenyl)ethanone) and various symmetrical and unsymmetrical derivatives
thereof. Here, this route was advanced towards technical scale synthesis using the basic (rac)-benzoin as
model system. The reaction employed stereoselective transesterification of (S)-benzoin with lipase TL®
from Pseudomonas stutzeri and racemization of (R)-benzoin with Metal-TUD-1, a metal-associated
acidic meso-porous silicate, in pure organic solvent. Enzyme performance was improved by
immobilization on Accurel MP1001 (yielding Acc-LipTL), and Zr-TUD-1 (Si/Zr ¼25) was identified as
most effective racemization catalyst. Compatibility in solvent and temperature dependency enabled
performance in only one pot. DKR in toluene at 50 C yielded conversions above 98% and an ee of >97%
in only five hours. Stability of Acc-LipTL was further improved with polyethylene imine and the reaction
system was then reused in five cycles, retaining a conversion of >99% and a product-ee of >98%. On a
semi-preparative scale, the isolated yield of enantiopure (S)-benzoin butyrate was >98%. Thus, the
system provides a good basis for synthesis of enantiopure benzoin, and potentially a broader range of
aromatic a-hydroxy ketones.
Introduction
Enantiomerically pure acyloins (a-hydroxy ketones) provide
important structural motifs for a number of ne chemicals and
pharmaceuticals.
1–3
Hence, various synthetic routes, chemo- or
biocatalytic, have been described to date.
2
They considerably
differ in selectivity and yield, as well as in the accessible range of
concrete structures (i.e. aliphatic, heterocyclic, aromatic,
symmetrical, unsymmetrical, R-/S-stereochemistry, etc.) giving a
portfolio of potentially interesting methods.
Dynamic kinetic resolution (DKR) of racemic starting mate-
rial by combination of a biocatalysed kinetic resolution (KR)
with in situ racemisation (RAC) of the undesired substrate
enantiomer (Scheme 1) has recently been described as a
promising route to optically pure (S)-benzoin (2-hydroxy-1,2-
di(phenyl)ethanone) and various symmetrical and unsymmet-
rical derivatives of this basic aromatic a-hydroxy ketone.
3–5
The method involves stereoselective transesterication of
(rac)-benzoin with a lipase from Pseudomonas stutzeri (com-
mercialised by Meito Sangyo Co., Japan, under the trade name
of Lipase TL®, and immobilised through entrapment of the
crude enzyme powder in a silicone matrix) and RAC with a
homogeneous ruthenium based transition metal catalyst
(Shvo's catalyst) and achieves substrate conversions above 90%.
As product, (S)-congured butyrate esters with an enantiomeric
excess above 99% are obtained, from which enantiopure (S)-
benzoin derivatives can easily be released through hydrolysis
(e.g. using Lipase TL® under suitably adapted conditions).
Scheme 1 Dynamic kinetic resolution of benzoin using vinyl butyrate
as acyl donor and Lipase TL® as catalyst.
a
Lehrstuhl f¨
ur Technische Chemie/Enzymtechnologie, Institut f¨
ur Chemie, Technische
Universit¨
at Berlin, 10623 Berlin, Germany
b
Gebouw voor Scheikunde, Biokatalyse, Afdeling Biotechnologie, Technische
Universiteit Del, Julianalaan 136, 2628 BL, Del, The Netherlands
c
Professur f¨
ur Molekulare Biotechnologie, Institut f¨
ur Mikrobiologie, Technische
Universit¨
at Dresden, 01062 Dresden, Germany. E-mail: marion.ansorge@tu-dresden.
de; Fax: +49 351 46339520; Tel: +49 351 46339518
d
Center for Environmentally Benecial Catalysis, The University of Kansas, Lawrence,
Kansas 66047, USA
†Electronic supplementary information (ESI) available. See DOI:
10.1039/c4ra06751g
Cite this: RSC Adv.,2014,4, 45495
Received 7th July 2014
Accepted 15th September 2014
DOI: 10.1039/c4ra06751g
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Despite this encouraging performance, however, the approach
has not been transferred to synthetic application on a larger
scale to date due to several unfavourable characteristics: (1)
reproducibility of catalytic efficiency between separate reaction
batches is low. Most probably this is connected to the prepa-
ration of the biocatalyst which must lead to an uneven and
uncontrollable distribution of the lipase within the particles
and hence to widely varying catalytic performance. Additionally,
sensitivity of the ruthenium catalyst for traces of molecular
oxygen in the reaction set-up, and interference of acetaldehyde
released from vinyl butyrate (i.e. the optimal acyl donor for
benzoin esterication
4
) with the ruthenium catalyst
6
could be
an issue. (2) The ruthenium catalyst cannot be recycled, which
increases both costs for reaction and costs for product workup.
(3) Reaction conditions required for optimal performance and
stability of bio- and chemo-catalyst (e.g. temperature) are hardly
compatible resulting in low total turnover numbers and
consequently large catalyst consumption. However, this specic
item could recently be overcome by introduction of a novel
ruthenium complex for racemization.
7
This paper describes the advancement of DKR for technical
scale hydroxy ketone synthesis with regard to reproducibility,
catalyst recyclability and performance in one pot using DKR of
(rac)-benzoin as model system. The study involved systematic
development of a controllable and reproducible protocol for
Lipase TL® immobilisation and introduction of a heteroge-
neous metal catalyst for racemization. Kinetic resolution and
racemization of benzoin with these catalysts were characterised
both separately and in a one-pot DKR. Optimisation was per-
formed with regard to reaction medium and temperature. The
synthetic potential of the approach was demonstrated on a
semi-preparative scale.
Results and discussion
Immobilisation of lipase TL®
For immobilisation of Lipase TL® (further annotated as LipTL)
adsorption to the porous polypropylene resin Accurel MP1001
was selected. Leaching of enzyme during reaction must not be
expected due to its insolubility in hydrophobic solvents making
covalent attachment unnecessary.
Accurel is a highly hydrophobic material, for which bene-
cial effects on the catalytic behaviour of bound enzymes has
been reported before.
8
The obtained material had a size distri-
bution of 400–1000 mm in diameter (Fig. 1) and a specic
surface of 35 m
2
(according to the BET method). The surface
topology of individual particles, especially regarding pore size,
was rather diverse. It can be assumed, however, that all micro-
scopically visible pores are macroporous (i.e. displaying a
diameter >500 nm) and are thus at least one to two orders of
magnitudes larger than LipTL. Size differences should therefore
be of only minor importance to the uptake of this enzyme.
Enzyme immobilisation by adsorption is a spontaneous
process running into a distinct thermodynamic equilibrium of
bound and unbound protein. The position of equilibrium is
inuenced by many factors including carrier constitution and
preparation conditions. A key role is played by the protein
concentration in the immobilisation set-up.
9
The amount of
protein bound to the carrier is usually proportional to the
available surface of the carrier until the binding capacity is
exceeded, the protein aggregates, or multilayers are formed.
Accordingly, optimum conditions for adsorption of LipTL on
Accurel MP1001 were determined by introducing different
concentrations of the crude preparation into the immobilisa-
tion process. Success was evaluated with regard to the specic
catalytic activity of the resulting immobilisates in the trans-
esterication of benzoin with vinyl butyrate using tetrahydro-
furan (THF) as solvent.
4,10
The immobilised preparation is
further denoted as Acc-LipTL.
The specic catalytic activity of Acc-LipTL increased with
increasing concentrations of LipTL in the immobilisation process
up to a steady value of 18.4 U g
Acc-LipTL
1
correlating to a powder
concentration of and above 60 mg
LipTL
mL
immobilisation medium
1
(Fig. 2). Loading at this concentration was determined as 15.4
mg
Protein
g
Accurel
1
and accordingly, the specic catalytic activity
related to the protein content was 1192 U g
Protein
1
,whichisbya
Fig. 1 SEM-picture of Accurel MP 1001.
Fig. 2 Influence of LipTL concentration in the immobilisation set-up
(10 mmol L
1
KP
i
buffer, pH 7.0) on the resulting specific activity of
immobilised Acc-LipTL (carrier concentration fixed at 50 mg mL
1
).
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factor of three higher than the specic activity of the crude LipTL
preparation (i.e. 350 U g
Protein
1
).
An increase in the specic catalytic activity of LipTL through
immobilisation (by factor 2.2) had also been observed by Hoyos
et al.
5
As an explanation the enhanced transfer of the hydro-
phobic substrates and products through the also hydrophobic
silicone matrix, and a stabilisation of the catalytic active
conformation of LipTL (generally recognised as hyperactivation
or interfacial activation) were suggested. This active conforma-
tion usually describes a state where an amphipathic peptide
chain (lid) at the active site of the enzyme is open due to the
interaction of its hydrophobic part with a hydrophobic interface
or surface,
11
and it has only lately been reported that LipTL
might contain even two such lids.
12
Both explanations could
equally apply to the behaviour of LipTL on Accurel MP1001 due
to the distinct hydrophobicity of this material. In addition –or
alternatively –a selective binding of LipTL to the carrier could
occur which would result in a purication of the enzyme.
According to Maraite et al.,
12
commercial LipTL contains an
assortment of side proteins that might (at least partly) have a
signicantly lower affinity to the hydrophobic support than the
lipase itself. Even simpler, the activity increase on Accurel
MP1001 could be connected with the more even distribution of
the enzyme, and therefore better accessibility, on the carrier
compared to suspended (and usually aggregated) solid enzyme
in pure organic solvents. Overall, the results strongly support
the suitability of Accurel MP1001 as a carrier for LipTL.
On the assumption that the observed maximum
specic activity of Acc-LipTL at a concentration of 60
mg
LipTL
mL
immobilisation medium
1
correlated to the arrival at the
maximum binding capacity and might therefore
prohibit detection of further effects on activity, further
experiments on the optimisation of the immobilisation
process were performed at a constant concentration of
30 mg
LipTL
mL
immobilisation medium
1
.Buffer (KP
i
) concentration
and pH were varied between 10 mmol L
1
and 400 mmol L
1
and around the isoelectric point of LipTL (i.e. 6.6) to pH 6.2,
pH 6.6 and pH 7.0, respectively.
Optimum conditions with regard to the specic catalytic
activity of Acc-LipTL were found at a buffer concentration of 200
mmol L
1
and pH 6.2, with the buffer concentration as the
decisive parameter (Fig. 3). The maximum specic activity was
24.7 U g
Acc-LipTL
1
. At a loading of 17.8 mg
Protein
g
Accurel
1
, this
corresponded to a protein related specic activity of 1.4
Ug
Protein
1
, which was an increase by factor 4.1 compared to
crude LipTL and was almost two-fold better than the effect of
silicone entrapment as described by Hoyos et al.
5
The ndings on buffer and pH effects are in good agreement
with the assumption that the adsorptive binding of proteins to
the nonionic Accurel MP1001 is predominantly promoted by
hydrophobic interactions: changes of pH have only minor
effects on such binding forces, while phosphate anions
comprising the buffer are kosmotropic in nature and are
therefore strengthening them. The same effects are exploited in
hydrophobic interaction chromatography, where high ionic
strengths are used to selectively bind hydrophobic proteins.
13
Transfer of these optimum conditions to LipTL immobili-
sation at an initial powder concentration of 60 mg
LipTL
mL
immobilisation medium
1
resulted in a minor improvement of
the specic catalytic activity to 28.2 U g
Acc-LipTL
1
, which was in
perfect agreement with the initial assumption on already satu-
rated loading at this protein concentration. As the poor addi-
tional benet did not justify consumption of the double amount
of the valuable enzyme, a LipTL concentration of 30 mg
LipTL
mL
immobilisation medium
1
was established in the nal immobi-
lisation protocol.
KR of (rac)-benzoin
Acc-LipTL was subjected to KR of (rac)-benzoin using dried THF,
2-MeTHF and toluene, respectively, as solvents. The selection
was based on the reported suitability of THF and its environ-
mentally benign derivative 2-methyl tetrahydrofuran (2-MeTHF)
for kinetic resolution of benzoin,
4,10,14
and of toluene for race-
mization.
15
At room temperature (21.5 C), Acc-LipTL displayed
good activity in all three solvents (Table 1, column 3). The time
to reach maximum conversion (i.e. 50%), however, was
considerably shorter in 2-MeTHF and toluene (2.5 h) than in
THF (>4 h) (Table 1, column 4). The half-life of Acc-LipTL under
process conditions (calculated from the decrease of conversion
aer reaching steady-state in a continuous reaction set-up) was
Fig. 3 Influence of KP
i
buffer concentration and pH in the immobili-
sation set-up on the resulting specific activity of immobilised Acc-
LipTL (LipTL concentration fixed at 30 mg
LipTL
mL
immobilisation medium
1
and carrier concentration at 50 mg mL
1
).
Table 1 Specific activity of Acc-LipTL in the KR of benzoin using
different (dry) solvents at room temperature or 50 C, respectively
(catalyst concentration fixed at 20 mg
Acc-LipTL
mL
1
; 47 mmol L
1
(rac)-benzoin)
Solvent
Temp.
[C]
Specic activity
[U g
Acc-LipTL
]
Time to max. conv.
(50%) [h] ee
a
[%]
THF 21.5 13.0 4 >99
50 36 3.5 >99
2-MeTHF 21.5 20.7 2.5 >99
50 47.8 1.5 >99
Toluene 21.5 15.2 2.5 >99
50 62 1.5 >99
a
ee of (S)-benzoin butyrate.
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about 1.6-fold and 2.9-fold higher in 2-MeTHF than in toluene
and THF, respectively.
Similar results were obtained at 50 C, which is the
temperature preferable for running the racemization catalyst.
Interestingly, the specic activity of Acc-LipTL in toluene now
exceeded the activity in 2-Me-THF, while the time to obtain full
conversion was still similar. The time to reach full conversion in
THF changed only slightly compared to the reaction at room
temperature. Both ndings can easily be explained by a lower
stability of Acc-LipTL in toluene and THF, respectively, than in
2-MeTHF conrming the observations on process stability
described above. Notably, in both 2-Me-THF and toluene KR can
obviously be run efficiently at 50 C.
Racemization of (R)-benzoin
Apart from the homogeneous Shvo's catalyst, several heteroge-
neous catalysts display activity on secondary alcohols including
transition metals supported on carbon, hydroxyapatite or
alumina, acidic resins, vanadium catalysts and zeolites.
16,17
As
demonstrated in earlier studies, commercial ion-exchangers
exert a detrimental effect on enzyme activity,
18
and are there-
fore not suitable for a one pot DKR as targeted in this study.
Heterogeneous (semi-) precious metal catalysts, on the other
hand, usually suffer from high costs and leaching of the active
species, while Zeolites have only small pore sizes limiting their
application to small substrates. Both disadvantages, however,
can be overcome by use of mesoporous material: framework
incorporation of active metals suppresses leaching and large
pores allow full accessibility even for large substrates.
19,20
TUD-1
is a mesoporous material with a sponge-like structure and
three-dimensional pores.
21
Its environmentally benign
synthesis allows for the straightforward introduction of a large
range of metals into the framework. High stability and recy-
clability has been demonstrated in many different reactions, in
particular in the Meerwein–Ponndorf–Verley reduction and the
Oppenauer oxidation.
19,21–23
Moreover, its induced acidic sites
make it ideal for the generation of carbenium ions, intermedi-
ates in the racemization of benzylic alcohols, while side reac-
tions oen observed with commercially available acid resins do
not occur. Accordingly, TUD-1 catalysts were tested for racemi-
zation of (R)-benzoin. As incorporated metals, aluminium,
24,25
zirconium,
15,26
and tungsten,
27
respectively, were involved.
Based on the ndings on KR performance with Acc-LipTL
(see previous section) and on the study of Ramanathan
et al.,
15
toluene was chosen as the solvent for racemization. The
racemization success was measured in terms of enantiomeric
excess (ee), for which in this case a value as small as possible
was targeted. Results are summarized in Table 2.
It was observed that purely siliceous TUD-1 (i.e. without any
metal incorporated) was already able to catalyse the racemiza-
tion of (R)-benzoin, although not very efficiently. An ee of 93.8%
was obtained aer 20 h at 50 C. Under the same conditions,
introduction of tungsten to TUD-1 (W-TUD-1) yielded a
maximum ee of 43.9% at a ratio of Si/W ¼20. Increases in the
metal content initially enhanced the reaction rate, while aer
surpassing a maximum racemization activity, high loadings of
tungsten were less efficient. As tungsten provides the acidic
reaction site for racemization, this behaviour is a reaction to the
actual availability of reactive centers. It increases with incor-
poration of tungsten until the formation of crystalline tungsten
oxide species with a reduced surface area and therefore lower
accessibility of the acidic metal occurs.
Excellent racemization results were obtained with aluminum
and zirconium as incorporated metals. Aer 20 h at 50 C both
Zr-TUD-1 (Si/Zr ¼25) and Al-TUD-1 (Si/Al ¼4) had completely
racemised (R)-benzoin. Aer a reaction time of only four hours
which is in the time range of KR with Acc-LipTL in toluene, (see
previous section) Zr-TUD-1 (Si/Zr ¼25) reduced the ee to 4.4%,
while Al-TUD-1 (Si/Al ¼4) managed a nal ee of 42.8%. Thus, Zr-
TUD-1 demonstrated a superior racemization activity, which is
most likely due to the purely Lewis acidic nature of zirconium in
the framework of TUD-1,
19
contrasting the mixed Lewis and
Brønsted acid character imparted by tungsten and
aluminum.
15,22,25
Importantly, Zr-TUD-1 showed no racemiza-
tion activity with (R)-benzoin butyrate (within two hours) even at
high temperature (70 C) and thus ensures that the enantio-
purity of the KR product cannot be affected.
For investigation of temperature effects on the racemization
efficiency both Zr-TUD-1 (Si/Zr ¼25) and Al-TUD-1 (Si/Al ¼4)
were subjected to higher reaction temperatures (50, 70 and 90
C). As expected from the Arrhenius relation, the racemization
rate increased with increasing temperature in both cases
(Fig. 4). With Zr-TUD-1 (Si/Zr ¼25) complete racemization was
achieved within 90 minutes at 70 C and only 30 minutes at 90
C. With Al-TUD-1 (Si/Al ¼4) considerably longer reaction times
were required. The minimum was 90 minutes at a temperature
of 90 C. Notably, only small amounts of benzil, the dicarbonyl
analogue of benzoin, were detected in all set-ups indicating that
dehydrogenation, which had been described in connection with
acidic racemization of hydroxyl compounds before,
17,18,28
plays a
minor role in the racemization of benzoin with the selected
TUD-1 catalysts.
The tendency of Zr-TUD-1 and Al-TUD-1 to catalyze unspe-
cic transesterication of benzoin in the nal reaction mixture
as a likely side reaction
17,18,28
was investigated by incubation of a
mixture of catalyst, (rac)-benzoin and vinyl butyrate in toluene
Table 2 Racemization of (R)-benzoin with TUD-1 catalysts incorpo-
rating different metals at varying concentration; the reaction was
performed in toluene at 50 C using 20 mg of catalyst per batch
Entry Catalyst Time [h] ee [%]
1 TUD-1 20 93.8
2 Zr-TUD-1 (Si/Zr ¼25) 20 1.3
3 Al-TUD-1 (Si/Al ¼4) 20 0.2
4 Al-TUD-1 (Si/Al ¼25) 20 23.2
5 W-TUD-1 (Si/W ¼10) 20 65.7
6 W-TUD-1 (Si/W ¼20) 20 43.9
7 W-TUD-1 (Si/W ¼30) 20 63.9
8 W-TUD-1 (Si/W ¼40) 20 73.7
9 W-TUD-1 (Si/W ¼50) 20 71.7
10 Zr-TUD-1 (Si/Zr ¼25) 4 4.4
11 Al-TUD-1 (Si/Al ¼4) 4 42.8
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at different temperatures. Under all conditions strong trans-
esterication activity was observed with Al-TUD-1 (Fig. 5),
yielding benzoin ester amounts comparable to the enzymatic
reaction. Due to the fully racemic nature of the ester, this would
diminish the product-ee in a one-pot DKR considerably. In
contrast, Zr-TUD-1 revealed strong transesterication activity
only at high temperature (90 C). Hardly any benzoin ester was
detected at 50 Cor70C. Thus, Zr-TUD-1 makes a selective
racemization catalyst up to a temperature of at least 70 C. Most
probably, this can again be explained by the Lewis acidic
character of the catalyst, which avoids initiation of trans-
esterication via Brønstedt protonation of the carbonyl group in
the acyl donor (which is then attacked by the nucleophilic
alcohol).
29
DKR of (rac)-benzoin
DKR of (rac)-benzoin was performed in one pot using a
combination of Acc-LipTL and metal-TUD-1 as catalysts and
vinyl butyrate as acyl donor. In contrast to the reaction set-up of
Hoyos et al.,
4
use of this most efficient acyl donor was possible,
because inactivation of metal-TUD-1 by the emerging volatile
acetaldehyde, as observed with Shvo's catalyst, was not to be
expected. For optimisation the reaction was investigated using
THF, 2-MeTHF and toluene, respectively, as solvents and at
temperatures up to 90 C.
Excellent results were obtained with Acc-LipTL and Zr-TUD-1
(Si/Zr ¼25) in toluene and equally in 2-MeTHF (Table 3). In
toluene 98.4% of the deployed (rac)-benzoin was converted
within 5 h yielding a product-ee of 98.5%. Within the same time
range, conversion was only slightly lower in 2-MeTHF (96.8%)
yielding a still remarkable product-ee of 97%. Interestingly, the
situation was controversial when Al-TUD-1 (Si/Al ¼4) was used
as racemization catalyst, which can most probably be explained
by the lower racemization activity of Al-TUD-1 in 2-MeTHF
compared to toluene (Table 3) entailing an also lower unspecic
transesterication activity.
In THF, the DKR was ineffective with both racemization
catalysts yielding lower (Al-TUD-1) or only slightly higher (Zr-
TUD-1) conversion than theoretically possible in KR (i.e. 50%).
This was surprising considering the good results of separate (R)-
benzoin racemization in this solvent (see Table 3). The behavior
might be connected with side activities of the acidic catalysts
under certain conditions (e.g. catalysis of polymerisation)
14,30
affecting their racemization activity.
Importantly, it becomes obvious from the performances of
the separate and combined catalysts (Tables 2 and 3) that
prediction of the best conditions for one pot DKR can hardly be
Fig. 4 Racemization of (R)-benzoin with (a) Zr-TUD-1 (Si/Zr ¼25) and
(b) Al-TUD-1 (Si/Al ¼4) in toluene at different temperatures (the
catalyst concentration was fixed at 40 mg mL
1
; the reaction was
performed in 500 mL solvent with 94 mM (R)-benzoin).
Fig. 5 Specific activity of Al-TUD-1 (Si/Al ¼4) and Zr-TUD-1 (Si/Zr ¼
25) in the transesterification of vinyl butyrate and benzoin in toluene at
different temperatures (catalyst concentration fixed at 40 mg
TUD-1
mL
1
; 500 mL solvent with 94 mM (rac)-benzoin as reaction medium).
Table 3 Racemization performance of Al-TUD-1 (Si/Al ¼4) and Zr-
TUD-1 (Si/Zr ¼25) and results of DKR with both metal catalysts and
Acc-LipTL in different solvents at 50 C (catalyst concentration fixed at
40 mg
TUD-1
mL
1
and 20 mg
Acc-LipTL
mL
1
; 500 mL solvent with 94 mM
(rac)-benzoin as reaction medium)
Solvent Rac. catalyst ee aer rac [%]
a
Conv. DKR [%]
b
ee DKR
b
[%]
THF Al-TUD-1 11.9 25.1
c
84.2
Zr-TUD-1 23.4 53.5
c
99.6
2-MeTHF Al-TUD-1 7.7 85.9 96.6
Zr-TUD-1 11.4 96.8 97
Toluene Al-TUD-1 0.2 93.2 83.3
Zr-TUD-1 1.3 98.4 98.5
a
ee of (R)-benzoin, 20 h at 50 C.
b
5 h at 50 C.
c
ee of (S)-benzoin
butyrate, increased side product formation indicating side activities of
the catalyst(s).
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based on the separate reactions. While Al-TUD-1 (Si/Al ¼4) in 2-
MeTHF leads to faster racemization of benzoin than Zr-TUD-1
(Si/Zr ¼25), Zr-TUD-1 is the catalyst of choice for the DKR.
Toluene turns out to be the best solvent (with 2-MeTHF as a
close second) even though it is only second choice for the KR on
its own. This highlights the complexity of combination reac-
tions with two types of catalyst, here heterogeneous and bio-
logical, as multi-parameter problems.
31
Considering that both KR and racemization, but also
unspecic transesterication and biocatalyst inactivation,
proceed faster at elevated temperature, DKR of benzoin using
Acc-LipTL and Zr-TUD-1 (Si/Zr ¼25) in toluene was studied at
50 C, 70 C and 90 C (Fig. 6). At 50 C, the conversion was over
98% aer 5 h with an ee of 98.5%. The reaction time for
reaching >98% conversion was reduced to 3.5–4 h when the
DKR was performed at 70 C, while a still very good product-ee
of 97.6% was obtained. The minor decrease in ee can certainly
be explained by the small unspecic transesterication activity
of Zr-TUD-1 at this temperature (see previous section). In
agreement with this assumption, the ee dropped to 65.2% at a
reaction temperature of 90 C, where the unspecic trans-
esterication activity of Zr-TUD-1 is considerable. Notably, full
conversion was still obtained at this temperature (within 1.5 h)
demonstrating a remarkable thermostability of the immobi-
lized biocatalyst under the applied conditions.
In view of application in technical synthesis benetting from
repetitive (or continuous) use of the catalysts,
31
recyclability of
Acc-LipTL and Zr-TUD-1 in DKR was investigated. Repeated
batch-cycles were performed in toluene and 2-MeTHF, respec-
tively, at 50 C. Aer each cycle the catalysts were separated by
ltration and washed with the respective solvent until no
residual substrates or product were detectable in HPLC anal-
ysis. In comparison of the two solvent systems the reaction time
was chosen in a way that both reactions were stopped when full
conversion (>99%) was rst accomplished in one of the reac-
tions. The maximum reaction time per batch was 24 h.
In 2-MeTHF full conversion of (rac)-benzoin (>99%) was
obtained over three batches (Fig. 7) with a runtime of 8 h, 16 h
and 24 h, respectively. In the same time intervals DKR in
toluene achieved >99% in the rst, 96% in the second and only
84% in the third cycle. Product-ee was >97% in both solvents
throughout all experiments. The increasing runtimes per cycle
required to obtain full conversion in at least one solvent as well
as the lower conversion in toluene than in 2-MeTHF within
comparable time intervals most probably indicate catalyst
deactivation, with a stronger effect exerted by toluene. In prin-
cipal, this deactivation could concern Acc-LipTL as well as Zr-
TUD-1. However, considering that an excellent reusability has
been reported for Zr-TUD-1 in the cyclisation of rac-citronellal,
15
deactivation of the enzyme catalyst is more likely. This also
agrees with the solvent effects on the activity and process
stability of Acc-LipTL observed in separate KR (see Table 1).
In fact, stability of the reaction system on repetitive use in 2-
MeTHF was considerably enhanced (Fig. 8) when the bio-
catalyst, Acc-LipTL, was stabilized by incubation in a PEI-buffer
solution prior to drying as suggested by Guisan et al.
32
While in
DKR with untreated Acc-LipTL, conversion decreased to 89% in
the fourth and 78% in the h cycle (each cycle with a runtime
of 24 h), use of Acc-LipTL treated with PEI enabled full
conversion (>99%) in all cycles. The product-ee remained at
>98% and was therefore unaffected by the treatment.
Fig. 6 DKR of (rac)-benzoin catalyzed by Acc-LipTL and Zr-TUD-1 (Si/
Zr ¼25) in toluene at different temperatures.
Fig. 7 Repeated DKR of (rac)-benzoin with Acc-LipTL and Zr-TUD-1
(Si/Zr ¼25) in toluene or 2-MeTHF, respectively, at 50 C. Each data
point is the endpoint of a cycle over accumulated time. Reactions were
stopped when full conversion was first accomplished in one of the
solvents.
Fig. 8 Repeated DKR of (rac)-benzoin with Zr-TUD-1 (Si/Zr ¼25) and
untreated Acc-LipTL or PEI-Acc-LipTL, respectively, in 2-MeTHF at 50
C. Each data point is the endpoint of a cycle over accumulated time.
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Finally, the actual practicability of the process for synthesis
was demonstrated by performing the DKR on a semi-
preparative scale using 150 mg racemic benzoin as substrate.
The reaction proceeded as smoothly as in small scale (data not
shown), reaching 92% conversion within 5 hours and full
conversion within less than 20 hours. The product was a col-
ourless oil consisting of enantiopure (ee > 98%) (S)-benzoin
butyrate (
1
H-NMR (400 MHz, CDCl
3
): 7.92 (m, 2H; Ar-H), 7.55–
7.3 (m, 8H; Ar-H), 6.85 (s, 1H; CHO), 2.51–2.39 (m, 2H; CH
2
),
1.74–1.60 (m, 2H; CH
2
), 0.98–0.90 (m, 3H; CH
3
); consult Fig. S1
in the ESI†for the spectrum). The isolated yield aer removal of
catalysts and solvent was 196 mg corresponding to more than
98%. Thus, the performance of the proposed one-pot system is
very promising with regard to synthetic application.
Experimental
Materials
Lipase TL from P. stutzeri was a gifrom Meito & Sangyo Co.,
Ltd. (Tokyo, Japan). Accurel MP1001 was obtained from Mem-
brana (Wuppertal, Germany). TUD-1 catalysts were activated via
calcination in the presence of air at up to 600 C at a temper-
ature ramp of 1 K min
1
and subsequent heating for 10 h at 600
C. Aer cooling down to 100 C the catalysts were stored at 100
C until use. All chemicals and liquid substrates were purchased
from Aldrich (Steinheim, Germany), VWR (Darmstadt, Ger-
many) or Carl-Roth (Karlsruhe, Germany) and used as received.
All solvents were dried over activated molecular sieves before
use.
Reaction analysis
Conversion and enantiomeric excess (ee) were determined via
HPLC analysis using a Daicel Chiralpak IA column at a ow of
2.0 mL min
1
of n-hexane/isopropyl alcohol (90/10) at 40 C.
Typical retention times were 2.9 min for (R)-benzoin butyrate,
3.3 min for (S)-benzoin butyrate, 5.8 min for (R)-benzoin and 6.6
min for (S)-benzoin. An exemplary spectrum is given in the ESI
(Fig. S1†).
Immobilisation of lipase TL
Lipase TL powder was stirred in KPi buffer at room temperature
until agglomerates were no more visible. Undissolved material
was separated by centrifugation at 5000 rpm for 10 minutes. 500
mg Accurel MP1001 were placed in 3.75 mL ethanol (abs.) for 30
minutes, then 10 mL of lipase solution was directly added. The
sealed vessel was placed on an orbital shaker at 340 rpm and 40
C for 4 h. The immobilisates were aerwards ltered from the
solution and washed 2 times with 10 mL immobilisation buffer.
Aer nal vacuum ltration the Acc-LipTL particles were dried
via storing over silica gel at reduced pressure and room
temperature until use.
Kinetic resolution (KR)
Typically 20 mg mL
1
(94 mM) (rac)-benzoin was dissolved in
dry organic solvent (usually THF). 10 mg of lipase were added to
1 mL of the benzoin solution and the mixture was incubated on
a thermoshaker at 1200 rpm and room temperature. Aer 10
min. KR was started by addition of 6 eq. (related to benzoin) of
vinyl butyrate. For sample analysis 10 mL reaction medium were
dissolved in 990 mL isopropyl alcohol. If crude lipase powder
was used, the diluted samples were centrifugated and the
supernatant was analysed. Conversion and enantiomeric excess
were determined via HPLC analysis.
Racemization of (R)-benzoin
20 mg metal-TUD-1 catalyst were loaded in a dried Schlenk ask
under nitrogen atmosphere. (R)-Benzoin was dissolved in
toluene at a concentration of 20 mg mL
1
; slight heating was
required for complete dissolvation. Racemization was started by
adding 500 mL of the substrate solution to the metal-TUD-1
catalysts in the Schlenk ask. The reaction vessel was immedi-
ately placed in a temperated oil bath (temperature depending
on the reaction temperature of following racemization reac-
tions) and mixed with a magnetic stir bar. For sample analysis
10 mL reaction medium were taken under nitrogen counter ow
in appropriate time intervals and were dissolved in 990 mL
isopropyl alcohol. Enantiomeric excess was determined via
HPLC analysis.
Dynamic kinetic resolution (DKR)
For characterisation and optimisation, 10 mg Acc-LipTL and 20
mg Metal-TUD-1 (Si/Zr ¼25) were loaded in a dried Schlenk
ask under nitrogen atmosphere. Benzoin was dissolved in
toluene at a concentration of 20 mg mL
1
; slight heating was
required for complete dissolvation. 500 mL of the substrate
solution were added to the lipase and metal-TUD-1 in the
Schlenk-ask and the mixture was incubated for 10 minutes.
DKR was started by addition of 6 eq. (related to benzoin) of vinyl
butyrate and the reaction vessel was immediately placed in a
temperature controlled oil bath (temperature depending on the
desired reaction temperature) and mixed with a magnetic stir
bar. For sample analysis, 10 mL reaction medium were taken
under nitrogen counter ow in appropriate time intervals and
were dissolved in 990 mL isopropyl alcohol. Conversion and
enantiomeric excess were determined via HPLC analysis. For
recycling experiments the catalysts (Acc-LipTL and Metal-TUD-
1) were separated from the reaction mixture by ltration and
washed two times with the solvent providing the reaction
medium. Aer nal ltration new substrate solution was added
and the reaction was restarted by adding vinyl butyrate under
nitrogen atmosphere. The runtime of reaction cycles was
adjusted for rst reaching a conversion of >98% in one of the
solvents. The maximum reaction time was limited to 24 h. For
scale-up, the reaction volume was increased to 7.5 mL toluene,
using 300 mg Zr-TUD-1 (Si/Zr ¼25), 150 mg Acc-LipTL, 150 mg
racemic benzoin and 400 mg vinyl butyrate. The reaction was
performed under stirring at 50 C. Samples were taken and
dened time intervals and reaction progress was followed via
HPLC analysis. Aer reaching full conversion the catalysts were
ltered offand the solvent was removed under reduced
pressure.
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Determination of LipTL stability
A sealed vessel with a volume of 6 mL was continuously oper-
ated by using a HPLC pump at a ow rate of 0.5 mL min
1
. The
benzoin solution (10 mg mL
1
(47 mM) in the given solvent
(THF as standard)) with 3 eq. vinyl butyrate was introduced
through a pipe and lethe reactor by a bleeder pipe. The
reaction medium was vigorously mixed with a magnetic stir bar.
100 mg of Acc-LipTL was loaded in the reactor and the reaction
was performed until a steady state was reached. For sample
analysis 10 mL reaction medium were taken from the outlet
stream and dissolved in 990 mL isopropyl alcohol. Conversion
and enantiomeric excess were determined via HPLC analysis.
From the decrease of the conversion (never exceeded 10%) over
time the half life of Acc-Lip TL could be determined.
Modication of Acc-LipTL
100 mg Acc-LipTL were incubated in 7.5 mL ethanol (abs.) for 30
minutes. 2 mL of polyethylene imine (PEI)-solution (1% in 200
mmol L
1
KPi) were added to the mixture and the sealed vessel
was incubated on an orbital shaker for 1 h at 340 rpm and room
temperature. The particles were separated by ltration, washed
two times with KPi buffer and dried via storing over silica gel at
reduced pressure and room temperature until use.
Conclusions
By appropriate preparation and choice of biocatalyst and che-
mocatalyst, respectively, dynamic kinetic resolution of (rac)-
benzoin has been considerably advanced towards preparative
scale synthesis. Particularly benecial were the targeted heter-
ogenization of LipTL through adsorption on Accurel MP1001
and the implementation of heterogeneous Si-based Zr-TUD-1 as
racemization catalyst. Both promoted reproducibility of appli-
cation and recycling of catalysts, and in combination yielded
full conversion (>99%) at very high product-ee (>98%) in less
than 20 hours. Compatibility in solvent and temperature
dependency enabled performance in only one pot. The appli-
cation range of the reaction system will have to be elucidated in
further studies, in view of the documented broad substrate
range of LipTL,
3–5
however, accessibility of various a-hydroxy
ketones via this route can actually be envisaged.
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