Process for the enantioselective enzymatic reduction of secodione derivatives

Abstract

The invention relates to a process for the enantioselective enzymatic reduction of secodione derivatives. The secodione derivative is reduced with an oxidoreductase/dehydrogenase in the presence of NADH or NADPH as a cofactor. The secodione derivative is used in the reaction batch at a concentration of ≧10 g/l and the oxidized cofactor NAD or NADP formed by the oxidoreductase/dehydrogenase is regenerated continuously.

1 . A process for the enantioselective enzymatic reduction of secodione derivatives of general formula I 
       
         
           
           
               
               
           
         
         wherein the ring structures comprise no, one or several heteroatoms, 
         R 1  is hydrogen or a C 1 -C 4  alkyl group, 
         R 2  is hydrogen, a C 1 -C 8  alkyl group or a protective group for OH known in prior art, such as an ester, 
         R 3  is hydrogen, a methyl group or a halide, 
         the structural element 
       
       
         
           
           
               
               
           
         
         represents a benzene ring or a C 6  ring having 0, 1 or 2 C—C double bonds, 
         a double bond is optionally included at positions 6/7 or 7/8, and 
         the carbon at positions 1, 2, 4, 5, 6, 7, 8, 9, 11, 12 and 16 is independently substituted with hydrogen, a C 1 -C 4  alkyl group, a halide or a phenyl group, 
         wherein the secodione derivative is reduced with an oxidoreductase/dehydrogenase in the presence of NADH or NADPH as a cofactor, 
         characterized in that the secodione derivative is used in the reaction batch at a concentration of ≧10 g/l and the oxidized cofactor NAD or NADP formed by the oxidoreductase/dehydrogenase is regenerated continuously. 
       
     
     
         2 . A process for the enantioselective enzymatic reduction of secodione derivatives of general formula I, wherein the secodione derivative is reduced with an oxidoreductase/dehydrogenase in the presence of NADH or NADPH as a cofactor, characterized in that the oxidoreductase/dehydrogenase
 a) comprises an amino acid sequence in which at least 50% of the amino acids are identical to those of amino acid sequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5,   b) is encoded by the nucleic acid sequence SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10, or   c) is encoded by a nucleic acid sequence which hybridizes to SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10 under stringent conditions.   
     
     
         3 . A process for the enantioselective enzymatic reduction of secodione derivatives of general formula I, wherein the secodione derivative is reduced with an oxidoreductase/dehydrogenase in the presence of NADH or NADPH as a cofactor, characterized in that the oxidoreductase/dehydrogenase has a length of from 230 to 260 amino acids and comprises one or several of the partial sequences selected from the group consisting of [sequences SEQ ID NO:18 to SEQ ID NO:42]
 nalvtgasrgig, nalvtggsrgig, nalitggsrgig, nalitgasrgig, nalitggsrgmg, halvtgasrgig,   gysvtla, gynvtla, gysvtlv, gynvtlv,   fkgaplpa, fkaaplpa,   fvsnag, ffsnag, fvcnag, fvanag,   spialtkal, spvaltkti, spialtktl, spvamtkal, sqialtkal,   avysask, avysatk,   pikgwi and pisgwi.   
     
     
         4 . A process according to  claim 1 , characterized in that the oxidoreductase/dehydrogenase
 a) comprises an amino acid sequence in which at least 50% of the amino acids are identical to those of amino acid sequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5,   b) is encoded by the nucleic acid sequence SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10, or   c) is encoded by a nucleic acid sequence which hybridizes to SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10 under stringent conditions.   
     
     
         5 . A process according to  claim 1 , characterized in that the oxidoreductase/dehydrogenase has a length of from 230 to 260 amino acids and comprises one or several of the partial sequences selected from the group consisting of [sequences SEQ ID NO:18 to SEQ ID NO:42]
 nalvtgasrgig, nalvtggsrgig, nalitggsrgig, nalitgasrgig, nalitggsrgmg, halvtgasrgig,   gysvtla, gynvtla, gysvtlv, gynvtlv,   fkgaplpa, fkaaplpa,   fvsnag, ffsnag, fvcnag, fvanag,   spialtkal, spvaltkti, spialtktl, spvamtkal, sqialtkal,   avysask, avysatk,   pikgwi and pisgwi.   
     
     
         6 . A process according to  claim 2  or  3 , characterized in that the oxidized cofactor NAD or NADP formed by the oxidoreductase/dehydrogenase is regenerated continuously. 
     
     
         7 . A process according to any of  claims 1  to  3 , characterized in that the oxidized cofactor NAD or NADP is regenerated by oxidation of an alcohol. 
     
     
         8 . A process according to  claim 7 , characterized in that a secondary alcohol having the general formula R X R Y CHOH is used for cofactor regeneration, wherein R X  and R Y  independently of each other are hydrogen, a branched or unbranched C 1 -C 8  alkyl group and C total ≧3. 
     
     
         9 . A process according to  claim 7 , characterized in that an alcohol from the group consisting of 2-propanol, 2-butanol, 2-pentanol, 4-methyl-2-pentanol, 2-hexanol, 2-heptanol, 5-methyl-2-hexanol, cyclohexanol or 2-octanol is used for cofactor regeneration. 
     
     
         10 . A process according to any of  claims 1  to  3 , characterized in that an oxidoreductase/dehydrogenase is additionally added for the regeneration of the cofactor. 
     
     
         11 . A process according to any of  claims 1  to  3 , characterized in that the TTN (total turn over number=mol of reduced secodione derivative/mol of cofactor used) is ≧10 3 . 
     
     
         12 . A process according to any of  claims 1  to  3 , characterized in that it is carried out in an aqueous organic two-phase system. 
     
     
         13 . A process according to any of  claims 1  to  3 , characterized in that an organic solvent, preferably diethyl ether, tertiary butyl methyl ether, diisopropyl ether, dibutyl ether, ethyl acetate, butyl acetate, heptane, hexane, toluene, dichloromethane, cyclohexane or mixtures thereof, is additionally used. 
     
     
         14 . A process according to any of  claims 1  to  3 , characterized in that the secodione derivative is used in the reaction batch in an amount of from 10 g/l to 500 g/l, based on the total volume, preferably from 25 g/l to 300 g/l, particularly preferably from 50 g/l to 200 g/l. 
     
     
         15 . A process according to any of  claims 1  to  3 , characterized in that 13-ethyl-3-methoxy-8,14-seco-gona-1,3,5(10),9(11)-tetraene-14,17-dione (ethyl secodione—Formula III) is used as the secodione derivative. 
     
     
         16 . A process according to any of  claims 1  to  3 , characterized in that 13-methyl-3-methoxy-8,14-seco-gona-1,3,5(10),9(11)-tetraene-14,17-dione (methyl secodione—Formula II) is used as the secodione derivative.