Screening of liver acetone powders in the resolution of 1-phenylethanols and 1-phenylpropanols derivatives

Article abstract of DOI:10.1016/j.tetasy.2008.01.041

Hydrolases from the liver acetone powders (LAPs) of bovine, cat, chicken, turkey, lamb, pig, rabbit, and rat were assessed for the enantioselective hydrolysis of acetates of 1-(4-chlorophenyl)ethanol, 1-(3-bromophenyl)ethanol, 1-(4-chlorophenyl)propanol, 1-(4-bromophenyl)propanol, and 1-(3-bromophenyl)propanol. The enantioselectivity of the hydrolytic reaction was dependent upon the liver hydrolase, substrate, pH of the reaction media, and the cosolvent. The most ester selective LAP was from chicken, and the resulting alcohols had the highest ee (80% to >99%). All of the LAPs tested catalyzed the hydrolysis of 1-(4-chlorophenyl)ethanol, except for lamb LAP.

Full text of DOI:10.1016/j.tetasy.2008.01.041

Available online at www.sciencedirect.com
Tetrahedron: Asymmetry 19 (2008) 549–553
Screening of liver acetone powders in the resolution of
1-phenylethanols and 1-phenylpropanols derivatives
*
´
´
´
´
Aida Solıs, Susana Garcıa, Herminia I. Perez, Norberto Manjarrez and Hector Luna
Departamento de Sistemas Biolo´gicos, Universidad Auto´noma Metropolitana, Unidad Xochimilco, Calz. del Hueso No. 1100,
Col. Villa Quietud, Deleg. Coyoacan, cp 04960 Me´xico DF, Mexico
Received 5 November 2007; accepted 31 January 2008
Abstract—Hydrolases from the liver acetone powders (LAPs) of bovine, cat, chicken, turkey, lamb, pig, rabbit, and rat were assessed for
the enantioselective hydrolysis of acetates of 1-(4-chlorophenyl)ethanol, 1-(3-bromophenyl)ethanol, 1-(4-chlorophenyl)propanol, 1-(4-
bromophenyl)propanol, and 1-(3-bromophenyl)propanol. The enantioselectivity of the hydrolytic reaction was dependent upon the liver
hydrolase, substrate, pH of the reaction media, and the cosolvent. The most ester selective LAP was from chicken, and the resulting
alcohols had the highest ee (80% to >99%). All of the LAPs tested catalyzed the hydrolysis of 1-(4-chlorophenyl)ethanol, except for lamb
LAP.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
Herein, LAPs from bovine (BLAP), cat (CALAP), chicken
(CLAP), turkey (TLAP), lamb (LLAP), pig (PLAP), rabbit
(RLAP) and rat (RALAP) were used in the resolution of
1-phenylethanols and 1-phenylpropanols by the hydrolysis
of their corresponding acetates. The influence of the
reaction conditions was evaluated to obtain high conver-
sion and enantiomeric excess.
Optically active secondary alcohols are useful intermedi-
ates in the synthesis of biologically active compounds, such
as drugs and agrochemicals. For example, chlorophenyl-
propanols have antifungal properties against Botrytis cine-
rea, which damages economically important crops,1 while
other phenylethanol derivatives also have antifungal prop-
erties.2,3 Optically active sec-alcohols can be prepared by
asymmetric reduction of the corresponding ketone,4,5 and
by resolution of the racemic sec-alcohols via enantioselec-
tive oxidation.6–8 Asymmetric hydrogen transfer is based
on alcohol dehydrogenases that require nicotinamide
cofactors, which have the disadvantage of requiring cofac-
tor recycling.9 The resolution of racemic sec-alcohols via
enantioselective hydrolysis of their corresponding esters
can be carried out using hydrolases that include lipases
from microbial origin10 and esterases from animal origin,
specifically from the liver.11 Liver acetone powders (LAPs)
from different animals were used as crude sources of ester-
ases,11 which have the advantage of not going through the
tedious and expensive process of purification. These LAPs
constitute inexpensive and accessible sources of this type of
enzymes.
2. Results and discussion
2.1. Effect of pH
Esterases from bovine, cat, chicken, turkey, lamb, pig, rab-
bit and rat LAPs were tested to perform the hydrolysis of
1a (Fig. 1). As pH is an important factor that can affect
the rate and enantioselectivity of enzymatic reactions, the
hydrolysis of 1a was investigated at pH values of 6.0, 7.0
and 8.0 using the LAPs previously mentioned. The results
are shown in Table 1.
The rate and enantioselectivity of the reaction varied mark-
edly depending on the source of the enzyme employed,
although the stereochemical preference remained un-
changed. The preferred absolute configuration of the
resulting alcohol was R, irrespective of the LAP used. Of
the esterases tested, LAPs from turkey, lamb, rat and
*
Corresponding author. E-mail: asolis@correo.xoc.uam.mx
0957-4166/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2008.01.041

550
A. Solı´s et al. / Tetrahedron: Asymmetry 19 (2008) 549–553
Table 2. Effect of the cosolvent on the biocatalyzed hydrolysis of 1a
OAc
OAc
OH
% Conv. 2aa
% ee 2ab
Hydrolysis
LAP
Entry
LAP
Cosolvent
pH
+
1
2
3
4
5
6
7
8
9
BLAP
BLAP
BLAP
BLAP
BLAP
CALAP
CALAP
CALAP
CALAP
CALAP
CLAP
CLAP
CLAP
CLAP
CLAP
TLAP
TLAP
TLAP
TLAP
TLAP
LLAP
LLAP
LLAP
LLAP
LLAP
PLAP
THF
7.0
7.0
7.0
7.0
7.0
8.0
8.0
8.0
8.0
8.0
6.0
6.0
6.0
6.0
6.0
7.0
7.0
7.0
7.0
7.0
7.0
7.0
7.0
7.0
7.0
6.0
6.0
6.0
6.0
7.0
7.0
7.0
7.0
7.0
7.0
7.0
7.0
7.0
7.0
7.0
8
34
48
23
10
9
32
36
16
23
25
28
29
74
25
5
nd
86
63
57
1
2
5
2
8
23
38
35
37
31
—
10
98
66
69
—
25
75
—
63
nd
88
69
89
76
70
81
78
74
82
75
71
78
40
80
nd
nd
11
22
11
nd
nd
nd
nd
nd
90
81
79
82
85
nd
nd
5
Cl
Cl
Cl
Dioxane
DMSO
DMF
Acetonitrile
THF
Dioxane
DMSO
DMF
Acetonitrile
THF
Dioxane
DMSO
DMF
Acetonitrile
THF
Dioxane
DMSO
DMF
Acetonitrile
THF
Dioxane
DMSO
DMF
Acetonitrile
THF
Dioxane
DMSO
DMF
Acetonitrile
THF
Dioxane
DMSO
DMF
Acetonitrile
THF
Dioxane
DMSO
DMF
2a
1a
Figure 1.
Table 1. Effect of pH and LAP source on the biocatalyzed hydrolysis of 1a
Entry
LAP
pH
t (h)
% Conv. 2aa
% ee 2ab
1
2
3
4
5
6
7
8
9
BLAP
BLAP
BLAP
CALAP
CALAP
CALAP
CLAP
CLAP
CLAP
PLAP
6.0
7.0
8.0
6.0
7.0
8.0
6.0
7.0
8.0
6.0
7.0
8.0
6.0
7.0
8.0
6.0
7.0
8.0
7.0
7.0
24
24
24
24
24
24
24
24
6
10
10
13
12
22
23
25
43
35
31
53
41
44
69
55
12
63
40
57
8
46
76
72
89
82
82
80
74
60
85
78
75
45
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
10
11
12
13
14
15
16
17
18
19
20
6
4
2
PLAP
PLAP
RLAP
RLAP
RLAP
RALAP
RALAP
RALAP
TLAP
LLAP
24
24
24
24
24
24
24
48
0
28
18
22
11
nd
PLAP
PLAP
PLAP
PLAP
Cosolvent: acetonitrile;
conversion.
T 25 °C; nd: not determined due to low
RLAP
RLAP
RLAP
RLAP
RLAP
RALAP
RALAP
RALAP
RALAP
RALAP
a Determined by GC.
b Determined by chiral HPLC.
18
3
nd
28
17
nd
18
rabbit biocatalyzed the hydrolysis of 1a, but the stereose-
lectivity of the reaction was very low (Table 1, entries
14–20). With regard to the other LAPs tested, the conver-
sion and enantioselectivity were dependent on the pH of
the reaction media. In the case of the LAPs from cat, chick-
en and pig, the highest ee was reached at pH 6.0 (entries 4,
7, and 10, which correspond to 89%, 80% and 85% ee for
cat, chicken and pig LAPs, respectively, Table 1); however,
the conversion was better at pH 7.0 for CLAP and PLAP
and at pH 8.0 for CALAP. With BLAP, the best enantiose-
lectivity was obtained at pH 7.0 (Table 1, 76% ee, entry 2);
however, the conversion remained low at all the pHs tested.
Acetonitrile
T 25 °C, 24 h; nd = not determined due to low conversion.
a Determined by GC.
b Determined by chiral HPLC.
toward the hydrolysis of 1a under the reaction conditions
used.
The biocatalytic behavior of CALAP, BLAP, CLAP and
PLAP depended on the cosolvent used. The enantioselec-
tivity of the reaction biocatalyzed by CALAP was favored
in the presence of dioxane and acetonitrile (81% and 82%
ee of 2a, entries 7 and 10, respectively, Table 2), by BLAP
in the presence of dioxane and DMF (88% and 89% ee of
2a, entries 2 and 4, respectively, Table 2), by CLAP in
the presence of DMSO and acetonitrile (78% and 80% ee
of 2a, entries 13 and 15, respectively, Table 2), and by
PLAP in the presence of THF and acetonitrile (90% and
85% ee of 2a, entries 26 and 30, respectively, Table 2).
Among the LAPs tested, BLAP, CLAP, CALAP and
PLAP proved to be the most enantioselective toward the
biocatalyzed hydrolysis of 1a and hence the next experi-
ments were conducted with these LAPs only.
2.2. Effect of the cosolvent
As 1a is water insoluble and the reaction was carried out in
aqueous media, it was necessary to use an organic solvent
to help the dissolution of 1a in the reaction media. Water
miscible solvents THF, 1,4-dioxane, DMSO, DMF and
acetonitrile were used to determine their influence on the
enantioselectivity of the hydrolysis of 1a. The pH of the
reaction media, for each LAP, was selected from the best
results shown in Table 1. The results shown in Table 2
enabled us to establish that the cosolvents tested had little
impact on the ee of 2a when TLAP, LLAP, RLAP and
RALAP were used as the biocatalysts. These facts confirm
that these sources of esterases were not stereoselective

A. Solı´s et al. / Tetrahedron: Asymmetry 19 (2008) 549–553
551
2.3. Effect of the structure of acetates 1a–1e
as a biocatalyst, the conversion was the highest possible in
this kind of resolution (51%, entry 6, Table 3).
Acetates 1a–1e (Fig. 2) were selected to determine the effect
of the alcohol structure on the enantioselectivity of the
hydrolytic reaction biocatalyzed by BLAP, CLAP, CA-
LAP and PLAP. The reactions were carried out under
the best conditions determined in the previous experiments
for each LAP (Tables 1 and 2), and the results are shown in
Table 3.
With the other meta-substituted, 3-bromophenylpropanol
acetate 1c, the results were irregular, the conversion was
high in the case of BLAP, but the enantioselectivity was
moderate (48% and 63% of 2c, respectively, entry 9, Table
3); in contrast to this, the hydrolysis biocatalyzed by CLAP
was highly enantioselective, but the conversion was lower
(>99% and 20% of 2c, respectively, entry 11, Table 3).
For the hydrolysis of acetate 1d using, as the source of
hydrolase, BLAP and CLAP, the enantioselectivity was
similarly high, but the conversion was lower with BLAP
and moderate with CLAP (ee >99%, E >100%, 17% and
30% of conversion of 2d, respectively, entries 13 and 15,
Table 3). In the case of acetate 1e, only CLAP gave a high
enantiomeric excess of alcohol 2e, but a low conversion
(91% and 12%, respectively, entry 19, Table 3). Although
PLAP is the most widely used source of esterase in syn-
thetic procedures, it proved to be the least active LAP in
this work.
Acetate 1a was well accepted by BLAP, CALAP, CLAP
and PLAP, the enantiomeric excess of alcohol 2a was
higher than 80% (Table 3, entries 1–4), but the conversion
extent depended on the LAP used; the highest enantioselec-
tivity and conversion were observed using BLAP as biocat-
alyst (88% and 34%, respectively, E = 34, entry 1, Table 3).
It was interesting to note that the biocatalyzed hydrolytic
reaction of 3-bromophenylethanol acetate 1b proved to
be highly enantioselective with three of the four LAPs
tested, BLAP, CALAP and CLAP (>99% ee 2b, E >100,
entries 5–7, Table 3); furthermore, when CALAP was used
In all cases, the configuration of the main enantiomer of
the resulting alcohols 2a–2e was (R), which was determined
by HPLC comparing the retention times against authentic
samples.7,8 The configuration of 2a was determined by
comparing the specific rotation values with data from the
literature.6
OAc
OAc
OH
R
R
R
Hydrolysis
LAP
+
G
G
G
2
1
R
G
a
b
c
d
e
4-Cl
3-Br
3-Br
4-Cl
4-Br
H
H
CH3
CH3
CH3
3. Conclusion
Most of the LAPs tested biocatalyzed the hydrolysis of ace-
tate 1a. RALAP and TLAP hydrolyzed the acetate to a
high extent but without enantioselectivity. LAP from lamb
Figure 2.
Table 3. Effect of the structure of acetates 1a–1e
Entry
Substrate
LAP
pH
Cosolvent
t (h)
% Conv. 2a
% ee 2b
Ec
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
1a
1a
1a
1a
1b
1b
1b
1b
1c
1c
1c
1c
1d
1d
1d
1d
1e
1e
1e
1e
BLAP
CALAP
CLAP
PLAP
BLAP
CALAP
CLAP
PLAP
BLAP
CALAP
CLAP
PLAP
BLAP
CALAP
CLAP
PLAP
7.0
8.0
6.0
6.0
7.0
8.0
6.0
6.0
7.0
7.0
6.0
6.0
7.0
8.0
6.0
6.0
7.0
7.0
6.0
6.0
Dioxane
Dioxane
Acetonitrile
THF
Dioxane
Dioxane
Acetonitrile
THF
Dioxane
Dioxane
Acetonitrile
THF
Dioxane
Dioxane
Acetonitrile
THF
24
24
24
24
73
50
49
50
24
48
49
73
73
49
73
73
24
48
49
73
34
32
25
23
29
51
12
12
48
22
20
6
17
40
30
7
57
5
88
81
80
34
13
11
24
>100
>100
>100
2.5
7.8
2.3
>100
—
>100
5.9
>100
—
16
—
23
—
90
>99
>99
>99
40
63
35
>99
nd
>99
60
>99
nd
76
BLAP
CALAP
CLAP
PLAP
Dioxane
Dioxane
Acetonitrile
THF
nd
91
nd
12
3
T 25 °C; nd = not determined due to low conversion.
a Determined by GC.
b Determined by chiral HPLC.
c Ref. 12.

552
A. Solı´s et al. / Tetrahedron: Asymmetry 19 (2008) 549–553
did not catalyze the hydrolytic reactions studied. Among
all the LAPs used, only BLAP, CALAP, CLAP and PLAP
proved to be enantioselective, but to different extents, to-
ward the hydrolysis of acetates 1a–1e. Their biocatalytic
activity was strongly influenced by the reaction conditions
such as pH and cosolvent. With regard to the alcohol struc-
ture, the most active LAP was CLAP because the resulting
alcohols 2a–2e had the highest enantiomeric excesses (81%
to >99%).
NMR (400 MHz, CDCl3) d 1.44 (3H, d, J = 6.4 Hz), 4.83
(1H, q, J = 6.4 Hz), 7.2–7.45 (4H, m).
4.5. 1-(3-Bromophenyl)ethanol, ( )-2b
GC, column HP5, 120 °C, 1 mL/min, t2b = 2.88 min,
t1b = 4.51 min; HPLC, OB-H column, hexanes–isopropa-
1
nol 98:2, 0.7 mL/min, tR = 19.93 min, tS = 23.39 min; H
NMR (400 MHz, CDCl3) d 1.45 (3H, d, J = 6.4 Hz), 4.84
(1H, q, J = 6.4 Hz), 7.19–7.48 (4H, m).
4. Experimental
4.6. 1-(3-Bromophenyl)propanol, ( )-2c
4.1. Materials and instruments
GC, column HP5, 120 °C, 0.8 mL/min, t2c = 4.66 min,
t1c = 8.34 min; HPLC, OJ-H column, hexanes–isopropanol
97:3, 0.7 mL/min, tR = 38.19 min, tS = 41.63 min; 1H
NMR (400 MHz, CDCl3) d 0.83 (3H, t, J = 7.2 Hz),
1.58–1.77 (2H, m), 4.45 (1H, t, J = 6.7 Hz), 7.12–7.40
(4H, m).
Infrared spectra were recorded on a Perkin–Elmer Paragon
1
1600 FT as liquid films, H NMR spectra on a Varian
400 MHz instrument in CDCl3 using tetramethylsilane as
internal reference and TLC on Silica Gel 60 GF254 Merck.
HPLC analysis was performed on an Agilent 1100 liquid
chromatograph, equipped with a diode array detector,
and using Chiracel OB-H and OJ-H columns. GC analysis
was performed on a Hewlett–Packard HP 6890 gas chro-
matograph, equipped with a flame ionization detector
and HP-5 column (30 m  0.33 mm) and nitrogen as
carrier.
4.7. 1-(4-Chlorophenyl)propanol, ( )-2d
GC, column HP5, 120 °C, 0.8 mL/min, t2d = 3.03 min,
t1d = 4.99 min; HPLC, OB-H column, hexanes–isopropa-
1
nol 99:1, 0.6 mL/min, tR = 15.99 min, tS = 17.79 min; H
NMR (400 MHz, CDCl3) d 0.88 (3H, t, J = 7.5 Hz),
1.62–1.84 (2H, m), 4.55 (1H, t, J = 6.5 Hz), 7.12–7.40
(4H, m).
All racemic alcohols were prepared from the corresponding
aldehyde and Grignard reagent, while the alcohols were
converted to their O-acetyl esters following the standard
procedure using acetic anhydride and triethylamine. All
compounds were purified by silica gel column chromato-
graphy using hexanes–ethyl acetate as eluent and were
4.8. 1-(4-Bromophenyl)propanol, ( )-2e
GC, column HP5, 120 °C, 0.8 mL/min, t2e = 5.0 min,
t1e = 8.133 min; HPLC, OJ-H column, hexanes–isopropa-
1
characterized by IR and H NMR.
1
nol 99:1, 0.6 mL/min, tR = 33.69 min, tS = 35.34 min. H
NMR (400 MHz, CDCl3) d 0.83 (3H, t, J = 7.2 Hz),
1.59–1.74 (2H, m), 4.44 (1H, t, J = 6.5 Hz), 7.11–7.41
(4H, m).
4.2. Liver acetone powders (LAPs)
The corresponding livers were purchased in local stores or
obtained as gifts from the University animal facilities.
First, the excess fat was removed from the liver, and then
washed with water. It was then ground three times with
acetone in a blender, and the powder was filtered, dried,
and stored at 5 °C.
Acknowledgements
1
The authors wish to thank Dr. Julia Cassani for the H
NMR spectra. They also thank the partial financial
´
4.3. General procedure for enzyme-mediated hydrolysis
support of Consejo Nacional de Ciencia y Tecnologıa
(CONACYT), Mexico, Grant 37272N.
About 0.4 mL of a buffer phosphate solution (0.1 M, pH
6.0, 7.0 and 8.0) and 10 mg of LAP were added to 5 mg
of 1a–1e in 0.1 mL of a cosolvent. The mixture was stirred
at 25 °C (for reaction times, refer to Tables 1–3), then ex-
tracted twice with methylene chloride. The organic phase
was dried over Na2SO4 and the solvent evaporated under
reduced pressure to dryness. Conversion % was determined
by GC ([peak area of alcohol/peak area of alcohol + peak
area of acetate] Â 100) and % ee by HPLC using a chiral
column.
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