Enzymatic synthesis of an important building block for the new nicotinyl pesticides

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Various pyridine derivatives have physiological activity. Very recently, the new powerful insecticides belonging to nicotinly group, have been found. These nicotinly insecticides are expected to be popular due to the powerful activity and low toxicity. However, it is not easy to synthesize the building block of 6-chloropyridyl through the conventional chemical synthesis. Thus, the development of the new bioprocess for the synthesis of the building block is required. To accomplish this purpose, we investigated the enzymatic regio-specific hydroxylation reaction of pyridine ring. Once the pyridine ring is hydroxylated, the halogenation of pyridine ring can be proceeded easily.We have focused on the regiospecific hydroxylation reaction of pyridine ring for two years. We attempted to isolate the microorganisms which catalyze the regiospecific hydroxylation of nicotinic acid. We found Pseudomonas fluorescens TN5 catalyzes the 6-position specific hydroxylation of nicotinic acid. We optimized … More the culture conditions to enhance the hydroxylation reaction. When the resting cells were added to the reaciton mixture containing nicotinic acid with aerobically shaking, and enormous amount of 6-hydroxynicotinic acid accumulated with the high molecular conversion yield. We also found the microorganism to catalyze the 6-position hydroxylation reaction of 3-cyanopyridine to form 3-cyano-6-hydroxypyridine. 6-Hydroxynicotinic acid and 3-cyano-6-hydroxypyridine are useful for the synthesis of the building block of the new nicotinyl insecticides. The enormous high yield suggests the high possibility of this process for industrail use.Further, we elucidated the enzymes to catalyze the regio-specific hydroxylation of nicotinic acid. The dehydrogenase, coupled to cytochrome oxidase, is responsible for this hydroxylation. When the resting cells and the substrate are incubated aerobically, the hydroxylation reaction proceeded efficiently.Further, we attempted to establish the new enzymatic production process of d-aminolevurilic acid, which is promising as the new plant hormon. P.fluorescens catalyzes the conversion of 6-hydroxynicotinic acid into 2,5-dihydroxypyridine, which can be synthesized easily through chemical way into delta-aminolevurilic acid. We purified and characterized the enzyme which catalyzed the conversion of 6-hydroxynicitnic acid into 2,5-dihydroxypyridine. We elucidated that the new monooxygenase is responsible for this decarboxylative hydroxylation reaction. We also optimized the reaction conditions for the production of 2,5-dihydroxypyridine using the monooxygenase. Thus, we have established the new biological production process for the synthesis of delta-aminolevurilic acid. Less