M. Pogore6c, K. Faber / Tetrahedron: Asymmetry 13 (2002) 1435–1441
1439
s), 4.40 (1H, m); 13C NMR (CD3OD): 13.6, 20.3, 23.0,
25.6, 29.6, 29.9, 32.2, 37.4, 76.6 (C-2); 78% yield.
17.0, 26.5 (2×C), 26.9, 28.5, 28.9, 44.0, 80.3 (C-1); 95%
yield.
1
1
rac-2-Decyl sulfate, 4a: H NMR (360 MHz, CD3OD):
rac-4-Phenyl-2-butyl sulfate, 16a: H NMR (500 MHz,
0.86 (3H, t, J=6.8 Hz), 1.25–1.47 (14H, m), 1.85 (3H,
s), 4.38 (1H, m); 13C NMR (CD3OD): 13.7, 20.4, 23.0,
23.5, 25.6, 29.7, 30.0, 30.0, 32.3, 37.4, 76.6 (C-2); 70%
yield.
CD3OD): 1.37 (3H, d, J=6.27 Hz), 1.85–1.93 (2H, m),
2.71–2.79 (2H, m) 4.52 (1H, m), 7.15–7.26 (5H, m); 13C
NMR (CD3OD): 20.4, 31.8, 39.3, 76.2 (C-2), 126.0,
128.5 (2×C), 128.6 (2×C), 142.6; 80% yield.
1
rac-2-Dodecyl sulfate, 5a: 1H NMR (500 MHz,
CD3OD): 0.87 (3H, t, J=6.8 Hz), 1.26–1.60 (18H, m),
1.87 (3H, s), 4.40 (1H, m); 13C NMR: (CD3OD) 11.6,
18.2, 20.9, 23.5, 27.6, 27.9 (4×C), 30.2, 35.3, 74.4 (C-2);
60% yield.
rac-1-Phenyl-2-propyl sulfate, 17a: H NMR (500 MHz,
CD3OD): 1.25 (3H, d, J=6.15 Hz), 1.95 (2H, m), 4.7
(1H, m), 7.22–7.30 (5H, m); 13C NMR (CD3OD): 19.3,
43.2, 76.8, 126.6, 128.5 (2×C), 130.0 (2×C), 138.2; 90%
yield.
1
rac-3-Octyl sulfate, 6a: H NMR (360 MHz, CD3OD):
3.3. Biocatalytic procedures
0.83–0.92 (6H, m), 1.28–1.40 (6H, m), 1.52–1.68 (4H,
m), 4.25 (1H, m); 13C NMR (CD3OD): 9.0, 13.8, 22.9,
25.0, 27.1, 32.3, 33.8, 81.6 (C-3); 87% yield.
3.3.1. Growth of strains. All bacteria strains were grown
under aerobic conditions in baffled Erlenmeyer flasks at
30°C and 130 rpm using the following medium: 10 g/l
glucose, 10 g/l peptone, 10 g/l yeast extract, 2 g/l NaCl,
1.5 g MgSO4·7 H2O, 1.3 g/l NaH2PO4 and K2HPO4.
Cell growth was monitored by measurement of the
optical density via the absorption at 546 nm.
1
rac-4-Octyl sulfate, 7a: H NMR (500 MHz, CD3OD):
0.85–1.0 (6H, m), 1.25–1.5 (6H, m), 1.55–1.7 (4H, m),
4.3 (1H, m); 13C NMR (CD3OD): 13.6, 13.7, 18.4, 23.0,
27.4, 34.2, 36.7, 80.0 (C-4); 78% yield.
1
rac-4-Decyl sulfate, 8a: H NMR (360 MHz, CD3OD):
1.00–1.08 (6H, m), 1.43–1.58 (10H, m), 1.70–1.78 (4H,
m), 4.47 (1H, m); 13C NMR (CD3OD): 11.2, 11.2, 16.0,
20.4, 22.8, 27.2, 29.7, 32.2, 34.4, 77.7 (C-2); 70% yield.
3.3.2. Screening for biocatalytic activity. Lyophilised
cells (50 mg) were rehydrated in Tris-buffer (pH 7.5, 0.1
M, 600 ml) and shaken for 1 h at 24°C. To the rehy-
drated cells, 200 ml of a 88 mM rac-2-octyl sulfate
(rac-1a) stock solution (20.5 mg/ml, 0.1 M Tris, pH 7.5
buffer) was added and the vials were shaken for 5 days.
Finally, 400 ml of the suspension were extracted with
400 ml of ethyl acetate. The reaction was monitored by
TLC and GC. For GC-analysis, 200 ml of the organic
phase was mixed with a stock solution of 5 ml (25 ml)
menthol in ethanol [c 15.4 mg/ml (77 mg/ml)], which
served as an internal standard. For each substrate, any
spontaneous non-enzymatic hydrolysis was checked by
blank-experiments in the absence of enzyme. With the
exception of rac-18a, which was prone to decomposi-
tion, the degree of spontaneous hydrolysis was
negligible.
1
rac-1-Octyl sulfate, 9a: H NMR (360 MHz, CD3OD):
0.85 (3H, t, J=7.0 Hz), 1.26–1.35 (8H, m), 1.58–1.63
(2H, m), 1.85 (2H, m), 3.95 (2H, t, J=6.6 Hz); 13C
NMR (CD3OD): 12.6 (C-8), 21.9, 25.1, 28.6 (3×C),
31.2, 67.4 (C-1); 96% yield.
rac-1-Octen-3-yl sulfate, 11a: 1H NMR (360 MHz,
CD3OD): 1.04 (3H, t, J=6.8 Hz), 1.46–1.55 (6H, m),
1.83–1.87 (2H, m), 4.87 (1H, m), 5.27 (1H, d, J=10.6
Hz), 5.42 (1H, d, J=17.1 Hz), 6.0 (1H, m); 13C NMR
(CD3OD): 11.1, 20.3, 22.3, 29.6, 33.0, 77.9 (C-3), 113.2
(C-1), 135,8 (C-2); 60% yield.
1
rac-2-Methyl-3-octyl sulfate, 12a: H NMR (360 MHz,
CD3OD): 1.06 (9H, m), 1.45 (6H, m), 1.74 (2H, m), 2.2
(1H, m), 4.29 (1H, m); 13C NMR (CD3OD): 11.1, 14.9,
20.3, 20.9, 22.7, 28.2, 28.7, 29.8, 82.3 (C-3); 57% yield.
3.3.3. Determination of conversion. The degree of con-
version and relative enzyme activities were measured
using 15 mM substrate in Tris buffer (0.1 M, pH 7.5).
Enzyme solution [400 ml, partially purified enzyme solu-
1
rac-6-Methyl-5-hepten-2-yl sulfate, 13a: H NMR (360
MHz, CD3OD): 1.27 (3H, d, J=6.3 Hz), 1.57 (3H, s),
1.62 (3H, s), 1.85–2.05 (4H, m), 4.40 (1H, m), 5.08 (1H,
m); 13C NMR (CD3OD): 16.1, 19.3, 23.2, 24.2, 36.5,
75.4 (C-2), 123.4 (C-5), 130.8 (C-6); 80% yield.
tion after chromatography on a Q6 column or
lyophilized enzyme powder, after chromatography on
Phenyl Sepharose, resuspended in Tris buffer (0.1 M,
pH 7.5)] was mixed with 400 ml of a 30 mM substrate
solution. This mixture was shaken at 24°C at 130 rpm
for 6 or 12 h, depending on the range of relative
activity. An aliquot of 200 ml from the reaction mixture
was extracted with 200 ml of ethyl acetate. After vigor-
ous vortexing (30 s) and centrifugation (13.000 rpm, 5
min), 100 ml of the organic phase was mixed with a
stock solution of 5 ml (25 ml) menthol in ethanol [c 15.4
mg/ml (77 mg/ml)], which served as an internal stan-
dard. The conversion was calculated from calibration
curves for rac-2-, 3- and 4-octyl sulfate.
1
rac-1-Bromo-7-octyl sulfate, 14a: H NMR (360 MHz,
CD3OD): 1.27 (3H, d, J=6.3 Hz), 1.33–1.86 (10H, s),
3.32 (2H, m, obscured by solvent peak), 4.41 (1H, m);
13C NMR (CD3OD): 20.4, 25.4, 28.4, 29.0, 33.2, 33.7,
37.2, 76.4 (C-2); 80% yield.
rac-1-Cyclohexylethyl sulfate, 15a: 1H NMR (500 MHz,
CD3OD): 1.33 (3H, d, J=6.39 Hz), 1.56–1.91 (11H, m),
2.71–2.79 (2H, m) 4.29 (1H, m); 13C NMR (CD3OD):