10.1002/cbic.201700316
ChemBioChem
FULL PAPER
be equal for isomeric metabolites. The coupling efficiency was calculated
as the percentage of NADH used to generate product.
131.13 (C3), 130.37 (C8), 130.33 (C4), 129.36 (C5), 127.98 (C7), 127.92
(C1), 68.27 (C11), 24.36 (C12).
Product isolation and characterisation
Data for 3-biphenylmethanol [15d]: 1H NMR (500 MHz, CDCl3) 7.64 7.57
(m, 3H), 7.53 (m, 1H), 7.64 – 7.57 (m, 3H), 7.39 7.31 (m, 2H), 4.77 (s,
2H).
Whole-cell turnovers were used to synthesise products on a larger scale
for characterisation when standards were not available. The plasmid
pETDuetArx/ArR was combined in E. coli with pRSFDuetArx/CYP101B1
[15d]
Data for 2-(7-methylnaphthyl)methanol
:
1H NMR (500 MHz, d6-
or pRSFDuetArx/H85FCYP101B1 and used in a whole-cell turnover to
acetone) δ 7.79 (s, 1H), 7.77 (m, 1H), 7.75 (m, 1H), 7.63 (s, 1H), 7.46 (m,
1H), 7.33 (m, 1H), 4.77 (s, 2H), 2.48 (s, 3H).
14a]
generate metabolites.[6b,
The growths and protein induction were
conducted on
a 100 ml scale using 2xYT media as described
previously.[15a, c, d] The cells were harvested by centrifugation (8 g of cell
wet weight per litre, P450 concentration 650 nM) and resuspended in 200
ml E. coli minimal media (EMM). The resuspended cells and substrate (0.5
- 2 mM) were added to a 2 L baffled flask and shaken at 150 rpm at 30 C
for 16 hours. Two further aliquots of substrate (same amount as the initial
addition were added after 3 and 6 hours). The supernatant was extracted
in ethyl acetate (3 x 100 mL), washed and dried with brine and magnesium
sulfate respectively. The organic extracts were collected and the solvent
was removed by vacuum distillation and then under a stream of nitrogen.
The products were purified using silica gel chromatography using a
hexane/ethyl acetate stepwise gradient ranging from 90:10 to 60:40
hexane to ethyl acetate (EtOAc) with a 2.5% increase in the amount of
EtOAc every 50 mL.
Data for 1-acenaphthol [29] 1H NMR (500 MHz, CDCl3) δ 7.76 – 7.74 (m,
:
1H, H6), 7.67 (d, J = 8.2 Hz, 1H, H8), 7.58 – 7.53 (m, 2H, H5 & H10), 7.52
– 7.47 (m, 1H, H9), 7.31 (d, J = 6.8 Hz, 1H, H4), 5.75 (d, J = 7.5 Hz, 1H,
H1), 3.82 (dd, J = 17.9, 7.5 Hz, 1H, H2), 3.26 (d, J = 17.8 Hz, 1H, H2). 13
C
NMR (126 MHz, CDCl3) δ 148.34 (C11), 144.19 (C3), 139.81 (C12),
133.86 (C7), 130.88 (C9), 130.69 (C5), 127.67 (C6), 125.39 (C8), 122.95
(C10), 122.51 (C4), 77.09 (C1), 44.59 (C2).
Acknowledgements
Md.R.S. thanks the University of Adelaide for an International PhD
scholarship. J.H.Z.L acknowledges an Australian Government
Research Training Program Scholarship (M. Phil). S.G.B.
acknowledges the ARC for a Future Fellowship (FT140100355).
The purified products (ranging from 110 mg) were dissolved in CDCl3 and
characterised by NMR spectroscopy (Fig. S5). Minor products were
identified via GC coelution or comparison of the MS spectra of standards
published by others (Fig. S4).
Keywords: biocatalysis • enzyme catalysis • protein engineering
• monooxygenases • regioselectivity
NMR Data
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Data for (2-ethylphenyl)methanol: 1H NMR (500 MHz, CDCl3) δ 7.35 (d, J
= 6.6 Hz, 1H, H6), 7.24 (d, J = 7.6 Hz, 1H, H3) 7.18-7.12 (m, 2H, H4 & H5),
4.52 (s, 2H, 2 x H7), 2.60 (q, J = 7.9 Hz, 2H, 2 x H8), 1.14 (t, J = 7.9 Hz,
3H, 3 x H9).
Data for 1-(3-methylphenyl)ethanol: 1H NMR (500 MHz, CDCl3) δ 7.23 (d,
J = 7.5 Hz, 1H, H6) 7.20 (s, 1H, H2), 7.18 – 7.16 (m, 1H, H5), 7.09 (d, J =
7.4 Hz, 1H, H4), 4.87 (q, J = 6.5 Hz, 1H, H7), 2.36 (s, 3H, 3 x H9), 1.5 (d,
J = 6.5 Hz, 3H, 3 x H8).
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7.6 Hz, 2H, 2 x H7), 2.22 (s, 3H, 3 x H9), 1.18 (t, J = 7.6 Hz, 3H, 3 x H8).
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Data for 1-methyl-2-naphthalenemethanol: 1H NMR (500 MHz, CDCl3) δ
8.08 (d, J = 8.6 Hz, 1H, H5), 7.83 (d, J = 8.8 Hz, 1H, H4), 7.72 (d, J = 8.4
Hz, 1H, H8), 7.54 – 7.45 (m, 3H, H6, H7, H3), 4.91 (s, 2H, H12), 2.71 (s,
3H, H11). 13C NMR (126 MHz, CDCl3) δ 134.87 (C2), 131.15 (C4), 129.16
(C8), 129.05 (C6), 128.68 (C7), 128.19 (C3), 127.78 (C1), 126.81 (C5),
67.01 (C12), 16.55 (C11).
Data for 5-methyl-1-naphthalenemethanol: 1H NMR (500 MHz, CDCl3) δ
8.04 – 8.00 (m, 2H, H4 & H8), 7.56 (d, J = 6.9 Hz, 1H, H2), 7.53 – 7.43 (m,
2H, H3 & H7), 7.37 (d, J = 7.6 Hz, 1H, H6), 5.18 (s, 2H, 2 x H11), 2.73 (s,
3H, H12).
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Data for 6-methyl-2-naphthalenemethanol: 1H NMR (500 MHz, CDCl3) δ
7.77 (s, 1H, H1), 7.76 (d, J = 3.1 Hz, 1H, H8), 7.75 – 7.73 (m, 1H, H4),
7.62 (s, 1H, H1, H5), 7.46 (dd, J = 8.3, 1.6 Hz, 1H, H7) 7.34 (dd, J = 8.3,
1.5 Hz, 1H, H3), 4.85 (s, 2H, 2 x H11), 2.53 (s, 3H, 3 x H12). 13C NMR
(126 MHz, CDCl3) δ 140.02 (C9), 138.25 (C10), 135.83 (C2), 134.24 (C6),
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