Methanophenazin: Strukturaufklärung und Totalsynthese eines neuartigen Cofaktors aus methanogenen Archaea

Abstract

Methanogenesis is the terminal step in the degradation of complex organic compounds and essential for the mineralization of organic material. The formation of methane from simple compounds such as H2 and CO2 , formic acid, methanol, methyamines or acetic acid is the characteristic feature of the strictly anaerobic methanogenic archaea. The metabolic pathways leading to the generation of methane are unique and involve a number of enzymes and co-factors that occur in methanogens only. This thesis reports on the purification, structure elucidation and total synthesis of Methanophenazine (1) the first phenazine derivative from methanogenic archea. Methanophenazine (1) was isolated from the cytoplasmic membranes of Methanosarcina mazei Gö1. HPLC-purification of the crude isooctane extracts of membranes provided small amounts of pure material. The elucidation of the structure is mainly based on NMR spectroscopic experiments (1H, 13C, H,H- and C,H-COSY) and mass spectrometry (EI, HRMS). The new natural compound 1 represents a linear sesterterpene ether of 2-hydroxy-phenazine (2). The lipophilic side chain consists of five isoprene units arranged in a head to tail manner. The C5 -unit directly connected to the aromatic system is fully saturated while the other isoprene units are unsaturated. They show (E)-configurated double bonds. The retrosynthetic analysis of 1 results in the building blocks 2-hydroxy-phenazine (2) and the terpenoid unit 3 which was divided into alkyl iodide 6 and (E)-vinyl iodide 7. Compound 2 was made accessible by condensation of 1,2,4-trihydroxybenzene (45) with o-phenylendiamine (5). In order to develop a stereodivergent approach to (R)-6 and (S)-6 , ethyl (R)-3-methylglutarate [(R)-11] was found to be suitable for the synthesis of both enantiomerically pure buildung blocks. First lactone (R)-12 was prepared by chemoselective reduction of the carboxyl group of (R)-11 which was then transformed to (R)-6 in enantiomerically pure form. The preparation of (S)-1 started with the transformation of (E,E )-farnesyl acetone (10) into the terminal alkyne 9. Carboalumination of 9 followed by a stereospecific quench of the resulting aluminium species with I2 afforded the (E)-vinyl iodide 7. A Pd(0) catalyzed sp2-sp3 cross coupling reaction of 7 with (R)-6 delivered alkohol (S)-78 after deprotection in enantio- and diastereomerically pure form. Etherification of 2-hydroxy-phenazine (2) with (S)-78 yielded the target molecule (S )-methanophenazine [(S)-1]. The spectral data for the synthetic methanophenazine are identical to those of the natural product. Since lactone (S)-12 can also be prepared from ethyl (R )-3-methylglutarate [(R)-11] by chemoselective reduction of its ester group this approach will also allow for the synthesis of (R )-methanophenazine [(R)-1]. In conclusion, methanophenazine [(S)-1] has been synthesized using a stereodivergent approach from three building blocks, i.e. 2-hydroxy-phenazine (2), ethyl (R)-3-methylglutarate [(R)-11] and (E,E)-farnesyl acetone (10) in a highly convergent manner. First experiments on the biological function of 1 show that the molecule is able to mediate the electron transport between membrane-bound enzymes in washed cytoplasmic membranes of Methanosarcina mazei Gö1. In this way Methanophenazine (1) was characterized as the first phenazine derivative involved in the electron transport of biological systems. These results suggest that its role in the energy metabolism of methanogens is similar to that of ubiquinone in mitochondria and bacteria.