An approach to chemoenzymatic DKR of amines in Soxhlet apparatus Dedicated to Prof. Maria José Calhorda on the occasion of her 65th birthday.

Article abstract of DOI:10.1016/j.jorganchem.2013.11.005

The coexistence of thermolabile enzyme and metal catalyst for racemization of amines in chemoenzymatic dynamic kinetic resolution, requiring high temperature of operation, is enabled by carrying out the reaction in modified Soxhlet extraction system. Init

Full text of DOI:10.1016/j.jorganchem.2013.11.005

Journal of Organometallic Chemistry 760 (2014) 161e166
Contents lists available at ScienceDirect
Journal of Organometallic Chemistry
journal homepage: www.elsevier.com/locate/jorganchem
An approach to chemoenzymatic DKR of amines in Soxhlet apparatus
*
Denys Mavrynsky, Reko Leino
Laboratory of Organic Chemistry, Åbo Akademi University, FI-20500 Åbo, Finland
a r t i c l e i n f o
a b s t r a c t
Article history:
The coexistence of thermolabile enzyme and metal catalyst for racemization of amines in chemo-
enzymatic dynamic kinetic resolution, requiring high temperature of operation, is enabled by carrying
out the reaction in modified Soxhlet extraction system. Initial insights into the scope and limitations of
the developed reaction system are discussed.
Received 20 August 2013
Received in revised form
1 November 2013
Accepted 6 November 2013
Ó 2013 Elsevier B.V. All rights reserved.
Dedicated to Prof. Maria José Calhorda on
the occasion of her 65th birthday.
Keywords:
DKR
Chemoenzymatic synthesis
Chiral amines
Shvo’s catalyst
Soxhlet extraction
1. Introduction
than the corresponding transformation from amine to imine [6b].
Furthermore, poisoning of metal-based racemization catalysts may
Chiral amines are valuable building blocks for synthesis of fine
chemicals including pharmaceuticals, fragrances and pesticides [1].
Consequently, several methods have been developed for their
preparation, based on e.g., alkylation [2] and hydrogenation [3] of
prochiral imines, enamines and aldimines, as well as reductive
amination of prochiral ketones [3c,4]. Efficient enzymatic methods
for kinetic resolution (KR) of various racemic amines utilizing li-
pases and proteases exist [5], while being hampered by the
maximum theoretical yield of 50%, characteristic for all KRs. By
converting such processes into dynamic kinetic resolutions (DKR)
where the slower reacting amine enantiomer is racemized in situ
by a chemocatalyst, often based on a transition metal [6], 100%
yields of pure enantiomers can be obtained, at least in theory
(Scheme 1).
While the field of chemoenzymatic DKR of secondary alcohols
has advanced remarkably in recent years [6b,c,7], the analogous
DKR of amines has remained challenging for various reasons. In
general, racemization of amines is more difficult than the racemi-
zation of sec-alcohols, typically requiring much harsher conditions.
Overall, the oxidation of an alcohol enantiomer to ketone and the
subsequent reaction back to racemic alcohol is much more facile
take place in the presence of potentially ligating amines. Additional
problems are encountered by the typically high temperatures
required for racemization of amines being often detrimental for
enzyme activity [8].
The first chemoenzymatic DKR of an amine utilizing Pd/C as the
racemization catalyst in combination with CAL-B was described by
Reetz [9]. The influence of support material has been studied with
Pd on modified silica providing the best results [10]. The reactions
were carried out in autoclave under slight excess of H2 pressure in
order to facilitate racemization and suppress the formation of side
products. A similar approach has been described for DKR of
selenium-containing amines over Pd supported on BaSO4 [11]. Pd
nanoparticles precipitated on Al(OH)3 have likewise been
employed [12], whereas attempts to use Raney Ni and Co have been
less successful [13]. Bäckvall and coworkers investigated the ho-
mogeneous Shvo’s catalyst [14] (Scheme 1) and its para-fluoro and
para-methoxy substituted analogs for racemization of amines [15],
with the last mentioned providing the best performance. The same
para-methoxy substituted Shvo’s catalyst has also been used by
other investigators [16]. Other examples of homogeneous catalysts
include half-sandwich iridium complexes investigated for DKR [17]
and iridacycle based systems used for racemization only [18].
Photo-induced radical racemization in the DKR of amines has also
been demonstrated [5b].
* Corresponding author.
E-mail address: reko.leino@abo.fi (R. Leino).
0022-328X/$ e see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jorganchem.2013.11.005

162
D. Mavrynsky, R. Leino / Journal of Organometallic Chemistry 760 (2014) 161e166
Scheme 1. Chemoenzymatic DKR of amines in the presence of Shvo’s (R ¼ Ph) catalyst
and its close analogs.
The earlier described strategies have mainly been devised in
order to circumvent the limitations for co-existence of the ther-
molabile enzyme and the amine racemization catalyst, requiring
high temperatures for its operation. The enzyme and the catalyst
can be separated in space, as exemplified by the MEDKR system
where the DKR is carried out in an apparatus containing a “hot”
racemization vessel and a “cold” enzyme vessel connected by
tubing [19]. Circulation of the reaction mixture through the vessels
was achieved by HPLC pump and leakage of enzyme into the “hot”
racemization vessel was prevented by an ultra-filtration unit. In this
note, we describe our initial approach towards an alternative re-
action and reactor setup for chemoenzymatic DKR of amines based
on a simple, modified Soxhlet extraction system. In this setup, the
racemization catalyst operates in solution or suspension in the
reaction flask under heating at reflux at high temperature, whereas
the enzyme is placed in the extractor socket under continuous
cooling. Prospects and limitations of the approach are discussed.
Fig. 1. Schematic illustration of the Soxhlet reactor setup.
Removal of an alcohol (ethanol or i-propanol) formed during the
enzymatic acylation reaction by evaporation shifts the equilibrium
towards the desired product [16]. Mixing of the extraction chamber
content takes place mainly by liquid flow back to the racemization
flask. In order to decrease the inner volume of the extraction
chamber 2 of the Soxhlet extractor, and the duration of the
extraction cycle, the chamber can be filled with glass beads. This
also, subsequently, decreases the total volume of solvent required
for the reaction.
Since most of the relevant target amines to be resolved have
boiling points above 150 ꢀC, the refluxing is ideally performed
under reduced pressure. In principle, the extent of reduced pres-
sure can be selected in such a way that the boiling point of the
reaction mixture is sufficiently high for the metal catalyzed race-
mization of amines to proceed, i.e., close to 100 ꢀC. During the
condensation, the volatile components will cool down to approxi-
mately 50e60 ꢀC temperature range which, in contrast to the reflux
temperature in the reaction flask, is viable for the resolving CAL-B
enzyme. In the case of thermolabile enzymes, the condensation
temperature can be lowered by applying external cooling to the
extractor vessel. After completion of the DKR, the solvent and
excess of acyl donor can be removed by vacuum distillation and
reused for a subsequent reaction with the same amine.
Further, for monitoring of the temperature in the racemization
flask, an inner thermometer (6) can be applied. The temperature in
the extraction chamber 2 can be determined by placing a ther-
mometer into the glass beads. In such case, the reflux condenser 4
should have an appropriate construction allowing the insertion of
thermometer into the center of the condenser. Uniform boiling can
be further facilitated by capillar (7). Temperature of the oil bath or
heating mantel used for heating of the flask 1 does not influence the
boiling temperature but will define the rate of evaporation. In order
to ensure that the local temperature in the enzyme envelope will
not exceed the upper limit for enzyme stability and function during
the reactions, disposable thermo-sensors can be incorporated to
the enzyme package, for example sealed capillars filled with crys-
talline solid with predetermined melting point, such as tosyl-
chloride (mp 69 ꢀC) or benzophenone (mp 50 ꢀC). After completion
of the reaction, the thermosensors can easily be removed for
inspection.
2. Results and discussion
2.1. Separation of the reaction vessels
A schematic illustration of the DKR Soxhlet reactor system is
shown in Fig. 1. The reaction flask (1) is filled with solvent, race-
mization catalyst, and the corresponding amine and acyl donor. The
enzyme, packed into porous polyethylene envelopes, is placed in
the extractor chamber (2). Ideally, the solvent and the acyl donor
may be selected in such a way that their boiling points are close to
the boiling point of the racemic amine to be resolved, allowing for
circulation and condensation of all compounds via the reflux
condenser and the extraction socket. Potential examples of such
combinations are diglyme (bp 165 ꢀC and iso-propylmethox-
yacetate (bp 160 ꢀC) for amines with boiling points below 180e
190 ꢀC, triglyme (bp 210 ꢀC) and ethyl caprylate (bp 208 ꢀC) for
amines with boiling points below 230 ꢀC, and tetraglyme (bp
275 ꢀC) and ethyl laurate (bp 269 ꢀC) for amines with boiling points
below 300 ꢀC. A wide range of low-priced “glymes” and fatty acid
esters for fine tuning of the system are commercially available.
Upon heating of the reaction flask to reflux, all volatile compounds
(solvent, acyl donor and the amine) will evaporate and pass
through a side arm (3) of the extractor, cooling down to suitable
temperature while condensing in the reflux condenser (4), and will
then collect in the chamber (2) of the extractor where the enzy-
matic resolution reaction takes place. Then, the chamber is filled
and the reaction mixture returns to the racemization flask (1)
through a siphon side-arm (5).

D. Mavrynsky, R. Leino / Journal of Organometallic Chemistry 760 (2014) 161e166
163
2.2. Proof-of-concept
mixture at 120 ꢀC/0.5 mbar for 48 h with increased catalyst loading
(2 mol%), providing moderate isolated yield of the target (R)-amide
at the expense of excessive formation of side products.
For investigating the initial feasibility of the Soxhlet system,
chemoenzymatic DKR of 1-(phenyl)ethylamine, was studied in the
presence of CAL-B and the Shvo’s catalyst (Scheme 2). First, regular
KRs using 20 mmol of rac-1-(phenyl)ethylamine (bp 187 ꢀC) in the
Soxhlet reactor were performed providing optimized loadings for
the acyl donor, enzyme and the solvent. By use of 200 mg of
enzyme, 36 mmol of acyl donor and 70 ml of diglyme under reflux
at 100 ꢀC and reduced pressure (130 mbar) for 18 h, close to 50%
conversion in the KR was observed after cooling down of the re-
action mixture and analysis by chiral GC. Next, for DKR under
similar conditions, the reaction loading was adjusted to 10 mmol
and 1 mol% of Shvo’s catalyst was added. In DKR, The reduced
pressure applied and the air-sensitivity of the catalyst makes
monitoring of the reaction by sampling difficult. For this reason, the
test reactions were run for 24 h and only the final samples after
cooling were analyzed showing in most cases conversions
exceeding 90%. From these reactions, the (R)-product was isolated
either by crystallization or column chromatography in 65e70%
yield and 99% ee.
For preliminary investigations towards extension of the
approach, also KR and DKR of 1-(para-fluorophenyl)ethylamine (bp
w175 ꢀC), 1-(para-chlorophenyl)ethylamine (bp w230 ꢀC) and 1-
(naphthyl)ethylamine (bp w280 ꢀC) were rapidly screened. For
DKR of the low-boiling 1-(para-fluorophenyl)ethylamine, reaction
conditions similar to those used for the unsubstituted parent
compound 1-(phenyl)ethylamine could be utilized. According to
chiral GC analysis, complete conversion of the starting material into
reaction products was achieved. While the amount of the desired
product did not exceed 65% by GC analysis, its enantiopurity was
high (97%). For preliminary screening of the medium-boiling 1-
(para-chlorophenyl)ethylamine, triglyme and ethyl caprylate were
used. For this compound, lower pressure (15 mbar) was applied in
order to adjust the boiling point. First, regular KR was performed
indicating full conversion after 48 h [20]. Upon application of the
DKR protocol to this system, full conversion of the starting material
provided only a minor amount (20% by NMR) of the target product
[21]. In the literature, dehalogenation of arylhalides with ruthe-
nium hydride has been reported [22], although traces of this side
reaction were not observed here.
In cases of the high-boiling amines 1-(para-chlorophenyl)eth-
ylamine and 1-(naphthyl)ethylamine, enantiomeric excesses of the
products were not determined in this work. Fatty acid derivatives of
(aryl)ethylamides are not volatile, and, therefore, not applicable for
chiral GC analysis. Hydrolysis of the products to the corresponding
more volatile amides, in turn, proved problematic due to the low
reactivities of the starting amides. Nevertheless, in earlier work,
similar amines have been shown to undergo enzymatic acylation to
yield products in excellent enantiopurities [14e16].
2.3. Suppression of side product formation
DKR of amines is often complicated by formation of side prod-
ucts [6a,10b,15b]. Racemization of secondary amines commonly
proceeds via an imine intermediate, which upon reaction with a
second amine molecule irreversibly results in the formation of di-
mers (Scheme 2). In the present case, reduced pressure facilitates
the oxidative dehydrogenation of amine to imine and suppresses
the reverse reaction. The subsequent increase in the concentration
of the imine intermediate then facilitates side product formation.
Comparison of the DKRs of 1-(phenyl)ethylamine at 130 mbar and
1-(naphthyl)ethylamine below 1 mbar demonstrates the strong
dependence of side product formation on the applied pressure in
the Soxhlet reaction vessel. In the case of 1-(phenyl)ethylamine,
10% of side product was formed as determined by GC analysis. In
the case of 1-(naphthyl)ethylamine, the amount of side product
formed increased significantly and reached the level of 30e40%, as
determined by NMR. In principle, different hydrogen donors can be
utilized for reduction of the imine intermediate generated and,
consequently, for suppression of the side product formation. For
example, 2,4-dimethyl-3-pentanol as a hydrogen donor has been
reported to suppress side product formation in one-pot DKR of
amines [16]. This compound easily forms ketones while releasing
hydrogen upon Ru-catalyzed transfer hydrogenation. The branched
structure of 2,4-dimethyl-3-pentanol, in turn, prevents its enzy-
matic acylation. Due to its relatively low boiling point (140 ꢀC),
however, this alcohol is not compatible with the Soxhlet reactor
setup when higher boiling components are used. We thus sought
out to screen other reducing agents for suppression of dimerization
under the presently described Soxhlet conditions. The model
racemization reaction for reducing agent screening under reduced
pressure is shown in Scheme 3 with the results collected in Table 1.
As evident from entry 1, racemization under reduced pressure in
the absence of reducing agents results in significant dimerization.
When the racemization reaction was performed at atmospheric
pressure (entry 2), only minor amounts of the side product were
detected. This observation is consistent with Le Chatelier’s principle
with inverse dependence of side product formation on the pressure
applied. The use of hydroquinone, a widely utilized reducing agent
(entry 3), did not suppress side product formation to any desired
extent. A possible explanation may be related to the relatively high
acidity of the OH group in hydroquinone (pKa1 ¼ 10), resulting in
decomposition of the ruthenium hydride catalyst intermediate and
the subsequent liberation of hydrogen gas. Promising results were
Finally, both the KR and DKR of the high-boiling 1-(naphthyl)
ethylamine using tetraglyme as the solvent and ethyl laurate as the
acyl donor were screened. In this case, the KR proceeded fairly
slowly (10% conversion in 9 h) due to lower activity of the bulk acyl
donor. DKR, in turn, was performed by refluxing of the reaction
Scheme 2. DKR of 1-(phenyl)ethylamine and 1-(naphthyl)ethylamine in the Soxhlet
reactor system.
Scheme 3. Model reaction studied for suppression of side product formation.

164
D. Mavrynsky, R. Leino / Journal of Organometallic Chemistry 760 (2014) 161e166
Table 1
3. Experimental section
Screening of reducing agents for suppression of side product formation.
3.1. Reactor setup
Entry
Reducing agent
Formation of
side product, %
General experimental conditions and results on the DKR re-
actions are collected in Table 2. First, 110 mg (1 mol%) of the Shvo’s
catalyst, 70 ml of the corresponding glyme, racemic 1-(aryl)ethyl-
amine and the corresponding acyl donor were placed in a three-
necked 100 ml round-bottom flask, equipped with a thermom-
eter, a magnetic stirrer bar, a capillar and a Soxhlet head (50 ml)
with a reflux condenser. The enzyme (Novozyme 435) was packed
in 4 porous polyethylene bags, 50 mg in each. In the case of the
slower reacting 1-(naphthyl)ethylamine, 6 bags containing 300 mg
of enzyme in total were utilized. The bags containing the enzyme
were placed into an extraction chamber of the Soxhlet extractor
together with 5 mm glass beads. In this way the “dead volume” of
the Soxhlet extractor was reduced to approximately 20 ml.
Disposable thermo-sensors were placed between the enzyme bags.
If desired, with proper construction of the reflux condenser, a
thermometer could also be inserted into the glass beads. The outlet
of the reflux condenser was connected to an inlet of a membrane
pump equipped with vacuum control unit. The argon inlet was
connected to the capillar and to the gas inlet of the pump. We
recommend the incorporation of a 1e2 l buffer flask between the
apparatus and the vacuum pump for controlling the vacuum
oscillation. The use of Teflon thermostable grease for the hot joints
is likewise recommended. The side arm of the extractor should be
thermally insulated. Loops of rubber tubing with cooling water
circulation can be applied around the extraction chamber for
additional cooling of the enzyme.
1
2
3
4
5
6
7
8
e
9
1
15
1
3
3
ea
Hydroquinoneb
2-Octanolb
2,2,4-Trimethyl-1,3-pentanediolb
2,2,4-Trimethyl-1,3-pentanediolc
3-hydroxy-2,2,4-trimethylpentyl dodecanoateb
Bubbling of H2
3
1
a
P ¼ 1 bar.
b
c
0.5 equiv.
1 equiv.
obtained by use of 2-octanol (entry 4). This compound has a boiling
point of 180 ꢀC and can only be utilized with low boiling amines in
the Soxhlet system. In the case of higher boiling substrates, octanol
will be carried together with the other vapors to the enzyme
chamber resulting in enzymatic acylation and a loss of reducing
properties. The economically viable and commercially readily
available 2,2,4-trimethyl-1,3-pentanediol, likewise, demonstrated
acceptable efficiency (entry 5). Increase in the loading of this
reducing agent did not improve the result (entry 6). The high
boiling point of this compound (232 ꢀC) enables its use and
compatibility with a broad range of amines. A model reaction on
enzymatic acylation of this compound showed acylation mainly at
the primary alcohol group with only minor amounts of secondary
alcohol group acylation observed. The isolated acyl product from
the enzymatic test reaction was evaluated for side product sup-
pression (entry 7), showing similar behavior and efficiency
compared to the non-acylated parent diol. Since the oxidation to
ketone only involves the secondary alcohol moiety, the primary
alcohol function does not influence the results of side product
suppression. The introduction of gaseous hydrogen into the reac-
tion mixture via capillar (entry 8) also efficiently suppresses side
product formation being, however, complicated due to the high
diffusion rate through the rubber tubing.
In accordance with earlier reports [10b,c], heterogeneous Pd/C
demonstrates excellent racemization activity towards 1-(phenyl)
ethylamine under atmospheric pressure without any detectable
formation of side products. Unfortunately, when Pd/C was here
employed instead of the Shvo’s catalyst in the Soxhlet reactor setup,
dimerization became the dominant reaction under reduced pres-
sure. The interaction which binds hydrogen to palladium metal is
weak compared to the covalent hydride bond in the Shvo’s catalyst.
Consequently, loss of hydrogen under reduced pressure followed by
dimerization is likely to become a major reaction pathway in the
case of heterogeneous catalysts.
3.2. Operation [pressure and temperatures given for 1-(phenyl)
ethylamine]
The reaction was performed at 130 mbar pressure and tem-
perature of 105 ꢀC. The reaction flask was heated on an oil bath.
Temperature of the oil bath influences the evaporation rate but not
the boiling temperature. Maintaining the bath temperature at
140 ꢀC allowed for 3e5 min duration of the extraction cycle. After
24 h, the heating was stopped and the apparatus was cooled down
under argon atmosphere. Completion of the reaction was verified
by chiral GC or NMR. The extraction chamber was washed thor-
oughly with toluene. The combined liquid phases were concen-
trated under reduced pressure and the residue was purified by
chromatography (hexane-ethylacetate) in the case of 1-(phenyl)
ethylamine or recrystallized from hexane in the case of 1-(naphtyl)
ethylamine to yield 1.25 g (65%, 99% ee) of (R)-2-methoxy-N-(1-
phenylethyl)acetamide, or 0.65 g (37%) of (R)-N-(1-(naphthalen-
2-yl)ethyl)dodecanamide. The amount of the side product formed
was quantified by chiral GC or 1H NMR. Physical and spectral
properties of (R)-2-methoxy-N-(1-phenylethyl)acetamide [mp 58e
Finally, it could be assumed that the imine intermediate formed
during the racemization reaction will be further stabilized by
conjugation of the C]NH double bond with the aromatic ring. To
investigate this, also 4-phenyl-2-aminobutane, an amine without
aromatic ring in the alpha-position to the nitrogen atom, was
tested. To our disappointment, significant side product formation
was observed in this case as well, possibly facilitated by the
decrease in steric hindrance (Scheme 4).
61 ꢀC, ½a 2D2
¼ þ78.4 (c ¼ 5%, CHCl3)] are in accordance with those
ꢁ
previously reported for this compound [23]. For (R)-N-[1-(naph-
thalen-2-yl)ethyl]dodecanamide, only the negative sign of rotation
without numerical value has been reported for the opposite
enantiomer in the literature [24]. Analytical data obtained here for
this compound: mp 77e80 ꢀC, ½a D22
ꢁ
¼ þ89.2 (c ¼ 5%, CHCl3); 1H
NMR (600.13 MHz, CDCl3): dH 7.81e7.78 (3H, m, ArH), 7.73 (1H, s,
ArH), 7.48e7.41 (3H, m, ArH), 5.86 (1H, br d, JHH ¼ 8.0 Hz, NH),
5.32e5.27 (1H, m, CHeNH), 2.17 (2H, t, JHH ¼ 7.0 Hz, COeCH2),1.65e
1.59 (2H, m, COeCH2eCH2), 1.56 (3H, d, JHH ¼ 7 Hz, CH(NH)eCH3),
1.32e1.20 (16H, m, aliphatic chain), 0.88 (3H, t, JHH ¼ 7.0 Hz,
(CH2)10eCH3). 13C{1H} NMR (150.9 MHz, CDCl3): dC 172.28, 140.72,
133.34, 132.71, 128.46, 127.88, 127.61, 126.22, 125.87, 124.82, 125.50,
Scheme 4. Dimerization of 4-phenyl-2-aminobutane.

D. Mavrynsky, R. Leino / Journal of Organometallic Chemistry 760 (2014) 161e166
165
Table 2
Reaction conditions for DKR of the 1-(aryl)ethylamines.a
Substrate/loading
Acyl donor/loading
Solvent
Temperatureb/pressure
105 ꢀC/130 mbar
Results
MeOCH2COO-i-Pr 18 mmol
Diglyme
Full conversion; Isol. yield 65%; ee 99%
Full conversion; GC yield 65%; ee 97%
Full conversion; NMR yield 20%
10 mmol [c]
10 mmol
10 mmol
MeOCH2COO-i-Pr 15 mmol
Diglyme
Triglyme
105 ꢀC/130 mbar
110 ꢀC/15 mbar
CH3(CH2)6COOEt 15 mmol
CH3(CH2)10COOEt 10 mmol
Tetraglyme
120 ꢀC/0.5 mbar
Slow reaction; Isol. yield 37%
5 mmol [d]
aUnless otherwise stated, 1 mol% of the Shvo’s catalyst, 4 ꢂ 50 mg of Novozym 435, 48 h reaction time.
bTemperature in the racemization vessel.
c24 h.
d2 mol% of catalyst and 6 ꢂ 50 mg of Novozyme 435.
48.57, 36.94, 31.92, 29.62, 29.52, 29.38, 29.34, 29.31, 25.81, 22.70,
21.63, 14.14.
however, further improvement and optimization. Other modifica-
tions of the system can, likewise, be envisioned for future investi-
gation. For example, when using the Shvo’s catalyst for
racemization, temperatures above 80 ꢀC are required for activation
of the dimeric catalyst precursor to form the catalytically active
monomer(s) [25]. In principle, a “catalyst saving mode” could be
applied in the beginning of the reaction by adjusting the pressure
inside the system to allow for refluxing below the activation tem-
perature of the racemization catalyst. Under such conditions only
conventional kinetic resolution should take place. After this initial
period, the pressure can be increased, simultaneously increasing
the boiling point and inducing activation of the racemization
catalyst and starting the DKR reaction.
3.3. Side product suppression experiments
Shvo’s catalyst (22 mg, 2 mol%), amine (2 mmol), diglyme
(40 ml) and the appropriate reducing agent (Table 1) were placed
into a 100 ml three-necked round-bottom flask equipped with a
thermometer, a capillar and a reflux condenser. Argon input was
connected to a capillary and a membrane pump intake was con-
nected to the exit of a reflux condenser. In case of using hydrogen as
the reducing agent, a heavy walled rubber balloon with hydrogen
was connected to a capillar. The reaction mixture was refluxed at
105 ꢀC/130 mbar for 48 h, cooled down under an argon atmosphere
and analyzed by chiral GC and 1H NMR.
Acknowledgments
Financial support from the Finnish Funding Agency for Tech-
nology and Innovation (TEKES) Technology Programme SYMBIO/
Project #40168/07 Developing New Chemoenzymatic Methods and
Biocatalysts is gratefully acknowledged. We thank professor Liisa
Kanerva and Mari Päiviö for fruitful discussions and experimental
assistance.
4. Summary and conclusions
To summarize, a flexible system for chemoenzymatic DKR of
amines based on space separated racemization and enzyme vessels
in a Soxhlet-type reactor setup has been developed and its feasi-
bility demonstrated in a proof-of-concept study. Mass transfer of
the reaction mixture is performed by repeating cycles of evapora-
tion, condensation and siphon flow. This approach allows the usage
of membrane pump instead of HPLC pump for liquid circulation.
Different solvents and acyl donors with a broad range of boiling
points are widely available from commercial sources and should
allow the further fine tuning of the system. Currently, the main
limiting factor with the developed system is the vacuum enhanced
loss of hydrogen which facilitates side product formation, compli-
cates the DKR process and decreases the yield of the desired
product to the level of conventional kinetic resolution. Preliminary
results demonstrate that such side product formation can be sup-
pressed by additional hydrogen donors in the system, requiring,
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.jorganchem.2013.11.005.
References
[1] V. Farina, J.T. Reeves, C.H. Senanayake, J.J. Song, Chem. Rev. 106 (2006) 2734e
2793.
[2] S.E. Denmark, O.J.C. Nicaise, in: E.N. Jacobsen, A. Pfaltz, H. Yamamoto (Eds.),
Alkylation of Imino Groups, vol. 1, Springer, Berlin, 1999, pp. 923e961.
[3] (a) T. Ohkuma, M. Kitamura, R. Noyori, in: O. Iwao (Ed.), Catalytic Asymmetric
Synthesis, second ed., Wiley-VCH, New York, 2000, pp. 1e110;

166
D. Mavrynsky, R. Leino / Journal of Organometallic Chemistry 760 (2014) 161e166
(b) C. Claver, E. Fernández, in: P.G. Andersson, I.J. Munslow (Eds.), Modern (c) L.K. Thalén, D. Zhao, J.-B. Sortais, J. Paetzold, C. Hoben, J.-E. Bäckvall, Chem.
Reduction Methods, Wiley-VCH, Weinheim, 2008, pp. 237e269;
(c) H. Adolfsson, in: P.G. Andersson, I.J. Munslow (Eds.), Modern Reduction
Methods, Wiley-VCH, Weinheim, 2008, pp. 341e361.
Eur. J. 15 (2009) 3403e3410.
[16] M.A.J. Veld, K. Hult, A.R.A. Palmans, E.W. Meijer, Eur. J. Org. Chem. (2007)
5416e5421.
[4] (a) T.C. Nugent, in: T.C. Nugent (Ed.), Chiral Amine Synthesis, Wiley-VCH,
Weinheim, 2010, pp. 225e245.
[5] (a) M. Höhne, U.T. Bornscheuer, ChemCatChem 1 (2009) 42e51;
(b) L. El Blidi, M. Nechab, N. Vanthuyne, S. Gastaldi, M.P. Bertrand, G. Gil, J. Org.
Chem. 74 (2009) 2901e2903.
[17] (a) M. Stirling, J. Blacker, M.I. Page, Tetrahedron Lett. 48 (2007) 1247e1250;
(b) A.J. Blacker, M.J. Stirling, M.I. Page, Org. Process Res. Dev. 11 (2007) 642e648.
[18] T. Jerphagnon, A.J.A. Gayet, F. Berthiol, V. Ritleng, N. Mrsic, A. Meetsma,
ꢀ ꢁ
M. Pfeffer, A.J. Minnaard, B.L. Feringa, J.G. de Vries, Chem. Eur. J. 15 (2009)
12780e12790.
[6] For reviews, see: (a) Y. Ahn, S.-B. Ko, M.-J. Kim, J. Park, Coord. Chem. Rev. 252
(2008) 647e658;
[19] (a) C. Roengpithya, D.A. Patterson, P.C. Taylor, A.G. Livingston, Desalination
199 (2006) 195e197;
(b) J.H. Lee, K. Han, M.-J. Kim, J. Park, Eur. J. Org. Chem. (2010) 999e1015;
(c) Y. Kim, J. Park, M.-J. Kim, ChemCatChem 3 (2011) 271e277;
(d) P. Hoyos, V. Pace, A.R. Alcántara, Adv. Synth. Catal. 354 (2012) 2585e2611.
[7] For reviews, see: (a) B. Martín-Matute, J.-E. Bäckvall, Curr. Opin. Chem. Biol. 11
(2007) 226e232;
(b) C. Roengpithya, D.A. Patterson, A.G. Livingston, P.C. Taylor, J.L. Irwin,
M.R. Parrett, Chem. Commun. (2007) 3462e3463.
[20] NMR analysis of the reaction mixture is ineffective here for monitoring of
the reaction progress due to overlapping of the solvent peak with char-
acteristic signals of reagents. Removal of solvent prior to preparation of the
NMR sample may influence the ratio of the starting material and the
product in the prepared NMR sample. Also, GC analysis is not challenging
due to poor volatility of the product. The starting material, however, is
volatile and can be traced by GC. Comparison of intensities of the peaks
from the starting material enantiomers allows estimation of the conversion
value. After 48 h, only one enantiomer of the starting material was
observed indicating complete KR.
[21] Conventional chiral GC analysis cannot be utilized for monitoring of the re-
action progress due to racemization of the starting material (compare with KR
[20]). Consequently, comparison of the starting material peak intensity with
an internal standard (N-(1-phenylethyl)propionamide) was performed.
[22] (a) M.E. Cucullu, S.P. Nolan, T.R. Belderrain, R.H. Grubbs, Organometallics 18
(1999) 1299e1304;
(b) N.J. Turner, Curr. Opin. Chem. Biol. 14 (2010) 115e121;
(c) B.L. Conley, M.K. Pennington-Boggio, E. Boz, T.J. Williams, Chem. Rev. 110
(2010) 2294e2312;
(d) M.C. Warner, J.-E. Bäckvall, Acc. Chem. Res. 46 (2013) 2545e2555.
[8] P. Halling, in: K. Drauz, H. Waldmann (Eds.), Enzyme Catalysis in Organic
Synthesis, vol. 1, Wiley-VCH, Weinheim, 2002, pp. 259e285.
[9] M.T. Reetz, K. Schimossek, Chimia 50 (1996) 668e669.
[10] (a) A. Parvulescu, D. De Vos, P. Jacobs, Chem. Commun. (2005) 5307e5309;
(b) A.N. Parvulescu, P.A. Jacobs, D.E. De Vos, Chem. Eur. J. 13 (2007) 2034e
2043;
(c) A.N. Parvulescu, P.A. Jacobs, D.E. De Vos, Appl. Catal. A: Gen. 368 (2009) 9e16.
[11] L.H. Andrade, A.V. Silva, E.C. Pedrozo, Tetrahedron Lett. 50 (2009) 4331e4334.
[12] M.-J. Kim, W.-H. Kim, K. Han, Y.K. Choi, J. Park, Org. Lett. 9 (2007) 1157e1159.
[13] A.N. Parvulescu, P.A. Jacobs, D.E. De Vos, Adv. Synth. Catal. 350 (2008) 113e
121.
(b) G. Csjernyik, K. Bogár, J.-E. Bäckvall, Tetrahedron Lett. 45 (2004) 6799e
6802.
[14] O. Pámies, A.H. Éll, J.S.M. Samec, N. Hermanns, J.-E. Bäckvall, Tetrahedron Lett.
43 (2002) 4699e4702.
[15] (a) J. Paetzold, J.-E. Bäckvall, J. Am. Chem. Soc. 127 (2005) 17620e17621;
(b) C.E. Hoben, L. Kanupp, J.-E. Bäckvall, Tetrahedron Lett. 49 (2008) 977e979;
[23] K. Han, J. Park, M.-J. Kim, J. Org. Chem. 73 (2008) 4302e4304.
[24] W.H. Pirkle, C.J. Welch, M.H. Hyun, J. Org. Chem. 48 (1983) 5022e5026.
[25] M.K. Thorson, K.L. Klinkel, J. Wang, T.J. Williams, Eur. J. Inorg. Chem. (2009)
295e302.