Immobilization does not influence the enantioselectivity of CAL-B catalyzed kinetic resolution of secondary alcohols

Article abstract of DOI:10.1016/j.tetasy.2004.11.081

Decreasing enantioselectivity (E-value) by increasing conversion has been observed in transesterification reactions of secondary alcohols catalyzed by a pure protein formulation of lipase B from Candida antarctica (Novozym 525 F). Addition of a range of e

Full text of DOI:10.1016/j.tetasy.2004.11.081

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 16 (2005) 847–850
Immobilization does not influence the enantioselectivity of
CAL-B catalyzed kinetic resolution of secondary alcohols
Elisabeth Egholm Jacobsen, Liv Siri Andresen and Thorleif Anthonsen*
Department of Chemistry, Norwegian University of Science and Technology, N-7491 Trondheim, Norway
Received 22 October 2004; accepted 16 November 2004
Available online 22 January 2005
Abstract—Decreasing enantioselectivity (E-value) by increasing conversion has been observed in transesterification reactions of sec-
ondary alcohols catalyzed by a pure protein formulation of lipase B from Candida antarctica (Novozym 525 F). Addition of a range
of enantiopure alcohols caused a temporary increase in selectivity of the transesterification reaction of 3-chloro-1-phenoxy-2-pro-
panol with vinyl butanoate. The corresponding immobilized lipase B, (Novozym 435) showed a similar relationship between the
E-value and degree of conversion.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
tion rates.3 This observation was first reported in 1930
when it was found that strychnine enhanced the human
liver esterase catalyzed hydrolysis of methyl L-mandelate
but not the D-isomer. These results indicated an alloste-
ric binding of the enantiopure additive.4,5
Previously we have reported that the enantioselectivity
(E) decreased during esterifications of a range of second-
ary alcohols 1–4 catalyzed by immobilized lipase B from
Candida antarctica (Novozym 435) and that addition of
enantiopure (R)-alcohols, (R)-1, (R)-2, (R)-5, (R)-6 and
(R)-7, induced an increase in the E-value of the esterifi-
cation of 3-chloro-1-phenoxy-2-propanol 4.1 We sug-
gested that the increase in enantioselectivity was
caused by inhibition of the slower reacting enantiomer
due to an allosteric binding of the enantiopure additive.
Enantioselective inhibition of Candida rugosa (cylindra-
cea) by dextromethorphan and levomethorphan result-
ing in an enhanced enantioselectivity has been
reported.2 Inhibition experiments revealed that the ac-
tion of the base was non-competitive inhibition, that
is, binding of the base to an allosteric site in the lipase.
This caused inhibition of the transformation of one
enantiomer leading to increased selectivity. Hydrolysis
of 3-acetoxy nitriles catalyzed by lipase PS (Pseudomo-
nas sp.) with the addition of L-methioninol showed an
increasing hydrolysis rate of the (R)-enantiomer and a
decreasing hydrolysis rate of the (S)-enantiomer. It
was suggested that the substrate and L-methioninol were
bound to the enzyme at different sites and consequently
that conformational changes provided a change of reac-
Recently it was reported that the changing E-value in
esterifications of 4-methyloctanoic acid catalyzed by
Novozym 435 was due to substrate sorption into the
polymer matrix of the immobilized enzyme.6 Hence it
was interesting to investigate whether immobilization
was the reason for the changing E-value in our experi-
ments. In addition to the immobilized CAL-B, Novozym
435, we used lipase B from C. antarctica, Novozym 525 F
that was not immobilized, for comparison.
2. Results and discussion
Esterifications of the alcohols 1-phenoxy-2-butanol 1, 1-
phenoxy-2-pentanol 2, 3-bromo-1-phenoxy-2-propanol
3 and 3-chloro-1-phenoxy-2-propanol 4 catalyzed by a
freeze dried pure preparation of lipase B from C. antarc-
tica, Novozym 525 F, with vinyl butanoate as acyl
donor, were performed in hexane. The predominantly
formed esters and remaining unreacted alcohols are
shown in Scheme 1.
As for the reactions with the immobilized CAL-B, Nov-
ozym 4351 the Novozym 525 F that was not immobi-
lized showed a decrease in E with conversion (Fig. 1).
*
Corresponding author. Tel.: +47 73596206; fax: +47 73550877;
e-mail: thorleif.anthonsen@chem.ntnu.no
0957-4166/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2004.11.081

848
E. E. Jacobsen et al. / Tetrahedron: Asymmetry 16 (2005) 847–850
O
OH
Novozym
525 F
OH
O
R
OPh
R
OPh
+
Vinyl butanoate
hexane
R
OPh
(R)-1a and (R)-2a
(S)-3a and (S)-4a
(S)-1 and (S)-2
(R)-3 and (R)-4
1
2
3
4
R = CH3
R = CH2CH3
R = Br
R = Cl
Scheme 1.
250
200
1000
4+(R)-1/435
800
600
400
200
0
1
150
4+(R)-1/525 F
E
E
100
4
3
2
50
0
10
20
30
40
50
60
0
10
30
40
50
60
20
% Conversion
% Conversion
Figure 1. Decrease of enantioselectivity with conversion in the
transesterification reactions of 1–4 with vinyl butanoate catalyzed by
CAL-B Novozym 525 F. ( ) = 1, ( ) = 2, ( ) = 3, ( ) = 4.
Figure 3. Transesterification of 3-chloro-1-phenoxy-2-propanol 4 cat-
alyzed by CAL-B 435 ( ) and CAL-B 525 F ( ) in hexane with vinyl
butanoate as acyl donor and with addition of (R)-1 at 30% conversion.
180
4
4/525 F
3.5
160
3
0.66
2.5
140
4/435
120
2
1.5
1
E
100
80
60
0.5
0
40
50
60
10
20
0
30
40 80 120 160 200 240 280 320 360
% Conversion
Enantioselectivity, E
Figure 2. Comparison of the decreasing enantioselectivity (E-value) in
the transesterification reactions of 4 catalyzed by CAL-B Novozym
525 F ( ) and CAL-B Novozym 435 ( ).
Figure 4. The relationship between the E-value and the difference in
free energy of activation, DDG#, for the reaction with the two
enantiomers (DDG# = ꢀRTlnE). The exemplified reaction is the
transesterification of 4 catalyzed by CAL-B Novozym 525 F (see
Fig. 2). The difference in the E-value of 180 at an early stage of the
reaction (15% conversion) and at the end of the reaction (E = 62, 55%
conversion) corresponds to the difference in free energy of activation of
0.66 kcal/mol.
The largest effect was observed with 1, however, it is dif-
ficult to measure extremely large values of E accurately,
hence we chose 4 as the candidate for comparative stud-
ies. The results of the esterification of 4 with the two dif-
ferent catalysts are shown in Figure 2. In fact, the graphs
relating the E-values with conversion were virtually
identical for the immobilized and the not immobilized
preparations of CAL-B.
version, a similar burst of increase in E-value was ob-
served (Fig. 3). Also when (R)-2, (R)-5, (R)-6 and (R)-
7 were used as additives, similar results were obtained.
Moreover, when enantiopure (R)-1 was added to the
Novozym 525 F catalyzed esterification of 4 at 30% con-
The drop in enantioselectivity in these reactions corre-
sponds to a change in free energy of activation, DDG#,
H
OH
H
CH3
OH
H
OH
OPh
OCH3
HO
(R)-7
(R)-5
(R)-6

E. E. Jacobsen et al. / Tetrahedron: Asymmetry 16 (2005) 847–850
849
for the reaction with the two enantiomers of 0.66 kcal/
mol (Fig. 4). The significance of this relatively small
number is uncertain. However, it indicates that a small
change in enzyme conformation may lead to consider-
able effects on the selectivity of the enzyme.
reaction conditions without an enzyme, no acylation
was observed.
4.2.2. Transesterification reactions with the addition of
enantiopure alcohols. Substrate 4 (1.31 · 10ꢀ4 mol),
an acyl donor (6.55 · 10ꢀ4 mol) and Novozym 525
F (30 mg) were added to hexane (3 mL) and per-
formed in the same way as the original reaction of
4 but with the addition of (R)-1 (0.0099 g, 5.96 ·
10ꢀ5 mol), (R)-2 (0.0097 g, 5.38 · 10ꢀ5 mol), (R)-1-
phenoxy-2-hexanol, (R)-5, (0.0045 g, 2.33 · 10ꢀ5 mol),
(R)-1-methoxy-2-propanol, (R)-6, (0.0117 g, 1.30 ·
10ꢀ4 mol) and (R)-2-methyl-1,4-butanediol, (R)-7,
(0.0083 g, 7.97 · 10ꢀ5 mol) at approximately 30%
conversion.
3. Conclusions
Resolutions of 1-phenoxy-2-butanol 1, 1-phenoxy-2-
propanol 2, 3-bromo-1-phenoxy-2-propanol 3 and 3-
chloro-1-phenoxy-2-propanol 4 catalyzed by Novozym
CAL-B 435 and Novozym CAL-B 525 F both showed
a significant decrease in E-values by increasing conver-
sion. Addition of the (R)-alcohols (R)-1, (R)-2, (R)-5,
(R)-6 and (R)-7 at 30% conversion to the resolution of
4 with both enzymes induced a temporary increase in
the enantioselectivity of the reactions. It can be con-
cluded that the decrease in E-value by increasing conver-
sion in resolutions of 1–4 is not due to the
immobilization preparation of the lipase B from C. ant-
arctica as in Novozym 435 as reported by Heinzman
et al. for the esterification of 4-methyloctanoic acid
catalyzed by Novozym 435.
4.3. Synthesis of enantiopure alcohols
(R)-1-Phenoxy-2-butanol (R)-1: The butanoate of 1-
phenoxy-2-butanol 1a, (0.8691 g, 3.68 mmol) was hydro-
lyzed by addition of CAL-B Novozym 435 (0.105 g) in
phosphate buffer (0.1 M, 183.5 mL). The enantiopure
alcohol (R)-1 was separated from the remaining butano-
ate on silica with acetone/hexane, 2:8, as eluent with a
yield of 0.137 g (15.75%), purity 100% (GLC), and an ee
of 96%, ½aꢁ ¼ ꢀ6:6 (c 1.369, CHCl3).
25
D
4. Experimental
4.1. General
(R)-1-Phenoxy-2-pentanol (R)-2: The butanoate of 1-
phenoxy-2-pentanol 2a, (1.47 g, 5.89 mmol) was hydro-
lyzed by addition of CAL-B Novozym 435 (0.20 g) in
phosphate buffer (0.05 M, 100 mL). The enantiopure
alcohol (R)-2 was separated from the remaining butano-
ate on silica with acetone/hexane, 3:7, as eluent with a
Immobilized lipase B from C. antarctica (CAL-B Novo-
zym 435) had an activity of 10 PLU/mg and a water
content of 2% w/w. The pure enzyme preparation of
lipase B from C. antarctica (CAL-B Novozym 525 F)
was a water solution with 1–10% protein content. Both
enzyme preparations were gifts from Novozymes, Bags-
værd, Denmark. Chemicals were purchased from
Fluka. Column and flash chromatography were per-
formed using silica gel 60 from Fluka, with pore size
0.0663–0.2000 mm and 0.035–0.070 mm, respectively.
Optical rotations were determined using an Optical
Activity Ltd. AA-10 automatic polarimeter, concentra-
tions are given in g/100 mL. Chiral analyses were
performed using a Varian 3400 gas chromatograph
equipped with CP-Chirasil-Dex CB columns from
Chrompack (25 m, 0.25 or 0.32 mm i.d., 0.25 lm film
density). For syntheses of racemic substrates with
NMR data and chromatographic parameters of the res-
olution products see Refs. 7 and 8. Enantiomeric ratios,
E, were calculated based on ping-pong bi-bi kinetics
using the computer program E&K Calculator 2.1b0
PPC.9
yield of 0.279 g (19%), purity 95% (GLC) and an ee of
30
D
99.3%, ½aꢁ ¼ ꢀ12:25 (c 1.142, CHCl3).
(R)-1-Phenoxy-2-hexanol (R)-5 was synthesized from
(R)-phenyl glycidyl ether as described in Ref. 7. The
yield was 0.630 g (65.5%) with a purity of 100%
25
(GLC) and an ee higher than 99% ½aꢁ ¼ ꢀ5:55
D
(c 0.90, CHCl3).
(R)-1-Methoxy-2-propanol (R)-6 and (R)-2-methyl-1,4-
butanediol (R)-7 were purchased from Fluka.
4.4. Determination of absolute configurations
The absolute configurations of the faster reacting enantio-
mers of 1–3 were determined by comparisons of the spe-
cific rotation and of the retention times on GLC with
(R)-1, (R)-2 and (S)-3 synthesized by a two-step proce-
dure from (R)-phenyl glycidyl ether made from (S)-
epichlorohydrin and phenol.10,11 The synthesized
4.2. Enzymatic reactions
enantiopure alcohols had the following properties: (R)-
1
4.2.1. Transesterification reactions. Substrates 1–4
(1.31 · 10ꢀ4 mol) and an acyl donor (6.55 · 10ꢀ4 mol)
were added to hexane (3 mL). The reactions were started
by the addition of Novozym 525 F (30 mg) and per-
formed in an Infors shaker incubator at 30 ꢁC. Chiral
GLC analyses gave ees- and eep-values from which the
degree of conversion was determined according to
c = ees/(ees + eep). In controlled experiments under the
25
D
ee >99%, ½aꢁ ¼ ꢀ6:4 (c 1.40, CHCl3), (R)-2:
22
20
ee >99%, ½aꢁ ¼ ꢀ6:9 (c 1.17, CHCl3) and (S)-3 ee =
D
96%, ½aꢁ ¼ þ5:3 (c 1.71, EtOH). The absolute configu-
D
ration of 4 was not determined directly, but assigned
by comparing relative retention times on chiral
GLC supported by the known enantiopreference of
CAL-B.

850
E. E. Jacobsen et al. / Tetrahedron: Asymmetry 16 (2005) 847–850
4. Ammon, R.; Fischgold, H. Biochem. Z. 1931, 234,
54.
Acknowledgements
5. Bamann, E.; Laeverenz, P. Z. Physiol. Chem. 1930, 193,
201–214.
6. Heinsman, N. W. J. T.; Schro¨en, C. G. P. H.; van der
We thank Novozymes, Bagsværd, Denmark, for kind
gifts of CAL-B Novozym 435 and CAL-B Novozym
525 F and Ahmed Nuriye, NTNU, for the synthesis of
racemic substrates.
`
Padt, A.; Franssen, M. C. R.; Boom, R. M.; van’t Riet, K.
Tetrahedron: Asymmetry 2003, 14, 2699–2704.
7. Jacobsen, E. E.; Hoff, B. H.; Anthonsen, T. Chirality 2000,
12, 654–659.
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