Microbial bioreductions of γ- and δ-ketoacids and their esters

Article abstract of DOI:10.1016/S0957-4166(01)00184-7

A series of yeasts were used in the bioreductions of aliphatic and aromatic γ- and δ-ketoacids and esters to investigate the preparation of enantiomerically pure γ- and δ-lactones through the intermediacy of their corresponding γ- and δ-hydroxyacids and esters. Bioreduction of ethyl 4-oxononanoate 2a with Pichia etchellsii afforded the γ-nonanolide (+)-5a with 99% e.e., while Pichia minuta proved to be the best choice for the bioreduction of ethyl 2-oxocyclohexylacetate 2e, which afforded cis-(-)-5e and trans-(-)-5e with 98 and 99% e.e., respectively. Reduction of 3-(2-oxocyclohexyl)propionic acid 3e with Pichia glucozyma gave predominantly the corresponding δ-lactone trans-(-)-6e with 94% e.e., whose absolute configuration was determined by means of CD spectroscopy.

Full text of DOI:10.1016/S0957-4166(01)00184-7

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 12 (2001) 1039–1046
Microbial bioreductions of g- and d-ketoacids and their esters
Cristina Forzato,a Raffaella Gandolfi,b Francesco Molinari,b Patrizia Nitti,a Giuliana Pitaccoa and
Ennio Valentina,*
aDipartimento di Scienze Chimiche, Universita` di Trieste, via Licio Giorgieri 1, I-34127 Trieste, Italy
bDipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Sezione Microbiologia Industriale, Universita` di Milano,
via Celoria 2, I-20133 Milan, Italy
Received 12 March 2001; accepted 23 April 2001
Abstract—A series of yeasts were used in the bioreductions of aliphatic and aromatic g- and d-ketoacids and esters to investigate
the preparation of enantiomerically pure g- and d-lactones through the intermediacy of their corresponding g- and d-hydroxyacids
and esters. Bioreduction of ethyl 4-oxononanoate 2a with Pichia etchellsii afforded the g-nonanolide (+)-5a with 99% e.e., while
Pichia minuta proved to be the best choice for the bioreduction of ethyl 2-oxocyclohexylacetate 2e, which afforded cis-(−)-5e and
trans-(−)-5e with 98 and 99% e.e., respectively. Reduction of 3-(2-oxocyclohexyl)propionic acid 3e with Pichia glucozyma gave
predominantly the corresponding d-lactone trans-(−)-6e with 94% e.e., whose absolute configuration was determined by means of
CD spectroscopy. © 2001 Elsevier Science Ltd. All rights reserved.
1. Introduction
2. Results and discussion
Substituted g- and d-lactone rings are present in many
of the optically active natural products isolated from
insects, plants, fungi and marine organisms.1,2 There-
fore, the synthesis of these heterocyclic subunits in
enantiomerically pure form has received particular
attention. Of the numerous synthetic possibilities,3 the
enantioselective reduction of their corresponding g- or
d-ketoacid or ester intermediates by means of enzymes,
either isolated or belonging to a whole cell system, has
found general applicability.4 It is widely accepted that
baker’s yeast plays the most traditional and useful
role,4 although yeasts belonging to other genera5 have
been found to be able to compete with the more
common Saccharomyces cerevisiae. In this paper, we
report the results obtained for the bioreduction of some
g- and d-ketoacids and their corresponding ethyl esters
with S. cerevisiae and other yeasts such as Pichia
minuta CBS 1708, Pichia fermentans DPVPG 2770,
Pichia glucozyma CBS 5766, Pichia etchellsii CBS 2011,
Candida boidinii CBS 2428, Candida utilis CBS 621 and
Kluyveromyces marxianus CBS 397, in order to make a
comparison of their respective efficiency in the
biotransformations.
The substrates chosen in this study are the linear
aliphatic and aromatic g-ketoacids 1a and 1c, their
respective ethyl esters 2a and 2c, and their homologs 1b
and 1d and 2b and 2d, all bearing a methyl group at the
b carbon atom. The cyclic g-ketoacid 1e, its ethyl ester
2e and the cyclic d-ketoacid 3e and its ester 4e were also
studied (Scheme 1). The aim was to examine the influ-
ence of the aromatic ring linked to the site of the
reaction and also the influence of the smallest of the
possible substituents at the carbon atom adjacent to the
carbonyl group on the enzymatic activity.
2.1. Bioreductions of 1a, 1b, 2a and 2b
Table 1 lists the main results obtained for bioreductions
of 1a, 1b, 2a and 2b. In the case of reductions with S.
* Corresponding author. Tel.: +39-040-6763917; fax: +39-040-
6763903; e-mail: valentin@dsch.univ.trieste.it
Scheme 1.
0957-4166/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(01)00184-7

1040
C. Forzato et al. / Tetrahedron: Asymmetry 12 (2001) 1039–1046
cerevisiae several conditions were examined,4a such as
the use of dry baker’s yeast, pre-treated raw baker’s
yeast and reactions with addition of glucose, while the
remaining yeasts were grown as reported before6 and
used as re-suspended cells.
ate in the synthesis of a prototype non-peptidic
inhibitor of the enzyme rennin.13 Ester 2d was reduced
successfully only by P. glucozyma, which afforded a 1:3
mixture of cis- and trans-lactones 5d both having low
e.e.s.
As can be seen in Table 1, the acids 1a and 1b were
reduced much less easily than the corresponding ethyl
esters 2a and 2b, but reductions of 2a and 2b were
complicated by the competing hydrolysis of the ester
group. In some cases, for instance in the transformation
of 2a with C. boidinii, the ketoacid was obtained as
nearly the sole product. Hydrolyses of 2b were only
slightly enantioselective and the resulting acid (+)-1b
could be recovered with 20–39% e.e. In contrast, when
the enzymes were effective in reducing the substrates,
the enantioselectivity was in general high, ranging from
62 to 99%, whereas diastereoselectivity was poor. In
fact from the reduction of 2b both cis- and trans-
diastereomers of 5b were obtained.
2.3. Bioreductions of 1e–4e
With the cyclic g- and d-ketoacids and esters 1e–4e
almost all bioreductions proceeded to fairly high con-
version values, with an enantioselectivity ranging from
26 to 99% and a diastereoselectivity ranging from 5 to
97% (Table 3). Baker’s yeast reductions of 2e,14 3e15
and 4e15 has already been reported to give very good
results in terms of conversion and enantioselectivity and
with 4e also in terms of diastereoselectivity. On the
contrary, the reduction of the ketoacid 1e with S.
cerevisiae was not so satisfactory affording both
diastereomers of 5e with moderate e.e.s. As to the other
yeasts, P. minuta and P. fermentans were active towards
2e affording the cis- and trans-diastereomers of 5e,
(3aS,7aS)-(−)-5e and (3aS,7aR)-(−)-5e, respectively,
both with high e.e.s, although a small amount of the
hydrolysis product was also detected in both cases.
In the reductions of 1a and 2a, good results were
obtained with baker’s yeast7,8 and with P. etchellsii,
which showed the same enantiopreference, affording
(R)-(+)-5a9 with very high e.e.s (Table 1). Interestingly,
the reduction of 2a performed with P. glucozyma
afforded the enantiomeric (S)-(−)-5a, although with low
enantiomeric purity.
Bioreductions of 3e and 4e allowed the isolation of
both cis- and trans-isomers of the d-lactone 6e in
enantiomerically pure form. P. minuta reduced 4e
rapidly and completely to the corresponding isomeric
lactones (4aS,8aS)-(−)-6e and (4aS,8aR)-(−)-6e in
about 1:1 ratio, both with 99% e.e. P. glucozyma was
also effective in reducing 3e and 4e affording the trans-
isomer (−)-6e with 99% e.e. although as a mixture with
the cis-isomer, whose enantiomeric purity was lower.
Reduction of 3e with P. glucozyma was then used on a
preparative scale to isolate trans-(−)-6e for CD spec-
troscopy analysis.
Reductions of 1b and 2b with baker’s yeast have
already been reported:7,8,10 the trans-lactone (4S,5R)-
(+)-5b was obtained from 1b as a single isomer and the
cis-lactone (4S,5S)-(−)-5b from 2b, although as a 7:3
mixture with trans-(−)-5b. Since these diastereomeric
g-lactones constitute the Cognac lactones11 their prepa-
ration in enantiomerically pure form is of interest. P.
glucozyma was the only other microorganism to give
fairly good results in the bioreduction of 2b, affording
trans-(−)-5b with 96% e.e., as a mixture with its enan-
tiomeric cis-(−)-5b having 62% e.e.
2.4. Determination of the absolute configuration
The (R)-configuration of the 4-nonanolide (+)-5a was
determined by comparison with an authentic commer-
cial sample. The absolute configurations of cis-(−)-5b,10
trans-(+)-5b,10 (−)-5c,12 cis-(−)-5e,14,16 trans-(−)-5e14,16
and cis-(−)-6e17 are already known, whereas those of
the diastereomeric lactones 5d could not be determined
because they were inseparable on column chromatogra-
phy. The d-lactone trans-(−)-6e was assigned
(4aR,8aS)-absolute configuration by comparing its CD
spectrum with those of the known lactones
(4aS,6S,8aS)-(+)-7e15 and (4aS,6S,8aS)-(−)-8e17 (Fig.
1). The different descriptor for C-(4a) in the three
lactones is due to a different priority sequence.
2.2. Bioreductions of 1c, 1d, 2c and 2d
Bioreductions of compounds 1c and 2c with baker’s
yeast have already been reported to furnish the lactone
(S)-(−)-5c with >95% e.e.12 Bioreductions of the same
substrates with the other yeasts were unsatisfactory, as
conversions were low and in the case of the ester 2c
hydrolysis was always a competing reaction. The only
good result was that obtained with K. marxianus on 1c
(Table 2), which gave (S)-(−)-5c with 70% yield and
99% e.e. All active yeasts showed the same enantiopref-
erence for the (S)-configuration of the carbinol carbon
atom.
Similarly, reductions of the more hindered systems 1d
and 2d were unsatisfactory and most yeasts did not
work at all. Only baker’s yeast was able to mediate the
reduction of 1d in a diastereoselective manner (71%
d.e.) affording trans-5d with 99% e.e., although in low
yield. The synthesis of the lactone trans-(−)-5d is nota-
ble since it has recently been proposed as an intermedi-
3. Conclusions
Among the yeasts examined, S. cerevisiae was the most
versatile because it was active with the aliphatic and
aromatic ketoacids and esters with only a few excep-
tions. The enantioselectivity was consistently high and
the diastereoselectivity was also good. In some cases,

Table 1. Bioreductions of 1a, 1b, 2a and 2ba
Yeasts
1a
Products
2a
1b
2b
Conv. (%)b
Conv. (%)b
Products
Conv. (%)b
Products
Conv. (%)b
Products
Sign of h
rel. (%)
Sign of h
rel. (%)
Sign of h
rel. (%)
Sign of h
rel. (%)
[e.e. (%)]c
[e.e. (%)]c
[e.e. (%)]c
[e.e. (%)]c
5a
1a
5a
cis-5b
trans-5b
1b
cis-5b
trans-5b
(+)
40
[99]
(+)
46
[99]
(+)
36
[99]
(−)
50
[99]
(−)
21
[83]
S. cere6isiaed
P. minutae
40
0
50
25
4
22
66
–
36
0
–
71
79
2
–
/
–
3
(+)
77
[20]
–
2
–
–
–
–
–
–
–
–
–
[n.d.]
[n.d.]
(+)
5
[99]
(+)
34
[99]
P. fermentanse
P. glucozymae
P. etchellsiie
C. boidiniie
C. utilise
5
100
93
0
–
–
2
[n.d.]
(−)
93
[36]
(−)
17
[62]
(−)
52
[96]
1
0
–
0
69
97
0
)
(+)
37
[90]
(+)
90
[99]
(+)
81
[39]
–
5
(+)
11
[90]
1
37
4
90
–
4
2
2
[n.d.]
[n.d.]
[n.d.]
6
–
4
–
4
95
91
29
53
0
–
–
–
–
–
–
–
–
–
–
[n.d.]
[n.d.]
(+)
58
[99]
–
1
–
1
0
–
87
0
2
[n.d.]
[n.d.]
(+)
10
(+)
38
–
4
–
1
(−)
18
K. marxianuse
10
91
4
19
[99]
[65]
[n.d.]
[n.d.]
[82]
a 7 days.
b Determined by HRGC.
c Determined by chiral HRGC on a g-CDX column for compounds 5a and cis-5b and on a b-CDX column for trans-5b;
d Compound 1a (0.5 mmol), raw baker’s yeast (5 g), glucose (3.1 g), water (10 mL); 2a (0.5 mmol), dry baker’s yeast (2.8 g), glucose (3.1 g), water (19 mL); 1b (1.8 mmol), baker’s yeast (56 g), glucose
(56 g), phosphate buffer (0.1 M, pH 7.4, 500 mL), 3 days; 2b (0.5 mmol), raw baker’s yeast (5 g), water (10 mL), 10 days.
e Yeast (21–27 mg/mL), substrate (12.5 mg), glucose (0.25 g), phosphate buffer (0.1 M, pH 6.0, 5.0 mL).

Table 2. Bioreduction of 1c, 1d, 2c and 2da
Yeasts
1c
Products
2c
1d
2d
Conv. (%)b
Conv. (%)b
Products
Conv. (%)b
Products
Conv. (%)b
Products
Sign of h
rel. (%)
Sign of h
rel. (%)
Sign of h
rel. (%)
Sign of h
rel. (%)
[e.e. (%)]c
[e.e. (%)]c
[e.e. (%)]c
[e.e. (%)]c
5c
1c
5c
cis-5d
trans-5d
1d
cis-5d
trans-5d
/
(−)
84
[\95]
(−)
31
[\95]
–
3
–
18
[99]
S. cere6isiaed
P. minutae
84
2
31
63
10
40
45
26
23
37
–
21
0
10
55
8
10
–
–
[n.d.]
–
2
–
2
–
5
63
10
35
38
16
10
10
–
–
–
–
–
–
–
48
8
[n.d.]
[n.d.]
[n.d.]
(−)
13
[99]
P. fermentanse
P. glucozymae
P. etchellsiie
C. boidiniie
C. utilise
13
21
18
0
0
–
–
1
(−)
21
[99]
(−)
5
[66]
–
3
–
18
[53]
–
50
[22]
(
3
68
2
–
[n.d.]
1
(−)
18
[96]
(−)
7
[74]
–
2
–
2
2
–
–
–
–
–
–
–
–
–
6
[n.d.]
[n.d.]
(−)
10
[92]
–
0
–
–
0
–
–
–
–
(−)
8
[95]
(−)
13
[91]
8
0
9
9
(−)
70
(−)
27
–
2
K. marxianuse
70
2
2
2
[99]
[30]
[n.d.]
a 7 days.
b Determined by HRGC.
c Determined by chiral HRGC on a b-CDX column.
d Ref. 12 for 1c and 2c; 1d or 2d (0.08 mmol), raw baker’s yeast (2.24 g), glucose (2.24 g), phosphate buffer (0.1 M, pH 7.4, 20 mL), 12 days.
e Yeast (21–27 mg/mL), substrate (12.5 mg), glucose (0.25 g), phosphate buffer (0.1 M pH 6.0, 5 mL).

Table 3. Bioreduction of 1e, 2e, 3e and 4ea
Yeasts
1e
2e
3e
4e
Conv. (%)b
Products
Conv. (%)b
Products
Conv. (%)b
Products
Conv. (%)b
Products
Sign of h
rel. (%)
Sign of h
rel. (%)
Sign of h
rel. (%)
Sign of h
rel. (%)
[e.e. (%)]c
[e.e. (%)]c
[e.e. (%)]c
[e.e. (%)]c
cis-5e
trans-5e
1e
–
cis-5e
trans-5e
cis-6e
trans-6e
cis-6e
trans-6e
/
(−)
28
[46]
(−)
12
[93]
(−)
6
[70]
(+)
26
[66]
–
2
[n.d.]
–
2
(+)
45
[70]
(−)
14
[73]
(+)
18
[98]
(−)
14
[56]
(−)
78
[28]
(−)
21
(−)
18
[97]
(−)
44
[98]
(−)
50
[97]
(−)
64
[26]
(−)
21
[26]
(−)
25
(−)
54
[97]
(−)
33
[99]
(−)
27
[94]
(−)
36
[98]
(−)
49
[98]
(−)
58
(−)
30
[99]
(−)
24
[98]
–
5
[n.d.]
(−)
28
[58]
(−)
20
[98]
–
6
(−)
45
[43]
(−)
32
[98]
(+)
18
[27]
(−)
61
[99]
(−)
80
[87]
(−)
85
(−)
80
[99]
(−)
47
[99]
(−)
64
[86]
(−)
55
[80]
(−)
47
[93]
–
S. cere6isiaed
P. minutae
73
26
24
40
80
23
31
74
72
100
93
75
56
80
100f
100
100f
100
83
–
(−)
53
[99]
(9)
36
[0]
(−)
45
[99]
(−)
53
[77]
(9)
78
23
16
–
P. fermentanse
P. glucozymae
P. etchellsiie
C. boidiniie
C. utilise
23
1
)
100
72
89
1
2
100g
81
6
83
–
5
[n.d.]
(−)
14
[64]
–
17
[59]
(−)
20
[81]
(−)
17
[n.d.]
–
3
[92]
(−)
67
[n.d.]
(−)
9
[0]
–
3
40
3
70
12
[94]
–
1
[n.d.]
(−)
73
[61]
(−)
39
[80]
(−)
59
[n.d.]
(9)
11
[72]
(−)
89
[60]
–
10
[n.d.]
(−)
83
K. marxianuse
a 7 days.
98
–
100
93g
[n.d.]
[86]
[96]
[55]
[0]
[30]
[n.d.]
[40]
b Determined by HRCG.
c Determined by chiral HRGC on a b-CDX column for cis-5e and a g-CDX column for trans-5e and 6e.
d Raw baker’s yeast (10 g), water (20 mL), mixture pre-incubated at 50°C for 30 min, 1e (1 mmol); Ref. 14 for 2e; Ref. 15 for 3e and 4e.
e Yeast (21–27 mg/mL), substrate (12.5 mg), glucose (0.25 g), phosphate buffer (0.1 M, pH 6.0, 5 mL).
f 1 day.
g 3 days.

1044
C. Forzato et al. / Tetrahedron: Asymmetry 12 (2001) 1039–1046
Figure 1. CD spectra of trans-(−)-6e, trans-(+)-7e and trans-(−)-8e.
other yeasts were found to be good alternatives; K.
marxianus, P. etchellsii, P. glucozyma and P. minuta
were effective in reducing 1c, 2a, 2b and 2e, respec-
tively, with high enantioselectivity but low diastereose-
ethyl 3-methyl-4-oxo-4-phenylbutanoate 2d derived
from an alkylation reaction of the pyrrolidino enamine
of propiophenone with ethyl bromoacetate;22 ethyl 2-
oxocyclohexylacetate 2e was purchased from Aldrich;
lectivity.
These
microorganisms
were
often
ethyl
3-(2-oxocyclohexyl)propionate
4e23,24
was
substrate-specific and, when active, did not show a
definite trend.
obtained from a Micheal-type reaction between ethyl
acrylate and the pyrrolidino enamine of cyclohex-
anone.17 The acids 1a,8,25 1b,10 1d,26 1e27 and 3e15,28
were obtained by basic hydrolysis of the corresponding
ethyl esters.
4. Experimental
4.1. General
4.2.1. Ethyl 4-oxononanoate 2a18. Oil; IR (film): 1735,
1
1710 cm−1; H NMR: l 4.12 (2H, q, J=7.3, CH2O),
IR spectra were recorded on a Jasco FT/IR 200 spec-
2.72 (2H, t, J=6.5, H-2), 2.57 (2H, t, J=6.5, H-3), 2.45
(2H, t, J=7.6, H-5), 1.61 (2H, quintet, J=7.3, H-6),
1.32 (4H, m), 1.25 (3H, t, J=7.1, CH3CH2O), 0.89 (3H,
t, J=7.1, CH3) ppm; 13C NMR: l 209.2 (s, C-4), 172.8
(s, C-1), 60.5 (t, CH2O), 42.7 (t, C-5), 36.9 (t, C-2), 31.3
(t), 27.9 (t, C-3), 23.4 (t, C-6), 22.4 (t), 14.1 (q,
CH3CH2O), 13.9 (q, C-9) ppm.
1
trophotometer. H and 13C NMR spectra were run on
a Jeol EX-400 spectrometer (400 MHz for proton),
using deuteriochloroform as a solvent and tetra-
methylsilane as the internal standard. Coupling con-
stants and WHs are given in Hz. Optical rotations were
determined on a Perkin–Elmer model 241 polarimeter.
CD spectra were obtained on a Jasco J-700A spectro-
polarimeter (0.1 cm cell). GLC analyses were run on a
Carlo Erba GC 8000 instrument and on a Shimadzu
GC-14B instrument, the capillary columns being OV
1701 (25 m×0.32 mm) (carrier gas He, 40 KPa, split
1:50) and a Chiraldex™ type G-TA, trifluoroacetyl
g-cyclodextrin (g-CDX) (40 m×0.25 mm) (carrier gas
He, 180 KPa, split 1:100) or DiMePe b-cyclodextrin
(b-CDX) (25 m×0.25 mm) (carrier gas He, 110 KPa,
split 1:50). TLC was performed on Polygram® Sil G/
UV254 silica gel pre-coated plastic sheets (eluant: light
petroleum–ethyl acetate). Flash chromatography was
run on silica gel 230–400 mesh ASTM (Kieselgel 60,
Merck). Light petroleum refers to the fraction with a
bp of 40–70°C and ether refers to diethyl ether.
4.2.2. Ethyl 3-methyl-4-oxo-4-phenylbutanoate 2d. Com-
pound 2d was prepared according to the literature.22
1
Oil; IR (film): 1715 (COOEt), 1670 (COPh) cm−1; H
NMR: l 7.98 (2H, m), 7.56 (1H, m), 7.45 (2H, m), 4.10
(2H, q, J=7.3, CH2O), 3.95 (1H, m, CHCH3), 2.96
(1H, dd, J1=8.5, J2=16.8, CH2COO), 2.46 (1H, dd,
J1=5.9, J2=16.8, CH2COO), 1.22 (3H, d, J=6.8,
CHCH3), 1.20 (3H, t, J=7.3, CH2CH3) ppm; 13C
NMR: l 202.7 (s), 172.2 (s), 135.8 (s), 132.9 (d), 128.5
(d), 60.5 (t), 37.4 (t), 37.1 (d), 17.7 (q), 14.0 (q) ppm.
4.3. General procedure for hydrolysis reactions
The ketoester (1 mmol) was added to a solution of
KOH (2 mmol) in methanol (1.6 mL). The mixture was
stirred for 2 days, acidified with 2N HCl and extracted
with ether, after evaporation of the methanol.
4.2. Synthesis of the substrates
Ethyl 4-oxononanoate 2a18 was obtained as a by-
product in the synthesis of hexanoylsuccinate;19,20 ethyl
3-methyl-4-oxononanoate 2b was prepared by radical
condensation of ethyl crotonate with hexanal;10 ethyl
4-oxo-4-phenylbutanoate 2c21 was prepared from com-
mercially available 4-oxo-4-phenylbutanoic acid 1c;
4.3.1. 2-Oxocyclohexylacetic acid 1e27. Oil; IR (film):
1
3000 (OH), 1749, 1710 cm−1; H NMR: l 8.40 (1H,
OH), 2.82 (2H, m), 2.39 (2H, m), 2.16 (3H, m), 1.89
(1H, m), 1.69 (2H, m), 1.43 (1H, m) ppm; 13C NMR: l

C. Forzato et al. / Tetrahedron: Asymmetry 12 (2001) 1039–1046
1045
211.3 (s), 177.9 (s), 46.8 (d), 41.6 (t), 34.2 (t), 33.7 (t), 27.6
(t), 25.0 (t) ppm.
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1
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