A two-step synthesis of ephedrine derivatives catalyzed by immobilized ThDP-dependent oxidoreductase and ω-transaminase

The research described in this thesis was carried out at “Istituto di Scienze e Tecnologie Chimiche (SCITEC)” of CNR (Milan) and at “Laboratorio di Biocatalisi” of the University of Pavia. The aim of this research was to synthesize (1S,2S)-norpseudoephedrine or (1S,2R)-norephedrine, through a bi-enzymatic reaction catalyzed by S-selective or R-selective ω-transaminases (E.C. 2.6.1.x), respectively, starting from S-phenylacetyl-carbinole (S-PAC). On the other hand, S-PAC can be synthesized by a stereocontrolled reaction catalyzed by the ThDP-dependent acetoin:dichlorophenolindophenol oxidoreductase (Ao:DCPIP OR, E.C. 2.3.1.190) from Bacillus licheniformis. This enzyme is the first example of S-selective ThDP-dependent oxidoreductase reported to date. (1S,2S)-Norpseudoephedrine (also called cathine) and (1S,2R)-norephedrine are two out of the four isomers of nor(pseudo)ephedrine that also include (1R,2R)-norpseudoephedrine and (1R,2S)-norephedrine. These compounds are used as nasal decongestants, appetite suppressants, blood pressure stabilizers and mydriatic agents. Most interestingly, from the organic synthesis viewpoint, ephedrine derivatives are valuable chiral building blocks. The first part of the research was focused on the screening of a collection of S- and R-selective transaminases against S-PAC using (S)- or (R)-alfa-methylbenzyl amine (MBA) as the amine donor, respectively. To this aim, 11 ω-transaminases (8 S-selective and 3 R-selective enzymes) were expressed, purified and characterized about their protein concentration and activity by a standard spectrophotometric assay before their use as biocatalysts in the biotransformation of S-PAC under different reaction conditions. The ω-transaminase from Streptomyces sp., namely Bv333, was selected for this reaction step and submitted to immobilization trials by using both an hydrophilic aldehyde-activated carrier (glyoxyl-agarose) and hydrophobic acrylic epoxy-activated carriers (Sepabeads™ EC-EP/S and Eupergit® C). In the last stage of the internship, preliminary immobilization trials were also carried out for Ao:DCPIP OR (glyoxyl-agarose). Both immobilized enzymes were successfully used in the planned biotransformation. Results obtained to date pave the way for the use of these biocatalysts in the preparative synthesis of (1S,2S)-norpseudoephedrine and (1S,2R)-norephedrine following a linear cascade mode that could be further implemented by setting-up a continuous flow system.