M. Fuchs et al. / Tetrahedron 68 (2012) 7691e7694
7693
ion source, positive mode, capillary voltage 2.6 kV) using a syringe
dehydrogenase (20
mL, 7.5 mg protein/mL stock solution), ammo-
pump to directly infuse the sample dissolved in MeCN. Chiral HPLC
nium formate (9.5 mg, 0.15 mmol), formate dehydrogenase (20
220 U/mL stock solution), -alanine (22 mg, 0.25 mmol) and sub-
strate 4 (11 mg, 0.05 mmol) were added and the suspension was
shaken for 24 h. Saturated K CO solution was added (200 L) and
the aqueous layer was extracted with EtOAc (2ꢁ500 L, denaturated
enzyme was removed by centrifugation). The combined organic
phase was dried over Na SO and subjected to GCeMS-analysis.
mL,
analysis of (S)-Rivastigmine (1) was performed on a Shimadzu HPLC
L
18
system using a modified method of Srinivasu et al. Optical rota-
tion values were measured on a Perkin Elmer Polarimeter 341.
Ethyl(methyl)carbamic chloride (2) was prepared as described
previously.6 The following enzyme preparations were obtained
2
3
m
m
ꢂ1
ꢂ1
from Codexis: Vf-
u
-TA (020207KVP, 49 mg mL , 7.3 U mg ), ATA-
2
4
ꢂ1
1
13 (102907WW, 0.46
U
mg ), ATA-114 (1091108 MW,
.7 U mg ), ATA-117 (102907WW,1.9 U mg ), LDH mix (PRM-102,
ꢂ1
ꢂ1
2
4.5. Derivatisation of compound 5 for chiral analysis
1
01807KVP) and FDH (24.11, H62411.01, 220 U/mL). Lyophilized
whole cells of E. coli containing overexpressed BM- -TA, CV- -TA
and AS-
-TA were obtained as previously reported.19 In case of Pd-
-TA, Pp- -TA_1 (Gen PP5182) and Pp- -TA_2 (Gen PP2180) the
u
u
EtOAc from the GCeMS sample was removed under a stream of
u
air. The residue was dissolved in CH
(20 mg), NaBH(OAc) (50 mg) and formaldehyde (37% in water,
30 L) were added and the mixture was shaken at rt for 24 h. The
reaction was quenched with HCl (1 M, 100 L), the pH was adjusted
to >10 (NaOH, 10 M) and the phases were separated. The organic
layer was dried over Na SO , filtered and the solvent was removed
under a stream of air. The residue was dissolved in HPLC eluent (n-
heptane/2-propanol/TFA 8/2/0.2, 1 mL) and subjected to chiral
HPLC analysis.
2 2 2 4
Cl (800 mL) and Na SO
12
u
u
u
3
plasmids were transformated into E. coli BL21(DE3) cells, which
were grown according to the manufactor’s manual in LB/Amp
medium. Induction was done using anhydrotetracycline according
to the manual of the used vector (pASK-IBA35þ) as recently
described.13
m
m
2
4
4
.2. 3-Acetylphenyl ethyl(methyl)carbamate (4)
Sodium hydride (180 mg, 7.5 mmol, prewashed with n-pentane
4.6. Prep-scale synthesis of (S)-3-(1-aminoethyl)phenyl
ethyl(methyl)carbamate [(S)-5]
to remove mineral oil) was suspended in dry THF (80 mL). Under
0
a stream of Ar 3 -hydroxyacetophenone (3, 234 mg, 1.7 mmol) was
added and the mixture was stirred for 20 min at rt. Ethyl(methyl)
Pd-
u
TA (200 mg whole cells) was rehydrated in sodium phos-
0
carbamic chloride (2, 310
mL, 322 mg, 2.7 mmol) was dissolved in
phate buffer (10 mL, 100 mM, pH 7.0) containing pyridoxal 5 -
dry THF (20 mL) and added to the reaction mixture, which was
stirred for 5 h. The reaction was quenched by addition of NaOH
phosphate (1 mM). LDH-mix (300 mg, containing LDH, GDH, glu-
þ
cose, NAD ),
L-alanine (220 mg, 2.5 mmol) and substrate 4 (99 mg,
(
1.1 M, 40 mL) and extracted with EtOAc (3x 20 mL). The combined
organic phase was washed with NaOH (1.1 M, 20 mL), dried over
Na SO and concentrated under reduced pressure to give 3-
acetylphenyl ethyl(methyl)carbamate (4) as yellow oil (337 mg,
.52 mmol, 89%): max 2973, 2935, 1716, 1683, 1587, 1476, 1426,
396, 1357, 1258, 1191, 1151 cm ; H NMR (DMSO) 7.84e7.80 (m,
H), 7.66 (bs, 1H), 7.54 (t, 1H, J¼8.1), 7.42e7.39 (m, 1H), 3.42 (q, 1H,
0.45 mmol) were added and the suspension was shaken for 24 h.
The mixture was extracted with EtOAc (2ꢁ10 mL) to remove traces
of remaining starting material. The pH was adjusted to >10 with
2
4
2 3
saturated K CO solution. Subsequent extraction with EtOAc
1
1
1
y
(3ꢁ10 mL), drying of the combined organic phase and concentra-
ꢂ1
1
tion under reduced pressure gave (S)-5 (75 mg, 0.34 mmol, 76%):
2
0
½
a
ꢃ
ꢂ11.2 (c 1.0, MeOH);
ymax 2968, 2930, 1710, 1609, 1589, 1476,
D
ꢂ1
1
J¼6.9, 1 rotamer), 3.32 (q, 1H, J¼7.2, 1 rotamer), 3.04 (s, 1.5H, 1
1433, 1397, 1236, 1217, 1158 cm
; H NMR (MeOD) 7.34 (t, 1H,
rotamer), 2.91 (s, 1.5H, 1 rotamer), 2.59 (s, 3H), 1.20 (t, 1.5H, J¼7.2),
J¼7.8), 7.24 (d, 1H, J¼7.5), 7.14 (t, 1H, J¼0.9), 7.01e6.98 (m, 1H), 4.07
(q, 1H, J¼6.9), 3.52 (q, 1H, J¼7.2, 1 rotamer), 3.41 (q, 1H, J¼7.2, 1
rotamer), 3.11 (s, 1.5H, 1 rotamer), 2.99 (s, 1.5H, 1 rotamer), 1.41 (s,
1.5H, 1 rotamer), 1.39 (s, 1.5H, 1 rotamer), 1.28 (t, 1.5H, J¼6.9, 1
13
1
.11 (t, 1.5H, J¼6.9); C NMR (DMSO) 197.7, 153.9 (1 rotamer), 153.8
(
1
1 rotamer), 152.0, 138.5, 130.1, 127.3 (1 rotamer), 127.3 (1 rotamer),
25.5, 121.8, 44.0, 34.4 (1 rotamer), 34.1 (1 rotamer), 27.3, 13.5 (1
13
rotamer), 12.7 (1 rotamer); GCeMS (EI): tret 13.02, m/z (relative
intensity [%]): 221 (8), 121 (5), 95 (4), 86 (100), 58 (54); HRMS (ESI):
MH found 222.1120, C12H16NO requires 222.1130.
rotamer), 1.20 (t, 1.5H, J¼7.2, 1 rotamer); C NMR (MeOD) 155.0,
151.6, 148.2, 129.1, 122.7, 120.1 (1 rotamer), 120.0 (1 rotamer), 119.1
(1 rotamer), 119.0 (1 rotamer), 50.6, 43.8, 33.2 (1 rotamer), 33.0 (1
rotamer), 23.6, 12.1 (1 rotamer), 11.3 (1 rotamer); GCeMS (EI): tret
þ
þ
3
4
.3. General procedure for enzymatic transamination using
12.65, m/z (relative intensity [%]): 222 (1), 180 (5), 120 (7), 86 (100),
þ
the LDH/GDH system
77 (5), 58 (60); HRMS (ESI): M-NH
requires 206.1181.
2
found 206.1188, C12
H16NO
2
Crude enzyme preparations (10 mg) were suspended for 5 min
in sodium phosphate buffer (1 mL, 100 mM, pH 7.0) containing
pyridoxal 5 -phosphate (1 mM). Whole lyophilised cells (20 mg)
4.7. (S)-3-[1-(Dimethylamino)ethyl]phenyl ethyl(methyl)
carbamate (Rivastigmine) [(S)-1]
0
were rehydrated for 20 min in the same buffer. LDH-mix (30 mg,
þ
containing LDH, GDH, glucose, NAD ),
0
L
-alanine (22 mg,
Amine (S)-4 (64 mg, 0.29 mmol) was dissolved in CH
and Na SO (42 mg, 0.30 mmol), NaBH(OAc) (504 mg, 2.4 mmol)
and formaldehyde (37% in water, 88 L, 1.0 mmol) were added and
the reaction mixture was stirred at room temperature for 16.5 h.
The reaction was quenched with satd K CO solution. The phases
were separated, the aqueous phase was washed with EtOAc
(2ꢁ10 mL) and the combined organic phase was dried over Na SO
filtered and concentrated under reduced pressure to give (S)-1
2 2
Cl (6 mL)
.25 mmol) and substrate 4 (11 mg, 0.05 mmol) were added and the
suspension was shaken for 24 h. Saturated K CO solution was
added (200 L) and the aqueous layer was extracted with EtOAc
L), denaturated enzymes were removed by centrifugation.
2
4
3
2
3
m
m
(
2ꢁ500
m
2
3
2 4
The combined organic phase was dried over Na SO and subjected
to GCeMS-analysis.
2
4
,
2
0
4
.4. General procedure for enzymatic transamination using
(72 mg, 0.29 mmol, >99%): ½
a
ꢃ
ꢂ32.8 (c 1.0, CH
2
Cl
2
), (lit. ꢂ28.5, c
D
5
c
the AlaDH/FDH system
1.0, CH
2
Cl
2
);
ymax 2974, 2934, 2817, 2768, 1716, 1607, 1589, 1454,
ꢂ1
1
1435, 1396, 1227, 1156 cm
;
H NMR (DMSO) 7.30 (t, 1H, J¼7.8),
Pd-u
TA (lyophilized whole cells, 20 mg) was rehydrated for
0 min in sodium phosphate buffer (1 mL, 100 mM, pH 7.0) con-
taining pyridoxal 5 -phosphate (1 mM) and NAD (1 mM). Alanine
7.12 (d, 1H, J¼7.8), 7.02e6.96 (m, 2H), 3.45e3.23 (m, 3H), 3.01 (s,
1.5H, 1 rotamer), 2.89 (s, 1.5H, 1 rotamer), 2.09 (s, 6H),1.26 (s, 1.5H, 1
rotamer),1.24 (s.1.5H,1 rotamer),1.15 (t,1.5H, J¼6.9,1 rotamer),1.10
2
0
þ