Biocatalytic reduction of nitro groups
AssigneeDiversa Corporation
InventorsWilliam Greenberg, David Paul Weiner, Brian Adger, Mark J. Burk
Life of this patent
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Abstract
The invention provides methods of discovering nitroreductases and biocatalytic methods for reducing compounds comprising a nitro group.
Claims76
A biocatalytic method for reducing a compound comprising a nitro group, the method comprising the following steps:
- (a) providing a nitroreductase;
- (b) providing a compound comprising a nitro group;
- and
- (c) contacting the nitroreductase with the compound under conditions wherein the nitro group is reduced to an amine group.
A method for selecting a nitroreductase that can catalyze the reduction of a nitro group to its corresponding amine comprising the following steps:
- (a) providing a test sample comprising a polypeptide;
- (b) providing a compound comprising a nitro group;
- and
- (c) contacting the test sample with the compound comprising a nitro group and detecting the generation of a corresponding amine, wherein the generation of the corresponding amine indicates the presence of a nitroreductase in the test sample.
A method for selecting a nitroreductase that can catalyze the reduction of an aliphatic nitro group comprising the following steps:
- (a) providing a test sample comprising a polypeptide;
- (b) providing a 4-nitrobuylamine or a 1,4-dinitro-butane;
- and
- (c) contacting the test sample with the 4-nitrobuylamine or the 1,4-dinitrobutane and detecting the generation of putrescine, wherein the generation of putrescine indicates the presence of a nitroreductase in the test sample.
The method for selecting a nitroreductase that can catalyze the reduction of an aliphatic nitro group comprising the following steps:
- (a) providing a test sample comprising a polypeptide;
- (b) providing a 4-nitrobuylamine or a 1,4-dinitro-butane;
- and
- (c) contacting the test sample with the 4-nitrobuylamine or the 1,4-dinitrobutane in an in vivo system or equivalent system and detecting the generation of succinate, wherein the generation of succinate indicates the presence of a nitroreductase in the test sample.
A method for selecting a nitroreductase that can catalyze the reduction of an aliphatic nitro group comprising the following steps:
- (a) providing a test sample comprising a polypeptide;
- (b) providing a 2-nitro-benzoic acid;
- and
- (c) contacting the test sample with the 2-nitro-benzoic acid system and detecting the generation of anthranilic acid, wherein the generation of anthranilic acid indicates the presence of a nitroreductase in the test sample.
A method for selecting a nitroreductase that can catalyze the reduction of an aliphatic nitro group comprising the following steps:
- (a) providing a test sample comprising a polypeptide;
- (b) providing a 2-nitro-benzoic acid;
- and
- (c) contacting the test sample with the 2-nitro-benzoic acid and detecting the generation of anthranilic acid, wherein the generation of anthranilic acid indicates the presence of a nitroreductase in the test sample.
A method for selecting a nitroreductase that can catalyze the reduction of an aliphatic nitro group comprising the following steps:
- (a) providing a test sample comprising a polypeptide;
- (b) providing a 2-nitro-benzoic acid;
- and
- (c) contacting the test sample with the 2-nitro-benzoic acid in an in vivo system or equivalent system and detecting the generation of tryptophan, wherein the generation of tryptophan indicates the presence of a nitroreductase in the test sample.
A method for selecting a nitroreductase that can catalyze the reduction of an aliphatic nitro group comprising the following steps:
- (a) providing a test sample comprising a polypeptide;
- (b) providing a nitroacid;
- and
- (c) contacting the test sample with the nitroacid and detecting the generation of an amino acid, wherein the generation of the amino acid indicates the presence of a nitroreductase in the test sample.
A method for selecting a nitroreductase that can catalyze the reduction of a halogenated nitroaromatic compound comprising the following steps:
- (a) providing a test sample comprising a polypeptide;
- (b) providing a halogenated nitroaromatic compound;
- and
- (c) contacting the test sample with the halogenated nitroaromatic compound and detecting the generation of a corresponding amine, wherein the generation of the corresponding amine indicates the presence of a nitroreductase in the test sample.
A method for selecting a nitroreductase that can catalyze the reduction of a styrene comprising the following steps:
- (a) providing a test sample comprising a polypeptide;
- (b) providing a styrene;
- and
- (c) contacting the test sample with the styrene and detecting the generation of a corresponding amine, wherein the generation of the corresponding amine indicates the presence of a nitroreductase in the test sample.
A method for selecting a nitroreductase that can catalyze the reduction of an aliphatic nitrocompound comprising the following steps:
- (a) providing a test sample comprising a polypeptide;
- (b) providing an aliphatic nitrocompound;
- and
- (c) contacting the test sample with the aliphatic nitrocompound and detecting the generation of a corresponding amine, wherein the generation of the corresponding amine indicates the presence of a nitroreductase in the test sample.
A method for selecting a nitroreductase that can catalyze the reduction of an asymmetric nitroaldol comprising the following steps:
- (a) providing a test sample comprising a polypeptide;
- (b) providing an asymmetric nitroaldol;
- (c) contacting the test sample with the asymmetric nitroaldol and detecting the generation of a corresponding amine, wherein the generation of the corresponding amine indicates the presence of a nitroreductase in the test sample.
A method for chemoselective reduction of a nitro group, the method comprising the following steps:
- (a) providing a nitroreductase;
- (b) providing a nitro group;
- and
- (c) contacting the nitroreductase with the nitro group under conditions wherein the nitroreductase catalyzes the reduction of the nitro group to an amine group.
A biocatalytic method for making a substituted aniline, the method comprising the following steps:
- (a) providing a nitroreductase;
- (b) providing a nitroaromatic compound;
- and
- (c) contacting the nitroreductase with the nitroaromatic compound under conditions wherein the nitroreductase catalyzes the reduction of the nitro group to an amine group and a substituted aniline is generated.
A biocatalytic method for reducing a styrene, the method comprising the following steps:
- (a) providing a nitroreductase;
- (b) providing a styrene;
- and
- (c) contacting the nitroreductase with the styrene under conditions wherein the nitroreductase catalyzes the reduction of the nitro group to an amine group and the styrene is reduced to its corresponding amine.
A biocatalytic method for making a chiral amine, the method comprising the following steps:
- (a) providing a nitroreductase;
- (b) providing a racemic nitroalkane compound;
- and
- (c) contacting the nitroreductase with the nitroalkane under conditions wherein the nitroreductases reduces the nitroalkane to generate a chiral amine.
A biocatalytic method for making putrescine, the method comprising the following steps:
- (a) providing a nitroreductase;
- (b) providing a 4-nitrobuylamine or a 1,4-dinitro-butane;
- and
- (c) contacting the nitroreductase with the 4-nitrobuylamine or 1,4-dinitrobutane under conditions wherein the nitroreductases reduces the 4-nitrobuylamine or 1,4-dinitro-butane to generate a putrescine.
A method for selecting a nucleic acid encoding a nitroreductase, the method comprising the following steps:
- (a) providing a plurality of nucleic acids;
- (b) expressing the nucleic acids in a system comprising a nitro compound;
- and
- (c) detecting the system where the nitro compound has been reduced to its corresponding amine, thereby selecting a nucleic acid encoding a nitroreductase.
A method for making a nucleic acid encoding a nitroreductase capable of catalyzing reduction of a nitro compound in a set of conditions, the method comprising the following steps:
- (a) providing a nucleic acid encoding a first nitroreductase;
- (b) modifying the nitroreductase-encoding nucleic acid to generate a plurality of modified nucleic acids;
- (c) expressing the modified nucleic acids in a system comprising a nitro compound in the set of conditions and detecting if the nitro compound has been reduced to its corresponding amine;
- and
- (d) detecting which modified nucleic acid encoded the nitroreductases that catalyzed the reduction of the nitro compound to its corresponding amine, thereby making a nucleic acid encoding a modified nitroreductase capable of catalyzing reduction of a nitro compound in a set of conditions.
A method for making a nitroreductase capable of catalyzing reduction of a nitro compound in a set of conditions, the method comprising the following steps:
- (a) providing a nucleic acid encoding a first nitroreductase;
- (b) modifying the nitroreductase-encoding nucleic acid to generate a plurality of modified nucleic acids;
- (c) expressing the modified nucleic acids in a system comprising a nitro compound in the set of conditions and detecting if the nitro compound has been reduced to its corresponding amine;
- and
- (d) detecting which nitroreductase catalyzed the reduction of the nitro compound to its corresponding amine, thereby making a modified nitroreductase capable of catalyzing reduction of a nitro compound in a set of conditions.
Dependencies are inferred from the claim text.