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130.32, 129.69, 129.44, 122.12, 118.59, 115.49, 39.58 ppm; HRMS
(APCI) [M+Na+] calcd for C14H11Cl2NO3: 334.0008, found: 334.0013.
isolated after silica-gel column chromatography using a petroleum
ether/EtOAc solvent system.
15b-Hydroxytestosterone (6): 1H NMR (500 MHz, CDCl3): d=5.75
(s, 1H), 4.23 (ddd, J=7.8, 5.7, 2.4 Hz, 1H), 3.59 (t, J=8.7 Hz, 1H),
2.64 (ddd, J=14.6, 8.6, 7.7 Hz, 1H), 2.53–2.28 (m, 4H), 2.18 (s, 1H),
2.14–1.95 (m, 3H), 1.85 (dt, J=12.3, 3.2 Hz, 1H), 1.72 (td, J=14.0,
4.7 Hz, 1H), 1.66–1.51 (m, 9H), 1.46 (ddd, J=13.0, 6.5, 3.9 Hz, 2H),
1.24 (d, J=5.8 Hz, 3H), 1.18–0.96 ppm (m, 7H); 13C NMR (126 MHz,
CDCl3): d=199.70, 171.12, 124.16, 81.31, 69.33, 55.36, 54.47, 43.63,
42.43, 38.97, 38.06, 35.98, 34.16, 32.86, 31.67, 31.24, 20.76, 17.52,
13.90 ppm; HRMS (APCI) [M+H+] calcd for C19H28O3: 305.2111,
found: 305.2111.
Oxidation of naproxen
The 50 mL reaction mixture in 200 mm phosphate buffer, pH 7.5,
contained 2 mm of the RT2/AP/PV variant, 100 mm glucose,
2 UmLÀ1 glucose dehydrogenase and naproxen sodium salt (1 mm,
12.6 mg, from a 100 mm methanol stock). NADP+ monosodium
salt was added to a final concentration of 80 mm (4 mm stock in
200 mm phosphate buffer, pH 7.5) to initiate the reaction. After stir-
ring at 500 rpm at room temperature for 2 h the reaction mixture
was extracted with EtOAc (50 mL3). The combined EtOAc ex-
tracts were dried over MgSO4 and the solvent was removed after
filtration. The residue was subjected to silica-gel column chroma-
tography, eluted with petroleum ether and then petroleum ether/
Et2O to give 6.2 mg (57%) of 6-desmethylnaproxen, 3, after remov-
al of solvent.
6-Desmethylnaproxen (3): 1H NMR (500 MHz, CD3OD): d=7.69–
7.57 (m, 3H), 7.35 (dd, J=8.5, 1.9 Hz, 1H), 7.09–7.01 (m, 2H), 3.81
(q, J=7.1 Hz, 1H), 1.51 ppm (d, J=7.3 Hz, 3H); 13C NMR (126 MHz,
CD3OD): d=177.18, 155.01, 135.40, 134.09, 128.89, 128.45, 126.13,
125.67, 125.53, 118.10, 108.34, 45.16, 17.63 ppm. HRMS (APCI) [M+
Na+] calcd for C12H18N2O: 239.0683, found: 239.0679.
The oxidation of testosterone to generate 2b,16b-dihydroxytestos-
terone (7), was performed following the same protocol as for 5 but
utilizing 1 mm of the RLYF/KSK19/IP variant. 2b,16b-Hydroxytestos-
terone, 7 (7.6 mg) was isolated after silica-gel column chromatog-
raphy using a petroleum ether/EtOAc solvent system.
2b,16b-Dihydroxytestosterone (7): 1H NMR (500 MHz, CDCl3): d=
5.83 (d, J=1.0 Hz, 1H), 4.19 (dd, J=13.1, 5.0 Hz, 1H), 3.60 (t, J=
8.7 Hz, 1H), 3.52–3.44 (m, 1H), 2.69–2.56 (m, 2H), 2.50 (dd, J=13.8,
5.6 Hz, 1H), 2.30 (dt, J=7.1, 6.0 Hz, 2H), 2.18 (s, 3H), 1.95–1.77 (m,
2H), 1.68–1.31 (m, 6H), 1.32–1.19 (m, 2H), 1.19–1.04 ppm (m, 4H);
13C NMR (126 MHz, CDCl3): d=199.69, 174.97, 118.71, 81.02, 69.23,
68.54, 55.16, 50.69, 43.62, 42.83, 41.62, 39.53, 37.89, 33.99, 32.95,
32.01, 22.84, 22.51, 13.92 ppm; HRMS (APCI) [M+H+] calcd for
C19H29O4: 321.2060, Found: 321.2062.
2-Acetyl-6-methoxynaphthalene (4), was generated and isolated
(11.6 mg, 62%) from a 100 mL reaction with 2 mm of the RT2/AP/
F81W variant by the same method as for 3.
2-Acetyl-6-methoxynaphthalene (4): 1H NMR (500 MHz, CDCl3):
d=8.41 (d, J=2.0 Hz, 1H), 8.02 (dd, J=8.7, 1.9 Hz, 1H), 7.87 (d, J=
8.8 Hz, 1H), 7.78 (d, J=8.7 Hz, 1H), 7.22 (dd, J=9.0, 2.5 Hz, 1H),
7.17 (d, J=2.5 Hz, 1H), 3.96 (s, 3H), 2.71 ppm (s, 3H); 13C NMR
(126 MHz, CDCl3): d=198.09, 159.96, 137.48, 132.82, 131.32, 128.01,
127.29, 124.87, 119.93, 105.94, 55.63, 26.76 ppm; HRMS (APCI) [M+
Na+] calcd for C13H12O3: 233.0730, found: 233.0731.
Oxidation of chlorzoxazone
The 50 mL reaction mixture in 200 mm phosphate buffer, pH 7.5,
contained 2 mm of the RT2/AP/A184I variant, chlorzoxazone
(17.0 mg, 2 mm added from a 100 mm ethanol stock), 2 UmLÀ1 glu-
cose dehydrogenase and 100 mm glucose. NADP+ monosodium
salt was added to a final concentration of 80 mm from a 4 mm
stock in 200 mm phosphate buffer (pH 7.5), to initiate the reaction.
The reaction mixture was stirred at 500 rpm at room temperature
for 2 h and then extracted with EtOAc (50 mL3). The combined
EtOAc extracts were dried over MgSO4 and the solvent was re-
moved after filtration. The residue was subjected to silica-gel
column chromatography eluting with petroleum ether and then
petroleum ether/Et2O to give 18.1 mg (98%) of 6-hydroxychlorzox-
azone (8), after removal of solvent.
Oxidation of testosterone
The 50 mL reaction mixture in 200 mm phosphate buffer, pH 7.5,
contained 1 mm of the RLYF/KSK19 variant, testosterone (14.4 mg,
1 mm added from a 100 mm methanol stock), 2 UmLÀ1 glucose de-
hydrogenase and 100 mm glucose. NADP+ monosodium salt was
added to a final concentration of 80 mm (4 mm stock in 200 mm
phosphate buffer, pH 7.5) to initiate the reaction. After stirring at
500 rpm at room temperature for 2 h the reaction mixture was ex-
tracted with EtOAc (50 mL3). The combined EtOAc extracts were
dried over Na2SO4 and the solvent was removed after filtration.
The residue was subjected to silica-gel column chromatography,
eluted with petroleum ether/EtOAc to give 8.4 mg (55%) of 2b-hy-
droxytestosterone (5). 1H NMR (500 MHz, CDCl3): d=5.81 (d, J=
1.2 Hz, 1H), 4.19 (dd, J=13.9, 5.6 Hz, 1H), 3.67 (t, J=8.6 Hz, 1H),
3.51–3.46 (m, 1H), 2.57–2.46 (m, 2H), 2.29–2.23 (m, 1H), 2.18 (s,
3H), 2.08 (tdd, J=9.3, 7.6, 4.2 Hz, 1H), 2.02–1.95 (m, 1H), 1.92–1.86
(m, 1H), 1.80 (ddd, J=13.4, 7.0, 3.8 Hz, 1H), 1.71 (ddd, J=13.9,
10.7, 4.1 Hz, 1H), 1.64–1.37 (m, 9H), 1.36–1.25 (m, 2H), 1.19 (s, 3H),
1.14 (td, J=12.8, 4.1 Hz, 1H), 1.06–0.96 (m, 2H), 0.80 ppm (s, 3H);
13C NMR (126 MHz, CDCl3): d=199.90, 175.28, 118.86, 81.66, 68.72,
50.67, 50.39, 43.58, 41.64, 39.64, 36.56, 36.05, 34.74, 33.18, 30.65,
23.53, 23.02, 22.73, 11.42 ppm; HRMS (APCI) [M+H+] calcd for
C19H28O3: 305.2111, found: 305.2111.
6-Hydroxychlorzoxazone (8): 1H NMR (500 MHz, CD3OD): d=6.94
(s, 1H), 6.75 ppm (s, 1H); 13C NMR (126 MHz, CD3OD): d=155.92,
148.86, 143.29, 123.25, 115.57, 110.21, 98.82 ppm; HRMS (APCI)
[M+Na+] calcd for C7H4ClNO3: 207.9772, found: 207.9767.
Oxidation of amitriptyline
The 50 mL reaction mixture in 50 mm Tris-HCl, pH 8.5, contained
2 mm of the KSK19 variant, amitriptyline hydrochloride monohy-
drate (31.3 mg, 2 mm, added as a 100 mm ethanol stock), 2 UmLÀ1
glucose dehydrogenase and 100 mm glucose. NADP+ monosodi-
um salt was added to a final concentration of 80 mm (4 mm stock
in 50 mm Tris-HCl, pH 8.5) to initiate the reaction. The reaction mix-
ture was stirred at 500 rpm under O2 at room temperature for 2 h,
adjusted to pH>12 with 5m KOH and extracted with Et2O
(50 mL3). The combined Et2O extracts were dried over MgSO4
and the solvent was removed after filtration. The residue was sub-
jected to silica-gel column chromatography, eluting with CHCl3 and
then CHCl3/MeOH/NH4OH (90:10:0.1) to give 9.78 mg (69%) of nor-
triptyline (9): 1H NMR (400 MHz, CDCl3): d=7.34–7.28 (m, 1H),
7.24–7.11 (m, 6H), 7.08–7.03 (m, 1H), 5.87 (t, J=7.4 Hz, 1H), 3.48–
The oxidation of testosterone to generate 15b-hydroxytestoster-
one, 6, was performed following the same procedure as for 5 but
utilizing 1 mm of the RT2/V78F variant. Compound 6 (11.8 mg) was
Chem. Eur. J. 2015, 21, 15039 – 15047
15045
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim