Formation of 2-Tropolones
Preparation of spiroepoxides 7a–d: Spiroepoxides
7a–d were prepared by using a procedure adapted
from Adler et al.[13] In order to minimise the dimerisa-
tion of the spiroepoxides, all procedures were carried
out at 48C in flasks wrapped with Al foil. A solution
of sodium periodate (2.41 g, 11 nmol) in water
(50 mL) was added dropwise to a stirred solution of
substituted salicyl alcohol 6a–d (10 mmol) in water
(75 mL). Ethyl acetate (50 mL) was then added to the
resulting yellow solution, and the reaction was stirred
at 48C for 1 h. The organic layer was separated, and
solvent was removed at reduced pressure (without
heating) to give spiroepoxides 7a–d. The rate of self-
dimerisation of each spiroepoxide was assessed by
NMR spectroscopy, in D2O at 258C, with sodium p-tol-
uenesulfonate as internal standard (see the Support-
ing Information).
Cyclohexa-2,4-dien-1-one 6-oxamethylene spiroepoxide
(7a) (prepared in ref. [13]): Yield 33%; dimerisation
1
t
1/2 =10 min; H NMR (400 MHz, CDCl3): d=7.43 (ddd,
J=10.5, 6.0, 1.4 Hz, 1H; H-3), 6.66 (dd, J=10.2, 6.0 Hz,
1H; H-4), 6.27 (dd, J=10.2, 1.4 Hz, 1H; H-5), 6.24 (d,
J=10.5 Hz, 1H; H-2), 3.39, 3.25 ppm (d, J=7.5 Hz, 2ꢁ
1H; CH2O).
Scheme 4. Mechanism for the 1,2-rearrangement of diols 8a–d to 2-tropolones 10a–d, and
comparison with the Criegee 1,2-rearrangement of the extradiol catechol dioxygenase reac-
tion mechanism.
4-Methyl-cyclohexa-2,4-dien-1-one 6-oxamethylene spi-
roepoxide (7b): Yield 40%; dimerisation t1/2 =12 h;
1H NMR (400 MHz, CDCl3): d=6.98 (d, J=10.0 Hz, 1H;
H-3), 6.13 (d, J=10.0 Hz, 1H; H-2), 5.74 (s, 1H; H-5),
that the carbocation intermediate in this mechanism would be
3.22, 3.05 (d, J=8.2 Hz, 2ꢁ1H; CH2O), 2.03 ppm (s, 3H; CH3);
13C NMR (100 MHz, CDCl3): d=195.3, 146.1, 136.0, 133.2, 125.8,
57.8, 56.5, 21.2 ppm; MS (ES +ve ion) m/z: 137 [M+H]+, 159
[M+Na]+; HRMS obsd for C8H8O2Na: 159.0410, calcd 159.0417.
stabilised by a 5-methyl substituent, but destabilised by a 5-
chloro substituent. The observation of alternative rearrange-
ment products in the case of the 5-methyl analogue could be
explained by the formation of a stable tertiary carbocation in
this case, leading to other possible rearrangements; however,
the efficient processing of the 5-chloro analogue would argue
against such a mechanism.
4-Chloro-cyclohexa-2,4-dien-1-one 6-oxamethylene spiroepoxide (7c):
Yield 50%; dimerisation t1/2 =40 min; H NMR (400 MHz, CDCl3): d=
7.27 (d, J=10.0 Hz, 1H; H-3), 6.31 (s, 1H; H-5), 6.29 (d, J=10.0 Hz,
1H; H-2), 3.38, 3.26 ppm (d, J=7.5 Hz, 2ꢁ1H; CH2O); MS (ES Àve
ion) m/z: 155 [MÀH]À; HRMS obsd for C7H4ClO2: 154.9911, calcd
154.9905.
1
To our knowledge, the only other enzymatic ring expansion
that has been demonstrated biochemically is that involved in
cephalosporin biosynthesis, which is also catalysed by a non-
haem-iron-dependent oxygenase, deacetoxycephalosporin C
synthetase.[19] This unusual transformation thus provides a ra-
tionalisation for the biosynthesis of tropolone natural products,
and offers another example of the novel chemistry that can be
catalysed by non-haem-iron-dependent enzymes in biology.
2-Oxo-1,2-dihydronaphthalene 1-oxamethylene spiroepoxide (7d):
1
Yield 68%; only monomer observed; H NMR (400 MHz, CDCl3): d=
7.53 (d, J=10.0 Hz, 1H; H-4), 7.6–7.3 (m, 3H), 7.19 (d, J=7.0 Hz,
1H), 6.27 (d, J=10.0 Hz, 1H; H-3), 3.32, 3.05 ppm (d, J=8.2 Hz, 2ꢁ
1H; CH2O); 13C NMR (100 MHz, CDCl3): d=197.3, 145.9, 133.6,
131.9, 130.5, 129.7, 128.7, 125.5, 123.6, 72.4, 66.5 ppm; MS (ES +ve
ion) m/z: 173 [M+H]+, 195 [M+Na]+; HRMS obsd for C11H8O2Na:
195.0434, calcd 195.0417.
Conversion of spiroepoxides to substituted tropolones
Experimental Section
Using dioxygenase MhpB: Spiroepoxide 7a, 7b or 7d (1 mmol,
10 mm) was dissolved in potassium phosphate buffer (50 mm,
100 mL, pH 8.0), to which was then added Aspergillus niger epoxide
hydrolase (5 mg), and the mixture was stirred at 48C for 30 min. To
each incubation was added MhpB enzyme (400 mL, 30 units),
which had been pre-activated for 1 min at 08C by the addition of
ammonium iron(II) sulfate (5 mm) and sodium ascorbate (5 mm).[20]
Aliquots (5 mL) were removed after 10, 60, 90, 180 min and 48 h,
and the reaction was stopped by adding 100% trichloroacetic acid
(TCA; 300 mL). The samples were centrifuged for 10 min at
13000 rpm, and then analysed by HPLC and GC-MS. From the re-
maining reaction mixture, products were extracted into ethyl ace-
Materials: Substituted salicyl alcohols 6a–f were prepared in 47–
76% yield by reduction of commercially available substituted ben-
zaldehydes by using sodium borohydride in methanol. Aspergillus
niger epoxide hydrolase was purchased from Fluka. Escherichia coli
2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB) was over-
expressed from a recombinant plasmid pIPB containing the mhpB
gene, as previously described,[12] and was purified to near homoge-
neity by hydrophobic interaction chromatography, as previously
described,[20] by using potassium phosphate buffer (50 mm, pH 7.0)
containing 0.07% (v/v) 2-mercaptoethanol as buffer. Other chemi-
cals and biochemicals were purchased from Sigma–Aldrich.
ChemBioChem 2010, 11, 272 – 276
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
275