Hydroxynitrile lyase in organic solvent-free systems to overcome thermodynamic limitations

Article abstract of DOI:10.1002/adsc.200700016

The overcoming of thermodynamic limitations in the synthesis of optically active ketone cyanohydrins by using organic solvent-free systems has been investigated. Therefore, substrates with known unfavorable results within hydroxynitrile lyase-catalyzed re

Full text of DOI:10.1002/adsc.200700016

FULL PAPERS
DOI: 10.1002/adsc.200700016
Hydroxynitrile Lyase in Organic Solvent-Free Systems to Over-
come Thermodynamic Limitations
Jan von Langermann,a Annett Mell,a Eckhard Paetzold,b Thomas Daußmann,c
and Udo Kragla,*
a
Institut für Chemie, Universität Rostock, Albert-Einstein-Str. 3a, 18059 Rostock, Germany
Phone: (+49)-381-498-6450; fax: (+49)-381-498-6452; e-mail: udo.kragl@uni-rostock.de
Leibniz-Institut für Katalyse e.V. an der Universität Rostock, Albert-Einstein-Str. 29a, 18059 Rostock, Germany
Julich Chiral Solutions GmbH, Prof.-Rehm-Straße 1, 52428 Jülich, Germany
b
c
Received: January 9, 2007; Revised: March 25, 2007
Dedicated to Prof. Dr. Maria-Regina Kula on the occasion of her 70th birthday.
Abstract: The overcoming of thermodynamic limita- exhibit only low grades of conversion also with sever-
tions in the synthesis of optically active ketone cya- al other hydroxynitrile lyases. With organic solvent-
nohydrins by using organic solvent-free systems has free systems under optimized reaction conditions
been investigated. Therefore, substrates with known conversions up to 78% with>99.0 ee (S) were ob-
unfavorable results within hydroxynitrile lyase-cata- tained. Finally, 5 mL of (S)-acetophenone cyanohy-
lyzed reactions were selected for the determination drin with an enantiomeric excess of 98.5% ee (S)
of limitations and bottlenecks in ketone cyanohydrin were synthesized.
synthesis. The highly (S)-selective hydroxynitrile
lyase from Manihot esculenta (MeHNL) has been Keywords: acetophenone cyanohydrin; enzyme catal-
chosen for the conversion of acetophenone and the ysis; hydroxynitrile lyase; Manihot esculenta; ther-
corresponding derivatives, which are substrates that modynamics
Introduction
matic and enzymatic techniques. In the field of enzy-
matic cyanohydrin synthesis several ketone substrates
Chiral cyanohydrins are versatile intermediates in the showed, for example, low conversions using several
synthesis of a-hydroxy acids, b-amino alcohols, amino sources of hydroxynitrile lyases.[12,15,16] On the one
nitriles, a-hydroxy ketones and aziridines.[1–4] Among hand, this behavior is often explained by low reactivi-
the known methods for the synthesis of enantiomeri- ties of ketone substrates. On the other hand, other
cally pure cyanohydrins, the use of hydroxynitrile ketone cyanohydrins are obtained with high yields
lyases for the synthesis of cyanohydrins is currently and excellent enantiomeric excess, for example, phe-
the most effective approach for this class of com- nylacetone cyanohydrin with a yield of 97% and an
pounds.[5,6] These hydroxynitrile lyases have been in- enantiomeric excess of 98% ee (S) using an immobi-
tensively studied during the last decades with excel- lized hydroxynitrile lyase from Manihot esculenta
lent results for a variety of unnatural substrates yield- (MeHNL).[17] This indicates that the limitations in en-
ing (R)- and (S)-cyanohydrins in high purities and zymatic ketone cyanohydrin synthesis are much more
high enantiomeric excess.[7–10] An interesting member complicated.
of this group is the hydroxynitrile lyase from Manihot
By contrast, several non-enzymatic pathways were
esculenta (E.C. 4.1.2.37) (MeHNL), which exhibits developed to overcome these limitations, for example,
high enantioselectivity for a broad spectrum of sub- catalytic asymmetric cyanosilylation using titanium
strates of aldehydes and ketones.[11–13] Moreover, the isopropoxide and trimethylsilyl cyanide as a cyanide
availability of several mutants is also beneficial due to donor.[18–20] However, the requirement of anhydrous
the conversion of substrates with very bulky substitu- conditions, including high pressure reactions condi-
ents, for example, 3-phenoxybenzaldehyde (mutant tions, and an additional hydrolysis step represents a
MeHNL-W128A).[14]
significant drawback, which is directly related to high
Unfortunately, low grades of conversion are still costs and low atomic efficiencies. Therefore, the enzy-
observed for many substrates using both non-enzy-
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Hydroxynitrile Lyase in Organic Solvent-Free Systems
FULL PAPERS
matic cyanohydrin synthesis is still the best pathway Homogeneous Aqueous-Ethanol System
to obtain enantiopure cyanohydrins.
To describe limitations in enzymatic ketone cyano- At the beginning, a one-phase-system with ethanol as
hydrin synthesis we have chosen acetophenone and co-solvent (to increase the solubility of acetophe-
the corresponding derivatives as substrates, which none) was chosen for the determination of possible
showed low grades of conversion and only moderate substrate and product inhibitions. However, first re-
enantioselectivities with several hydroxynitrile sults showed clearly that an inhibition of the enzyme
lyases.[12,16,21–23] Enantiopure acetophenone cyanohy- is not the limiting effect. On the contrary, the unfav-
drin is a very useful building block in the atrolactic orable position of the thermodynamic equilibrium
acid synthesis.[24,25]
prohibits high grades of conversions, which can be ex-
plained by a higher stability of the substrate aceto-
phenone compared to the product acetophenone cya-
nohydrin. Within the homogenous aqueous-ethanol
system, the initial substrate concentration is directly
Results and Discussion
In biocatalytic reactions several limitations, like sub- correlated with the thermodynamic equilibrium. An
strate or product inhibition, may occur, which directly acetophenone concentration of only 10 mmol/L yields
lead to a limitation in productivity. On the one hand, only a very low grade of conversion, while an initial
one way to overcome the problem of inhibition is the acetophenone concentration of 500 mmol/L allows
right choice of reactor type, where a substrate inhibi- the highest grade of conversion (Figure 2). This in-
tion can be overcome, for example, by a fed-batch crease of the thermodynamic equilibrium by an in-
process or by performing the reaction in a continu- crease of the substrate concentration can be explained
ously operated stirred-tank reactor (CSTR). In case by the principle of Le Châtelier. In this case, 2 mole-
of a product inhibition an in situ product removal cules (the substrates: acetophenone and hydrogen cy-
would be the best choice. Usually aqueous/organic anide) are transformed into 1 molecule (the product:
two-phase systems and XAD-resins (for adsorption of
the product) are used for this approach.[26] Additional-
ly, thermodynamic limitations can by overcome be the
removal of the product.
On the other hand, the hazardous properties of hy-
drogen cyanide allow for large-scale applications usu-
ally only simple batch reactions. Only small-scale ap-
plications, for example, small continuously operated
membrane reactors, have proven their practicability
under the safety circumstances of hydrogen cya-
nide.[27] Additionally, a continuous removal of the
product (cyanohydrin) is not known so far in enzy-
matic cyanohydrin synthesis.
Finally, the undesired chemically side reaction
(non-enzymatic formation of racemic cyanohydrins)
can be reduced by decreasing the pH value within the
aqueous phase[27,44] or by decreasing the reaction tem-
perature.[28] The mass transfer limitation is also a pow-
erful tool to enhance the enantiomeric excess.[29,30]
In order to understand the underlying limitations
within hydroxynitrile lyase-catalyzed ketone cyanohy-
drin synthesis, we investigated several different reac-
tion systems to overcome the limitations (Figure 1).
Figure 2. Acetophenone cyanohydrin synthesis in one-phase-
system consisting of a mixture of ethanol and citrate buffer
pH 4.0: (-<-) 10 mmol/L acetophenone: 100% (v/v) buffer,
~
(- -) 50 mmol/L acetophenone: 25% (v/v) ethanol, 75%
*
(v/v) buffer, (- -) 100 mmol/L acetophenone: 35% (v/v)
ethanol, 65% (v/v) buffer, (-&-) 500 mmol/L acetophenone
50% (v/v) ethanol, 50% (v/v) buffer. Reaction conditions:
58C, 50–500 mmol/L citrate buffer pH 4.0/ethanol, 400 rpm.
Equilibrium constant: K=0.0006 L/mmol. Due to the deter-
mination of the thermodynamic equilibrium no enzyme was
[a]
required (reaction yielding the racemate).
[Bühler et al.
2003][12] – 200 mmol/L acetophenone, 400 mmol/L hydrogen
cyanide (ratio 1:2) in diisopropyl ether with immobilized
MeHNL on nitrocellulose (pure organic one-phase system).
Figure 1. (S)-Acetophenone cyanohydrin synthesis using the
hydroxynitrile lyase from Manihot esculenta (MeHNL).
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Table 1. Enzyme stability and enzyme activity with the addi-
tion of ethanol at 58C.[a]
Table 2. Acetophenone cyanohydrin synthesis in a two-
phase system.[a]
Addition of etha- Enzyme stability, half Relative enzyme
Overall acetophenone concentration
within the two-phase system (DIPE
buffer)
Equilibrium con-
version [%]
nol [(v/v)%]
life period [h]
activity [%]
0
5
10
15
25
50
>500
>500
>500
n.d.
40
<1
100.0
18.1Æ3.8
17.3Æ1.2
16.1Æ2.5
8.0Æ3.5
9.6Æ1.9
10 mmol/L acetophenone
100 mmol/L acetophenone
2.0
2.5
[a]
Reaction conditions: substrate to HCN ratio 1:2; diiso-
propyl ether to buffer ratio 1:1; citrate buffer pH 4.0;
108C, 400 rpm, 20 UmLÀ1 MeHNL
[a]
Reaction conditions: 58C, 50-500 mmol/L citrate buffer
pH 4.0, 400 rpm; n.d. - not determined
This very low equilibrium conversion is associated
with the results from the homogeneous aqueous-etha-
acetophenone cyanohydrin), which is a decrease of nol system. Due to the high partition coefficient of
the overall concentration of the reactants. Therefore, acetophenone, the acetophenone concentration within
the reaction system responds with an increase of the the aqueous phase is very low (both experiments
equilibrium conversion when the acetophenone con- <5 mmol/L). Hence, these low substrate concentra-
centration is increased. This behavior was also found tion result in low grades of conversion for the two-
with the (better soluble) substrate benzaldehyde.[31]
Interestingly, Bühler et al. reported similar results
by using pure organic one-phase systems, whereas the
phase system.
enzyme
was
immobilized
on
nitrocellulose Organic Solvent-Free System
(Figure 2).[12] By using an HCN to acetophenone ratio
of only 2 and an initial acetophenone concentration In contrast to the one- and two-phase systems an or-
of 200 mmol/L, 13% conversion was obtained, which ganic solvent-free system consists only of the sub-
correlates with our findings. Some authors discussed a strate, which represents in this special case also the
difference of the equilibrium constants between enan- organic phase, and the aqueous phase, containing the
tioselective and racemic cyanohydrin formation.[28]
On the other hand, the high concentration of etha-
hydroxynitrile lyase and the buffer salts.
By using this unusual reaction system the equilibri-
nol has a negative effect on the enzyme stability and um conversion can be enhanced significantly. At a
activity (Table 1). With increasing amount of ethanol fixed acetophenone to buffer ratio of 1:20 (equivalent
the enzyme stability decreases dramatically from a to 0.41 mol/L acetophenone) and an increasing aceto-
half life period of over 500 h to only 40 h at 25% phenone to HCN ratio up to 1:20, the thermodynamic
(v/v) and less than 1 hour at 50% (v/v) ethanol. Un- equilibrium conversion has been raised up to 46%
fortunately, due to the extremely low enzyme stability (Table 3). Unfortunately, the enzyme stability de-
at an ethanol content of 50% (v/v), the enantioselec- creases simultaneously with increasing hydrogen cya-
tive catalysis is completely suppressed and only the nide and acetophenone concentrations from>500 h in
non-enzymatic addition of hydrogen cyanide occurs, pure buffer (Table 1) via 29 h in the organic solvent
yielding the racemate.
free system with acetophenone and finally to 7 h with
2 mol/L hydrogen cyanide (also organic solvent-free
system with an acetophenone to HCN ratio of 1:5).
This prevents high grades of enantioselective conver-
sion. The same behavior was also found for the close-
Two-Phase System
Two-phase systems are well established and investi- ly related hydroxynitrile lyase from Hevea brasilien-
gated within hydroxynitrile lyase-catalyzed reactions sis.[35–37]
using a variety of substrates and hydroxynitrile lyases
Nevertheless, for an acetophenone to HCN ratio of
from several sources.[29,32–34] Unfortunately, only low 1:5 an enzyme stability of 7 h still enables 22% con-
grades of conversion were obtained for acetophenone version with excellent 97% ee (S). Higher hydrogen
cyanohydrin synthesis by using a two-phase system cyanide concentrations permanently deactivate the
consisting of diisopropyl ether as the organic phase enzyme and thus prevent higher grades of conversion,
and citrate buffer as the aqueous phase. Even an in- which is a general problem in hydroxynitrile lyase-cat-
crease of the substrate concentration leads only to a alyzed cyanohydrin synthesis.[37,38]
slight increase of the equilibrium conversion
(Table 2).
Furthermore, the acetophenone to buffer ratio has
also a significant influence on the equilibrium conver-
sion. By adjusting the acetophenone to buffer ratio
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Table 3. Variation of acetophenone to HCN ratio.[a]
Acetophenone to Enzyme stability (half Equilibrium con-
pH 4.8
pH 4.0
HCN ratio
life time) [h]
version [%]
Conversion Enantiomeric
Conversion Enantiomeric
[%]
excess [%]
[%]
excess [%]
1:0
29
0
0
0
0
0
1:2
1:5
1:10
1:15
1:20
8
7
10
22
31
37
46
10
22
6
5
5
95
96
6
0
0
n.d.
22
22
0
-
97
97
0
<0.5
<0.5
n.d.
0
0
[a]
Reaction conditions: reaction time: 6 h; acetophenone to buffer ratio 1:20 (overall acetophenone concentration equivalent
to 0.41 mol/L), citrate buffer pH 4.0 or pH 4.8, 58C, 400 rpm; 450 UmLÀ1 for pH 4.0; 150 UmLÀ1 for pH 4.5; n.d.=not
determined.
Table 4. Variation of acetophenone to buffer ratio.[a]
Acetophenone to
buffer ratio
Overall acetophenone
concentration [mol/L]
pH 4.8; 150 UmLÀ1 MeHNL
pH 4.0; 450 UmLÀ1 MeHNL
Conversion
[%]
Enantiomeric
excess [%]
Conversion
[%]
Enantiomeric
excess [%]
1:100
1:33
1:20
1:10
1:6.6
0.085
0.25
0.41
0.78
1.13
n.d.
11
22
36
31
-
9
86
94
97
98
95
96
91
75
11
22
15
n.d.
n. d.
[a]
Reaction conditions: reaction time: 6 h; acetophenone to HCN ratio 1:5; citrate buffer pH 4.0 or pH 4.8, 58C, 400 rpm;
n.d.=not determined.
from 1:100 up to 1:10 the equilibrium conversion has hydrin only in 14% yield, while 2’-fluoroacetophe-
been increased from 9% up to 36% with a slight de- none showed a conversion of 71% (as already stated
cline of the enantioselectivity (Table 4).
above). This can be explained by an intramolecular
Noteworthy, the hydrogen cyanide concentration hydrogen bond, which stabilizes the cyanohydrin and
increases simultaneously with the increasing aceto- facilitates high grades of conversion (Figure 3).[40] In
phenone concentration at a constant acetophenone to contrast, by applying a two-phase system (diisopropyl
HCN ratio of 1:5. This leads again to a loss of enzyme ether-citrate buffer) for 2’-fluoroacetophenone the
stability and prevents higher grades of conversion at conversion decreases to 8%, the effect was pointed
even lower acetophenone to buffer ratios (1:6.6).
out in the section ꢁTwo-Phase Systemꢂ. The intramo-
lecular stabilization by a hydrogen bond occurs also
for 2-nitroacetophenone cyanohydrin. In contrast to
this, 2’-methoxyacetophenone cyanohydrin and 2’-hy-
droxyacetophenone result still only in low grades of
Reactions with Acetophenone Derivatives
Finally, the position and kind of substituents at the ar- conversion. Potentially, the positive mesomeric effect
omatic ring also influence the maximal conversion of the oxygen atom is still too strong and thus pre-
and enantiomeric excess within hydroxynitrile lyase- vents higher equilibrium conversions.
catalyzed reactions.[39] Therefore, several acetophe-
Additionally, the conversions of 2’-chloroacetophe-
none derivatives were tested under the optimized none and 2’-bromoacetophenone result in lower
conditions, but only substrates being that were liquid yields and enantiomeric purities, which can be ex-
at 58C were applied.
plained by steric effects due to the size of the sub-
On the one hand, substrates with electronegative stituent.
substituents like fluoro, chloro and nitro groups
On the other hand, electropositive substituents like
showed the best results, for example, yields up to methyl and amino groups resulted in extremely low
71% could be obtained for the substrate 2’-fluoroace- conversions of less than 1%. 2’,3’,4’,5’,6’-Fluoroaceto-
tophenone (Table 5). Additionally, the position of the phenone affords also a very low conversion, while the
substituents also displays a significant influence on reasons for this behavior are not completely known
the maximum conversion. For instance, 4’-fluoroace- till now.
tophenone was converted to the corresponding cyano-
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Table 5. Acetophenone derivative cyanohydrin formation.[a]
Firstly, the substrate concentration within the aque-
ous phase correlates directly with the enzyme activity.
Especially at substrate concentrations below the KM
value (Michaelis–Menten constant) the enzyme activi-
ty will be reduced dramatically. In the case of an
aqueous organic two phase-system, for example, with
diisopropyl ether as the organic solvent and a buffer
as the aqueous layer, the partition coefficient of the
substrate generates only a low substrate concentration
within the aqueous phase, which leads directly to a
low enzyme activity. The kinetics within the two-
phase system were intensely studied during the last
years.[33,36] On the other hand, the organic solvent-free
system affords always a maximum enzyme activity,
because the substrate concentration is only limited by
the solubility of the substrate in the aqueous phase.
Secondly, an increase of the substrate concentration
within the aqueous phase results also in higher grades
of conversion, as pointed out in the section ꢁHomoge-
nous Aqueous-Ethanol Systemꢂ. Therefore the organ-
ic solvent-free system is extremely advantageous, be-
cause the substrate concentration is only limited by
the solubility of the substrate, resulting in very high
grades of conversion.
Acetophenone (AP) de-
rivative
Time
[h]
Conversion
[%]
ee (S)
[%]
AP
4’-F-AP
3’-F-AP
2’-F-AP
2’,3’,4’,5’,6’-F-AP
4’-Cl-AP
3’-Cl-AP
2’-Cl-AP
4’-Br-AP
3’-Br-AP
2’-Br-AP
4’-I-AP
6
1.5
3
3
6
6
6
4.5
solid
6
22
14
48
71
<1
18
23
6
96
>99
>99
>99
-
97
97
80
10
9
>99
68
6
solid
6
6
6
6
solid
6
6
solid
solid
1.5
solid
solid
6
solid
solid
6
2’-I-AP
<1
<1
<1
<1
-
-
-
-
4’-Me-AP
3’-Me-AP
2’-Me-AP
4’-MeO-AP
3’-MeO-AP
2’-MeO-AP
4’-NO2-AP
3’-NO2-AP
2’-NO2-AP
4’-NH2-AP
3’-NH2-AP
2’-NH2-AP
4’-OH-AP
3’-OH-AP
2’-OH-AP
<1
<1
-
-
40
>99
Thirdly, the organic layer (in this case mainly the
substrate) acts for the product (cyanohydrin) as an or-
dinary organic phase, resulting in a simple partition
coefficient of the product. Therefore the product is
selectively extracted into the organic phase, whereas
the substrate concentration in the aqueous phase
<1
<1
-
-
[a]
Reaction conditions: reaction time: 1.5–6 h; 0.4 mmol (where the enzymatic reaction takes place) is still
acetophenone derivative: 2 mmol hydrogen cyanide;
1 mL citrate buffer pH 4.0, 58C, 450 UmLÀ1, 400 rpm;
solid=not determined due to the absence of an organic
layer.
maximal due to the limited water miscibility of aceto-
phenone. This in situ product removal shifts the equi-
librium to higher grades of conversion, depending
also on the type and position of the substituents.
Which effect is more relevant to explain the ob-
served higher conversion can only be confirmed with
a thorough investigation. This would include exact de-
termination of phase ratios, distribution coefficients
and equilibrium constants, but this is complicated due
to the volatility of hydrogen cyanide. That such stud-
ies are indeed possible has been shown recently for
the enantioselective ketone reduction using alcohol
dehydrogenases.[41,42]
Figure 3. Possible intramolecular hydrogen bond of (S)-2’-
fluoroacetophenone.
The potential of the approach presented here was
proven by synthesis of 5 mL of (S)-acetophenone cya-
nohydrin with an enantiomeric excess of 98.5% ee
Conclusions
We investigated the use of organic solvent-free sys- (S). This included also a distillation step without any
tems for their practicability to overcome limitations decomposition or racemization of the product.[43]
and bottlenecks within acetophenone cyanohydrin
In summary, the organic solvent-free system ena-
synthesis. We pointed out that organic solvent-free bles moderate to high conversions, even for substrates
systems enable good conversions also for substrates which were not obtained by conventional reaction
which otherwise showed unsatisfactory results using systems, for example, the two-phase system using di-
conventional methods, like aqueous-organic two- isopropyl ether and buffer or immobilized enzyme in
phase systems.
The organic solvent free system has several advan- is reduced by the very high concentrations of aceto-
tages. phenone and hydrogen cyanide, but the hydroxynitrile
diisopropyl ether. Unfortunately, the enzyme stability
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Hydroxynitrile Lyase in Organic Solvent-Free Systems
FULL PAPERS
lyase from Manihot esculenta still exhibited good Homogenous Aqueous-Ethanol System
enzyme stability and excellent enantioselectivity.
The required amount of ethanol (co-solvent) was added to
50–500 mM citrate buffer pH 4.0 in order to obtain the de-
sired solubility of acetophenone in buffer. Afterwards aceto-
phenone and hydrogen cyanide were added. Due to the de-
termination of the thermodynamic equilibrium no enzyme
was required.
Experimental Section
Chemicals
2’-Methylacetophenone, 2’-fluoroacetophenone, 2’-iodoace-
tophenone, 2’-nitroacetophenone, 3’-chloroacetophenone
and 3’-methylacetophenone were purchased from Sigma–Al-
drich Chemie GmbH, Steinheim, Germany. 2’-Hydroxyace-
tophenone, 2’-bromoacetophenone, 3’-methoxyacetophe-
none, 3’-fluoroacetophenone, 3’-bromoacetophenone, 4’-flu-
oroacetophenone and 2’,3’,4’,5’,6’-pentafluoroacetophenone
were obtained from Acros Organics, Geel, Belgium. 2’-
Chloroacetophenone, 4’-chloroacetophenone, racemic man-
delonitrile and acetophenone were products of Merck-Schu-
chardt, Hohenbrunn, Germany. 4’-Methylacetophenone, 2’-
methoxyacetophenone and sodium cyanide were purchased
from Fluka Chemie GmbH, Buchs, Switzerland.
Two-Phase System
The required amounts of acetophenone and hydrogen cya-
nide were added to the biphasic system, consisting of diiso-
propyl ether and 50–500 mM citrate buffer (pH 4.0, ratio
1:1). The enzymatic reaction was started by the addition of
the enzyme solution (20 UmLÀ1).
Organic Solvent-Free System, General Procedure
Acetophenone and hydrogen cyanide were added to 1 mL
buffer, the reaction was initiated by the addition of the
enzyme solution (450 UmLÀ1).
Comparison of the Acetophenone Derivatives
Enzyme
0.4 mmol of the acetophenone derivative and 2 mmol of hy-
drogen cyanide were added to 1 mL 50–500 mM citrate
buffer (pH 0.0 or pH 4.8). The enzymatic reaction was start-
ed by the addition of the required enzyme solution (150 or
450 UmLÀ1).
The (S)-hydroxynitrile lyase from Manihot esculenta (re-
combinant in E. coli) was obtained from Julich Chiral Solu-
tions GmbH (http://www.julich.com/), Jülich, Germany.
Enzyme Assay
The enzyme activity was determined by following the cleav-
age of rac-mandelonitrile into benzaldehyde and HCN at
258C. The formation of benzaldehyde was measured spec-
trometrically at 280 nm. The non-enzymatic cleavage reac-
tion was monitored under identical conditions and subtract-
ed. One unit of enzyme activity was defined as the amount
of enzyme that catalyzes the cleavage of 1 mmol mandeloni-
trile per minute under assay conditions.
Assay conditions: 700 mL citrate-phosphate buffer pH 5.0,
100 mL enzyme solution (dilution if required) and 200 mL
mandelonitrile stock solution (60 mmol/L in citrate-phos-
phate pH 3.5) were mixed in a cuvette with 1 cm pathlength
and the increase of absorbance at 280 nm was measured for
2 min.
Sample Taking
Homogeneous aqueous-ethanol system: A sample (100 mL)
was extracted with 100 mL n-hexane. 50 mL of the organic
phase (n-hexane) were added to a mixture of 500 mL di-
chloromethane, 50 mL trifluoroacetic anhydride and 50 mL
pyridine for the acetylation procedure.
Two-phase system: 50 mL of the organic phase were added
to a mixture of 500 mL dichloromethane, 50 mL trifluoroace-
tic anhydride and 50 mL pyridine for the acetylation proce-
dure.
Organic solvent-free system: A sample (100 mL) of the
suspension was added to 100 mL of diisopropyl ether for the
extraction. 50 mL of the organic phase were added to a mix-
ture of 500 mL dichloromethane, 50 mL trifluoroacetic anhy-
dride and 50 mL pyridine for the acetylation procedure.
HCN Formation, General Procedure
The required amount of HCN was freshly distilled in a well
ventilated hood. 4 grams of sodium cyanide were dissolved
in 10 mL de-ionized water and 10 mL of 5 mol/L sulfuric
acid were added dropwise within 2 min. Afterwards the
combined solutions were heated up to 758C and formed
HCN was trapped and stored at 58C. For the removal of
traces of water traces a spatula tip of sodium sulfate was
added. All waste solutions were collected and disposed.
An electrochemical HCN detector (Micro III G203, GfG-
Gesellschaft für Gerätebau mbH, Dortmund, Germany) was
placed in the hood for continuous monitoring.
GC Analysis for Determination of Conversion and
Enantiomeric Excess
The conversion of acetophenone (and the derivatives) to
acetophenone cyanohydrin (derivatives) as well as their en-
antiomeric excess were determined by gas chromatographic
analysis with a Chiraldex capillary gas chromatography
column (G-PN – g-Cyclodextrin, Propionyl) from astec
using a CP3800 (Varian) with a flame ionization detector
(FID). Carrier gas was helium at 2 mLminÀ1. Temperature
gradient: 808C for 0.5 min, rise with 108CminÀ1 to 1308C
and hold 1308C for 15 min . The injector and detector tem-
peratures were set to 2508C.
Reaction Conditions for the Enzymatic Synthesis of
Cyanohydrins
All reactions were carried out at 58C.
Adv. Synth. Catal. 2007, 349, 1418 – 1424
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1423

Jan von Langermann et al.
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Acknowledgements
This research was supported by the BMBF-Project -Biohy-
droform- (grant number: 0313402E). The authors also thank
the other members of the BMBF-Project for fruitful discus-
sions: Dr. Martina Pohl and Jan-Karl Guterl from the Insti-
tute for Molecular Enzymetechnology (IMET), University of
Düsseldorf at the Research Centre Jülich; Dr. Harald Wajant
from the University of Würzburg; Dr. Richard Wisdom, Dr.
Andreas Meudt and Dr. Matthias Helms from Archimica
GmbH, Frankfurt/Main and Hans-Georg Hennemann from
Julich Chiral Solutions GmbH, Jülich. The authors also
thank Julich Chiral Solutions GmbH, Jülich (http://www.ju-
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Adv. Synth. Catal. 2007, 349, 1418 – 1424