4
I. C. R. Costa et al. / Tetrahedron: Asymmetry xxx (2017) xxx–xxx
combined and dried over anhydrous sodium sulfate. The chloro-
form solution was filtered and the solvent was removed by evapo-
ration under reduced pressure. The residual oil was distilled under
vacuum (fraction boiling at 114–116 °C) and the purified com-
pound was characterized by NMR and IR. 1a (oily liquid, 50% yield),
1H NMR (DMSO-d6, 300 MHz) d (ppm) = CH3 0.86 (3H); CH2 1.27
(4H); CH2 1.52 (2H); CH2 2.18 (2H); CH2 4.54 (2H); CH2 4.85
(2H). 13C NMR (DMSO-d6; 300 MHz) d (ppm) = CH3 13.79; CH2
21.14; CH2 24.14; CH2 30.74; CH2 33.60; CH2 65.62; CH2 65.62;
C@O 174.45. FT-IR (neat): 3400, 2958, 2901, 2662, 1710, 1465,
30 °C. The production of acetophenone was followed at 245 nm,
as described by Schätzle et al. 22 The activities were calculated
as U/mL enzyme solution. One unit is defined as the formation of
1 mmol product per minute. Values and standard deviations given
are based on three measurements.
4.6. Biocatalysis (analytical scale)
The biocatalysis reactions had a final volume of 1.0 mL: 100 mL
enzyme solution, 5 mM 1a and 5 mM
a-phenylethylamine were
1380, 1246, 1111, 619 cmꢀ1
.
dissolved in HEPES buffer (50 mM, pH 7.5) including 1 mM pyri-
doxal-50-phosphate and 10% DMSO. The reaction was performed
at 30 °C and 600 rpm. For the analysis of the reaction, a sample
of the reaction mixture was taken (200 mL) and treated with a sat-
urated solution of Na2CO3 (300 mL). The extraction of substrates
and products was performed with ethyl acetate (3 ꢁ 200 mL) after
mixing (30 s). Separation of the two phases was obtained via cen-
trifugation (2 min, 13,000 rpm). The organic phase was transferred
into a clean Eppendorf tube and dried over MgSO4. The solution
was mixed again and centrifuged for the removal of the MgSO4.
4.3. Synthesis of the racemic mixture of 2-amino-3-
hydroxypropyl hexanoate 2a
A racemic mixture of the serinol-monoester 2a was prepared by
chemical reductive amination. Ammonium acetate (4.50 mmol,
0.350 g) and sodium cyanoborohydride (1.50 mmol, 0.094 g) were
added to
a
solution of dihydroxyacetone-monoester 1a
(0.38 mmol, 0.071 g) in anhydrous methanol (1.1 mL). The reaction
mixture was then stirred for 24 h at room temperature. The solvent
was evaporated and the remaining residue was suspended in 0.5 M
NaOH (6 mL) and extracted with ethyl acetate (3 ꢁ 2 mL). The
organic layer was dried over MgSO4, filtered and the solvent evap-
orated under reduced pressure. The crude amine was purified by
column chromatography (CH2Cl2/MeOH 90:10). 1H NMR (CDCl3,
300 MHz) d (ppm) = CH3 0.90 (3H); CH2 1.29 (2H); 1.31 (2H);
CH2 1.63 (2H); NH2 2.0 (2H); CH2 2.32 (2H); CH 3.15 (H); OH
3.63 (H); CH2 3.74 (2H); CH2 4.22 (2H).
4.7. Analytical methods
4.7.1. TLC
The reactions were monitored by thin-layer chromatography.
The mobile phase used was dichloromethane/methanol (9:1). The
spots were identified by UV light (254 nm). Amines were stained
using ninhydrin and for staining the ketones, 2,4-dinitrophenylhy-
drazine was used. The retention factors (Rf) found were:
a-phenylethylamine (Rf = 0.29), 2a (Rf = 0.51); 1a (Rf = 0.67) and
4.4. Preparation of enzymes
acetophenone (Rf = 0.88).
The amine transaminases used herein include the variants of
4.7.2. HPLC
14
the Vibrio fluvialis enzyme described by Nobili et al.
and Genz
Determination of the conversion and enantiomeric excess was
performed by HPLC analysis.26 400 mL of an extracted sample (in
ethyl acetate) was placed in an Eppendorf tube. The ethyl acetate
was evaporated using a continuous flow of nitrogen. Then the sam-
ple was diluted in 1 mL 2-propanol (HPLC grade), 10 mg 9-
anthraldehyde and 60 mg MgSO4 were added. This mixture was
stirred at 600 rpm at room temperature for 12 h. After this time,
the supernatant was separated by centrifugation (2 min,
13000 rpm) and the sample was directly injected into the HPLC.
The separation of the chiral amines as 9-anthraldimine derivatives
using a coated covalently bonded polysaccharide-derived chiral
column was achieved. Chromatography was performed at room
temperature using the LaChrom Elite HPLC system, with automatic
injector, UV detector at 254 nm and a Lux Cellulose-1 column
(250 mm L ꢁ 4.6 mm I.D., 5 mm). The volume injected was 10 mL.
The mobile phase and the flow were optimized. The best condi-
tions for the separation were 10% 2-propanol in hexane (v/v) and
a flow rate of 0.3 mL/min. Under these conditions the retention
times (tR) were: acetophenone: 17.2 min; 1a: 19.5 min; (S)-2a:
et al.,15 Vibrio fluvialis wild-type and the (R)-amine transaminases:
Aspergillus fumigatus wild-type (AspFum)11 and amine transami-
nase-11718 from Codexis (Table 5).
The enzymes were expressed as previously described.14,15 The
crude lysates obtained were frozen overnight and lyophilized for
24 h. The solid obtained from each variant was maintained in the
fridge until use.
4.5. Acetophenone assay
For each lyophilized enzyme a 10 mg/mL solution in HEPES buf-
fer (50 mM, pH 7.5) was prepared and 10 mL of this solution were
used to determine the activity. The activity assay was performed
in 96 well plates at the following conditions: HEPES buffer
(50 mM, pH 7.5) and 0.1 mM pyridoxal-50-phosphate, DMSO 10%,
2.5 mM of
a-phenylethylamine and 2.5 mM amine acceptor
(pyruvate, pentanal or 1 a), 200 mL final reaction volume and
20.5 min; (R)-2a: 22.5 min; (S)-
a-phenylethylamine: 25.9 min
Table 5
and (R)- -phenylethylamine: 28.2 min, see also Figure 1.
a
Variants of Vibrio fluvialis amine transaminases used herein
Entry
Mutations
Entry
Mutations
4.8. Preparative scale
1
2
3
4
5
6
7
8
9
F85La
10
11
12
13
14
15
16
17
18
F19Ya
Y150Fa
Y150M/V153Aa
The volume of the reactions was 100 mL and 5.3 mM of 1-hex-
anoyloxy-3-hydroxyacetone 1a, were used. The reaction mixtures
contained HEPES buffer (50 mM, pH 7.5), pyridoxal-50-phosphate
Y150Ma
V153Aa
F85L/Y150M/V153Aa
L56V/W57F/F85Va
L56V/W57Cb
F85L/Y150Fa
F85L/Y150Ma
F85L/V153Aa
F19Ca
(1 mM), DMSO (10% v/v) and 100 mM
a-phenylethylamine. The
L56V/W57C/F85Vb
L56V/W57C/F85V/V153Ab
L56V/W57F/F85V/V153G/R415Cb
L56V/W57C/V153Ab
reactions were performed in a shaker at 30 °C, 180 rpm and were
monitored by TLC. After 72 h, the reactions were acidified with
HCl to pH 2.0 to precipitate protein, which was removed by
centrifugation. Acetophenone and 1a were extracted with ethyl
acetate. The pH was then increased to 10 by the addition of
F19Va
a
14
Variant described by Nobili et al.
Variant described by Genz et al.
.
b
15
.