An enantiodivergent trend in microbial Baeyer-Villiger oxidations of functionalized pentalenones by recombinant whole cells expressing monooxygenases from Acinetobacter and Pseudomonas

Article abstract of DOI:10.1002/ejoc.200300023

We have investigated the microbial Baeyer-Villiger oxidations of pentalenones by recombinant whole cells expressing cyclohexanone monooxygenase from Acinetobacter and cyclopentanone monooxygenase from Pseudomonas. The two enzymes display a distinguishable

Full text of DOI:10.1002/ejoc.200300023

FULL PAPER
An Enantiodivergent Trend in Microbial Baeyer؊Villiger Oxidations of
Functionalized Pentalenones by Recombinant Whole Cells Expressing
Monooxygenases from Acinetobacter and Pseudomonas
Marko D. Mihovilovic,*[a] Bernhard Müller,[a] Alexander Schulze,[a][‡] Peter Stanetty,[a] and
Margaret M. Kayser[b]
Keywords: BaeyerϪVilliger Oxidation / Biocatalysis / Biotransformation / Enantiodivergence / Enantioselective
Synthesis / Monooxygenase
We have investigated the microbial Baeyer−Villiger oxida- A significant influence of the conformation of the fused-ring
tions of pentalenones by recombinant whole cells expressing
cyclohexanone monooxygenase from Acinetobacter and
cyclopentanone monooxygenase from Pseudomonas. The
two enzymes display a distinguishable enantiodivergent
trend in the biotransformation of some prochiral substrates.
system and the polarity of the substituents on the conversion
and enantioselectivity of the microbial oxidations was ob-
served and is discussed.
( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2003)
Introduction
rities obtained by microbial BaeyerϪVilliger oxidations
have yet to be attained by artificial catalytic entities.
Since the discovery of an oxidative ring expansion from
ketones to lactones by Adolf Baeyer and Victor Villiger in
1899,[1] many aspects of the reaction, which later was
named after these pioneers in chemistry, have received con-
siderable attention. Substantial progress has been made in
understanding the mechanism, predicting the migratory
preference, and applying this conversion to preparative
chemistry.[2,3] The BaeyerϪVilliger oxidation is of consider-
able value to synthetic chemists because it is a powerful
method for breaking carbonϪcarbon bonds with concomi-
tant oxygen atom insertion.
Chiral BaeyerϪVilliger oxidation of cyclic ketones allows
rapid access to asymmetric lactones, which are valuable in-
termediates in organic chemistry and frequently encoun-
tered precursors in enantioselective synthesis. In recent ye-
ars, the use of organometallic catalysts has allowed continu-
ous improvements in this reaction and represents a promis-
ing approach for its future process development.[4]
Complementing this strategy, biocatalysis offers a ‘‘green
chemistry’’ alternative for this transformation. Optical pu-
Nature has developed a variety of flavin-dependent
BaeyerϪVilliger monooxygenases (BVMOs) that catalyze
this biotransformation, which plays a key role in degrad-
ative pathways of microorganisms.[5Ϫ10] While the metabolic
action of such enzymes in the cell is restricted to a single
compound, these biocatalytic entities often display a re-
markably broad acceptance for nonnatural substrates. This
feature, together with high enantioselectivity, are prerequi-
sites for a powerful catalytic system for chiral synthesis.
Artificial organometallic catalysts can allow access, in a
straight forward manner, to both enantiomeric products
through chiral catalysis merely by inverting the chirality of
the inducing ligands. In contrast, this aspect represents one
of the key challenges for biotransformations. Enzymes con-
sist of hundreds of -amino acids that are responsible for
strong chiral induction. With the present state-of-the-art,
inverting the enantioselectivity of an enzyme, with a well-
defined substrate profile, is not feasible by the act of chang-
ing the stereochemistry of its building blocks. Currently, the
only options to access the enantiomeric product in bio-
transformations are directed evolution and screening for an
alternative enzyme. As a consequence, such an alternative
enzyme does not necessarily possess matching substrate ac-
ceptance profiles. Nevertheless, the identification and sub-
sequent characterization of enantiodivergent enzymes with
largely overlapping substrate profiles is a crucial aspect for
the successful application of biocatalysts in synthetic chem-
istry.
[a]
Vienna University of Technology Ϫ Institute of Applied
Synthetic Chemistry,
Getreidemarkt 9/163-OC, 1060 Vienna, Austria
Fax: (internat.) ϩ 43-1/58801-15499
E-mail: mmihovil@pop.tuwien.ac.at
[b]
University of New Brunswick, Department of Physical Sciences,
Saint John, N.B., Canada
[‡]
Current address: University of Leipzig, Institute of Organic
Chemistry,
Leipzig, Germany
Eur. J. Org. Chem. 2003, 2243Ϫ2249
DOI: 10.1002/ejoc.200300023
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M. D. Mihovilovic, B. Müller, A. Schulze, P. Stannetty, M. M. Kayser
FULL PAPER
Recently, we started to investigate the oxidation of func- ism, safe and simple to cultivate. By performing whole-cell
tionalized pentalenones using a recombinant overexpres- biotransformations the laborious process of enzyme iso-
sion system for cyclohexanone monooxygenase (CHMO)[11] lation, which is often complicated by limited protein sta-
from Acinetobacter sp. NCIMB 9871 (E.C. 1.14.13.22).[12] bility, can be avoided. In addition, all cofactors required by
This enzyme represents the best studied BaeyerϪVilligerase the biocatalyst (NADPH in the case of the above BVMOs)
to date and transforms more than 100 nonnatural sub- are recycled by the living cells. Hence, the construction of
strates by both kinetic resolution and desymmetrization artificial regeneration systems[29Ϫ31] is not required and an
with good enantioselectivity.[13] With the recent construc- easy-to-use methodology can be offered to preparative
tion of a whole-cell overexpression system for cyclopenta- chemists who lack special expertise in microbiology.
none monooxygenase (CPMO)[14] from Pseudomonas sp.
NCIMB 9872 (EC 1.14.13.16),[15] we became interested in
Results and Discussion
expanding this profiling program. CPMO had received con-
siderably less attention in biocatalytic BaeyerϪVilliger oxi-
dations, probably because its biocatalytic behavior was as-
sumed to be similar to CHMO.[16,17] In a previous com-
parative study with cells expressing CHMO and CPMO by
one of us (MMK), a matching trend in enantioselectivity
was found for the resolution of 2-substituted cyclopenta-
nones with similar optical purity.[18] To our surprise, re-
cently we observed an enantiodivergent trend for CHMO
and CPMO in the biotransformation of some mesomeric
perhydroindenones, key intermediates for the synthesis of
some indole alkaloids.[19]
These findings prompted us to investigate this substrate
class further and to study the effects of conformational and
electronic aspects on fused-ring systems. Our approach, de-
signed to provide simple catalytic entities for enantioselec-
tive BaeyerϪVilliger oxidations, utilizes recombinant whole
cells that were engineered to produce the required enzyme
in substantial quantities. Several such systems for CHMO
based on Saccharomyces cerevisiae[20Ϫ22] and Escherichia
coli[23Ϫ28] host organisms have been developed within the
We considered ketones 1 bearing various substituents in
the 5-position as interesting probes to investigate the effects
of conformation and polarity of functional groups on the
microbial BaeyerϪVilliger oxidation (Scheme 1). Moreover,
enzymatic asymmetrization of the prochiral substrates gen-
erates optically active products with a theoretical yield of
100%. In contrast, the classical kinetic resolution of racemic
samples leads only to a maximum 50% yield of enantiopure
product.[32] Lactones of type 2 have been used previously in
various natural product syntheses and represent interesting
chiral intermediates.[33Ϫ36]
Scheme 1. Biotransformation of functionalized fused systems with
CHMO- and CPMO-expressing recombinant cells
For the investigation of conformational effects, we were
past few years and are, often in contrast to the native organ- interested in both endo (1a/c) and exo (1b/d) substitution
Scheme 2. Synthetic route to methoxy-substituted ketones 1a/b
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Microbial BaeyerϪVilliger Oxidations of Functionalized Pentalenones
FULL PAPER
patterns at the sp3-hybridized C-5 carbon atom, together
Racemic reference samples of compounds 2aϪd were
with an sp2-hybridized C-5 center (1e). Methoxy and chloro prepared by abiotic BaeyerϪVilliger oxidation with
substituents in the 5-position served as representative func- mCPBA in dry methylene chloride. Microbial transform-
tional groups to investigate the aspect of polarity on the ation was performed in shake-flask cultures in rich media.
biotransformation.
Protein production in the two expression systems was under
The synthetic strategy towards these substrates followed the control of a T7 promoter[46] and was triggered by ad-
a straightforward route: Compound 3, which is readily dition of isopropyl β--thiogalactopyranoside (IPTG).
available by a Weiss-condensation[37] and subsequent selec- Equimolar amounts of β-cyclodextrin were supplemented
tive ketalization,[38] was reduced diastereoselectively to prior to addition of the substrate to enhance solubility and
endo-alcohol 4a (Scheme 2). We obtained superior selec- membrane penetration and decrease its toxicity toward the
tivity by using sodium borohydride[39,40] instead of LAH.[41] living cells. Cyclic sugars of this type[47] are known to exert
Methylation yielded ketal 6a and subsequent deprotection this effect, and we observed improved conversion in most
gave endo-methoxy substrate 1a in good yield as a represen- cases.
tative polar substrate.
Table 1 summarizes the results of the biotransformations
Alcohol 4a also served as a key intermediate to access of substrates 1aϪe with recombinant E. coli cells expressing
the exo series: Inversion of the hydroxy group was per- CHMO and CPMO. All substrates were converted into the
formed by a Mitsunobu[42] protocol with nitrobenzoic acid corresponding lactones with excellent chemoselectivity.
as nucleophile, via the corresponding exo-ester 5. Sub- Starting from prochiral ketones, two (2e) or three (2aϪd)
sequent basic hydrolysis of the crude intermediate afforded new chiral centers were introduced in one biocatalytic step.
exo-alcohol 4b. Methylation and deprotection was per- CHMO and CPMO displayed a trend to enantiodivergent
formed analogously to the procedure in the endo series to conversion for several substrates.
give exo-methoxy substrate 1b in excellent overall yield.
Some general trends for the two expression systems are
This sequence is superior to previous inversion strategies deduced from the data on biotransformations with the
that proceeded through tosylation to access compound model substrates:
4b.[41]
In the case of the CHMO-producing strain, the methoxy-
Synthesis of the chloro-substituted pentalenones as less- substituted substrates with higher polarity are poorly ac-
polar substrates utilized the protected alcohols 4a/b cepted by CHMO and complete conversions were not ob-
(Scheme 3). Halogenation through a Mitsunobu protocol served for substrates 1a and 1b. The catalytic activity of
using ZnCl2 as the halogen donor[43] gave a clean inversion recombinant cells usually dropped significantly after reach-
of the desired stereocenter to yield the desired products 7a/ ing the stationary growth phase (24Ϫ48 h). This effect was
b. This reaction was accompanied, however, by formation recently described by Stewart and co-workers for the
of substantial amounts of elimination product 8. This unde- CHMO-producing strain.[48] We attribute the poor sub-
sired side reaction was reduced to a yield of approx. 10% strate acceptance to a feature of the active site of CHMO,
of 8 by changing the halogenation protocol to the use of since the related E. coli host strain producing CPMO gave
triphenylphosphine and NCS.[44] By this method, endo-7a excellent conversion for these substrates. At the current
and exo-7b ketals were obtained in 52 and 50% yields, state of research, we give two possible explanations for this
respectively. Subsequent acidic deprotection conditions af- observation: (i) the steric limitations of the active site are
forded substrates 1c and 1d.
reached, and/or (ii) the active site possesses a polar group
in the region of the C-5 substituent, imposing a repulsive
electrostatic interaction. While the less-polar chloro-sub-
strates 1c and 1d were readily accepted by both CHMO and
CPMO to give excellent conversions of the corresponding
lactones 2c and 2d, olefin 1e also showed only sluggish con-
version with CHMO-expressing cells.
The stereochemistry at the 5-position in substrate ketones
1aϪe substantially influenced the selectivity of the CHMO-
producing strain. Generally, endo compounds were con-
verted into chiral lactones with only low-to-moderate en-
antiomeric excess. For CHMO, the combination of polar
substituent and endo substitution pattern is especially unfa-
vorable, leading to almost-racemic product (Ϫ)-(S)-2a (9%
ee). The assignment of absolute configuration is based on
our previous results for perhydroindenones.[19]
Scheme 3. Synthesis of chloro ketones 1c/d
Changing to exo ketones, however, led to a significant
improvement in the optical purity of products. The nature
Synthesis of the exocyclic olefin 1e utilized precursor 3 of the functional group in the 5-position of the ketone
and proceeded through a sequence of a Wittig reaction fol- seems to have only a minor effect on the enantioselectivity
lowed by deprotection according to the literature.[38,45]
of the biotransformation. (S)-Methoxy compound 2b was
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M. D. Mihovilovic, B. Müller, A. Schulze, P. Stannetty, M. M. Kayser
Table 1. Biotransformation of functionalized fused substrates with CHMO- and CPMO-expressing recombinant cells
FULL PAPER
Substrate
R
Product
Config.
Strain
Yield[a]
ee[b]
[α]2D0
1a
1a
1b
1b
1c
1c
1d
1d
1e
1e
MeO
MeO
MeO
MeO
Cl
Cl
Cl
Cl
ϭCH2
ϭCH2
2a
2a
2b
2b
2c
2c
2d
2d
2e
2e
endo
endo
exo
CHMO
CPMO
CHMO
CPMO
CHMO
CPMO
CHMO
CPMO
CHMO
CPMO
24% (71%)
81%
40% (81%)
75%
75% (98%)
79%
78% (97%)
92%
9%
Ϫ3.0 (c ϭ 0.16, EtOAc)
ϩ13.2 (c ϭ 0.50, EtOAc)
Ϫ36.2 (c ϭ 1.0, CHCl3)
Ϫ4.3 (c ϭ 1.0, CHCl3)
Ϫ23.7 (c ϭ 1.0, CHCl3)
Ϫ17.2 (c ϭ 1.0, CHCl3)
Ϫ38.9 (c ϭ 0.5, CHCl3)
ϩ33.2 (c ϭ 0.5, CHCl3)
Ϫ3.2 (c ϭ 0.3, EtOAc)
ϩ2.2 (c ϭ 1.0, CHCl3)
34%
96%
11%
80%
60%
Ͼ99%
Ͼ99%
61%
41%
exo
endo
endo
exo
exo
n.a.
n.a.
43% (88%)
85%
[a] Isolated yield after chromatographic purification; yield in parenthesis is based on consumed starting material. [b] Value of ee determined
by chiral-phase gas chromatography; racemic reference material was prepared by oxidation of ketones 1aϪd with mCPBA.
obtained in good enantiomeric excess for CHMO-express- optical purity and with matching sign of optical rotation as
ing cells. Again, the endo-chloro ketone 1c was converted was obtained for the CHMO product, both 1d and 1e were
with moderate stereoselectivity (note, that the chloro sub- converted into antipodal lactones by CPMO and CHMO.
stituent requires a change in priority of numbering, which The exo-chloro compound (ϩ)-2d was obtained in excellent
leads to a reversal of the R/S assignment; in all cases, the optical purity as determined by chiral-phase GC. This sub-
sense of chirality for CHMO products was the same) into strate (1d) is the first example of an efficient enantiodiverg-
(R)-lactone 2c by CHMO, while the best results were ob- ent BaeyerϪVilliger oxidation by CHMO and CPMO.
tained for exo substrate 1d, where both strains gave excel-
lent chemical yields together with Ͼ99% optical purity .
When comparing the conversions and yields of CHMO
and CPMO biotransformations, it is noteworthy that
The sp2-hybridized compound 1e gives enantioselectivi- CPMO seems to be rather tolerant toward steric demands
ties between those of the endo and exo substrates. Biooxida- by the substrate.
tions with CHMO-producing cells did not proceed to com-
pletion, which indicates a limiting effect imposed by the ac- zymes involved in camphor degradative pathways from
tive site. Pseudomonas putida NCIMB 10007. On some fused sub-
Enantiodivergent behavior has been observed for two en-
Summarizing the results of the CHMO biotransform- strate compounds, 2-oxo-∆3-4,5,5-trimethylcyclopentylace-
ations, a ‘‘stretched’’ geometry (exo) for bicycloketones of tyl coenzyme A monooxygenase (MO2) and 2,5-diketocam-
type 1 is a prerequisite for high optical purity of product phane monooxygenase (2,5-DKCMO) yielded antipodal
lactones 2. The enzyme displays a decreased enantioselec- enantiomeric products when used in an isolated form.[50]
tivity for the oxidation of substrates adopting a sterically The application of this process in a native whole-cell system
more-demanding ‘‘angled’’ configuration (endo). The pres- was compromised by overlapping substrate acceptance.[51]
ence of less-polar functional groups is favorable for both With the overexpression systems available, we have been
high conversion and stereoselectivity of the biocatalyst. Re- able to study microbial biotransformations of enantiocom-
cently, we have observed a strong influence of confor- plementary BVMOs from two different natural sources for
mational aspects on the recognition and induction of chiral- the first time.
ity by CHMO.[49] The data presented in this study further
support this theory.
Biotransformations with CPMO-producing cells do not
display such a significant trend that is governed by confor-
mational aspects. In some cases, however, the enzyme pro-
Conclusions
Expanding previous substrate-profiling studies, we disco-
duces lactones 2 that are antipodal as compared to those vered enantiodivergent BaeyerϪVilliger oxidation of func-
from the CHMO-mediated biotransformations. We have tionalized pentalenones by CHMO and CPMO in some
observed this behavior of CPMO recently for the cases. We identified these substrates as valuable probes to
BaeyerϪVilliger oxidation of perhydroindenones.[19]
investigate the effects of conformational and polar interac-
Polar interactions within the active site seem to play a tions in determining the yields and optical purities of the
relevant role in the biooxidation of substrates 1, since both product lactones. Our results complement recent findings
methoxy-substituted ketones 1a and 1b gave only poor en- of an enantiodivergent oxidation by CPMO and CHMO in
antioselectivities for the production of lactones 2. In the the 4-substituted cyclohexanone series.[15]
case of the endo compound 2a, we have observed the forma-
CHMO displayed a significant trend for the enantioselec-
tion of the enantiomeric lactone (R) when compared to tive conversion of ketones of type 1, governed predomi-
CHMO, while exo-1b gave almost racemic product with a nantly by conformational aspects, with beneficial effects ex-
slight preference for the (S)-enantiomer. While the less-po- hibited by exo geometry and low substrate polarity. Predic-
lar endo substrate 1c gave rise to (Ϫ)-lactone 2c in moderate tions of the optical purities obtained by CPMO-mediated
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Microbial BaeyerϪVilliger Oxidations of Functionalized Pentalenones
FULL PAPER
solution of potassium hydroxide (2.0 g) in water (15 mL). The mix-
ture was kept at 40 °C for 15 h, then water was added and the
mixture was extracted with CH2Cl2. The combined organic layers
were dried over anhydrous potassium carbonate and concentrated.
The residue was submitted to column chromatography (silica gel;
light petroleum/EtOAc, 10:1) to afford 4b[39] (1.4 g, quant., 77%
overall from 4a) as colorless crystals. M.p. 78Ϫ80 °C. 1H NMR
(200 MHz, CDCl3): δ ϭ 0.97 (s, 6 H), 1.47 (br. s, 1 H), 1.51Ϫ1.93
(m, 6 H), 2.15Ϫ2.30 (m, 2 H), 2.60Ϫ2.80 (m, 2 H), 3.45 (s, 2 H),
3.48 (s, 2 H), 4.36Ϫ4.47 (m, 1 H) ppm. 13C NMR (50 MHz,
CDCl3): δ ϭ 22.5 (q), 30.1 (s), 38.0 (d), 40.1 (t), 42.2 (t), 71.8 (t),
72.3 (t), 74.8 (d), 110.0 (s) ppm.
biooxidation are complicated by obvious additional interac-
tions. Additional substrate profiling is required to draw
further conclusions.
Considering the absolute enantiodivergence of CHMO
and CPMO for the formation of lactones 2d, the investi-
gation of the potential of this pair of ‘‘antipodal’’ biocata-
lysts is an important contribution for the further prolifer-
ation of enzyme-mediated BaeyerϪVilliger oxidations,
which is currently under way in our laboratory.
Experimental Section
Methylation of Alcohols 4a/b
General: Unless otherwise noted, chemicals and microbial-growth
media were purchased from commercial suppliers and used without
further purification. All solvents were distilled prior to use. Flash
column chromatography was performed on silica gel 60 from
Merck (40Ϫ63 µm). Kugelrohr distillation was carried out using a
Büchi GKR-51 apparatus. Melting points were determined using a
Kofler-type Leica Galen III micro-hot-stage microscope and are
uncorrected. Elemental analyses were carried out in the Microana-
lytical Laboratory, University of Vienna. Chiral-phase GC was per-
formed on a ThermoFinnigan Trace-GC and an HP 6890 Series
chromatograph using a BGB 175 cross-linked column (30 m ϫ
0.25 mm ID, 0.25 µm film). Specific rotation was determined with a
PerkinϪElmer Polarimeter 241. NMR spectra were recorded from
CDCl3 solutions with a Bruker AC 200 (200 MHz) or Bruker Av-
ance UltraShield 400 (400 MHz) spectrometer and chemical shifts
are reported in ppm using Me4Si as internal standard.
Oil-free sodium hydride (4 equiv.; washed with light petroleum) was
suspended in dry THF under nitrogen and treated with a 5% solu-
tion of alcohol 4a/b (1 equiv.) in dry THF. After heating under
reflux for 2 h the mixture was cooled to room temp. and dimeth-
ylsulfate (1 equiv.) was added. The mixture was heated under reflux
for 48 h and then quenched with satd. NH4Cl solution. The aque-
ous layer was extracted with diethyl ether (3 ϫ 50 mL) and the
combined organic layers were washed with brine. After drying over
sodium sulfate and evaporation, crude 6a/b was isolated and was
used without further purification.
(3Јaα,5Јβ,6Јaα)-Hexahydro-5Ј-methoxy-5,5-dimethylspiro[1,3-
dioxane-2,2Ј(1ЈH)-pentalene] (6a): Alcohol 4a (0.950 g, 4.46 mmol)
was converted according to the above procedure to give 6a (0.900 g,
1
90%) as a colorless oil. H NMR (200 MHz, CDCl3): δ ϭ 0.97 (s,
6 H), 1.47Ϫ1.80 (m, 4 H), 1.95Ϫ2.10 (m, 2 H), 2.22Ϫ2.36 (m, 2
H), 2.37Ϫ2.53 (m, 2 H), 3.30 (s, 3 H), 3.46, 3.49 (2s, 4 H),
3.68Ϫ3.84 (m, 1 H) ppm. 13C NMR (50 MHz, CDCl3): δ ϭ 22.5
(q), 30.0 (s), 37.8 (d), 38.1 (t), 40.3 (t), 56.7 (q), 71.5 (t), 72.5 (t),
84.3 (d), 110.0 (s) ppm.
(3Јaα,5Јβ,6Јaα)-Hexahydro-5,5-dimethylspiro[1,3-dioxane-2,2Ј(1ЈH)-
pentalen]-5Ј-ol (4a): Ketone 3 (5.19 g, 23.1 mmol) was dissolved in
dry methanol (80 mL) and treated portionwise with NaBH4 (1.73 g,
45.8 mmol, 2.0 equiv.) between Ϫ2 and 0 °C. The mixture was
stirred for 4 h, and then 0.1  NaOH (100 mL) was added while
keeping the temperature at 0Ϫ10 °C. The aqueous layer was ex-
tracted with diethyl ether and then the combined organic layers
were dried over sodium sulfate and concentrated to give 4a[39,40] as
colorless crystals (4.85 g, 93%). M.p. 73Ϫ74 °C. 1H NMR
(200 MHz, CDCl3): δ ϭ 0.97 (s, 6 H), 1.45Ϫ1.60 (m, 2 H),
1.85Ϫ1.98 (m, 2 H), 2.03Ϫ2.35 (m, 5 H), 2.45Ϫ2.65 (m, 2 H), 3.48
(s, 2 H), 3.52 (s, 2 H), 4.16Ϫ4.30 (m, 1 H) ppm. 13C NMR
(50 MHz, CDCl3): δ ϭ 22.5 (q), 30.0 (s), 38.6 (d), 40.8 (t), 42.5 (t),
71.9 (t), 72.2 (t), 74.7 (d), 110.5 (s) ppm.
(3Јaα,5Јα,6Јaα)-Hexahydro-5Ј-methoxy-5,5-dimethylspiro[1,3-
dioxane-2,2Ј(1ЈH)-pentalene] (6b): Alcohol 4b (1.20 g, 5.3 mmol)
was converted according to the above procedure to give 6b (1.26 g,
1
98%), as a colorless oil. H NMR (200 MHz, CDCl3): δ ϭ 0.96 (s,
6 H), 1.50Ϫ1.65 (m, 4 H), 1.78Ϫ1.95 (m, 2 H), 2.13Ϫ2.27 (m, 2
H), 2.50Ϫ2.70 (m, 2 H), 3.25 (s, 3 H), 3.43 (s, 2 H), 3.47 (s, 2 H),
3.68Ϫ3.95 (m, 1 H) ppm. 13C NMR (50 MHz, CDCl3): δ ϭ 22.5
(q), 30.0 (s), 37.8 (d), 38.5 (t), 40.2 (t), 56.3 (q), 71.7 (t), 72.3 (t),
83.3 (d), 109.9 (s) ppm.
Chlorination with PPh3/NCS
(3Јaα,5Јα,6Јaα)-Hexahydro-5,5-dimethylspiro[1,3-dioxane-2,2Ј-
(1ЈH)-pentalen]-5Ј-yl 4-Nitro-benzoate (5): A solution of alcohol 4a
(2.20 g, 9.72 mmol) and triphenylphosphane (2.55 g, 9.72 mmol) in
dry diethyl ether (15 mL) was added dropwise at room temp. to
a solution of DEAD (1.69 g, 9.72 mmol) and p-nitrobenzoic acid
(1.63 g, 9.72 mmol). The resulting yellow mixture was stirred at 20
°C for 14 h. After evaporation of the solvent, the residue was sub-
jected to flash chromatography (silica gel; light petroleum/EtOAc,
10:1) to give 5 (13.30 g, 77%) as colorless crystals. M.p. 160Ϫ162
°C. 1H NMR (200 MHz, CDCl3): δ ϭ 0.99 (s, 6 H), 1.61Ϫ1.93 (m,
4 H), 2.06Ϫ2.32 (m, 4 H), 2.71Ϫ2.90 (m, 2 H), 3.50 (s, 4 H),
5.50Ϫ5.60 (m, 1 H), 8.17 (d, J ϭ 8 Hz, 2 H), 8.29 (d, J ϭ 8 Hz, 2
H) ppm. 13C NMR (50 MHz, CDCl3): δ ϭ 22.5 (q), 30.1 (s), 38.3
(d), 39.3 (t), 39.9 (t), 72.0 (t), 72.1 (t), 80.3 (d), 110.1 (s), 123.4 (d),
130.6 (d), 136.2 (s), 150.4 (d), 164.2 (s) ppm.
Triphenylphosphane (1 equiv.) in dry THF (10% solution) was
treated dropwise with NCS (1 equiv.) in dry THF (5% solution).
The corresponding alcohol (1 equiv.) in dry THF (10% solution)
was added to the resulting suspension. The mixture was stirred at
room temp. until the solution became clear (approx. 1.5 h). After
evaporation of the volatiles, the residue was taken up in water and
extracted with diethyl ether. The organic layer was washed with
brine, dried over anhydrous sodium sulfate, and then the solvents
were evaporated. The crude product was submitted to flash chro-
matography (silica gel; light petroleum/EtOAc, 100:1).
(3Јaα,5Јβ,6Јaα)-5Ј-Chlorohexahydro-5,5-dimethylspiro[1,3-dioxane-
2,2Ј(1ЈH)-pentalene] (7a): exo-Alcohol 4b (0.50 g, 2.2 mmol) was
converted according to the above procedure to give endo-7a (0.25 g,
1
50%) as a colorless oil. H NMR (200 MHz, CDCl3): δ ϭ 0.97 (s,
6 H), 1.61Ϫ1.91 (m, 4 H), 2.12Ϫ2.59 (m, 6 H), 3.46 (s, 4 H),
(3Јaα,5Јα,6Јaα)-Hexahydro-5,5-dimethylspiro[1,3-dioxane-2,2Ј-
3.98Ϫ4.15 (m, 1 H) ppm. 13C NMR (50 MHz, CDCl3): δ ϭ 22.5
(1ЈH)-pentalen]-5Ј-ol (4b): The purified benzoate
6.4 mmol) was dissolved in methanol (30 mL) and treated with a
5
(2.2 g, (q), 30.0 (s), 38.6 (d), 39.9 (t), 43.6 (t), 58.8 (d), 71.8 (t), 72.2 (t),
110.1 (s) ppm.
Eur. J. Org. Chem. 2003, 2243Ϫ2249
www.eurjoc.org
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2247

M. D. Mihovilovic, B. Müller, A. Schulze, P. Stannetty, M. M. Kayser
FULL PAPER
(3Јaα,5Јα,6Јaα)-5Ј-Chlorohexahydro-5,5-dimethylspiro[1,3-dioxane-
2,2Ј(1ЈH)-pentalene] (7b): endo-Alcohol 4a (0.50 g, 2.2 mmol) was
converted according to the above procedure to give exo-7b (0.28 g,
Biotransformations with Recombinant Cells
Fresh LB-amp medium (250 mL; 1% Bacto-Peptone, 0.5% Bacto-
Yeast Extract, 1% NaCl supplemented by 200 ppm ampicillin) was
1
52%) as a colorless oil. H NMR (200 MHz, CDCl3): δ ϭ 0.90 (s,
inoculated with
a 1:100 aliquot of an overnight preculture
6 H), 1.55Ϫ1.72 (m, 2 H), 1.73Ϫ1.94 (m, 2 H), 1.97Ϫ2.20 (m, 4
H), 2.67Ϫ2.87 (m, 2 H), 3.38 (s, 2 H), 3.40 (s, 2 H), 4.32Ϫ4.48 (m,
1 H) ppm. 13C NMR (50 MHz, CDCl3): δ ϭ 22.5 (q), 30.0 (s), 38.1
(d), 39.3 (t), 43.7 (t), 62.6 (d), 72.0 (t), 109.8 (s) ppm.
of the corresponding construct BL21(DE3)(pMM4) or
DH5α(pCMO206) in a baffled Erlenmeyer flask. The culture was
incubated at 37 °C on an orbital shaker at 120 rpm for 2 h, then
IPTG was added so as to obtain a final concentration of 0.025 m.
The substrate 1aϪe (100 µL) was added neat along with β-cyclo-
dextrin (1 equiv.). The culture was incubated at room temperature
for 18Ϫ48 h and the conversion was monitored by GC. The bio-
mass was removed by centrifugation (3500 rpm, 10 min), the
aqueous layer was passed through a bed of Celite, and then the
product was isolated by repeated extraction with EtOAc. The com-
bined organic layers were dried with sodium sulfate and concen-
trated. Lactones 2aϪe were purified by flash column chromatogra-
phy.
cis-(1Ј,3Јa,6Ј,6Јa)-Tetrahydro-5,5-dimethylspiro[1,3-dioxane-
2,2Ј(1ЈH)-pentalene] (8): Compound 8[39] was obtained as by-prod-
uct (10Ϫ15%) in the chlorination according to the procedure above.
1H NMR (200 MHz, CDCl3): δ ϭ 0.97 (s, 6 H), 1.48Ϫ1.73 (m, 2
H), 2.11Ϫ2.19 (m, 1 H), 2.25Ϫ2.45 (m, 2 H), 2.50Ϫ2.72 (m, 2 H),
3.10Ϫ3.28 (m, 1 H), 3.45 (s, 2 H), 3.52 (s, 2 H), 5.52Ϫ5.70 (m, 2
H) ppm. 13C NMR (50 MHz, CDCl3): δ ϭ 22.48 (q), 22.53 (q),
30.1 (s), 36.7 (d), 38.37 (t), 38.45 (t), 40.0 (t), 46.6 (d), 71.8 (t), 72.0
(t), 109.2 (s), 128.5 (d), 133.8 (d) ppm.
(4aα,6β,7aα)-Hexahydro-6-methoxycyclopenta[c]pyran-3(1H)-one
(2a): Biotransformation of 1a (108 mg, 0.7 mmol) with recombi-
nant cells according to the above biotransformation protocol gave
lactone 2a as a colorless oil after chromatographic purification (sil-
ica gel; light petroleum/EtOAc, 10:1) in the yield and enantiomeric
purity specified in Table 1. 1H NMR (400 MHz, CDCl3): δ ϭ
1.50Ϫ1.65 (m, 2 H), 1.82Ϫ2.10 (m, 2 H), 2.40Ϫ2.65 (m, 4 H), 3.23
(s, 3 H), 3.72Ϫ3.83 (m, 1 H), 4.08 (dd, J ϭ 14, 7 Hz, 1 H), 4.31
(dd, J ϭ 14, 7 Hz, 1 H) ppm. 13C NMR (100 MHz, CDCl3): δ ϭ
32.8 (d), 33.8 (t), 34.9 (t), 35.2 (d), 38.0 (t), 56.4 (q), 69.9 (t), 82.3
(d), 173.8 (s) ppm. C9H14O3 (170.21): calcd. C 63.51, H 8.29; found
C 63.67, H 8.04.
Cleavage of the 2,2-Dimethylpropanediol Protecting Group
The pertinent ketal was stirred overnight at room temp. in a 1:1
mixture of 0.1  H2SO4 and acetone. The solution was neutralized
with satd. NaHCO3 solution and extracted with diethyl ether. The
combined organic layers were washed with brine, dried over sodium
sulfate, and concentrated in vacuo.
(3aα,5β,6aα)-Hexahydro-5-methoxy-2(1H)-pentalenone (1a): Ketal
6a (0.880 g, 3.66 mmol) was deprotected according to the above
procedure to give 1a (0.371 g, 66%) as a colorless oil after Kugel-
rohr distillation. B.p. 160Ϫ163 °C/13 mbar. 1H NMR (200 MHz,
CDCl3): δ ϭ 1.58Ϫ1.73 (m, 2 H), 2.02Ϫ2.20 (m, 4 H), 2.23Ϫ2.28
(m, 2 H), 2.43Ϫ2.60 (m, 2 H), 3.26 (s, 3 H), 3.83Ϫ3.96 (m, 1 H)
ppm. 13C NMR (50 MHz, CDCl3): δ ϭ 37.7 (d), 39.1 (t), 45.1 (t),
56.3 (q), 83.5 (d), 220.3 (s) ppm. C9H14O2 (154.21): calcd. C 70.10,
H 9.15; found C 69.86, H 9.31.
(4aα,6α,7aα)-Hexahydro-6-methoxycyclopenta[c]pyran-3(1H)-one
(2b): Microbial oxidation of 1b (108 mg, 0.7 mmol) yielded lactone
2b as a colorless oil after chromatographic purification (silica gel;
light petroleum/EtOAc, 10:1) in the yield and enantiomeric purity
1
specified in Table 1. H NMR (400 MHz, CDCl3): δ ϭ 1.24Ϫ1.44
(m, 1 H), 1.52Ϫ1.68 (m, 1 H), 2.00Ϫ2.90 (m, 6 H), 3.29 (s, 3 H),
3.80Ϫ3.92 (m, 1 H), 4.10 (dd, J ϭ 14, 7 Hz, 1 H), 4.35 (dd, J ϭ
14, 7 Hz, 1 H) ppm. 13C NMR (100 MHz, CDCl3): δ ϭ 32.1 (d),
34.1 (t), 34.2 (t), 34.4 (d), 38.4 (t), 55.7 (q), 69.7 (t), 81.4 (d), 173.6
(s) ppm. C9H14O3 (170.21): calcd. C 63.51, H 8.29; found C 63.17,
H 8.16.
(3aα,5α,6aα)-Hexahydro-5-methoxy-2(1H)-pentalenone (1b): Com-
pound 6b (0.750 g, 3.12 mmol) was converted according to the
above protocol to yield 1b (0.383 g, 80%) as a colorless oil after
flash column chromatography (silica gel; light petroleum/EtOAc,
10:1). 1H NMR (200 MHz, CDCl3): δ ϭ 1.50Ϫ1.68 (m, 2 H),
1.95Ϫ2.20 (m, 4 H), 2.43Ϫ2.65 (m, 2 H), 2.78Ϫ3.00 (m, 2 H), 3.30
(s, 3 H), 3.94 (m, 1 H) ppm. 13C NMR (50 MHz, CDCl3): δ ϭ 37.5
(d), 39.1 (t), 44.4 (t), 56.1 (q), 82.9 (d), 220.2 (s) ppm. C9H14O2
(154.21): calcd. C 70.10, H 9.15; found C 69.96, H 9.23.
(4aα,6β,7aα)-6-Chlorohexahydrocyclopenta[c]pyran-3(1H)-one (2c):
Biooxidation of 1c (116 mg, 0.73 mmol) yielded lactone 2c as color-
less crystals after chromatographic purification (silica gel; light
petroleum/EtOAc, 10:1) in the yield and enantiomeric purity speci-
fied in Table 1. M.p. 90Ϫ92 °C. 1H NMR (400 MHz, CDCl3): δ ϭ
1.58Ϫ1.68 (m, 1 H), 1.70Ϫ1.81 (m, 1 H), 2.30Ϫ2.70 (m, 6 H),
4.05Ϫ4.15 (m, 1 H), 4.20Ϫ4.30 (m, 2 H) ppm. 13C NMR
(100 MHz, CDCl3): δ ϭ 32.8 (d), 34.2 (t), 35.1 (d), 38.8 (t), 43.1
(t), 56.6 (t), 69.2 (t), 172.7 (s) ppm. C8H11ClO2 (174.63): calcd. C
55.02, H 6.35; found C 54.87, H 6.55.
(3aα,5β,6aα)-5-Chloro-hexahydro-2(1H)-pentalenone (1c): Precursor
7a (0.27 g, 1.1 mmol) was hydrolyzed by the above procedure to
give 1c (0.10 g, 58%) as a colorless oil after flash column chroma-
tography (silica gel; light petroleum/EtOAc, 5:1). 1H NMR
(200 MHz, CDCl3): δ ϭ 1.73Ϫ1.92 (m, 2 H), 2.20Ϫ2.40 (m, 2 H),
2.45Ϫ2.70 (m, 4 H), 2.72Ϫ2.95 (m, 2 H), 4.18Ϫ4.37 (m, 1 H) ppm.
13C NMR (50 MHz, CDCl3): δ ϭ 38.1 (d), 44.2 (t), 44.8 (t), 58.6
(d), 219.3 (s) ppm. C8H11ClO (158.63): calcd. C 60.57, H 6.99;
found C 60.54, H 6.83.
(4aα,6α,7aα)-6-Chlorohexahydrocyclopenta[c]pyran-3(1H)-one (2d):
Biotransformation of 1d (116 mg, 0.73 mmol) according to the gen-
eral protocol yielded lactone 2d[34] as colorless crystals after chrom-
atographic purification (silica gel; light petroleum/EtOAc, 10:1) in
the yield and enantiomeric purity specified in Table 1. M.p. 88Ϫ90
°C. 1H NMR (400 MHz, CDCl3): δ ϭ 1.60Ϫ1.80 (m, 1 H),
1.92Ϫ2.10 (m, 1 H), 2.20Ϫ2.88 (m, 6 H), 4.10 (dd, J ϭ 14, 7 Hz,
(3aα,5α,6aα)-5-Chloro-hexahydro-2(1H)-pentalenone (1d): Ketal 7b
(257 mg, 1.05 mmol) gave ketone 1d (112 mg, 68%) as a colorless
oil following the above procedure after flash column chromatogra-
1
phy (silica gel; light petroleum/EtOAc, 10:1). H NMR (200 MHz,
CDCl3): δ ϭ 1.73Ϫ2.12 (m, 4 H), 2.30Ϫ2.72 (m, 4 H), 3.00Ϫ3.25 1 H), 4.30 (dd, J ϭ 14, 7 Hz, 1 H), 4.45Ϫ4.60 (m, 1 H) ppm. 13C
(m, 2 H), 4.50Ϫ4.60 (m, 1 H) ppm. 13C NMR (50 MHz, CDCl3): NMR (100 MHz, CDCl3): δ ϭ 32.2 (d), 33.7 (t), 34.5 (d), 39.5 (t),
δ ϭ 37.1 (d), 43.9 (t), 44.5 (t), 61.7 (d), 219.3 (s) ppm. C8H11ClO 43.5 (t), 61.4 (d), 69.1 (t), 172.8 (s) ppm. C8H11ClO2 (174.63): calcd.
(158.63): calcd. C 60.57, H 6.99; found C 60.74, H 6.73.
C 55.02, H 6.35; found: C 55.03, H 6.73.
2248
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2003, 2243Ϫ2249

Microbial BaeyerϪVilliger Oxidations of Functionalized Pentalenones
FULL PAPER
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orless oil after chromatographic purification (silica gel; light petro-
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Table 1. 1H NMR (400 MHz, CDCl3): δ ϭ 1.98Ϫ2.70 (m, 8 H),
3.93 (dd, J ϭ 14, 7 Hz, 1 H), 4.21 (dd, J ϭ 14, 7 Hz, 1 H), 4.80
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Acknowledgments
This project was funded by the Oesterreichische Nationalbank
(grant no. JF-7619). The visit of A.S. as a visiting scientist at Vi-
enna University of Technology was supported by the Deutsche For-
schungsgemeinschaft DFG (German Research Council). Contri-
butions by Baxter Immuno Austria and Novartis Donation and
Sponsoring are gratefully acknowledged. We thank Dr. Erwin Ro-
senberg (Vienna University of Technology) for his assistance in the
determination of enantiomeric purities.
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Received January 17, 2002
Eur. J. Org. Chem. 2003, 2243Ϫ2249
www.eurjoc.org
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2249