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using l-amino acid oxidases[14] and deaminases (l-AAO/l-AADs)
employed as freeze-dried E. coli cell preparations containing
the overexpressed enzyme. Possible formed H2O2 will be dis-
proportionated by the catalase(s) present in E. coli. Using air as
oxygen source, best results were obtained for the l-AAD from
Proteus myxofaciens (56% conversion, 18 h), which produces
ammonia as a side product.[15] Improved conversion (95%) was
achieved when the reaction was conducted under 2 bar of
oxygen pressure.
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Figure 2. Percentage of 1–3a versus time ( : 1a; : 2a; : 3a). Reaction
conditions: (S)-1a (50 mm), O2 (1 bar), l-AAD (20 mg, 1 U), l-Hic (15 mg,
6.6 U), FDH (2 mg, 4.2 U), HCO2NH4 (150 mm, pH 7), 218C, 170 rpm.
For the asymmetric reduction of 2a different S and R selec-
tive reductases were tested. Best results were obtained em-
ploying the stereocomplementary l- and d-isocaproate reduc-
tases (HicDHs) from Lactobacillus paracasei DSM 20008 (l-
Hic)[16] and Lactobacillus confuses DSM 20196 (d-Hic).[17] Apply-
ing these reductases, the S as well as the R enantiomer of 3a
were accessible in enantiopure form (>99% ee) at substrate
concentrations of up to 100 mm.
In a next step, the oxidation and reduction steps were run
simultaneously. To identify suitable reaction conditions, the
amount of l-AAD was varied while the other reaction parame-
ters were kept constant. Stopping the reaction after one hour,
the amount of final product (S)-3a increased with higher
amount of the deaminase (Figure 1). In all these experiments,
the amount of the intermediate ketoacid 2a remained below
5%, demonstrating the ideal coupling of the system and indi-
cating that the oxidation is the limiting step of the set up.
3a were isolated with high yields (78–84%) after a simple ex-
traction step without requiring chromatographic purification.
To demonstrate the scope of the method a representative
panel of natural amino acids and l-norleucine (S)-1b–f was
transformed possessing different functionalities, such as ali-
Table 1. Simultaneous oxidation and asymmetric reduction for the inver-
sion and retention of (S)-1a to yield enantiopure hydroxy acids (S)- or (R)-
3a.
Entry
Hic
[1a][a]
c [%][b]
2a [%][b]
3a [%][b]
ee 3a [%][c]
1
2
3
l-Hic
l-Hic
l-Hic
50
100
200
>99
>99
>99
3
2
1
97
>99 (S)
>99 (S)
>99 (S)
98 (81)[d]
99 (78)[d]
4
5
6
d-Hic
d-Hic
d-Hic
50
100
200
>99
>99
>99
3
1
1
97
>99 (R)
>99 (R)
>99 (R)
99 (84)[d]
99 (79)[d]
Reaction conditions: (S)-1a (50–200 mm), HCO2NH4 (3 equiv, pH 7), NAD+
(1 mm), FDH (42 UmmolÀ1), 1 bar O2, 7 h, 218C, 170 rpm; l-AAD:
15 UmmolÀ1. Hic: 66 UmmolÀ1 for l-Hic and 96 UmmolÀ1 for d-Hic.
[a] Concentration of starting material. [b] Determined by HPLC, reverse
phase. [c] Determined by HPLC on a chiral phase. [d] Isolated yields in
brackets.
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Figure 1. Percentage of 1–3a versus the amount of l-AAD ( : 1a; : 2a;
3a). Reaction conditions: (S)-1a (50 mm), O2 (2 bar), l-AAD, l-Hic (15 mg,
6.6 U), FDH (2 mg, 4.2 U), HCO2NH4 (150 mm, pH 7), 1 h, 218C, 170 rpm.
phatic, aromatic and heteroaromatic moieties. The substrate
concentration was optimized in each case considering the sol-
ubility of the amino acid aiming to obtain complete conversion
(see Supporting Information Table S2 for details). For instance,
excellent results were obtained for tryptophan (1b) bearing
a sensitive indole moiety. At 50 mm substrate concentration
the hydroxy acids (R)- as well as (S)-3b were isolated in enan-
tiopure form (>99% ee) and high isolated yield (83%, Table 2,
entries 1 and 2). The transformation was also successfully per-
formed for amino acids bearing aliphatic chains (1c–d, en-
tries 3–6). In these cases the reactions were followed by NMR
spectroscopy (see the Supporting Information for details). At
100 mm substrate concentration enantiopure (R)- as well as (S)-
3c–d were successfully isolated with high yields. In the case of
(R)-3c, the biocatalytic inversion was performed with 0.5 g of
substrate (entry 4) demonstrating the scalability of the meth-
odology. Using methionine (S)-1e as starting material, the bio-
retention and inversion were performed at 200 mm substrate
concentration allowing the isolation of enantiopure (S)- as well
Consequently, the O2 pressure was investigated showing
that at 1 bar O2 comparable results were obtained to those at
2 bar (Supporting Information Figure S3). Consequently, one
bar O2 was used for further experiments.
Following the biocatalytic transformation under these condi-
tions over time, the transformation of (S)-1a to (S)-hydroxy
acid (S)-3a went to completion within 7 h without formation
of any side products (Figure 2). Notably, the amount of keto
acid 2a remained constant and below 3% along the reaction.
Preparative inversion as well as retention of l-amino acid (S)-
1a (66 mg, 0.40 mmol, 50–200 mm) was achieved by combin-
ing the l-AAD with the l- or d-selective HicDHs at 50–200 mm
concentration (Table 1), leading to (S)- or (R)-3a with complete
conversion and in enantiopure form (>99% ee). At 100–
200 mm substrate concentration the hydroxy acids (S)- and (R)-
Chem. Eur. J. 2014, 20, 11225 – 11228
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