Efficient biocatalytic synthesis of (R)-pantolactone

Article abstract of DOI:10.1002/adsc.200800354

Screening for stereoselective cyanohydrin synthesis in 96-well plates was employed in the development of an efficient, pH-stable hydroxynitrile lyase for the conversion of sterically hindered aliphatic aldehydes. Site-saturation mutagenesis (SSM) resulted in a powerful catalyst for the stereoselective conversion of hydroxypivalaldehyde and pivalaldehyde to their corresponding (R)-cyanohydrins (ee >97%) which are used as chiral building blocks (e.g., for pantothenic acid production). Furthermore, redesigning the PaHNL5 gene and improving its expression by Pichia pastoris with the help of a new P _AOX1 promoter variant and the helper protein PDI (protein disulfide isomerase) led to elevated amounts of today's most efficient biocatalyst for vitamin B5 synthesis.

Full text of DOI:10.1002/adsc.200800354

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DOI: 10.1002/adsc.200800354
Efficient Biocatalytic Synthesis of (R)-Pantolactone
Beate Pscheidt,a Zhibin Liu,a,b Richard Gaisberger,a Manuela Avi,c
Wolfgang Skranc,d Karl Gruber,a Herfried Griengl,a,c and Anton Gliedera,*
a
Research Centre Applied Biocatalysis GmbH, Petersgasse 14, 8010 Graz, Austria
Fax : (+43)-316-873-9302; e-mail: glieder@glieder.com
Current address: A*STAR Institute of Chemical and Engineering Sciences, Pesek Road 1, Jurong Island, 627833
b
Singapore
c
Institute of Organic Chemistry, Graz University of Technology, Stremayrgasse 16, 8010 Graz, Austria
DSM Fine Chemicals Austria Nfg. GmbH & Co KG, R & D Centre Linz, St.-Peter-Strasse 25, 4021 Linz, Austria
d
Received: June 6, 2008; Published online: August 19, 2008
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/adsc.200800354.
Abstract: Screening for stereoselective cyanohydrin
synthesis in 96-well plates was employed in the de-
velopment of an efficient, pH-stable hydroxynitrile
lyase for the conversion of sterically hindered ali-
phatic aldehydes. Site-saturation mutagenesis
(SSM) resulted in a powerful catalyst for the stereo-
selective conversion of hydroxypivalaldehyde and
pivalaldehyde to their corresponding (R)-cyanohy-
drins (ee >97%) which are used as chiral building
blocks (e.g., for pantothenic acid production). Fur-
thermore, redesigning the PaHNL5 gene and im-
proving its expression by Pichia pastoris with the
help of a new PAOX1 promoter variant and the
helper protein PDI (protein disulfide isomerase)
led to elevated amounts of todayꢀs most efficient
biocatalyst for vitamin B5 synthesis.
pharmaceutical
ingredients.
The
(R)-selective
hydroxynitrile lyase [(R)-HNL] based synthesis of hy-
A
droxypivalaldehyde cyanohydrin [(R)-2],[2,3] followed
by acid-catalyzed hydrolysis, provides a highly attrac-
tive alternative.
Focusing on process and reaction engineering, Ef-
fenberger et al.[3] succeeded in achieving the highest
ee (89%) and yield (84%) reported so far, employing
large amounts of highly purified and immobilized
native PaHNL. Screening for new natural enzymes
did not result in any further improvements.[10,11]
Herein, we communicate the semi-rational redesign
of acid-stable almond (R)-HNL isoenzyme
5
(PaHNL5)[12] to realize high selectivity and yield in
one of the most challenging biocatalytic routes for
todayꢀs fine chemical industry.
Hydroxypivalaldehyde (1a), exists as an equilibrium
(Scheme 1)[13] between the dimer and the monomer
with the exposed aldehyde functionality for HCN ad-
dition. Heating at 658C in THF[14] or without solvent
at elevated temperatures (ꢀ1008C)[15] are common
procedures to shift the equilibrium to the monomer.
As reported previously,[16] we observed unwanted by-
Keywords: biocatalysis; cyanohydrin; enzyme engi-
neering; hydroxynitrile lyase; pantolactone
(R)-Pantolactone is the most important chiral build- product formation via an ꢁanomalous Tishchenko re-
ing block for the synthesis of (R)-pantothenic acid, actionꢀ when the aldehyde was stirred at elevated
also called vitamin B5 – a constituent of coenzyme A, temperatures.
and its provitamin (R)-pantothenol. Since the first
Alternatively, acid-catalyzed hemi-acetal cleavage
successful synthesis in 1940,[1] great efforts have been of 1b (Figure 1) can be used for efficient substrate
made to develop efficient techniques for the technical monomerization. Thus, only gentle substrate heating
preparation
of
the
(R)-enantiomer
[(R)-3, was necessary prior to the biocatalytic reaction. A pH
Scheme 1].[2–10]
value ꢁ2.5 provided the preferred compromise be-
Starting with hydroxypivalaldehyde (1a), common tween substrate monomerization, enzyme stability/ac-
stereoselective synthesis routes depend on either ad- tivity and stability of the chiral cyanohydrin [(R)-2].
ditional process steps (e.g., enzymatic kinetic resolu-
Recently, PaHNL5 was successfully engineered for
tion) or the employment of highly complex metal cat- superior conversion of non-natural aromatic sub-
alysts (selective reduction) which are often not desira- strates following a structure-guided, rational ap-
ble for the production of food and feed, cosmetic and proach.[12,17] Thereupon, we expanded into the chal-
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Beate Pscheidt et al.
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Scheme 1. Asymmetric synthesis of (R)-pantolactone ((R)-3) from hydroxypivalaldehyde (1a) by the (R)-HNL route[2,3,10]
(gray background). Other methods comprise the chemical resolution or enantioselective hydrolysis of (RS)-pantolactone
[(RS)-3] or (RS)-pantolactone esters [(RS)-4],[4,5] asymmetric hydrogenation of ketopantolactone (5),[6,7] Sharpless asymmet-
ric dihydroxylation of 6[8] and the proposed enantioselective hydrolysis of the corresponding a-hydroxynitrile using nitrilas-
es,[9] respectively, as key step for the introduction of chirality. (N.e.p.=no example provided)
lenging group of sterically hindered aliphatic sub-
strates. However, as the modeled PaHNL5/(R)-2 and
(S)-2 complexes did not display any clearly unfavora-
ble interactions, all hydrophobic residues of the sub-
strate binding pocket, namely F72, V113, V317, V329,
L331, L343 and V360, were considered as targets for
saturation mutagenesis combined with screening for
cyanohydrin synthesis.
For saturation library generation, Pichia pastoris
was transformed with linear expression cassettes
made by overlap extension PCR employing the
PaHNL5 gene ligated into the plasmid pGAPZA as a
template. Therefore, the basic method established by
Liu et al.[18] was adapted for site-saturation mutagene-
sis. For each of the 7 targeted sites, ꢀ200 transform-
ants were assayed for improved conversion of 1a. Fol-
lowing the first law of directed evolution ꢁyou get
what you screen for’[19] – whose consequences we had
Figure 1. Investigation of the amount of monomer available
after incubation in buffered aqueous solution at different
to encounter quite recently when engineering
pH values. A pH ꢁ2.5 is advantageous for efficient sub-
PaHNL5 for improved (R)-2-chloromandelonitrile
strate monomerization prior to the stereoselective, enzymat-
synthesis[18] – we searched for a method which for the
ic hydrocyanation of 1a to (R)-2. At neutral or basic pH
first time provided the possibility to screen specifical-
ly for the cyanohydrin synthesis reaction, namely the
conversion of 1a to 2 at pH 2.4 in a 96-well format.
For detection, a colorimetric high-throughput screen-
ing method from Bornscheuer and co-workers[20] was
values, the desired cyanohydrin (R)-2 would racemize again
and non-selective chemical addition of HCN to the aldehyde
would be favored [180 mg gently liquefied 1b were dissolved
in 5 mL of a 30 mM potassium phosphate buffer (pH 2.0–
7.0)]
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Efficient Biocatalytic Synthesis of (R)-Pantolactone
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Table 1. Comparison of V317A and V317G with the unmodified PaHNL5 for the hydrocyanation of 1a.
Entry
Enzyme
Reaction time (conv. [%]/ee [%])[a]
TOF[b] [sꢂ1]
2.5 h
5.0 h
20.5 h
1
2
3
4
Blank
5.6/rac[c]
14.9/22.2
15.1/21.8
42.4/87.1
8.4/rac[c]
n.d./n.d.
24.4/25.9
59.2/86.7
37.8/rac[c]
64.9/22.2
53.5/10.6
86.5/81.8
–
PaHNL5
V317G
V317A
0.83ꢃ0.02
n.d.[d]
11.08ꢃ0.19
[a]
Reaction conditions: 1.17 mmol of 1a and 1 mg of enzyme after 2-L shake flask cultivation (0.85 mg enzyme mmolꢂ1 sub-
strate), 48C, pH 2.4.
[b]
Reaction conditions: 1.17 mmol of substrate and necessary amount of purified enzyme after production in a 5-L bioreac-
tor so that conversion of 1a was in a linear range during the first 30 min.
rac: racemic.
[c]
[d]
n.d.: not determined since ee was not improved.
adapted for lyases and an aqueous system. All var- previous results with the wild-type enzyme,[11] the
iants were screened for improved conversion (see Ex- mutant V317A even retained stability at the very low
perimental Section). Subsequently, a 96-well plate- pH 2.0 where the unselective chemical background re-
based GC method was used to determine the enantio- action is repressed in a large part (Figure 2).
meric excess of preselected variants. Rescreening of
Reconsidering the modeled enzyme-cyanohydrin
the best clones led to the identification of one signifi- complexes in detail, we recognized three possible in-
cantly improved variant with the amino acid exchange teractions between valine 317 and a methyl group of
V317A (GTA!GCT).
the (S)-enantiomer but only one hydrophobic interac-
This was surprising, because mutation V317G tion with the (R)-enantiomer (Figure 3). Thus, ex-
caused negative effects for the synthesis of another changing the isopropyl moiety of V317 for a methyl
bulky compound [i.e., (R)-2-chloromandelonitrile].[12] group most probably accounts for a loss of stabilizing
However, mutation V317A seemed to be beneficial interactions with (S)-2. Wanting to confirm the impact
for sterically hindered aliphatic aldehydes. Employing of this qualitative structural analysis, we used the pro-
1 mg of protein after shake-flask cultivation resulted gram Amber to elucidate differences between the
in elevated conversion and almost four-fold improved native PaHNL5 and mutein V317A. Complexes of
enantiopurity (Table 1). At the same time, the intro- the native enzyme resulted in comparable energies
duction of alanine at position 317 did not negatively for the binding of both enantiomers, whereas mutein
influence the enzymeꢀs stability at low pH. This is es- V317A provided a more favorable binding site for
pecially important for (R)-2 production. In contrast to (R)-2 with a (potential) energy difference of 2 kcal
Figure 2. Testing different pH values for the synthesis of (R)-2 employing PaHNL5V317A in buffered aqueous solution at
pH 1.5 to 8.0. The PaHNL5-variant V317A retained stability even at pH 2.0 where the unselective chemical background re-
action is repressed in a large part. Reaction conditions: 1.17 mmol of 1a, 170 U of enzyme PaHNL5V317A (~1.7 mgmmolꢂ1
substrate) and 100 mL of anhydrous HCN were diluted in 5 mL of 2M potassium phosphate buffer at pH 1.5–8.0.
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Beate Pscheidt et al.
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Figure 3. Superposition of the modelled complexes with the substituents (R)-2 (green) and (S)-2 (purple) at the active site of
PaHNL5 provides an insight into the substrate/product binding pocket at the active site and indicates possible interactions
of V317 with a methyl group of (S)-2. Amino acid side chains are depicted in gray, the FAD cofactor in yellow and N and O
atoms in blue and red, respectively. V317 is shown in cyan. The image was created using the software PyMol v0.99 (http://
www.pymol.org).
molꢂ1 compared to (S)-2. Calculations thus supported
To explore turnover rates, purified enzyme pro-
our interpretation, but again did not exclude addition- duced with a 5-L bioreactor was employed. Compared
al hydrophobic residues to be possible engineering to the unmodified PaHNL5, mutein V317A accelerat-
targets. In contrast to our previous studies with other ed the conversion of 1a approximately 13-fold. In this
substrates,[12,17] not the removal of steric hindrance case, modeling results did not provide further insight.
but the reduction of hydrophobic interactions with Then, we examined V317Aꢀs performance in different
the wrong enantiomer resulted in increased stereose- reaction systems (Table 2). Using Celiteꢃ as enzyme
lectivity. As in the case of (R)-2-chloromandelonitrile support in a microaqueous, organic solvent system
synthesis,[12] the variant V317G, providing even more also resulted in high enantiopurities but compared to
space at this position, resulted in decreased enantio- a pure aqueous reaction solution, conversion was low.
meric excess and conversion (Table 1).
The buffered aqueous system also surpassed a bipha-
sic (H2O/TBME) reaction system. Finally, the ability
Table 2. Synthesis of (R)-2 employing PaHNL5V317A in different reaction systems.
Entry
Conditions
pH
Enzyme amount [U/mmol 1a]
Reaction time (conv. [%]/ee [%])
2 h
6 h
22 h
1[a]
2[a]
3[a]
H2O
2.5
2.5
2.5
140
310
1340
77.0/94.4
87.9/94.5
99.8/95.5
96.2/95.5
100/95.0
100/97.0
99.1/94.9
100/94.9
100/97.5
4 h
16/60.8
31/86.6
24 h
29/58.2
54/87.5
4[b]
5[b]
H2O/TBME
H2O
3.4
3.4
100
100
2 h
22/94.0
6 h
26/95.5
20 h
37/96.9
6[c]
Celiteꢃ/DIPE
3.4
100
[a]
Buffered aqueous system.
[b]
[c]
Comparison of biphasic system (H2O/TBME) with aqueous system.
V317A immobilized on Celiteꢃ: microaqueous, organic (DIPE) system.
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Efficient Biocatalytic Synthesis of (R)-Pantolactone
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the pH to <1.0 removing the remaining HCN and prevent-
ing a continuing enzymatic reaction. All steps including the
possible evaporation of HCN were carried out under a well
ventilated hood.
for scale-up of the aqueous system was investigated.
1.2 g of 1a were converted employing only 218 U
V317Ammolꢂ1 1a in a 100-mL reactor at 48C. After
20 h the reaction was completed and subsequent
work-up supplied the crude product with 88% yield
and 96% ee.
Furthermore, variant V317A proved to be benefi-
cial for another bulky aliphatic substrate, namely piv-
alaldehyde, resulting in quantitative conversion with
an approximately 15% elevated enantiopurity (97.3%
ee compared to 82.5% ee for PaHNL5, employing
1.1 mg protein per mmol of pivalaldehyde).
In order to screen for conversion, a fluorimetric assay was
carried out in 96-well PP-microplates. 4-Hydrazino-7-nitro-
benzofurazane (NBDH) was employed to detect the remain-
ing amount of aldehyde in the reaction solution. The in-
crease in the strongly fluorescent hydrazone was tracked
and the slope of this chemical reaction was evaluated. It
could be directly correlated to the amount of aldehyde 1a
remaining in the reaction solution after a distinct reaction
time.
In order to boost the biocatalytic system from an-
other side, in parallel, a synthetic Pahnl5V317A gene
Subsequently, preselected variants were rescreened em-
ploying a 96-well plate based GC method in order to deter-
mine enantiomeric excess. After the hydrocyanation reac-
tion in 96-well deep well plates, 1a and the corresponding
cyanohydrins (R)-2 and (S)-2 were extracted with tert-butyl
methyl ether (TBME). Aliquots of the organic phase were
transferred to a new 96-well PP-microplate (V-bottom,
clear) and after derivatization with acetic anhydride in the
presence of pyridine and dichloromethane the enantiomeric
purities were analyzed using a Hewlett–Packard 6890 instru-
ment equipped with an FID and a Chirasil-DEX CB
column.
was designed and expressed with the help of the im-
[21,22]
proved promoter variant PAOX1D1
and the co-ex-
pression of PDI.[23] Thus, a further 4-fold improved
enzyme activity per volume of yeast culture was ob-
tained.
Stepwise redesign of PaHNL5 gene and enzyme
thus led to elevated amounts of todayꢀs most efficient
biocatalyst for the vitamin B5 synthesis route and
seemingly, for the conversion of sterically hindered
aliphatic aldehydes in general. Screening for cyanohy-
drin synthesis at low pH, resulted in a highly stable
biocatalyst for the synthesis of the chiral key inter-
mediate for (R)-pantolactone synthesis with ee >97%
via the efficient HNL route. Furthermore, the aque-
ous system was shown to be superior to alternatives
employing organic solvents.
Biotransformations for the characterization of PaHNL5
V317A were performed on a 5-mL reaction scale. Varying
amounts of enzyme were diluted to 5 mL using a citrate
phosphate or potassium phosphate buffer at different pH
values (pH 1.5–8.0). Then, for example, 120 mg of 1a and,
for example, 100 mL of anhydrous HCN were added. Reac-
tions were performed at 48C and 600 rpm applying the mag-
netic stirrer Variomag Electronicrührer Poly 15. After the
scheduled time, samples were taken, extracted with TBME
and derivatized for chiral GC analysis.
Experimental Section
Further experimental details are available in the Support-
ing Information.
Site-saturation mutagenesis libraries (F72X, V113X, V317X,
V329X, L331X, L343X, V360X) were created by overlap ex-
tension PCR (oePCR) using template plasmid pGAPZA
PaHNL5[24] and partly complementary mutagenic primers
containing a degenerated codon [N N (G/T)] at the desired
site. The resulting oePCR products were purified and direct-
ly employed for Pichia pastoris X33 transformation. Zeocin
was used as selection marker.
Acknowledgements
This research was supported by DSM, the FFG, the Province
of Styria, the SFG and the City of Graz. We want to thank
Prof. Manfred Reetz for providing valuable information
about robotized high-throughput screening instruments. We
acknowledge H. Mandl and B. Krenn for excellent technical
support.
The resultant P. pastoris X33 transformants were cultivat-
ed in 96-well deep well plates and screened for improved
conversion of 1a, also on the 96-well deep well plate scale.
Therefore, 400 mL of the culture supernatant were mixed
with 150 mL of a 3M citrate phosphate buffer (pH 2.4) and
200 mL of a 1a-stock solution [1.2 mL of 1a were added in
46.8 mL of a 3M citrate phosphate buffer (pH 2.4)]. 22 mL
of a 12M NaCN solution were added to start the reaction. References
To prevent HCN evaporation, deep well plates were finally
covered with
a
SILVERsealTM sealer aluminium foil
[1] E. T. Stiller, S. A. Harris, J. Finkelstein, J. C. Keresztesy,
K. J. Folkers, J. Am. Chem. Soc. 1940, 62, 1785–1790.
[2] V. I. Ognyanov, V. K. Datcheva, K. S. Kyler, J. Am.
Chem. Soc. 1991, 113, 6992–6996.
(Greiner Bio-One GmbH, Germany). To agitate the reac-
tion solution, a magnetic stirrer for 96-well deep-well plates
(Alligator Tumble Stirrer from V&P Scientific) was used to-
gether with one PTFE encapsulated stir stick (VP 734–2)
per well. After one hour, the reaction was stopped by re-
moving the silver seal foil and adding a 50% (v/v) solution
of H2SO4 in H2O. The addition of the acidic solution shifted
[3] F. Effenberger, J. Eichhorn, J. Roos, Tetrahedron:
Asymmetry 1995, 6, 271–282.
[4] L. Haughton, J. M. J. Williams, J. A. Zimmermann, Tet-
rahedron: Asymmetry 2000, 11, 1697–1701.
Adv. Synth. Catal. 2008, 350, 1943 – 1948
ꢂ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
1947

Beate Pscheidt et al.
COMMUNICATIONS
[5] a) M. Kesseler, B. Hauer, T. Friedrich, R. Mattes,
(BASF AG, Germany), WO Patent 0132890, 2001;
Chem. Abstr. 2001, 134, 352360m; b) S. Shimizu, Bita-
min 1999, 73, 713–720.
[12] A. Glieder, R. Weis, W. Skranc, P. Poechlauer, I. Dre-
veny, S. Majer, M. Wubbolts, H. Schwab, K. Gruber,
Angew. Chem. Int. Ed. 2003, 42, 4815–4818.
[13] E. Spath, I. v. Szilagyi, Ber. Dtsch. Chem. Ges. 1943,
76B, 949–956.
[14] O. P. Toermaekangas, A. M. P. Koskinen, Tetrahedron
Lett. 2001, 42, 2743–2746.
[15] O. P. Toermaekangas, P. Saarenketo, A. M. P. Koskinen,
[6] a) W. Bonrath, R. Karge, T. Netscher, J. Mol. Catal. B:
Enzym. 2002, 19–20, 67–72; b) H. Yamada, S. Shimizu,
Angew. Chem. Int. Ed. 1988, 27, 622–642; c) R. Eck,
H. Simon, Tetrahedron: Asymmetry 1994, 5, 1419–
1422; d) S. Shimizu, S. Hattori, H. Hata, H. Yamada,
Enzyme Microb. Technol. 1987, 9, 411–416; e) K.
Achiwa, T. Kogure, I. Ojima, Tetrahedron Lett. 1977,
18, 4431–4432; f) K. Achiwa, Heterocycles 1978, 9,
1539–1543; g) E. A. Broger, Y. Crameri, (F. Hoffmann-
La Roche & Co. AG, CH), EP Patent 0218970, 1987;
Chem. Abstr. 1987, 107, 7394m; h) A. Schaefer, D.
Arntz, (Degussa AG, Germany), US Patent 5,177,220,
1991; Chem. Abstr. 1991, 115, 183072m; i) M. Maris, T.
Mallat, A. Baiker, J. Mol. Catal. A: Chem. 2005, 242,
151–155.
Org. Process Res. Dev. 2002, 6, 125–131.
[16] G. K. Finch, J. Org. Chem. 1960, 25, 2219–2220.
[17] R. Weis, R. Gaisberger, W. Skranc, K. Gruber, A.
Glieder, Angew. Chem. Int. Ed. 2005, 44, 4700–4704.
[18] Z. Liu, B. Pscheidt, M. Avi, R. Gaisberger, F. S. Hart-
ner, C. Schuster, W. Skranc, K. Gruber, A. Glieder,
ChemBioChem. 2008, 9, 58–61.
[19] F. H. Arnold, Acc. Chem. Res. 1998, 31, 125–131.
[20] a) U. T. Bornscheuer, Eng. Life Sci. 2004, 4, 539–542;
b) M. Konarzycka-Bessler, U. T. Bornscheuer, Angew.
Chem. Int. Ed. 2003, 42, 1418–1420.
[7] C. Pasquier, S. Naili, L. Pelinski, J. Brocard, A. Mor-
treux, F. Agbossou, Tetrahedron: Asymmetry 1998, 9,
193–196.
[21] F. Hartner, A. Glieder, (Technische Universitaet Graz,
Austria), WO Patent 06089329A2, 2006; Chem. Abstr.
2006, 145, 264347m.
[8] T. T. Upadhya, S. Gurunath, A. Sudalai, Tetrahedron: [22] F. S. Hartner, C. Ruth, D. Langenegger, S.N. Johnson,
Asymmetry 1999, 10, 2899–2904.
P. Hyka, G.P. Lin-Cereghino, J. Lin-Cereghino, K.
Kovar, J. M. Cregg, A. Glieder, Nucleic Acids Res.
2008, 36 (12), e76.
[9] G. DeSantis, J. M. Short, M. J. Burk, K. Wong, R. Far-
well, K. Chatman, (Diversa Corporation, USA), US
Patent 2004,014195, 2004; Chem. Abstr. 2004, 140,
124551m.
[23] M. Inan, D. Aryasomayajula, J. Sinha, M. M. Meagher,
Biotechnol. Bioeng. 2006, 93, 771–778.
[10] L. Synoradzki, T. Rowicki, M. Włostowski, Org. Pro-
cess Res. Dev. 2006, 10, 103–108.
[11] B. Pscheidt, M. Avi, R. Gaisberger, F. S. Hartner, W.
Skranc, A. Glieder, J. Mol. Catal. B: Enzym. 2008, 52–
53, 183–188.
[24] H. Schwab, A. Glieder, C. Kratky, I. Dreveny, P. Poe-
chlauer, W. Skranc, H. Mayrhofer, I. Wirth, R. Neuhof-
er, R. Bona, (DSM Fine Chemicals, Austria), EP
Patent 1223220, 2005; Chem. Abstr. 2005, 143,
244072m.
1948
asc.wiley-vch.de
ꢂ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2008, 350, 1943 – 1948