Enantioselective hydrolyses with Yarrowia lipolytica: A versatile strain for esters, enol esters, epoxides, and lactones

Article abstract of DOI:10.1016/S0957-4166(01)00463-3

Racemic secondary esters 1-3, γ-lactones 8-9, and styrene oxide 7 are kinetically resolved via hydrolysis with Yarrowia lipolytica YL2 strain. The enantioselective hydrolysis of prochiral enol esters 4-6 to the corresponding homochiral carbonyl compounds 13-15 is also described. Subsequent reduction of the ketone 13 and of the aldehyde 15 can be avoided using lyophilised cells.

Full text of DOI:10.1016/S0957-4166(01)00463-3

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 12 (2001) 2709–2713
Enantioselective hydrolyses with Yarrowia lipolytica: a versatile
strain for esters, enol esters, epoxides, and lactones
Giancarlo Fantin, Marco Fogagnolo, Alessandra Guerrini, Alessandro Medici,* Paola Pedrini and
Silvia Fontana
Dipartimento di Chimica, Universita` di Ferrara, Via L. Borsari 46, I-44100 Ferrara, Italy
Received 18 September 2001; accepted 19 October 2001
Abstract—Racemic secondary esters 1–3, g-lactones 8–9, and styrene oxide 7 are kinetically resolved via hydrolysis with Yarrowia
lipolytica YL2 strain. The enantioselective hydrolysis of prochiral enol esters 4–6 to the corresponding homochiral carbonyl
compounds 13–15 is also described. Subsequent reduction of the ketone 13 and of the aldehyde 15 can be avoided using
lyophilised cells. © 2001 Elsevier Science Ltd. All rights reserved.
1. Introduction
2. Results and discussion
Among biocatalyzed reactions the hydrolytic transfor-
mations involving amide and ester bonds are most easy
to perform and are widely used to obtain homochiral
secondary alcohols by proteases, esterases and most of
all lipases.1 Other types of applications of hydrolytic
enzymes involving the cleavage of phosphate esters,
nitriles and epoxides are generally more complicated.2
Whole microbial cells have also been used to catalyse
some esterolytic reactions: interesting examples can be
found for both bacteria and fungi, i.e. Bacillus subtilis,3
Brevibacterium ammoniagenes,4 Bacillus coagulans,5
Pichia miso,6 Rhizopus nigricans,7 and Rhizopus oryzae.8
On the other hand, the examples of microbial hydroly-
sis of epoxides9–12 and nitriles13–15 are not so common,
while only a few papers describe the hydrolytic conver-
sions by baker’s yeast,16–18 although the use of this
microorganism is very simple and obviates the need for
sterile fermentation equipment. Herein, we describe the
very efficient microbial hydrolysis of compounds 1–9
with Yarrowia lipolytica strains (Scheme 1). These
microorganisms are yeasts, distributed over a wide
range of food systems, that use oils and fats as the sole
carbon source and previously have been efficiently
employed in the enantioselective reduction of prochiral
ketones19 and in the kinetic resolution of racemic sec-
ondary alcohols via oxidation.20
Seventeen strains of Y. lipolytica19 have been employed
in hydrolytic screening of compounds 1–9 and the
results have been monitored by GLC. These prelimi-
nary results have focused our attention on Y. lipolytica
YL2, that had been the strain of choice in the oxidation
of secondary alcohols20 and that, also in this case, show
higher efficiency in the hydrolysis of all substrates.
Most of the hydrolyses with Y. lipolytica YL2 are
repeated on preparative scale and the best results, both
on analytical and preparative scale, are summarised in
Table 1.
Scheme 1.
* Corresponding author. E-mail: mda@dns.unife.it
0957-4166/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(01)00463-3

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G. Fantin et al. / Tetrahedron: Asymmetry 12 (2001) 2709–2713
Table 1. Hydrolysis of compounds 1–9 with Y. lipolytica YL2

G. Fantin et al. / Tetrahedron: Asymmetry 12 (2001) 2709–2713
2711
The hydrolysis of the endo-bicyclo[3.2.0]hept-2-en-6-ol
acetate 1, after 1 h incubation with growing cells of Y.
lipolytica YL2, produces the (1S,5R,6S)-endo-alcohol
10 in 50% yield (ee 60%) and leaves the (1R,5S,6R)-
endo-acetate 1 (50%, ee 67%). The same acetate is
recovered enantiomerically pure (21%) after 2 h incuba-
tion and the hydrolysis is complete after 4 h. The
screening reveals, however, two strains, i.e. Y. lipolytica
YL4 and YL10, that give the opposite enantiomer
(1R,5S,6R)-endo-alcohol 10 in 32% yield (ee 100%, 6 h
incubation) and in 46% yield (ee 66%, 4 h incubation),
respectively. Obviously in both cases the (1S,5R,6S)-
endo-acetate 1 is recovered in 68% yield (ee 47%) and in
45% yield (ee 34%), respectively.
buffer at pH 7. After 30 min incubation the (S)-alde-
hyde 15 was obtained (76% yield, ee 57%) and no
reduction product was detected even after 1 h (100%
yield, ee 34%). In order to avoid the easy racemization,
the absolute configuration of the aldehyde was deter-
mined by reduction to the corresponding (S)-alcohol
16.
Styrene oxide 7 was totally hydrolysed after 2 h incuba-
tion with growing cells of Y. lipolytica YL2 in
Sabouraud. On the contrary, the reaction carried out
with the cells, suspended in phosphate buffer at pH 7,
leaves, after 8 h, the (R)-styrene oxide 7 (57% yield, ee
20%). Obviously when only the 22% of the starting
product remains (24 h) the enantiomeric excess of the
(R)-isomer increases (52%).
Y. lipolytica YL2 is also efficient in the hydrolysis of
racemic cis- and trans-2-methylcyclohexanol acetates 2
and 3: the rate is different but both racemic acetates
afford the 1S-alcohols 11 and 12 leaving the 1R-
acetates unreacted. In particular the (1R,2S)-cis acetate
2 is resolved after 18 h incubation (yield 44%, ee 95%)
producing the (1S,2R)-alcohol 11 (56%, ee 60%), while
the (1R,2R)-trans-acetate 3 is obtained in 44% yield
only after 2 h but with a lower enantiomeric excess
(42%): in this case the (1S,2S)-alcohol 12 (56%, ee 39%)
is produced.
The hydrolysis, moreover, of the a-methyl-g-butyrolac-
tone 8 with Y. lipolytica YL2 kinetically resolves (48 h
incubation) the (S)-enantiomer (48% yield, ee 72%). On
the other hand, the hydrolysis of the a-bromo deriva-
tive 9 is faster but less selective. After 2 h incubation
the (S)-enantiomer 9 was recovered in 56% yield but
with low enantiomeric excess (12%) and with 36% yield
(ee 22%) after 4 h.
On the other hand, 1-acetoxy-2-methylcyclohexene 4 is
enantioselectively hydrolysed by Y. lipolytica YL2 pro-
ducing, after 2 h incubation, (S)-2-methylcyclohex-
anone 13 (78% yield, ee >95%) together with the pure
reduction product (1S,2R)-cis-2-methylcyclohexanol 11
(22%). Under these conditions (Sabouraud medium) the
reduction cannot be avoided because the ratio between
the hydrolysis and reduction product is also the same
after 10 min incubation and keeps uniform during the
biotranformation. In order to obtain only the (S)-
ketone, Y. lipolytica YL2 cells were harvested by cen-
trifugation and suspended in phosphate buffer at pH 6,
7 and 8. The incubation at pH 7 gave the best result but
similar (see Table 1) to that obtained in Sabouraud,
while in the other cases only lower yields were obtained.
The cis-alcohol 11 was not produced carrying out the
hydrolysis only with lyophilised cells of Y. lipolytica
YL2 suspended in phosphate buffer at pH 6: the enan-
tiomeric excess of the (S)-2-methylcyclohexanone 13
(91%, ee 53%) was lower, while at pH 7 it increased (ee
64% of S-13) but again the cis-alcohol 11 (15%, ee
100%) was obtained. No reduction product was
detected in the Y. lipolytica hydrolysis of 1-acetoxy-2-
methylcyclopentene 5: the reaction is very fast and
affords, after 30 min incubation, only the (R)-ketone 14
with low enantiomeric excess (100% yield, ee 22%). The
ee does not change during the biotranformation.
3. Conclusion
In this screening the yeast strain Y. lipolytica YL2,
already amply utilized in reduction of prochiral car-
bonyls and in oxidation of racemic secondary alcohols,
has showed a great versatility, efficiency and aptitude
for hydrolysing various substrates. The possibility of
utilizing lyophilised cells with only minor lack of activ-
ity will be studied in order to use this strain in organic
solvents both in hydrolysis and in esterification. Y.
lipolytica YL2 appears as a candidate to become a valid
choice towards hydrolytic enzymes.
4. Experimental
The acetyl derivatives of the commercially available
endo-bicyclo[3.2.0]hept-2-en-6-ol,
cis-2-methyl-cyclo-
hexanol, and trans-2-methylcyclohexanol were prepared
with acetic anhydride and pyridine. 1-Acetoxy-2-
methylcyclohexene and 1-acetoxy-2-methylcyclopentene
were prepared by treatment of the corresponding
ketone with perchloric acid and acetic anhydride.21
2-Phenyl-2-propen-1-ol acetate was prepared from the
corresponding aldehyde with acetic anhydride, potas-
sium carbonate and sodium acetate.22 Styrene oxide,
a-methyl- and a-bromo-g-butyrolactone, and 4-methyl-
cyclohexanone are commercially available. The fermen-
tation was obtained in a Bioindustrie Mantovane
bioreactor Model BM 3000 CLP. Lyophilization was
achieved with an Edwards E-C Modulyo lyophilizator.
However, the hydrolysis of 2-phenyl-2-propen-1-ol acet-
ate 6 to the corresponding aldehyde 15 (54%) followed
by reduction to 2-phenyl-1-propanol 16 (31%) was
practically immediate (30 s). In fact, after 20 min
incubation only 6% of (S)-aldehyde 15 was detected (ee
28%) together with the racemic alcohol 16 (92%). Also
in this case, the reduction was inhibited using
lyophilised cells of Y. lipolytica YL2 in phosphate
4.1. Enantiomer separation
Gas chromatographic analyses were performed on a
Carlo Erba GC 6000 Vega series 2. Enantiomer separa-

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G. Fantin et al. / Tetrahedron: Asymmetry 12 (2001) 2709–2713
4.3. Screening of hydrolysis with Y. lipolytica strains of
compounds 1–9 on analytical scale. General procedure
tion for compound 1 was achieved on Megadex 5
column (25 m×0.25 mm) containing n-pentyl dimethyl
b-cyclodextrin in OV 1701 from Mega s.n.c.: carrier
gas: helium 82 kPascal. Enantiomer separation for
compounds 2–9 was achieved on a Megadex DETTBSb
column (25 m×0.25 mm) containing diethyl-tert-butyl-
silyl b-cyclodextrin in OV 1701 from Mega s.n.c.: car-
rier gas: helium 100 kPascal. The yield of the reaction
on analytical scale was determined by GC using 4-
methylcyclohexanone as internal standard.
The sterilized (120°C for 20 min) culture medium
(Sabouraud, 10 mL), containing glucose (40 g/L) and
peptone (10 g/L), was inoculated with a loopful of the
selected Y. lipolytica and grown for 48 h at 28°C. To
the culture was added the selected substrate solution
(100 mL, 10 mg) (the solution was prepared dissolving
0.1 g of the selected substrate in 1 mL of DMF).
Aliquots were withdrawn at 2, 4, 6, 24 h, extracted with
diethyl ether, dried over anhydrous Na2SO4 and moni-
tored by GLC on chiral column using 4-methylcyclo-
hexanone as internal standard. In Table 1 are reported
the results obtained with Y. lipolytica YL2 that is the
more efficient strain in the hydrolysis of all screened
substrates.
For the hydrolysis of 1, temperature 90–200°C (1°C/
min), retention time (min): (1R,5S,6R)-10, 19.04;
(1S,5R,6S)-10, 19.29; (1S,5R,6S)-1, 20.50; (1R,5S,6R)-
1, 22.30. For the hydrolysis of 2, temperature 70–200°C
(1.5°C/min), retention time (min): 11, 10.31; (1R,2S)-2,
10.77; (1S,2R)-2, 12.01. The enantiomeric excess of the
cis-alcohol 11 was calculated after separation and
acetylation with acetic anhydride and pyridine. For the
hydrolysis of 3, temperature 70–200°C (1.5°C/min),
retention time (min): (1S,2S)-12, 9.60; (1R,2R)-12, 9.78;
(1R,2R)-3, 10.61; (1S,2S)-3, 10.78. For the hydrolysis
of 4, temperature 70–200°C (1.5°C/min), retention time
(min): (S)-13, 8.27; (R)-13, 8.53; 11, 10.48; 4, 13.99. For
the hydrolysis of 5, temperature 80–200°C (1°C/min),
retention time (min): (S)-14, 3.93; (R)-14, 4.17; 5, 6.45.
For the hydrolysis of 6, temperature 100–200°C (5°C/
min), retention time (min): (S)-15, 5.10; (R)-15, 5.23;
16, 7.18; 6, 9.28. For the hydrolysis of 7, temperature
100°C, retention time (min): (R)-7, 5.96; (S)-7, 6.48.
For the hydrolysis of 8, temperature 100°C, retention
time (min): (R)-8, 4.64; (S)-8, 5.36. For the hydrolysis
of 9, temperature 90–200°C (5°C/min), retention time
(min): (R)-9, 9.07; (S)-9, 9.43.
Some biotransformations were carried out suspending
the cells harvested by centrifugation in phosphate
buffer (10 mL) (see Table 1).
4.4. Hydrolysis of compounds 1–4, 6, 8 with Y. lipolytica
YL2 on preparative scale. General procedure
The reaction was carried out as above starting from 200
mL of the culture medium and 0.2 g of the substrate in
2 mL of DMF (see Table 1). After the appropriate time
(monitored by GLC) the reaction mixture was extracted
with diethyl ether (200 mL) by a continuous liquid
extractor and dried over anhydrous Na2SO4. The crude
reaction products and the enantiomeric excesses were
analysed by GLC. Chromatography of the crude reac-
tion mixture (silica gel, cyclohexane/diethyl ether,
80:20) gave the purified products (see Table 1).
4.5. Lyophilisation of Y. lipolytica cells
The absolute configurations of the compounds were
determined comparing the sign of their specific rotation
with those of the literature: for (1S,5R,6S)-123 [h]D=
−36.3 (c 2.27, CHCl3); for (1R,5S,6R)-1024 [h]D=−68 (c
1.1, CHCl3); for (1R,2S)-225 [h]D=−37.4 (c 1.50,
CHCl3); for (1S,2R)-1126 [h]D=18 (c 1.0, MeOH); for
(1S,2S)-327 [h]D=69.9 (c 0.64, EtOH); for (1R,2R)-1228
[h]D=−38.2 (c 9.6, EtOH); for (R)-1326 [h]D=14 (c
0.23, MeOH); for (S)-1628 hD=−17 (neat); for (S)-729
hD=−33 (neat); for (R)-830 [h]D=21.2 (c 8.6, EtOH).
The absolute configurations of compounds 931 and 1432
were assigned comparing the signs of their specific
rotation with those of the literature: (R)-(−)-9 and
(R)-(−)-14.
The sterilized (120°C for 20 min) culture medium (10
mL), containing glucose (40 g/L) and peptone (10 g/L),
was inoculated with a loopful of Y. lipolytica YL2 and
grown for 24 h at 28°C. This culture (1 mL) was
inoculated in 50 mL of the same broth (freshly steril-
ized) and grown for a further 24 h at 28°C. The last
culture (50 ml) was added to the same culture medium
(2 L) in a bioreactor and the grown is continued for
further 48 h at 28°C monitoring the following parame-
ters: pH range 2–9; O2 pressure, 30 mmHg; gas flow,
1.0 Nl/min; stirring 300–900 rpm. The cells (43 g) were
harvested by centrifugation (5 min, 9000 rpm) and
saccharose 20% (140 mL) was added. The suspension
was frozen and lyophilised to obtain 37 g of lyophilised
cells.
4.2. Microorganisms
Y. lipolytica strains19 were isolated from various habi-
tats and belong to DPVA (Dipartimento di Protezione
4.6. Hydrolysis of 1-acetoxy-2-methylcyclohexene 4 with
lyophilised cells of Y. lipolytica YL2
e
Valorizzazione Agroalimentare, University of
Bologna, Italy). Seventeen strains were tested in the
oxidation of racemic alcohols: YL1 (Y2), YL2 (Y9),
YL4 (Y21), YL5 (PO5), YL6 (Y5), YL7 (RO3), YL8
(1A), YL9 (PO19), YL10 (Y10), YL12 (RO13), YL13
(RO18), YL14 (RO21), YL15 (16B), YL16 (27D),
YL17 (PO6), YL18 (PO17), YL19 (PO23).33
Y. lipolytica lyophilised cells (1 g) in phosphate buffer
pH 7 (10 mL) were stirred for about 10 min and then
the enol acetate solution (100 mL, 10 mg) (the solution
was prepared dissolving 0.1 g of the substrate in 1 mL
of DMF) was added. After 2 h incubation at 28°C, the
suspension was extracted with diethyl ether and dried

G. Fantin et al. / Tetrahedron: Asymmetry 12 (2001) 2709–2713
2713
over anhydrous Na2SO4. GLC analysis on chiral
column using 4-methylcyclohexanone as internal stan-
dard showed the presence of (S)-methylcyclohexanone
13 in 85% yield (ee 64%), together with the pure
(1S,2R)-cis methylcyclohexanol 11 (15% yield). The
reaction was repeated in phosphate buffer at pH 6 and
after 2 h only the (S)-ketone 13 was obtained (91%
yield, ee 53%).
8. Demir, A. S.; Hamamci, H.; Tanyeli, C.; Akhmedov, I.
M.; Doganel, F. Tetrahedron: Asymmetry 1998, 9, 1673.
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639.
10. Weijers, C. A. G. M.; Botes, A. L.; van Dyk, M. S.; de
Bont, J. A. M. Tetrahedron: Asymmetry 1998, 9, 467.
11. Goswami, A.; Totleben, M. J.; Singh, A. K.; Patel, R. N.
Tetrahedron: Asymmetry 1999, 10, 3167.
12. Botes, A. L.; Weijers, C. A. G. M.; Botes, P. J.; van Dyk,
M. S. Tetrahedron: Asymmetry 1999, 10, 3327.
13. Crosby, J.; Moilliet, J.; Parratt, J. S.; Turner, N. J. J.
Chem. Soc., Perkin Trans. 1 1994, 1679.
4.7. Hydrolysis of 2-phenyl-2-propen-1-ol acetate 6 with
lyophilised cells of Y. lipolytica YL2
14. Meth-Cohn, O.; Wang, M.-X. J. Chem. Soc., Perkin
Y. lipolytica lyophilised cells (10 g) in phosphate buffer
pH 7 (100 mL) were stirred for about 10 min and then
the enol acetate 6 (0.1 g) in 1 mL of DMF was added.
After 30 min and usual work up, GLC analysis on
chiral column using 4-methylcyclohexanone as internal
standard showed the presence of the (S)-aldehyde 15 in
76% yield (ee 57%). After 1 h the hydrolysis was
complete (100% yield, ee 34%) with no reduction
product.
Trans. 1 1997, 1099.
15. Matoishi, K.; Sano, A.; Imai, N.; Yamazaki, T.;
Yokoyama, M.; Sugai, T.; Ohta, H. Tetrahedron: Asym-
metry 1998, 9, 1097.
16. Servi, S. Synthesis 1990, 1.
17. Csuk, R.; Gla¨nzer, B. I. Chem. Rev. 1991, 91, 49.
18. Fouche´, G.; Horak, R. M.; Meth-Cohn, O. J. Chem.
Soc., Chem. Commun. 1993, 119.
19. Fantin, G.; Fogagnolo, M.; Giovannini, P. P.; Medici,
A.; Pedrini, P.; Gardini, F.; Lanciotti, R. Tetrahedron
1996, 52, 3547.
The absolute configuration of the aldehyde 15 was
determined by reduction to the corresponding alcohol
16. The suspension obtained after 1 h incubation was
extracted with ethyl acetate. The organic layer was
dried over anhydrous Na2SO4 and the solvent was
removed under vacuum. The crude reaction mixture
was dissolved in methanol (5 mL) and NaBH4 (1.5
equiv.) was added at 0°C. Usual work and chromatog-
raphy (silica, petroleum ether/diethyl ether, 8:2)
afforded the pure alcohol 16 in quantitative yield.
20. Fantin, G.; Fogagnolo, M.; Medici, A.; Pedrini, P.;
Fontana, S. Tetrahedron: Asymmetry 2000, 11, 2367.
21. Gall, M.; House, H. Org. Synth. Vol. 1 1988, 121.
22. Barbier, P.; Benezra, C. J. Org. Chem. 1983, 48, 2705.
23. Klempier, N.; Geymayer, P.; Stadler, P.; Faber, K.;
Griengl, H. Tetrahedron: Asymmetry 1990, 1, 111.
24. Fantin, G.; Fogagnolo, M.; Medici, A.; Pedrini, P.;
Rosini, G. Tetrahedron: Asymmetry 1994, 5, 1635.
25. Naemura, K.; Fukuda, R.; Murata, M.; Konishi, M.;
Hirose, K.; Tobe, Y. Tetrahedron: Asymmetry 1995, 6,
2385.
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