Sphingomonas Sp. Hxn-200

Figure 15.2 Improvement of the hydroxylation activity and enantioselectivity with Sphingomonas sp. HXN-200 by substrate modification.

hydroxylation with Sphingomonas sp. HXN-200 gave 62% of (S)-enantiomer in 53% ee with six times higher activity.

It was found that Sphingomonas sp. HXN-200 contains a soluble alkane mono-oxygenase and accepts a broader range of substrates. The enantioselectivity and activity were further improved by introducing a "docking/protecting" group into the pyrrolidine substrates. As shown in Figure 15.2, changing the N-substitution from a benzyl to a benzyloxycarbonyl group resulted in a three-fold increase of the activity and an improvement of product ee from 53% (S) to 75% (R).26 We found that the ee can be further increased by simple crystallization of the biopro-duct from a mixture of n-hexane and ethyl acetate. Thus, it is possible to produce (R)- and (S)-3-hydroxypyrrolidine in 98% and 96% ee, respectively.

We optimized the growth of strain HXN-200 on n-octane and produced the cells in a large amount. It was found that the cells can be stored at — 80°C for two years without significant loss of activity. The frozen/thawed cells, that are easy to handle for the organic chemist, can be used for routine hydroxylation in an organic chemistry laboratory.

15.4 SYNTHESIS OF HYDROXYPYRROLIDINES, HYDROXYPYRROLIDINONES, AND HYDROXYPIPERIDINES

3-Hydroxypyrrolidines were easily prepared in 62-94% yield and in 1-2 g/L by hydroxylation with the frozen/thawed cells. Even higher productivity was obtained by hydroxylation with growing cells of HXN-200,26 which is obviously the best choice for an industrial hydroxylation. It was found that Sphingomonas sp. HXN-200 can also catalyze the hydroxylation of 2-pyrrolidinone.27 As shown in Figure 15.3, hydroxylation of N-benzylpyrrolidinone gave the corresponding (S)-4-hydroxypyrrolidine in >99.9% ee, demonstrating the best enantioselectivity ever reported for biohydroxylations. Changing the "docking/protecting" group to a tert-butoxycarbonyl group increased the activity to 11 U/g cell dry weight (cdw). Although the enantioselectivity was slightly lower, simple crystallization

99.9% ee (after crystalization)

CS-834 (Sankyo) f NH

Carbapenems 2

Figure 15.3 Biohydroxylation of 2-pyrrolidinones with Sphingomonas sp. HXN-200.

from n-hexane/ethyl acetate improved the ee from 92% to 99.9%. Here again, (S)-4-hydroxypyrrolidines are useful intermediates for the preparation of several pharmaceuticals.

Sphingomonas sp. HXN-200 was also able to accept a six-member ring substrate. Hydroxylation of N-benzyl- and N-teri-butoxycarbonyl-2-piperidinone gave the corresponding (R)-4-hydroxy-piperidin-2-ones in 31% and 68% ee, respectively28 (Figure 15.4). This provides a simple synthesis of such types of useful synthons.

Further investigation demonstrated that Sphingomonas sp. HXN-200 is also the best biocatalyst known thus far for the hydroxylation of N-substituted piperidines.29 Even small cyclic compounds such as azetidines, which are difficult to be hydroxyl-ated by other system, are also good substrates for strain HXN-200.29 Excellent regioselectivity and high activity were obtained in all hydroxylations shown in Figure 15.5. This provides simple and efficient synthesis of 4-hydroxypiperidines and 3-hydroxyazetidines, which are useful pharmaceutical intermediates.

Sphingomonas sp. -

O HXN-200

Sphingomonas sp. -

O HXN-200

Activity (U/g cdw)

31 68

Figure 15.4 Biohydroxylation of 2-piperidinones with Sphingomonas sp. HXN-200.

Ebastine, drug for allergic rhinilis

Dezinamide

Antiepilephlic (Phase II) American Home Products

Ebastine, drug for allergic rhinilis

^LH p COOR

Oral carbapenem antibiotics

Dezinamide

Antiepilephlic (Phase II) American Home Products

Nadifloxacin, antibacterial drugs

Figure 15.5 Biohydroxylation of piperidines and azetidines with Sphingomonas sp. HXN-200.

Nadifloxacin, antibacterial drugs

Me N NH2 H

Azelnidipine, antihypertensive Sankvo: Libe

Figure 15.5 Biohydroxylation of piperidines and azetidines with Sphingomonas sp. HXN-200.

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