Previously reported syntheses

(Trudgill et al, 1990) demonstrated that the enzyme has a high substrate specificity for a variety of ketones, from cyclobutanone to complex cyclic molecules such as (+)-camphor and dihydrocarvonone.

(Schwab et al, 1983) showed that even with small substitutions on the ring structure such as is found on 2-methyl cyclohexanone enantioselectivity was shown by the enzyme. After 50% conversion the ee dropped due to the other enantiomer of the ketone being processed. Examples do exist however when CHMO performs a Baeyer-Villiger reaction with no selectivity (Gagnon et al, 1994), where the reaction using a racemic norbanone produced the lactone with no enantiomeric excess, in a similar fashion to that which can be achieved using a peracid in the classical chemical Baeyer-Villiger reaction.

(Abril et al, 1989) examined a range of substrates derived from cyclohexanone, including 4-phenylcyclohexanone and 4-rerf-butylcyclohexanone as well as more complicated molecules such as 7-benzyloxymethyl-2-norban-5-one using immobilised CHMO. It was found that substrate specificity was broad but CHMO did not accept a,p-unsaturated ketones or 1,3-diketones. High yields were obtained with most ketones, although no data on enantioselectivity was reported.

The focus of much work in recent years has been on various bicyclic ketones; (Sandey and Willetts 1992; Alphand et al, 1989; Shipstone et al, 1992; Lenn and Knowles 1994; Alphand and Furstoss, 1992, and Grogan et al, 1992). All have studied the oxidation of bicyclo[3.2.0]hept-2-en-6-one and found that each isomer undergoes oxygenation at a different position, forming in high yield and enantiomeric purity (>90% ee) two regioisomers. It was also found that Acinetobacter TD63 and Pseudomonas putida can also catalyse this reaction. Lenn and Knowles also found that substitutions at the 1-endo position can increase selectivity; whereas substitutions at the l~exo position has the opposite effect.

(Camell et al, 1991) also studied derivatives of bicyclo[3.2.0]hept-2-en-6-one, and found that regioselectivity and enantioselectivity varied considerably, possibly due to the enzyme’s preferred method of approach to the substrate being via the exo face, which is more open. (Sandey et al, 1992) also describe this reaction, additionally reporting that 1% o f the products formed are as a result o f an alternative, reductive pathway. Bicyclo[2.2.1]hepten-7-ones have also been investigated, with the usual excellent enantioselectivity (Taschner and Peddada, 1992; Konigsberger et al, 1991).

(Taschner and Chen, 1991) successfully v&Qà. A.calcoaceticiis to generate the correct stereochemistry in an intermediate of the antibiotic ionomycin, a key step to developing a competitive synthetic route to the antibiotic.

Investigations using substituted cyclohexanones have been reported, (e.g. Alphand and Furstoss, 1992^; Tascher et al, 1993). The latter produced (S)-lactones in high ee (>98%) using 4-substituted cyclohexanones when the substituted group was ethyl, propyl or fer/-butyl; but n-butyl generated only 52% ee with the (R)-conflguration. This suggests this could be a point of changeover in enantioselectivity, but no high alkyl groups were used to verify this. (Alphand and Furstoss, 1990) worked with disubstituted cyclohexanones, menthone and dihydrocarvone. High ee’s were reported for both reactions, the only unusual result being the formation o f 3R,6S- lactone from 2-methyl, 6-propenyl cyclohexanone with the oxygen inserted between the carbonyl group and the least substituted carbon atom; normally the insertion is between the carbonyl group and the most substituted. This was explained by steric interactions in the active site only allowing the more unusual configuration to be produced.

(Gagnon et al, 1995^) reported CHMO transformations o f various 3-substituted cyclobutanones from 3-butyl to 3-phenylethylacetyl cyclobutanone producing (S)- lactones exclusively, with ee’s between 50 and 95%.

Work has also been carried out with various 2-alkyl substituted (C5-C11)

cyclopentanones presented to A.calcoaceticus (Alphand et al, 1990), it was found that both yield and enantiomeric excess depended on the length o f substitution.

Yields increased with increasing chain length; explained by the possibility o f better positioning in the active site, or the ease of passage across the cell membrane due to increased hydrophobicity. Also possible is the existence o f a second metabolic pathway which acts only on the shorter chain ketones or their lactone derivatives. This is supported by the fact that adding 1,2-cyclohexanediol, a proposed intermediate in this pathway, increases yields of short chain lactones. Enantiomeric excess seems to act in the opposite way; increasing as chain length decreases. 97% ee was reported for C5 down to 73% with Cn. EE drops after 50% conversion as the unfavoured enantiomer of the ketone is reacted. Interestingly it seems that the lactones produced are all (S)-configuration, the same result as has been found previously with 3-substituted cyclobutanones and 4-substituted cyclohexanones. 2- and 3- substituted cyclopentanones have also been studied using whole cell S.cerevisiae, producing S-lactones in high ee (>95%) for 2- substituted and poorer ee (30-40%) using 3-substituted cyclopentanones (Kayser et al, 1998).

It has also been shown that along with nucleophillic attack on carbonyl species producing the Baeyer-Villiger reaction, unusual substrates containing species such as iodine, boron, selenium and sulphur allow the oxygen to act as an electrophile to a nucleophillic electron pair on the substrate (Branchaud and Walsh 1985). This makes CHMO unique among flavin monooxygenases in it’s ability to act as both a nucleophile and an electrophile.

Numerous oxygenation reactions o f sulphides and sulphoxides have been reported, often producing products with ee’s close to 100% (Donaghue et al,, 1976; Carrea et al, 1992; Secundo et al,, 1993; Pasta et al,, 1995, Kelly et al, 1996, Colonna et al, 1997). This reaction has also been reported in Helminthosporium sp. (Holland et al, 1997) and rabbit monooxygenase (Fisher and Rettie, 1997). Recently the range of sulphur containing substrates has been increased, such as the synthesis of thioacetal and thioketal sulphoxides (Alphand et al, 1997) and the conversion o f organic sulphites to sulphates (Colonna et al, 1998). Molecules with a chiral centre in proximity to a S=0 bond are useful pharmaceutical intermediates (Davies and Reider, 1996; Collins et al, 1997).