Sequential resolution of ethyl 3-aminobutyrate with carboxylic acid esters by Candida antarctica lipase B

Article abstract of DOI:10.1016/S0957-4166(99)00261-X

The reactions of ethyl 3-aminobutyrate 1 with carboxylic acid esters, catalyzed by lipases from Candida antarctica, Pseudomonas cepacia and Pseudomonas fluorescens, have been studied. The reactions take place on the amino and ester functions of the substrate provided that the alkyl group of the achiral ester differs from ethyl. This property has been exploited for the Candida antarctica lipase B-catalyzed resolution of 1 in butyl butyrate, leading to the unreacted enantiomer (S)-1 and butyl 3-aminobutyrate, and to the butanamide of butyl (R)-3-aminobutyrate.

Full text of DOI:10.1016/S0957-4166(99)00261-X

Pergamon
Tetrahedron: Asymmetry 10 (1999) 2573–2581
Sequential resolution of ethyl 3-aminobutyrate with carboxylic
acid esters by Candida antarctica lipase B
a
a
b
a,∗
Szilvia Gedey, Arto Liljeblad, Ferenc Fülöp and Liisa T. Kanerva
a
Departments of Chemistry and Biomedicine, University of Turku, FIN-20014 Turku, Finland
b
Institute of Pharmaceutical Chemistry, Albert Szent-Györgyi Medical University, Szeged, Hungary
Received 7 May 1999; accepted 23 June 1999
Abstract
The reactions of ethyl 3-aminobutyrate 1 with carboxylic acid esters, catalyzed by lipases from Candida
antarctica, Pseudomonas cepacia and Pseudomonas fluorescens, have been studied. The reactions take place on the
amino and ester functions of the substrate provided that the alkyl group of the achiral ester differs from ethyl. This
property has been exploited for the Candida antarctica lipase B-catalyzed resolution of 1 in butyl butyrate, leading
to the unreacted enantiomer (S)-1 and butyl 3-aminobutyrate, and to the butanamide of butyl (R)-3-aminobutyrate.
©
1999 Elsevier Science Ltd. All rights reserved.
1. Introduction
β-Amino acids are important constituents of many natural products, and their free forms and deriva-
1
tives exhibit interesting pharmacological effects. β-Amino acids are also useful tools in the synthesis
of modified peptides. A number of syntheses and transformations leading to β-amino acids in diastereo-
merically and enantiomerically enriched forms have been reviewed.1 The potential of lipases as chiral
catalysts is widely exploited for the kinetic resolution of racemic mixtures.3 Use was made previously
of Pseudomonas cepacia (lipase PS) and Candida antarctica lipase A (Chirazyme L5)-catalyzed asym-
metric amide formation to prepare the enantiomers of various alicyclic β-amino acids from the racemic
amino acid esters with the aid of an appropriate 2,2,2-trifluoroethyl carboxylate in diisopropyl ether.5
Additionally, the lipase-catalyzed acylation of N-hydroxymethylated β-lactams afforded optically active
precursors for the preparation of alicyclic β-amino acids.7
,2
,4
,6
Good enantioselectivity (with an enantiomeric ratio of E=74) was also described for the preferential
acetylation of the ethyl (R)-3-aminobutyrate 1 in the presence of Candida antarctica lipase B (Chirazyme
∗
Corresponding author. E-mail: lkanerva@utu.fi
0957-4166/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(99)00261-X

2574
S. Gedey et al. / Tetrahedron: Asymmetry 10 (1999) 2573–2581
L2) in ethyl acetate.6,8 For an E=74, the highest theoretical enantiomeric excess (ee ) value of the (R)-
P
9
amide produced (P , R=Me) was 97% at the very beginning of the reaction. The ee value gradually
1
decreased with increasing conversion and resulted in an ee =87% when the unreacted substrate reached
P
a value of ee =99% at 53% conversion, i.e. the method allows the preparation of the less reactive (S)-
S
enantiomer in a reasonable chemical yield. As synthons or intermediates for the synthesis of natural
products, e.g. for the synthesis of indolizine alkaloids, the stereochemical structure of β-amino acids
often corresponds to the structure of the (R)-enantiomer.10
The primary aim of this work was to study the lipase-catalyzed resolution of racemic 1, focus-
ing on enzymatic transformations which produce the (R)-enantiomer in particular. Compound 1 can
be seen as a simple structural analogue of alicyclic β-amino esters such as ethyl cis- and trans-
β-aminocyclopentanecarboxylates. Consequently, the resolution of 1 in the presence of lipase PS or
5
Chirazyme L5 was expected to proceed in a highly chemo- and enantioselective manner. Surprisingly,
the resolution tended to lead to low chemoselectivity with the formation of multiple products. This
opened up possibilities for studies of sequential resolution and for the enantiopurity enhancement of
the enzymatically obtained product. The conventional in situ sequential resolutions involve compounds
containing two identical reactive functional groups (e.g. the acylation–acylation sequence of diols or
the esterification–esterification sequence of carboxylic acid derivatives) or compounds containing one
functional group which twice visits the active site of an enzyme (e.g. an ester transforming to another
ester through a hydrolysis–esterification sequence).1
1–13
In the present work, the resolution exploits the
two different functional groups of 1 through routes A -→ B or C -→ D (Scheme 1). The reactions in the
presence of various achiral esters have been studied in order to optimize the resolution conditions.
0
Scheme 1. R=Me, Et, Pr or CH
2
Cl and R =Et, Bu or CH
2
CF
3
2. Results and discussion
2.1. Enzymes and products
5
An expectation according to the previous work was that in the presence of 2,2,2-trifluoroethyl butyrate
or chloroacetate lipase PS leads to the highly (R)-selective transformation of 1 to the corresponding
R)-butyramide (P , R=Pr) or (R)-chloroacetamide (P , R=CH Cl) as the only detectable product,
(
1
1
2
respectively (Scheme 1, route A). However, in the present work the products P (Table 1, rows 1 and
1
2) were contaminated by racemic amide, resulting from the simultaneous chemical reaction, which
became especially important during long reaction times. Moreover, 2,2,2-trifluoroethyl butyrate as the

S. Gedey et al. / Tetrahedron: Asymmetry 10 (1999) 2573–2581
2575
acyl donor led to a mixture of products (row 1). In the case of Chirazyme L5 catalysis, the formation
of (R)-butyramide (P , R=Pr; row 7) proceeded with low enantioselectivity. For the above reasons, the
1
reaction of racemic 1 with 2,2,2-trifluoroethyl butyrate was further screened using various immobilized
Pseudomonas lipases and Chirazyme L2 in diisopropyl ether. The tendency for the formation of multiple
products according to Scheme 1 is obvious (Table 1).
Table 1
Enzyme screening for the reaction of 1 (0.1 M) with 2,2,2-trifluoroethyl butyrate (0.2 M) in diiso-
propylether at room temperature within 25 h
A GLC–MS method was used to determine the structures of products P –P . For P (R=Pr), P
2
1
3
1
0 0
R =Bu) and P (R=Pr, R =Bu) the identification was confirmed by preparing the corresponding race-
3
(
0
0
mates. Efforts to prepare pure products P (R =CH CF ) and P (R=Me or Pr, R =CH CF ) were less
2
2
3
3
2
3
successful. Good baseline separations for each stereoisomer in Scheme 1 were achieved when 1 and P2
0
(
R =Bu or CH CF ) were chemically acylated before the chiral GLC analysis. Attention was paid to the
2
3
0
fact that, in Chirazyme L2 catalysis, product (R)-P (R=Pr, R =CH CF ) was obtained in an enantiopure
3
2
3
form under the given screening conditions. Accordingly, this enzyme was chosen for further studies.
2.2. Structural effects for Candida antarctica lipase B
0
The structure of an achiral ester RCO R may affect the course and the enantioselectivity and reactivity
2
of Chirazyme L2-catalyzed transformations (Scheme 1, Table 2, Figs. 1–3). The esters RCO Et serve as
2
achiral reagents and the reaction media. The reaction route is A (Scheme 1). Accordingly, the solvent
effects of RCO Et (R=Me, Et or Pr) are inextricably included in the results. If this fact is accepted, it
2
can be concluded that the time needed to reach a certain conversion increases with increasing length
0
0
of the carbon chain R in RCO R (R =Et or CH CF ; Table 2, rows 1–4 and rows 7 and 8). The
2
2
3
enantioselectivity of Chirazyme L2, on the other hand, favours long-chains (R=Pr) rather than short-
chains or chloro-substituted ethyl carboxylates, as is seen by comparing the E values. For the 2,2,2-

2576
S. Gedey et al. / Tetrahedron: Asymmetry 10 (1999) 2573–2581
Table 2
Chirazyme L2 (40 mg/ml)-catalyzed reactions of 1 (0.1 M) with esters in the ester as solvent at room
temperature
0
trifluoroethyl esters (R =CH CF , rows 79), it is not possible to discuss enantioselectivity in terms of
2
3
0
the E values because R now differs from ethyl in 1. The structural effects caused by R are seen in the
proportions of the various products and in the ways in which the ee values for each are affected by the
conversion. The results in Table 2 demonstrate that butyrates in general and 2,2,2-trifluoroethyl and butyl
butyrates in particular are the most promising candidates for further studies.
Figure 1. Relative amounts of various products vs time for the Chirazyme L2 (30 mg/ml)-catalyzed reaction of 1 (0.1 M) in
0 0
1 2 3
butyl butyrate: 1 (ꢀ); P (R=Pr) (ꢁ); P (R =Bu) (ꢂ) and P (R=Pr, R =Bu) (ꢃ)

S. Gedey et al. / Tetrahedron: Asymmetry 10 (1999) 2573–2581
2577
Figure 2. Enantiomeric excess vs conversion (%) for the Chirazyme L2 (30 mg/ml)-catalyzed reaction of 1 (0.1 M) with
0 0
1 2 2 3 3 2 3
(R=Pr) (ꢁ); P (R =CH CF ) (ꢂ) and P (R=Pr, R =CH CF )
2,2,2-trifluoroethyl butyrate (0.2 M) in diisopropyl ether: 1 (ꢀ); P
(ꢃ)
Figure 3. Enantiomeric excess vs conversion (%) for the Chriazyme L2 (30 mg/ml)-catalyzed reaction of 1 (0.1 M) in butyl
0 0
1 2 3
butyrate: 1 (ꢀ); P (R=Pr) (ꢁ); P (R =Bu) (ꢂ) and P (R=Pr, R =Bu) (ꢃ)
2.3. Sequential resolution by Candida antarctica lipase B
Chirazyme L2 is a commonly used lipase for the enantioselective acylation of amines.14 Accordingly,
the formation of product P (R=Pr; Scheme 1, route A; Tables 1 and 2) is possible. On the other hand,
1
the enzyme also catalyzes enantioselective reactions of carboxylic acid derivatives. With dicarboxylic
acids and esters, such as N-protected D-glutamic acid diesters, the regioselectivity of Chirazyme L2 can
be directed by adding nucleophiles to the less hindered carbonyl groups.1
2,15,16
Clearly, the ester group
in 1 corresponds to such a less hindered position, allowing the enzymatic reaction to occur at the ester
0
function and the formation of product P (R =CH CF or Bu; route C). Since routes A and C are possible,
2
2
3
it is natural that the further reactions through routes B and D can lead to the formation of product P3.
For sequential resolution of 1 by Chirazyme L2, the two functional groups are simultaneously
susceptible to enzymatic transformation, the activation energy differences for their reactions with 2,2,2-
trifluoroethyl or butyl butyrate determining regioselectivity/chemoselectivity. For 2,2,2-trifluoroethyl
butyrate, the observed product distribution P >P >P [e.g., the area ratio 1:P :P :P =4.0:1.5:0.3:1.0
1
3
2
1
2
3

2578
S. Gedey et al. / Tetrahedron: Asymmetry 10 (1999) 2573–2581
0
R=Pr and R =CH CF ) at 60% conversion] differs significantly from that for butyl butyrate where P
2 3 3
(
(
0
R=Pr, R =Bu) is the major and P (R=Pr) the minor product (Fig. 1). These observations indicate that
1
the activated 2,2,2-trifluoroethyl butyrate favours butanamide formation (product P , R=Pr), followed by
1
0
the formation of product P (R=Pr, R =CH CF ) through route A+B (Scheme 1). In support of this, the
concentration of P (R =CH CF ; route C) is low and that of P (R=Pr, route A) relatively high all the
3
2
3
0
2
2
3
1
way up to 80% conversion. The separate reaction of racemic P (R=Pr) with 2,2,2-trifluoroethyl butyrate
1
(
route B) was relatively fast and highly enantioselective (27% conversion in 40 min, ee =98%), further
P3
0
indicating that the formation of P must be favourable when 1 reacts with (R)-P (R=Pr, R =CH CF ).
1
3
2
3
On the other hand, for sequential resolution of 1 with butyl butyrate the formation of product P
2
0 0
R =Bu) (route C) is favoured (Fig. 1). In order to confirm this, racemic P (R=Pr) and P (R =Bu) were
1 2
(
separately subjected to reactions with butyl butyrate under the resolution conditions. The formation of
0
P (R=Pr, R =Bu; route B) from racemic P (R=Pr) in butyl butyrate proceeded extremely slowly (2–3%
3
1
conversion in 6 h). Because the concentration of P stayed negligible throughout the reaction route A+B
1
0
cannot be significant. In contrast, amide formation from racemic P (R =Bu; route D) was favourable and
2
5
0% conversion was reached in 6 h with ee /ee =97%/95%.
P2 P3
The ee values vs the corresponding total conversion for the Chirazyme L2-catalyzed reactions of 1
with 2,2,2-trifluoroethyl butyrate and with butyl butyrate are shown in Figs. 2 and 3, respectively. In
8
accordance with the previously observed (R)-selectivity of Chirazyme L2, the (R)-enantiomer reacts
faster in the formation of product P (route A) or P (route C). According to the chiral GLC method,
1
2
these (R)-enantiomers are also the reactive enantiomers in the formation of product P through route B or
3
D. This means that the products (S)-P and (S)-P start to accumulate with time. This is seen as the change
1
2
of the prevailing (R)-enantiomer to the (S) one at higher conversions and is shown by the change of the
solid lines to the dotted ones in Figs. 2 and 3. With 2,2,2-trifluoroethyl butyrate as achiral reagent (route
0
A+B favoured), the favourable lipase-catalyzed ethanolysis of the activated ester P (R=Pr, R =CH CF )
3
2
3
back to P (R=Pr) and the consequent equilibrium nature of step B can explain the racemization of P3
1
after about 60% conversion (Fig. 2). This information related to the above-mentioned product distribution
(
P >P >P ) suggests that the resolution of 1 in butyl butyrate is more favourable. Thus, in butyl butyrate,
1 3 2
0
both product P (R =Bu) and the unreacted substrate fraction have an (S)-stereocentre at conversion
close to 80%, whereas product P (R=Pr and R =Bu) at this point represents the corresponding antipode
2
0
3
at 97% ee (Scheme 2, Fig. 3). By stopping the reaction before 70% conversion eeP3 >97% is obtained.
The main aim of this work was the preparation of the (R)-enantiomer. To this end, a successful gram-
0
scale resolution of 1 in butyl butyrate was performed, allowing the preparation of (R)-P (R=Pr, R =Bu)
3
(ee >99%) when the reaction was stopped at 65% conversion, as is shown in the Experimental.
Scheme 2.
An interesting point concerning the Chirazyme L2-catalyzed reactions of 1 with 2,2,2-trifluoroethyl
and butyl butyrates is the process leading to the observed products P –P (Scheme 1). Routes A and
1
3
0
D are simple amide formations (aminolyses, CH CH(NH )CH CO X+RCO R ), whereas the lipase-
catalyzed transesterifications (CH CH(NHX)CH CO Et+RCO R ) need more attention. With 2,2,2-
3
2
2
2
2
0
3
2
2
2
0
trifluoroethyl butyrate as achiral reagent, the formation of products P (R =CH CF ; route C) or P
3
2
2
3
0
R=Pr, R =CH CF ; route B) was not expected because of the poorly nucleophilic nature of 2,2,2-
2 3
(
trifluoroethanol, liberated when 2,2,2-trifluoroethyl butyrate forms a butyryl–enzyme intermediate. In-

S. Gedey et al. / Tetrahedron: Asymmetry 10 (1999) 2573–2581
2579
deed, no reaction took place between 1 or P (R=Pr) and 2,2,2-trifluoroethanol (0.1 M) in diisopropyl
1
ether or in neat 2,2,2-trifluoroethanol within 2–3 days in the presence of Chirazyme L2. However, as
0
0
already mentioned, product P (R=Pr, R =CH CF ) was smoothly produced from P (R=Pr) and 2,2,2-
3
2
3
1
trifluoroethyl butyrate. We propose that intramolecular hydrogen bonding (N–H···O_C) and the liber-
ated alcohol already situated at or in the proximity of the active site aid the present transesterifications.
In the case of butyl butyrate, on the other hand, the Chirazyme L2-catalyzed butanolysis (0.01–0.1 M)
of 1 and P (R=Pr) in diisopropyl ether proceeded efficiently and accordingly enzymatic butanolysis by
1
liberated butanol is more natural.
3. Conclusions
Chirazyme L2 has proved its potential for the resolution of racemic amino ester 1 with an appropriate
0
0
achiral carboxylic acid ester (RCO R , with R differing from the alkyl group of the racemic ester)
2
exploiting sequential resolution. One of the enantiomers is then obtained as product (R)-P through route
3
0
A+B or C+D (Scheme 1). With butyl butyrate as RCO R and as solvent (route C+D), the concen-
2
tration of P (R=Pr) is negligible, allowing the preparation of two highly enantiopure fractions [(S)-P
(
1
2
0 0
R =Bu)+(S)-1 and (R)-P (R=Pr, R =Bu)] at ca. 80% total conversion. In order to understand the reasons
3
for the observed enantioselectivities and reactivities (Figs. 1–3), the enzyme-catalyzed butyrylation of
racemic butyl 3-aminobutyrate (route D) and the transesterification/butanolysis of butyramide P (route
1
B) in butyl butyrate were separately investigated. Excellent enantioselectivities (E>100) were generally
observed, indicating that these reactions can serve as tools for resolution as well. Route D corresponds
8
to the previous Chirazyme L2-catalyzed resolution of ethyl 3-aminobutyrate in ethyl acetate (E=74 or
38; Table 2) and in connection with the present results indicates the importance of structural and solvent
effects on enantioselectivity.
4. Experimental
4.1. Materials and methods
6
Chirazyme L2 and L5 were purchased from Boehringer Mannheim. Lipase PS, PSC-I and PSC-II
from Pseudomonas cepacia and lipase AK and AKC-I from Pseudomonas fluorescens were purchased
or were generous gifts from Amano Pharmaceuticals. Lipase preparations from lipases PS, AK and
5
Chirazyme L5 were adsorbed on Celite in the presence of sucrose. The final preparations contained 20%
(
lipases PS and AK) or 10% (w/w) (Chirazyme L5) of the lipase. 3-Aminobutyric acid was prepared
17
by known methods. Racemic ethyl and butyl 3-aminobutyrates were prepared from the amino acid
by standard procedures, using thionyl chloride and the corresponding dry alcohol. Achiral esters were
prepared from the corresponding acid chlorides and alcohols. The solvents were of the best analytical
grade from Lab Scan.
In a typical small-scale experiment, ethyl 3-aminobutyrate (0.1 M) and hexadecane (0.01 M, an
internal standard) were dissolved in 2.5 ml of diisopropyl ether, and the acyl donor (0.2 M) was added,
or the solution was made in alkyl carboxylate, which served as acyl donor and solvent. The enzyme
preparation (30–75 mg/ml) was added in order to start the reaction. The reaction mixture was shaken
at room temperature. The progress of the reaction and the ee values of the products were followed by
taking samples (0.1 ml) at intervals and analyzing them by gas chromatography on a Chrompack CP-

2580
S. Gedey et al. / Tetrahedron: Asymmetry 10 (1999) 2573–2581
cyclodextrin-β 2,3,6-M-9 column (25 m). Before the analysis, the unreacted ethyl ester 1 in the sample
was derivatized with acetic or butyric anhydride in the presence of 4-N,N-dimethylaminopyridine and
pyridine.
1
H NMR spectra were measured on a Bruker 200/Aspect 3000 spectrometer in CDCl (tetramethylsi-
3
lane as internal standard). Mass spectra were recorded on a VG Analytical 7070E instrument equipped
with a Vaxstation 3100 M 76 computer. Elemental analyses were performed with a Perkin–Elmer CHNS-
2
400 Ser II elemental analyzer. Optical rotations were measured with a Jasco DIP-360 polarimeter, and
−1
2
−1
[α] values are given in units of 10 deg cm g .
D
4.2. Gram-scale resolution of ethyl 3-aminobutyrate
Racemic ethyl 3-aminobutyrate (2.33 g, 17.7 mmol) was dissolved in butyl butyrate (178 mmol) and
Chirazyme L2 (5.3 g) was added. The mixture was stirred at room temperature for 2 h. The reaction was
stopped by filtering off the enzyme at 65% conversion. In order to facilitate the separation in the column,
0
the unreacted 1 and product P (R =Bu) were transformed to the corresponding acetamides with acetic
2
anhydride (2 ml, 0.021 mol) in the presence of 4-N,N-dimethylaminopyridine and pyridine (1 ml) by
stirring the reaction mixture overnight. After evaporation, the products were separated on silica gel by
0
elution with petroleum ether:propan-1-ol (100:6), the elution sequence being P (R=Pr, R =Bu) before
3
0
the acetamide of P (R =Bu) and P (R=Pr). After the three products had eluted, the eluent was changed
2
1
to propanol and the acetamide of unreacted (S)-1 was obtained. Evaporation of the solvent gave the
products and the unreacted substrate, except that the amount of P (R=Pr) was too small to be isolated.
1
0
4
.2.1. (R)-P (R=Pr, R =Bu)
1
3
20
1
.32 g, 5.77 mmol; [α]D +16.1 (c=1.0, MeOH); ee 99%. M=229 according to MS. H NMR (200
MHz) δ (ppm): 0.9 (6H, m, 2×CH CH ), 1.2 (3H, d, J=6.6, CHCH ), 1.3 (2H, m, CH CH CH ), 1.6
2
3
3
2
2
2
(
4H, m, CH CH CH , CH CH CH ), 2.1 (2H, t, J=7.4, COCH ), 2.5 (2H, d, J=5.3, CH CO ), 4.0 (2H,
2 2 2 2 2 3 2 2 2
t, J=6.5, CO CH ), 4.3 (1H, m, CH). Analysis: calculated for C H NO : C, 62.85; H, 10.11; N, 6.11;
2
2
12 23
3
found: C, 62.49; H, 10.41; N, 6.11.
0
4
.2.2. Acetamide of (S)-P (R =Bu)
2
0
.46 g, 2.28 mmol; contains 10% of the unreacted substrate 1 and 4% of the produced P . M=201
3
1
according to GLC–MS. H NMR (200 MHz) δ (ppm): 0.9 (3H, t, J=7.25, CH CH ), 1.2 (3H, d, J=6.72,
CHCH ), 1.3–1.7 (4H, m, 2CH ), 1.9 (3H, s, COCH ), 2.5 (2H, d, J=5.16, CH CO ), 4.0 (2H, t, J=6.7,
2
3
3
2
3
2
2
CO CH ), 4.3 (1H, m, CH).
2
2
4
.2.3. Acetamide of (S)-1
20
1
0.83 g, 4.82 mmol; [α]D −22.5 (c=1.0, MeOH); ee 96%. M=173 according to MS. H NMR (200
MHz) δ (ppm) 1.1–1.3 (6H, m, 2CH ), 1.9 (3H, s, COCH ), 2.5 (2H, d, J=5.4, CH CO ), 4.1 (2H, m,
J=7.0, CO CH ), 4.3 (1H, m, CH). Analysis: calculated for C H NO : C, 55.47; H, 8.73; N, 8.09;
3
3
2
2
2
2
8
15
3
found: C, 54.06; H, 8.74; N, 7.54.

S. Gedey et al. / Tetrahedron: Asymmetry 10 (1999) 2573–2581
2581
Acknowledgements
S.G. is grateful for a grant from the Centre for International Mobility (CIMO), and F.F. expresses his
thanks for support from grand FKFP 0535/1999. The authors thank Laszlo Lazar for the synthesis of
compound 1.
References
1
. Zilkha, A.; Cole, D. C. Tetrahedron 1994, 50, 9517; Juaristi, E. In Enantioselective Synthesis of β-Amino Acids; Wiley-
VHC: New York, 1997.
2. Fülöp, F.; Bernáth, G.; Pihlaja, K. Adv. Heterocyclic Chem. 1998, 69, 349.
3. Kanerva, L. T. In Enzymatic Reactions in Organic Media; Koskinen, A. M. P.; Klibanov, A. M., Eds.; Chapman & Hall:
London, 1996; Chapter 7, p. 170.
4. Kanerva, L. T.; Vänttinen, E.; Kiljunen, E. In Recent Research Developments in Organic & Bioorganic Chemistry;
Transworld Research Network: India, 1997; Vol. 1, p. 55.
5
6
. Kanerva, L. T.; Csomós, P.; Sundholm, O.; Bernáth, G.; Fülöp, F. Tetrahedron: Asymmetry 1996, 7, 1705.
. Chirazyme L2 is immobilized Candida antarctica lipase B, which, according to Boehring Mannheim, is a catalyst
corresponding to Novozym 435 (the trade name of immobilized Candida antarctica lipase B or SP 525) from Novo-
Nordisk. Chirazyme L5 is Candida antarctica lipase A which is similar to the former lipase SP 526 from Novo-Nordisk.
Novo-Nordisk no longer supplies Candida antarctica lipases.
7
8
9
. Csomós, P.; Kanerva, L. T.; Bernáth, G.; Fülöp, F. Tetrahedron: Asymmetry 1996, 7, 1789.
. Sánchez, V. M.; Rebolledo, F.; Gotor, V. Tetrahedron: Asymmetry 1997, 8, 37.
1 1 P 1 1 P 1 1 P
. E=ln[(1−ee )/(1−ee /ee )]/ln[(1−ee )/(1+ee /ee )] with c=ee /(ee +ee ) as derived from the original equations in Chen,
C.-S.; Sih, C. J. Angew. Chem., Int. Ed. Engl. 1989, 28, 695.
10. Chalard, P.; Remuson, R.; Gelas-Mialhe, Y.; Gramain, J.-C.; Canet, I. Tetrahedron Lett. 1999, 40, 1661.
11. Guo, Z.-W.; Wu, S.-H.; Chen, C.-S.; Girdaukas, G.; Sih, C. J. J. Am. Chem. Soc. 1990, 112, 4942.
12. Ozegowski, R.; Kunath, A.; Schick, H. Liebigs Ann. Chem. 1994, 1019.
13. Macfarlane, E. L. A.; Roberts, S. M.; Turner, J. N. J. Chem. Soc., Chem. Commun. 1990, 569.
14. Kazlauskas, R. J.; Bornscheuer, U. T. Biotechnology, 2nd ed.; Biotransformations I; Kelly, D. R., Ed.; Wiley-VCH:
Weinheim, 1998; Vol. 8a, 37.
1
1
1
5. Chamorro, C.; Gonzáles-Muniz, R.; Conde, S. Tetrahedron: Asymmetry 1995, 6, 2343.
6. Liljeblad, A.; Kanerva, L. T., unpublished results.
7. Rivlin, J. J. Org. Chem. 1958, 23, 94.