La mística del compromiso definitivo

Monooxygenases are enzymes that incorporate one oxygen atom from dioxygen into a substrate, with the remaining oxygen atom converted into H2O. The Baeyer- Villiger monooxygenases are monooxygenase enzymes which catalyse the oxygen insertion of C-C bonds beside ketone groups. The oxygenation by monooxygenases usually requires NADH or NADPH as a coenzyme during the reaction. The oxygenation reaction is very interesting especially within non-activated compounds whose oxygenated forms may be potentially very useful.

Biotransformation is defined as a process that is exploited to convert a chemical compound by using biological systems. Benefits of biotransformation are, for instance, in the pharmaceutical industry and in organic synthesis.

In the pharmaceutical industry, the production of a single enantiomer is crucial. Adverse side effects and toxicity of a drug may occur if another enantiomer is included. In any application of organic synthesis in which regio- or enantiospecificity is desired, biotransformation techniques play an important role in producing a variety of organic compounds. The advantages of biotransformation are not only that regio and enantiomeric specific molecules are obtained; it may also lowers the cost of manufacture.

The organic synthesis industry has shown a growing trend in using biotransformation, especially in amino acid, steroid and antibiotic manufacture (Grogan

et al., 1992; Grogan et al., 1993; Gagnon et al., 1995; Furstoss and Petit, 1995 and Kelly

et al., 1998). The biotransformation are performed in mild conditions and considered to be environmentally friendly (Hanson, 1995).

In extending the range of useful biotransformations, oxygenation by Baeyer- Villiger monooxygenases has been employed in organic synthesis. These monooxygenases can be used to perform chemeoenzymatic synthesis on various chemicals. For example, cyclohexanone monooxygenase (CHMO) from Acinetobacter

calcoaceticus is a NADPH dependent Baeyer-Villiger monooxygenase. This CHMO has been used in the organic synthesis of many useful intermediate compounds. By CHMO, the biotransformation of ketone, bicyclo (3.2.0) hept-2-en-6-one yeilds the chiral syntons, which can subsequently enter a variety of routes to chemoenzymatic synthesis of valuable compounds such as pheromones, antibiotic sarkomycin A, viridine, ionomycin and derivatives with interesting antileukaemic activities (Willetts, 1997). Also, employing the oxygen insertion reaction to the ketone of bicyclo (3.2.0) hept-2-en-6-one can be a potential route to the synthesis of prostaglandins and nucleosides (Newton and Roberts, 1980). Moreover, using either cyclopentanone monooxygenase or 2-oxo-4,5,5- trimethylcyclopentenylacetyl-CoA monooxygenase in the chemoenzymatic synthesis of the intermeadiate (S)-methyl 6,8-dihydroxyoctanoate can lead to the production of (+)-

(R)-lipoic acid (Adger et aL, 1997). In ketone synthesis, Acenetobacter calcoaceticus has been used to oxidise dihalogenoketone, yielding optically pure ketone (40%) that has been oxidised to lactone by chemical process and further steps to an AZT analogue (Roberts and Willetts, 1993) (see Figure 1.14). However, there have to be clear benefits to employing the CHMO and cyclopentanone monooxygenase, because of the cost and cofactor involvements since the CHMO and cyclopentanone monooxygenase utilise NADPH which is expensive and difficult to recycle (Roberts and Willetts, 1993). This introduces the thought of employing an alternative enzyme which might lower the cost and be put to practical use.

The diketocamphane monooxygenases from the camphor degradation pathway utilising NADH as a cofactor can be useful in organic synthesis compared to the monooxygenases CHMO (Roberts and Willetes, 1993). There are at least three Baeyer- Villiger monooxygenases in the camphor degradation pathway: 2,5-diketocamphane 1,2- monooxygenase, 3,6-diketonecamphane 1,6-monooxygenase and 2-oxo-A^-4,5,5- trimethylcyclopentenylacetyl-CoA monooxygenase. Pilot studies using these Baeyer- Villiger monooxygenases in the biotransformation of chemical compounds have been carried out. Preliminary studies of the Baeyer-Villiger monooxygenases in microbial transformation using whole cells of P. putida NCIMB10007 which carried the CAM plasmid, were used to transform a ketone into two isomeric optically active lactones

(Roberts and Willetts, 1993) (Figure 1.13). The products of this enzymatic reaction represent a mirror image of cyclohexanone monooxygenase (CHMO) biotransformation of the same substrate.

,0 >95% e.e. >95% e.e. .0 / ' " T ^ r s C H M O / '' - | f 2,5-DKCMO A " "— P o + \ >89% e.e. >99% e.e.

Figure 1.13

Biotransformation of bicyclo (3.2.0) hept-2-en-6-one by cyclohexanone monooxygenase (CHMO) and 2,5-diketocamphane 1,2-monooxygenase (2,5-DKCMO) (Willetts, 1997)

Therefore, diketocamphane monooxygenases may hold great promise and can be considered as alternatives to existing Baeyer-Villiger monooxygenases in biotransformation to produce useful compounds.

B D

\ \‘f /

E ' 1 CHMO I CHMO

% B

inetobacter / / \ \ CHMO I CHMO 1

Icoaceticus /r A ' v ^ i y ^ C O - o C C ? ° + o q * * M-"'

Cdo

tQ 0 > I

o

I I

OAc Cyclopentanone monooxygenase prostagrandins O steps OH OH N3/ AZT analogue

I

c c . 4:,,

_Multifidene

—A

+ c c _ COjH Sarkomycin A Viridiene (+)-(R)-Lipoic acid 0.J A c ^ Q Clerodin

Figure

1.14

Chemoenzymatic approaches that employ Baeyer-Villiger monooxygenases to yield target molecules in order to use these intermediates in the synthesis of useful compounds (Newton and Roberts, 1980; Willetts, 1997; and Adger et aL, 1997).

A: Biotransformation of dihalogenoketone by A. calcoaceticus strain NCIMB 9871, which gave lactone that can be used in the synthesis of an AZT analogue. B: Oxygenation of bicyclo[3.2.0]hept-2-en-6-one to compounds that can be used in preparing prostraglandins. C, D and E: Biotransformation of bicyclo[3.2.0]hept-2en-6- one by cyclohexanonone monooxygenase in A. calcoaceticus which yields intermediate molecules that can be useful in the organic synthesis of pheromones (multifidene and viridine), antibiotic (sarkomycin A) and insect antifeedant (clerodin).

Figure 1.14

(continued). F: The use of cyclopentanone monooxygense in the chemoenzymetic synthesis of the intermediate of (S)-methyl 6,8-dihydroxyoctanoate to prepare (+)-(/?)-lipoic acid.

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