Asymmetric synthesis of an (R)-cyanohydrin using enzymes entrapped in lens-shaped gels.

Article abstract of DOI:10.1021/ol015920g

[structure: see text] A novel synthesis of (R)-cyanohydrins is described which is based on the use of cross-linked and subsequently poly(vinyl alcohol)-entrapped (R)-oxynitrilases. These immobilized lens-shaped biocatalysts have a well-defined macroscopic

Full text of DOI:10.1021/ol015920g

ORGANIC
LETTERS
2
001
Vol. 3, No. 13
969-1972
Asymmetric Synthesis of an
R)-Cyanohydrin Using Enzymes
Entrapped in Lens-Shaped Gels
1
(
,†
‡
§
,‡,|
Harald Gr o1 ger,* Emine Capan, Anita Barthuber, and Klaus-Dieter Vorlop*
Degussa AG, Project House Biotechnology, Rodenbacher Chaussee 4, 63457
Hanau-Wolfgang, Germany, Bundesforschungsanstalt f u¨ r Landwirtschaft (FAL),
Institut f u¨ r Technologie und Biosystemtechnik, Bundesallee 50, 38116 Braunschweig,
Germany, and Degussa AG, R&D Department FC-FEA4, Dr.-A.-Frank-Strasse 32,
83308 Trostberg, Germany
harald.groeger@degussa.com
Received April 1, 2001
ABSTRACT
A novel synthesis of (R)-cyanohydrins is described which is based on the use of cross-linked and subsequently poly(vinyl alcohol)-entrapped
R)-oxynitrilases. These immobilized lens-shaped biocatalysts have a well-defined macroscopic size in the mm range, show no catalyst leaching,
(
and can be recycled efficiently. Furthermore, this immobilization method is cheap and the entrapped (R)-oxynitrilases gave similar good
results compared with those of free enzymes. The (R)-cyanohydrin was obtained in good yields and with high enantioselectivities of up to
>99% ee.
Among industrial production of chiral building blocks,
cyanohydrins play an important role, since these molecules
find a wide range of pharmaceutical and agrochemical
et al., and other groups3h using metal-based complexes or
organic catalysts, respectively. The formation of (R)-cyano-
hydrins can be also realized by means of a biocatalytic
hydrocyanation using (R)-oxynitrilases. This enzymatic ap-
proach has been developed by Effenberger, Kula, and other
1
applications. Highly efficient asymmetric methods to these
2
3a-e
3f,g
molecules have been realized by Shibasaki
et al., Inoue
2
,4a-d
groups.
Immobilization was used to try to prevent the
†
Degussa AG, Project House Biotechnology.
Institut f u¨ r Technologie und Biosystemtechnik.
Degussa AG, R&D Department FC-FEA4.
E-mail: klaus.vorlop@fal.de.
disadvantages associated with “free” (R)-oxynitrilases (e.g.,
‡
2,4a-d,5
rapid deactivation).
However, many known immobi-
§
|
lization methods with oxynitrilases have practical problems
on a large scale. Among the main problems are the
(1) Important industrial applications of chiral cyanohydrins are the
manufacture of pyrethroids (insecticides), e.g., deltamethrin, as well as the
production of R-hydroxy carboxylic acids as intermediates for pharmaceu-
ticals and resolving agents. For example, (R)-mandelic acid (a derivative
of (R)-mandelonitrile) represents a fine chemical which is produced on multi-
hundred ton scale.
2405-2409. (d) Hamashima, Y.; Kanai, M.; Shibasaki, M. J. Am. Chem.
Soc. 2000, 122, 7412-7413. (e) Hamashima, Y.; Kanai, M.; Shibasaki, M.
Tetrahedron Lett. 2001, 42, 691-694. (f) Tanaka, K.; Mori, A.; Inoue, S.
J. Org. Chem. 1990, 55, 181-185. (g) Mori, A.; Nitta, H.; Kudo, M.; Inoue,
S.; Tetrahedron Lett. 1991, 32, 4333-4336. (h) For reviews, see ref 2.
(4) (a) Effenberger, F.; Ziegler, T.; F o¨ rster, S. Angew. Chem. 1987, 99,
491-492; Angew. Chem., Int. Ed. Engl. 1987, 26, 458-460. (b) Wehtje,
E.; Adlercreutz, P.; Mattiasson, B. Biotechnol. Bioeng. 1990, 36, 39-46.
(c) Wehtje, E.; Adlercreutz, P.; Mattiasson, B. Biotechnol. Bioeng. 1993,
41, 171-178. (d) Gill, I.; Ballesteros, A. J. Am. Chem. Soc. 1999, 120,
8587-8598. (e) At pH 5.5, a low ee of 77% was obtained.
(2) For excellent reviews about the asymmetric synthesis of cyanohydrins,
see: (a) Effenberger, F. Angew. Chem. 1994, 106, 1609-1619; Angew.
Chem., Int. Ed. Engl. 1994, 33, 1555-1564. (b) Gregory, R. J. H. Chem.
ReV. 1999, 99, 3649-3682.
(3) (a) Hamashima, Y.; Sawada, D.; Kanai, M.; Shibasaki, M. J. Am.
Chem. Soc. 1999, 121, 2641-2642. (b) Sawada, D.; Shibasaki, M. Angew.
Chem. 2000, 112, 215-218; Angew. Chem., Int. Ed. 2000, 39, 209-213.
(c) Kanai, M.; Hamashima, Y.; Shibasaki, M. Tetrahedron Lett. 2000, 41,
1
0.1021/ol015920g CCC: $20.00 © 2001 American Chemical Society
Published on Web 05/30/2001

nonsatisfied long-term stability, catalyst leaching, abrasion
of the immobilizate, and a wide particle distribution (instead
of a well-defined macroscopic size) in the case of powdered
first the cross-linking process was carried out only with
glutaraldehyde, but the resulting cross-linked enzymes
showed a drastically decreased activity (probably due to
deactivation of the enzyme). However, a combination of
glutaraldehyde and chitosan led to an improved cross-linked
enzyme which showed 89% of its original activity.
In a subsequent step, this cross-linked enzyme was
entrapped in a hydrogel matrix which is (mainly) based on
poly(vinyl alcohol). In this step a lens-shaped catalyst with
a well-defined particle diameter of 3 or 5 mm and a thickness
of 0.3-0.4 mm is produced. These lens-shaped hydrogels
are highly elastic and flexible toward mechanical treatment.10
The concept of this two-step immobilization method is shown
in Scheme 1, and a corresponding synthetic protocol is given
in the Supporting Information.
5
,6
immobilizates.
On the other hand, Vorlop et al. has recently developed a
highly efficient immobilization method for cells by entrap-
ping them in a hydrogel matrix mainly based on poly(vinyl
7
alcohol). This method represents a cheap, efficient, and
industrially feasible immobilization technique, which not only
shows negligible catalyst leaching but also gives macroscopi-
cally well-defined and highly flexible lens-shaped particles
with a high activity (type, LentiKats; diameter, 3-5 mm;
thickness, 300-400 µm).7 Several successful applications
of this concept have been previously reported by Vorlop and
co-workers, e.g., for the synthesis of itaconic acid and 1,3-
propanediol.7 It is noteworthy from an industrial point of
view that these lens-shaped hydrogels can easily be separated
a
c,d
(
due to a diameter of 3-5 mm), are suitable for stirred tank
Scheme 1. Two-Step Procedure for Entrapping
reactors, and show no abrasion as well as minimized
(R)-Oxynitrilases
diffusion limitations (due to low thickness of <0.5 mm). This
8
technology has been already commercialized by geniaLab.
We envisioned that this concept could be extended to a
suitable immobilization method for oxynitrilases (and more-
over for enzymes in general).
In the following we report the first application of LentiKat-
entrapped enzymes (here: oxynitrilases) in organic catalysis,
namely in the synthesis of (R)-mandelonitrile. For our studies
we used a nonpurified (R)-oxynitrilase for economical
9
reasons. To prepare an efficient entrapped oxynitrilase, a
two-step procedure has been chosen. In a first step, a cross-
linking process was carried out which led to an increased
molecular weight of the (cross-linked) enzymes. This cross-
linking procedure is necessary since enzymes with a mo-
lecular weight of 50000 maximum (as in the case of
oxynitrilases) would not be restrained in the hydrogels. At
(5) One of the rare exceptions of a very practical immobilization method
represents the production of (R)-mandelonitrile with the continuous
membrane reactor technique which is mainly applied for continuous
processes, see: (a) Vasic-Racki, D.; Jonas, M.; Wandrey, C.; Hummel, W.;
Kula, M.-R. Appl. Microbiol. Biotechnol. 1989, 31, 215-222. (b) Bom-
marius, A. S.; Drauz, K.; Groeger, U.; Wandrey, C. In Chirality in Industry;
Collins, A. N., Sheldrake, G. N., Crosby, J., Eds.; John Wiley & Sons Ltd:
New York, 1992; Chapter 20, pp 371-397.
(6) (a) Despite the high potential of immobilized biocatalysts, in a United
Nations publication from 1989, only eight industrial processes have been
reported which are based on immobilized enzymes or microbial cells, see:
Klyosov Report, A. A. UNIDO/IPTC. 93, V-89-61316. Order No. PB90-
Regarding the synthetic potential of the “catalytic cap-
sules”, investigations have been carried out using “free” and
entrapped oxynitrilases. In the presence of the free, non-
purified oxynitrilase with an activity of 15 U/mmol a
satisfactory result (95% ee, 85% yield) was obtained at pH
4.5 under biphasic conditions (Table 1, entry 1).4e
2
10360, 111 pp. (b) The manufacture of 7-aminocephalosporanic acid
represents (probably) the most important industrial process which is based
on the use of an immobilized enzyme.
(
7) (a) Jekel, M.; Buhr, A.; Willke, T.; Vorlop, K.-D. Chem. Eng.
Technol. 1998, 21, 275-278. (b) Wittlich, P.; Schlieker, M.; Jahnz, U.;
Willke, T.; Vorlop, K.-D. Proc. 9th Eur. Congr. Biotechnol. 1999, No.
P2762, ISBN 805215-1-5. (c) Welter, K.; Willke, T.; Vorlop, K.-D. SchrR
Nachwachsende Rohstoffe 1999, 14, 520-521. (d) Wittlich, P.; Schlieker,
M.; Lutz, J.; Reimann, C.; Willke, T.; Vorlop, K.-D. SchrR Nachwachsende
Rohstoffe 1999, 14, 524-532. (e) Durieux, A.; Nicolay, X.; Simon, J.-P.
Biotechnol. Lett. 2000, 22, 1679-1684.
11
When applying the entrapped catalyst (8.16 U/g) in the
model reaction, we were pleased to find thatsdespite
modifying the enzyme by cross-linking and entrappingsa
comparable result was obtained regarding enantioselectivity
as well as yield. The desired (R)-mandelonitrile was obtained
with 94% ee and 93% yield (Table 1, entry 2) which
(8) For information about commercial applications of this type of
immobilization technology as well as commercial products from geniaLab,
see: http://www.geniaLab.de.
(9) The experiments described herein have been carried out using a
nonpurified oxynitrilase purchased from ASA Spezialenzyme GmbH,
Braunschweig (specific activity, 13.6 U/mg proteine; amount of proteine,
(10) The physical properties of such type of lens-shaped hydrogels in
general have been described earlier; for details, see ref 7.
7
.6 mg/mL). Although purity and activity are somewhat lower compared
with those of other commercially available, highly purified oxynitrilases,
this enzyme was chosen on basis of cost.
(11) The reaction conditions for the preparation of the lens-shaped
hydrogels can be varied widely, e.g., resulting in a different catalyst loading.
1970
Org. Lett., Vol. 3, No. 13, 2001

Table 1. Entrapped Biocatalysts in Asymmetric
Scheme 2. Recycling of Entrapped (R)-Oxynitrilases
Hydrocyanation
type of
oxynitrilasea
U per
mmolb
organic
solventc
yield
[%]d
ee
[%]e
entry
1
2
3
4f
5f
6g
free
15
15
150
75
75
100
MTBE/hex.
MTBE/hex.
MTBE/hex.
EtOAc
MTBE
i-Pr2O
85
93
74
62
70
84
95
94
91
93
92
99
entrapped (8)
entrapped (40)
entrapped (40)
entrapped (40)
entrapped (40)
a
The catalyst loading is given in U per g of capsules in parentheses; a
general procedure is as follows. To a solution of the PVAL-entrapped (R)-
oxynitrilases (lens-shaped capsules; g of lenses per mmol of substrate:
entries 2 and 4-5, 1.84 g; entry 3, 3.67 g; entry 6, 2.47 g) in 4 mL of a
citric buffer, subsequently 4 mL of the organic solvent, 1 mmol of
benzaldehyde, and 3.5 mmol of HCN (as an 20% aqueous solution) are
added. After stirring of the reaction mixture for 2 h, the organic layer is
separated, and the aqueous layer is washed with 2 × 20 mL MTBE
In addition, we further investigated a potential catalyst
leaching. These experiments were carried out with respect
to the dehydrocyanation reaction of mandelonitrile. The
results underline the recycling abilities of the entrapped
oxynitrilase catalyst (Scheme 3): No catalyst leaching was
observed during a time of 143 h. This conclusion is supported
by the (nearly) identical course of the enzyme activities
determined from the reactions with “fresh” hydrogels and
hydrogels after stirring for 143 h, respectively.
12
(subsequently, the aqueous layer is treated with NaOCl solution in order to
decompose HCN which has been used in excess amount). The organic
phases are dried over magnesium sulfate, and after filtration the volatile
materials are removed in vacuo. The product (R)-mandelonitrile is obtained
with a purity of >90-95% (in some cases of up to 99%). CAUTION:
HCN is very toxic and must be handled with high caution. Safety instructions
are given in the material safety data sheet (MSDS) of HCN. For further
safety information, see the international chemical safety card of HCN
(
ICSC0492) which is available from the Internet: http://www.cdc.gov/niosh/
b
ipcsneng/neng0492.html. The catalytic amount is defined as U per mmol
of substrate (benzaldehyde). MTBE/hex. ) mixture of MTBE and hexane
in a ratio of 4:6. The yield was determined from proton NMR spectroscopic
c
d
Scheme 3. Investigation about Leaching of the Entrapped
Enzyme via Photometrical Detection of the Concentration of
Benzaldehyde (Extinction E) during the Reaction Course (for
data of the crude product (after the evaporation step) which contains (R)-
mandelonitrile as the main product with a purity of >90-95% (in some
cases of up to 99%). e The enantiomeric excess (ee) was determined by
HPLC or by proton NMR spectroscopic data after derivatization with a
1
2
Details of This Experiment, see Supporting Information)
f
chiral carboxylic acid chloride. This experiment has been carried out at
g
pH 4.25. The reaction time was 3 h.
corresponds to a volumetric productivity of 1.4 mol/(d‚L).
It is noteworthy that all reaction parameters remained
unchanged when compared with the standard reaction using
the free enzyme. Using entrapped oxynitrilases with a
somewhat higher catalyst loading of 40 U per g, lens-shaped
capsules led to 74% yield and slightly decreased 91% ee
(Table 1, entry 3).
Next we were interested in the recycling abilities of the
entrapped enzymes. These experiments were carried out on
the basis of the reaction parameters described in Table 1,
entry 3. In total, the lens-shaped hydrogels were recycled
2
0 times. The results, shown in Scheme 2, indicate the
In addition, no activity of the supernatant (of the im-
mobilized enzyme after stirring for 143 h in aqueous solution)
was found, indicating that there is no diffusion of the enzyme
into the solution. Thus, neither a catalyst leaching nor a
decrease of the catalyst activity (due to deactivation) has been
observed.
potential to recycle the catalyst efficiently. Even after reusing
the lens-shaped catalysts 20 times, no decrease of enantio-
selectivity was observed. In contrast, the ee slightly increased
from 91% ee to 95% ee. This might be due to an increased
stabilization of the enzyme within the hydrogel matrix. Also,
the yield remains in the same range of ca. 80%. Furthermore,
the oxynitrilase-containing hydrogels do not change their
elasticity, size, and flexibility. The long-term-stability of the
enzyme, however, might mainly result from cross-linking
the enzyme.
(12) In general, the catalytic activity of oxynitrilases is determined via
this cleavage reaction of mandelonitrile (dehydrocyanation reaction). The
reaction course has been determined according to the increase of the
concentration of benzaldehyde (extinction E) which has been detected with
a photometer (at 250 nm). For further details, see Supporting Information.
Org. Lett., Vol. 3, No. 13, 2001
1971

With an efficient entrapped catalyst in hand, we focused
on the optimization of the reaction conditions. In particular,
a high enantioselectivity even when using a nonpurified,
entrapped enzyme was desirable.
A remarkable dependence of enantioselectivity and yields
on the nature of the solvent has been observed. The catalytic
amount also plays a significant role. It is noteworthy that an
enantioselectivity of 99% ee was obtained when carrying
out the reaction in diisopropyl ether and with a catalytic
amount of 100U/mmol (Table 1, entry 6). Interestingly,
g99% ee was also found in the subsequent recycling stages
filtration steps possible; (iv) suitable for technical reactors
due to high elasticity; (v) very low price of the encapsulation
materials; (vi) nontoxicity of the encapsulation materials; (vii)
simple encapsulation technique; and (viii) a high synthetic
efficiency of the lens-shaped catalyst capsules. The use of
these entrapped oxynitrilases enables an efficient synthesis
of (R)-mandelonitrile, which is a starting material for the
manufacture of the industrially important R-hydroxy car-
boxylic acid (R)-mandelic acid.
Acknowledgment. The authors thank Dr. Benedikt Ham-
mer, Dr. Reinhard Kr o¨ ner, and Dr. Stefan Weiss for their
support and help.
(in total seven reactions). The average yields are in the range
of >80%.
In conclusion, we reported a new type of entrapped
oxynitrilase catalyst which not only shows a long-term-
stability, high activity, and good recycling rates but also gives
high enantioselectivities of up to 99% ee. This new type of
entrapped biocatalyst is further characterized by the following
properties: (i) recyclable without loss of enzymatic activity;
Supporting Information Available: An experimental
protocol for the preparation of the (R)-oxynitrilase-containing
lens-shaped PVAL-hydrogels (according to Scheme 1) as
well as experimental protocols and data referring to Schemes
2
and 3, respectively. This material is available free of charge
via the Internet at http://pubs.acs.org.
(ii) no catalyst leaching; (iii) macroscopic well-defined size
with a diameter in the mm range, thus making simple
OL015920G
1972
Org. Lett., Vol. 3, No. 13, 2001