Reversibility of asymmetric catalyzed C-C bond formation by benzoylformate decarboxylase

Article abstract of DOI:10.1039/c4cy00171k

Benzoylformate decarboxylase (BFD) from Pseudomonas putida catalyzed the formation of 2-hydroxy-1-phenylpropanone (2-HPP), a 2-hydroxy ketone, from the kinetic resolution of rac-benzoin in the presence of acetaldehyde. The formation rate of 2-HPP via kinetic resolution of benzoin was 700-fold lower compared to the formation via direct carboligation of benzaldehyde and acetaldehyde. Further investigations revealed that BFD not only accepts (R)-benzoin but also 2-HPP as the substrate. A typical Michaelis-Menten type kinetics was observed starting from enantiopure (S)- or (R)-2-HPP. The formation of racemic 2-HPP while using benzoin as the donor in the presence of acetaldehyde and the racemization of (R/S)-2-HPP were detected. The equilibrium constant determined, showed favoured conditions towards the product side i.e. (R)-benzoin and 2-HPP. In the end, an extended reaction mechanism was proposed by supplementing the already known mechanism with the C-C bond cleavage activity of BFD towards 2-hydroxy ketones. This journal is

Full text of DOI:10.1039/c4cy00171k

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Reversibility of asymmetric catalyzed C–C bond
formation by benzoylformate decarboxylase†
Cite this: DOI: 10.1039/c4cy00171k
a
ab
a
*
*
and Andreas Liese
Marco Berheide, Selin Kara
Benzoylformate decarboxylase (BFD) from Pseudomonas putida catalyzed the formation of 2-hydroxy-1-
phenylpropanone (2-HPP), a 2-hydroxy ketone, from the kinetic resolution of rac-benzoin in the presence
of acetaldehyde. The formation rate of 2-HPP via kinetic resolution of benzoin was 700-fold lower com-
pared to the formation via direct carboligation of benzaldehyde and acetaldehyde. Further investigations
revealed that BFD not only accepts (R)-benzoin but also 2-HPP as the substrate. A typical Michaelis–
Menten type kinetics was observed starting from enantiopure (S)- or (R)-2-HPP. The formation of racemic
2-HPP while using benzoin as the donor in the presence of acetaldehyde and the racemization of IJR/S)-2-
HPP were detected. The equilibrium constant determined, showed favoured conditions towards the prod-
uct side i.e. (R)-benzoin and 2-HPP. In the end, an extended reaction mechanism was proposed by
supplementing the already known mechanism with the C–C bond cleavage activity of BFD towards
2-hydroxy ketones.
Received 10th February 2014,
Accepted 27th January 2015
DOI: 10.1039/c4cy00171k
www.rsc.org/catalysis
of acetaldehyde but not for benzaldehyde hence yielding (S)-
2-HPP and (R)-benzoin, respectively.
Introduction
Synthesis of chiral 2-hydroxy ketones has attracted great
attention as these compounds are applied broadly for the syn-
thesis of biologically active compounds such as pharmaceuti-
cals, agrochemicals, and pheromones.1 Thiamine diphos-
phate (ThDP) dependent enzymes have been widely applied
for the synthesis of these crucial compounds and among
them, benzoylformate decarboxylase (BFD)2 and benzaldehyde
lyase (BAL)3 have been investigated in detail.
It was reported that BFD naturally catalyzes the non-
oxidative decarboxylation of benzoylformate to form benzal-
dehyde and carbon dioxide.4 Later, in the beginning of the
1990s, IJS)-2-hydroxy-1-phenylpropanone ((S)-2-HPP) was
described as the product of benzoylformate decarboxylation
in the presence of acetaldehyde.5 Since the 1990s, enantio-
selective C–C bond formation, termed carboligation, has
gained increased interest in the research community. After
the crystal structure of Pseudomonas putida BFD (EC 4.1.1.7)6
was known, stereoselectivity of BFD-catalyzed carboligations
was investigated by molecular modelling studies revealing an
‘S-pocket’ responsible for the (S)-selectivity of BFD.7 The size
of the ‘S-pocket’ is found to be large enough for the binding
Another ThDP-dependent enzyme is BAL from Pseudomo-
nas fluorescens Biovar I (EC 4.1.2.38), reported on for the first
time in 1989.8 It was shown that Pseudomonas fluorescens Bio-
var I can grow on benzoin as sole carbon source owing to the
BAL-catalyzed cleavage of acyloin linkage of (R)-benzoin. It
took a decade until the potential activity of BAL for the
reverse acyloin condensation was reported.3 Since then, BAL
has been commonly applied for the synthesis of chiral
2-hydroxy ketones, the catalytic mechanism of which involves
two steps: (1) the nucleophilic attack of the ylide form of
ThDP on the carbonyl C-atom of (R)-benzoin (or an
araliphatic compound) yielding an enamine–carbanion inter-
mediate after the release of the aromatic aldehyde and (2)
the attack of the enamine–carbanion intermediate on an
acceptor aldehyde yielding a 2-hydroxy ketone.3a,b,h
The C–C bond cleavage of benzoin catalyzed by BFD has
been already a topic discussed in the literature.7a,9 As (R)-
benzoin is formed from two molecules of benzaldehyde cata-
lyzed by BFD, it is clear that the active site of BFD can accom-
modate benzoin. In fact, the reversible benzoin synthesis by
BFD was reported to be possible but disfavored due to the
low solubility of benzoin in the aqueous medium (i.e. ~1.4 mM
at 25 °C), which hinders the binding of BFD to benzoin.9
On the other hand, BFD-catalyzed cleavage of benzoin was
reported to be impossible from the mechanistic point of view
as benzoin cannot sterically fit into the active site of BFD.7a
The present study was based on initial evidences that hint to
a carbolyase activity also present in BFD. Therefore, in this
a Institute of Technical Biocatalysis, Hamburg University of Technology,
Denickestrasse 15, 21073, Hamburg, Germany. E-mail: liese@tuhh.de;
Fax: (+) 49 40 42878 2127; Tel: (+) 49 40 42878 3018
b Institute of Microbiology, Chair of Molecular Biotechnology, Technische
Universität Dresden, 01062 Dresden, Germany. E-mail: selin.kara@tu-dresden.de;
Fax: (+) 49 351 463 39520; Tel: (+) 49 351 463 39517
† In memory of Ayhan S. Demir (1950–2012).
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work the catalytic activity of BFD on the cleavage of C–C bond
of 2-hydroxy ketones was investigated assuming a similar
mechanism as for BAL.
An experimental evidence for the BFD-catalyzed cleavage
of (R)-benzoin is however difficult to obtain, due to the low
solubility of benzoin in aqueous buffer and the thermody-
namic equilibrium, which strongly favors the formation of
benzoin and the presumable low activity of BFD for the cleav-
age of (R)-benzoin. However, the equilibrium can be shifted
towards benzaldehyde formation by in situ removal of the
benzaldehyde formed. Therefore, acetaldehyde was added to
the reaction medium to form 2-HPP (4) (Scheme 1).
Fig. 1 The ee value of (S)-benzoin as a function of the conversion of
benzoin in the kinetic resolution of rac-benzoin (1) (with 50 mM acet-
aldehyde). Reaction conditions: 1.6 mM rac-benzoin, 50 mM acetalde-
hyde 50 mM TEA, 30% IJv/v) DMSO, 0.5 mM ThDP, 2 mM Mg2+, wtBFD
(81 U2-HPP mL−1, 9.8 mg mL−1 wtBFD) at pH 7.5 and 25 °C. Lines indi-
cate visual aids. Data points are average values of duplicates. The curve
represents an ideal kinetic resolution (i.e. 50% conversion and 100% ee
IJS)-benzoin).
Results and discussion
Earlier, BFD-mediated C–C bond formations were conducted
in phosphate buffer, whereby no C–C bond cleavage activity
of BFD could be detected under these conditions so far. Alter-
natively, triethanolamine (TEA) was also applied for ThDP-
dependent enzymes, which is in fact the buffer of choice for
BAL-catalyzed reactions.3 Therefore, we became interested in
using TEA buffer to evaluate the C–C bond cleavage activity
of BFD. In addition, due to the limited solubility of benzoin,
DMSO, a common cosolvent used for BFD and BAL,2,3 was
applied.
benzoin is incubated with BAL in the presence of acetalde-
hyde enantiopure (R)-4 is formed.3a,10 Regarding the kinetic
parameters and microscopic rate constants, it was shown that
BFD and BAL exhibit differences.11 Nevertheless, in addition
to their catalytic activities, BFD and BAL are structurally simi-
lar, since (i) both enzymes are homotetrameric and (ii) both
bind to the cofactor ThDP in the active site.6,12 In contrast to
BFD, no ‘S-pocket’ was observed in the structure BAL and
hence the strict (R)-enantioselectivity (>99%) of BAL for the
formation of (R)-2-HPP is attributed to only one possible
arrangement of acetaldehyde in the active site prior to C–C
bond formation with ThDP-bound benzaldehyde.7a In this
study, formation of benzaldehyde from benzoin catalyzed by
BFD could not be verified, as benzaldehyde immediately
reacts with the added acetaldehyde to afford 4. For BAL it
was postulated that there is a direct reaction involving an
enzyme–benzoin complex with acetaldehyde and benzalde-
hyde to form 4,13,14 which might also be valid for BFD.
Kinetic resolution of rac-benzoin catalyzed by BFD
Firstly, the kinetic resolution of rac-benzoin was investigated,
whereby the enantiomeric excess (ee) of benzoin was moni-
tored during the course of reaction. Since BFD catalyzes the
formation of (R)-benzoin,2a only (R)-benzoin can be accepted
as substrate in a kinetic resolution. In an ideal situation the
ee of (S)-benzoin would continuously increase as the cleavage
of (R)-benzoin proceeds, reaching 100% at 50% of conver-
sion. Indeed, our results revealed an ideal kinetic resolution
catalyzed by BFD as shown in Fig. 1.
Starting from 1.6 mM rac-benzoin the concentrations as
well as the ee values of benzoin (1) and 2-HPP (4) were moni-
tored during the course of the reaction, showing a perfect
kinetic resolution. As shown in Fig. 2A, the concentration of
benzoin reached 0.8 mM after 2 h. Only (S)-benzoin was
detectable in the reaction mixtures. Simultaneously, 1.6 mM
of rac-2-HPP (Fig. 2A) were formed in the presence of 50 mM
acetaldehyde.
Next, we determined the formation rate of 4 from 1.6 mM
rac-benzoin and 50 mM acetaldehyde by linear regression.
Table 1 illustrates the formation rate of 4 via kinetic resolu-
tion of benzoin compared with that of via direct
carboligation. Here, it was clearly seen that the formation of
4 starting from benzoin and acetaldehyde was ~700-fold
The BFD-catalyzed kinetic resolution of benzoin shown
here is very similar to that catalyzed by BAL. When (R)-
Fig. 2 Kinetic resolution of rac-benzoin (1.6 mM) catalyzed by wtBFD
in the presence of 50 mM acetaldehyde. Plots show the concentrations
of benzoin and 2-HPP (left) and the respective ee-values for (S)-
benzoin and 2-HPP (right). Reaction conditions: 50 mM TEA, 30% IJv/v)
DMSO, 0.5 mM ThDP, 2 mM Mg2+, wtBFD (81 U2-HPP mL−1, 9.8 mg mL−1
wtBFD) at pH 7.5 and 25 °C. Lines indicate visual aids. Data points
are average values of duplicates.
Scheme 1 Postulated reaction sequence for the cleavage of benzoin
(1) to benzaldehyde (2) and further carboligation with acetaldehyde (3)
yielding 2-HPP (4) catalyzed by BFD.
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Table 1 Comparison of formation rates of 2-HPP via direct carboligation
and via kinetic resolution of (R)-benzoin
VDirect carboligation [U2-HPP mg−1 a
]
VKinetic resolution [U2-HPP mg−1 b
11.3 × 10−3
]
8.3
a
Conditions for direct carboligation: 40 mM benzaldehyde, 400 mM
acetaldehyde, 50 mM phosphate buffer, 0.5 mM ThDP, 2 mM Mg2+ at
pH 7.5 and 30 °C. Conditions for kinetic resolution of benzoin:
b
Fig.
3
Formation rates of (R)-4 from enantiopure (S)-4 (A) and
formation rates of (S)-4 from enantiopure (R)-4 (B). Reaction
conditions: (R)- or (S)-4 (0–170 mM), 25 mM acetaldehyde, 50 mM TEA
buffer, 0.5 mM ThDP, 2 mM Mg2+, wtBFD (51 U2-HPP mL−1, 6 mg mL−1
wtBFD) at pH 7.5 and 25 °C. Data points are average values of
duplicates.
1.6 mM rac-benzoin, 50 mM acetaldehyde, 50 mM TEA buffer, 0.5 mM
ThDP, 2 mM Mg2+, 30% IJv/v) DMSO at pH 7.5 and 25 °C. Results are
average values of duplicates.
slower than the direct carboligation of benzaldehyde and
acetaldehyde.
Fig. 4, maximum rates for the formation of (R)-4 or (S)-4 were
detected at low acetaldehyde concentrations (<50 mM). Here,
of particular importance is the course of the respective for-
mation rates of 4 at higher acetaldehyde concentrations. An
exponential decay in the formation rates of (R)- or (S)-4 from
the cleavage of their enantiocomplementray forms was
observed when higher acetaldehyde concentrations (>50 mM)
were applied. This observed decrease in the enzyme activity
with increasing acetaldehyde concentrations was not in
agreement with the data reported for the formation of 4 for
the direct carboligation of benzaldehyde and acetaldehyde by
Wilcocks and Ward (1992).5a Iding et al. (2000)2a also
reported a decreased activity of BFD at higher acetaldehyde
concentrations for the direct carboligation of benzaldehyde
and acetaldehyde to afford 4, however, first when acetalde-
hyde concentrations exceeded ~500 mM.
Next, we investigated the formation of benzaldehyde as a
cleavage product of (S)- or (R)-4 in the presence of different
acetaldehyde concentrations (Fig. 5). Similar to the behavior
shown in Fig. 4, formation rates of benzaldehyde also
decreased with increasing acetaldehyde concentrations. For
concentration values of acetaldehyde higher than 400 mM,
benzaldehyde formation rates were ~0.2 mU mg−1. It is
important to mention that benzaldehyde formation rates
presented here are only apparent values, since only free benz-
aldehyde in the reaction solution could be detected.
Cleavage of 2-HPP catalyzed by BFD
Encouraged by the results obtained for the kinetic resolution
of rac-benzoin, we evaluated the cleavage of 4 as it might also
be accepted as the substrate by BFD (Scheme 2).
To evaluate 4 as a substrate for BFD and to investigate the
formation of surprising rac-4 in detail, we applied enantio-
pure (>99.9% ee) (S)-4 or (R)-4 and monitored the ee values.
Our results showed that low concentrations of acetaldehyde
are required to accelerate the racemization of 4 (data not
shown); hence, we applied 25 mM acetaldehyde. The forma-
tion of the enantiocomplementary form of 4 was analyzed
starting from different concentrations of 4 (≤170 mM)
(Fig. 3). The maximum solubility of 4 in aqueous medium
was determined to be 180 mM at 25 °C (measured for both
enantiomers). The BFD-mediated cleavage of 4 showed a
‘classic’ Michaelis–Menten activity depending on the sub-
strate concentration. The KM values were determined as 62
9 mM for (R)-4 and 62 4 mM for (S)-4 and the Vmax values
were found as 1.0 mU mg−1 for the formation of (R)-4 and
2.1 mU mg−1 for the formation of (S)-4. Despite of the high
enantioselectivity of BFD for the formation of (S)-4 in the
direct carboligation of benzaldehyde and acetaldehyde (ee =
92% (S)),2a we observed a similar affinity of BFD for (R)- and
(S)-4 in the cleavage reaction. Presently, there is no fully satis-
factory explanation for this observation and further kinetic
and molecular modelling investigations are required to
explain this observation.
Overall, the here presented BFD-catalyzed formation of
(R)-4 from (S)-4, and vice versa, follows a two-step process: (1)
Further on, we investigated the formation rates of (R)- or
(S)-4 at different acetaldehyde concentrations. As shown in
Fig. 4 Formation rates of (R)-4 from 30 mM of enantiopure (S)-4 (A)
and formation rates of (S)-4 from 30 mM of enantiopure (R)-4 (B) as a
function of the acetaldehyde concentration (0–700 mM). Reactions
were carried out in 50 mM TEA buffer, 0.5 mM ThDP, 2 mM Mg2+
,
Scheme 2 Postulated reaction scheme for the cleavage of (S)- or (R)-
4 to benzaldehyde and acetaldehyde, with subsequent reformation of
rac-4.
wtBFD (21 U2-HPP mL−1 (2.5 mg mL−1 wtBFD) for (S)-4 and 33 U2-HPP
mL−1 (4 mg mL−1 wtBFD) for IJR)-4), at pH 7.5 and 25 °C. Data points
are average values of duplicates.
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its low boiling point (20.4 °C). Therefore, it is highly
recommended to run the reactions as well as to perform sam-
pling under slightly pressurized conditions to prevent evapo-
ration of acetaldehyde. As shown in Fig. 6, benzaldehyde and
(R)-benzoin concentrations increased during the course of
reaction. After 90 h, benzoin and benzaldehyde reached
0.4 mM and 1.2 mM, respectively, whereby the concentration
of 4 was almost constant at 140 mM during the course of
reaction.
Based on the aforementioned results showing: (i) the for-
mation of 4 from (R)-benzoin and acetaldehyde and (ii) the
racemization of 4, we postulate a mechanism shown in
Scheme 3, whereby the substrates (e.g. benzaldehyde and
acetaldehyde) and the carboligation products (e.g. 4 and (R)-
benzoin) are in equilibrium.
Fig. 5 Formation of benzaldehyde from 30 mM (S)-4 (A) or (R)-4 (B) in
the presence of varying acetaldehyde concentrations catalyzed by
wtBFD. Reaction conditions: 30 mM (R)- or (S)-4, 0–700 mM
acetaldehyde, 50 mM TEA buffer, 0.5 mM ThDP, 2 mM Mg2+, wtBFD
(21 U2-HPP mL−1 (2.5 mg mL−1 wtBFD) for (S)-4 and 33 U2-HPP mL−1
(4 mg mL−1 wtBFD) for IJR)-4), at pH 7.5 and 25 °C. Data points are
average values of duplicates.
C–C bond cleavage: formation of benzaldehyde and acetalde-
hyde from 4 and (2) C–C bond formation: reformation of (R)-
or (S)-4 from benzaldehyde and acetaldehyde (Scheme 2).
To describe the whole reaction (Scheme 3), as a reversible-,
coupled- and isolated system, the following equation was
used:
Negative control for the kinetic resolution of rac-benzoin
3 benzaldehyde + acetaldehyde ⇌ (R)-benzoin + 2-HPP
Thus, the equilibrium constant “K” can be calculated as:
To exclude a non-enzymatic cleavage of benzoin, three nega-
tive control experiments were performed: (1) without BFD to
examine a possible cleavage of benzoin catalyzed by buffer
components, (2) using heat-inactivated BFD (a variant with
similar activity) (30 min incubation at 80 °C) and lastly (3)
using BFD (a variant with similar activity) with additional 20
mM of 3-chloromethylbenzoyl-phosphonate (3-Cl-MBP) added
as inhibitor.15 The amount of enzyme used in negative con-
trols (2 and 3) was 30 U2-HPP mL−1 (3.6 mg mL−1) BFD defined
under standard synthesis conditions (see experimental sec-
tion). In all negative controls 1.6 mM of rac-benzoin and
50 mM of acetaldehyde were used and no formation of 4 was
detected over 72 h.
c
R -benzoin  c 2-HPP






KEq.

c benzaldehyde 3  c acetaldehyde




When the equilibrium concentrations of all reaction com-
ponents are known, the equilibrium constant can be deter-
mined based on the below given assumptions:
1. The equilibrium concentration of 4 is the average value
of the data measured during the reaction course, as no signif-
icant change in the concentrations of 4 was detectable
(Fig. 6).
Negative control for the racemization of 4
2. The concentration of acetaldehyde at equilibrium
equals to its initial concentration since the formation of
acetoin and/or evaporation of acetaldehyde from the reaction
medium is neglected.
3. The equilibrium concentrations of (R)-benzoin and
benzaldehyde are calculated from exponential regression for
t → ∞.
In order to exclude a potential auto-catalytical racemization
of 4 in the absence of enzyme two negative controls (starting
from 30 mM (R)-4 (>99% ee) and 25 mM acetaldehyde) were
performed: (1) with heat-inactivated 33 U2-HPP mL−1 (4 mg
mL−1) of wtBFD (30 min incubation at 80 °C) and (2) with
33 U2-HPP mL−1 (4 mg mL−1) of wtBFD in the presence of 20 mM
of 3-Cl-MBP used as inhibitor.15 In the positive control, the
ee dropped to 78% (R)-4 after 24 h, whereas no change was
observed in the negative controls. Even no formation of benz-
aldehyde was detected in the negative controls. Whereas,
0.17 mM benzaldehyde were found after 24 h in the positive
control.
Equilibrium conditions for 2-HPP, benzoin, benzaldehyde
and acetaldehyde
In order to determine the equilibrium concentrations of the
reaction components, a racemic mixture of 4 (at 140 mM)
was incubated in the presence of acetaldehyde (at 50 mM).
Although equimolar amounts of acetaldehyde are formed due
to the cleavage of 4, the supply of 50 mM acetaldehyde was
necessary since formed acetaldehyde may evaporate due to
Fig. 6 Concentrations of 2-HPP, benzaldehyde and (R)-benzoin at
approached equilibrium. The reaction was catalyzed by wtBFD. Reac-
tion conditions: 140 mM rac-4, 50 mM acetaldehyde, 50 mM TEA
buffer, 0.5 mM ThDP, 2 mM Mg2+, BFD (204 U2-HPP mL−1, 25 mg mL−1
wtBFD), at pH 7.5 and 25 °C. Data points are average values of
duplicates.
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complex, 1,2-dihydroxy-1,2-diphenylethyl-ThDP IJtTS(R)-benzoin),
which reacts to the ThDP-ylide by reversible elimination of
(R)-benzoin (route 4).
6. The chiral information of the carboligation products 4
and (R)-benzoin at the tetrahedral transition states is
included, since these complexes already have the chirality of
the products (route 3 and 4).
7. All reaction steps are in principle reversible, only the
decarboxylation activity of BFD can be regarded as quasi-
irreversible.
The BFD-mediated racemization of (S)-4 can be explained
by the above postulated reaction mechanism (Scheme 4).
Based on the considerations given above, (S)-4 binds first to
the ThDP-ylide forming the tTS2-HPP adduct (route 3) which is
followed by the elimination of acetaldehyde to afford the
enamine–carbanion intermediate. However, acetaldehyde can
rebind to the enamine–carbanion and thus reforms the
tTS2-HPP adduct (route 3) which predominantly yields (S)-4
(e.g. 92% (S)5a and 90% (S)17) but tiny amounts of (R)-4 are
also formed. While the reaction proceeds these ‘selectivity
mistakes’ accumulate and hence give a racemic mixture at the
end, since the formation of (S)-4 is highly preferred over (R)-4
whereas the kinetic parameters for the BFD-catalyzed cleavage
reaction of both enantiomers are similar. In principle, the
same might also be true for (R)-benzoin; however, since only
(R)-benzoin is formed via BFD catalysis, racemization of
(R)-benzoin is not possible. This is due to the fact that benzal-
dehyde cannot fit into the ‘S-pocket’7 found in BFD due to
steric hindrance and hence only (R)-benzoin is formed.
Herein, the orientation of the acceptor aldehyde to the
enamine–carbanion has to be justified since binding of the
acceptor aldehyde to the prochiral enamine–carbanion inter-
mediate is the crucial step. Iding et al. (2000) analyzed the ee
of the carboligation products (e.g. 4 or benzoin) based on Re-
or Si-attack on the acceptor aldehyde (e.g. acetaldehyde in the
synthesis of 4 or benzaldehyde in the synthesis of benzoin).2a
The above described extended reaction model can also
explain the observed BFD-catalyzed racemization of 4. Race-
mization takes place since the tetrahedral transition state
IJtTS2-HPP) is cleaved into the enamine–carbanion (EC) and
acetaldehyde (AA). Subsequently, 4 is formed from the cleav-
age products. Therefore, this elementary step can be
expressed as:
Scheme
3 Schematic representation of equilibrium between
benzaldehyde (2), acetaldehyde (3), 2-HPP (4) and (R)-benzoin (1).
The concentrations of reaction components at equilibrium
and the equilibrium constant are illustrated in Table 2. A very
high equilibrium constant of 6.3 × 105 L2 mol−2 clearly indi-
cates that the reaction is favored to the side of 2-hydroxy
ketones (e.g. 4 and IJR)-benzoin), whereby 4 is the main prod-
uct. The very low concentration of benzaldehyde at equilib-
rium shows also that the formation of 2-HPP proceeds very
slowly due to the high KM value for benzaldehyde (~80 mM
ref. 2a, 9 and 16). The same is also true for benzoin forma-
tion, since for (R)-benzoin formation no substrate saturation
was achieved up to ~40 mM of benzaldehyde.16 As mentioned
above, the synthesis of acetoin by BFD catalysis was neglected
due to the previously reported low activity of BFD for this
reaction.2a,16,17
Based on our results, the existing reaction mechanism for
decarboxylation of benzoylformate6b and for the formation of
(S)-4 ref. 2a can be extended as shown in Scheme 4. In the
proposed enhanced reaction mechanism the following princi-
ples and reaction steps are considered:
1. The cofactor ThDP is in equilibrium with its reactive,
deprotonated ylide-form.
2. The mandelyl-ThDP complex15 is formed from benzo-
ylformate and ThDP-ylide, followed by an irreversible forma-
tion of enamine–carbanion intermediate via elimination of
CO2 (route 1).
3. Benzaldehyde can reversibly bind to ThDP-ylide and
thus forms hydroxybenzyl-ThDP15 and the enamine–carbanion
intermediate is formed by reversible deprotonation (route 2).
4. The binding of acetaldehyde as an acceptor substrate at
C2-atom of the enamine–carbanion forms a new tetrahedral
c EC  c acetaldehyde




transition
state,
1-phenyl-1,2-dihydroxypropyl-ThDP
KEq.

c tTS

2-HPP 
IJtTS2-HPP), which reversibly yields the ThDP-ylide by elimina-
tion of 4 (route 3).
5. The binding of benzaldehyde as an acceptor at C2-atom
of the enamine–carbanion complex provides another tetrahedral
At equilibrium if cIJacetaldehyde) is increased then cIJEC)
should decrease or cIJtTS2-HPP) should increase. However, a
Table 2 Equilibrium concentrations of 2-HPP, acetaldehyde (AA), benzaldehyde (BA) and (R)-benzoin and the determined equilibrium constant. Reac-
tion conditions: 140 mM rac-4, 50 mM acetaldehyde, 50 mM TEA buffer, 0.5 mM ThDP, 2 mM Mg2+, wtBFD (204 U2-HPP mL−1, 25 mg mL−1 wtBFD) at
pH 7.5 and 25 °C.
2-HPP [mM]
141.3 4.2
AA [mM]
50
BA [mM]
(R)-Benzoin [mM]
KEq. [L2 mol−2
6.3 × 105
]
1.37 0.04
0.58 0.04
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Scheme 4 Extended reaction mechanism of BFD based on the reaction mechanisms given in literature.2a,5b In addition to the irreversible
decarboxylation of benzoylformate (route 1), the binding of benzaldehyde (route 2), 2-HPP (4) (route 3) and (R)-benzoin (route 4) to the ylide-form
of the cofactor ThDP is possible. Starting from a respective tetrahedral transition state (tTS) and followed elimination of acceptor electrophile (e.g.
proton (route 2), acetaldehyde (route 3) and benzaldehyde (route 4); irreversible elimination of CO2 (route 1)), the enamine–carbanion intermediate
is formed. As the tetrahedral transition states: mandelyl-ThDP (route 1), hydroxybenzyl-ThDP (route 2), tTS2-HPP (route 3) and tTS(R)-benzoin (route 4)
are formed.
decrease of the average concentration of cIJEC) leads to
decreased reaction rates from EC to tTS2-HPP, which means
decreased racemization rates. Furthermore, reduced racemi-
zation rates of 4 in the presence of low acetaldehyde concen-
trations (0–25 mM) (Fig. 4) can result from a substrate-
to form (R)-benzoin and 2-HPP is favored by BFD as also indi-
cated by the very high equilibrium constant. A racemic mix-
ture of 2-HPP was detected while using benzoin as the donor
in the presence of acetaldehyde. Not only (R)-benzoin but
also both enantiomers of 2-HPP are accepted by BFD as a
substrate for the cleavage reaction. The similar affinity of
BFD observed for the cleavage of (R)- and (S)-2-HPP necessi-
tates further kinetic investigations. Herein, in silico substrate
docking for both enantiomers to investigate the orientation
of phenyl- and methyl groups of 2-HPP in the ‘S-pocket’
might be useful.
The formation of (R)-2-HPP from IJS)-2-HPP, and vice versa,
was demonstrated and the formation rates were shown to be
dependent of the acetaldehyde concentration. The formation
of benzaldehyde as the cleavage product of 2-HPP was suc-
cessfully monitored. Lastly, the reaction mechanism reported
previously was supplemented with the observed C–C bond
cleavage activity of BFD, which explains the ‘reversibility’ of
the BFD-catalyzed C–C bond formations.
limited reaction of the EC and acetaldehyde towards tTS2-HPP
.
In addition, increased reaction rates by increasing the con-
centrations of 4 (Fig. 3) can be simply explained by the
increased amounts of 4 reacting with the ThDP-ylide to form
tTS2HPP
.
Further on, the formation of 4 from (R)-benzoin and acet-
aldehyde can be explained through our postulated mecha-
nism. Here, (R)-benzoin binds first to ThDP-ylide to form the
enamine–carbanion (route 4). Subsequently, the enamine–
carbanion can further react with acetaldehyde yielding 4
(route 3). Starting from rac-benzoin, whereby only (R)-benzoin
is accepted as the substrate, a classical resolution yields in 4
and (S)-benzoin (Fig. 2). Consequently, tTS(R)-benzoin can only
be formed in the (R)-form since only (R)-benzoin is synthe-
sized from benzaldehyde. As aforementioned, due to steric
hindrance benzaldehyde cannot fit into the previously defined
S-pocket7a and thus no proper alignment of enamine–carbanion
The practical usefulness of the BFD-catalyzed C–C cleavage
in the kinetic resolution of benzoin can be improved by
increasing the solubility of benzoin. To overcome this chal-
lenge the reaction can be performed under low water activity
conditions (e.g. neat substrate, organic media etc.), where
BFD is applied in an immobilized form or as in the whole
cells.
and benzaldehyde is possible to form tTS(S)-benzoin
.
Conclusion
This work represents a first time report of the C–C bond
cleavage activity of benzoylformate decarboxylase from
Pseudomonas putida towards 2-hydroxy ketones. This has
been demonstrated by the formation of rac-2-HPP from
rac-benzoin via the in situ removal of benzaldehyde in the
presence of acetaldehyde. However, the carboligation reaction
Experimental
The chemicals used in this study were commercially obtained
in analytical-grade quality and were used as received. Alde-
hydes were distilled prior to their use.
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Paper
Preparation of wtBFD
of 4 TEA buffer in the presence of 25 mM acetaldehyde was
used. The enzyme solutions were prepared by dissolving
lyophilized wtBFD in TEA buffer in the presence of 25 mM
acetaldehyde. Reactions were started by addition of the
enzyme solution (51 U2-HPP wtBFD) to the substrate solution.
Samples were taken over a period of 30 h, quenched with
stop-solution (as above described) and subsequently analyzed
by HPLC. Total reaction volume was 1 mL. The Michaelis–
Menten kinetic parameters were determined using Origin 9.1
(Origin Lab Corporation, Northampton, MA, USA).
To analyze the reactions using varying acetaldehyde con-
centrations, a stock solution of 2 M acetaldehyde in 50 mM
TEA buffer, 0.5 mM ThDP, 2 mM MgCl2 at pH 7.5 and 25 °C
was prepared. The concentration of (S)- or (R)-4 was 30 mM.
The final concentrations of acetaldehyde were adjusted to 0,
25, 50, 200, 400 and 700 mM. The concentration of enzyme
used per reaction was 21 U2-HPP mL−1. Only in the case of
30 mM (R)-4 as substrate, 33 U2-HPP mL−1 of wtBFD was
applied (Fig. 4(B)). Samples were taken over a period of 30 h and
handled as described before. Total reaction volume was 1 mL.
Detailed description of cell cultivation, gene expression, and
enzyme purification are found in the PhD thesis of M.
Berheide18 and Berheide et al. (2010).19
Determination of the activity of BFD for direct carboligation
of benzaldehyde and acetaldehyde
Activity analysis was performed in 50 mM phosphate buffer,
0.5 mM ThDP, 2 mM MgCl2 at pH 7.5 and 30 °C. The sub-
strate stock of benzaldehyde and acetaldehyde was prepared
in the above given buffer, respectively. The substrate solution
(4.9 mL) was incubated at 30 °C for 3 min in a glass vessel
with a volume of 10 mL. The reaction was started by the addi-
tion of wtBFD stock solution (0.1 mL) which was prepared in
50 mM phosphate buffer, 0.5 mM ThDP, 2 mM MgCl2 at pH
7.5. Final concentrations of benzaldehyde and acetaldehyde
were 40 mM and 400 mM, respectively. Samples were taken
at definite time intervals and quenched with stop-solution
IJacetonitrile : H3PO4 (95 : 5), v/v) at a 2 : 1 ratio (sample : stop-
solution, v/v). After centrifugation of the precipitate (13 000 rpm,
3.5 min), samples were analyzed by HPLC.
Analysis of reaction equilibrium starting from rac-4 and
Here, one unit of activity IJU2-HPP) is defined as the amount
of enzyme which catalyzes the formation of 1 μmol of 2-HPP
in 1 min at 30 °C under the conditions given above. Protein
amounts were determined by the standard Bradford
method20 using bovine serum albumin (BSA) as a standard.
acetaldehyde
A stock solution containing 180 mM of (S)-and (R)-4 and
50 mM of acetaldehyde was prepared in TEA buffer. The lyophi-
lized enzyme was dissolved in 222 μL TEA buffer which also
contained 50 mM of acetaldehyde. Reactions were started by
addition of 778 μL of rac-4 solution (containing 50 mM acet-
aldehyde) so that the final concentrations were 140 mM rac-4
and 50 mM acetaldehyde. Reactions contained 204 U2-HPP of
wtBFD. Samples were taken over a period of 92 h, processed
as previously described and analyzed by HPLC. Total reaction
volume was 1 mL.
Analysis of kinetic resolution of rac-benzoin
The experiments for kinetic resolution of benzoin were
performed in 1.93 mL of total volume using 30% IJv/v) DMSO.
The stock solution of rac-benzoin was prepared in DMSO at a
concentration of 5.2 mM in the presence of 50 mM acetalde-
hyde. The enzyme solution was prepared by dissolving lyophi-
lized wtBFD (157 U2-HPP, 19 mg wtBFD) in 1.35 mL of 50 mM
TEA buffer, 0.5 mM ThDP, 2 mM MgCl2 at pH 7.5 and 25 °C,
which also contained 50 mM of acetaldehyde. The enzyme
solution was cooled on ice and the reactions were started by
addition of 0.58 mL of rac-benzoin stock solution (also
containing acetaldehyde) into the enzyme solutions. There-
fore, the final concentrations were 1.6 mM of rac-benzoin
and 50 mM of acetaldehyde. Samples were taken at definite
time intervals over a period of 312 h and quenched with
stop-solution IJacetonitrile : H3PO4 (95 : 5), v/v) at a 2 : 1 ratio
(sample : stop-solution, v/v). This was followed by pelleting of
the precipitate (13,000 rpm, 3.5 min) and subsequent HPLC
analysis.
Standard deviations observed in duplicated experiments
In case of determination of conversion values (e.g. Fig. 2A,
5 and 6) the deviation between duplicated experiments was
3–15%, whereas in case of the determination of ee values
(Fig. 1, 2B, 3, and 4) the standard deviation between experi-
ments was 3–5%.
Negative control for kinetic resolution of rac-benzoin
Information on the BFD variant: the used BFD variant (BFD
A460I) showed cleavage activity like wild type BFD under the
same reaction conditions.19
Synthesis of enantiopure (S)-4
Analysis of cleavage of 2-HPP (4)
Substrate solution containing benzaldehyde (47.25 g, 445 mmol)
and acetaldehyde (195 g, 4.42 mol) was prepared in 10.5 L
potassium phosphate buffer (20 mM), ThDP (0.2 mM) and
MgCl2 × 6H2O (2 mM). The pH was adjusted to 7.5 using con-
centrated H3PO4 and NaOH. Reaction was started by the addi-
tion of 1400 U2-HPP of freshly purified wtBFD to the substrate
solution and run at room temperature. After 36 h the total
All experiments were performed in 50 mM TEA buffer,
0.5 mM ThDP, 2 mM MgCl2 at pH 7.5 and 25 °C. The stock
solutions of (S)- and (R)-4 were prepared at a concentration
of 180 mM which also contained 25 mM of acetaldehyde and
the final concentrations were adjusted to be 5, 50, 80, 110,
140 and 170 mM of (S)- or (R)-4. To dilute the stock solutions
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reaction mixture was extracted with dichloromethane Acknowledgements
(5 × 200 mL). The organic phase was washed with water and
Special thanks to Prof. Dr. Martina Pohl (Institute of Molecu-
lar Enzyme Technology, Heinrich-Heine University of
Düsseldorf, Research Center Juelich) for generously providing
the wild type BFD. Dr. A. Bornadel is acknowledged for proof-
reading parts of the manuscript. We thank anonymous ref-
erees for critically reading our manuscript and for providing
valuable inputs and comments.
brine then dried over using magnesium sulfate and lastly the
solvent was removed with a rotary evaporator. The crude prod-
uct (68.3 g, ee(S)-2-HPP = 85%) was obtained as a yellow viscous
oil. After recrystallization using isohexane, (S)-4 (ee > 99.9%,
yield = 44.85 g, 67.2%) was isolated as needle-shaped crystals
(~3 cm long).
1H-NMR (400 MHz, CDCl3) δ (ppm): 1,47 (d, 3H, ,3J = 7,1
3
Hz; CH3); 3,82 (br, 1H; OH); 5,19 (q, 1H, J = 7,1 Hz; CHOH);
7,52 (“t”, 2H, 3J = 7,47 Hz; Ar–H); 7,64 (tt, 1H, 3J = 7,47 Hz, 4J =
1,3 Hz; Ar–H); 7,95 (dd, 2H, 3J = 7,07 Hz, 4J = 1,3 Hz; Ar–H).
13C-NMR (100 MHz, CDCl3) δ (ppm): 22,32 (CH3); 69,32
(CHOH); 128,67/128,89 (CH); 133,31 (Cq); 134,01 (CH); 202,4
(CO).
Notes and references
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Synthesis of enantiopure (R)-4
Benzaldehyde (4.6 g, 43 mmol) and acetaldehyde (17.64 g,
0.4 mol) were dissolved in 1 L of potassium phosphate buffer
(50 mM), ThDP (0.5 mM) and MgCl2 × 6H2O (2 mM) and 5%
MTBE IJv/v). The pH was adjusted to 8.0 using concentrated
H3PO4 and NaOH and the reaction was started by addition of
40 mg of BAL (provided by Dr. Nils Kurlemann). Reaction
was run at room temperature and after 130 h the reaction
mixture was extracted with dichloromethane (5 × 100 mL).
The combined organic phase was washed with water and
brine, dried over magnesium sulfate and lastly the solvent
was removed with a rotary evaporator. The crude product
(9.69 g) was obtained in pale yellow, viscous form and
recrystallized using isohexane. In the end, (R)-4 (ee > 99.9%,
yield = 6.3 g, 97%) was isolated as needle-shaped crystals
(~4 cm long).
1H-NMR (400 MHz, CDCl3) δ (ppm): 1,47 (d, 3H, 3J = 7,05 Hz;
CH3); 3,82 (br, 1H; OH); 5,19 (q, 1H, 3J = 7,05 Hz; CHOH);
3
3
4
7,52 (“t”, 2H, J = 7,4 Hz; Ar–H); 7,64 (tt, 1H, J = 7,4 Hz, J =
3
4
1,3 Hz; Ar–H); 7,95 (dd, 2H, J = 7,0 Hz, J = 1,3 Hz; Ar–H).
13C-NMR (100 MHz, CDCl3) δ (ppm): 22,32 (CH3); 69,32
(CHOH); 128,67/128,89 (CH); 133,31 (Cq); 134,01 (CH); 202,4
(CO).
Analytics
Reactions were analyzed by HPLC (Agilent 1100, Hewlett
Packard) equipped with
a LiChrosphere RP-8 column
(Hypersil, 250 × 4 mm, Merck) and detections were at
254 nm. Triethanolamine (0.2%, pH 3.0):(60 : 40, v/v) was
used as an eluent at a flow rate of 1.0 mL min−1 at 30 °C.
Retention times were t2-HPP = 5.0 min, tbenzaldehyde = 8.1 min
and tbenzoin = 11.9 min. For ee determination, samples were
extracted with isohexane and analyzed by HPLC using a
Daicel Chiralcel OD-H column (5 μm). Isohexane : iso-
propanol (98 : 2, v/v) was used as a mobile phase at a flow
rate of 0.75 mL min−1 at 20 °C and detections were at
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21.8 min, t(S)-benzoin = 30.0 min and t(R)-benzoin = 48.0 min.
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