Immobilized Manihot esculenta preparation as a novel biocatalyst in the enantioselective acetylation of racemic alcohols

Article abstract of DOI:10.1016/j.tetasy.2008.05.018

The enzymatic preparation obtained from a discard of Manihot esculenta roots has been successfully immobilized on calcium alginate hydrogels. This preparation has been tested as a chiral biocatalyst in the enzymatic acylation of a set of racemic aromatic alcohols. Depending on the reaction conditions, excellent enantioselectivities can be achieved. Some parameters that can alter the biocatalytic properties of the enzyme, such as solvent, temperature, acyl donor and substrate structure have been studied exhaustively in order to establish a deeper knowledge of this novel biocatalyst.

Full text of DOI:10.1016/j.tetasy.2008.05.018

Tetrahedron: Asymmetry 19 (2008) 1419–1424
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Immobilized Manihot esculenta preparation as a novel biocatalyst
in the enantioselective acetylation of racemic alcohols
a
a
Luciana L. Machado a, Telma L. G. Lemos a, , Marcos Carlos de Mattos , Maria da Conceicão F. de Oliveira ,
*
Gonzalo de Gonzalo b, Vicente Gotor-Fernández b, Vicente Gotor b,
*
a Departamento de Química Orgânica e Inorgânica, Laboratorio de Biotransformaçôes e Produtos Naturais (LBPN), Universidade Federal do Ceará, 60451-970 Fortaleza, Ceará, Brazil
b Departamento de Química Orgánica e Inorgánica, Instituto Universitario de Biotecnología de Asturias, Universidad de Oviedo, c/ Julián Clavería 8, 33006 Oviedo, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 March 2008
Accepted 23 May 2008
The enzymatic preparation obtained from a discard of Manihot esculenta roots has been successfully
immobilized on calcium alginate hydrogels. This preparation has been tested as a chiral biocatalyst in
the enzymatic acylation of a set of racemic aromatic alcohols. Depending on the reaction conditions,
excellent enantioselectivities can be achieved. Some parameters that can alter the biocatalytic properties
of the enzyme, such as solvent, temperature, acyl donor and substrate structure have been studied
exhaustively in order to establish a deeper knowledge of this novel biocatalyst.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
been employed in the bioreduction of different aldehydes and ke-
tones, and also in hydrolytic enzymatic processes.4
Nowadays, the use of biotransformations presents a great inter-
est for organic chemists in order to develop the preparation of high
added value compounds.1 It has been recognized that enzymes are
able to accept a wide set of substrates catalyzing chemo-, regio-
and enantioselective processes and represent an effective and
sometimes preferable alternative to chemical synthesis. As a result,
biocatalysts can be used in the synthesis of enantiomerically pure
compounds with high efficiencies and low by-product formation.
Over the last few years, the search for inexpensive biocatalysts
has become one of the main objectives in the field of biotransfor-
mations. From several easily available sources, plant cell cultures
and whole plant cells have gained an increasing interest as a source
from which to obtain novel enzymes.2 These biocatalysts are easily
obtained from markets and manipulated by organic chemists.
Manihot esculenta (cassava) is one of the major sources of carbo-
hydrates in the world. During the treatment of these plant roots in
order to obtain flour, several liters of an aqueous phase called
manipueira are separated from the solid and later discarded. In
the last years, it has been discovered that this discard presents
some interesting properties as a surfactant and is an important
source of enzymes. For example, one (S)-hydroxynitrile lyase from
M. esculenta has been cloned, overexpressed, and studied as a chiral
biocatalyst in the synthesis of cyanohydrins, which are interesting
synthons in organic chemistry.3 Recently, crude manipueira has
A practical technique to improve enzyme efficiency, operational
stability, and performance in organic solvents is biocatalyst immo-
bilization.5 Among all the immobilization methods that have been
described, the entrapment of biocatalysts on calcium alginate
hydrogels is a well-known methodology.6 By forming a solidified
water phase in an aqueous-organic two-phase system, hydrogels
protect the entrapped biocatalyst against detrimental effects of
the environment. It provides an easy, fast, non-toxic, inexpensive,
and versatile immobilization technique.
As a part of our ongoing research in the preparation and appli-
cation of novel enzymatic systems, calcium alginate hydrogels ob-
tained from the roots of M. esculenta have been prepared and
studied as a biocatalyst in the selective enzymatic acylation of
racemic secondary alcohols.
2. Results and discussion
The biocatalyst contained in the manipueira discarded from the
treatment of the tuber M. esculenta was immobilized by the sodium
alginate (1% w/v)-calcium chloride (5% w/v) method,7 and obtained
as an active enzyme form encapsulated in alginate spheres. The
yield of the immobilization was determined through the concen-
tration of protein present in the initial extract in comparison to
the solution of calcium chloride used to precipitate the spheres,
using the Hartree method.8 Only a 3.8% of the active enzyme from
the initial extract was not incorporated. The size of the beads was
determined by Electronic Microscopy Sweeping, spheres of 1.0 mm
diameter, as shown in Figure 1.
* Corresponding authors. Tel./fax: +34 985 103448 (V.G.); tel.: +55 85 33669366;
fax: +55 85 33669782 (T.L.G.L.).
E-mail addresses: tlemos@dqoi.ufc.br (T. L. G. Lemos), vgs@fq.uniovi.es (V. Gotor).
0957-4166/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2008.05.018

1420
L. L. Machado et al. / Tetrahedron: Asymmetry 19 (2008) 1419–1424
Table 1
Acetylation of racemic phenylethan-1-ol catalyzed by immobilized Manihot esculenta
employing vinyl acetate as acyl donora
Entry
Solvent
Equiv
VA
T
(°C)
Time
(h)
ee
ee
cc
(%)
Ed
(S)-1ab
(R)-1bb
1
2
3
4
5
6
7
8
9
Hexane
Toluene
iPr2O
5
5
5
5
5
—
5
5
5
5
45
45
45
45
45
45
45
45
45
45
45
45
60
45
45
118
116
118
118
118
118
116
116
116
92
34
10
17
18
36
15
12
7
90
94
96
94
94
90
72
85
88
93
95
94
92
92
74
27
14
15
16
28
14
14
6
27
34
54
39
46
23
7
13
16
40
51
50
30
31
8
tBuOMe
Et2O
Vinyl acetate
THF
1,4-Dioxane
MeCN
Et2O
4
5
10e
11e
12
13
14f
15g
36
18
40
26
21
17
28
16
30
25
20
19
iPr2O
5
92
Et2O
10
10
10
10
116
116
116
116
iPr2O
Et2O
Et2O
Figure 1. Scanning electron microscopy of Manihot esculenta hydrogels prepared by
the sodium alginate-calcium chloride method.
a
For reaction conditions, see Section 4.
Determined by HPLC or GC.
Conversion, c = ees/(ees + eep).
b
c
d
e
f
Once obtained, after the immobilized enzyme preparation, the
system was subjected to study as a chiral catalyst in a set of biocat-
alyzed acylations of racemic aromatic alcohols. Firstly, the enzy-
matic acetylation of phenylethan-1-ol ( )-1a was studied using
5 equiv of acetic anhydride with immobilized M. esculenta. Reac-
tions were performed in different organic solvents at 45 or 60 °C,
being observed after 120 h conversions from 5% in 1,4-dioxane to
37% when using toluene. Very low enantioselectivities were
achieved (E ꢀ 1.0)9 for all the conditions employed. The acetyla-
tions performed in absence of the biocatalyst led to the recovery
of the starting material, indicating that the lack of enantioselectiv-
ity is due to the enzyme biocatalytic properties.
In view of these results, we decided to employ a less reactive re-
agent, vinyl acetate (5 equiv) as an acyl donor in the enzymatic res-
olution of ( )-1a. First, we studied the influence of the organic
solvent on the biocatalytic properties of the enzyme. In all the
tested conditions, the (R)-enantiomer of the starting material was
acetylated, to give an alcohol with an (S)-configuration. Thus it
can be established that this enzyme preparation follows Kazlaus-
kas’ rule for the acylation of secondary alcohols.10 Enzymatic reac-
tions were performed at 45 °C, due to the low activity shown by
the hydrogels of M. esculenta at lower temperatures.
In order to determine if alginate beads could have any contribu-
tion in the acetylation reactions, experiments were carried out
using only this polymer in the absence of manipueria. When the
enzymatic acylation of ( )-1a was performed with vinyl acetate,
only starting material was recovered after long reaction times.
Compound ( )-1b was obtained with only 4.0% conversion after
120 h when acetic anhydride was used as the acyl donor, hence
the presence of the alginate beads can affect slightly the enantiose-
lectivities achieved when using this latest compound.
Enantiomeric ratio, E = ln[1 ꢁ c(1 + eep)]/ln[1 ꢁ c(1 ꢁ eep)].
Reactions were performed with double amount of biocatalyst.
Resolution was performed with 0.7 equiv of NEt3.
g
The reaction was carried out in the presence of 0.7 equiv of 4-DMAP.
high hydrophilic character such as THF, 1,4-dioxane, or acetoni-
trile (entries 7–9).
i
Once Pr2O and Et2O were established as the best solvents for
the enzymatic resolution of ( )-1a, other parameters that could
influence the biocatalytic properties of M. esculenta were studied.
As shown in entries 10 and 11, the enzymatic resolution was per-
formed using double the amount of biocatalyst hydrogels. Under
these conditions, only a slight increase was observed in the conver-
sion. The enzymatic acylation of ( )-phenylethanol was developed
at 45 °C with 10 equiv of vinyl acetate (entry 12) resulting in a
slight increase in enzymatic activity (c = 30% after 116 h). Taking
into account that the temperature can have a marked effect on
the activity and selectivity of enzymes, the resolution of ( )-1a
i
was performed at 60 °C when using Pr2O as a solvent (entry 13),
it being possible to obtain an increase in the enzymatic activity
(c = 25% after 116 h) but with a significant loss in its selectivity.
Finally, in order to improve the reaction rate obtained when
performing the acetylations in Et2O, different amines were used
as additives in the resolution of ( )-1a.11 The presence of 0.7 equiv
of triethylamine or 4-DMAP (entries 14 and 15) led to a decrease in
both conversion and selectivity with repect to the reaction without
amine. This loss is especially significant when using 4-DMAP, a
very low E value in the resolution of phenylethan-1-ol being
observed.
Next, the influence of the type of acyl donor used in the biocata-
lytic acetylation of ( )-1a was analyzed (Table 2). Thus, it was
observed that isopropenyl acetate was also a suitable donor for
Table 1 summarizes the results achieved in the enzymatic
acetylation of racemic phenylethan-1-ol 1a employing different
organic solvents. As shown in entry 3, the highest enantioselectiv-
i
the transesterification of racemic phenylethan-1-ol, both in Pr2O
i
ity was obtained with Pr2O (E = 54) in a process with moderate
and Et2O (entries 1 and 2), lower activities and selectivities were
measured when compared with vinyl acetate. By using this acyl
donor, a slightly higher conversion and E value were obtained in
Et2O (E = 26 and conversion of 14% after 116 h). Ethyl methoxyac-
etate has also been employed as an acetylating reagent in the
enzymatic resolution of alcohols and amines,12 but when
phenylethan-1-ol was treated with 10 equiv of this compound in
Et2O at 45 °C in the presence of immobilized M. esculenta, no reac-
tion was observed after long times (entry 3). Finally, a less reactive
vinyl ester such as vinyl benzoate was tested in the acylation of
conversion (c = 15% after 118 h). The use of Et2O (entry 5) led to
the formation of (R)-1b. Good selectivity (E = 46), was observed
with an increase in activity (c = 28%) when compared with Pr2O.
i
Immobilized M. esculenta also exhibited good enantioselectivities
when working with either toluene or tBuOMe (entries 2 and 4,
respectively), but moderate reaction rates (conversions close to
15% after 5 days). When employing vinyl acetate as an acyl donor
and solvent in the resolution of ( )-1a (entry 6), moderate con-
version and enantioselectivity were measured. For the rest of
the solvents tested, lower selectivities were found, in addition
to a loss in the enzymatic activity when employing solvents with
i
( )-1a in both Pr2O and Et2O (entries 4 and 5), but no formation
of the corresponding ester was achieved.

L. L. Machado et al. / Tetrahedron: Asymmetry 19 (2008) 1419–1424
1421
Table 2
116 h, while for the meta-substituted alcohols ( )-6a and ( )-9a,
an important loss in both conversions and enantioselectivities
was observed (E values close to 50). The effect of the ortho-substi-
tution was also analyzed by reacting racemic 2-chlorophenyl-
ethan-1-ol 7a with vinyl acetate and immobilized M. esculenta in
Et2O. After 116 h, (R)-7b was obtained with an 18% conversion
and a low enantioselectivity. We can establish that there is a clear
influence of the halide atom position on the biocatalytic properties
of the enzyme, higher enantioselectivities for para-derivative, and
low E values for the ortho-one being obtained.
Next, the enzymatic acylation of racemic 3-methoxyphenyl-
ethan-1-ol 10a was performed with good enantioselectivity and
moderate conversion. When a phenylethan-1-ol derivative with
an electron-donating group at the para-position was employed as
a substrate of Manihot esculenta [( )-11a], a moderate E value
and conversion were achieved. The enzymatic acetylation of 4-
hydroxyphenylethan-1-ol ( )-12a led to the formation of the
monoester (R)-12b with good selectivity (E = 50) and moderate
conversion. Only trace amounts of the diacetylated product (less
than 1%) were observed when the crude reaction was analyzed
by GC.
Enzymatic resolution of ( )-phenylethan-1-ol catalyzed by immobilized Manihot
esculenta with different acyl donorsa
O
O
R
1
OH
OH
O
R
2
M. esculenta
Org. solvent
+
+
R
O
1
45 ºC/116 h
250 rpm
(
)-1a
(S)-1a
(R)-1b-d
cc (%)
b
b
Entry
R1
R2
Solvent
eeS (%)
eeP (%)
E
1
2
3
4
5
Me
Me
MeOCH2
Ph
CH3
CH3
H
iPr2O
Et2O
Et2O
Pr2O
Et2O
12
14
—
—
—
90
92
—
—
—
11
14
61.0
61.0
61.0
21
26
—
—
—
H
H
Ph
a
Reactions were carried out employing 10 equiv of acyl donor, except for entries
1 and 2 (5 equiv). For other details, see Section 4.
b
Determined by GC or HPLC.
Conversion, c = ees/(ees + eep).
c
The effect of the substrate structure on the biocatalytic proper-
ties of the enzyme was also analyzed by testing phenyl alcohols
possessing larger alkyl chains than the methyl group, as shown
in Table 4. When racemic 1-phenylpropan-2-ol 13a was acetylated
in the presence of immobilized M. esculenta, (S)-13a and the corre-
sponding ester (R)-13b were obtained in a process with low con-
version (c = 12% after 116 h) and moderate enantioselectivity,
lower values than those observed with racemic phenylethan-1-ol.
The acetylation of longer chain alcohols, as for example ( )-14a,
led to the formation of (S)-14a and (R)-14b with a higher conver-
sion (c = 20%) and lower enantioselectivity. The acetylation of an
allyl alcohol such as ( )-1-phenylbut-3-en-1-ol gave a low activity
and selectivity, with only 6% of (R)-15b after 116 h being obtained.
The biocatalyzed acylation of racemic 2-hydroxy-2-phenylaceto-
nitrile took place with low conversion and enantioselectivity, with
(S)-16b being obtained only 4% after a long reaction time. When
the alcohol moiety is located further away from the aromatic ring,
there was also an important loss in the biocatalytic properties of
immobilized M. esculenta, as shown in Table 4 for 1-phenylpro-
pan-2-ol ( )-17a. Finally, the immobilized form of Manihot was
tested in the enzymatic acetylation of a tertiary alcohol, but no
reaction was observed when 2-phenylpropan-2-ol 18a was treated
with vinyl acetate in Et2O at 45 °C for 116 h.
At this point, we decided to study the behavior of M. esculenta
hydrogels in the biocatalyzed acetylation of racemic phenyl-
ethan-1-ols presenting different substituents in the aromatic moi-
ety. The best reaction conditions found for the enzymatic
resolution of ( )-1a (10 equiv of vinyl acetate, Et2O, and 45 °C)
were applied in the acetylation of different substituted phenyle-
than-1-ols, as summarized in Table 3. When employing com-
pounds with strong electron-withdrawing groups (4-NO2 and
3-NO2) as substrates, it was observed that the presence of the nitro
group at the meta-position increased to a great extent the enanti-
oselectivity of the process, being achieved an excellent E value
(E > 200) in the resolution of ( )-3a with a conversion of 12% after
116 h. When the nitro group was located at the para-position, the
enantioselectivity decreased (E = 53) in a reaction with a higher
conversion (c = 18%). Also, the acetylation of 3-trifluoromethyl-
phenylethan-1-ol ( )-4a led to the formation of (S)-4a and (R)-4b
with excellent enantioselectivity in a process with moderate con-
version. Some halide-substituted 1-phenylethan-1-ols were ana-
lyzed; the 4-bromo- and 4-chloro-derivatives [( )-5a and ( )-8a]
gave high E values (E > 100) with conversions close to 30% after
Table 3
To prove the efficiency of this biocatalyst, we have also investi-
gated the feasibility of reusing the immobilized M. esculenta under
Immobilized M. esculenta-catalyzed acetylation of substituted phenylethan-1-olsa
OH
OH
OAc
M. esculenta
Vinyl acetate/ Et2O
+
45 ºC / 250 r.p.m.
116 h
Table 4
X
X
X
Immobilized Manihot esculenta-catalyzed acetylation of phenyl alcoholsa
( )-2-12a
(
S)-2-12a
(R)-2-12b
M. esculenta
Vinyl acetate/Et2O
b
b
Compound
X
ees (%)
eep (%)
cc (%)
E
n
n
n
R
R
R
+
1a
2a
3a
4a
5a
6a
7a
8a
9a
H
40
94
95
99
99
97
96
88
97
96
95
91
95
30
18
12
12
28
12
18
28
16
27
28
16
50
53
45 ºC / 250 r.p.m.
OH
OH
OAc
4-NO2
3-NO2
3-CF3
4-Cl
3-Cl
2-Cl
4-Br
3-Br
3-OMe
4-OMe
4-OH
21
13
9
>200
>200
105
51
( )-13-17a
(
S
)-13-17a
(R)-13-17b
33
11
19
35
13
31
31
18
b
b
Compound
R
n
ees (%)
eep (%)
cc (%)
E
13a
14a
15a
16ad
17a
Et
Bu
Allyl
CN
Me
0
0
0
0
1
9
13
5
2
12
93
58
85
62
9
12
20
6
4
6
32
4
12
4
19
111
59
10a
11a
12a
55
29
50
1
a
For reaction details, see Section 4.
Determined by GC or HPLC.
Determined by 1H NMR.
b
c
a
Reactions were carried out employing 10 equiv of vinyl acetate. For other
details, see Section 4.
d
b
Absolute configuration is reversed due to a change in the substituent priority
Determined by GC or HPLC.
c
Determined by 1H NMR.
according to the sequence rules.

1422
L. L. Machado et al. / Tetrahedron: Asymmetry 19 (2008) 1419–1424
Table 5
kin–Elmer 1720-X infrared Fourier transform spectrophotometer
using KBr pellets. Optical rotations were measured using a Per-
kin–Elmer 241 polarimeter and are quoted in units of
Reuse of immobilized Manihot esculenta-catalyzed in the enzymatic acetylation of
4-bromophenylethan-1-ola
10ꢁ1 deg cm2 gꢁ1 1H NMR, 13C NMR, and DEPT spectra were re-
.
OH
OH
OAc
M. esculenta
Vinyl acetate/ Et2O
corded with TMS (tetramethylsilane) as the internal standard using
Bruker AC-300 (1H, 300.13 MHz and 13C, 75.4 MHz) and Bruker AC-
300-DPX (1H, 300.13 MHz and 13C: 75.4 MHz) spectrometers. The
chemical shift values (d) are given in ppm and the coupling con-
stants (J) in Hertz (Hz). APCI+ using a Hewlett Packard 1100 chro-
matograph mass detector or EI+ with a Hewlett Packard 5973 mass
spectrometer were used to record mass spectra (MS).
+
45 ºC /250 r.p.m.
Br
Br
Br
116 h
( )-8a
(S
)-8a
(R)-8b
b
b
Cycle no.
ees (%)
eep (%)
cc (%)
E
1
2
3
4
5
6
7
30
32
35
20
24
8
98
97
97
97
91
71
71
25
25
26
17
17
10
6
103
94
90
72
24
7
GC analyses were performed on a Hewlett Packard 6890 Series
II chromatograph equipped with a CP-Chiralsil DEX CB column
(30 m ꢂ 0.25 mm ꢂ 0.25
ing the enantiomeric purities and a HP-1 (crosslinked methyl silox-
m, 1.0 bar N2) from Hewlett–Packard
lm, 1.0 bar N2) from Varian for determin-
5
6
ane, 30 m ꢂ 0.25 mm 0.25
l
a
Reactions were carried out employing 10 equiv of vinyl acetate. For other
details, see Section 4.
for measuring the conversion values. For all the analyses, the injec-
tor temperature is 225 °C and the FID temperature is 250 °C. HPLC
analyses were developed with a Hewlett Packard 1100 LC liquid
chromatograph equipped with a Chiralcel OD (0.46 cm ꢂ 25 cm),
Chiralcel OB-H (0.46 cm ꢂ 25 cm), or Chiralpak IA (0.46 cm ꢂ
25 cm) chiral column from Daicel.
b
Determined by GC or HPLC.
c
Determined by 1H NMR.
the optimal reaction conditions found for the acetylation of 1-(4-
bromophenyl)ethan-1-ol, ( )-8a: Et2O as organic solvent, 10 equiv
of vinyl acetate as acylating agent, and 116 h of reaction time at
45 °C at 250 rpm. As shown in Table 5, the reuse of the immobi-
lized biocatalyst afforded almost the same enantioselectivity and
activity in the second and third cycles, with only a slight loss in
these biocatalytic properties for the fourth one (E = 72 and
c = 17%). When the biocatalyst was employed in the fifth cycle,
the conversion obtained was the same as the previous cycle, but
the enantioselectivity in the resolution of ( )-8a decreased by a
large extent (E = 24). For the next two cycles, the enzyme seemed
to be almost completely inactivated [(S)-8b was obtained with only
6% of conversion and E = 6 for the seventh cycle].
Alcohols ( )-1a, ( )-8a, ( )-9a, ( )-11a, ( )-13a, ( )-15–17a,
18a, and 4-hydroxyacetophenone were purchased from Sigma–
Aldrich–Fluka. Compounds ( )-2a, ( )-5-7a, ( )-14a, and
3-nitroacetophenone were products from Alfa Aesar. 3-Methoxy-
acetophenone and ( )-4a are available at Acros Organics. Starting
racemic alcohols 3a, 10a, and 12a were prepared from commercial
ketones by treatment with NaBH4 in methanol. In all cases, high
yields were achieved. Racemic esters ( )-1–17b and 18b were syn-
thesized by the chemical acetylation of the corresponding alcohols
(yields higher than 80%) employing acetic anhydride and triethyl-
amine in CH2Cl2. Preparation of racemic 1c and 1d was performed
by the addition of pyridine and benzoic anhydride or methoxyace-
tyl chloride, respectively, to a solution of ( )-1a in CH2Cl2, with
good yields being obtained. Solvents were distilled over an appro-
priate desiccant under nitrogen. All other reagents and solvents
were of the highest quality grade available, supplied by Sigma–
Aldrich–Fluka.
Absolute configurations of alcohols 1a, 8a, and 16a were deter-
mined by comparison of their GC chromatograms with samples
obtained from the commercial chiral alcohols (S)-1a, (S)-8a, and
(R)-16a. For compounds 2a,2a 3a,13a 4a,12b 5a,2a 6a,13b 7a,13b
9a,13c 10a,13d 11a,2a 12a,2a 13a,13e 14a,13e 15a,13f and 17a,13g the
absolute configurations were established by comparison of the
specific rotation measured with the ones previously reported.
3. Conclusion
In this paper, an enzymatic preparation obtained from the dis-
card waters of the treatment of M. esculenta roots was immobilized
with high efficiency by the alginate-calcium chloride method in
order to form the corresponding hydrogels. This preparation has
been employed as a chiral biocatalyst in the enzymatic resolution
of different alcohols. Analysis of the different reaction parameters
in the resolution of phenylethan-1-ol led to the highest activities
and selectivities when using 10 equiv of vinyl acetate as an acyl
donor in Et2O or iPr2O and 45 °C. Depending on the substrate struc-
ture, excellent enantioselectivities can be achieved with certain
substituted phenylethan-1-ols presenting electron-withdrawing
groups, while the enzyme presented high preference for the
para-position when using bromo- and chlorophenylethan-1-ols.
The enzymatic resolution of longer alkyl chain alcohols took place
with moderate to low selectivities. This enzymatic preparation has
been demonstrated to be reusable for four cycles with only a slight
loss in the activity and selectivity. Hence, by properly immobilizing
a food manufacture discard, we have been able to obtain a really
promising biocatalyst for the enzymatic resolution of racemic
alcohols.
4.2. Plant material and extraction
M. esculenta roots were collected at the Ematerce farm (Caucaia,
CE, Brazil). Identification was performed by Professor Edson P.
Nunes. Voucher specimen number 34831 has been deposited at
the Herbarium Prisco Bezerra (Departamento de Biologia, Univer-
sidade Federal do Ceará, Brazil). The roots were rinsed with a 5%
aqueous solution of sodium hypochlorite and distilled water and
after this, 5.0 kg of M. esculenta was grated and then pressed in
order to separate the aqueous phase manipueira (2.5 L) from the
solid residue (3.0 kg).
4. Experimental
4.1. General
4.3. Immobilization process
Biocatalyst from M. esculenta was immobilized using the cal-
cium alginate technique. Manipueira (300 mL) was shaken for 3 h
in the presence of 1.0% w/v sodium alginate. After homogenization,
this mixture was dropped into 400 mL of a 5.0% w/v solution of cal-
cium chloride, using a syringe with a 1.0 mm internal diameter
Chemical reactions were monitored by analytical TLC, per-
formed on Merck Silica Gel 60 F254 plates, and visualized by UV
irradiation. Flash chromatography was carried out with Silica Gel
60 (230–240 mesh, Merck). IR spectra were recorded on a Per-

L. L. Machado et al. / Tetrahedron: Asymmetry 19 (2008) 1419–1424
1423
needle. Upon contact with the solution, the drops were gelled to
form constant and defined-size spheres, which remained in the
solution for 12 h, under mild agitation, to complete gel formation.
After hardening, microspheres were washed several times with
water and acetone, and dried at room temperature until constant
weight (5.4 g). Determination of the protein content using the Har-
tree method has been done in triplicate at the initial and final
stages of the immobilization process. Experiments allowed the cal-
culation of protein content not incorporated into the beads (3.8%).
4.6.4. (R)-1-(4-Nitrophenyl)ethyl acetate, (R)-2b
Determination of the ee by GC analysis. 70 °C (10 min), 2 °C/
min, 200 °C; tR (S) 54.9 min; tR (R) 55.9 min.
4.6.5. (S)-3-Nitrophenylethan-1-ol, (S)-3a
Determination of the ee by HPLC analysis: Chiralcel OB-H, 20 °C,
n-hexane/2-propanol (95:5), 0.8 mL/min; tR (S) 23.8 min; tR (R)
27.3 min.
4.6.6. (R)-1-(3-Nitrophenyl)ethyl acetate, (R)-3b
Determination of the ee by GC analysis. 70 °C (10 min), 2 °C/
min, 200 °C; tR (S) 50.4 min; tR (R) 51.5 min.
4.4. Size of beads
By varying the orifice used for dropping the enzyme-sodium
alginate mixture, the dimension of the beads was varied. In this
case, the spherical diameter of the particles was determined as
1.0 mm by measuring microscopically the dimensions of beads
using Electronic Microscopy of Sweeping (MEV), with the aid of a
Philips XL-30 microscope.
4.6.7. (S)-3-Trifluoromethylphenylethan-1-ol, (S)-4a
Determination of the ee by GC analysis. 70 °C (10 min), 2 °C/
min, 200 °C; tR (S) 35.6 min; tR (R) 34.2 min.
4.6.8. (R)-1-(3-Trifluoromethylphenyl)ethyl acetate, (R)-4b
Determination of the ee by GC analysis. 70 °C (10 min), 2 °C/
min, 200 °C; tR (S) 22.4 min; tR (R) 26.1 min.
4.5. Typical procedure for the enzymatic acetylation of racemic
alcohols catalyzed by immobilized M. esculenta
4.6.9. (S)-4-Chlorophenylethan-1-ol, (S)-5a
Unless otherwise stated, the immobilized preparations of M.
esculenta (30 mg) and vinyl acetate (5 or 10 equiv, except when
used as the solvent) were added to a solution of the corresponding
racemic alcohol ( )-1a–17a, and 18a (30 mg, 1.0 equiv) in the
selected organic solvent (4.0 mL) at room temperature under a
nitrogen atmosphere. The resulting mixture was shaken at the
established temperature and at 250 rpm on an orbital shaker.
The progress of the reaction was monitored by TLC analysis. Once
the reaction was finished, the enzyme was filtered off, washed with
ethyl acetate, and the solvent evaporated under reduced pressure.
The crude residue was then purified by flash chromatography
using n-hexane/ethyl acetate 7:3 (for compounds 2–4a, 12a, and
14a) or 8:2 (1a, 5–11a, 13a, and 15–18a) in order to obtain the
optically active alcohols (S)-1–15a, (R)-16a, (S)-17a, and 18a and
the corresponding esters (R)-1–15a, (S)-16b, (R)-17b, and 18b.
Determination of the ee by GC analysis. 70 °C (10 min), 2 °C/
min, 200 °C; tR (S) 45.6 min; tR (R) 44.4 min.
4.6.10. (R)-1-(4-Chlorophenyl)ethyl acetate, (R)-5b
Determination of the ee by GC analysis. 70 °C (10 min), 2 °C/
min, 200 °C; tR (S) 37.8 min; tR (R) 39.9 min.
4.6.11. (S)-3-Chlorophenylethan-1-ol, (S)-6a
Determination of the ee by HPLC analysis: Chiralcel OB-H, 20 °C,
n-hexane/2-propanol (95:5), 0.8 mL/min; tR (S) 9.8 min; tR (R)
12.5 min.
4.6.12. (R)-1-(3-Chlorophenyl)ethyl acetate, (R)-6b
Determination of the ee by GC analysis. 70 °C (10 min), 2 °C/
min, 200 °C; tR (S) 34.6 min; tR (R) 36.9 min. ½a D25
¼ þ15:8 (c 0.80,
ꢃ
CH2Cl2), ee 96%.
4.6. Study of enzyme recycling in the acetylation of ( )-8a
catalyzed by immobilized M. esculenta
4.6.13. (S)-2-Chlorophenylethan-1-ol, (S)-7a
Determination of the ee by HPLC analysis: Chiralcel OB-H, 20 °C,
n-hexane/2-propanol (95:5), 0.8 mL/min; tR (S) 7.4 min; tR (R)
5.7 min.
To a solution of ( )-8a (30 mg, 0.15 mmol) in Et2O (4 mL) were
added 50 mg of immobilized M. esculenta and vinyl acetate
(138 lL, 1.5 mmol) under a nitrogen atmosphere. The reaction
was stirred at 45 °C and 250 rpm for 116 h. After this, the enzyme
was filtered off, washed with dry Et2O, and stored under nitrogen
atmosphere in order to start a new cycle. The biocatalyst was used
repeatedly in the subsequent enzymatic resolutions, following in
all cases the same procedure. In order to determine the conver-
sions and the enantiomeric excesses of the alcohol (S)-8a and the
ester (R)-8b, flash chromatography was performed using n-hex-
ane/ethyl acetate 8:2 as eluent in each cycle.
4.6.14. (R)-1-(2-Chlorophenyl)ethyl acetate, (R)-7b
Determination of the ee by GC analysis. 70 °C (10 min), 2 °C/
min, 200 °C; tR (S) 43.4 min; tR (R) 45.7 min. ½a D25
¼ þ9:6 (c 0.64,
ꢃ
CH2Cl2), ee 88%.
4.6.15. (S)-4-Bromophenylethan-1-ol, (S)-8a
Determination of the ee by GC analysis. 70 °C (10 min), 2 °C/
min, 200 °C; tR (S) 50.6 min; tR (R) 49.7 min.
4.6.1. (S)-Phenylethan-1-ol, (S)-1a
4.6.16. (R)-1-(4-Bromophenyl)ethyl acetate, (R)-8b
Determination of the ee by GC analysis. 70 °C (10 min), 2 °C/
min, 200 °C; tR (S) 43.2 min; tR (R) 45.0 min.
Determination of the ee by HPLC analysis: Chiralcel OB-H, 20 °C,
n-hexane/2-propanol (95:5), 0.8 mL/min; tR (S) 10.2 min; tR (R)
15.5 min.
4.6.17. (S)-3-Bromophenylethan-1-ol, (S)-9a
Determination of the ee by HPLC analysis: Chiralcel OB-H, 20 °C,
n-hexane/2-propanol (95:5), 0.8 mL/min; tR (S) 11.0 min; tR (R)
14.2 min.
4.6.2. (R)-1-Phenylethyl acetate, (R)-1b
Determination of the ee by GC analysis. 70 °C (5 min), 3 °C/min,
200 °C; tR (S) 17.5 min; tR (R) 19.1 min.
4.6.3. (S)-4-Nitrophenylethan-1-ol, (S)-2a
Determination of the ee by HPLC analysis: Chiralpak IA, 20 °C,
n-hexane/2-propanol (96:4), 0.6 mL/min; tR (S) 35.2 min; tR (R)
33.7 min.
4.6.18. (R)-1-(3-Bromophenyl)ethyl acetate, (R)-9b
Determination of the ee by GC analysis. 70 °C (10 min), 2 °C/
min, 200 °C; tR (S) 39.5 min; tR (R) 41.3 min. ½a D25
¼ þ13:7 (c 0.75,
ꢃ
CH2Cl2), ee 96%.

1424
L. L. Machado et al. / Tetrahedron: Asymmetry 19 (2008) 1419–1424
4.6.19. (S)-3-Methoxyphenylethan-1-ol, (S)-10a
Determination of the ee by HPLC analysis: Chiralcel OB-H, 20 °C,
n-hexane/2-propanol (95:5), 0.8 mL/min; tR (S) 21.5 min; tR (R)
31.1 min.
4.6.33. (S)-1-Phenylpropan-2-ol, (S)-17a
Determination of the ee by HPLC analysis: Chiralpak IA, 25 °C,
n-hexane/2-propanol (98:2), 0.3 mL/min; tR (S) 38.9 min; tR (R)
41.3 min.
4.6.20. (R)-1-(3-Methoxyphenyl)ethyl acetate, (R)-10b
4.6.34. (R)-1-Phenylprop-2-yl acetate, (R)-17b
Determination of the ee by GC analysis. 70 °C (5 min), 3 °C/min,
200 °C; tR (S) 21.8 min; tR (R) 22.8 min.
Determination of the ee by GC analysis. 70 °C (10 min), 2 °C/
min, 200 °C; tR (S) 38.8 min; tR (R) 40.0 min. ½a D25
¼ þ27:8 (c 0.94,
ꢃ
CH2Cl2), ee 95%.
4.6.35. 2-Phenylpropan-2-ol, 18a
4.6.21. (S)-4-Methoxyphenylethan-1-ol, (S)-11a
Determination of the degree of conversion by GC analysis, HP-1,
60 °C (5 min) 2 °C/min 200 °C; tR 6.7 min.
Determination of the ee by HPLC analysis: Chiralcel OB-H, 20 °C,
n-hexane/2-propanol (95:5), 0.8 mL/min; tR (S) 25.2 min; tR (R)
37.7 min.
4.6.36. 2-Phenylprop-2-yl acetate, 18b
4.6.22. (R)-1-(4-Methoxyphenyl)ethyl acetate, (R)-11b
Determination of the ee by GC analysis. 70 °C (10 min), 2 °C/
min, 200 °C; tR (S) 41.0 min; tR (R) 42.5 min.
tR 14.2 min.
Acknowledgments
4.6.23. (R)-4-Hydroxyphenylethan-1-ol, (S)-12a
Determination of the ee by HPLC analysis: Chiralcel OB-H, 20 °C,
n-hexane/2-propanol (95:5), 0.8 mL/min; tR (S) 34.8 min; tR (R)
41.1 min.
This work was supported by the Ministerio de Educación y
Ciencia of Spain (Project CTQ 2007-61126). G. de G. and V.G.F.
thank MEC for personal grants (Juan de la Cierva and Ramon y Cajal
program, respectively). The authors thank the Brazilian and Span-
ish agencies CNPq, FUNCAP, PRONEX, CAPES-DGU (Process: 149/
07) for fellowships and financial support. The authors also thank
Dr. Pedro de L. Neto and Msc. Regilany P. Colares (UFC-Brazil) for
assistance in the scanning electron microscopy of M. esculenta
hydrogels.
4.6.24. (R)-1-(4-Hydroxyphenyl)ethyl acetate, (R)-12b
Determination of the ee by HPLC analysis: Chiralcel OB-H, 20 °C,
n-hexane/2-propanol (95:5), 0.8 mL/min; tR (S) 32.3 min; tR (R)
22.3 min. ½a 2D5
¼ þ11:2 (c 1.00, CH2Cl2), ee 95%.
ꢃ
4.6.25. (S)-1-Phenylpropan-1-ol, (S)-13a
Determination of the ee by HPLC analysis: Chiralcel OB-H, 20 °C,
n-hexane/2-propanol (95:5), 0.8 mL/min; tR (S) 7.7 min; tR (R)
9.2 min.
References
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4.6.26. (R)-1-Phenylprop-1-yl acetate, (R)-13b
Determination of the ee by GC analysis. 70 °C (5 min), 3 °C/min,
200 °C; tR (S) 20.0 min; tR (R) 20.9 min.
4.6.27. (S)-1-Phenylpentan-1-ol, (S)-14a
Determination of the ee by HPLC analysis: Chiralcel OB-H, 20 °C,
n-hexane/2-propanol (97:3), 0.5 mL/min; tR (S) 15.0 min; tR (R)
17.0 min.
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4.6.28. (R)-1-Phenylpentan-1-yl acetate, (R)-14b
Determination of the ee by HPLC analysis: Chiralcel OB-H, 20 °C,
n-hexane/2-propanol (97:3), 0.5 mL/min; tR (S) 12.2 min; tR (R)
9.0 min.
4.6.29. (S)-1-Phenylbut-3-en-1-ol, (S)-15a
Determination of the ee by HPLC analysis: Chiralcel OB-H, 20 °C,
n-hexane/2-propanol (98:2), 0.6 mL/min; tR (S) 17.3 min; tR (R)
22.8 min.
8. Hartree, E. F. Anal. Biochem. 1972, 48, 422–427.
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4.6.30. (R)-1-Phenylbut-3-en-1-yl acetate, (R)-15b
Determination of the ee by HPLC analysis: Chiralcel OB-H, 20 °C,
n-hexane/2-propanol (98:2), 0.6 mL/min; tR (S) 10.8 min; tR (R)
9.8 min.
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4.6.31. (R)-2-Hydroxy-2-phenylacetonitrile, (S)-16a
Determination of the ee by GC analysis after derivatization by
acetylation with acetic anhydride. 70 °C (5 min), 3 °C/min,
200 °C; tR (S) 26.9 min; tR (R) 28.7 min.
4.6.32. (S)-Cyano(phenyl)methyl acetate, (R)-16b
Determination of the ee by GC analysis. 70 °C (5 min), 3 °C/min,
200 °C; tR (S) 26.9 min; tR (R) 28.7 min.