Biohydroxylation of N-Benzoylspirooxazolidines - Masked Cyclic Ketone Substrates

G. Braunegg[2], A. de Raadt*[1], H. Griengl[1], I. Kopper[2], M. Kreiner[2], M. Petsch[1], P. Plachota[1], N. Schoo[1], H. Weber[1], A. Zeiser[2]

[1] Christian Doppler Laboratorium fuer Chirale Verbindungen Institut fuer Organische Chemie der Technischen Universitaet Graz, Stremayrgasse 16, A-8010 Graz, Austria; [2] 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 substrates. 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 very promising.

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) with chromatography.

References and Footnotes:
  1. D. H. R. Barton, D. Doller, Acc. Chem. Res., 1992, 25, 504 - 512
  2. H. L. Holland, Organic Synthesis with Oxidative Enzymes; VCH / USA, 1992.

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