Conversion of a pentane-1,3,5-triol derivative using lipases as chiral catalysts and possible function of the lid for the regulation of substrate selectivity and enantioselectivity

Article abstract of DOI:10.3109/10242422.2012.661726

The enantioselective desymmetrization of a prochiral 3-O-silyl protected pentane-1,3,5-triol derivative was achieved by lipase-catalysed hydrolysis. The lipase from B. cepacia led to 95.4% enantiomeric excess (ee)of the (R)-configured compound (R)-4 at a

Full text of DOI:10.3109/10242422.2012.661726

Biocatalysis and Biotransformation, March–April 2012; 30(2): 217–225
ORIGINAL ARTICLE
Conversion of a pentane-1,3,5-triol derivative using lipases as chiral
catalysts and possible function of the lid for the regulation
of substrate selectivity and enantioselectivity
JENS KÖHLER & BERNHARD WÜNSCH
Institut für Pharmazeutische und Medizinische Chemie,WestfälischeWilhelms-Universität Münster,
Hittorfstraße 58-62, 48149 Münster, Germany
Abstract
The enantioselective desymmetrization of a prochiral 3-O-silyl protected pentane-1,3,5-triol derivative was achieved by
lipase-catalysed hydrolysis. The lipase from B. cepacia led to 95.4% enantiomeric excess (ee)of the (R)-configured com-
pound (R)-4 at a theoretical yield of 79% and was isolated with 98.2% ee and 27% yield. Furthermore, it was found
that the ee switched from an excess of (R)-4 to an excess of (S)-4 during the course of the reaction using crude lipase
from C. rugosa under the same conditions. This finding was investigated in detail and compared to the change of sub-
strate selectivity known for the lipase from M. miehei. It is supposed that both phenomena may result from a movement
of the lid.
Keywords: lipase, enantioselectivity, pentane-1,3,5-triol, lid
Introduction
form. It can easily switch between the open and
closed conformation and thereby regulate the bind-
ing of substrates (Cygler & Schrag 1999; Grochulski
et al. 1994; Overbeeke et al. 2000; Peters & Bywater
1999; Vasel et al. 1993). It is widely accepted that
the lid is opened by contact of the enzyme with an
oil-water interface, which is called interfacial activa-
tion (Bornscheuer & Kazlauskas 2005; Derewenda
et al. 1992; Grochulski et al. 1993; Schmid &Verger
1998). Furthermore it is known that the binding site
consists of two hydrophobic regions, a large hydro-
phobic region and the smaller stereoselectivity
pocket. The latter contains the catalytic triad as the
active centre of the lipase with the OH-group of ser-
ine that accepts the acyl residue within the catalytic
cycle (Bornscheuer & Kazlauskas 2005; Brady et al.
1990; Dodson & Wlodawer 1998; Pleiss et al. 1998;
Schmid & Verger 1998).
Lipases are classified as hydrolases, as they are pre-
dominantly responsible for the catalytic hydrolysis
of triglycerides, which are the main components of
nutritional and storage fat.The hydrolysis of triglyc-
erides provides free fatty acids that serve as a source
of metabolic energy. Lipases have been extensively
employed in organic synthesis (Bornscheuer &
Kazlauskas 2005) and used in industrial-scale
conversions (Gunstone 1999; Schmid et al. 2001).
Within such processes, lipases are used not only for
hydrolysis of esters but also for acylation of alcohols
(Schmid &Verger 1998).Particular interest is focused
on enantioselective conversions, which result from
the chiral environment of the active site of these
enzymes (Ghanem 2007). Next to the active site, a
flexible part of the protein is located, which is known
as the lid (Bornscheuer & Kazlauskas 2005; Schmid
& Verger 1998; Cambillau & van Tilbeurgh 1993;
Secundo et al. 2006). The lids of different lipases
have individual characteristics and can cover the
active site to a greater or lesser extent in its closed
We are interested in the production of chiral
building blocks with a pentane-1,3,5-triol scaffold
for the synthesis of enantiomerically pure NMDA
and σ receptor ligands. The required prochiral
Correspondence: Prof. Bernhard Wünsch, University of Münster, Institute of Pharmaceutical and Medicinal Chemistry, Hittorfstr.58–62, 48149 Münster,
Germany. Email: wuensch@uni-muenster.de
(Received 23 August 2011; revised 22 December 2011; accepted 25 January 2012)
ISSN 1024-2422 print/ISSN 1029-2446 online © 2012 Informa UK, Ltd.
DOI: 10.3109/10242422.2012.661726

218 J. Köhler & B.Wünsch
(S)
starting material pentane-1,5-diol 3 was prepared
from 3-hydroxyglutarate 1 by silylation with Me2Ph-
SiCl resulting in compound 2, which was subse-
quently reduced with LiBH4. Whereas the racemic
monoacetate rac-4 was produced non-enzymatically
by reaction of diol 3 with 1.1 equivalents of Ac2O,
lipases were used as chiral catalysts for the prepara-
tion of the enantiomerically pure monoacetates
(S)-4 and (R)-4 (Figure 1). The key step of the
synthesis was the enantioselective derivatization of
just one of the two enantiotopic OH-groups of diol
3 or OAc-groups of diacetate 5 (Figure 1).This was
achieved using two different strategies. For produc-
tion of the (S)-configured monoacetate (S)-4 diol
3 was enantioselectively acetylated by the lipase
from B. cepacia. On the other hand, hydrolysis of
diacetate 5 catalysed by the same lipase resulted in
the (R)-configured monoacetate (R)-4, because the
enzyme preferentially transformed the same
enantiotopic ORgroup in both cases (Figure 2).
The lipase-catalysed acetylation of diol 3 has already
been intensively investigated. In Figure 3 (a, b) the
course of the reaction is shown using lipase Amano
PS-CII® from Burkholderia cepacia and isopropenyl
acetate (IPA) as acylating agent in tert-butyl methyl
ether (TBME). Under these reaction conditions the
(S)-configured monoacetate (S)-4 was produced
with Ͼ99.9% enantiomeric excess (ee) and Ͼ50%
yield (Köhler & Wünsch 2006). The lipase Fluka
Lipozyme® from Mucor miehei, however, led to quite
a different reaction course using the same reaction
conditions (Figure 3c, d). Clearly the substrate
selectivity of the lipase from M. miehei changed
during the progress of the reaction. As shown in
Figure 3 (c, d) during time period A the M. miehei
lipase converted only diol 3 but not the monoace-
tates 4.Then, the substrate selectivity of the enzyme
HO
OH
OSiMe2Ph
HO
OAc
OSiMe2Ph
(a)
3
(S)-4
(R)
AcO
OH
AcO
OAc
(b)
OSiMe2Ph
OSiMe2Ph
5
(R)-4
Figure 2. Reaction scheme of the lipase catalysed reactions. (a)
Acetylation of diol 3 using isopropenyl acetate as acylating agent
in tert-butyl methyl ether led to monoacetate (S)-4 in enantiomeric
excess and diacetate 5 as a side product. (b) Hydrolysis of diacetate
5 in aqueous buffer solution gave (R)-4 in enantiomeric excess.
changed and during time period B both diol 3
and monoacetate 4 were converted resulting in the
formation of diacetate 5. This observation was
explained by an allosteric modulation of the lipase
(Köhler & Wünsch 2007). However, the causative
mechanism that is responsible for the change of
selectivity has not been addressed so far.
Methods
Instruments
Thin layer chromatography (tlc) was performed on
Silica gel 60 F254 (E. Merck) and flash chromatog-
raphy (fc) with Silica gel 60, 40–63 μm (E. Merck)
(Still et al. 1978). HPLC-analysis was done using
Merck Hitachi equipment with an UV-Detector
L-7400, Autosampler L-7200, Pump L-7100 and
data acquisition via HSM-Software. Optical rotation
was measured with a Polarimeter 341 from Perkin–
Elmer in a 1.0 dm tube at the given concentration
c [g/100 mL] and 20 °C temperature (the unit
[deg · mL · dmϪ1 · gϪ1] is omitted).
Chemical compounds and lipases
MeO2C
CO2Me
MeO2C
CO2Me
OSiMe2Ph
Me2PhSiCl
CH2Cl2
imidazole
OH
Dimethyl 3-hydroxyglutarate 1 (CAS 7250-55-7)
was purchased from Sigma–Aldrich® and compounds
2, 3, (S)-4 and 5 were synthesised and characterized
as described in the literature (Köhler & Wünsch
2006).The following lipases were also obtained from
Sigma–Aldrich®: Amano AK (Pseudomonas fluore-
scens), Amano PS (Burkholderia cepacia), Amano PS-
CII® (Burkholderia cepacia), Chirazyme L-2 (Candida
antarctica B), Chirazyme L-3 pur. (Candida rugosa),
Chirazyme L-5 (Candida antarctica A), Chirazyme
L-7(Porcinepancreas),FlukaLipase(Candidarugosa)
and Fluka Lipozyme® (Mucor miehei).
/
1
2
R1O
OR2
OSiMe2Ph
HO
OH
LiBH4
Et2O
lipase
compare
Fig. 2
OSiMe2Ph
3
(S)- : R =H,R 2=Ac
1
4
(R)- : R =Ac,R 2=H
1
4
Ac2O CH2Cl2/pyridine
1
2
5
: R =R =Ac
HO
OAc
OSiMe2Ph
rac-4
Monoacetate rac-4 was produced by dissolving
diol 3 (955 mg, 3.75 mmol) in CH2Cl2 (50 mL) and
addition of pyridine (332 μL, 4.13 mmol), acetic
Figure 1. Synthesis of chiral building blocks with a pentane-1,3,5-
triol scaffold.

Conversion of a pentane-1,3,5-triol derivative 219
3
4
5
(S)-4
(a)
(b)
100
80
60
40
20
0
100
99
98
0
20
40
60
80
100
120
140
0
20
40
60
80
100
120
140
Time [h]
Time [h]
100
80
60
40
20
0
100
95
(c)
(d)
time
period B
time
period B
time
period A
time
period A
90
85
0
50
100
150
Time [h]
200
250
0
50
100
150
Time [h]
200
250
Figure 3. Reaction course of the acetylation of diol 3 carried out with lipase from B. cepacia (a, b) and with lipase from M. miehei (c, d);
a, c: Amount of compounds 3, 4 and 5 (n [%]); b, d: Enantiomeric excess of (S)-4 [% ee].
anhydride (390 μL, 4.13 mmol) and a small amount
of 4-(dimethylamino)pyridine. After stirring the reac-
tion mixture for 40 h at room temperature, a saturated
solution of NaHCO3 (50 mL) was added.The aque-
ous layer was separated and extracted with CH2Cl2
(2ϫ30 mL).The combined organic layers were dried
over Na2SO4, concentrated in vacuo and the residue
purified by flash chromatography (Ø 3 cm; h 20 cm;
cyclohexane/EtOAc 1:1; fraction size 10 mL); Rf 0.29;
colourless oil; yield 454 mg (41%). Compounds
(R)-6, (R)-7 and (R)-8 (Figure 5) were synthesized
and the CD-spectrum of (R)-8 (Figure 6) recorded
as described in the literature for the (S)-configured
enantiomers (Köhler & Wünsch 2006).
5ϭ12.4 min; scaling factors 3ϭMe2PhSiOHϭ
4ϭ5ϭ1.00 [n (%)/area (%)].The enantiomeric ratio
of the monoacetates (S)-4:(R)-4 was determined
with the chiral HPLC method 2: Daicel Chiralpak
AD-H (5 μm), 250-4.6 mm; n-hexane/2propanol
51:1, 1 mL/min (after 40 min the column was purged
with n-hexane/2-propanol 9:1 and re-equilibrated.);
λϭ264 nm for 40 min; (S)-4ϭ25.0 min, (R)-4ϭ
27.5 min; scaling factors (S)-4ϭ(R)-4ϭ1.000 [n (%)/
area (%)].
Optimization of the reaction conditions for hydrolysis
of diacetate 5 and screening of lipases
In a 50 mL round-bottomed flask the given amounts
of lipase and diacetate 5 were mixed (Table I
and II). Then the solvent was added and the reac-
tion mixture was stirred intensively with a magnetic
bar at room temperature. The progress of the reac-
tion was monitored by thin layer chromatography
and the reaction stopped after the given time. The
reaction mixture was extracted with CH2Cl2, the
CH2Cl2 layer dried over Na2SO4, the filtrate was
concentrated in vacuo and the residue analysed by
HPLC methods 1 and 2.
HPLC methods
Analytical methods were required for separation and
quantification of the involved compounds 3, (S)-4,
(R)-4 and 5 (Köhler & Wünsch 2006). The ratio of
diol 3:monoacetates 4:diacetate 5 was determined
with the achiral HPLC method 1: Merck LiChro-
spher 100 RP-18e (5 μm), 125-4 mm; acetonitrile/
water 50:50, 1 mL/min; λϭ264 nm for 16 min;
3ϭ1.9 min, Me2PhSiOHϭ3.0 min, 4ϭ4.1 min,

220 J. Köhler & B.Wünsch
Table I. Optimization of the reaction conditions for the hydrolysis of diacetate 5 using lipase Amano PS-CII (B. cepacia) at room temperature.
Lipase
Substrate
Time
Remaining
Reaction conditions
m [mg]
5 m [mg]
[hours]
substrate 5 n [%]
77.3
72.6
67.2
67.9
73.3
144.0
141.5
139.0
205.2
192.6
180.7
182.5
188.3
364.0
376.8
367.6
25
20
17
18
19
22
24
24
93.5
100.0
96.7
95.6
99.9
74.6
51.0
53.8
TBME (20 mL)ϩPB (20 mL)
CH2Cl2 (20 mL)ϩPB (20 mL)
acetone (20 mL)ϩPB (20 mL)
TBME (20 mL)ϩPB (1 mL)ϩH2O (19 mL)ϩCaCO3 (243 mg)
THF (40 mL)ϩH2O (1 mL)ϩNaHCO3 (429 mg)
CB (30 mL)ϩbutan-1-ol (1 mL)
CB (30 mL)ϩpropan-1-ol (1 mL)
CB (30 mL)
PB, phosphate buffer solution (7.5 % Na2HPO4·H2O); CB, carbonate buffer solution (5 % NaHCO3); m [mg]ϭamount of substance
[mg]; n [%]ϭrelative amount of substance [%].
Reaction courses
NaHCO3-solution (5%, 30 mL) at room tempera-
ture. An additional amount of diacetate 5 (181.5 mg,
0.536 mmol) was added to the reaction mixture after
53.0 h. The reaction course shown in Figure 8 was
recorded using the same lipase from Candida rugosa
(151.4 mg) and monoacetate rac-4 (184.8 mg, 0.623
mmol) as substrate in NaHCO3-solution (5%, 15
mL) at room temperature.
The hydrolytic conversions were performed in a
50 mL two-necked flask. After mixing substrate and
lipase, the solvent was added and the reaction mix-
ture stirred intensively with a magnetic bar to give a
homogeneous emulsion. The temperature was kept
constant using a cryostat and samples were taken
through a septum without stopping the stirrer to
ensure that the reaction mixture was emulsified con-
tinuously. Samples of ∼200 μL were taken from the
reaction mixture, saturated with NaCl and extracted
with ∼400 μL of TBME. After separation of the lay-
ers,∼200 μL of the organic layer were filtered through
a membrane filter (0.45 μm) and the solvent evapo-
rated in a nitrogen stream.The residue was dissolved
in 100 μL of acetonitrile for HPLC method 1 and
in 100 μL of n-hexane/2-propanol 9:1 for HPLC
method 2. The course of the reaction displayed in
Figure 4 resulted from reaction of diacetate 5 (360.7
mg, 1.07 mmol) using lipase Amano PS-CII® from
Burkholderia cepacia (360.4 mg) in NaHCO3-solu-
tion (5%, 40 mL) atϩ3 °C. Figure 7 shows the reac-
tion course with diacetate 5 (361.5 mg, 1.07 mmol)
and Fluka lipase from Candida rugosa (360.4 mg) in
Synthesis of monoacetate (R)-4
Production of monoacetate (R)-4 for synthetic use
was carried out in a 1000 mL three-necked flask as
follows. Diacetate 5 (7.18 g, 21.2 mmol) was mixed
with Amano PS Lipase (7.25 g) and NaHCO3-
solution (5%, 800 mL), cooled toϩ3 °C, was added.
The mixture was stirred intensively with a KPG-
stirrer to give a homogeneous emulsion and the tem-
perature kept constant at ϩ3 °C using a cryostat.
The reaction was monitored by taking samples as
described for the reaction course. After 96 h an ee
of Ͼ98% was measured for monoacetate (R)-4 and
the reaction was stopped.The mixture was carefully
extracted with CH2Cl2 (1ϫ300 mL, 2ϫ200 mL),
Table II. Screening of lipases for the hydrolysis of diacetate 5 using standard conditions: 30 mL of NaHCO3-solution (5%) at room
temperature.
Lipase
Lipase m [mg]
Substrate 5 m [mg]
Time [hours]
Product 4 n [%]
ee of 4 [%]
Amano AK
Amano PS
364.1
358.0
357.1
354.3
361.0
357.7
364.1
358.1
372.0
360.1
360.6
362.6
360.5
359.8
358.9
369.6
360.2
358.3
372.2
357.1
16
22
23
23
1
91
20
22
16
46
61.2
52.1
55.7
0.1
20.1
1.5
19.0
33.5
25.2
5.8
84.2 % ee of (R)-4
96.6 % ee of (R)-4
94.6 % ee of (R)-4
Ϫ
Amano PS-CII*
Chirazyme L-2
Chirazyme L-2
Chirazyme L-3 pur.
Chirazyme L-5
Chirazyme L-7
Fluka Lipase
11.2 % ee of (R)-4
5.4 % ee of (S)-4
1.6 % ee of (S)-4
97.0 % ee of (S)-4
4.8 % ee of (R)-4
83.6 % ee of (R)-4
Fluka Lipozyme*
m [mg]ϭamount of substance [mg]; n [%]ϭrelative amount of substance [%]; *immobilized.

Conversion of a pentane-1,3,5-triol derivative 221
3
SiR
4
5
(R)-4
(a)
(b)
100
80
60
40
20
0
100
98
B
A
96
B
A
94
92
0
50
100
Time [h]
150
200
0
50
100
150
200
Time [h]
Figure 4. Reaction course of the hydrolysis of diacetate 5 carried out with lipase from B. cepacia; a: Amount of compounds 3,
SiRϭMe2PhSiOH, 4 and 5 (n [%]); b: Enantiomeric excess of (R)-4 [% ee]; Sample A (27.3 h): 79.2% of 4, 95.4% ee of (R)-4; Sample
B (150.8 h): 21.8% of 4, 99.0% ee of (R)-4.
the combined organic layers were dried over K2CO3,
filtered and concentrated in vacuo. The residue was
purified by flash chromatography (Ø 8 cm; h 20 cm;
cyclohexane/EtOAc 1:1; fraction size 30 mL); Rf
0.29; colourless oil; yield 1.68 g (27%); [α]20D: Ϫ3.7
(cϭ0.83, CH2Cl2); 98.2% ee (HPLC method 2).
lished HPLC methods were used. However, it was
found that in contrast to monoacetates 4 and diacetate
5, diol 3 was not stable under hydrolytic conditions,
as its silyl ether moiety was hydrolysed non-enzymat-
ically during the progress of the reaction. Since diol 3
was only an undesired side-product in this process, its
decomposition was not relevant for the outcome of
the reaction. Nevertheless, it was necessary to deter-
mine the amount of silanol Me2PhSiOH resulting
from decomposition of diol 3 with HPLC method 1
as well, because the amounts of the silylated com-
pounds were calculated as relative amounts, and in
Figures 3, 4, 7 and 8, they are given as percentage of
the total amount of silylated compounds.
Results
Hydrolysis of diacetate 5
Since the lipase catalysed acetylation of diol 3 pro-
vided only the (S)-configured monoacetate (S)-4, the
(R)-configured monoacetate (R)-4 was prepared by
enantioselective hydrolysis of diacetate 5. Diacetate 5
was available in high amounts as a result of the acety-
lation of diol 3 with lipase from B. cepacia and could
also be produced using the lipase Chirazyme L-2 from
Candida antarctica B or by non-enzymatic acylation
with acetic anhydride (Köhler & Wünsch 2006). For
analysis of the hydrolytic transformations the estab-
Reaction conditions
In order to find reasonable conditions for the screen-
ing of different lipases, the reaction conditions were
10
+10
CD
235 nm
Zn(N3)2
PPh3/DIAD
AcO
HO
OH
AcO
N3
0
Δε
0
OSiMe2Ph
OSiMe2Ph
toluene
4
6
(R)-
(R)-
252 nm
-10
–10
3
1.MeOH
K2CO3
2.HCl
0.1M
O
O
N3
N3
4-BrBzCl
CH2Cl2
UV
O
O
OH
245 nm
ε · 10–4
-20
7
(R)-
Br
(R)-8
Br
-30
0
350
200
250
300
200
250
300
350
Figure 5. In order to determine the absolute configuration of
(R)-4 by CD-spectroscopy, (R)-8 was produced as described in
literature for the (S)-configured enantiomer (Köhler & Wünsch
2006).
λ[nm]
Figure 6. CD and UV spectra of (R)-8 in n-hexane.

222 J. Köhler & B.Wünsch
3
SiR
4
5
(S)-4
(R)-4
100
60
55
50
45
40
(a)
(b)
80
60
C
D
40
C
D
20
0
0
10
20
30
Time [h]
40
50
0
10
20
30
Time [h]
40
50
100
80
60
40
20
0
60
55
50
45
40
(c)
(d)
E
F
E
F
53
63
73
83
Time [h]
93
103
53
63
73
83
Time [h]
93
103
Figure 7. Reaction course of the hydrolysis of diacetate 5 carried out with lipase from C. rugosa; c, d: after 53.0 h an additional amount
of diacetate 5 was added (50% of the starting amount); a, c: Amount of compounds 3, SiRϭMe2PhSiOH, 4 and 5 (n [%]); b, d:
Enantiomeric ratio of (S)-4 [%] : (R)-4 [%]; Sample C (1.0 h): 14.1% of 4, 41.7% (S)-4 and 58.3% (R)-4; Sample D (52.8 h): 13.7%
of 4, 52.0% (S)-4 and 48.0% (R)-4; Sample E (61.0 h): 14.0% of 4, 50.9% (S)-4 and 49.1% (R)-4; Sample F (106.5 h): 11.0% of 4,
52.4% (S)-4 and 47.6% (R)-4.
optimized using lipase Amano PS-CII® (Burkholderia
cepacia) for all experiments (Table I). As it is known
that lipases are activated at interfaces, biphasic sys-
tems with organic layers above as well as below the
aqueous phase were tested. However, the substrate
5 was not converted, probably since it was dissolved
in the organic layer and did not come into contact
with the lipase in the aqueous phase (Lines 1ϩ2,
Table I). Although it is miscible with water, acetone
did not lead to better conversion but crystallization
of the buffer salts (Line 3, Table I) and using
solid buffer salts was also ineffective (Lines 4ϩ5,
3
SiR
4
5
(S)-4
(R)-4
(a)
(b) 60
100
80
60
40
20
0
55
50
45
40
G
H
G
H
0
5
10
0
5
10
Time [h]
Time [h]
Figure 8. Reaction course of the hydrolysis of a racemic mixture of the monoacetates rac-4 carried out with lipase from C. rugosa; a:
Amount of compounds 3, SiRϭMe2PhSiOH, 4 and 5 (n [%]); b: Enantiomeric ratio of (S)-4 [%]:(R)-4 [%]; Sample G (3.0 h): 36.4%
of 4 (52.5% (S)-4 and 47.5% (R)-4) and 12.8% of 5; Sample H (11.2 h): 14.0% of 4 (51.5% (S)-4 and 48.5% (R)-4).

Conversion of a pentane-1,3,5-triol derivative 223
Table I). Small amounts of additives (butan-1-ol,
propan-1-ol) had a slight benefit for acceleration of
the reaction rate but entail the risk of producing side
products and are not really necessary (Lines 6, 7, 8,
Table I). The enzyme itself led to sufficient emulsi-
fication of the substrate. Therefore, the ratio
enzyme:substrate was increased to 1:1 for further
experiments (Table II). NaHCO3-solution (5%) was
preferred as buffer solution, since the pH-value was
kept more stable during the reaction process than
was possible using phosphate buffer.
of (R)-4 with an ee Ͼ98% and the yield was of minor
importance, large scale conversions were carried out
until the monoacetate (R)-4 reached Ͼ98% ee. Fur-
thermore, comparison between experiment C in
Table II at ϩ20 °C and the experiment shown in
Figure 4 using the same reaction conditions at ϩ3
°C demonstrate the benefit of lowering the reaction
temperature for the selectivity of the lipase. In exper-
iment C the reaction was faster due to the higher
temperature. The ee of (R)-4, however, was only
94.6% at a concentration of 55.7% of monoacetate
4. In Figure 4, at a concentration of about 56% of
monoacetate 4 the ee of (R)-4 was about 97.7%.
Preparative synthesis of monoacetate (R)-4 for
synthetic use was performed with up to 20 mmol of
diacetate 5 and resulted in an ee of 98.2% (R)-4.The
absolute configuration of monoacetate (R)-4 was
determined by CD-spectroscopy as described in
the literature for the (S)-configured enantiomer
(Köhler & Wünsch 2006). For this, (R)-4 was con-
verted to the azide (R)-6 by a Mitsunobu reaction
and subsequently hydrolysed to give azidodiol (R)-7,
which was acylated with 4-bromobenzoyl chloride
resulting in dibenzoate (R)-8 (Figure 5). The CD-
spectrum of (R)-8 shown in Figure 6 is the mirror
image of the corresponding enantiomer (S)-8. Fur-
thermore, the bisignate curve of the spectrum that
results from exciton-coupling of the benzoyl chro-
mophores is in accordance with analogous (R)-
configured compounds (Soriente et al. 1995; Harada
et al. 1991).
Screening of lipases
Diacetate 5 was hydrolysed using different lipases
in NaHCO3-solution atϩ20 °C. The results are
given inTable II. Most of the lipases that were inves-
tigated produced an ee of the desired (R)-configured
monoacetate (R)-4. Lipase Chirazyme L-7 from
Porcine pancreas, however, led to formation of the
(S)-configured monoacetate (S)-4 in high ee. This
was an interesting result, since it demonstrates
that both enantiomers could theoretically be pro-
duced hydrolytically using different enzymes. Lipase
Chirazyme L-2 (Candida antarctica B) led to a very
fast but non-selective hydrolysis.The highest enanti-
oselectivity for the production of the (R)-configured
monoacetate (R)-4 was found for the lipase from
B. cepacia in pure form and immobilized on ceramic
particles (C, Table II). Due to the better emulsifica-
tion of the reaction mixture the native enzyme was
used for further experiments.
Hydrolytic conversions using lipase from C. rugosa
Synthesis of monoacetate (R)-4
Within the screening process two lipase prepara-
tions originating from the same organism Candida
rugosa were investigated (Chirazyme L-3 pur. and
Fluka Lipase). Unexpectedly, Chirazyme L-3 pur.
gave an ee of (S)-4 and Fluka Lipase an ee of (R)-4
(Table II). Since it is known that commercial prep-
arations of crude lipase from C. rugosa usually
consist of a mixture of several hydrolases (Lalonde
et al. 1995; Brocca et al. 1998), the question arose
whether the enantioselectivity of the preparations
was different or changed during the progress of the
reaction. Due to its higher activity, Fluka Lipase was
used for all further experiments. The course of the
hydrolysis of diacetate 5 catalysed by this prepara-
tion is shown in Figure 7 (a, b). At the beginning,
an excess of (R)-4 was generated (Sample C).Then,
during the reaction an increasing amount of (S)-4
was produced and the ee switched from an excess
of (R)-4 to an excess of (S)-4 (Sample D). In order
to demonstrate that this phenomenon did not result
from altered reaction conditions, additional diacetate
5 (50% of the starting amount) was added to the
In order to find the optimal reaction time, the course
of the hydrolytic conversion of diacetate 5 was
recorded using lipase Amano PS from Burkholderia
cepacia. The reaction temperature was lowered
fromϩ20 °C toϩ3 °C, since in a previous study it
was shown that lowering the reaction temperature
led to increased enantioselectivity of the lipase dur-
ing acetylation (Köhler & Wünsch 2006). Figure 4
demonstrates that the hydrolysis of diacetate 5 gave
monoacetate (R)-4 very rapidly at the beginning of
the reaction. Sample A was taken after 27.3 h and
contained 79.2% of monoacetate 4 with an ee of
95.4% of (R)-4. Due to hydrolysis of the second ester
group and since the lipase hydrolysed (S)-4 faster
than (R)-4, the ee of (R)-4 increased during the
course of the reaction and reached 99.0% ee after
150.8 h (sample B). For the same reason, however,
the total amount of monoacetate 4 was rather low at
that time (21.8%). Stopping the reaction after a short
time would give a high yield (almost 80%, sample A)
with an ee of ∼95%. Since our aim was the production

224 J. Köhler & B.Wünsch
reaction mixture (Figure 7c, d). As a result (R)-4
was preferentially produced again (Sample E), then
in the course of the reaction, the enantiomer (S)-4
(sample F).
An additional experiment with Fluka lipase from
C. rugosa was performed. Instead of diacetate 5, a
racemic mixture of the monoacetates rac-4 was used
for hydrolysis (Figure 8). Contrary to expectations,
a substantial amount of diacetate 5 was produced
(sample G) indicating that monoacetate 4 was not
only hydrolysed but also acylated in aqueous medium
due to transesterfication. Furthermore, the ee did
not steadily increase.
several hydrolases as most commercial preparations
of crude lipase from C. rugosa (Lalonde et al. 1995;
Brocca et al. 1998). One of the isoenzymes may con-
vert diacetate 5 faster than monoacetates 4 and may
preferentially give (R)-4 at the beginning of the reac-
tion. Another isoenzyme may catalyse the second
step of the hydrolysis from mono-acetate 4 to diol 3
faster than the first step and with opposite and higher
enantiopreference. Hence, in combination, the phe-
nomenon observed during course of the reaction
(Figure 7) could theoretically result from a coopera-
tion of these isoenzymes. The reaction course of the
acetylation of diol 3 using the lipase from M. miehei
(Figure 3c, d), however, cannot be explained by the
existence of isoenzymes,since the lipase (Lipozyme®)
was expressed as a single enzyme in Aspergillus oryzae
available from Novo Nordisk A/S Corp.(Rodrigues
& Fernandez-Lafuente 2010).Therefore, we suggest
that the switch of enantioselectivity observed for the
lipase preparation from C. rugosa as well as the
change of substrate selectivity observed for the lipase
from M. miehei may result from an connatural move-
ment of the lid partially covering the stereoselectivity
pocket of the active site. Verification of this hypoth-
esis, however, will need a wide range of further
kinetic investigations and biochemical studies.
Discussion
Synthesis of monoacetate (R)-4
Whereas (S)-configured pentane-1,3,5-triol deriva-
tives are available by acetylation with lipase from
B. cepacia in organic solvent (Köhler & Wünsch
2006), the same lipase can be used to produce the
(R)-configured enantiomers in aqueous buffer solu-
tion, because the enzyme preferentially transforms
the same enantiotopic group in organic as well as in
aqueous solution. Though lipases are activated at
interfaces, the use of biphasic systems did not lead to
hydrolysis. It was necessary to emulsify the substrate
in aqueous solution. This was achieved using NaH-
CO3-solution (5%) and intensive stirring.The hydro-
lysis of diacetate 5 provided monoacetate (R)-4 in
high ee.The decisive factor for a successful synthesis,
however, is finding the optimal reaction time, since
the possible yield as well the ee alters to a large extent
during the progress of the reaction (Figure 4).
Declaration of interest: The authors report no
declarations of interest.The authors alone are respon-
sible for the content and writing of the paper.
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