Front Matter

A major feature of these experiments was that only low oxygen pressures were

needed to conduct high-yield lipoxygenation. This prompted the authors (Martini et

al., 1994) to use a microbial fermentor for the oxygenation of LA on a 100-fold

greater scale (0.1 mol). This was applied to the chemo-enzymatic synthesis of a

natural product, (+)-coriolic acid (13(S)-HODE). A 2-L Biolafitte (St. Germain

en Laye, France) fermentor was filled with 950 mL of borate buffer (pH 11) and

maintained at 5 8 C. LA (28 g, 0.1 mol) dissolved in ethanol (50 mL) was then

added, together with 4 g of SBLOX-1. The reaction was started by pressurization

of the fermentor (1.5 bar of O 2 ) and mixing (1000 rpm). The reaction was completed

in 30 min (95 % yield), and the HPODs reduced and purified on a medium-pressure

chromatography column (silica gel). Depending on the fractionation, (+)-coriolic

acid (97 % ee) was obtained nearly pure (99 %) in 54 % yield or as an isomeric

mixture (96.5 %) in 78 % overall yield. This final experiment showed that lipoxy-

genation could be conducted, using readily available vessels, on a 0.1 mol scale for

the multigram synthesis of (n-6)-(S)-PUFA-HPODs and their corresponding alco-

hols; moreover, this was a process that could easily be scaled-up.

16.2.4 Oxygen bubbling

As an alternative method to ‘under-pressure lipoxygenation’, the group of Roberts

developed a practical method involving common vessels, such as Erlenmeyer flasks,

combined with oxygen bubbling and magnetic agitation in order to perform a correct

oxygenation of the reaction mixture (Maguire et al., 1993). By using a 2-L conical

flask, over-foaming was avoided, and 208 mg of LA placed in 200 mL of buffer was

oxygenated by 17 mg of crude SBLOX-1. After 1 h the reaction was stopped, and the

formed HPODs reduced, leading to the formation of (+)-coriolic acid in 75–80 %

yield. This method was easy to perform, and allowed the generation of hundred of

milligrams of PUFA-HPODs of high purity with low-cost vessels and equipment.

16.2.5 Use of a biphasic system

As PUFAs are poorly soluble in aqueous media, the use of a biphasic system has been

tested, the substrate being dissolved in an organic solvent (Drouet et al., 1994).With

SBLOX-1 as catalyst and LA as substrate, a 8 : 1 borate buffer:octane ratio was

shown to be the most appropriate, and substrate concentrations in the range 10–

40 g L–1have been tested. The final yield in HPODs varied from 78 % to 30 %

as LA concentration increased. At 10 g L–1, 13(S)-HPODE was the major isomer

formed (92 %). In this work, the need for strong agitation and oxygen bubbling was

also claimed to achieve satisfactory yields in HPODs. As for the previous method,

this technique was well suited for the generation of hundred of milligrams of PUFA-

HPODs.

342 16 Properties and Applications of Lipoxygenases