Supporting Information
© Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2009
Minor Enantiomer Recycling Metal Catalyst and Biocatalyst Working in Concert
Erica Wingstrand,† Anna Laurell,† Linda Fransson,‡ Karl Hult,‡ Christina Moberg*,†
†
KTH School of Chemical Science and Engineering, Organic Chemistry, SE 100 44 Stockholm, Sweden,
and ‡KTH School of Biotechnology, Department of Biochemistry, AlbaNova University Center, SE 106
91 Stockholm, Sweden
*To whom correspondence should be addressed. E-mail: [email protected]
General remarks: All aldehydes were distilled (benzaldehyde from CaH2) before
use. Solvents were collected from a Glass-contour solvent dispensing system. Internal
standard, Candida antarctica lipase B, CALB (Novozyme 435), Candida rugosa
lipase (Sigma, Type VII), and Candida cylindracea lipase (Fluka) were purchased and
used without further purification. (S,S)- and (R,R)-[(salen)Ti(µ-O)]21, acetyl cyanide
and butanoyl cyanide2 were prepared following published procedures. Yields and
enantiomeric ratios were determined by GC/MS using a chiral column (Chiraldex, GTA (gamma cyclodextrin trifluoroacetyl, 30 m 0.25 mm), helium as carrier gas
(flow: 1 mL/min, split: 50:1), and n-undecane as internal standard. 1H NMR spectra
were recorded at 500 or 400 MHz. The chemical shifts are reported in ppm relative to
the solvent as internal standard. A syringe pump was used for additions of acyl
cyanides; for small scale reactions it is important that the needle tip is placed in the
organic phase.
L*2Ti2 =
N O
Ti
N O
O
O
O N
Ti
O N
Determination of the enantioselectivity in the Lewis Acid-Lewis Base catalyzed
reaction in the absence of enzyme at 40 °C: To a solution of (S,S)-[(salen)Ti(µ-O)]2
(6.7 mg, 0.0055 mmol), DBU (10-20 mol %), internal standard C11H24 (10 µL, 0.047
mmol), and benzaldehyde (12 µL, 0.12 mmol) in toluene (0.3 mL) was added 1 M
phosphate buffer pH 8 (0.5 mL). Acetyl cyanide (17 µL, 0.24 mmol) diluted to 0.2
mL with toluene was then added over 3.5 h to the vigorously stirred reaction mixture
at 40 °C using a syringe pump. The reaction was monitored by taking samples from
the organic phase (20 µL), which were filtered through a plug of silica, eluted with
diethyl ether, and analyzed by GC. With 10 and 20 mol % DBU er values of 62:38
and 53:47, respectively, were observed.
Determination of the enantioselectivity in the Lewis Acid-Lewis Base catalyzed
reaction in the absence of enzyme using various Lewis bases: To a solution of
(S,S)-[(salen)Ti(µ-O)]2 (6.7 mg, 0.0055 mmol), Lewis base (2.5-5 mol %), internal
standard C11H24 (10 µL, 0.047 mmol), and benzaldehyde (12 µL, 0.12 mmol) in
toluene (0.5 mL) was added 1 M phosphate buffer pH 8 (0.5 mL). Acetyl cyanide (17
µL, 0.24 mmol) was then added to the stirred reaction mixture at room temperature.
The reaction was monitored by GC as described above.
entry
1
Lewis base
DMAP 5%
2
DABCO 5%
3
DIPEA 5%
4
Quinidine 5%
5
DMAP 2.5%
6
DABCO 2.5%
7
DIPEA 2.5%
8
Quinidine 2.5%
9
DBU 2.5%
time (h)
1
2.5
7
1
2.5
7
1
2.5
7
1
5
7.5
1
5
7.5
1
5
7.5
1
5
7.5
1
3
19.5
1
4
6.25
% yield (GC)
24
31
41
18
22
25
35
57
66
66
68
68
27
36
36
28
33
34
18
22
26
56
59
59
66
74
76
er
76.5:23.5
74:26
69:31
78:22
76:24
72.5:27.5
67.2:32.5
61.5:38.5
60.5:39.5
65.5:34.5
64:36
63.5:26.5
74.5:25.5
69.5:30.5
69:31
71.5:28.5
68.5:31.5
68:32
77.5:22.5
70:30
68.5:31.5
67.5:32.5
66.5:33.5
66.5:33.5
62.5:67.5
61.5:38.5
61:39
Determination of the enantioselectivity in the Lewis Acid-Lewis Base catalyzed
reaction in the absence of enzyme and buffer using various Lewis bases: Acetyl
cyanide (17 µL, 0.24 mmol) was added to a solution of (S,S)-[(salen)Ti(µ-O)]2 (6.7
mg, 0.0055 mmol), Lewis base (2.5-5 mol %), internal standard C11H24 (10 µL, 0.047
mmol), and benzaldehyde (12 µL, 0.12 mmol) in toluene (0.5 mL). The reaction was
monitored by GC as described above.
entry
1
Lewis base
DIPEA 2.5%
2
Quinidine 2.5%
3
DMAP 5%
4
DMAP 2.5%
5
DBU 2.5%
6
DABCO 2.5%
time (h)
1
3
19.5
1
3
19.5
1
3
19.5
1
4
6.25
1
4
6.25
1
4
6.25
% yield (GC)
27
29
37
38
40
54
34
63
105
21
45
57
57
81
87
22
41
49
er
79.5:20.5
79:21
74:26
82.5:17.5
81:19
77.5:22.5
81:19
81.5:18.5
80.5:19.5
79.5:20.5
81.5:18.5
81:19
73:27
73:27
73:27
80.5:19.5
80.5:19.5
81:19
General procedure for synthesis of racemic O-acetylated cyanohydrins: Acetyl
cyanide (0.53 mL, 7.4 mmol) was added to a solution of aldehyde (4.9 mmol) and
triethylamine (0.14 mL, 1 mmol) in dry dichloromethane (10 mL). The reaction
mixture was stirred under nitrogen overnight at room temperature. The solvent was
removed by evaporation and the crude product was purified by flash chromatography
(silica gel, heptane/ethyl acetate).
General
procedure
for
CALB
catalyzed
hydrolysis
of
O-acetylated
cyanohydrins: Racemic O-acetylated cyanohydrin (0.12 mmol) and internal standard
C11H24 (10 µL, 0.047 mmol) were dissolved in toluene (0.5 mL). Immobilized CALB
(10 mg) was added followed by 1 M phosphate buffer pH 8 (0.5 mL). The reaction
mixture was vigorously stirred at 40 °C. Yields and er were monitored by GC as
described above.
CRL/CCL catalyzed hydrolysis of O-acetyl-2-hydroxy-2-phenylacetonitrile:
Racemic O-acetyl-2-hydroxy-2-phenylacetonitrile (0.12 mmol) and internal standard
C11H24 (10 µL, 0.047 mmol) were dissolved in toluene (0.5 mL). Enzyme (CRL or
CCL) was added to 1 M phosphate buffer pH 8 (0.5 mL) until the solution was
saturated (approximately 5 mg). The slurry was stirred for 10 min and then
centrifuged. Undissolved material was filtered off and the solution was added to the
organic reaction mixture. The reaction mixture was vigorously stirred at 40 °C. Yields
and er were monitored by GC as described above.
General procedure for Lewis Acid-Lewis Base-CALB catalyzed synthesis of Oacylated cyanohydrins: A solution was made of (S,S)-[(salen)Ti(µ-O)]2 (6.7 mg,
0.0055 mmol), DBU (3.4 µL, 0.024 mmol), internal standard C11H24 (10 µL, 0.047
mmol), aldehyde (0.12 mmol) and acyl cyanide (0.014 mmol) in toluene (0.25 mL).
Immobilized CALB (10 mg) was added followed by 1 M phosphate buffer pH 8 (0.5
mL). Acyl cyanide (0.35 mmol) diluted to 0.25 mL with toluene was then added over
5-22 h to the vigorously stirred reaction mixture at 40 °C using a syringe pump. The
reaction was monitored by GC while stirring was continued until high enantiomeric
ratio was reached.
Alternative procedure for Lewis Acid-Lewis Base-CALB catalyzed synthesis of
O-acylated cyanohydrins: A solution of (S,S)-[(salen)Ti(µ-O)]2 (6.7 mg, 0.0055
mmol), DMAP (6.7 mg, 0.006 mmol), internal standard C11H24 (10 µL, 0.047 mmol),
benzaldehyde (12 µL, 0.12 mmol) and acetyl cyanide (9.4 µL, 0.13 mmol) in toluene
(0.3 mL) was stirred at room temperature for 9 h. Immobilized CALB (10 mg) was
then added followed by 1 M phosphate buffer pH 8 (0.5 mL). More acetyl cyanide
(16.2 µL, 0.23 mmol) diluted to 0.2 mL with toluene was then added over 16.5 h to
the vigorously stirred reaction mixture at 40 °C using a syringe pump. The reaction
was monitored by GC while stirring was continued.
time (h) % yield
er
9*
83
79:21
25.75
95
98:2
29
93
99.4:0.6
30.5
92
99.5:0.5
32.5
92
99.5:0.5
*Before addition of CALB and buffer
Preparative scale Lewis Acid-Lewis Base-CALB Catalyzed Synthesis and
purification of O-acetyl-(R)-2-hydroxy-2-phenylacetonitrile: A solution was made
of (S,S)-[(salen)Ti(µ-O)]2 (70 mg, 0.06 mmol), DBU (34 µL, 0.24 mmol),
benzaldehyde (122 µL, 1.2 mmol) and acetyl cyanide (5 µL, 0.071 mmol) in toluene
(4.25 mL). Immobilized CALB (100 mg) was added followed by 1 M phosphate
buffer pH 8 (5 mL). Acetyl cyanide (250 µL, 3.5 mmol) diluted to 1 mL with toluene
was then added over 18 h to the vigorously stirred reaction mixture at 40 °C using a
syringe pump. The reaction was monitored by GC while stirring was continued for 3
h. After dilution with Et2O, the reaction mixture was filtered through silica and the
solvent was evaporated under vacuum. The residue was purified with flash
chromatography (hexane/ethyl acetate 7:1) to give O-acetyl-(R)-2-hydroxy-2phenylacetonitrile (180 mg, 86%, 99.67:0.33 er). Spectral data are in accordance with
those previously published.3
O-Acetyl-(R)-2-hydroxy-2-phenylacetonitrile (3a), optimized conditions: To a
solution of (S,S)-[(salen)Ti(µ-O)]2 (6.7 mg, 0.0055 mmol), DMAP (0.73 mg, 5 mol
%), internal standard C11H24 (10 µL, 0.047 mmol), and benzaldehyde (12 µL, 0.12
mmol) in toluene (0.3 mL) was added acetyl cyanide (9.4 µL, 0.132 mmol). The
reaction mixture was stirred in room temperature for 14 h, then immobilized CALB
(10 mg) followed by 1 M phosphate buffer pH 8 (0.5 mL) were added. Acetyl cyanide
(16 µL, 0.228 mmol) diluted to 200 µL with toluene was then added over 24 h to the
vigorously stirred reaction mixture at 40 °C using a syringe pump. When the addition
was finished the reaction mixture was stirred for 4.5 h, then more acetyl cyanide (8.5
µL, 0.12 mmol diluted to 100 µL with toluene) was added in the same manner. After
completed addition the reaction mixture was stirred for 18 h before the last addition of
acetyl cyanide was made (4.25 µL, 0.06 mmol diluted to 50 µL in toluene). The
reaction was monitored by GC as described above.
time
(h)
%
AcCN
er
%yield
0
14
15
16
18
19
20
21
22
26
36.5
38
40
42.5
72
90
97
113
137
0
110
140
150
175
205
210
225
235
297
297
300
300
300
400
400
400
450
450
05:05
88.67:19.33
89.11:10.89
93.4:6.6
96.21:3.79
95.33:4.67
94.59:5.41
97.05:2.95
96.1:3.9
96.45:3.55
99.62:0.38
96.98:3.02
99.58:0.42
99.74:0.26
99.42:0.58
99.51:0.48
99.63:0.37
99.56:0.44
99.78:0.22
0
111
90.5
91.5
89.09
94.8
94.3
93.2
97.99
95.6
85.2
91.3
90.6
89.1
93.2
96.3
94.7
93.9
96.9
O-Acetyl-(R)-2-hydroxy-2-pentylacetonitrile (3d), optimized conditions: To a
solution of (S,S)-[(salen)Ti(µ-O)]2 (6.7 mg, 0.0055 mmol), DMAP (0.73 mg, 5 mol
%), internal standard C11H24 (10 µL, 0.047 mmol), and hexanal (14 µL, 0.114 mmol)
in toluene (0.3 mL) was added acetyl cyanide (9.7 µL, 0.137 mmol). The reaction
mixture was stirred at room temperature for 3 h, then immobilized CALB (10 mg)
followed by 1 M phosphate buffer pH 8 (0.5 mL) were added. Acetyl cyanide (14.5
µL, 0.205 mmol) diluted to 200 µL with toluene was then added at intervals over a
total time of 75.5 h to the vigorously stirred reaction mixture at 40 °C using a syringe
pump. A new addition of acetyl cyanide (10 µL, 0.14 mmol diluted to 100 µL with
toluene) was added over 18 h. After complete additions the reaction mixture was
stirred for 22 h. The reaction was monitored by GC as described above.
time
(h)
%
AcCN
er
%yield
0
2.5
17
20.5
22.5
25
27.5
40
45.5
68.5
92.5
110.5
117
133
0
120
120
162
162
162
205
260
260
260
300
420
420
420
0
63.37:36.63
98.71:1.29
90.53:9.47
92.16:7.84
96.16:3.84
91.75:8.55
96.16:3.84
98.24:1.78
99.66:0.34
99.54:0.46
95.68:4.32
98.33:1.67
99.67:0.33
0
95.3
59.9
73.1
73.2
66.8
78.9
82.9
79.3
78.3
83.7
94.6
90.3
88.3
O-Acetyl-(R)-2-hydroxy-2-2-furylacetonitrile, (3f), optimized conditions: To a
solution of (S,S)-[(salen)Ti(µ-O)]2 (6.7 mg, 0.0055 mmol), DMAP (0.73 mg, 5 mol
%), internal standard C11H24 (10 µL, 0.047 mmol), and 2-furaldehyde (10 µL, 0.12
mmol) in toluene (0.4 mL) was added acetyl cyanide (17 µL, 0.24 mmol). The
reaction mixture was stirred at -40°C for 12 h followed by 1 h at room temperature.
Immobilized CALB (10 mg) followed by 1 M phosphate buffer pH 8 (0.5 mL) were
added. Acetyl cyanide (8.5 µL, 0.12 mmol) diluted to 100 µL with toluene was then
added over 5 h to the vigorously stirred reaction mixture at 40 °C using a syringe
pump. When the addition was finished the reaction mixture was stirred for 4 h, then
additional acetyl cyanide (4.25 µL, 0.06 mmol diluted to 50 µL with toluene) was
added in the same manner. After complete addition the reaction mixture was stirred
for 8 h. The reaction was monitored by GC as described above.
time
(h)
%
AcCN
er
%ee
%yield
0
12
19
21
23
36
200
200
300
300
300
350
0
88.11:11.89
96.77:3.23
98.82:1.18
99.03:0.97
98.48:1.52
0
76.2
93.5
97.6
98.1
97
0
81.1
99.9
92.8
85.6
86.9
Lewis Acid-Lewis Base-CRL (or CCL) catalyzed synthesis of O-acylated
cyanohydrins: Acetyl cyanide (9.4µL, 0.132mmol) was added to a solution of (S,S)[(salen)Ti(µ-O)]2 (6.7 mg, 0.0055 mmol), DMAP (0.73 mg, 0.006 mmol), internal
standard C11H24 (10 µL, 0.047 mmol) and benzaldehyde (12 µL, 0.12 mmol) in
toluene (0.3 mL). The reaction mixture was stirred at room temperature overnight.
Enzyme (CRL or CCL) was added to 1 M phosphate buffer pH 8 (0.5 mL) until the
solution was saturated (ca 5 mg). The slurry was stirred for 10 min and then
centrifuged. Undissolved material was filtered off and the solution was added to the
organic reaction mixture. Acetyl cyanide (17µL, 0.228 mmol) diluted to 0.25 mL with
toluene was then added over 20 h to the vigorously stirred reaction mixture at 40 °C
using a syringe pump. Additional acetyl cyanide (8.5µL, 0.12 mmol) diluted to 0.1
mL with toluene, was then added over 10 h. The reaction was monitored by GC.
CRL
time
(h)
approx. %
AcCN added
er (R:S)
%ee
%yield
0
13
17
25
110
110
112
121
00:00
18.48:81.52
7.48:92.52
2.62:97.38
0
63.04
85.04
94.8
0
99.8
78.2
77.8
37
49
53
61
76
84
100.5
107.5
174
300
400
400
400
400
400
400
400
400
2.13:97.87
14.85:85.15
7.65:92.35
4.83:95.17
3.39:96.61
2.98:97.02
2.48:97.52
1.89:98.11
1.83:98.17
95.7
70.3
84.7
90.3
93.2
94.04
95.04
96.2
96.3
72.4
165
120
92.3
86.6
81.5
74.5
70.8
70.7
CCL
time
(h)
approx. %
AcCN added
er (R:S)
%ee
%yield
0
13
17
25
37
39
49
53
61
76
84
100.5
107.5
174
110
110
112
121
300
300
400
400
400
400
400
400
400
400
19.43:80.57
10.27:89.73
5.48:94.52
9.53:90.47
23.06:76.94
18.41:81.59
8.2:91.8
6.02:93.98
4.32:95.68
4.18:95.82
3.12:96.88
2.88:97.12
1.94:98.06
61.1
79.5
89
80.9
53.9
63.2
83.6
87.9
91.4
91.7
93.8
94.3
96.1
99.8
91.2
137
97.8
183
163
106
92.7
85.6
77
76.8
76.1
71.9
O-Acetyl-(R)-2-hydroxy-2-phenylacetonitrile
O
O
N
er >99.85:0.15
GC parameters: 70 °C (hold 1 min), 15 °C/min up to 120 °C (19 min)
Retention times: 3.8 min (n-undecane), 4.5 min (benzaldehyde), 14.4 min (major
enantiomer), 19.3 (minor enantiomer).
1
H NMR of isolated product:
Racemic sample:
er >99.85:0.15:
O-Acetyl-(R)-2-hydroxy-2-(4-chlorophenyl)acetonitrile:
O
O
N
Cl
er 99.07:0.93
GC parameters: 70 °C (hold 1 min), 15 °C/min up to 130 °C (35 min)
Retention times: 3.8 min (n-undecane), 5.9 min 4-Cl-benzaldehyde), 22.5 min (major
enantiomer), 33.5 (minor enantiomer).
Spectral data are in accordance with those previously published.3a, 4
Racemic sample:
er 99.1:0.9:
O-Acetyl-(S)-2-hydroxy-(2-furyl)acetonitrile:
O
O
O
N
er 98.56:1.44
GC parameters: 70 °C (hold 1 min), 4 °C/min up to 95 °C, 15 °C/min up to 120 °C
(15 min)
Retention times: 4.8 min (n-undecane), 5.2 min (2-furylaldehyde), 12.5 min (major
enantiomer), 16.3 (minor enantiomer).
Spectral data are in accordance with those previously published.5
Racemic sample:
er 98.6:1.4:
O-Acetyl-(R)-2-hydroxy-2-(4-methoxyphenyl)acetonitrile:
O
O
N
O
er 99.6:0.4
GC parameters: 70 °C (hold 1 min), 15 °C/min up to 130 °C (45 min)
Retention times: 3.8 min (n-undecane), 8.6 min (4-MeO-benzaldehyde), 31.8 min
(major enantiomer), 43.3 (minor enantiomer).
Spectral data are in accordance with those previously published.6
Racemic sample:
er 99.6:0.4:
O-Butyryl-(R)-2-hydroxy-2-phenylacetonitrile:
O
O
N
er 97.71:2.28
GC parameters: 70 °C (hold 1 min), 15 °C/min up to 120 °C (30 min)
Retention times: 3.8 min (n-undecane), 4.5 min (benzaldehyde), 24.7 min (major
enantiomer), 27.3 (minor enantiomer).
Spectral data are in accordance with those previously published.4
Racemic sample:
er 97.7:2.3:
O-Acetyl-(R)-2-hydroxy-2-(hexyl)acetonitrile:
O
O
N
er 99.6:0.4
GC parameters: 70 °C (hold 1.80 min), 4 °C/min up to 80 °C (hold 0 min), 15 °C/min
up to 120 °C (hold 10 min), 15°C/min up to 130 °C (hold 2 min) (20 min).
Retention times: 3.19 min (hexanal), 4.99 min (n-undecane), 10.52 min (major
enantiomer), 13.36 (minor enantiomer).
Spectral data are in accordance with those previously published.5b
Racemic sample
er 99.6:0.4
O-Acetyl-(R)-2-hydroxy-2-(octyl)acetonitrile:
O
O
N
er 99.3: 0.7
GC parameters: 70 °C (hold 1 min), 4 °C/min up to 100 °C (hold 0 min), 15 °C/min
up to 130 °C (hold 13 min) (23.5 min)
Retention times: 4.78 min (n-undecane), 5.27 min (octanal), 16.67 min (major
enantiomer), 20.45 (minor enantiomer).
Spectral data are in accordance with those previously published.5b
Racemic sample
er 99.3: 0.7
O-Acetyl-(R)-2-hydroxy-2-phenylacetonitrile:
O
O
N
er 98.6:1.4
GC parameters: 70 °C (hold 1 min), 15 °C/min up to 120 °C (19 min)
Retention times: 3.8 min (n-undecane), 4.5 min (benzaldehyde), 14.4 min (minor
enantiomer), 19.3 (major enantiomer).
er 98.6:1.4
References
[1] a) Y. N Belokon', S. Caveda-Cepas, B. Green, N. S. Ikonnikov, V. N. Khrustalev,
V. S. Larichev, M. A. Moscalenko, M. North, C. Orizu, V. I. Tararov, M.
Tasinazzo, G. I. Timofeeva, L. V. Yashkina, J. Am. Chem. Soc. 1999, 121, 39683973; b) Y. N. Belokon, P. Carta, A. V. Gutnov, V. Maleev, M. A. Moskalenko,
L. V. Yashkina, N. S. Ikonnikov, N. V. Voskoboev, V. N. Khrustalev, M. North,
Helv. Chim. Acta 2002, 85, 3301-3312.
[2] a) S. Lundgren, E. Wingstrand, C. Moberg, Adv. Synth. Catal. 2007, 349, 364372; b) P. Roth, A. Hädener, C. Tamm, Helv. Chim Acta 1990, 73, 476-482.
[3] a) M. Inagaki, J. Hiratake, T. Nishioka, J. Oda, J. Org. Chem. 1992, 57, 56435649; b) H. S. Bevinakatti, A. A. Banerji, R. V. Newadkar, J. Org. Chem. 1989,
54, 2453-2455.
[4] L. Veum, M. Kuster, S. Telalovic, U. Hanefeld, M. Maschmeyer, Eur. J. Org.
Chem. 2002, 1516-1522.
[5] a) L. Veum, L. T. Kanerva, P. J. Halling, T. Maschmeyer, U. Hanefeld, Adv.
Synth. Catal. 2005, 347, 1015-1021; b) L. Veum, U. Hanefeld, Synlett 2005,
2382-2384.
[6] M. Schmidt, S. Hervé, N. Klempier, H. Griengl. Tetrahedron 1996, 52, 78337840.