UPDATES
DAADH Activity Assay
J=8 Hz), 7.19–7.27 (m, 3H, ArH), 3.50–3.53 (1H, m,
CHNH ), 3.06 (dd, 1H, CHH, J=12, 8 Hz), 2.89 (dd, 1H,
2
The reductive amination activity of purified DAADH to-
wards the panel of keto acids was determined by monitoring
the decrease of NADPH concentration at 340 nm. The reac-
tion mixture (100 mL) contained NH Cl buffer (300 mM,
pH 9.0), NADPH (1 mM) and the appropriate substrate
13
CHH, J=12, 8 Hz); C NMR (100 MHz, D O+NaOH):
2
d=182.32, 135.98, 133.82, 131.61, 129.38, 128.21, 127.02,
+
+
5
6.50, 38.63; HR-MS (ESI ): m/z=200.0497 (MH ), calcu-
4
+
lated for C H ClNO (MH ): 200.0478.
9
11
2
(
R)-2-Amino-3-(4-bromophenyl)propanoic acid (d-2d):
H NMR (400 MHz, D O+NaOH): d=7.34 (d, 2H, ArH,
(
2 mM, added as a stock solution in DMSO). Reactions
1
2
were initiated by the addition of enzyme to the mix (10 mL,
J=8 Hz), 7.00 (d, 2H, ArH, J=8 Hz), 3.28–3.33 (m, 1H,
À1
0
.3 mgmL stock). Measurements were carried out in UV
CHNH ), 2.75 (dd, 1H, CHH, J=16, 8 Hz), 2.65 (dd, 1H,
2
transparent 96-well microplates (Greiner) using a Magellan
Infinity Pro 200 microplate reader. One unit of DAADH is
defined as the amount of protein that oxidised 1 mmol
NADPH per minute.
13
CHH, J=16, 8 Hz); C NMR (100 MHz, D O+NaOH):
2
d=182.13, 137.39, 131.32, 131.22, 119.73, 57.28, 40.21; HR-
+
+
MS (ESI ): m/z=243.9985 (MH ), calculated for
+
C H BrNO (MH ): 243.9895.
9
11
2
(
R)-2-Amino-3-(2-methoxyphenyl)propanoic acid (d-2g):
1
General Procedure for Preparative Scale
Biotransformations
H NMR (400 MHz, D O+NaOH): d=7.26 (t, 1H, ArH,
J=8 Hz), 7.16 (d, 1H, ArH, J=8 Hz), 6.99 (d, 1H, ArH, J=
Hz), 6.93 (t, 1H, ArH, J=8 Hz), 3.79 (s, 3H, OCH ), 3.46
dd, 1H, CHNH , J=8, 8 Hz), 2.95 (dd, 1H, CHH, J=12,
Hz), 2.73–2.78 (dd, 1H, CHH, J=12, 8 Hz); C NMR
100 MHz, D O+NaOH): d=182.70, 157.54, 131.19, 128.17,
2
+
126.64, 120.79, 111.38, 55.49, 55.48, 35.46; HR-MS (ESI ):
m/z=196.1017 (MH ), calculated for C10H14NO (MH ):
3
2
8
(
8
(
3
Method A (from keto acids): The suitable keto acid 1a–n
2
(
200 mg, final conc. approx. 40 mM) was dissolved in MeOH
1
3
(
1.0 mL, 4% v/v) and added to the reaction buffer (25 mL,
1
00 mM Na CO , 200 mM NH Cl, 100 mM d-glucose,
2 3 4
+
0.5 mM NADP , pH 9.0). E. coli cells co-producing
+
+
À1
DAADH and GDH (30 mgmL wet weight) were added
and the mixture was incubated at room temperature with
gentle agitation (50 rpm) for 24 h. Biotransformation sam-
ples (300 mL) were mixed with MeOH (300 mL), thoroughly
shaken and centrifuged (13000 rpm, 1 min), then the super-
natant was transferred to a filter vial and used directly for
HPLC analysis. For product isolation, the reaction mixture
was acidified to pH <2.0 by addition of aqueous H SO
1
96.0895.
R)-2-Amino-3-(4-methoxyphenyl)propanoic acid (d-2h):
(
1
H NMR (400 MHz, D O+NaOH): d=7.16 (d, 2H, ArH,
J=9 Hz), 6.91 (d, 2H, ArH, J=9 Hz), 3.78 (s, 3H, OCH ),
.42 (dd, 1H, CHNH , J=4 Hz), 2.87 (dd, 1H, CHH, J=12,
Hz), 2.75 (dd, 1H, CHH, J=12, 4 Hz);
2
3
3
4
(
2
1
3
C NMR
100 MHz, D O+NaOH): d=182.46, 157.44, 130.82, 130.53,
2
+
2
4
1
14.00, 57.42, 55.36, 39;79. HR-MS (ESI ): m/z=196.1045
+ +
(
10% w/v) and centrifuged (8000 rpm, 10 min) to remove
ꢃ
(MH ), calculated for C H NO (MH ): 196.0895.
10 14 3
cells and insoluble components. Dowex 50WX8 hydrogen
form (2.5 g) was washed with deionised water (50 mL) and
aqueous H SO (25 mL, 10% w/v). The supernatant from
(
R)-2-Amino-3-(benzo[d][1,3]dioxol-5-yl)propanoic acid
1
(
d-2i): H NMR (400 MHz, D O+NaOH): d=6.79 (d, 1H,
ArH, J=8 Hz), 6.74 (s, 1H, ArH), 6.68 (d, 1H, ArH, J=
8 Hz), 5.88 (s, 2H, CH ), 3.39 (dd, 1H, CHNH , J=8 Hz),
.84 (dd, 1H, CHH, J=12, 8 Hz), 2.71 (dd, 1H, CHH, J=
2, 8 Hz); C NMR (100 MHz, D O+NaOH): d=182.35,
46.98, 145.55, 132.08, 122.51, 109.67, 108.35, 100.80, 57.48,
0.43; HR-MS (ESI ): m/z=201.0875 (MH ), calculated for
2
2
4
the biotransformation was loaded onto the resin
À1
(
1 mLmin ). The resin was washed repeatedly with deion-
2
2
2
1
1
4
ised water and the product was eluted with aqueous
1
3
NH OH (30 mL, 10% w/v). Fractions containing the product
2
4
were pooled and evaporated overnight, to afford the corre-
sponding d-phenylalanine d-2a–n.
Method B (from racemic or l-amino acids): The suitable
racemic or optically pure amino acid 2a–n (100 mg, final
conc. approx. 20 mM) was dissolved in the reaction buffer
+
+
+
C H NO (MH ): 201.0688.
10 12
4
(
R)-2-Amino-3-[4-(methylthio)phenyl]propanoic acid (d-
1
2
j): H NMR (400 MHz, D O+NaOH): d=7.22 (d, 2H,
ArH, J=8 Hz), 7.14 (d, 2H, ArH, J=8 Hz), 3.39 (dd, 1H,
CHNH , J=8, 4 Hz), 2.87 (dd, 1H, CHH, J=12, 4 Hz),
.71–2.76 (dd, 1H, CHH, J=12, 8 Hz); C NMR (100 MHz,
2
(
30 mL, 100 mM Na CO , 200 mM NH Cl, 100 mM d-glu-
2 3 4
+
cose, 0.5 mM NADP , pH 9.0). E. coli cells producing
LAAD (30 mgmL wet weight) and E. coli cells co-produc-
ing DAADH and GDH (30 mgmL wet weight) were
2
1
3
À1
2
À1
D O+NaOH): d=182.30, 135.54, 135.22, 130.10, 126.72,
2
+
+
5
7.34, 40.20, 14.91; HR-MS (ESI ): m/z=212.0820 (MH ),
calculated for C H NO S (MH ): 212.0667.
added and the mixture was incubated at room temperature
with gentle agitation (50 rpm) for 24 h. HPLC analysis and
product isolation were carried out as described in Method
A.
+
10 14
2
1
(R)-2-Amino-3-(o-tolyl)propanoic acid (d-2k): H NMR
(400 MHz, D O+NaOH): d=7.11–7.17 (m, 4H, ArH), 3.38
(t, 1H, CHNH , J=8 Hz), 2.95 (dd, 1H, CHH, J=16, 8 Hz),
2.72 (dd, 1H, CHH, J=16, 8 Hz), 2.27 (s, 3H, CH );
O+NaOH): d=182.70, 137.14,
136.81, 130.30, 130.20, 126.76, 125.97, 56.78, 38.34, 18.62;
2
(
R)-2-Amino-3-(4-fluorophenyl)propanoic acid (d-2b):
2
1
H NMR (400 MHz, D O+NaOH): d=7.16–7.20 (m, 2H,
3
2
1
3
ArH), 7.00–7.05 (m, 2H, ArH), 3.40–3.43 (m, 1H, CHNH ),
C NMR (100 MHz, D
2
2
2
1
1
8
.89 (dd, 1H, CHH, J=12, 8 Hz), 2.78 (dd, 1H, CHH, J=
13
+
+
2, 8 Hz); C NMR (400 MHz, D O+NaOH): d=182.32,
HR-MS (ESI ): m/z=180.0933 (MH ), calculated for
2
1
4
3
+
61.45 ( J =240 Hz), 133.92 ( J =3 Hz), 130.84 ( J =
C
10
H
14NO
2
(MH ): 180.0946.
(R)-2-Amino-3-(p-tolyl)propanoic acid (d-2l): H NMR
(400 MHz, D O+NaOH): d=7.12 (d, 2H, ArH, J=8 Hz),
7.08 (d, 2H, ArH, J=8 Hz), 3.39 (dd, 1H, CHNH , J=8,
C,F
C,F
C,F
2
+
1
Hz), 115.02 ( J =21 Hz), 57.42, 39.87; HR-MS (ESI ):
C,F
+
+
m/z=184.0864 (MH ), calculated for C H FNO (MH ):
2
9
11
2
184.0774.
2
(
R)-2-Amino-3-(2-chlorophenyl)propanoic acid (d-2c):
4 Hz), 2.87 (dd, 1H, CHH, J=12, 8 Hz), 2.72 (dd, 1H,
1
13
H NMR (400 MHz, D O+NaOH): d=7.39 (d, 1H, ArH,
CHH, J=12, 8 Hz), 2.23 (s, 3H, CH ); C NMR (100 MHz,
2
3
Adv. Synth. Catal. 0000, 000, 0 – 0
8
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÞÞ
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