J. H. Schrittwieser et al. / Tetrahedron: Asymmetry 20 (2009) 483–488
487
incubated for 4 h at 30 °C and 120 rpm. After that HheB (50
l
L,
4.3.8. Analytics
4.3.8.1. GC, achiral stationary phase. A Varian CP 1301
126 U) and MTO-DowexÒ SBR LCNG OH-form (35 or 50 mg) were
added. Afterwards all samples were incubated for another 20 h, ex-
(30 m ꢃ 250
lm ꢃ 0.25
lm) 6% cyanopropyl-phenylpolysiloxane
tracted with EtOAc (600
l
L) and dried over Na2SO4. The conversion
phase column was employed and H2 (column flow: 1.2 mL minꢁ1
was used as carrier gas.
)
was determined by GC analysis.
The reaction conditions are summarised in Table 5.
1a–3a: temperature programme: 100–150 °C/25 °C minꢁ1
–
270 °C/10 °C minꢁ1/hold 2 min. tr [min] = 4.4 1a, 4.6 2a, 3.1 3a.
1b–3b: temperature programme: 90–145 °C/5 °C minꢁ1–205 °C/
30 °C minꢁ1/hold 3 min. tr [min] = 4.5 1b, 4.9 2b, 2.3 3b. 1c–3c:
temperature programme: 90–120 °C/10 °C minꢁ1–270 °C/15 °C
minꢁ1/hold 2 min. tr [min] = 5.2 1c, 5.0 2c, 2.8 3c. 1d–3d: temper-
ature programme: 90–130 °C/5 °C minꢁ1–250 °C/30 °C minꢁ1/hold
2 min. tr [min] = 3.6 1d, 4.1 2d, 2.2 3d.
Table 5
Reaction conditions for the cascade reaction sequence using LBADH and HheB
Substrate
Procedure
LBADH (
lL)
HheB (
l
L)
IEa (mg)
1a
1b
1c
1d
A
A
B
B
10
10
20
20
30
30
50
50
50
30
35
50
4.3.8.2. GC, chiral stationary phase. A Varian CP-Chiralsil-DEX CB
a
(25 m ꢃ 320
l
m ꢃ 0.25
lm) cyclodextrin on dimethylpolysiloxane
IE = ion exchanger MTO-DowexÒ SBR LCNG OH-form.
phase column was employed and H2 was used as carrier gas.
3b: column flow: 0.4 mL minꢁ1, temperature programme: 65 °C
isothermal. tr [min] = 13.1 (R), 13.5 (S). 3c: column flow:
4.3.7. Procedures for the preparative scale cascade reaction
sequence using E. coli TunerTM (DE3)/pET22b-’ADH-A’ and HheC
4.3.7.1. Procedure A—addition of all reagents at t = 0 h. Lyoph-
ilised cells of E. coli TunerTM (DE3)/pET22b-’ADH-A’ (50 mg) were
rehydrated in Tris–SO4 buffer (5 mL, 200 mM, pH 7.5) for 1 h at
0.4 mL minꢁ1
, temperature programme: 80 °C isothermal. tr
[min] = 15.1 (R), 16.8 (S). 3d: column flow: 0.3 mL minꢁ1, temper-
ature programme: 60 °C isothermal. tr [min] = 23.1 (R), 23.5 (S).
30 °C and 120 rpm. 2-Propanol (50
19.5 U), MTO-DowexÒ SBR LCNG OH-form (350 or 500 mg) and
the substrate (1a, 40 L, 0.27 mmol or 1b, 50 L, 0.31 mmol) were
added. The mixture was incubated for 24 h at 30 °C and 120 rpm,
extracted with EtOAc (2 ꢃ 10 mL), dried over Na2SO4 and evapo-
rated under reduced pressure to give the crude product. Flash chro-
matography (Al2O3, petrol ether/EtOAc = 4/1) furnished the pure
epoxides.
l
L, 0.65 mmol), HheC (300
l
L,
4.3.8.3. HPLC, chiral stationary phase. A Daicel Chiralpak AD col-
umn (0.46 cm ꢃ 25 cm, column oven temperature: 12 °C) was em-
ployed and n-heptane (flow: 0.4 mL minꢁ1) was used as the eluent.
3a: tr [min] = 34.5 (R), 41.8 (S).
l
l
4.3.9. Determination of absolute configurations
Absolute configurations were either assigned by comparison of
specific rotation with literature data (see above), and/or compari-
son of elution order with the literature on a chiral stationary phase.
(S)-3a (19 mg, 47%) colourless liquid. 1H NMR (360 MHz, CDCl3)
d: 2.78 (1H, dd, J1 = 4.7 Hz, J2 = 2.5 Hz, CH2), 2.92 (1H, t, J = 4.5 Hz,
CH2), 3.36–3.39 (1H, m, CH), 3.98 (1H, dd, J1 = 11.0 Hz, J2 = 5.4 Hz,
CH2), 4.23 (1H, dd, J1 = 11.0 Hz, J2 = 3.2 Hz, CH2), 6.93–7.01 (3H, m,
Ar), 7.29–7.33 (2H, m, Ar). 13C NMR (90 MHz, CDCl3) d: 44.7, 50.1,
68.7, 114.7, 121.2, 129.5, 158.5. m/z (EI, 70 eV): 150 (M+), 120, 107,
94, 77, 65, 57, 51, 39. HPLC (Chiralpak AD, n-Heptan): ee >99% (S).
Acknowledgements
Financial support by University of Graz and FWF (project num-
ber P18537-B03) is gratefully acknowledged.
½
a 2D0
ꢂ
¼ þ4:7 (c 1, CHCl3), lit.32
½
a 2D0
ꢂ
¼ þ4:5 (c 0.4, CHCl3, ee = 93.2%).
(R)-3b (16 mg, 40%) colourless liquid. 1H NMR (360 MHz, CDCl3)
References
d: 0.85–0.90 (3H, m, CH3), 1.24–1.63 (10H, m, CH2), 2.48 (1H, dd,
J1 = 5.0 Hz, J2 = 2.7 Hz, OCH2), 2.77 (1H, t, J = 4.5 Hz, OCH2), 2.90–
2.94 (1H, m, OCH). 13C NMR (90 MHz, CDCl3) d: 14.0, 22.5, 25.9,
29.1, 31.7, 32.5, 47.1, 52.4. m/z (EI, 70 eV): 128 (M+), 113, 85, 57,
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¼ þ4:8 (c 1, CHCl3),
ꢂ
lit.33
½
a 2D0
ꢂ
¼ þ7:4 (c 1.15, CHCl3, ee >99%).
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0.65 mmol) and -chloroacetophenone (1c, 54 mg, 0.35 mmol)
were added. The solution was incubated for 4 h at 30 °C and
120 rpm. After that HheC (300
L, 19.5 U) and MTO-DowexÒ SBR
lL,
x
´
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l
LCNG OH-form (350 mg) were added. Afterwards the mixture
was incubated for another 20 h, extracted with EtOAc
(2 ꢃ 10 mL), dried over Na2SO4 and evaporated under reduced
pressure to give the crude product as a yellowish liquid. Flash chro-
matography (Al2O3, petrol ether/EtOAc = 4/1) furnished (R)-3c
(19 mg, 45%) as a colourless liquid. 1H NMR (360 MHz, CDCl3) d:
2.82 (1H, dd, J1 = 5.5 Hz, J2 = 2.6 Hz, OCH2), 3.17 (1H, dd,
J1 = 5.4 Hz, J2 = 4.1 Hz, OCH2), 3.88 (2H, dd, J1 = 3.8, J2 = 2.7, OCH),
7.29–7.39 (5H, m, Ar). 13C NMR (90 MHz, CDCl3) d: 51.2, 52.3,
125.5, 128.2, 128.5, 137.6. m/z (EI, 70 eV): 120 (M+), 119, 104, 91,
77, 65, 51, 39. GC (Chiralsil Dex-CB): ee >99% (R). ½a D20
¼ ꢁ19:5 (c
ꢂ
1, CHCl3), lit.34
½
a 2D3
ꢂ
¼ þ23:6 (c 0.83, CHCl3, ee = 94.4%).