Enantioselective hydrolysis of 1-aryl ethyl acetates and reduction of aryl methyl ketones using carrot, celeriac and horseradish enzyme systems

Article abstract of DOI:10.1016/S0957-4166(02)00635-3

Enantioselective hydrolyses of racemic 1-phenyl (or naphthyl) ethyl acetates and the reduction of methylphenyl (or naphthyl) ketones have been conducted using the comminuted roots of carrot (Daucus carota L.), celeriac (Apium graveolens L., var. rapaceum), and horseradish (Armoracia lapatifolia Gilib.). (S)-(-)-1-(2-Naphthyl)ethanol (100% yield, ee=100%) has been obtained by reduction of 2-acetonaphthone using the comminuted roots of carrot.

Full text of DOI:10.1016/S0957-4166(02)00635-3

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 13 (2002) 2299–2302
Enantioselective hydrolysis of 1-aryl ethyl acetates and reduction
of aryl methyl ketones using carrot, celeriac and horseradish
enzyme systems
Wanda K. Ma˛czka and Agnieszka Mironowicz*
Department of Chemistry, Agricultural University, ul. Norwida 25, 50-375 Wrocław, Poland
Received 26 July 2002; revised 6 September 2002; accepted 27 September 2002
Abstract—Enantioselective hydrolyses of racemic 1-phenyl (or naphthyl) ethyl acetates and the reduction of methylphenyl (or
naphthyl) ketones have been conducted using the comminuted roots of carrot (Daucus carota L.), celeriac (Apium graveolens L.,
var. rapaceum), and horseradish (Armoracia lapatifolia Gilib.). (S)-(−)-1-(2-Naphthyl)ethanol (100% yield, ee=100%) has been
obtained by reduction of 2-acetonaphthone using the comminuted roots of carrot. © 2002 Elsevier Science Ltd. All rights
reserved.
1. Introduction
(Apium graveolens L., var. rapaceum—from the same
family Appiaceae) and horseradish (Amroracia lapatifo-
lia Gilib.) from the taxonomically distant family Brassi-
caceae were selected for the biotransformation.
Optically active alcohols may be obtained either by the
well known method of asymmetric reduction of a
prochiral ketone, or alternatively, by enantioselective
hydrolysis of racemic esters. Plant cell enzymes, like
those from microorganisms, are able to catalyse reac-
tions with high regio- and stereospecificity. Hence, both
the reduction of acetophenone 41,2 (or other ketones3)
and the enantioselective hydrolysis of racemic 1-
phenylethyl acetate 14 using the enzyme systems of
plant cells result in 1-phenylethanol enantiomers 1a (or
other alcohols).3
The application of comminuted tissue of ripe vegetable
roots in the biotransformation (which have the advan-
tages of low cost, short reaction times of ca. 48 h)
instead of isolated enzymes is possible thanks to the
group of enzymes that are able to accept xenobiotic
substrates. However, the course of the transformation
can become complex because the use of groups of
enzymes enables many processes to occur simulta-
neously in the course of the biotransformation. For
example, ester hydrolysis, oxidation of the resulting
alcohol into a ketone and its subsequent reduction.
In our previous research, Jerusalem artichoke
(Helianthus tuberosus L.) and carrot (Daucus carota L.)
were used (among other vegetables) for the biotransfor-
mation of racemic 1-phenylethyl acetate 1, acetophe-
none 4 and their analogues. Jerusalem artichoke was
used both in the form of cell cultures and comminuted
root tissue, while carrot was used in the form of cell
cultures only.5,6 On comparing the reactions of
Jerusalem artichoke cell cultures with those where the
comminuted tissues of its root were used, the efficiency
of the biostransformation was found to be 20 times
higher when the comminuted root tissue was used.
Hence, we anticipated the same results using the com-
minuted roots of carrot. As well as carrot cells, celeriac
The 1-phenylethyl analogues were selected as substrates
for this study due to the utility of these compounds as
chiral auxiliaries and synthons when in enantiomeri-
cally pure form.
2. Results and discussion
The racemic acetates 1-phenylethyl 1, 1-(1-naph-
thyl)ethyl 2, 1-(2-naphthyl)ethyl 3 and prochiral aro-
matic-aliphatic ketones 4, 5 and 6 were used as
substrates to obtain optically active alcohols by bio-
transformation using the vegetable (celeriac, carrot and
horseradish) enzyme systems (Scheme 1).
* Corresponding author. Tel.: (+4871) 3205468; fax: (+4871) 3284124;
e-mail: amiron@ozi.ar.wroc.pl
0957-4166/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(02)00635-3

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W. K. Ma˛czka, A. Mironowicz / Tetrahedron: Asymmetry 13 (2002) 2299–2302
Scheme 1.
Besides the acetates 1, 2 and 3 and the ketones 4, 5 and
6, the racemic alcohols 1a, 2a and 3a were subjected to
separate biotransformations. The results from all the
transformations (average results from repeated bio-
transformations) are presented in Table 1.
Considering the abilities of these biocatalysts to carry
out enantioselective processes, it was observed that the
carrot enzymatic system exhibits poor enantioselectivity
in the hydrolysis of the racemic acetate 1 (Fig. 1b and
Table 1, entry 2) to afford the enantiomerically pure
alcohols 1a. However, the same biocatalyst is capable
of converting 2-acetonaphthone 6 with 100% efficiency
into (S)-(−)-alcohol 3a with ee=100% (Table 1, entry
17 and Fig. 1d).
These results allow comparison of the presently
obtained results from biotransformations using the
comminuted carrot root tissue enzymatic system with
the results achieved previously using a suspension cell
culture.5,7 This comparison shows that using a com-
minuted catalyst increases the efficiency of the reaction
by a factor of 18.
In summary, the following aspects of the biotransfor-
mations presented are of note.
Hydrolysis of acetates. (a) The carrot enzymatic system
effects hydrolysis of the acetates 1, 2, and 3 to higher
degrees and with greater stereoselectivity than the enzy-
matic systems of the other vegetables studied here. (b)
Oxidation of the alcohols obtained as a result of the
hydrolysis cannot be treated as a rule as it depends on
the structure of the substrate and the biocatalyst used.
(c) The horseradish enzymatic system effects the
hydrolysis of acetates 1, 2 and 3 to afford alcohols of
the opposite configuration compared to the reactions
completed using the carrot and celeriac enzyme
systems.
The progress of the reaction and its enantioselectivity
were investigated by monitoring (GC) the components
in the reaction mixture every few hours. The results
of representative biotransformations are presented in
Fig. 1.
Following the example of ( )-1-phenylethyl acetate 1,
biotransformation by the celeriac enzymatic system
(Table 1, entry 1 and Fig. 1a), it is possible to examine
the successive stages of the reaction: hydrolysis, oxida-
tion and reduction. During the first hours of the bio-
transformation of 1, the hydrolysis delivers more
(S)-(−)-alcohol, (S)-(−)-1a. But at the end of the bio-
transformation the amount of (S)-(−)-1a drops signifi-
cantly because it is the main substance to be oxidized
(Table 1, entry 19). Nevertheless the level of (S)-(−)-1a
in the mixture never equals zero because the ketone
obtained is rapidly reduced, initially to (S)-(−)-1a, as
confirmed by the results presented in entry 10 of Table
1, and in Fig. 1(c). This reversible reaction: ketone
4 ? alcohol 1a, leads to the continuous formation of
(S)-(−)-1-phenylethanol (S)-(−)-1a, which makes the
preparation of pure (R)-(+)-alcohol impossible via this
method.
Reduction of ketones. The performance of the carrot
enzymatic system is highly substrate dependent: 2-acet-
onaphthone 6 is completely reduced but 1-acetonaph-
thone 5 is not transformed (Table 1, entries 14 and 17).
High enantioselectivity was observed in only one case
(Table 1, entry 17).
Oxidation of alcohols. The celeriac enzymatic system is
highly sensitive to the substrate structure (Table 1,
entries 22 and 25). The oxidation of ethanols with a
naphthyl substituent by the carrot enzymatic system did

W. K. Ma˛czka, A. Mironowicz / Tetrahedron: Asymmetry 13 (2002) 2299–2302
2301
Table 1. Results from transformations of (9)-1, (9)-2, (9)-3, 4, 5, 6, (9)-1a, (9)-2a and (9)-3a with celeriac, carrot and
horseradish. (The contents of the reaction mixture was determined by gas chromatographic analysis of the crude extracts)
Entry substrate
Vegetable
Contents of the reaction mixture after biotransformation
Alcohol
Ketone (%)
Unreacted substrate
Quantity (%)
ee (%)
Quantity (%)
ee (%)
Acetates
(9)-1
(9)-2
(9)-3
Ketones
4
1
2
3
4
5
6
7
8
9
Celeriac*
Carrot*
Horseradish
Celeriac
Carrot
Horseradish
Celeriac
61
91
62
48
64
20
55
74
14
47 (R)-(+)
11 (R)-(+)
41 (S)-(−)
68 (R)-(+)
75 (R)-(+)
53 (S)-(−)
33 (R)-(+)
50 (R)-(+)
0
39
0
0
0
0
0
0
0
0
0
9
–
67 (S)-(−)
66 (R)-(+)
71 (S)-(−)
82 (S)-(−)
70 (R)-(+)
32 (S)-(−)
91 (S)-(−)
0
38
52
36
80
45
26
86
Carrot
Horseradish
10
11
12
13
14
15
16
17
18
Celeriac*
Carrot
Horseradish
Celeriac
Carrot
Horseradish
Celeriac
20
4
5
8
0
85 (S)-(−)
82 (S)-(−)
88 (S)-(−)
0
–
–
72 (S)-(−)
100 (S)-(−)
–
80
96
95
92
5
100
100
81
0
100
0
19
100
0
6
Carrot*
Horseradish
Alcohols
(9)-1a
(9)-2a
(9)-3a
19
20
21
22
23
24
25
26
27
Celeriac
Carrot
Horseradish
Celeriac
Carrot
Horseradish
Celeriac
Carrot
29
11
Trace
0
0
0
60
0
71
89
69 (R)-(+)
0
–
–
–
–
35 (S)-(−)
–
–
100
100
100
40
100
100
Horseradish
0
* Reaction enantioselectivity see Fig. 1.
not proceed in our hands (Table 1, entries 23 and 26);
the presence of a phenyl substituent in the substrate
allows the oxidation to occur more readily with all
three biocatalysts (Table 1, entries 19, 20 and 21).
3.2. Biocatalysts
Fresh celeriac (Apium graveolens L. var. rapaceum),
carrot (Daucus carota L.) and horseradish (Armoracia
lapathifolia Gilib.) were purchased from a local market.
On comparing the transformations of 1- and 2-naph-
thalene derivatives it can be said that the b-position is
more suitable for enzymatic reaction. Additionally, sub-
strates with one aromatic ring are transformed more
easily and with higher enantioselectivity than molecules
containing biaryl systems.
3.3. Biotransformation conditions
Healthy vegetable roots were comminuted using an
electric mixer for 2 min and vegetable pulp (20 ml,
1.0–1.5 g of dry wt, 100°C, 24 h) was placed in Erlen-
mayer flasks with phosphate buffer (0.1 M, 50 ml, pH
6.2 (celeriac), pH 6.5 (carrot), pH 4.5 (horseradish)).
This pulp was mixed with a solution of the substrate
(20–30 mg) in acetone (0.5 mL) and the resulting mix-
ture was shaken for 48 h. The course of the biotransfor-
mation was monitored by means of TLC and GC
analysis. Biotransformed mixtures were extracted with
CHCl3. The enantiomeric composition of the product
3. Experimental
3.1. Substrates 1a, 4, 5 and 6
These substrates were purchased from Aldrich Chemi-
cal Co., the others were obtained in our laboratory.

2302
W. K. Ma˛czka, A. Mironowicz / Tetrahedron: Asymmetry 13 (2002) 2299–2302
Figure 1. Changes of the reaction mixture contents during the biotransformation: ( )-1-phenylethyl acetate ( )-1 by celeriac (a)
and carrot (b); acetophenone 4 by celeriac (c) and 2-acetonaphthone 6 by carrot (d). The contents of the reaction mixture was
determined by gas chromatographic analysis of the crude extracts.
mixture was established by GC by application of chiral
columns, as mentioned in Section 3.4. All substrates in
the buffer solution were stable under these conditions.
References
1. Akakabe, Y.; Naoshima, Y. Phytochemistry 1994, 35,
661–664.
2. Villa, R.; Molinari, F.; Levati, M.; Aragozzini, F. Bio-
technol. Lett. 1998, 20, 1105–1108.
3.4. Analytical methods
GC: Hewlett–Packard 5890, FID, carrier gas—H2 at 2
ml min−1, using following Chrompack WCOT capillary
columns: Chirasil-L-Val (25 m×0.25 mm×0.12 mm) for 2,
2a, 3, 3a, 5, 6 (column temp. 120°C/30 min; gradient
20°C/min, injector temp. 200°C, detector temp. 250°C),
Chirasil Dex CB (25 m×0.25 mm×0.12 mm) for 1, 1a, and
4 (column temp. 125°C/10 min; gradient 15°C/min,
injector temp. 200°C, detector temp. 250°C); TLC: silica
gel 60 F254 pre-coated aluminium sheets (layer thickness
0.2 mm, Merck) with n-hexane–Et2O (5:1) for 2, 2a, 3,
3a, 5, 6 and n-hexane–acetone (14:1) for 1, 1a and 4.
3. Naoshima, Y.; Akakabe, Y. J. Org. Chem. 1989, 54,
4237–4239.
4. Mironowicz, A.; Kromer, K. Collect. Czech. Chem.
Commun. 1998, 1655–1662.
5. Mironowicz, A.; Jarosz, B.; Siewinski, A. Acta Soc. Bot.
Poloniae. 1995, 64, 281–285.
6. Mironowicz, A. Phytochemistry 1998, 47, 1531–
1534.
7. Mironowicz, A.; Kromer, K.; Pawłowicz, P.; Siewinski,
A. Acta Soc. Bot. Poloniae. 1994, 63, 43–48.