F.M. Perna et al. / Journal of Molecular Catalysis B: Enzymatic 124 (2016) 29–37
35
carried out in MRS medium [39] containing: 20 g/L glucose, 10 g/L
121 (10), 113 (36), 112 (15), 111 (14), 103 (25), 78 (12), 77 (93), 75
(17), 74 (13), 51 (20), 50 (12), 43 (33).
peptone, 8 g/L meat extract, 4 g/L yeast extract, 1 g/L Tween 80,
•
•
2
2
0
g/L di-potassium hydrogen phosphate, 5 g/L sodium cetate 3H O,
(R)-1-(3-Chlorophenyl)ethanol (2c): [52,53] (99%) er = 99:1
2
◦
◦
◦
g/L tri-ammonium citrate, 0.2 g/L of magnesium sulfate 7H O,
[GC, T (oven program) 80 C (2 min) to 100 C at 10 C/min]:
2
◦
•
20
.05 g/L manganese sulfate 2H O. Cells were incubated at 37 C
(R)-isomer: 20.30 min, (S)-isomer: 20.51 min. [˛]D = +56.1 (c 1,
CHCl ); ıH (400 MHz, CDCl3 25 C) 7.38 (1H, m, aromatic pro-
2
◦
for 24 h, statically. Cell density was monitored using optical den-
sity at 620 nm (OD620) with a Genesys TM 20 spectrophotometer
3
ton), 7.32–7.20 (3H, m, aromatic protons), 4.88 (1H, q, J 6.4 Hz);
(
Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA).
1.90–1.88 (1H, bs, OH: exchanges with D O);1.48 (3H, d, J 6.4 Hz,);
2
Blank experiments: A 1 L flask containing 50 mL of the cul-
ı (100 MHz, CDC1 ) 148.1, 134.6, 123.0, 127.8, 125.8, 123.7, 70.0,
25.4.
C
3
◦
ture medium was stirred at 37 C on an orbital shaker at 200 rpm.
Ketones 1a-o (100 mg) were added. The content of the flask was
extracted with diethyl ether and analyzed. After 4 h no reaction
occurred, as monitored by NMR analysis.
Bioreduction of acetophenone (1a) by using Lactobacillus
reuteri growing cells: Lactobacillus reuteri DSM 20016 was grown
in MRS medium overnight. The cells were inoculated at OD620 = 0.2
in flasks containing 50 mL of MRS, in which the ketone 1a was added
at the final concentration of 1 g/L. To ensure the anaerobic condi-
(R)-1-(2-Chlorophenyl)ethanol (2d): [15,50,54] (89%) er = 99/1
(HPLC LUX Cellulose-1, Hexane/IPA = 9:1, 0.5 mL/min, ꢁ = 230 nm;
20
(R)-isomer: 10.4 min, (S)-isomer: 10.9 min); [˛]D = +68.0 (c 1,
−
1
CHCl ); ꢂmax/cm
(neat): 3351, 3069, 2976, 2929, 2872, 1722,
3
1596, 1575, 1473, 1436, 1369, 1348, 1266, 1203, 1133, 1094, 1073,
◦
1048, 1036, 1009, 946, 900, 754, 729, 693. ıH (400 MHz, CDCl 25 C)
3
7.60 (1H, d, J 7.7 Hz, aromatic proton); 7.34–7.18 (3H, m, aromatic
protons); 5.29 (1H, q, J 6.4 Hz); −2.15-2.05 (1H, bs, OH: exchanges
with D O); 1.49 (3H, d, J 6.4 Hz). ı (100 MHz, CDC1 ) 143.3, 131.8,
tions, flasks were degassed with N for 3 min. The reaction mixture
2
2
C
3
+
◦
37
was incubated at 37 C, 200 rpm. The reactions were monitored by
129.6, 128.6, 127.4, 126.6, 67.1, 23.7; m/z : 158 [M ( Cl), 6], 156
[M ( Cl), 19], 143 (32), 141 (100), 113 (24), 77 (66), 75 (10), 51
(12), 43(9).
+
35
collecting suspension aliquots in time course: after extraction with
diethyl ether, the organic phase was analyzed by NMR or HPLC.
Bioreduction of 1a-o by using Lactobacillus reuteri resting
cells: L. reuteri strains were grown in MRS medium. This pre-
(R)-1-(4-Fluorophenyl)ethanol (2e): [55] (98%) er = 99:1 [GC,
◦
◦
◦
T (oven program) 80 C (2 min) to 100 C at 10 C/min]: (R)-
20
culture was inoculated in 500 mL of MRS and incubated for 24 h
isomer: 48.4 min, (S)-isomer: 48.9 min; [˛]D = +42.0 (c 1, CHCl ).
3
◦
◦
(
37 C). Cells were harvested by centrifugation (2800 × g, 10 min)
ıH (400 MHz, CDCl3 25 C) 7.34–7.28 (2H, m, aromatic protons);
and washed twice with phosphate buffered saline pH 7.4 (PBS,
Sigma–Aldrich). Then the cells were suspended in the same buffer
and adjusted for cell density. To this cell suspension, 1% glucose and
the desired concentration of arylketone were added (total volume
7.05–6.97 (2H, m, aromatic protons); 4.85 (1H, q, J 6.4 Hz), 2.19 (1H,
br s, OH, exchanges with D O), 1.45 (3H, d, J 6.4 Hz). ı (100 MHz,
2
C
CDCl ) 162.1 (d, J 245 Hz), 141.5 (d, J 3 .1 Hz), 127.0 (d, J 8.1 Hz),
3
115.2 (d, J 21.3 Hz), 69.7, 25.3.
5
0 mL). To ensure the anaerobic conditions, flasks were degassed
(R)-1-[4-(Trifluoromethyl)phenyl]ethanol
(2f):
[56,57]
◦
◦
with a N flux for 3 min. The reaction mixture was incubated at
3
before the extraction of the bioconversion products. The reactions
progress was monitored by collecting suspension aliquots in a time
course: after extraction with diethyl ether, the organic phase was
analyzed by NMR or HPLC. After the appropriate conversion time
(99%) er = 98:2 [GC, T (oven program) 80 C (2 min) to 100 C
2
◦
◦
7 C, 200 rpm. Sugars and organic acids were analyzed by HPLC
at 10 C/min]: (R)-isomer: 38.5 min, (S)-isomer: 38.9 min.
2
0
−1
(neat): 3355, 1620, 1415,
[˛]D = +24.4 (c 1, CHCl ); ꢂmax/cm
3
◦
1125, 841. ıH (400 MHz, CDCl3 25 C) 7.65–7.60 (2H, m, aromatic
protons); 7.55–7.45 (2H, m, aromatic protons); 5.0 (1H, q, J 6.4 Hz),
1.95 (1H, br s, OH, exchanges with D O), 1.50 (3H, d, J 6.4 Hz). ı
2
C
(
4
see Table 3), the suspension was centrifuged (2800 × g, 10 min,
(100 MHz, CDCl ) 149.8, 129.6, 125.7, 122.7, 69.7, 25.5.
(R)-1-(4-Aminophenyl)ethanol (2g): [58] (70%) er = 51/49
(HPLC LUX Cellulose-1, Hexane/IPA = 90:10, 0.4 mL/min,
3
◦
C), and the aqueous phase was extracted with diethyl ether
(
3 × 15 mL). The organic phase was dried over Na SO , filtered, and
2
4
evaporated under reduced pressure. The product was isolated by
silica gel column chromatography using hexane and ethyl acetate
ꢁ = 230 nm); (R)-isomer: 7.17 min, (S)-isomer: 7.50 min; ıH
◦
(400 MHz, CDCl3 25 C) 7.20–7.15 (m, 2H, aromatic protons);
(
90:10 or 80:20) as eluents to yield the desired alcohols (2a-o,
6.70–6.60 (m, 2H, aromatic protons); 4.80 (1H, q, J 6.4 Hz);
Table 2). Absolute configurations of alcohols 2a-o obtained from
the bioprocess were determined by comparison of their optical spe-
cific rotation signs and/or retention times with known data (See
Supporting informations).
3.70–3.60 (2H, br s, NH , exchange with D O), 1.75–1.50 (1H, br s,
2
2
OH, exchanges with D O), 1.47 (3H, d, J 6.4 Hz, CH ); ı (100 MHz,
2
3
C
CDC1 ) 145.7, 136.2, 126.7, 115.2, 70.0, 24.9.
3
(R)-1-(2-Methoxyphenyl)ethanol (2i): [15,59] (50%) er = 83/17
(HPLC LUX Cellulose-1, Hexane/IPA = 90:10, 1 mL/min, ꢁ = 230 nm);
(R)-1-Phenylethanol (2a) [15,49,50,51] (94%) er = 99/1 (HPLC
2
0
LUX Cellulose-1, Hexane/IPA = 90:10, 1 mL/min, ꢁ = 254 nm) (R)-
(S)-isomer: 8.9 min, (R)-isomer: 9.4 min; [˛]D = +16.0 (c 1, CHCl );
3
2
0
−1
isomer: 6.00 min, (S)-isomer: 6.99 min; [˛]D = +45.3 (c 1, CHCl );
ꢂmax/cm (neat): 3391, 3107, 3067, 3035, 2971, 2930, 2837, 1602,
3
−
1
ꢂmax/cm 3346, 3080, 3063, 3030, 2973, 2927, 2875, 1602, 1493,
1588, 1489, 1464,1456, 1393, 1366, 1285, 1243, 1194, 1175, 1162,
1
7
451, 1410, 1369, 1304, 1262, 1204, 1077, 1029, 1011, 997, 898,
1125, 1079, 1050, 1031, 1011, 935, 899, 852, 802, 756; ıH (400 MHz,
◦
60, 699. ıH (400 MHz, CDCl ) 7.39–7.26 (5H, m, ArH), 4.9 (1H, q,
CDCl , 25 C) 7.33 (1H, d, J 7.3 Hz); 7.22 (1H, m); 6.94 1H, m); 6.85
3
3
J 6.5, CH), 1.93–1.88 (1H, br s, OH, exchanges with D O), 1.66 (3H,
(1H, d, J 8.2, Hz); 5.08 (1H, q, J 6.4 Hz); 3.82 (3H, s); 2.89–2.71 (1H,
br s, OH: exchanges with D O); 1.48 (3H, d, J 6.4 Hz). ı (100 MHz,
2
d, J 6.5, CH ); ı (100 MHz, CDCl ) 145.8, 128.5, 127.4, 125.4, 70.4,
3
C
3
2
C
+
2
(
5.1; m/z: 122 (M , 4%), 107 (10), 105 (1), 79 (10), 78 (2), 77 (6), 51
2), 43 (1).
R)-1-(4-Chlorophenyl)ethanol (2b): [15,49,50,51] (99%)
CDCl ) 156.5, 133.5, 128.2, 126.0, 120.8, 110.4, 66.3, 55.2, 22.9; m/z
3
+
: 152 (M , 26), 137 (100), 121 (18), 109 (27), 107 (63), 94 (14), 91
(
(13), 77 (26), 65 (11), 51 (9), 43 (9).
◦
◦
er = 99/1 [GC, T (oven program) 80 C (2 min) to 100 C
(R)-1-(4-Methoxyphenyl)ethanol
(2j):
[15,60,61,62]
◦
at 10 C/min]: (R)-isomer: 23.3 min, (S)-isomer: 23.6 min.;
(93%); er = 95/5 (HPLC LUX Cellulose-1, Hexane/IPA = 90:10,
−1
[
˛]D20 = +42.1 (c 1, CHCl ); ꢂmax/cm (neat): 3326, 2966, 2920,
1 mL/min, ꢁ = 230 nm); (R)-isomer: 8.1 min, (S)-isomer: 8.5 min;
3
20
−1
1
596, 1403, 1373, 1260, 1200, 1088, 1013, 896, 826; ıH (400 MHz,
[˛]D = +50.9 (c 1, CHCl ). ꢂmax/cm (neat): 3391, 3064, 2971,
3
◦
CDCl3 25 C) 7.31–7.26 (4H, m, aromatic protons); 4.84 (1H, q, J
2932, 2837, 1614, 1586, 1515, 1463, 1455, 1418, 1369, 1302, 1247,
6
6
2
.4 Hz); 2.26–2.23 (1H, bs, OH: exchanges with D O); 1.45 (3H, d, J
1205, 1177, 1116, 1087, 1036, 1006, 898, 833, 809, 739, 713. ıH
2
◦
◦
.4 Hz); ıC (100 MHz, CDCl , 25 C) 144.2, 128.5, 126.8, 94.4, 69.7,
(400 MHz, CDCl , 25 C) 7.27–7.23 (2H, m, aromatic protons);
3
3
+
5.2; m/z : 156 (M , 22), 143 (30), 141 (100), 139 (22), 138 (23),
6.86–6.82 (2H, m, aromatic protons); 4.79 (1H, q, J 6.4 Hz); 3.76