Biotransformation of aromatic ketones and ketoesters with the non-conventional yeast Pichia glucozyma

Article abstract of DOI:10.1016/j.tetlet.2014.10.133

The non-conventional yeast Pichia glucozyma CBS 5766 has been used for the biotransformation of different aromatic ketones and ketoesters. The growth and biotransformation conditions were optimised for the reduction of acetophenone and under optimised conditions, propiophenone, butyrophenone and valerophenone were reduced to the corresponding (S)-alcohols with high yields and enantioselectivity. Ketoreductase(s) of Pichia glucozyma showed high catalytic activities also towards aromatic β- and γ-ketoesters, being often competitive with esterase(s). These concurrent activities allowed for the preparation of hydroxyesters, hydroxyacids and lactones often in a very selective manner.

Full text of DOI:10.1016/j.tetlet.2014.10.133

Accepted Manuscript
Biotransformation of aromatic ketones and ketoesters with the non-conventional
yeast Pichia glucozyma
Martina Letizia Contente, Francesco Molinari, Paolo Zambelli, Valerio De
Vitis, Raffaella Gandolfi, Andrea Pinto, Diego Romano
PII:
S0040-4039(14)01851-6
DOI:
http://dx.doi.org/10.1016/j.tetlet.2014.10.133
TETL 45362
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 August 2014
21 October 2014
27 October 2014
Please cite this article as: Contente, M.L., Molinari, F., Zambelli, P., Vitis, V.D., Gandolfi, R., Pinto, A., Romano,
D., Biotransformation of aromatic ketones and ketoesters with the non-conventional yeast Pichia glucozyma,
Tetrahedron Letters (2014), doi: http://dx.doi.org/10.1016/j.tetlet.2014.10.133
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical Abstract
Biotransformation of aromatic ketones and
ketoesters with the non-conventional yeast
Pichia glucozyma
Leave this area blank for abstract info.
Martina Letizia Contente, Francesco Molinari, Paolo Zambelli , Valerio De Vitis, Raffaella Gandolfi, Andrea
Pinto, Diego Romano

1
Tetrahedron Letters
journal homepage: www.els evier.com
Biotransformation of aromatic ketones and ketoesters with the non-conventional yeast
Pichia glucozyma
Martina Letizia Contente a, Francesco Molinaria, Paolo Zambellia, Valerio De Vitisa, Raffaella Gandolfib,
Andrea Pintob, Diego Romanoa*
a Department of Food, Environmental and Nutritional Sciences (DEFENS), University of Milan, via Mangiagalli 25, 20133 Milan, Italy.
b Department of Pharmaceutical Sciences (DISFARM), University of Milan, via Mangiagalli 25, 20133 Milan, Italy.
ARTICLE INFO
ABSTRACT
Article history:
Received
The non-conventional yeast Pichia glucozyma CBS 5766 has been used for the
biotransformation of different aromatic ketones and ketoesters. The growth and
biotransformation conditions were optimised for the reduction of acetophenone and, under
optimised conditions, propiophenone, butyrophenone and valerophenone were reduced to the
corresponding (S)-alcohols with high yields and enantioselectivity. Ketoreductase(s) of Pichia
glucozyma showed high catalytic activities also towards aromatic ꢀ- and ꢁ-ketoesters, being
often competitive with esterase(s). These concurrent activities allowed for the preparation of
hydroxyesters, hydroxyacids, lactones often in a very selective manner.
Received in revised form
Accepted
Available online
Keywords:
Aromatic ketones
Aromatic ketoesters
Reduction
2014 Elsevier Ltd. All rights reserved
.
Enantioselective
Ketoreductase
Esterase
The asymmetric reduction of prochiral ketones using isolated
or cell-bound ketoreductases is a well-recognized method for the
preparation of chiral alcohols.1 The major drawback encountered
with enzymatic reduction using isolated enzymes is the necessity
for cofactor recycling, which can be circumvented by two-
enzyme or one-enzyme recycling methodologies.2 Microbial
whole cells exhibit as a major advantage that cofactors are
already present and can be recycled via the oxidation of a second
substrate (i.e., glucose or glycerol).3 Beside the easily available
Saccharomyces cerevisiae,4 other microbial species have been
widely employed for asymmetric reductions or more generally as
sources for new selective ketoreductases.5 However, there is still
a need for new biocatalysts able to perform stereoselective
reductions with different or ameliorated chemo-, regio- and
stereoselectivity, especially towards bulky substrates.6 Non-
conventional yeasts are plentiful sources of different carbonyl
reductases and during our past works on enantioselective
carbonyl reduction we have found that whole cells of the yeast
Pichia glucozyma CBS 5766 (now reclassified as Ogataea
glucozyma) catalysed the stereoselective reduction of different
ketones, often showing remarkable results in terms of
stereoselectivity.7 In this work, we have studied in details the
potential of whole cells of Pichia glucozyma CBS 5766 for the
biotransformation of various aromatic ketones and ketoesters
under optimised conditions.
Reduction of acetophenone (1a) was firstly studied with
whole cells of Pichia glucozyma grown using different culture
media. Experiments were performed with cells harvested after 24
and 48 hours and suspended in phosphate buffer (0.1 M, pH 7) in
the presence of 5% glucose. The best results were observed when
P. glucozyma was grown in a medium composed with malt
extract and 0.5% yeast extract for 24 hours: reduction of 1a with
Pichia glucozyma grown under these conditions, furnished (S)-1-
phenylethanol (2a) with good enantioselectivity (92%) and yield
(70%). The conditions of the sequential experimental trials aimed
at the optimization of the biotransformation conditions were
selected employing the Multisimplex® 2.0 software;8 control
variables were biocatalyst and substrate concentrations, pH and
temperature. The best results in terms of yields and
enantioselectivity were observed employing 25 mg of dry cells
and 20 mM substrate concentration, in the presence of glucose
(277 mM, 50 g/L) at pH 7.0 and 28 °C; whole cell based specific
activity towards acetophenone under these conditions was 1.33
mmol/gdry cells min (see Supplementary data for definition). The
influence of different co-substrates (ethanol, glucose, glycerol,
sucrose xylose at 50 g/L) was independently assayed. The results
obtained using glucose, glycerol and xylose were very similar,
while a slight decrease of enantioselectivity was detected with
ethanol.
Reduction of acetophenone (1a), propiophenone (1b),
butyrophenone (1c) and valerophenone (1d) under optimised
conditions (resting cells 20 mgdry weight/ml suspended in phosphate
buffer 0.1 M, pH 7 in the presence of 50 g/L of glucose) is
reported in Table 1.9 Reductions occurred with moderate to high
conversion and with reaction rates increasing when
*Corresponding author.Tel. +39 0250319134/fax: +39 0250319191
E-mail address: diego.romano@unimi.it (D. Romano)

2
Tetrahedron
hydrophobicity of the substrates was increased; high
reduced
to
ethyl-2-(benzamidomethyl)-3-hydroxy-
enantioselectivity was always observed, giving the corresponding
(S)-alcohol (Prelog rule). The absolute configuration was
determined by comparison with reported chiral HPLC data.6a, 7a
phenylpropanoate (2g); the biotransformation occurred with
higher conversion rate and yield than what reported before with
cells used under non-optimised conditions of growth and
biotransformation (Table 2, entry 4).7g
Table 2. Reduction of aromatic ethyl ꢀ ketoesters (20 mM) with
Pichia glucozyma CBS 5766. Biotransformations were carried
out with freshly prepared cells (50 mgdry weight/ml) suspended in
phosphate buffer (0.1 M, pH 7) in the presence of 50 g/L glucose.
Stereochemical compositions were determined by chiral HPLC
analysis (see Supplementary Data for details).
Table 1. Reduction of aromatic ketones (20 mM) with
Pichia glucozyma CBS 5766. Biotransformations were carried
out with freshly prepared cells (50 mgdry weight/ml) suspended
in phosphate buffer (0.1 M, pH 7) in the presence of 5%
glucose. Enantiomeric excesses were determined by chiral
HPLC analysis (see Supplementary Data for details).
2
3
Entry Substrate Yield
(%)
e.e.
d.e.
Yield
(%)
< 5
75
e.e.
Time
(h)
4
Entry
Substrate
Yield (%)
e.e. 2 (%)
Time (h)
(%)
(%)
(%)
1
2
3
4
5
6
7
8
1a
1a
1b
1b
1c
1c
1d
1d
39
90
28
97
41
96
46
97
97 (S)
97 (S)
4
18
1
1
2
3
4
1e
1e
1f
87
14
96
95
86(S)
95(R)
80(S)
-
-
-
>97(S)
24
97 (S)
34(2S,3S)
0
-
-
24
97 (S)
6
1g
>98(S) 70(2R,3S)
0
16
> 98 (S)
> 98 (S)
> 98 (S)
> 98 (S)
1
4
Biotransformation of ethyl ꢁ-ketoesters 1h-i gave a mixture
of lactones and ketoacids (conversion and stereochemical
composition after 12 and 24 h, Table 3).
1
4
Aromatic ꢀ-ketoesters were also assayed (Table 2). It is
known that biotransformations of ketoesters with yeasts occur
with competition between ester hydrolysis and carbonyl
reduction, due to the occurrence of cell-bound esterase
activities;10 this competitive enzymatic activity can be modulated
by using appropriate substrate/co-substrate concentrations: with
high co-substrate concentrations (e.g. glucose > 50 g/L) the
activity of ketoreductases is generally predominant, as a
consequence of redox cofactor regeneration, while esterase
activity is mostly observed in the absence of cosubstrates.7g The
biotransformations were performed in the optimal conditions for
favouring ketone reduction (50 g/L glucose; Table 2).
Table 3. Biotransformation of ethyl ꢁ-ketoesters with P.
glucozyma. Biotransformations were carried out with freshly
prepared cells (50 mgdry weight/ml) suspended in phosphate
buffer (0.1 M, pH 7) in the presence of 50 g/L glucose.
Stereochemical compositions were determined by chiral
HPLC analysis (see Supplementary Data for details).
The biotransformation of ethyl benzoylacetate 1e showed that
ketoreductase activity was predominant in the first part of the
3
4
biotransformation
leading
to
(S)-ethyl-3-hydroxy-3-
Entry Substrate Yield
(%)
e.e.
(%)
.
Yield
(%)
40
e.e.
(%)
14(S)
70
d.e.
(%)
Time
(h)
phenylpropanoate (2e, yield 87%, e.e. 86% after 4h; Table 2,
entry 1), which was further enzymatically hydrolyzed at
prolonged time (24h), giving optically pure (S)-3-hydroxy-3-
phenylpropionic acid (3e) and leaving low amounts of (R)-2e
(e.e. 95%; Table 2, entry 2). The absolute configuration of 2e and
1
2
1h
1i
55
44
12
82(R)
40
92(3S,5S)
24
3e was determined by comparison with reported chiral
12
These results can be explained by
chromatographic data.11,
Biotransformation of ethyl 4-oxo-4-phenylbutanoate 1h gave
a prevalence of the corresponding ketoacid 3h (55%), whereas
lactone (S)-4h was obtained in 40% yield and low
enantioselectivity (14%; Table 3, entry 1). Racemic ethyl 2-
methyl-4-oxo-4-phenylbutanoate 1i was resolved as a result of
the action of enantioselective esterase(s) with formation of the
ketoacid (R)-3i (e.e. 82% at 44% conversion), while reduction of
suggesting the occurrence of an enantioselective esterase able to
preferentially hydrolyze (S)-2e, therefore leaving unreacted (R)-
2e with high e.e. Ethyl 2-methyl-3-oxo-3-phenylpropanoate 1f
was reduced to the corresponding alcohol 2f with limited
enantiomeric and diastereomeric excesses (Table 2, entry 3);
absolute configuration of 2f was determined by comparison with
reported chiral chromatographic data.6b, No traces of ester
hydrolysis were found even at prolonged times, most likely due
the steric hindrance caused by the methyl in 2-position. Ethyl-2-
(benzamidomethyl)-3-oxo-phenylpropanoate (1g) was selectively
the
phenyldihydrofuran-2(3H)-one (4i) with high diastereoselectivity
carbonyl
afforded
lactone
(3S,5S)-3-methyl-5-
and good enantioselectivity (Table 3, entry 2). Stereochemical

3
1. (a) Matsuda, Y.; Yamanaka, R.; Nakamura K. Tetrahedron:
Asymmetry 2009, 20, 513–557. (b) Hall, M,; Bommarius A. S. Chem.
Rev. 2011, 111, 4088–4110.
compositions were determined by combining chiral HPLC
analysis and 1H NMR.13a-c
2. Monti, D.; Ottolina, G.; Carrea, G.; Riva, S. Chem. Rev. 2011, 111,
4111-4140.
3. Goldberg, K.; Schroer, K.; Lütz, S.; Liese, A. Appl. Microbiol.
Biotechnol. 2007, 76, 249–255.
For the preparation of ꢁ-hydroxyester by microbial reduction,
we decided to test the activity of Pichia glucozyma using
(enzymatically) difficult to hydrolyze tert-butyl ꢁ-ketoesters
(Table 4).
4. Kaluzna, I. A.; Matsuda, T.; Sewell, A. K.; Stewart, J. D. J. Am. Chem.
Soc. 2004, 126, 12827–12832.
5. (a) Moore, J. C.; Pollard, D. J.; Kosjek, B.; Devine, P. N. Acc. Chem.
Res. 2007, 40, 1412–1419; (b) Moore, J. C.; Pollard, D. J.; Kosjek, B.;
Devine, P. N. Acc. Chem. Res. 2007, 40, 1412–1419.
Table 4. Biotransformation of tert-butyl ꢁ-ketoesters with P.
glucozyma. Biotransformations were carried out with freshly
prepared cells (50 mgdry weight/ml) suspended in phosphate
buffer (0.1 M, pH 7) in the presence of 50 g/L glucose.
Stereochemical compositions were determined by chiral
HPLC analysis (see Supplementary Data for details).
6. a) Lavandera, I.; Kern, A.; Ferreira-Silva, B.; Glieder, A.; de
Wildeman, S.; Kroutil, W. J. Org. Chem. 2008, 73, 6003–6005. (b)
Cuetos, A., Rioz-Martínez, A., Bisogno, F.R.; Grischek, B.;
Lavandera, I.; Gonzalo, G.; Kroutil, W.; Gotor V. Adv. Synth. Catal.
2012, 354, 1743-1749..
7. (a) Molinari, F.; Gandolfi, R.; Villa, R.; Occhiato, E. G. Tetrahedron:
Asymmetry 1999, 10, 3515–3520. (b) Forzato, C.; Gandolfi, R.;
Molinari, F.; Nitti, P.; Pitacco, G. Tetrahedron 2001. (c) Hoyos, P.;
Sansottera, G.; Fernández, M.; Molinari, F.; Sinisterra, J. V.;
Alcántara, A. R. Tetrahedron 2008, 64, 7929-7936. (d) Gandolfi, R.;
Cesarotti, E.; Molinari, F.; Romano, D. Tetrahedron 2009, 20, 411–
414. (e) Rimoldi, I.; Pellizzoni, M.; Facchetti, G.; Molinari, F.; Zerla,
D.; Gandolfi, R. Tetrahedron: Asymmetry 2011, 22, 2110–2116. (f)
Husain, S. M.; Stillger, T.; Dünkelmann, P.; Lodige, M.ꢂꢃ Walter, L.ꢂꢃ
Breitling, E.ꢂꢃ Pohl, M.ꢂꢃ Bürchner, M.ꢂꢃ Krossing, I.ꢂꢃ Müller, M.;
Romano, D.; Molinari F. Adv. Synth. Catal. 2011, 353, 2359-2362. (g)
Rimoldi, I.; Cesarotti, E.; Zerla, D.; Molinari, F.; Albanese D.;
Castellano, C.; Gandolfi R. Tetrahedron: Asymmetry 2011, 22, 597-
602. (h) Fragnelli, M. C.; Hoyos, P.; Romano, D.; Gandolfi, R.;
Alcántara, A. R.; Molinari, F. Tetrahedron 2012, 68, 523–528. (i)
Contente, M.L.: Zambelli, P.; Galafassi, S.; Tamborini, L.; Pinto, A.;
Conti, P.; Molinari, F.; Romano D. J. Mol. Catal. B: Enzym.
http://dx.doi.org/10.1016/j.molcatb.2014.05.022.
8. (a) Molinari, F.; Cavenago, K.S.; Romano, A.; Romano, D.; Gandolfi,
R. J. Biotechnol. 2004, 15, 1945-1947. (b) Romano, D.; Gandolfi, R.;
Guglielmetti, S.; Molinari, F. Food Chem. 2011, 124, 1096-1098.
9. Typical procedure for the preparation of the biocatalysts and for
biotransformations: Pichia glucozyma CBS 5766 was cultured using
malt extract + 0.5% yeast extract medium (malt broth, yeast extract 5
g/L, pH 6.0) in a 3.0 L fermenter with 1.0 L of liquid medium for 24 h,
at 28 °C and agitation speed 100 rpm. Cells from submerged cultures
were harvested by centrifugation and washed with 0.1 M phosphate
buffer, pH 7.0. Reductions were carried out in 100 mL screw-capped
test tubes with a reaction volume of 50 mL with cells (2.5 g, dry
weight) suspended in 0.1 M phosphate buffer, pH 7.0 containing 50
g/L of glucose. After 30 min of incubation, substrates (20 mM) were
added and the incubation continued for 24 h under magnetic stirring.
When the reaction was over, pH was brought to pH 1 by the addition
of 1 M HCl and 35 mL of EtOAc were added and the resulting mixture
was shaken and centrifuged; the aqueous phase was extracted twice
more with 35 ml of EtOAc. The organic phases were collected and
dried over Na2SO4 and the solvent was evaporated. The crude residues
were purified by flash chromatography.
2
Entry
Substrate
Yield (%)
e.e. (%)
10(S)
d.e. (%)
< 5
Time (h)
18
1
2
1l
60
70
1m
75(S)
24
Reduction of tert-butyl 4-oxo-4-phenylbutanoate (1l) gave
(S)-tert-butyl 4-hydroxy-4-phenylbutanoate (2l) with sluggish
enantioselectivity (Table 4, entry 1), whereas formation of the
corresponding ꢁ-lactone was not observed; tert-butyl 2-methyl-4-
oxo-4-phenylbutanoate (1m) was also converted into tert-butyl 4-
hydroxy-2-methyl-4-phenylbutanoate 2m, as the only detectable
product (70% yield after 24 h) affording the syn- and anti-
diastereomers (2R,4S)-2m and (2S,4S)-2m with 75% e.e. (Table
4, entry 2). The stereochemical composition of 2m was
determined after purification of the four stereoisomers by
preparative chiral HPLC. Relative configuration of the syn- and
anti-diastereomers was assigned by 1H-NMR,13c whereas
comparison of the retention times in chiral HPLC with literature
data13d allowed for assignment of the absolute configuration.
In conclusion, we have shown that whole cells of Pichia
glucozyma CBS 5766 can be used for the enantioselective
reduction of different aromatic ketones; enantioselectivity
towards simple aromatic ketones under optimised conditions is
sometimes remarkable. Enantioselectivity was generally lower
with ꢁ-ketoesters respect to ꢀ-ketoesters; the occurrence of
competitive esterase activities can be modulated by choosing the
best biotransformation conditions and/or the suited substrate,
turning whole cells of P. glucozyma into a useful system for
multistep reactions.
10. (a) Kayser, M.M.; Mihovilovic, M.D.; Kearns, J.; Feicht, A.; Stewart,
J.D. J.Org Chem. 1999, 64, 6603-6608. (b) Milagre, C.D.F.; Milagre,
H.M.S.; Moran, P.J.S.; Rodrigues, J.A.R. J. Mol. Catal. B: Enzym.
2009, 56, 55-60.
11. Ratovelomanana-Vidal, V.; Girard, C.; Touati, R.; Tranchier, J.P.; Ben
Hassine, B.; Gênet, J.P. Adv. Synth. Catal. 2003, 345, 261-274.
12. Pamies, O.; Bäckvall, J.E. Adv. Synth. Catal., 2002, 344, 947-952.
13. (a) Hoffman, R.V.; Kim, H.O. J. Org. Chem. 1995, 60, 5107-5113. (b)
Wurz, N.E.; Daniliuc, C.G.; Glorius, F. Chem. Eur. J. 2012, 18, 16297-
16301. (c) Nagano, H.; Yokota, M.; Iwazaki, Y. Tethahedron Lett.
2004, 45, 3035-3037. (d) Kallemeyn, J.M.; Mulhern, M.M.; Ku, Y.Y.
Synlett, 2011, 4, 535-538.
References and notes

Highlights
-
-
The reduction of aromatic ketones using the non-conventional yeast Pichia glucozyma was
optimised and high yields and enantioselectivity were obtained
the biotransformation of different ethyl β-and γ-ketoesters was studied, observing a
competition between reductase and esterase activity, leading to hydroxyacids and
hydroxyesters (for ethyl β-ketoesters) and to ketoacids and lactones (for ethyl γ-ketoesters);
these concurrent reactions allowed sometimes for remarkable stereoselectivity.
-
the biotransformation of different tert-butyl γ-ketoesters furnished only hydroxysters, but with
limited enantioselectivity