Biohydroxylation of N-Benzoylspirooxazolidines - Masked Cyclic Ketone Substrates
G. Braunegg, A. de Raadt*, H. Griengl, I. Kopper, M. Kreiner, M. Petsch,
P. Plachota, N. Schoo, H. Weber, A. Zeiser
 Christian Doppler Laboratorium fuer Chirale Verbindungen Institut fuer Organische Chemie der Technischen
Universitaet Graz, Stremayrgasse 16, A-8010 Graz, Austria;
 Institut fuer Biotechnologie der Technischen Universitaet Graz, Petersgasse 10, A-8010 Graz, Austria
Apart from the relatively recent Gif System introduced by D.H.R. Barton 1, it is very difficult to introduce a hydroxyl moiety into an
unactivated carbon atom using purely chemical means. A valuable alternative is
microbial hydroxylation 2 and this, in fact, is of great
synthetic and commercial importance with respect to steroid and terpenoid
On the other hand, the applicability of this approach to compounds commonly dealt
with in organic chemistry, for example cyclic ketones, remains limited. This is
highlighted when the substances in question contain a functional group which can
also be transformed by the microorganism.
The general strategy of protecting these sensitive groups, carrying out the
biohydroxylation, and subsequently deprotecting to release the desired compound
is depicted below. These protecting groups not only prevent undesired side
reactions, but ease detection and aid docking into the active site. In
particular, results from employing N-benzoylspirooxazolidines as anchor /
protecting groups for ketones (Scheme below) will be presented in this poster.
Results and Discussion
The problems of employing unprotected cyclic ketones as substrates for
hydroxylation with microorganisms is illustrated in the following example. Using
the well known fungus Beauveria bassiana ATCC 7159, cyclohexanone affords
cyclohexanol and not the desired hydroxylated ketone (Scheme 1).
This stumbling block has been overcome by using the concept of reversible
protecting / anchor groups. These protecting groups have been found not only
prevent undesired side reactions, but also ease detection and aid docking into
the active site. In particular, for ketones, results employing
N-benzoylspirooxazolidines as anchor / protecting groups have been found to be
Step 1: Protecting / Anchor Group Introduction
This step was easily carried out in a one pot, two step synthesis (Scheme 2).
After a simple work up procedure, the crude residue was chromatographed to
produce the desired substrate which was then ready for the next step.
Step 2: Biohydroxylation Employing Beauveria Bassiana
After dissolving the substrate in ethanol (100mg/ml), this was introduced (400mg
substrate/l culture) into the second stage of a standard two stage culture of
Beauveria bassiana ATCC 7159. After 48 hours, all of the spirooxazolidine
derivative was converted into a single, more polar compound. Extraction of the
culture with equal volumes of EtOAc (3 times) and chromatography of the residue
furnished the desired compound in good yield (Scheme 3, 49% isolated).
Step 3: Anchor / Protecting Group Cleavage
This last step was easily realised by simply dissolving the hydroxylated compound
in acetonitrile and adding acidic ion exchange resin (IR 120) at room temperature
with vigorous stirring (Scheme 4). After about 24 hours the cleavage was complete
and the hydroxylated ketone could be isolated in very good yield (83% isolated)
References and Footnotes:
- D. H. R. Barton, D. Doller, Acc. Chem. Res.,
1992, 25, 504 - 512
- H. L. Holland, Organic Synthesis with Oxidative
Enzymes; VCH / USA, 1992.
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