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Keywords:

  • cofactor recycling;
  • fatty acids;
  • green chemistry;
  • hydroxylation;
  • macrolide antibiotics;
  • P450 monooxygenase

Abstract

Cytochrome P450 CYP102A1 from Bacillus megaterium is a fatty acid hydroxylase which catalyses the highly regioselective hydroxylation of branched fatty alcohols like (2R,4R,6R,8R)-tetramethyldecanol (4). The product of this reaction (2R,4R,6S,8S)-tetramethyldecane-1,9-diol (3) can be used in synthesis of macrolide antibiotics. For setting up the biooxidation process on a preparative scale a monophasic aqueous reaction system has been established. The system was optimised using an NADH-dependent CYP102A1 mutant, dodecanoic acid as a model substrate and takes advantage of randomly methylated beta-cyclodextrins for the solubilisation of hydrophobic substrates. In the reaction with 50 mM of dodecanoic acid a total turnover number of 66,700 and substrate conversion of 66.7 % could be reached. The total turnover number of a CYP102A1 mutant in the reaction with 23.4 mM (2R,4R,6R,8R)-tetramethyldecanol 4 was 17,290 and substrate conversion reached 74 %. The reaction on a preparative scale yielded 420 mg of (2R,4R,6S,8S)-tetramethyldecane-1,9-diol (3) in 60 % de. The major diastereomer 3a has the (2R,4R,6S,8S,9R)-configuration. The configuration of 3a was determined by an X-ray single crystal structure analysis of the corresponding bis-dinitrobenzoate 5a.