Biotransformation of Aromatic Ketones by Linum usitatissimum

Full text of DOI:10.1007/s10600-015-1400-y

DOI 10.1007/s10600-015-1400-y
Chemistry of Natural Compounds, Vol. 51, No. 4, July, 2015
B R I E F C O M M U N I C A T I O N S
BIOTRANSFORMATION OF AROMATIC KETONES
BY Linum usitatissimum
1
1*
Leonardo C. Tavares, Angela M. C. Arriaga,
1
1
Telma L. G. de Lemos, Juliana M. O. Souza,
Maria V. S. Teixeira, and Gilvandete M. P. Santiago
1
1,2
Acetophenone is a component of castoreum, the exudate from the castor sacs of the mature beaver [1]. Linum
usitatissimum L. (Linaceae), known as flax or linseed, is a good source of oil and meal, which is rich in fiber, protein, and fat.
Its oil is arguably one of the richest source of polyunsaturated fatty acids, used in food and in the prevention of cardiovascular
diseases [2, 3].
The use of natural catalysts offers a clean way to perform chemical process under mild reaction conditions with less
or no usage of organic solvents, a high degree of selectivity due to the high chemo-, regio- and enantioselectivity of enzymes,
while working under mild and environmental friendly conditions linked to the principles of green chemistry [4–8]. Thus, the
biocatalysts can be an interesting alternative to prepare chiral alcohols and can also act as chiral auxiliaries in asymmetric
synthesis of chiral molecules, and as a result the production of single enantiomers of molecules has become increasingly
important in the food, cosmetic, and pharmaceutical industries, from the corresponding prochiral ketones [6, 9–13]. Therefore,
researchers have strived to find solutions for the replacement of conventional chemical routes by application of enzymes.
Reports in the literature describe the use of different plant species for biotransformation, with several advantages
such as their disposal after use, their biodegrability, as well as their wide availability at low cost [6, 8–11, 14–17].
In particular, the potential source of enzymes from Brazilian northwestern plants and their use as biocatalysts in
bioreduction reactions have been related [10, 11, 14, 17, 18]. In order to contribute to the study of vegetables as natural
catalyst, this paper reports the use of whole plant cell of seeds of L. usitatissimum as a biocatalyst for the reduction reactions
of aromatic ketones.
The enzymatic reduction of acetophenone (1) as standard substrate prochiral ketone and derivatives 2–14 using
L. usitatissimum as biocatalyst is reported. All reactions were carried out using 50 mg of substrate and 20 g of biocatalyst in
a buffer solution (Na HPO –KH PO ), pH 6.0, over a period of 72 h at 25ꢀC without a co-solvent which are the optimal
2
4
2
4
reaction conditions among the tested parameters.
The bioreduction pattern of acetophenone (1) by L. usitatissimum occurred with a bioconversion value of 70.4%,
a very good enantioselectivity (93.7% ee), and a satisfactory yield of (S)-alcohol. The study of reduction enzymatic reaction
of acetophenone 1 and other prochiral ketones derived from acetophenones 2 to 12, includingꢁꢁꢂ-tetralone 13 and 1-acetonaphthone
1
4, was performed (Scheme 1).
The results showed that L. usitatissimum exhibited a broad spectrum of enantionselectivity among the
acetophenones and their derivatives, with a very good enantioselectivity (>75% ee) for acetophenone (1) and its derivatives
, 6, 8, 9, and 10 (Table 1). It was observed that the best enantiomeric excess (ee) compared with acetophenone (1) was
obtained from 4-methylacetophenone (6) and 2-methoxyacetophenone (10), with an ee of 93.8 and ꢃ 99.0, respectively.
2
1
) Programa de Pos-Graduacao em Quimica, Universidade Federal do Ceara, Cx Postal 6036, CEP 60451-970,
Fortaleza-Ceara, Brazil, e-mail: angelamcarriaga@yahoo.com.br, ang@ufc.br; 2) Departamento de Farmacia, Universidade
Federal do Ceara, Rua Capitao Francisco Pedro 1210, CEP 60430-370, Fortaleza-Ceara, Brazil. Published in Khimiya Prirodnykh
Soedinenii, No. 4, July–August, 2015, pp. 645–647. Original article submitted October 23, 2013.
0
752
0009-3130/15/5104-0752 2015 Springer Science+Business Media New York

TABLE 1. Linum usitatissimum Seeds as Biocatalyst in Reduction of Acetophenone and Its Derivatives
Substrate
Bioconversion, %
ee, %
Substrate
Bioconversion, %
ee, %
1
2
3
4
5
6
7
70.4
99.2
97.5
39.1
2.9
92.1
73.4
93.7 (S)
84.5 (S)
62.2 (S)
41.3 (S)
64.4 (S)
93.8 (S)
46.8 (S)
8
20.6
40.8
1.1
N.d.
80.3
N.d.
83.1
81.0 (S)
85.1 (S)
ꢃ 99 (S)
9
10
11
12
13
14
76.5 (S)
78.9 (S)
_
_____
N.d.: not determined.
O
OH
1
2
a
4
R
1
R
3
R
1
R
3
R
2
R
2
1
1
- 10, 12
(S)-1-10, 12
O
O
1' OH
O
1
2
1
a
a
and
4
n.d.
OH
1
1
3
14
(S)-14
1
5
8
1
: R = R = R = H; 2: R = Br, R = R = H; 3: R = F, R = R = H; 4: R = Cl, R = R = H
1 2 3 1 2 3 1 2 3 1 2 3
: R = OCH , R = R = H; 6: R = CH , R = R = H; 7: R = R = H, R = Br
1
3
2
3
1
3
2
3
1
3
2
: R = R = H, R = OCH ; 9: R = R = H, R = Br; 10: R = R = H, R = OCH
1
3
2
3
1
2
3
1
2
3
3
2: R = R = H, R = Ph
1
2
3
a. Linum usitatissimum
Scheme 1
Additionally we observed that 2-methoxyacetophenone (10) was reduced to an (S)-alcohol with excellent ee (ꢃ 99%) but
with a very low bioconversion of 1.1%. The bioconversion values and ee are given in Table 1. The configurations of the
enantiomers 1–10, 12, and 14 were established as “S” after comparison of their specific rotation with those described in the
literature, which is in agreement with the Prelog model for bioreduction [18]. The bioreduction of the 2-hydroxyacetophenone
(
11) and ꢂ-tetralone (13) was not observed in 11, probably because of the ortho-position of the hydroxyl group in the aromatic
ring, which favors the formation of intramolecular hydrogen bonds and makes the carbonyl group less reactive. In derivative
1
3 we can attribute this to steric hindrance, making enzyme-substrate interaction more difficult.
Values obtained from the seeds of L. usitatissimum were poor when compared to the use of immobilized yeast cells
such as Cryptococcus laurentii as biocatalyst [19]. Therein a higher percentage of bioconversion and an enantiomeric excess
was observed for compounds 1–7 and 9, but there was no change in enantioselectivity of the reaction, yielding the corresponding
(
S)-enantiomer of the alcohol, which is in agreement with Prelogꢄs rule. This was also related to the low values of bioconversion
in acetophenone derivatives containing electron-donating substituents at the para-position [19, 20].
In conclusion, this paper reports a novel, facile, efficient, and green biocatalyst for reducing acetophenone, a natural
compound, and some of its derivatives. Seeds of Linum usitatissimum L. can be very effective in the stereoselective reduction
of this aromatic carbonyl compound with enantioselectivity from medium to high conversions.
Commercial seeds of Linum usitatissimum were purchased from a local market. Botanical identification data were
provided by the Celeiro Alimentos Co. (celeiro@celeiroalimentos.com.br).
Studies were previously performed to determine the optimal reaction conditions among the parameters: amount of
biocatalyst (2 g, 5 g, 10 g, and 20 g), time of reaction (24 h, 48, 72, and 96 h), use of co-solvent isopropyl alcohol in the
proportions (v/v) 2, 5, and 10% in distilled water and a buffer solution previously prepared from Na HPO –KH PO , with
2
4
2
4
pH 6.0, 7.0, and 8.0, using 50 mg of acetophenone as pattern substrate. Therefore, the reactions were carried out in the best
reaction conditions among the parameters tested, using 50 mg of substrate and 20 g of biocatalyst in a buffer solution
753

(
Na HPO –KH PO ), pH 6.0, over a period of 72 h at 25ꢀC without a co-solvent. In these reaction conditions, 70.4% of
2 4 2 4
bioconversion and an ee of 93.7% were obtained for pattern acetophenone. All biotransformation reactions were performed
using a modified methodology proposed by Machado et al. [11]. Whole seeds of Linum usitatissimum L. were washed with 5%
sodium hypochlorite and rinsed with sterile distilled water. Each individual carbonyl substrate, 1–14 (50 mg), was added to a
suspension of 20 g of L. usitatissimum seeds in 40 mL of a buffered solution (Na HPO –KH PO ), pH 6.0, and incubated at
2
4
2
4
2
5ꢀC in an orbital shaker (175 rpm) for 72 h. Controls were similarly processed, except that no substrates were added. All
reactions were performed in triplicate. The course of all reactions was monitored by TLC (Merck, silica gel 60 F254) and the
substances revealed by spraying with vanillin solution. After completion of the reaction, each suspension was filtered and
washed with water, and the aqueous solutions were extracted with CH Cl (3 ꢅ 50 mL). The organic phases were dried with
2
2
Na SO and removed in a rotator evaporator. The reaction products were purified by column chromatography on silica gel
2
4
6
0 VETEC with a binary mixture of hexane–ethyl acetate (8:2, v/v) as eluent to afford the (S)-alcohols (Scheme 1). The optical
rotations were measured on a PerkinElmer 241 digital polarimeter.
All reaction products were analyzed by high-performance liquid chromatography (HPLC) using an L201147 Shimadzu
pump equipped with chiral columns OB-H and OD-H, a mobile phase binary mixture of n-hexane–isopropyl alcohol (90:10 to 98:2)
varying in composition according to the requirements of the sample, and a Shimadzu UV-Vis detector SPD-M20A. The mass
spectra were obtained using a Shimadzu GC-2010 plus model gas chromatograph coupled to a Shimadzu GCMS-QP model
SE 2010 mass spectrometer, using a column with a stationary phase of 95% dimethylpolysiloxane–5% diphenylpolysiloxane
with a length of 30 m, internal diameter 0.25 mm, outer diameter of 0.39 mm, and 0.25 ꢆm film thickness and a Shimadzu
auto-injector, modelAOC-20i. The mobile phase was helium. The levels of conversion and enantiomeric excess of biocatalytic
reactions were evaluated by a calibration curve (HPLC) made in the column corresponding to the starting ketone 1–14 and its
corresponding alcohol (Table 1).
2
0
+
(
S)-1-Phenylethanol (1). [ꢂ] –33.1ꢀ (c 0.01, CHCl ). GC-MS m/z (%): 122 (M , C H O; 25), 107 (100), 79 (90),
D 3 8 10
4
4
9
3 (30); HPLC: Chiralcel OB-H, 30ꢀC, n-hexane–isopropyl alcohol (95:5), 0.5 mL/min, t (min): 9.8 (S), 14.4 (R).
R
20
+
(
S)-1-(4-Bromophenyl)ethanol (2). [ꢂ]
–24.6ꢀ (c 0.015, CHCl ). GC-MS m/z (%): 200 (M , C H BrO; 20), 185 (60),
D 3 8 9
3 (60), 77 (100); HPLC: Chiralcel OB-H, 30ꢀC, n-hexane–isopropyl alcohol (95:5), 0.3 mL/min, t (min): 15.4 (S), 17.4 (R).
R
2
0
+
(
S)-1-(4-Fluorophenyl)ethanol (3). [ꢂ] –18.2ꢀ (c 0.02, CH Cl ). GC-MS m/z (%): 140 (M , C H FO; 20), 125 (100),
D 2 2 8 9
7 (70), 77 (30); HPLC: Chiralcel OB-H, 30ꢀC, n-hexane–isopropyl alcohol (95:5), 0.3 mL/min, t (min): 15.2 (S), 16.5 (R).
R
0
+
(
S)-1-(4-Chlorophenyl)ethanol (4). [ꢂ] 2D –19.6ꢀ (c 0.01, CH Cl ). GC-MS m/z (%): 156 (M , C H ClO; 30),
2 2 8 9
1
9
41 (100), 113 (40), 77 (100); HPLC: Chiralcel OB-H, 30ꢀC, n-hexane–isopropyl alcohol (98:2), 0.8 mL/min, t (min):
R
.4 (S), 10.5 (R).
+
(
S)-1-(4-Methoxyphenyl)ethanol (5). [ꢂ] 2D 0 –20.1ꢀꢁ(c 0.01, CH Cl ). GC-MS m/z (%): 152 (M , C H O ; 30), 137
2
2
9 12 2
(
100), 109 (50), 77 (30); HPLC: Chiralcel OB-H, 30ꢀC, n-hexane–isopropyl alcohol (92:8), 0.8 mL/min, t (min): 10.5 (S),
R
1
4.0 (R).
(
S)-1-(4-Methylphenyl)ethanol (6). [ꢂ] 2D 0 –23.7ꢀ (c 0.012, CH Cl ). GC-MS m/z (%): 136 (M , C H O; 40), 121
+
2 2 9 12
(
100), 93 (80), 77 (35); HPLC: Chiralcel OB-H, 30ꢀC, n-hexane–isopropyl alcohol (98:2), 0.5 mL/min, t (min): 17.0 (S),
R
1
9.0 (R).
20
+
(
S)-1-(3-Bromophenyl)ethanol (7). [ꢂ] –29.2ꢀ (c 0.011, CHCl ). GC-MS m/z (%): 200 (M , C H BrO; 20), 185
D 3 8 9
(
50), 77 (100), 43 (60); HPLC: Chiralcel OB-H, 30ꢀC, n-hexane–isopropyl alcohol (95:5), 0.5 mL/min, t (min): 10.4 (S),
R
1
4.0 (R).
20
+
(
S)-1-(3-Methoxyphenyl)ethanol (8). [ꢂ] –26.1° (c 0.01, CHCl ). GC-MS m/z (%): 152 (M , C H O ; 50), 135
D 3 9 12 2
(
70), 109 (100), 77 (50); HPLC: Chiralcel OB-H, 30ꢀC, n-hexane–isopropyl alcohol (92:8), 0.8 mL/min, t (min): 8.6 (S), 11.5 (R).
R
2
0
+
(
S)-1-(2-Bromophenyl)ethanol (9). [ꢂ]D –24.7ꢀ (c 0.013, CHCl ). GC-MS m/z (%): 200 (M , C H BrO; 10), 185
3 8 9
(
80), 77 (100), 43 (30); HPLC: Chiralcel OB-H, 30ꢀC, n-hexane–isopropyl alcohol (95:5), 0.5 mL/min, t (min): 7.2 (S), 11.9 (R).
R
S)-1-(2-Methoxyphenyl)ethanol (10). [ꢂ] 2D 0 –22.4ꢀ (c 0.02, CHCl ). GC-MS m/z (%): 152 (M , C H O ; 30), 137
+
(
3
9 12 2
(
100), 107 (70), 77 (30); HPLC: Chiralcel OB-H, 30ꢀC, n-hexane–isopropyl alcohol (95:5), 0.5 mL/min, t (min): 10.2 (S).
R
2
0
+
(
S)-1-(2-Biphenylyl)ethanol (12). [ꢂ] –22.4ꢀ (c 0.015, CHCl ). GC-MS m/z (%): 198 (M , C H O; 10), 107
D 3 14 14
(
70), 92 (100), 79 (60); HPLC: Chiralcel OB-H, 30ꢀC, n-hexane–isopropyl alcohol (95:5), 1 mL/min, t (min): 7.0 (S), 8.1 (R).
R
2
0
+
(
S)-1-(1ꢄ-Napthyl)ethanol (14). [ꢂ] –16.1ꢀ (c 0.02, CH Cl ). GC-MS m/z (%): 172 (M , C H O; 30), 157 (30),
D 2 2 12 12
1
29 (100), 43 (10); HPLC: Chiralcel OD-H, 30ꢀC, n-hexane–isopropyl alcohol (90:10), 0.5 mL/min, t (min): 11.5 (S), 15.8 (R).
R
754

ACKNOWLEDGMENT
The authors thank Brazilian agencies CNPq, PRONEX, and CAPES for fellowships and financial support.
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