Synthesis Of Epoxystyrene With Very High Enantioselectivity

Another useful bioconversion is the epoxidation of styrene to epoxystyrene. Styrene oxide is a valuable building block, because the epoxide function allows versatile synthetic chemistry, and the benzene ring is part of the majority of today's drugs (Figure 15.10). It is used, for example, in the production of the anti-helmintic drug Levamisole.42 However, to be an attractive building block for drug synthesis, the styrene oxide needs to be enantiopure. There are some chemical asymmetric synthesis routes described, but they usually deliver only moderate to good enantiomeric excesses. Kinetic resolution of styrene oxide employing the Jacobsen catalyst affords enantiopure styrene oxide, but this method has the inherent drawback of being limited to a maximum chemical yield of 50%.43 In order to circumvent these various limitations, we attempted to design an asymmetric synthesis route from styrene to styrene oxide using biocatalysis. Asymmetric synthesis in principle allows a 100% ee/100% yield process, and the enantioselectivity of enzymes should aid in overcoming the problem just mentioned in achieving satisfactory ee's.

We have used two enzyme systems for this purpose. The first is the xylene monooxygenase system of P. putida mt2, which is capable of utilizing xylene and toluene derivatives for growth. In the first enzymatic step, xylene monooxygenase introduces an oxygen atom in a toluene or xylene methyl substituent group. This monooxygenase can also introduce an epoxide in the vinyl double bond of styrene and substituted styrenes.44 The reaction was carried out using E. coli recombinants carrying only the xylene monooxygenase system, encoded by xylMA, resulting in efficient formation of epoxystyrene with an enantiomeric excess of 92%45 (see Figure 15.10).

Still, a biocatalyst that performs this reaction with an even higher ee was desirable, which led us to investigate styrene degradation in various bacterial soil strains. One of these, Pseudomonas sp. strain VLB120 was selected for further a o.

Styrene a o.


Figure 15.10 Biological oxidation of styrene to (S)-styrene oxide.


stySc t>


Pg Nc








pSPW 1







Figure 15.11 Styrene degradation in Pseudomonas sp. strain VLB120.

study, because it appeared to degrade styrene via styrene oxide and phenylacetal-dehyde (Figure 15.11). After preparation of a genomic library in E. coli, clones that could convert indole to indigo were selected and analyzed for their ability to transform styrene to styrene oxide. One such clone contained a 5.7-kb DNA fragment that encoded the major part of a styrene degradation pathway, the first step of which consists of the oxidation of styrene to epoxystyrene with a cytoplasmic two-component styrene monooxygenase (Figure 15.11). The genes encoding the styrene monooxygenase were used to construct a recombinant biocatalyst in E. coli JM101, which achieved the conversion of styrene into (S)-styrene oxide with an ee of more than 99% (Figure 15.10). In other words, natural biodiversity was sufficient to increase the ee to more than satisfactory levels.33,46

In designing a production process for (S)-styrene oxide, several issues must be considered. First, styrene monooxygenase activity depends on the availability of NADH, making the use of an enzyme reactor with (partially) purified enzyme complex and expensive because the cofactor needs to be regenerated while product is produced. Second, the substrate as well as the product of the reaction are not very soluble in water and, even at such low concentrations, toxic to living cells, complicating the development of a whole-cell biocatalytic system. One way to still create a potentially economically attractive process is to use growing cells in a two-liquid phase culture. The partition coefficients of substrate and product dictate that both compounds remain preferentially in the organic phase and the aqueous concentrations remain below toxic levels, while the overall concentrations of substrate and product in the reactor can be increased far beyond what would be possible if only the aqueous phase was present. In the present example, the overall styrene concentration could be increased from 2 mM for a one-liquid phase culture to 135 mM for a two-liquid phase culture that consisted of 50 vol % of aqueous medium with the biocatalyst and 50 vol % of the organic phase with 2 vol % styrene. During the reaction, a small amount of the styrene partitions out of the organic phase, is oxidized, and is then reextracted into the organic phase. The organic phase serves as substrate pool and product sink at the same time. We investigated this mode of production on a 2-L scale with recombinant E. coli JM101 cells that carried the styrene monooxygenase genes on the expression vector pSPZ10 under control of the alk regulatory system. This vector carries the styrene monooxygenase genes under control of the alkBp promoter, which is induced by octane and is not repressed by glucose in E. coli. The pBR322-based vector has been optimized by introducing transcriptional terminators to transla-tionally shield regions important for plasmid propagation and by replacing the tetracycline and ampicillin resistance genes with a kanamycin resistance gene.33 Such a genetic system allows easy induction in a two-liquid-phase culture and the use of a cheap carbon source for the cultivation.

We optimized the cultivation protocol with respect to aqueous medium composition, organic phase, and phase ratio (the ratio of the volume of the organic phase to the total liquid volume). The best system consists of a defined mineral medium, with glucose as the carbon source and diethylhexylphthalate as the organic phase at a phase ratio of 0.5. The organic phase contained 2 vol % styrene and 1 vol % of octane, which we added as an inducer for gene expression.

Synthesis Styrene Oxide
Figure 15.12 Two-liquid-phase-based biooxidation of styrene to styrene oxide with a recombinant whole-cell biocatalyst.

With this system we converted 135 mM styrene (relative to the total liquid volume) to styrene oxide in 10 h at a cell dry weight of around 10g/L aqueous phase, with an average activity of 152 U/L total liquid volume. This corresponds to a space-time yield of 1.1 g (S)-styrene oxide per liter and hour. These are the highest specific activities reported thus far for a microbial epoxidation process.33

The bioconversion was carried out in a two-liquid phase system (Figure 15.12), which was developed at the 2-L level, and scaled up to the 30-L level to produce almost 400 g of product. Several apolar phases were used, of which bis(2-ethylhexyl)phthalate (BEHP) was preferred because it showed a better partitioning of epoxystyrene toward the apolar phase and away from the aqueous phase than did hexadecane. This was important because the product was quite toxic to the recombinant biocatalyst when it appeared in the aqueous phase. This bioconversion illustrates that apolar compounds like styrene and its epoxide, which are quite toxic to microorganisms, can be handled successfully in two-liquid-phase cultures. The toxicity of the substrate and product are not significant issues here.

Again, a recombinant E. coli strain performs quite well in this system. Subsequent phase separation and distillation permit the simple purification of the product. The final product had an ee >99%, which is significantly better than that seen for the XylMA based system (Figure 15.10).

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  • Fethawi
    What is Sstyrene oxide used for?
    9 years ago

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