A one-pot remote allylic hydroxylation and Baeyer-Villiger oxidation of a bicyclo[3.2.0]hept-2-en-6-one by Cunninghamella echinulata NRRL 3655.

Article abstract of DOI:10.1039/b406016d

7-exo-Methyl-7-endo-phenylbicyclo[3.2.0]hept-2-en-6-one 3 undergoes Baeyer-Villiger and allylic oxidation, to yield novel hydroxylactone 8 in good yield by Cunninghamella echinulata NRRL 3655, representing a one step biocatalytic access to a cyclopentanoid scaffold with three chiral centers. Interesting, allylic oxidation occurs with transposition of the double bond.

Full text of DOI:10.1039/b406016d

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A one-pot remote allylic hydroxylation and Baeyer–Villiger
oxidation of a bicyclo[3.2.0]hept-2-en-6-one by
Cunninghamella echinulata NRRL 3655
Ian J. S. Fairlamb,*a Stephanie Grant,a Gideon Grogan,*b David A. Maddrellb
and Josephine C. Nicholsb
a Department of Chemistry, University of York, Heslington, York, UK YO10 5DD.
E-mail: ijsf1@york.ac.uk; Fax: 44 1904 432516; Tel: 44 1904 434091
b York Structural Biology Laboratory, Department of Chemistry, University of York,
Heslington, York, UK YO10 5YW. E-mail: gg12@york.ac.uk
Received 22nd April 2004, Accepted 26th May 2004
First published as an Advance Article on the web 10th June 2004
7-exo-Methyl-7-endo-phenylbicyclo[3.2.0]hept-2-en-6-one
3 undergoes Baeyer–Villiger and allylic oxidation, to yield
novel hydroxylactone 8 in good yield by Cunninghamella
echinulata NRRL 3655, representing a one step biocatalytic
access to a cyclopentanoid scaffold with three chiral centers.
Interesting, allylic oxidation occurs with transposition of
the double bond.
Baeyer–Villiger oxidation and the formation of an allylic
alcohol in the cyclopentene ring have occurred both regio- and
stereo-selectively.
In order to investigate the potential of Cunninghamella
echinulata 3655 for the resolution of the bicyclo[3.2.0] ketones,
resting cells of the fungus were first incubated with three
bicyclo[3.2.0]hept-2-en-6-one derivatives; the unsubstituted
parent compound 1 (Fig. 1); the 7,7-dimethyl derivative 2 and
the 7-endo-phenyl-7-exo-methyl derivative 3, each at a con-
centration of 0.5 g LϪ1. Biotransformations of 1 and 3 were
continued until the residual ketone constituted approximately
50% of the reaction mixture, in an effort to achieve resolution
of the racemic starting materials.
Whole cell preparations of filamentous fungi have been used
widely since the 1950s as biocatalysts often because of superior
regio- and enantioselectivities observed in a range of oxidation
reactions, compared to abiotic chemical methods. In addition
to hydroxylation1 and N-2 and O-demethylation3 reactions,
organisms such as Beauveria bassiana4 and Cunninghamella
echinulata have been used to catalyse preparatively useful
Baeyer–Villiger reactions.5 Cunninghamella echinulata NRRL
3655 has proved most useful in the latter case, particularly
in the transformation of bicyclo[3.2.0]hept-2-en-6-one 1,6
derivatives of which can serve as important synthons in the
preparation of prostanoids7 and pheromones.8 The interest in
this approach lies in the multifunctional potential of the
bicyclic heptanone nucleus, which can, after enantioselective
biological Baeyer–Villiger oxidation and hydrolysis, yield chiral
hydroxy acids and cyclopentanoid intermediates with two
new chiral centers. In the wake of current interest in the one
step preparation of multifunctionalised chiral cyclopentanoid
scaffolds,9 we were interested to use biological Baeyer–Villiger
reactions as a means of resolving racemic mixtures of bicyclo-
[3.2.0]hept-2-en-6-one derivatives such as 1 (Fig. 1). The
resolved ketones would serve as intermediates in our syntheses
of a range of ligands for palladium complexes.10 Cunning-
hamella echinulata NRRL 3655 was the catalyst of choice as
it had been reported to catalyse the apparent resolution of
bicyclo[3.2.0]hept-2-en-6-one to yield the 3-oxa lactone 5
exclusively,6 in contrast to the bacterial Baeyer–Villiger bio-
catalysts, noted for their ability to catalyse the enantiodivergent
oxidative biotransformation of this ketone series to yield
approximately equimolar quantities of optically active 2- and
3-oxa lactones (5 and 6).11 In this paper, in addition to
well-documented reductions and lactone formations of
the bicyclo[3.2.0]hept-2-en-6-one system, we have unearthed a
surprising and high yielding double oxidation of 7-exo-methyl-
7-endo-phenylbicyclo[3.2.0]hept-2-en-6-one 3, wherein both
The contrast in product profiles as determined by GC for
substrates 1, 2 and 3 after 22 h, 48 h and 22 h is illustrated in
Fig. 3. Compound 1 was transformed to two major metabolites
on GC, being approximately equal amounts of the reduction
product 6-endo-hydroxybicyclo[3.2.0]hept-2-ene
4
and
a
mixture of lactone products, not resolved by GC, but which
1H NMR confirmed to be a 1 : 1.2 mixture of both 2-oxa- 5 and
3-oxa- 6 lactones resulting from non-regioselective oxygen
insertion (Fig. 2). These results contrast with those previously
reported for biotransformation of 1 with the same organism
under equivalent conditions, where only the lactone 5 was
obtained.6 The residual ketone 1 was shown to be racemic by
chiral GC analysis. 7,7-Dimethylbicyclo[3.2.0]hept-2-en-6-one
2 proved to be a poor substrate for any transformation, but
Fig. 2 Baeyer–Villiger, allylic oxidation and reduction products from
the biotransformation studies.
Fig. 1 7,7-Disubstituted bicyclo[3.2.0]hept-2-en-6-ones.
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 8 3 1 – 1 8 3 3
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Fig. 4 Possible route to the hydroxylactone 8.
This hypothesis appeared to be supported by the inhibition
of biotransformation activity in the presence of 1 mM 1-amino-
benzatriazole, a cytochrome P450 inhibitor. However, when 7
was isolated, and resubmitted to active fungus under the same
reaction conditions, no biotransformation was observed,
suggesting that 7 is not an intermediate to 8. The active fungus
was then challenged with the 3-oxa lactone derivative 9. As
abiotic methods proved unsuccessful at producing 9 from 3, it
was produced by a biocatalytic process which will be described
elsewhere. Lactone 9 was not transformed to 8 using a fungal
culture whose capacity for the transformation of 3 to 8 was
monitored in parallel as a control. It is difficult at this stage to
suggest whether the lactone 9 is produced first, thence hydroxyl-
ated, or allylic hydroxylation to give 15, followed by Baeyer–
Villiger oxidation occurs, as there were no other observable
abundant intermediates by GC analysis during accumulation of
product 8. Subjecting 7-phenyl-7-chlorobicyclo[3.2.0]hept-2-
en-6-one 10 to the assay produced a number of products which
were not separable by chromatography.
7,7-Dichlorobicyclo[3.2.0]hept-2-en-6-one 12 was rapidly
converted to the racemic endo-alcohol 13, with no oxidation
products observed. It is perhaps unsurprising that 7,7-di-
phenylbicyclo[3.2.0]hept-2-en-6-one 14 was not biotransformed
under these conditions. Notwithstanding the poor solubility of
this compound under the reaction conditions, it may prove
sterically demanding for enzymes catalysing either reduction,
Baeyer–Villiger oxidation or indeed allylic hydroxylation.
In conclusion, a biocatalytic method for the one step double
oxidation of 7-phenylbicyclo[3.2.0]hept-2-en-6-one derivatives
has been described, leading to hydroxylactones which may
furnish potentially useful chiral cyclopentanone scaffolds
for further synthetic elaboration. In addition to this, the
investigation has highlighted a hitherto undescribed bio-
transformation of the bicyclo[3.2.0]hept-2-en-6-one nucleus,
which may involve previously undescribed enzyme activities
of potential synthetic utility. In due course the use of 8
as an intemediate to various novel chiral ligands will be
reported.
Fig. 3 Contrasting product profiles of biotransformation of bicyclo-
[3.2.0]hept-2-en-6-one derivatives by Cunninghamella echinulata NRRL
3655: a) GC chromatogram of biotransformation mixture of 1 after 22
h; b) GC chromatogram of biotransformation mixture of 2 after 48 h;
c) GC chromatogram of biotransformation mixture of 3 after 22 h.
after 48 h incubation, a GC trace showed four products,
but of insufficient abundance for characterisation by NMR
spectroscopy.
When C. echinulata was challenged with 7-endo-phenyl-7-
exo-methylbicyclo[3.2.0]hept-2-en-6-one 3, one minor and one
major product were observed after 22 h biotransformation.
NMR spectoscopy confirmed the formation of epoxide 7 (we
further prepared this compound independently by treatment of
3 with mCPBA in CH2Cl2, see experimental) and the hydroxyl-
actone 8 (Fig. 2), which was isolated in 41% yield. The latter
product represents a hitherto undescribed microbial transform-
ation of the bicyclo[3.2.0]hept-2-en-6-one nucleus and would
allow access to a highly elaborated cyclopentanoid ring con-
taining three chiral centres. Hydroxylation in the 2-position
yields a hydroxylactone of a substitution pattern previously
shown to be of use in the synthesis of, for example, cyclo-
sarkomycin.6 The residual ketone was obtained in 40% yield,
but chiral GC analysis showed it to be racemic.
The hydroxylactone 8 is evidently the result of both Baeyer–
Villiger oxidation and allylic hydroxylation, accompanied by
transposition of the double bond. We postulated that 8 may
have arisen first from cytochrome P450 dependent epoxidation
of the double bond of 3, followed by base catalysed elimination
on the resulting identified epoxide 7 followed by oxygenation
(Fig. 4).
Experimental
Procedure for biotransformation of 3
Cunninghamella echinulata NRRL 3655 was obtained from
the US Department of Agriculture, Agricultural Research
Service Culture Collection, Peoria, Illinois USA. A 250 mL
flask charged with 60 mL growth medium (corn steep solids, 7.5
g LϪ1; glucose 10 g LϪ1, pH 4.85) was inoculated with 0.5 mL of
a suspension derived from a 5 mm × 5 mm square of the fungus
grown on potato dextrose agar homogenised in 1 mL of sterile
water. After three days growth on an orbital shaker at 28 ЊC,
this culture was used to inoculate a 2 L flask charged with 500
mL growth medium. After a further three days growth at 28 ЊC,
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 8 3 1 – 1 8 3 3
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the mycelium was filtered off and washed with 50 mM phos-
phate buffer pH 8.0. The mycelium was resuspended in 500 mL
buffer and to this was added substrate (250 mg, 1.26 mmol)
dissolved in ethanol (2 mL). The biotransformation was moni-
tored by GC and after 22 h, the mycelium was removed by
filtration and the liquor extracted with diethyl ether (3 × 150
mL). The combined organic phases were dried over anhydrous
magnesium sulfate and the solvent removed to leave a residue,
which was chromatographed over silica to yield, in order of
elution, 3 (40%), 7 (7%) and 8 (41%).
(؊)-6-exo-Hydroxy-4-endo-phenyl-4-exo-methyl-3-oxabicyclo-
[3.3.0]oct-7-en-2-one (8)
20
[α]D = Ϫ12.0 (c 0.02, CH2Cl2).14 νmax (CH2Cl2)/cmϪ1 3591,
2927, 2856, 1772, 1603, 1496, 1006. δH (270 MHz, CDCl3) 7.18
(5H, m, Ph), 5.88 (1H, ddd, 3J8H = 5.6, 3J6H = 3.3, 4J5H = 1.9, 7-
3
3
4
H), 5.81 (1H, ddd, J1H = 5.9, J7H = 5.6, J6H = 2.2, 8-H), 4.87
( )-7-endo-Phenyl-7-exo-methylbicyclo[3.2.0]hept-2-en-6-one
3
4
3
(1H, dd, J7H = 3.3, J8H = 2.2, 6-H), 3.98 (1H, d, J8H = 5.9,
1-H), 3.67 (1H, m, 5-H), 1.68 (3H, s, C4(C-H3)). δC (68 MHz,
CDCl3) 180.30 (4Њ, C-2), 140.71 (4Њ, Ph), 136.91, 135.75, 128.33
(2C), 126.66, 125.80, 76.53 (C-6), 68.71 (4Њ, C-4), 67.22 (C-1),
50.43 (C-5), 26.91 (C-4(CH3)). LRMS (CI) m/z 248 (Mϩ = NH4,
30) 230 (Mϩ, 16), 214 (34), 196 (10), 186 (33), 169 (100), 132
(11), 120 (5). HRMS (CI) calculated for C14H14O3, 230.26588;
Found 230.26594.
(3)12
2-Methylphenyl acetyl chloride (3.01 g, 17.9 mmol) was reacted
with freshly distilled 1,3-cyclopentadiene (2.6 g, 39.3 mmol, 2.2
equiv.) and Et3N (2.7 g, 26.8 mmol, 1.5 equiv.) in hexane (100
mL) at room temperature. After 6 h the mixture was poured
through Celite® and washed with hexane. The hexane was
removed in vacuo to afford a dark brown oil. Purification by
flash chromatography using 15% diethyl ether/hexane gave the
title compound (1.27 g, 36%) as a lime green oil. νmax (neat)/cmϪ1
Acknowledgements
I. J. S. F. and G. G. thank the University of York for funding
through the I. R. P. F. We thank the EPSRC for a studentship
(S.G.) and Stylacats for CASE funding.
3059, 2960, 2922, 2852, 1771 (C᎐O), 1602 (C᎐C). δ (270 MHz,
᎐
᎐
H
CDCl3) 7.15 (5H, m, Ph), 5.55 (1H, dddd, 3J3H = 6.5, 3J1H = 1.8,
4J4Hendo = 1.8, J4Hexo = 1.8, 2-H), 5.47 (1H, dddd, J2H = 6.5,
3J4Hendo = 1.8, 3J4Hexo = 1.8, 4J1H = 1.8, 3-H), 3.99 (1H, ddd, 3J4Hexo
= 8.06, 3J1H = 7.2, 3J4Hendo = 1.1, 5-H), 3.55 (1H, dddd, 3J5H = 7.2,
4
3
4J4Hendo = 3.1, J4Hexo = 1.8, J2H = 1.8, 1-H), 2.63 (1H, ddddd,
4
3
Notes and references
1 For a recent review see L. R. Lehman and J. D. Stewart, Curr. Org.
Chem., 2001, 4, 439–470.
2 A. M. Abel, G. R. Allan, A. J. Carnell and J. A. Davis, Chem.
Commun., 2002, 1762.
3 A. R. S. Ibrahim, A. M. Galal, M. S. Ahmed and G. S. Mossa,
Chem. Pharm. Bull., 2003, 51, 203.
4 G. J. Grogan and H. L. Holland, J. Mol. Catal. B—Enzym., 2000,
9, 1.
5 M. D. Mihovilovic, B. Muller and P. Stanetty, Eur. J. Org. Chem.,
2002, 22, 3711.
6 L. Andrau, J. Lebreton, P. Viazzo, V. Alphand and R. Furstoss,
Tetrahedron Lett., 1997, 38, 825.
2J4Hexo = 17.6, J1H = 3.1, J3H = 1.8, J2H = 1.8, J5H = 1.1, 4-H
4
3
4
3
2
3
3
endo), 2.41 (1H, ddddd, J4Hendo = 17.6, J5H = 8.06, J3H = 1.8,
4J2H = 1.8, J1H = 1.8, 4-H exo), 1.67 (3H, s, C7(C-H3)). δC (68
4
MHz, CDCl3) 211.1 (4Њ, C-6), 140.7 (C-1Ј), 133.2, 128.2 (2C),
127.4 (2C), 126.2, 126.1, 71.5 (4Њ, C-7), 57.2 (C-5), 51.6 (C-1),
34.2 (C-4), 27.1 (C7(CH3)). LRMS (EI) m/z 198 (Mϩ, 15), 183
(Mϩ Ϫ CH3, 12), 170 (Mϩ Ϫ CO, 10), 132 (Ph(CH3)CCO, 45),
104 (Ph(CH3)C, 100), 77 (Ph, 40), 66 (C5H6, 33), 51 (C4H3, 14).
( )-2,3-exo-Epoxy-7-endo-7-exo-methyl-phenylbicyclo[3.2.0]-
hept-6-one (7)13
7 E. J. Corey, N. M. Weishenker, T. K. Schaaf and W. Huber,
J. Am. Chem. Soc., 1969, 91, 5675; C. B. Chapleo, M. A. Finch,
T. V. Lee, S. M. Roberts and R. F. Newton, J. Chem. Soc.,
Perkin Trans. 1, 1980, 2084.
8 J. Lebreton, V. Alphand and R. Furstoss, Tetrahedron, 1997, 53,
145.
9 M. E. B. Smith, M. C. Lloyd, N. Derrien, R. C. Lloyd, S. J. C. Taylor,
D. A. Chaplin, G. Casy and R. McCague, Tetrahedron Lett., 2001,
12, 703.
10 I. J. S. Fairlamb, S. Grant and S. M. Roberts, 2004, unpublished
results.
11 V. Alphand, A. Archelas and R. Furstoss, Tetrahedron Lett., 1989,
30, 3663; R. Gagnon, G. Grogan, M. S. Levitt, S. M. Roberts,
P. W. H. Wan and A. J. Willetts, J. Chem. Soc., Perkin Trans. 1, 1994,
2537.
12 I. J. S. Fairlamb, J. M. Dickinson, R. O’Connor, S. Higson,
L. Grieveson and V. Marin, Bioorg. Med. Chem., 2002, 10, 2641.
13 I. J. S. Fairlamb, Ph. D. Thesis, Manchester Metropolitan
University (UK), 2000.
Independent synthesis of 7. Alkene 3 (0.8 g, 4.04 mmol) in
dry CH2Cl2 (30 mL) was stirred at 0 ЊC. MCPBA (1.09 g,
4.44 mmol) was added in small portions over twenty minutes.
The reaction was left to warm to room temperature and stirred
overnight. The mixture was diluted with diethyl ether (40 mL)
and washed successively with saturated aqueous sodium
hydrogen carbonate solution (3 × 30 mL) and water (2 × 30
mL). The aqueous phases were back extracted with diethyl
ether (2 × 30 mL) and combined with the main organic extract.
The combined organic extracts were dried over anhydrous
MgSO4 and concentrated in vacuo to yield a clear oil. Purifi-
cation by flash chromatography using 30% ethyl acetate/hexane
gave the title compound as a colourless oil (0.80 g, 93%). δH (270
MHz, CDCl3) 7.22 (5H, m, Ph-H), 3.60 (1H, m, 5-H), 3.31 (1H,
m, 3-H), 3.16 (1H, d, 3J3H = 2.2, 2-H), 3.10 (1H, d, 3J5H = 7.3, 1-
3
4
H), 2.15 (2H, dd, J5H = 5.8 and J1H = 1.5, 4-H2), 1.63 (3H, s,
C7(C-H3)). δC (68 MHz, CDCl3) 213.8 (4Њ, C-6), 139.0, 128.7,
127.1, 125.1, 66.4 (4Њ, C-7), 60.9 (C-3), 60.0 (C-2), 59.2 (C-5),
46.8 (C-1), 29.8 (C-4), 26.3 (C7(CH3)). LRMS (EI) m/z 214
(Mϩ, 15), 199 (Mϩ Ϫ CH3, 10), 129 (100), 115 (84), 104
(PhCMe, 72), 77 (Ph, 34), 55 (21).
14 We have so far been unable to determine the optical purity or
absolute configuration of compound 8. We are currently identifying
a general synthetic route to racemic 8, which will help us to
determine this. Furthermore, we hope to derivatise 8 to facilitate
a determination of the absolute configuration. These studies are
ongoing and will be reported in a full paper.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 8 3 1 – 1 8 3 3
1833