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Eduardo Busto et al.
Small-scale reactions were shaken in a 2-mL thermo shaker
Eppendorfꢂ Comfort. Preparative scale reactions were per-
formed in an Infors Unitron shaker.
Biotransformations (AlaDH; Analytical Scale)
The ketone 2 (4.6 mg, 25 mmol) was suspended in K-phos-
phate buffer (pH 7, 100 mM, 550 mL) and the following re-
agents dissolved in K-phosphate buffer (pH 7, 100 mM)
were added: l-alanine (250 mL, 1 M), NH4HCOO (150 mL,
1.5 M), PLP (20 mL, 50 mM), NAD+ (20 mL, 50 mM) Ala-
DH (10 mL, 11 U) FDH (5 mg, 11 U) and the lyophilised
cells containing w-TA (30 mg). The reductive amination was
carried out at 308C in an orbital shaker (750 rpm, Eppen-
dorf thermo shaker) for 24 h. After that time the reaction
was stopped by adding a saturated solution of Na2CO3
(300 mL). The crude was extracted with CH2Cl2 (2ꢄ700 mL),
dried over Na2SO4 and injected in the GC for conversion
measurement.
Figure 1. Comparison between the biocatalytic amination
and the chemoenzymatic route (ADH reduction plus Mitsu-
nobu) in terms of optical purity, time, yield, E-factor and
number of steps.
the use of hazardous reagents such as DPPA or
DEAD.
Biotransformations (2-Propylamine; Analytical Scale)
In summary, an efficient one-step protocol was de-
veloped for the amination of ketone 2 to the enantio-
pure amine (R)-4, which is a key intermediate for the
synthesis of the anti-allergic drug ramatroban. The
practical applicability of the approach has been dem-
onstrated by performing the amination on a 500-mg
scale affording the enantiopure amine with excellent
chemical purity and very high isolated yield (96%).
Last but not least, the total yield of the formal total
synthesis of the drug has been significantly improved
from 30% in the previous route to 50% and the
number of steps was reduced from 6 to 4.
The ketone 2 (4.6 mg, 25 mmol) was suspended in a K-phos-
phate buffer (pH 7, 100 mM, 750 mL) containing PLP
(0.5 mM) and 2-propylamine (0.75 M). Then, semi-purified
Armut11 wTA (250 mL) was added and the mixture was
shaken at 458C in an Eppendorf thermo shaker (750 rpm,)
for 24 h. The reaction was quenched and worked-up as de-
scribed above.
Biotransformations [(R)-1-Phenylethylamine;
Analytical Scale]
The ketone 2 (4.6 mg, 25 mmol) was suspended in K-phos-
phate buffer (pH 7, 100 mM, 750 mL) containing PLP
(0.5 mM) and different concentrations of (R)-1-phenylethyl-
amine. Then, semi-purified Armut11 wTA (250 mL) was
added to the suspension and the mixture was shaken at
458C in an Eppendorf thermo shaker (750 rpm) for 4 h. The
reaction was quenched and worked-up as described above.
Experimental Section
General Remarks
All starting materials were obtained from commercial sup-
pliers and were used as received. TLC analyses were carried
out with precoated aluminium sheets (TLC silica gel 60
F254, Merck) with detection by UV or staining with potassi-
um permanganate. Preparative chromatographic separations
were performed by flash chromatography on Merck silica
gel (0.063–0.200 mm). Optical rotation was measured at
208C with a Perkin–Elmer polarimeter at the sodium d-line.
GC-MS were recorded with an Agilent 7890 A GC system
mass selective detector and HP-5 MS column [30 mꢄ
0.25 mmꢄ0.25 mm with helium as carrier gas (flow=
Biotransformations on Preparative Scale using w-TA
from C. violaceum
The ketone 2 (27.6 mg, 149 mmol) was suspended in K-phos-
phate buffer (pH 7, 100 mM, 3.3 mL) and the following
aqueous solutions were added to the suspension: l-alanine
(1.5 mL, 1M), NH4COOH (900 mL, 1.5 M), PLP (120 mL,
50 mM), NAD+ (120 mL, 50 mM) Ala-DH (100 mL, 110 U)
FDH (50 mg, 110 U) and the lyophilised cells containing w-
TA (180 mg). The reductive amination was carried out at
308C in a 15-mL Falcon tube and shaken in an Infors orbital
shaker (120 rpm) for 24 h. After that time the reaction was
stopped by adding a saturated solution of Na2CO3 (1.8 mL)
and H2O (10 mL) and the crude was centrifuged to remove
the protein. The aqueous phase was extracted with CH2Cl2/
EtOH (95:5) (3ꢄ20 mL). The organic phases were com-
bined, dried over Na2SO4, and the solvent evaporated under
vacuum. The crude was purified by flash chromatography
(0–2% NH3/MeOH) affording the amine as a white solid;
yield: 17.3 mg (0.093 mmol, 62%); Rf (1% NH3/MeOH):
0.20. 1H NMR (CD3OD, 300.13 MHz): d=1.68–1.81 (m,
1H), 2.07–2.11 (m, 1H), 2.37–2.45 (m, 1H), 2.77–2.83 (m,
1
0.55 mLminÀ1)]. H and 13C NMR spectra were recorded at
208C with a Bruker 300 MHz unit; chemical shifts are given
in ppm relative to the resonance of the solvent. Formate de-
hydrogenase from Candida boidinii catalogue no. FDH 002
[lyophilised powder 2.2 U/mg] and NAD+ free acid were
purchased from Codexis. Lyophilised E. coli cells containing
overexpressed w-transaminases were prepared as previously
reported.[13a,22] Activity for alanine-dependent w-TAs and
Armut11 was determined for the deamination of (R)- or
(S)-1-phenylethanamine with pyruvate. Purified l-alanine
dehydrogenase was prepared as described recently.[12a]
1940
ꢃ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2014, 356, 1937 – 1942