Determination of incorporation of deuterium into bioproducts dur-
ing labelling experiments
Cycloaddition of 4-phenyl-1,2,4-triazoline-3,5-dione to arene oxide
2dS/2dR to yield ( )-anti-10-carbomethoxy-1-(trifluoromethyl)-4-
phenyl-9-oxa-2,4,6-triazatetracyclo[5.3.2.02,6.08,10]dodec-11-ene-3,
5-dione 8
Percentage incorporation from methyl 1,2,3,4,5-pentadeuterio-
benzoate 1a-D5, methyl 2,6-dideuteriobenzoate 1a-D2 and trideu-
teriomethyl benzoate 1a-D3 into oxepine 2ꢀa and methyl salicylate
4-Phenyl-1,2,4-triazoline-3,5-dione (18 mg, 0.1 mmol) was added
to a solution of arene oxide 2dS/2dR (15 mg, 0.068 mmol) in
CH2Cl2 (4 mL) and the reaction mixture stirred at ambient
temperature for 3 days. The crude product obtained after removal
of the solvent was purified by PLC (7% MeOH in CHCl3) to give
the cycloadduct 8 as colourless plates (19 mg, 70%); mp 164–65 ◦C
(EtOAc–hexane); Rf 0.62 (7% MeOH in CHCl3); [a]D 0.0 (c 1.0,
CHCl3); (Found: M+, 395.0749. C17H12 F3N3O5 requires 395.0730);
dH (500 MHz, CDCl3) 3.91 (3 H, s, CO2Me), 4.02 (1 H, d, J8,7 4.5,
8-H), 5.51 (1 H, ddd, J7,11 1.7, J7,8 4.5, J7,12 6.0, 7-H), 6.32 (1 H, dd,
J12,11 8.4, J12,7 6.0, 12-H), 6.49 (1 H, dd, J11,12 8.4, J11,7 1.7, 11-H),
7.5 (5 H, m, C6H5); m/z (EI) 395 (M+, 33%), 308 (25), 220 (39),
189 (20), 161 (14), 133 (19), 119 (100), 91 (28), 59 (31).
1
6a was determined by both GC–MS, H-NMR and 13C-NMR
analyses. For GC–MS, the GC operating conditions were as
described above but the mass spectrometer was employed in the
selected ion monitoring mode measuring ion currents at m/e 152,
153, 154, 155, 156 and 157. Incorporation of label into oxepine
2ꢀa and methyl salicylate 6a was determined as the ratio of the
various ion currents, after correction for natural abundances,
measured at the expected retention time of the authentic standard
compounds.
Methyl 2-oxepinecarboxylate 2ꢀa
Crystal data for compound 8. C17H12F3N3O5, M = 395.3, mon-
Yellow oil (0.335 g, 65%); Rf 0.5, mmax/cm−1 1726 (C O); dH
(500 MHz, CDCl3) 3.82 (3 H, s, CO2Me), 5.78 (1 H, dd, J6,5 6.2,
J6,7 5.2, 6-H), 5.94 (1 H, d, J7,6 5.2, 7-H), 6.35 (1 H, dd, J4,5 10.5,
=
˚
oclinic, a = 10.584(3), b = 10.448(3), c = 14.677(4) A, b = 90.31(1),
3
˚
˚
U = 1623.1(8) A , T = 150(2) K, Mo-Karadiation, k = 0.71073 A,
space groupP21/c (no. 14), Z = 4, F(000) = 808, Dx = 1.618 g cm−3,
l = 0.143 mm−1, Bruker SMART CCD-detector diffractometer,
φ/x scans, 4.8◦ < 2h < 56◦, measured/independent reflections:
4219/2371, Rint = 0.046, direct methods solution, full-matrix least
J
4,3 6.1, 4-H), 6.47 (1 H, dd, J5,4 10.5, J5,6 6.2, J5,3 0.5, 5-H), 6.85 (1
H, dd, J3,4 5.6, J3,5 0.5, 3-H); dC (125 MHz, CDCl3) 52.45, 118.08,
123.91, 128.56, 132.90, 133.59, 137.02, 163.25; m/z (EI) 154 (M+,
85%), 121 (12), 93 (26), 65 (100). The isolated bioproduct 2ꢀa was
spectroscopically indistinguishable from a chemically synthesised
sample.20,21
2
squares refinement on F0 , anisotropic displacement parameters
for non-hydrogen atoms; all hydrogen atoms located in a difference
Fourier synthesis but included at positions calculated from the
geometry of the molecules using the riding model, with isotropic
vibration parameters. R1 = 0.072 for 1828 data with F0 > 4r(F0),
Biotransformation of methyl 2-(trifluoromethyl)benzoate 1d with
254 parameters, xR2 = 0.233 (all data), GoF = 1.14, Dqmin,max
=
−3
˚
P. ribis
−0.67/0.09 e A . CCDC reference number 668670.†
Methyl 2-(trifluoromethyl)benzoate 1d (0.5 mmol) was added to
each of six flasks each containing a growing culture of P. ribis
(10 mL, day 14); the culture media were left for 8 days at ambient
temperature. The combined culture broth was then centrifuged
and the aqueous phase extracted with Et2O (3 × 50 cm3). The
Et2O extract was dried (Na2SO4) and carefully concentrated to
yield the crude product as a yellow oil. 1H-NMR analysis of
the crude product showed evidence of the presence of natural
metabolites 2ꢀa, 5a/6a and 7a, derived from methyl benzoate
1a, and also metabolites 2dS/2dR, 5d/6d and 7d, derived from
methyl 2-(trifluoromethyl)benzoate 1d. The arene oxide metabolite
2dS/2dR was found to be one of the major metabolites; it was
purified by PLC (20% Et2O in pentane).
Base-catalysed hydrolysis of arene oxide 2dS/2dR to yield
( )-trans-6-(trifluoromethyl)-3,5-cyclohexadien-1,2-diol-1-
carboxylic acid methyl ester 9
t
A mixture of arene oxide 2dS/2dR (16 mg, 0.073 mmol), BuOH
(2 mL) and pH 8.1 buffer solution (4 mL) was heated under reflux
until the starting material had been consumed (6 h). The solvents
were removed under reduced pressure, the concentrate diluted with
water (5 mL), the mixture extracted (3 × 5 mL) with EtOAc and
the extract dried (Na2SO4). The residue, left after the removal
of solvent, was purified by PLC (50% EtOAc in hexane) to give
trans-diol 9 as colourless oil (6 mg, 35%); Rf 0.48 (50% EtOAc
in hexane); [a]D 0.0 (c 0.5, CHCl3); dH (500 MHz, CDCl3) 3.89
(3 H, s, CO2Me), 5.09 (1 H, br s, 2-H), 6.05 (1 H, m, 4-H), 6.18 (1
H, d, J3,4 10.4, 3-H), 6.67 (1 H, d, J5,4 5.5, 5-H). m/z (EI) 238 (M+,
48%), 179 (53), 161 (100), 150 (28), 142 (48), 133 (75), 102 (47).
2-(Trifluoromethyl)-7-oxabicyclo[4.1.0]hepta-2,4-diene-1-carboxy-
lic acid methyl ester 2dS/2dR
Epoxidation of arene oxide 2dS/2dR to yield ( )-anti-5-(trifluoro-
methyl)-3,8-dioxatricyclo[5.1.0.02,4]oct-5-ene-4-carboxylic acid
methyl ester 10anti/10syn
Colourless oil (73 mg, 11%); Rf 0.20 (20% Et2O in pentane); [a]D
−14 (c 0.35, CHCl3); (Found: M+, 220.0351. C9H7O3F3 requires
220.0347); dH (500 MHz, CDCl3) 3.86 (3 H, s, CO2Me), 4.23 (1 H,
m, J6,5 3.0, J6,4 1.8, 6-H), 6.59 (1 H, m, J4,6 1.8, 4-H), 6.70 (1 H,
m, J5,6 3.0, J5,4 6.4, 5-H), 6.97 (1 H, m, J3,4 6.4, J3,5 1.3, 3-H); dC
(125 MHz, CDCl3) 51.67, 59.59, 126.59, 128.82, 128.90, 129.42,
166.58; m/z (EI) 220 (M+, 100%), 189 (18), 173 (16), 161 (19), 145
(18), 133 (49), 113 (39), 83 (23), 59 (40).
A solution of arene oxide 2dS/2dR (17 mg, 0.077 mmol) in
acetone (1 mL) maintained at 0 ◦C was treated with an excess
of dimethyldioxirane solution in acetone (10 mL, 0.03 M). The
reaction mixture was kept at 0 ◦C until almost all the starting
compound had reacted (∼ 7 days). The solvents were carefully
1258 | Org. Biomol. Chem., 2008, 6, 1251–1259
This journal is
The Royal Society of Chemistry 2008
©