Enzymatic reduction for producing optically active alcohols
A process for preparing optically active alkanols of the formula I in which n is an integer from 0 to 5; Cyc is an optionally substituted, mono- or polynuclear, saturated or unsaturated, carbocyclic or heterocyclic ring, and R 1 is halogen, SH, OH, NO 2 , NR 2 R 3 or NR 2 R 3 R 4+ X − , with R 2 , R 3 and R 4 independently of one another being hydrogen or a lower alkyl or lower alkoxy radical and X − being a counterion, which process comprises incubating in a medium comprising alkanone of the formula II in which n, Cyc and R 1 are as defined above, an enzyme having a polypeptide sequence (i) SEQ ID NO: 1 or (ii) in which, compared to SEQ ID NO:1, up to 25% of the amino acid radicals have been altered by deletion, insertion, substitution or a combination thereof and which retains at least 50% of the enzymic activity of SEQ ID NO:1, with the compound of the formula II being enzymically reduced to give the compound of the formula I, and isolating the product formed.
1. A process for preparing an optically active alkanol of formula I
wherein
n is an integer from 0 to 5;
Cyc is an optionally substituted, mono- or polynuclear, saturated or unsaturated, carbocyclic or heterocyclic ring; and
R 1 is a halogen, SH, OH, NO 2 , NR 2 R 3 or NR 2 R 3 R 4+ X − , wherein R 2 , R 3 and R 4 independently are hydrogen or a lower alkyl or lower alkoxy radical, and X − is a counterion
comprising incubating the alkanone of formula II
in which n, Cyc and R 1 are as defined above,
in a medium comprising
(i) an enzyme comprising the amino acid sequence of SEQ ID NO: 1, or
(ii) an enzyme comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:1, and wherein the enzyme retains at least 50% of the dehydrogenase activity of the enzyme comprising the amino acid sequence of SEQ ID NO:1,
wherein the alkanone of formula II is enzymatically reduced to the compound of formula I;
and isolating the product formed.
2. The process of claim 1 , wherein the optically active alkanol of formula I is a 1-(2-thienyl)-(S)-propanol of formula III,
wherein R 1 is Cl or NHCH 3 ,
the alkanone of formula II is a 1-(2-thienyl)propanone of formula IV
wherein R 1 is as defined above,
and the isolated product is essentially enantiomerically pure.
3. The process of claim 1 , wherein the enzyme is isolated from a microorganism of the genus Lactobacillus.
4. The process of claim 1 , wherein the incubation further comprises adding reduction equivalents or regenerating reduction equivalents consumed in the reaction.
5. The process of claim 4 , further comprising regenerating cofactors with a C 2 -C 10 -alkanol.
6. The process of claim 1 , wherein the medium comprises a microorganism that has been transformed with a nucleic acid construct encoding an enzyme comprising
(i) the amino acid sequence of SEQ ID NO: 1, or
(ii) an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:1, and wherein the enzyme retains at least 50% of the dehydrogenase activity of the enzyme comprising the amino acid sequence of SEQ ID NO:1.
7. An isolated enzyme having dehydrogenase activity comprising
(i) the amino acid sequence of SEQ ID NO: 1, or
(ii) an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:1, and wherein the enzyme retains at least 50% of the dehydrogenase activity of the enzyme comprising the amino acid sequence of SEQ ID NO:1.
8. A method for preparing an optically active alkanol of formula I
wherein
n is an integer from 0 to 5;
Cyc is an optionally substituted, mono- or polynuclear, saturated or unsaturated, carbocyclic or heterocyclic ring; and
R 1 is a halogen, SH, OH, NO 2 , NR 2 R 3 or NR 2 R 3 R 4+ X − , wherein R 2 , R 3 and R 4 independently are hydrogen or a lower alkyl or lower alkoxy radical, and X − is a counterion
or an optically active alkanol of formula III
wherein R 1 is Cl or NHCH 3 ,
comprising incubating the alkanone of formula II
in which n, Cyc and R 1 are as defined for formula I above,
or the alkanone of formula IV
in which R 1 is as defined for formula III above,
with the enzyrne of claim 7 .
9. The method of claim 8 , further comprising additional conversion of the optically active alkanol of formula I to duloxetine.
10. The process of claim 6 , wherein the microorganism is selected from the group consisting of the bacterial families Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Streptomycetaceae and Nocardiaceae.