Quantitative gas chromatography analysis
were then maintained at 40 ◦C and 100 mbar until no change in
mass occurred.
The concentration of valencene oxidation products in an
incubation mixture was determined by calibrating the FID
response to monooxygenated derivatives using (+)-nootkatone
as a representative compound. The reasonable assumption
was that these isomeric compounds would have near identical
responses on the FID. Mixtures containing all components of a
standard valencene oxidation incubation except NAD(P)H and
the substrate, but additionally 5, 10, 50, 100, and 250 lM of
(+)-nootkatone, were extracted and analysed as for a normal
incubation. The ratio of the area of the (+)-nootkatone peak
to that of the internal standard was plotted against the (+)-
nootkatone concentration to give a straight-line calibration plot
that passed through the origin. The use of an internal standard
significantly reduced the spread of the data. The calibration
plot based on (+)-nootkatone did not apply for the further
oxidation products such as nootkaton-9-ol and nootkatone-
13,14-epoxide; these doubly oxygenated compounds had much
higher FID responses than the monooxygenated valencenes. (+)-
trans-Nootkaton-9-ol, which was isolated and purified from
a preparative scale whole-cell reaction, was used as a rep-
resentative example of these further oxidation products and
a calibration was repeated. The two calibration plots were
used to calculate the concentration of the products formed
in a reaction. For P450BM-3 there were products that were not
characterised; those that had similar retention times to the
nootkatols were assumed to be monooxygenated valencenes
while the ones with longer retention times than the valencene-
1,10-epoxide (>13 min) were assumed to be doubly oxygenated
compounds, and the appropriate calibration plots were used to
estimate their concentrations. Since the enzyme concentration
is 1 lM, if the total product concentration is a lM and the
NAD(P)H was consumed in t min, the substrate oxidation
rate is defined as a/t nmol (nmol P450)−1 min−1. If the initial
NAD(P)H concentration is b lM, the yield is a/b and expressed
as a percentage. This calculation ignores the fact that more than
one molecule of NAD(P)H may be required for the formation
of one molecule of a product. For example, if nootkatone was
the only product and every molecule of NAD(P)H consumed
was utilised for nootkatone formation, the yield would be 50%.
(+)-trans-Nootkaton-9-ol 4 (15 mg, 13.5% based on the total
amount of nootkatone added to the whole-cell reaction) was
obtained as a pale yellow oil; dH(500 MHz; C6D6; Me4Si) 0.53
(3 H, d, J 6.7, 4-Me), 0.82 (1 H, dd, J 12.5, 12.6, 6-Hax), 1.10
(3 H, s, 5-Me), 1.21 (1 H, ddd, J 3.2, 12.7, 13.5, 8-Hax), 1.54 (1 H,
ddq, J 5.5, 6.7, 12.0, 4-H), 1.62 (3 H, dd, J 0.9, 1.3 13-Me),
1.74 (1 H, ddd, J 2.7, 2.8, 12.9, 6-Heq), 1.98 (1 H, dddd, J 2.7,
2.8, 2.8, 13.5, 8-Heq), 2.09–2.12 (2 H, m, 2 × 3-H), 2.22 (1 H,
br s, 9-OHeq), 2.83 (1 H, dddd, J 2.8, 2.8, 12.5, 12.6, 7-Hax), 4.03
(1 H, dd, J 2.9, 2.9, 9-Hax), 4.77 (1 H, dq, J 0.9, 1.6, 14-H-cis-Me),
4.79 (1 H, dq, J 1.3, 1.6, 14-H-trans-Me), 5.79 (1 H, s, 1-H);
dC(125.7 MHz; C6D6; Me4Si) 13.99 (q, 4-Me), 17.58 (q, 5-Me),
20.56 (q, 13-Me), 34.16 (d, 7), 37.91 (t, 8), 38.62 (s, 5), 40.89 (d,
4), 42.29 (t, 3), 43.70 (t, 6), 72.95 (d, 9), 109.23 (t, 14), 127.06
(d, 1), 149.11 (s, 13), 167.65 (s, 10) and 199.32 (s, 2). m/z (EI)
234.1613 (M+, C15H22O2 requires 234.1620), 216 (9%), 190 (3).
(+)-cis-Valencene-1,10-epoxide 5 (19.1 mg, 16% based on the
total amount of valencene added to the whole-cell reaction) was
obtained as a pale yellow oil; m/z (EI) 220.1768 (M+, C15H24O
requires 220.1827).
(+)-Nootkatone-13S,14-epoxide 6 (52.1 mg, 47% based on the
total amount of nootkatone added to the whole-cell reaction)
was obtained as a pale yellow oil; dH(500 MHz; C6D6; Me4Si)
0.50 (3 H, d, J 6.7, 4-Me), 0.57 (3 H, d, J 0.4, 5-Me), 0.71 (1 H,
dd, J 12.8, 12.8, 6-Hax), 0.77 (1 H, dddd, J 4.6, 12.6, 12.6, 13.6,
8-Hax), 0.94 (3 H, d, J 0.7, 13-Me), 1.21 (1 H, dddd, J 3.2, 3.3,
12.6, 12.7, 7-Hax), 1.37 (1 H, ddddd, J 2.5, 3.1, 3.3, 5.2, 12.6,
8-Heq), 1.55 (1 H, ddq, J 4.0, 6.7, 14.2, 4-H), 1.76 (1 H, ddd, J
2.7, 2.8, 12.8, 6-Heq), 1.81 (1 H, ddd, J 2.7, 4.3, 14.9, 9-Heq), 1.87
(1 H, dddd, J 2.0, 5.0, 13.6, 14.9, 9-Hax), 2.00 (1 H, dd, J 14.2,
16.7, 3-Hax), 2.14 (1 H, ddd, J 1.0, 4.0, 16.7, 3-Heq), 2.16 (1 H, d,
J 5.0, 14-H-cis-Me), 2.21 (1 H, dq, J 0.7, 5.0, 14-H-trans-Me),
5.79 (1 H, d, J 0.6, 1-H); dC(125.7 MHz; C6D6; Me4Si) 14.30 (q,
4-Meax), 15.86 (q, 5-Meeq), 17.43 (q, 13-Me), 28.37 (t, 8), 31.81 (t,
9), 38.61 (s, 5), 39.16 (d, 7), 40.10 (t, 6), 40.10 (d, 4), 41.82 (t, 3),
52.30 (t, 14), 58.09 (s, 13), 125.02 (d, 1), 167.41 (s, 10) and 197.45
(s, 2). m/z (EI) 234.1626 (M+, C15H22O2 requires 234.1620), 216
(12%), 206 (14), 176 (4).
Whole-cell substrate oxidation
Acknowledgements
A number of (+)-valencene and (+)-nootkatone oxidation
products were prepared by whole-cell oxidation by E. coli
expressing the P450BM-3 holoenzyme.42 In a typical reaction, a
500 cm3 culture of the E. coli DH5a harbouring the expression
plasmid was grown at 30 ◦C until the turbidity at 600 nm
reached ∼1.0 and then induced with 0.5 mM isopropyl-b-D-
thiogalactopyranoside (IPTG). The cultures were then grown
fo◦r another 16 h. Cells were harvested by centrifugation (1000g,
4 C, 5 min) and resuspended in 500 cm3 E. coli minimal media
(per litre: 7 g K2HPO4, 3 g KH2PO4, 0.5 g sodium citrate, 0.1 g
MgSO4, 1 g (NH4)2SO4, 1 cm3 glycerol, 50 cm3 1 M Tris buffer,
pH 7.4, 0.1% w/v glucose, and 10 mg thiamine). Hexadecane was
added to 2% v/v to partition the valencene oxidation products
and organics that might be harmful to the bacterial host, into the
organic phase. The (+)-valencene or (+)-nootkatone substrate
was added in 50 mg aliquots in 1 cm3 ethanol and the cultures
returned to the orbital shaker (120 rpm, 30 ◦C). After 24 h, more
substrate and glucose were added. Cells were pelleted after 48 h
and the supernatant extracted with EtOAc (3 × 200 cm3). The
combined extracts were dried and the solvent was evaporated
off at 40 ◦C and 240 mbar. The oily residue was dissolved
in CH2Cl2 and applied to a silica column (0.06–0.2 mm, 70–
230 mesh). Organics were eluted with hexane–EtOAc mixtures
containing increasing concentrations of EtOAc and fractions
were analysed by GC to locate the products. The appropriate
fractions containing the target compounds were combined and
RJS thanks the EPSRC, UK for a studentship and SY thanks
the Lady Noon Foundation for a scholarship.
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