Process for the stereoselective enzymatic reduction of keto compounds

In a process for the stereoselective, in particular enantioselective enzymatic reduction of keto compounds to the corresponding chiral hydroxy compounds, wherein the keto compounds are reduced with an enantioselective, NADH-specific oxidoreductase, a polypeptide is used for reducing the keto compounds, which polypeptide exhibits an R-ADH-signature H-[P; A]-[I; A; Q; V; L]-[G; K]-R at positions 204-208 and the following further structural features in their entirety: (i) an N-terminal Rossmann-Fold GxxxGxG, (ii) an NAG-motif at position 87, (iii) a catalytic triad consisting of S 139, Y 152 and K 156, (iv) a negatively charged amino acid moiety at position 37, (v) two C-terminal motifs in the dimerization domain [A; S]-S-F and [V; I]-DG-[G; A]-Y-[T; C; L]-[A; T; S]-[Q; V; R; L; P], (vi) Val or Leu at position 159 (4 positions downstream of K 156), (vii) Asn at position 178, and (viii) a proline moiety at position 188.

Novel polypeptide having esterase activity and recombinant esterase and use thereof

Polypeptide and recombinant protein having esterase activity which exhibit the amino acid sequence SEQ. ID. No. 1 and the use thereof.

Novel amidase, gene for the same, vector, transformant, and method for production of optically active carboxylic acid amide and optically active carboxylic acid by using any one of those items

The present invention has its object to provide a novel polypeptide having amidase activity to selectively hydrolyze S-enantiomer in racemic nipecotamide, a DNA encoding the polypeptide, a vector containing the DNA, a transformant transformed with the vector, and a method for producing an optically active carboxylic acid amide and an optically active carboxylic acid in which a racemic carboxylic acid amide is hydrolyzed with the polypeptide or the transformant.

Process for Producing Aliskiren

A new route of synthesis of the compound Aliskiren of formula (I), used in the treatment of hypertension, is described.

ENZYMATIC RESOLUTION OF RACEMIC (2R,S)-2-(ACETYLAMINO)-3-METHOXY-N-(PHENYLMETHYL)PROPANAMIDE

The present invention is concerned with a process of preparing (R)-lacosamide. The process comprises providing an (R,S)-lacosamide precursor and contacting the same with at least an enzyme in the presence of a solvent. The enzyme either stereoselectively hydrolyzes or acetylates an (R)- or (S)-enantiomer of the (R,S)-lacosamide precursor. The process further comprises where appropriate also concurrently, or successively, employing one or more reagents capable of converting the hydrolysed or acetylated (R)- or (S)-enantiomer to (R)-lacosamide. 140.-. (canceled)42. The process of claim 41 , wherein step (ii) comprises contacting the (R claim 41 ,S)-compound of formula (II) with one or more enzymes in the presence of a solvent and optionally an acetyl donor compound to form an enantiomerically enriched or enantiomerically pure (R)-enantiomer of the compound formula (II) wherein Ris CHC(═O)— claim 41 ,{'sub': '1', 'wherein the one or more enzymes is capable of stereoselectively hydrolyzing the (R)- or (S)-enantiomer of a compound of formula (II) when the solvent is a protic solvent or is capable of stereoselectively acetylating the (R)-enantiomer of a compound of formula (II) when Ris a first intermediate moiety and when an acetyl donor compound is present, and'}{'sub': '2', 'wherein step (iii) comprises optionally further comprising contacting the (R,S)-compound of formula (II) or the enriched or enantiomerically pure (R)-enantiomer of the compound formula (II) with one or more reagents capable of converting a second intermediate moiety into —NHCHPh.'}45. The process of claim 41 , wherein the enzyme capable of stereoselectively acetylating either the (R)- or (S)-enantiomer of a compound of formula (II) is a lipase.46Candida antarcticaCandida antarcticaPseudomonas cepacia. The process of claim 45 , wherein the lipase is selected from the group consisting of lipase A (CAL-A) claim 45 , lipase B (CAL-B) claim 45 , and lipase.47. The process of claim 43 , wherein the ...

NOVEL BACTERIAL STRAIN OF ACHROMOBACTER SP. MTCC 5605 AND A HIGHLY ENANTIOSELECTIVE EPOXIDE HYDROLASE ISOLATED THEREFROM

The present invention relates to a novel epoxide hydrolase enzyme which aims to achieve a high degree of resolution towards a broader range of substrates with high enantioselectivity and yields with minimal product inhibition. The invention further relates to a new bacterial strain sp. MTCC 5605 isolated from a petroleum-contaminated sludge sample, capable of producing the said enzyme. It is notable that the enzyme can be used as whole bacterial cell preparation, which allows continuous hydrolysis of substrates at even higher concentration and have an advantage of being recycled. The invention further relates to a process for the hydrolysis of different aryl epoxides which are potential synthons of intermediates for the synthesis of chiral amino alcohols and bioactive compounds like β-blockers. 1Achromobacter. A highly enantioselective epoxide hydrolase from sp. MTCC 5605 , having molecular weight of 95 KDa , active in the pH range of 5 to 11 and temperature ranging between 30 to 50 degree C. , exhibiting hydrolysis of racemic aryl epoxides with >99% eeat substrate concentration up to 500 mM , yielding up to 42% of 100% optically pure aryl epoxides with a final concentration of up to 209 mM.2. The epoxide hydrolase as claimed in claim 1 , wherein it is highly enantioselective towards pharmaceutically important aryl epoxides selected from the group consisting of styrene oxide claim 1 , benzyl glycidyl ether claim 1 , phenyl glycidyl ether claim 1 , methoxyphenyl glycidyl ether claim 1 , limonene epoxide claim 1 , phenyl ethyl glycidate and indene oxide claim 1 , which yields valuable synthons for chiral amino acids and β-blocker drugs.3. A process for the resolution of racemic aryl epoxides using the epoxide hydrolase as claimed in claim 1 , comprising the steps of:{'i': 'Achromobacter', '(a) incubating an enantiomeric mixture of epoxide along with the whole resting cells of sp. MTCC 5605 containing an active epoxide hydrolase at a temperature of 35 to 40 degree C. ...

KETOREDUCTASE POLYPEPTIDES FOR THE SYNTHESIS OF CHIRAL COMPOUNDS

The present disclosure provides engineered ketoreductase enzymes having improved properties as compared to a naturally occurring wild-type ketoreductase enzyme. Also provided are polynucleotides encoding the engineered ketoreductase enzymes, host cells capable of expressing the engineered ketoreductase enzymes, and methods of using the engineered ketoreductase enzymes to synthesize a variety of chiral compounds. 1. An engineered polypeptide comprising an amino acid sequence with at least 80% sequence identity to SEQ ID NO:2 and at least one substitution at a position selected from X68 , X94 , X102 , X110 , X114 , X135 , X144 , X145 , X147 , X149 , X150 , X153 , X158 , X173 , X175 , X190 , X196 , X197 , X198 , X199 , X201 , X202 , X203 , X205 , X206 , X207 , X209 , X210 , X211 , X212 , X213 , X217 , X233 , X249 , X250 , and X252 , and wherein said polypeptide has greater 2a:2c selectivity as compared to SEQ ID NO:2.2. The engineered polypeptide of claim 1 , wherein said amino acid sequence comprises at least one of the following:the residue corresponding to X68 is a non-polar, or aliphatic residue;the residue corresponding to X94 is a polar, or non-polar residue;the residue corresponding to X102 is an acidic residue;the residue corresponding to X110 is an acidic, or aromatic residue;the residue corresponding to X114 is a non-polar residue;the residue corresponding to X135 is a basic residue;the residue corresponding to X144 is a non-polar, or aromatic residue;the residue corresponding to X145 is an aromatic residue;the residue corresponding to X147 is a non-polar, or aliphatic residue;the residue corresponding to X149 is a polar residue;the residue corresponding to X150 is an aromatic, or acidic residue;the residue corresponding to X153 is a polar, or aromatic residue;the residue corresponding to X158 is a non-polar, or aliphatic residue;the residue corresponding to X173 is a non-polar, or aliphatic residue;the residue corresponding to X175 is a polar residue;the ...

BIOCATALYSTS AND METHODS FOR THE SYNTHESIS OF ARMODAFINIL

The present invention relates to non-naturally occurring polypeptides useful for preparing armodafinil, polynucleotides encoding the polypeptides, and methods of using the polypeptides. The non-naturally occurring polypeptides of the present invention are effective in carrying out biocatalytic conversion of the (i) 2-(benzhydrylsulfinyl)acetamide to (−)-2-[(R)-(diphenylmethyl)sulfinyl]acetamide (armodafinil), or (ii) benzhydryl-thioacetic acid to (R)-2-(benzhydrylsulfinyl)acetic acid, which is a pivotal intermediate in the synthesis of armodafinil, in enantiomeric excess. 1. A non-naturally occurring polynucleotide encoding a non-naturally occurring polypeptide having cyclohexanone monooxygenase (CHMO) activity wherein the amino acid sequence of the polypeptide has at least 90% sequence identity to SEQ ID NO: 136 , and one or more amino acid substitutions compared to the naturally occurring polypeptide at one or more positions corresponding to positions in SEQ ID NO: 136 , selected from the group consisting of 75 , 79 , 82 , 99 , 110 , 166 , 172 , 208 , 216 , 273 , 324 , 364 , 395 , 412 , 491 , 503 , and 504.3. The non-naturally occurring polynucleotide encoding the non-naturally occurring polypeptide of claim 1 , wherein said non-naturally occurring polypeptide further comprises one or more amino acid substitutions relative to SEQ ID NO: 136 claim 1 , wherein the polypeptide comprises an alanine claim 1 , glutamic acid claim 1 , glycine claim 1 , isoleucine claim 1 , lysine claim 1 , proline claim 1 , serine claim 1 , threonine claim 1 , or valine at a position corresponding to position 246 of SEQ ID NO: 136.5. The non-naturally occurring polynucleotide encoding the non-naturally occurring polypeptide of claim 1 , wherein said non-naturally occurring polypeptide further comprises one or more amino acid differences relative to SEQ ID NO: 136 claim 1 , wherein said polypeptide further comprises one or more substitutions corresponding to substitutions in SEQ ID NO: ...

BIOCATALYSTS FOR THE PREPARATION OF HYDROXY SUBSTITUTED CARBAMATES

The present disclosure relates to engineered ketoreductase polypeptides for the preparation of hydroxyl substituted carbamate compounds, and polynucleotides, vectors, host cells, and methods of making and using the ketoreductase polypeptides. 2. The engineered ketoreductase polypeptide of claim 1 , wherein the amino acid sequence of said ketoreductase polypeptide comprises the substitution X40R claim 1 , and one or more residue differences as compared to SEQ ID NO:4 selected from: X7S; X17M; X17Q; X17R; X23V; X27L; X29G; X60I; X64V; X71P; X87L; X94A; X94P; X94S; X95M; X105G; X113I; X122A; X127R; X131S; X144V; X145L; X147I; X147L; X147Q; X150Y; X152G; X153G; X157C; X173L; X196M; X198S; X208R; X216R; X221S; X243S; X245I; X249F; X249G; and X249Y.3. The engineered ketoreductase polypeptide of claim 1 , wherein the amino acid sequence of said ketoreductase polypeptide comprises X40R claim 1 , and at least one or more residue differences as compared to SEQ ID NO:4 selected from: X17Q/R/M; X64V; X94P; X96L/Y; X144V; X147Q/UL; X157C; X195A/G; X196M; X199H; and X206L/F.4. The engineered ketoreductase polypeptide of claim 1 , wherein the ketoreductase polypeptide is capable of converting the substrate compound (2) to the product compound (1) with at least 10 fold the activity of the reference polypeptide of SEQ ID NO:4 claim 1 , wherein the amino acid sequence comprises the substitution X40R claim 1 , and one or more residue differences as compared to SEQ ID NO:4 selected from: X601; X71P; X94P; X94A; X95M; X96L; X96Y; X127R; X144V; X145I; X150Y; X152G; X153G; X157C; X195A; X195G; X196M; X198S; X199H; X206F/L claim 1 , X216R claim 1 , X2451 claim 1 , X245F; X249Y; and X249F.5. The engineered ketoreductase polypeptide of claim 1 , wherein the ketoreductase polypeptide has increased thermal stability as compared to the reference polypeptide of SEQ ID NO:4 or 32 claim 1 , wherein the amino acid sequence comprises the substitution X40R claim 1 , and one or more residue ...

PROCESS FOR PREPARING (CYCLOPENTYL[d]PYRIMIDIN-4-YL)PIPERAZINE COMPOUNDS

The present disclosure relates to processes for preparing (cyclopentyl[d]pyrimidin-4-yl)piperazine compounds, and more particularly relates to processes for preparing (R)-4-(5-methyl-7-oxo-6,7-dihydro-5H-cyclopenta[d] pyrimidin-4-yl)piperazine and N-protected derivatives thereof, which may be used as an intermediate in the synthesis of Ipatasertib (i.e., (S)-2-(4-chlorophenyl)-1-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-(isopropylamino)-propan-1-one). The present disclosure additionally relates to various compounds that are intermediates employed in these processes. 2. The process of claim 1 , wherein the cyclization is achieved by contacting the compound or salt of Formula VIwith a formamidine salt.3. The process of claim 1 , wherein the enzymatic resolution is achieved by contacting the isomeric mixture comprising the compound of Formula VIand Formula VI claim 1 , or salts thereof claim 1 , with a nitrilase enzyme or a lipase enzyme.4. The process of claim 3 , wherein the enzymatic resolution is achieved by contacting the isomeric mixture comprising the compound of Formula VIand Formula VI claim 3 , or salts thereof claim 3 , with a nitrilase enzyme.5. The process of claim 3 , wherein the enzymatic resolution is achieved by contacting the isomeric mixture comprising the compound of Formula VIand Formula VI claim 3 , or salts thereof claim 3 , with a lipase enzyme.6. The process of any one of to claim 3 , wherein the steps (ii) and (iii) are performed through-process.7. The process of claim 1 , wherein step (ii) is performed at a pH of about 7.8. The process of claim 1 , wherein step (ii) is performed at a pH of about 9. This application is a continuation of U.S. patent application Ser. No. 15/514,188, filed Mar. 24, 2017, which is a national phase entry of International Patent Application No. PCT/US2015/052143, filed Sep. 25, 2015, which claims the benefit of priority U.S. Provisional Application No. 62/055,893, ...

Ketoreductase polypeptides

The present disclosure provides engineered ketoreductase enzymes having improved properties as compared to a naturally occurring wild-type ketoreductase enzyme including the capability of reducing 5-((4S)-2-oxo-4-phenyl (1,3-oxazolidin-3-yl))-1-(4-fluorophenyl) pentane-1,5-dione to (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one. Also provided are polynucleotides encoding the engineered ketoreductase enzymes, host cells capable of expressing the engineered ketoreductase enzymes, and methods of using the engineered ketoreductase enzymes to synthesize the intermediate (4S)-3-[(5S)-5 -(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one in a process for making Ezetimibe.

ENZYMATIC PROCESS FOR THE PREPARATION OF (1S,2R)-2-(DIFLUOROMETHYL)-1-(PROPOXYCARBONYL)CYCLOPROPANECARBOXYLIC ACID

Disclosed are methods of synthesizing enantioenriched difluoroalkylcyclopropyl amino esters and their salts, such as the dicyclohexylamine salt of (1S,2R)-2-(difluoromethyl)-1-(propoxycarbonyl)cyclopropane carboxylic acid. These compounds are useful intermediates in the synthesis of viral protease inhibitors. 157-. (canceled)59. The method of claim 58 , wherein R is ethyl claim 58 , propyl claim 58 , or butyl.60. The method of claim 59 , wherein R is n-propyl.62. The method of claim 58 , wherein the hydrolysis step comprises treatment with aqueous LiOH.65. The compound of claim 64 , wherein R is ethyl claim 64 , propyl claim 64 , or butyl.66. The compound of claim 65 , wherein R is n-propyl.69. The compound of claim 68 , wherein R is ethyl claim 68 , propyl claim 68 , or butyl.70. The compound of claim 69 , wherein R is n-propyl.72. The compound of claim 71 , wherein R is ethyl claim 71 , propyl claim 71 , or butyl.73. The compound of claim 72 , wherein R is n-propyl.75. The compound of claim 74 , wherein R is ethyl claim 74 , propyl claim 74 , or butyl.76. The compound of claim 75 , wherein R is n-propyl. This application is a continuation of U.S. patent application Ser. No. 15/010,557, filed Jan. 29, 2016, now U.S. Pat. No. 10,316,338, which claims the benefit of priority to U.S. Provisional Patent Application Ser. No. 62/109,943, filed Jan. 30, 2015.Complex biologically active molecules are challenging, expensive, and time-consuming to synthesize. Synthesizing chiral, non-racemic compounds with good enantio- and diastereoselectivity is even more challenging. Doing so generally involves isolating or synthesizing an enantioenriched intermediate whose stereochemistry can be preserved in the required subsequent synthetic transformations.An example of a useful intermediate in the synthesis of a biologically active molecule is (1R,2R)-1-((tert-butoxycarbonyl)amino)-2-(difluoromethyl)cyclopropanecarboxylic acid (1, Boc-DFAA). In the past, this intermediate was ...

NOVEL HYDROLASE AND METHOD FOR PRODUCING (1S,2S)-1-ALKOXYCARBONYL-2-VINYLCYCLOPROPANE CARBOXYLIC ACID USING SAME

The present invention provides a novel hydrolase that can industrially produce optically highly pure (1S,2S)-1-alkoxycarbonyl-2-vinylcyclopropane carboxylic acid with high efficiency at low costs, and a production method using the hydrolase. 2. The hydrolase according to claim 1 , wherein R in the formula (1) is an ethyl group.3. A nucleic acid encoding hydrolase according to or .5. A method for producing (1S claim 1 ,2S)-1-alkoxycarbonyl-2-vinylcyclopropane carboxylic acid claim 1 , comprising bringing the hydrolase according to or claim 1 , a microorganism or cell having an ability to produce the aforementioned enzyme claim 1 , a treated product of the aforementioned microorganism or cell claim 1 , and/or a culture solution containing the aforementioned enzyme obtained by culturing the aforementioned microorganism or cell into contact with dialkyl 2-vinylcyclopropane-1 claim 1 ,1-dicarboxylic acid represented by the formula (I) to produce (1S claim 1 ,2S)-1-alkoxycarbonyl-2-vinylcyclopropane carboxylic acid represented by the formula (II).6. The production method according to claim 1 , wherein the aforementioned microorganism or cell is a microorganism or cell transformed with the nucleic acid according to or .7. The recombinant vector comprising the nucleic acid according to or .8. A transformant comprising the recombinant vector according to . The present invention relates to a novel hydrolase (esterase) that can be used for the production of (1S,2S)-1-alkoxycarbonyl-2-vinylcyclopropane carboxylic acid and use thereof.(1S,2S)-1-alkoxycarbonyl-2-vinylcyclopropane carboxylic acid is an intermediate useful for the production of various HCV NS3 protease inhibitors under development as a therapeutic drug for hepatitis C and the like.Non-patent document 1, patent document 1 and patent document 2 describe a method for obtaining (1S,2S)-1-methoxycarbonyl-2-vinylcyclopropane carboxylic acid by hydrolyzing dimethyl 2-vinylcyclopropane-1,1-dicarboxylic acid by an enzyme. ...

NUCLEIC ACID ENCODING AN ISOMERASE AND USES THEREOF

The present invention provides for a nucleic acid encoding an isomerase and uses of the isomerase for bioconversion of sugar substrates. The invention represents an advancement in the field of enzyme engineering and discloses a modified nucleic acid for achieving optimum expression of a protein having isomerase activity in a heterologous host. The invention also discloses vectors carrying the modified nucleic acid and recombinant host cells carrying the vectors. The invention also discloses the process for producing a recombinant host cell, process for production of the recombinant enzyme and the process for bioconversion of sugars into their respective isomers using the recombinant protein. 1. A modified nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1 , wherein the nucleic acid encodes a polypeptide having isomerase activity in a prokaryotic host cell.2. A vector comprising the nucleic acid as claimed in claim 1 , wherein the modified nucleic acid is operably linked to a T7 promoter.3. The vector as claimed in claim 2 , wherein the vector is selected from a group comprising pET11 vector and pET23 vector.4. The vector as claimed in claim 3 , wherein the pET11 vector comprises the nucleotide sequence of SEQ ID NO: 3 and the pET23 vector comprises the nucleotide sequence of SEQ ID NO: 4.5. A recombinant prokaryotic host cell comprising the vector as claimed in .6Escherichia coli. The recombinant host cell as claimed in claim 5 , wherein the host cell is JM 109.7. A process for producing a recombinant host cell capable of expressing a polypeptide having isomerase activity claim 5 , the said process comprising the steps of:a. constructing a recombinant vector harbouring the nucleic acid of SEQ ID NO: 1, wherein the nucleic acid is operably linked to a T7 promoter; andb. transforming a prokaryotic host cell with the recombinant vector to obtain a recombinant host cell.8. The process as claimed in claim 7 , wherein the vector is selected from a group ...

Methods for Making L-Glufosinate

Methods for the production of L-glufosinate (also known as phosphinothricin or (S)-2-amino-4-(hydroxy(methyl)phosphonoyl)butanoic acid) are provided. The methods comprise a two-step process. The first step involves the oxidative deamination of D-glufosinate to PPO (2-oxo-4-(hydroxy(methyl)phosphinoyl)butyric acid). The second step involves the specific amination of PPO to L-glufosinate, using an amine group from one or more amine donors. By combining these two reactions, the proportion of L-glufosinate in a mixture of L-glufosinate and D-glufosinate can be substantially increased. 1. A composition comprising D-glufosinate , PPO , and L-glufosinate , wherein L-glufosinate is present in the composition at an amount of 80% by weight or greater based on the total amount of D-glufosinate , PPO , and L-glufosinate.2. The composition of claim 1 , wherein the amount of L-glufosinate is 90% by weight or greater based on the total amount of D-glufosinate claim 1 , PPO claim 1 , and L-glufosinate.3. The composition of claim 1 , wherein the amount of L-glufosinate is 95% by weight or greater based on the total amount of D-glufosinate claim 1 , PPO claim 1 , and L-glufosinate.4. The composition of claim 1 , wherein the composition is a dried powder.5. A method for selectively controlling weeds in an area comprising applying an effective amount of the composition of .6. A method for selectively controlling weeds in an area comprising applying an effective amount of the composition of .7. A method for selectively controlling weeds in an area comprising applying an effective amount of the composition of . This application is a division of U.S. application Ser. No. 15/445,254, filed Feb. 28, 2017, which claims priority to U.S. Provisional Application No. 62/302,421, filed Mar. 2, 2016; U.S. Provisional Application No. 62/336,989, filed May 16, 2016; and U.S. Provisional Application No. 62/413,240, filed Oct. 26, 2016. These applications are incorporated herein by reference in their ...

Optical Resolution Methods for Bicyclic Compounds Using Enzymes

An object of the present invention is to efficiently produce an optically active bicyclic compound. The optically active bicyclic compound is efficiently produced using an enzyme. 3. The method of claim 1 , wherein the reductase or the alcohol dehydrogenase is E039 claim 1 , and the coenzyme is nicotinamide adenine dinucleotide (NADor NADH).4. The method of claim 1 , wherein the step (2) comprises reacting the compound of formula (I′) or the compound of formula (II′) with a cyclic acid anhydride in the presence of a base claim 1 , followed by the separation.5. The method of claim 1 , wherein Ris a hydrogen atom claim 1 , a methyl group claim 1 , or an ethyl group.6. The method of claim 1 , wherein the reaction in the step (1) is performed at a temperature of 20 to 40° C.7. The method of claim 1 , wherein in the step (1) claim 1 , the reductase or the alcohol dehydrogenase is in an amount of 4 to 20% by weight with respect to the compound of formula (I) or the compound of formula (II) claim 1 , and the coenzyme is in an amount of 0.001 equivalents or fewer with respect to the compound of formula (I) or the compound of formula (II).8. The method of claim 1 , wherein the formic acid or the salt thereof is sodium formate.9. The method of claim 1 , wherein the buffer solution is a phosphate buffer solution having a concentration of 50 mmol or higher.10. The method of claim 3 , wherein in the step (2) claim 3 , the cyclic acid anhydride is succinic anhydride claim 3 , maleic anhydride claim 3 , or phthalic anhydride claim 3 , and the base is a tertiary amine.12. The method of claim 2 , wherein the reductase or the alcohol dehydrogenase is E039 claim 2 , and the coenzyme is NADor NADH.13. The method of claim 12 , wherein in the step (2) claim 12 , the cyclic acid anhydride is succinic anhydride claim 12 , maleic anhydride claim 12 , or phthalic anhydride claim 12 , and the base is a tertiary amine.14. The method of claim 2 , wherein the step (2) comprises reacting the ...

BIOCATALYSTS AND METHODS FOR THE SYNTHESIS OF ARMODAFINIL

The present invention relates to non-naturally occurring polypeptides useful for preparing armodafinil, polynucleotides encoding the polypeptides, and methods of using the polypeptides. The non-naturally occurring polypeptides of the present invention are effective in carrying out biocatalytic conversion of the (i) 2-(benzhydrylsulfinyl)acetamide to (−)-2-[(R)-(diphenylmethyl)sulfinyl]acetamide (armodafinil), or (ii) benzhydryl-thioacetic acid to (R)-2-(benzhydrylsulfinyl)acetic acid, which is a pivotal intermediate in the synthesis of armodafinil, in enantiomeric excess. 1. A non-naturally occurring polypeptide having cyclohexanone monooxygenase (CHMO) activity wherein the amino acid sequence of the polypeptide has at least 90% sequence identity to SEQ ID NO:136 , and one or more amino acid substitutions at one or more positions in SEQ ID NO: 136 , selected from the group consisting of 37 , 277 , 278 , 280 , 281 , 326 , 432 , 433 , 435 , and 490.3. The non-naturally occurring polypeptide of claim 1 , wherein said non-naturally occurring polypeptide further comprises one or more amino acid substitutions relative to SEQ ID NO: 136 claim 1 , wherein the polypeptide comprises an alanine claim 1 , glutamic acid claim 1 , glycine claim 1 , isoleucine claim 1 , lysine claim 1 , proline claim 1 , serine claim 1 , threonine claim 1 , or valine at a position corresponding to position 246 of SEQ ID NO:136.4. The non-naturally occurring polypeptide of claim 1 , wherein said non-naturally occurring further is capable of converting the acid substrate of compound (1b) to the R-enantiomer compound (2b) in at least 50% enantiomeric excess.5. The non-naturally occurring polypeptide of claim 1 , wherein said non-naturally occurring polypeptide further comprises one or more amino acid differences relative to SEQ ID NO: 136 claim 1 , wherein said polypeptide further comprises one or more substitutions selected from the group consisting of a glycine at position 143 claim 1 , glycine at ...

SOLID FORMS OF A THIENOPYRIMIDINEDIONE ACC INHIBITOR AND METHODS FOR PRODUCTION THEREOF

The present invention provides solid forms of compounds useful as inhibitors of Acetyl CoA Carboxylase (ACC), compositions thereof, methods of producing the same, and methods of using the same in the treatment of ACC-mediated diseases. 17.-. (canceled)910.-. (canceled)11. A pharmaceutical composition comprising a therapeutically effective amount of an amorphous Compound 1 according to claim 8 , and a pharmaceutically acceptable carrier claim 8 , adjuvant claim 8 , or diluent.12. A method of treating an ACC-mediated disorder comprising administering to a patient in need thereof the pharmaceutical composition of .1324.-. (canceled)28. The compound of claim 27 , wherein Ris optionally substituted Caliphatic.34. The compound of claim 31 , where Ris halogen or sulfonate.35. The compound of claim 31 , where Ris bromo.36. The compound of claim 31 , where Ris mesylate.3738.-. (canceled)41. (canceled)44. (canceled) This application is a continuation of U.S. application Ser. No. 16/598,411, filed Oct. 10, 2019, which is a divisional of U.S. application Ser. No. 16/217,935, filed Dec. 12, 2018, now U.S. Pat. No. 10,487,090, which is a divisional of Ser. No. 15/446,873, filed Mar. 1, 2017, now U.S. Pat. No. 10,183,951, which claims the benefit under 35 U.S.C. § 119(e) to U.S. Application No. 62/302,755, filed Mar. 2, 2016, and U.S. Application No. 62/303,237, filed Mar. 3, 2016, each of which are incorporated herein by reference.Obesity is a health crisis of epic proportions. The health burden of obesity, measured by quality-adjusted life-years lost per adult, has surpassed that of smoking to become the most serious, preventable cause of death. In the U.S., about 34% of adults have obesity, up from 31% in 1999 and about 15% in the years 1960 through 1980. Obesity increases the rate of mortality from all causes for both men and women at all ages and in all racial and ethnic groups. Obesity also leads to social stigmatization and discrimination, which decreases quality of life ...

METHODS FOR MAKING L-GLUFOSINATE

Methods for the production of L-glufosinate (also known as phosphinothricin or (S)-2 -amino-4-(hydroxy(methyl)phosphonoyl)butanoic acid) are provided. The methods comprise a two-step process. The first step involves the oxidative deamination of D-glufosinate to PPO (2-oxo-4-(hydroxy(methyl)phosphinoyl)butyric acid). The second step involves the specific amination of PPO to L-glufosinate, using an amine group from one or more amine donors. By combining these two reactions, the proportion of L-glufosinate in a mixture of L-glufosinate and D-glufosinate can be substantially increased.

METHOD TO PRODUCE ENANTIOMERS OF UNDECAVERTOL

A method for increasing the proportion of an enantiomer of undecavertol in an enantiomeric mixture of undecavertol, a method for stereoselectively synthesising undecavertol, and the products thereof. 1. A method of increasing the proportion of an enantiomer of undecavertol in an enantiomeric mixture of undecavertol , the method comprising contacting the enantiomeric mixture of undecavertol with an alcohol dehydrogenase (ADH) and an ADH-cofactor.2. A method of stereoselectively synthesising undecavertol , the method comprising contacting undecavertone with an alcohol dehydrogenase (ADH) and an ADH-cofactor.3. The method of claim 1 , wherein the ADH-cofactor is selected from NADPH claim 1 , NADH claim 1 , quinoid cofactors claim 1 , zinc or a combination of one or more thereof.4. The method of claim 1 , wherein the method further comprises contacting the ADH-cofactor with an ADH-cofactor regeneration system.5. The method of claim 4 , wherein the ADH-cofactor regeneration system is a substrate-coupled regeneration system.6. The method of claim 4 , wherein the ADH-cofactor regeneration system is an enzyme-coupled regeneration system.7. The method of claim 1 , wherein the ratio of (R)-enantiomers to (S)-enantiomers in the enantiomeric mixture of undecavertol prior to contacting with the ADH and ADH-cofactor ranges from about 45:55 to about 55:45.8. The method of claim 1 , wherein the enantiomeric mixture of undecavertol prior to contacting with the ADH and ADH-cofactor is a racemic mixture.9. The method of claim 1 , wherein the method results in a product having an enantiomeric excess equal to or greater than about 94%.10. The method of claim 1 , wherein the method results in a product having an enantiomeric excess of (R)-enantiomer equal to or greater than about 94%.11. The method of claim 1 , wherein the contacting step occurs for a period of time ranging from about 30 minutes to about 3 days.12. The method of claim 1 , wherein the contacting step occurs at a ...

Synthetic route to anti-viral agents

The invention provides methods of synthesizing a viral protease inhibitor in high yield, without using expensive catalysts or challenging reaction conditions.

BIOCATALYSTS FOR THE PREPARATION OF HYDROXY SUBSTITUTED CARBAMATES

The present disclosure relates to engineered ketoreductase polypeptides for the preparation of hydroxyl substituted carbamate compounds, and polynucleotides, vectors, host cells, and methods of making and using the ketoreductase polypeptides. 2. The engineered polynucleotide encoding an engineered ketoreductase polypeptide of claim 1 , wherein the amino acid sequence of said ketoreductase polypeptide comprises the substitution X206F/L and one or more residue differences as compared to SEQ ID NO:4 selected from: X7S; X17M; X17Q; X17R; X23V; X27L; X29G; X40R; X60I; X64V; X71P; X87L; X94A; X94P; X94S; X95M; X105G; X113I; X122A; X127R; X131S; X144V; X145L; X147I; X147L; X147Q; X150Y; X152G; X153G; X157C; X173L; X196M; X198S; X208R; X216R; X221S; X243S; X245I; X249F; X249G; and X249Y.3. The engineered polynucleotide encoding an engineered ketoreductase polypeptide of claim 1 , wherein the amino acid sequence of said ketoreductase polypeptide comprises X206F/L claim 1 , and at least one or more residue differences as compared to SEQ ID NO:4 selected from: X17Q/R/M; X40R; X64V; X94P; X96L/Y; X144V; X147Q/I/L; X157C; X195A/G; X196M; and X199H.4. The engineered polynucleotide encoding an engineered ketoreductase polypeptide of claim 1 , wherein the ketoreductase polypeptide is capable of converting the substrate compound (2) to the product compound (1) with at least 10 fold the activity of the reference polypeptide of SEQ ID NO:4 claim 1 , wherein the amino acid sequence comprises the substitution X206F/L claim 1 , and one or more residue differences as compared to SEQ ID NO:4 selected from: X40R; X60I; X71P; X94P; X94A; X95M; X96L; X96Y; X127R; X144V; X145I; X150Y; X152G; X153G; X157C; X195A; X195G; X196M; X198S; X199H; X216R claim 1 , X245I claim 1 , X245F; X249Y; and X249F.5. The engineered polynucleotide encoding an engineered ketoreductase polypeptide of claim 1 , wherein the ketoreductase polypeptide has increased thermal stability as compared to the reference ...

TRANSAMINASES

The invention relates to transaminases and their use. The ATAs according to the invention are particularly useful for catalyzing the conversion of amines to ketones and/or vice versa. Preferably, the transaminase (ATA) according to the invention comprises an amino acid sequence with at least 80% homology to SEQ ID NO:1, wherein the amino acid sequence is engineered compared to SEQ ID NO:1 such that it comprises at least a substitution selected from the group consisting of F255L, F255A, F255C, F255D, F255E, F255G, F255H, F255K, F255M, F255N, F255P, F255Q, F255R, F255S, F255T, F255V, F255W, and F255Y. 1. A transaminase comprising an amino acid sequence with at least 80% homology to SEQ ID NO: 1 , wherein the amino acid sequence is engineered compared to SEQ ID NO: 1 such that it comprises at least a substitution selected from the group consisting of F255L , F255A , F255C , F255D , F255E , F255G , F255H , F255K , F255M , F255N , F255P , F255Q , F255R , F255S , F255T , F255V , F255W , and F255Y.2. The transaminase according to claim 1 , which is engineered compared to SEQ ID NO: 1 in at least two positions such that it comprises the substitutions F255L and N268A.3. The transaminase according to claim 1 , which is engineered compared to SEQ ID NO: 1 in at least two positions such that it comprises(i) a substitution selected from the group consisting of F255L, F255A, F255C, F255D, F255E, F255G, F255H, F255K, F255M, F255N, F255P, F255Q, F255R, F255S, F255T, F255V, F255W, and F255Y; and{'sub': 268', '268, '(ii) a substitution selected from the group consisting of NA, NC, N268D, N268E, N268F, N268G, N268H, N268I, N268K, N268L, N268M, N268P, N268Q, N268R, N268S, N268T, N268V, N268W, and N268Y or a substitution selected from the group consisting of N268A, N268C, N268E, N268F, N268G, N268H, N268I, N268K, N268L, N268M, N268P, N268Q, N268R, N268S, N268T, N268V, N268W, and N268Y, or'}a substitution selected from the group consisting of N268A, N268F, N268H, N268I, N268K, N268L, ...

Enzymatic processes for the preparation of (±)-2-(difluoromethyl)-1-(alkoxycarbonyl)-cyclopropanecarboxylic acid and (±)-2-(vinyl)-1-(alkoxycarbonyl)-cyclopropanecarboxylic acid

Disclosed are methods of synthesizing racemic 2-(difluoromethyl)-1-(alkoxycarbonyl)-cyclopropanecarboxylic acids and 2-(vinyl)-1-(alkoxycarbonyl)-cyclopropanecarboxylic acids and their salts, such as the dicyclohexylamine salt. Also disclosed are methods for preparing enantioenriched (1R,2R)-1-((tert-butoxycarbonyl)amino)-2-(difluoromethyl)cyclopropane-1-carboxylic acid and esters of the same. These compounds are useful intermediates in the synthesis of viral protease inhibitors.

SYNTHETIC ROUTE TO ANTI-VIRAL AGENTS

The invention provides methods of synthesizing a viral protease inhibitor in high yield, without using expensive catalysts or challenging reaction conditions. 118-. (canceled)20. The method of claim 19 , wherein the solvent comprises toluene claim 19 , dichloromethane claim 19 , THF claim 19 , acetone claim 19 , heptane claim 19 , hexane claim 19 , methyl tert-butyl ether claim 19 , ethyl acetate claim 19 , dioxane claim 19 , DMF claim 19 , DMA claim 19 , acetonitrile claim 19 , or DMSO claim 19 , or a mixture thereof.21. The method of claim 20 , wherein the solvent comprises toluene claim 20 , acetonitrile claim 20 , or dichloromethane or a mixture thereof.22. The method of claim 19 , wherein the reaction of scheme B takes place at a temperature from about −20° C. to about 10° C.23. The method of claim 22 , wherein the temperature is about −20° C. claim 22 , about −15° C. claim 22 , about −10° C. claim 22 , about −5° C. claim 22 , about 0° C. claim 22 , about 5° C. claim 22 , or about 10° C.24. The method of claim 22 , wherein the temperature is about −10° C. to about 10° C.25. The method of claim 22 , wherein the temperature is about 0° C. This application claims the benefit of priority to U.S. Provisional Patent Application Ser. No. 62/026,412, filed Jul. 18, 2014, the contents of which are hereby incorporated by reference.Complex biologically active molecules are challenging, expensive, and time-consuming to synthesize. Synthesizing chiral, non-racemic compounds with good enantio- and diastereoselectivity is even more challenging. An example of such a molecule is Compound 1:This compound is a potent inhibitor of the hepatitis C virus (HCV) NS3/4A protease; it shows broad genotype activity and substantially improved in vitro profile compared to earlier generation HCV NS3/4A protease inhibitors.The original synthesis of this compound requires a ring closing metathesis (RCM) reaction for synthesis of the macrocycle (see WO 2012/040167). However, this RCM reaction ...

KETOREDUCTASE POLYPEPTIDES

The present disclosure provides engineered ketoreductase enzymes having improved properties as compared to a naturally occurring wild-type ketoreductase enzyme including the capability of reducing 5-((4S)-2-oxo-4-phenyl (1,3-oxazolidin-3-yl))-1-(4-fluorophenyl) pentane-1,5-dione to (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one. Also provided are polynucleotides encoding the engineered ketoreductase enzymes, host cells capable of expressing the engineered ketoreductase enzymes, and methods of using the engineered ketoreductase enzymes to synthesize the intermediate (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one in a process for making Ezetimibe. 1. An engineered polypeptide having ketoreductase activity , wherein said engineered polypeptide comprises a region with an amino acid sequence having at least 90% sequence identity to residues 90 to 211 of SEQ ID NO: 130 , comprising a substitution at position X117 , and further comprising one or more of the following features selected from:residue corresponding to X94 is asparagine, glycine, serine, or a polar residue;residue corresponding to X95 is an aliphatic residue;residue corresponding to X96 is glutamine, asparagine, or threonine;residue corresponding to X101 is an acidic, non-polar, or a polar residue;residue corresponding to X105 is an acidic or non-polar residue;residue corresponding to X108 is a hydrophilic, polar or constrained residue;residue corresponding to X111 is a non-polar or aliphatic residue;residue corresponding to X112 is an acidic or polar residue;residue corresponding to X113 is a non-polar or aliphatic residue;residue corresponding to X127 is a basic residue;residue corresponding to X147 is a non-polar, aliphatic, aromatic, or hydrophobic residue;residue corresponding to X152 is a non-polar, basic, or hydrophilic residue;residue corresponding to X157 is a polar residue;residue corresponding to X163 is a non-polar or aliphatic ...

PROCESSES USING AMINO ACID DEHYDROGENASES AND KETOREDUCTASE-BASED COFACTOR REGENERATING SYSTEM

The present disclosure relates to the use of an amino acid dehydrogenase in combination with a cofactor regenerating system comprising a ketoreductase. In particular embodiments, the process can be used to prepare L-tert-leucine using a leucine dehydrogenase. 2. The process of claim 1 , wherein R is a substituted or unsubstituted —(C-C)alkyl claim 1 , —(C-C)alkenyl claim 1 , —(C-C)alkynyl claim 1 , —(C-C)cycloalkyl claim 1 , heterocycloalkyl claim 1 , aryl claim 1 , or heteroaryl.3. The process of claim 1 , wherein the lower secondary alcohol is isopropanol and the ketone is acetone.4. The process of claim 1 , further comprising the step of removing the lower secondary alcohol formed from the lower alkyl ketone from the reaction medium.5. The process of claim 1 , wherein the reaction medium is at a pH of about 8.5 to about 10.6. The process of claim 5 , wherein the reaction medium is at a pH of about 8.5 to about 9.0.7. The process of claim 1 , wherein the reaction medium is at a temperature of about 25° C. to about 45° C.8. The process of claim 7 , wherein the reaction medium is at a temperature of about 35° C. to about 40° C.9. The process of claim 1 , wherein the secondary alcohol is present in at least 1.5 fold stoichiometric excess of substrate.10. The process of claim 1 , wherein the ketoreductase is capable of recycling cofactor by converting isopropanol (IPA) to acetone in a reaction medium of 3 to 20% IPA at a pH of about 9.0 to 10.5 with an activity at least 2.0-fold greater than a reference ketoreductase encoded by the polynucleotide of SEQ ID NO:1.11. The process of claim 1 , wherein the activity of the ketoreductase with compound of formula I is less than 2% claim 1 , 1% claim 1 , 0.5% claim 1 , 0.1% claim 1 , 0.05% claim 1 , or 0.01% of the activity of the amino acid dehydrogenase with the compound of formula I claim 1 , or optionally no activity with the compound of formula I.12. The process of claim 1 , wherein the compound of formula IIa is 3 claim ...

BIOCATALYSTS AND METHODS FOR THE SYNTHESIS OF ARMODAFINIL

The present invention relates to non-naturally occurring polypeptides useful for preparing armodafinil, polynucleotides encoding the polypeptides, and methods of using the polypeptides. The non-naturally occurring polypeptides of the present invention are effective in carrying out biocatalytic conversion of the (i) 2-(benzhydrylsulfinyl)acetamide to (−)-2-[(R)-(diphenylmethyl)sulfinyl]acetamide (armodafinil), or (ii) benzhydryl-thioacetic acid to (R)-2-(benzhydrylsulfinyl)acetic acid, which is a pivotal intermediate in the synthesis of armodafinil, in enantiomeric excess. 1. A non-naturally occurring polypeptide having cyclohexanone monooxygenase (CHMO) activity wherein the amino acid sequence of the polypeptide has at least 90% sequence identity to SEQ ID NO: 136 , and one or more amino acid substitutions compared to the naturally occurring polypeptide at one or more positions corresponding to positions in SEQ ID NO: 136 , selected from the group consisting of 75 , 79 , 82 , 99 , 110 , 166 , 172 , 208 , 216 , 273 , 324 , 364 , 395 , 412 , 491 , 503 , and 504.3. The non-naturally occurring polypeptide of claim 1 , wherein said non-naturally occurring polypeptide further comprises one or more amino acid substitutions relative to SEQ ID NO: 136 claim 1 , wherein the polypeptide comprises an alanine claim 1 , glutamic acid claim 1 , glycine claim 1 , isoleucine claim 1 , lysine claim 1 , proline claim 1 , serine claim 1 , threonine claim 1 , or valine at a position corresponding to position 246 of SEQ ID NO: 136.5. The non-naturally occurring polypeptide of claim 1 , wherein said non-naturally occurring polypeptide further comprises one or more amino acid differences relative to SEQ ID NO: 136 claim 1 , wherein said polypeptide further comprises one or more substitutions corresponding to substitutions in SEQ ID NO: 136 selected from the group consisting of a glycine at position 143 claim 1 , glycine at position 278 claim 1 , an arginine at position 326 claim 1 , and ...

TRANSAMINASE POLYPEPTIDES

The present disclosure provides engineered transaminase enzymes having improved properties as compared to a naturally occurring wild-type transaminase enzyme. Also provided are polynucleotides encoding the engineered transaminase enzymes, host cells capable of expressing the engineered transaminase enzymes, and methods of using the engineered transaminase enzymes to synthesize a variety of chiral compounds. 1. An engineered transaminase polypeptide , wherein said transaminase polypeptide comprises a polypeptide sequence at least 80% identical to SEQ ID NO: 2 , and wherein the transaminase polypeptide comprises a substitution at one or more positions selected from the group consisting of X86 , X211 , X294 , X324 , and X391.2. The engineered transaminase polypeptide of claim 1 , wherein said polypeptide has at least about 10% residual activity in the conversion of pyruvate to L-alanine in presence of amino donor isopropylamine after treatment of the polypeptide at 50° C. for 23 h.3. The engineered transaminase polypeptide of claim 1 , wherein the transaminase polypeptide further comprises at least one additional substitution at a residue position selected from: X4 claim 1 , X12 claim 1 , X21 claim 1 , X157 claim 1 , X208 claim 1 , X253 claim 1 , X272 claim 1 , X302 claim 1 , X316 claim 1 , X398 claim 1 , X418 claim 1 , X420 claim 1 , X431 claim 1 , X444 claim 1 , and X446.4. The engineered transaminase polypeptide of claim 3 , wherein the transaminase polypeptide sequence comprises the substitution X233L and at least one further substitution selected from: X4 is R claim 3 , Q claim 3 , or L; X12 is K; X21 is N; X45 is H; X86 is Y; X157 is T; X177 is L; X208 is I; X211 is K; X253 is M; X272 is A; X294 is V; X302 is K; X316 is K; X324 is G; X391 is A; X398 is R; X418 is V; X420 is N; X431 is D; X444 is V; and X446 is V.5. The engineered transaminase polypeptide of claim 1 , wherein the transaminase polypeptide has increased transaminase activity as compared to SEQ ID NO ...

DIFLUOROALKYLCYCLOPROPYL AMINO ACIDS AND ESTERS, AND SYNTHESES THEREOF

The invention provides methods of synthesizing compounds in an asymmetric or enantioenriched fashion, wherein the compounds are useful intermediates in the synthesis of viral protease inhibitors. 116-. (canceled)18. The method of claim 17 , wherein the first metal comprises TiCl claim 17 , TiOR claim 17 , CeCl claim 17 , Ce(SO) claim 17 , CaCl claim 17 , MgCl claim 17 , Ti(Oi-Pr)Cl or Ti(OEt)Cl.19. The method of claim 17 , wherein the first base is present; and the first base comprises (i-Pr)EtN claim 17 , triethylamine claim 17 , EtNH claim 17 , EtNH claim 17 , or (iPr)NH.20. The method of claim 19 , wherein the first base is triethylamine.21. The method of claim 17 , wherein the first metal is CeClor MgCl; and the first base is absent.22. The method of claim 17 , wherein the first metal is CeClor MgCl; and the first metal is present in a catalytic quantity.23. The method of claim 17 , wherein the first metal is TiCl claim 17 , TiOR claim 17 , Ti(Oi-Pr)Cl claim 17 , or Ti(OEt)Cl; and the first metal is present in a stoichiometric quantity.24. The method of claim 17 , wherein the first solvent comprises MeOH claim 17 , EtOH claim 17 , n-PrOH claim 17 , i-PrOH claim 17 , tetrahydrofuran (THF) claim 17 , methyl tert-butyl ether claim 17 , ethyl acetate claim 17 , dioxane claim 17 , DMF claim 17 , acetonitrile claim 17 , or DMSO.25. The method of claim 24 , wherein the first metal is TiCl claim 24 , TiOR claim 24 , Ti(Oi-Pr)Cl claim 24 , or Ti(OEt)Cl.26. The method of claim 24 , wherein the first metal is CeClor MgCl.27. The method of claim 17 , wherein the first temperature is from about −10° C. to about 15° C.28. The method of claim 17 , wherein the first period of time is from about 6 h to about 18 h.29. The method of claim 17 , further comprising heating the first product mixture at a second temperature.30. The method of claim 29 , wherein the first product mixture is maintained at the second temperature for a second period of time.31. The method of claim 17 , ...

PRODUCTION OF OPTICALLY PURE PROPANE-1,2-DIOL

A method for producing optically pure propane-1,2-diol, including the method steps: a. hydrogenation of lactides, metal-catalysed heterogenous catalysis being carried out in the presence of hydrogen, a crude product containing propane-1,2-diol being produced, and b. dynamic, kinetic racemate resolution, propane-1,2-diol of an optical purity in the range of ≧99% e.e. being produced. 1. Process for the production of optically pure propane-1 ,2-diol comprising the following process steps:Hydrogenation of lactides, wherein a metal-catalysed heterogeneous catalysis is carried out in the presence of hydrogen, a raw product containing propane-1,2-diol being produced, andDynamic kinetic racemic resolution, in which optically pure propane-1.2-diol is produced within a range of ≧99% e.e.2. Process in accordance with claim 1 , wherein the lactides are selected from the group comprising D claim 1 ,D-lactide claim 1 , L claim 1 ,L-lactide claim 1 , meso-lactide and L claim 1 ,L/D claim 1 ,D-lactide.3. The process in accordance with claim 1 , wherein the metal-catalysed heterogeneous catalysis in step a) is carried out in the liquid phase.4. Process in accordance with claim 3 , wherein the liquid phase is selected from a group of solvents comprising water claim 3 , aliphatic or aromatic hydrocarbons with a chain length of up to 10 C-atoms claim 3 , and mixtures thereof claim 3 , wherein the aliphatic hydrocarbons are preferably alcohols with particular preference being given to methanol and/or ethanol being used.5. The process in accordance with wherein the heterogeneous catalysis in step a) is carried out using a catalyst from the metals group claim 1 , wherein the metal is selected from a group comprising ruthenium claim 1 , rhodium claim 1 , rhenium claim 1 , palladium claim 1 , platinum claim 1 , nickel claim 1 , cobalt claim 1 , molybdenum claim 1 , wolfram claim 1 , titanium claim 1 , zirconium claim 1 , niobium claim 1 , vanadium claim 1 , chromium claim 1 , manganese ...

PRODUCTION METHOD FOR L-CYCLIC AMINO ACIDS

An object of the present invention is to provide a method of industrially producing a high-purity L-cyclic amino acid more inexpensively and with a high efficiency, from a cyclic amino acid having a double bond at the 1-position. The present invention provides a method in which an L-cyclic amino acid is produced by allowing a cyclic amino acid having a double bond at the 1-position to react with a specific enzyme having a catalytic ability to reduce a cyclic amino acid having a double bond at the 1-position to produce an L-cyclic amino acid. 2. The method of producing an L-cyclic amino acid according to claim 1 , wherein the polypeptide is encoded by a nucleic acid shown in (D) claim 1 , (E) or (F) below:(D) a nucleic acid comprising the nucleotide sequence represented by SEQ ID NO: 1, 3, 5, 7, 9 or 11;(E) a nucleic acid which comprises the nucleotide sequence represented by SEQ ID NO: 1, 3, 5, 7, 9 or 11 except that one or several nucleotides are substituted, deleted and/or added, and which encodes a polypeptide having the ability to catalyze the reaction represented by the formula (1) to produce the L-cyclic amino acid; or(F) a nucleic acid which comprises a nucleotide sequence that hybridizes with a complementary strand of the nucleotide sequence represented by SEQ ID NO: 1, 3, 5, 7, 9 or 11 under stringent conditions, and which encodes a polypeptide having the ability to catalyze the reaction represented by the formula (1) to produce the L-cyclic amino acid.4. The method of producing an L-cyclic amino acid according to claim 3 , wherein the enzyme capable of converting the amino group at the α-position of the diamino acid to a keto group and producing an α-keto acid is one or more enzymes selected from the group consisting of a D-amino acid oxidase claim 3 , an L-amino acid oxidase claim 3 , a D-amino acid dehydrogenase claim 3 , an L-amino acid dehydrogenase claim 3 , a D-amino acid aminotransferase and an L-amino acid aminotransferase.5. The method of ...

PROCESS FOR THE PREPARATION OF TRIPLE-BOND-CONTAINING OPTICALLY ACTIVE CARBOXYLIC ACIDS, CARBOXYLATE SALTS AND CARBOXYLIC ACID DERIVATIVES

The invention provides a new enzimatic process for the preparation of chiral carboxylic acids, their salts and acid derivatives of the general formula (I) by enzymatic hydrolysis of racemic carboxylic acid ester of the general formula (II) and optionally subsequent esterification or acylation. 2. Process as defined in claim 1 , wherein the enzymatic hydrolysis is carried out with hydrolase enzyme.3Candida rugosa, Pseudomonas fluorescens, Candida antarctica A, Candida antarctica B, Burkholderia cepacia, Rhizopus arrhizus, Rhizomucor miehei, Pseudomonas cepacia lipase. Process as defined in claim 2 , wherein as hydrolase enzyme lipase enzyme claim 2 , such as or pig pancrease lipase claim 2 , calf pancrease lipase are applied.4Candida rugosa. Process as defined in claim 3 , wherein as lipase enzyme enzyme is applied.5. Process as defined in claim 1 , wherein the hydrolysis is carried out in the presence of solvent.6. Process as defined in claim 5 , wherein as solvent ethers claim 5 , hydrocarbon-type and aromatic solvents claim 5 , such as diisopropyl ether claim 5 , methyl tert.-butyl ether claim 5 , n-hexane claim 5 , toluene are applied.7. Process as defined in claim 6 , wherein as solvent methyl tert.-butyl ether is applied.8. Process as defined in claim 1 , wherein depending on the amount and activity of the enzyme claim 1 , the reaction time is 2-48 hours.9. Process as defined in claim 1 , wherein the reaction is performed at 20-40° C.10. Process as defined in claim 2 , wherein the hydrolysis is carried out in the presence of solvent.11. Process as defined in claim 3 , wherein the hydrolysis is carried out in the presence of solvent.12. Process as defined in claim 4 , wherein the hydrolysis is carried out in the presence of solvent.13. Process as defined in claim 2 , wherein depending on the amount and activity of the enzyme claim 2 , the reaction time is 2-48 hours.14. Process as defined in claim 3 , wherein depending on the amount and activity of the enzyme ...

Method for the preparation of (3e,7e)-homofarnesic acid or (3e,7e)-homofarnesic acid ester

The invention provides an improved method of isolating the 3-(E)-isomer of an unsaturated carboxylic acid from a mixture of corresponding (E/Z)isomers. More particularly, the present invention relates to an improved method for the biocatalytic preparation of (3E,7E)-homofarnesylic acid; as well as a novel biocatalytic method for the improved preparation of homofarnesol, in particular of (3E,7E)-homofarnesol and homofarnesol preparations having an increased content of (3E,7E)-homofarnesol. The present invention also relates to methods of preparing(−)-ambroxby applying (3E,7E)-homofarnesylic acid or (3E,7E)-homofarnesol as obtained according to the invention as starting material.

PROCESS FOR THE ENANTIOSELECTIVE ENZYMATIC REDUCTION OF HYDROXY KETO COMPOUNDS

In a process for the enantioselective enzymatic reduction of a hydroxy ketone of general formula I 2. The process according to claim 1 , wherein the cofactor is continuously reduced with a co-substrate.3. The process according to claim 1 , wherein NAD(P)H is used as a cofactor.4. The process according to claim 2 , wherein at least one member selected from the group consisting of 2-propanol claim 2 , 2-butanol claim 2 , 2-pentanol claim 2 , 4-methyl-2-pentanol claim 2 , 2-heptanol and 2-octanol is used as the co-substrate.5. The process according to claim 1 , wherein the hydroxy ketone is used in an amount of from 5 to 50% by weight based on the total reaction volume.6. The process according to claim 1 , wherein at least one member selected from the group consisting of tert. butyl (5R)-6-cyano-5-hydroxy-3-oxohexanoate claim 1 , tert. butyl (5S)-6-chloro-5-hydroxy-3-oxohexanoate and tert. butyl (5S)-5 claim 1 ,6-dihydroxy-3-oxohexanoate is used as the hydroxy ketone of general formula (I).7. The process according to claim 1 , wherein the TTN (total turn over number=mol of reduced hydroxy ketone/mol of cofactor used) is ≧10.8. The process according to claim 1 , wherein the process is carried out in an aqueous organic two-phase system.9. The process according to claim 1 , wherein claim 1 , in addition claim 1 , at least one organic solvent selected from the group consisting of diethyl ether claim 1 , tertiary butyl methyl ether claim 1 , diisopropyl ether claim 1 , dibutyl ether claim 1 , ethyl acetate claim 1 , butyl acetate claim 1 , heptane claim 1 , hexane and cyclohexane is used.11. The process according to claim 1 , wherein the hydroxy ketone is used in an amount of from 8-40% by weight claim 1 , based on the total reaction volume.12. The process according to claim 1 , wherein the hydroxy ketone is used in an amount of from 10-25% by weight claim 1 , based on the total reaction volume. This application is a Divisional application of co-pending U.S. application Ser ...

Process for producing optically active 2-alkyl-1,1,3-trialkoxycarbonylpropane

A process for producing an optically active 2-alkyl-1,1,3-trialkoxycarbonylpropane (2), comprising a step of asymmetric hydrolysis of 2-alkyl-1,1,3-trialokoxycarbonylpropane (1) by using an enzyme capable of selectively hydrolyzing an ester moiety of either one enantiomer of 2-alkyl-1,1,3-trialkoxycarbonylpropane (1), or by using a culture of a microorganism capable of producing the enzyme or a treated object thereof.

Bradyrhizobium monooxygenase and use thereof for preparation of chiral sulfoxide

A Bradyrhizobium monooxygenase, a gene for encoding the monooxygenase, a recombinant expression vector comprising the gene and a recombinant transformant, a method of preparing the monooxygenase by the recombinant expression transformant, and a method of preparing an optically pure chiral sulfoxide by the monooxygenase, in particular to a method of preparing prazole drugs by means of catalyzing the asymmetric oxidation of thioether, a prazole precursor. As compared with other methods of preparing an optically pure sulfoxide, the product produced by the monooxygenase of the present invention as a catalyst has high optical purity, avoids the generation of the byproduct sulfone, and has advantages of mild reaction conditions, simple and convenient operations, easy amplification, etc.

Lactate Production Process

A process for producing alkyl R-lactate from an initial compound comprising substructure (I) 2. (canceled)3. The process of claim 1 , wherein the initial compound comprises oligomeric S-lactic acid claim 1 , S-lactic acid or a salt thereof claim 1 , alkyl S claim 1 ,S-lactyllactate and/or alkyl S-lactate claim 1 , and wherein the intermediate is produced by:(i) subjecting the initial compound to stereoisomerisation conditions; and(ii) converting at least a portion of the product of step (i) into the intermediate.4. The process of claim 3 , further comprising converting S-lactic acid into the initial compound which comprises oligomeric-S-lactic acid claim 3 , and wherein (i) comprises contacting the oligomeric S-lactic acid with a tertiary amine stereoisomerisation catalyst at a temperature of from about 100° C. to about 200° C. to produce oligomeric lactic acid.5. The process of claim 4 , wherein (ii) comprises converting at least a portion of the oligomeric lactic acid into R claim 4 ,R- and S claim 4 ,S-lactide by heating at a temperature of from about 160° C. to about 240° C. in the presence of a Lewis acid catalyst comprising an oxide claim 4 , alkoxide or carboxylate salt of a metal.6. The process of claim 2 , wherein tertiary amine is recovered and recycled to the process.7. The process of claim 1 , wherein R claim 1 ,R- and S claim 1 ,S-lactide are separated from R claim 1 ,S-lactide claim 1 , and R claim 1 ,S-lactide is recycled to the process.811-. (canceled)1211. The process of claim claim 1 , wherein alkyl S claim 1 ,S-lactyllactate is recovered.1320-. (canceled)21. The process of claim 1 , in which the alkyl R-lactate prepared is subsequently converted into R-lactic acid claim 1 , oligomeric R-lactic acid claim 1 , R claim 1 ,R-lactide claim 1 , polyR-lactic acid claim 1 , or stereocomplex lactic acid.22. The process of claim 1 , wherein alkyl S claim 1 ,S-lactyllactate is recovered and recycled to the process. The present invention relates to the ...

SOLID FORMS OF A THIENOPYRIMIDINEDIONE ACC INHIBITOR AND METHODS FOR PRODUCTION THEREOF

The present invention provides solid forms of compounds useful as inhibitors of Acetyl CoA Carboxylase (ACC), compositions thereof, methods of producing the same, and methods of using the same in the treatment of ACC-mediated diseases. 119.-. (canceled)22. The process of claim 20 , wherein the [acyl] donor is an optionally substituted 4-7 membered lactone or 4-7 membered optionally substituted cyclic anhydride; or a compound of the formula RC(O)OR claim 20 , wherein Ris optionally substituted Caliphatic; and Ris optionally substituted Caliphatic or optionally substituted Cacyl.23. The process of claim 22 , wherein [acyl] is a Cacyl group.24Candida antarctica. The process of claim 22 , wherein the lipase enzyme is lipase B.2544.-. (canceled) This application is a continuation of U.S. application Ser. No. 16/217,935, filed Dec. 12, 2018, which is a divisional of Ser. No. 15/446,873, filed Mar. 1, 2017, now U.S. Pat. No. 10,183,951, which claims the benefit under 35 U.S.C. § 119(e) to U.S. Application No. 62/302,755, filed Mar. 2, 2016, and U.S. Application No. 62/303,237, filed Mar. 3, 2016, each of which are incorporated herein by reference.Obesity is a health crisis of epic proportions. The health burden of obesity, measured by quality-adjusted life-years lost per adult, has surpassed that of smoking to become the most serious, preventable cause of death. In the U.S., about 34% of adults have obesity, up from 31% in 1999 and about 15% in the years 1960 through 1980. Obesity increases the rate of mortality from all causes for both men and women at all ages and in all racial and ethnic groups. Obesity also leads to social stigmatization and discrimination, which decreases quality of life dramatically. The chronic diseases that result from obesity cost the U.S. economy more than $150 billion in weight-related medical bills each year. Furthermore, about half of the obese population, and 25% of the general population, have metabolic syndrome, a condition associated with ...

Process for the enzymatic synthesis of (7s)-3,4-dimethoxybicyclo[4.2.0]octa-1,3,5-triene-7-carboxylic acid and application in the synthesis of ivabradine and salts thereof

Process for the enzymatic synthesis of the compound of formula (I): Application in the synthesis of ivabradine and addition salts thereof with a pharmaceutically acceptable acid.

METHODS OF PRODUCING EPIMERASES AND BENZYLISOQUINOLINE ALKALOIDS

A method of epimerizing an (S)-1-benzylisoquinoline alkaloid to an (R)-1-benzylisoquinoline alkaloid is provided. The method comprises contacting the (S)-1-benzylisoquinoline alkaloid with at least one enzyme. Contacting the (S)-1-benzyl-isoquinoline alkaloid with the at least one enzyme converts the (S)-1-benzylisoquinoline alkaloid to an (R)-1-benzylisoquinoline alkaloid. 1. A method of epimerizing an (S)-1-benzylisoquinoline alkaloid to an (R)-1-benzylisoquinoline alkaloid , comprising:contacting the (S)-1-benzylisoquinoline alkaloid with at least one enzyme,wherein contacting the (S)-1-benzylisoquinoline alkaloid with the at least one enzyme converts the (S)-1-benzylisoquinoline alkaloid to an (R)-1-benzylisoquinoline alkaloid.2. The method of claim 1 , wherein the at least one enzyme is produced by culturing an engineered non-plant cell having a coding sequence for encoding the at least one enzyme.3. The method of claim 2 , further comprising:adding an (S)-1-benzylisoquinoline alkaloid to the cell culture.4. The method of claim 3 , further comprising:recovering the (R)-1-benzylisoquinoline alkaloid, or a derivative thereof, from the cell culture.5. The method of claim 1 , wherein the at least one enzyme comprises an oxidase.6. The method of claim 1 , wherein the at least one enzyme comprises a reductase.7. The method of claim 1 , wherein the at least one enzyme comprises an epimerase.8. The method of claim 7 , wherein the epimerase comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 1 claim 7 , 2 claim 7 , 3 claim 7 , 4 claim 7 , 5 claim 7 , 6 claim 7 , 7 claim 7 , 8 claim 7 , 9 claim 7 , 10 claim 7 , 11 claim 7 , 12 claim 7 , 13 claim 7 , 14 claim 7 , and 15.9. The method of claim 7 , wherein the epimerase comprises an oxidase domain and a reductase domain.10. The method of claim 9 , wherein the oxidase domain is a cytochrome P450 oxidase-like domain.11. The method of claim 9 , wherein the reductase domain is a codeinone ...

KETOREDUCTASE POLYPEPTIDES

The present disclosure provides engineered ketoreductase enzymes having improved properties as compared to a naturally occurring wild-type ketoreductase enzyme including the capability of reducing 5-((4S)-2-oxo-4-phenyl (1,3-oxazolidin-3-yl))-1-(4-fluorophenyl) pentane-1,5-dione to (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one. Also provided are polynucleotides encoding the engineered ketoreductase enzymes, host cells capable of expressing the engineered ketoreductase enzymes, and methods of using the engineered ketoreductase enzymes to synthesize the intermediate (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one in a process for making Ezetimibe. 1. An engineered polynucleotide encoding an engineered polypeptide having ketoreductase activity , wherein said engineered polypeptide comprises a region with an amino acid sequence having at least 90% sequence identity to residues 90 to 211 of SEQ ID NO: 130 , comprising a substitution at position X190 , and further comprising one or more of the following features selected from:residue corresponding to X94 is asparagine, glycine, serine, or a polar residue;residue corresponding to X95 is an aliphatic residue;residue corresponding to X96 is glutamine, asparagine, or threonine;residue corresponding to X101 is an acidic, non-polar, or a polar residue;residue corresponding to X105 is an acidic or non-polar residue;residue corresponding to X108 is a hydrophilic, polar or constrained residue;residue corresponding to X111 is a non-polar or aliphatic residue;residue corresponding to X112 is an acidic or polar residue;residue corresponding to X113 is a non-polar or aliphatic residue;residue corresponding to X117 is a non-polar or a polar residue;residue corresponding to X127 is a basic residue;residue corresponding to X147 is a non-polar, aliphatic, aromatic, or hydrophobic residue;residue corresponding to X152 is a non-polar, basic, or hydrophilic residue;residue ...

BIOCATALYSTS AND METHODS FOR THE SYNTHESIS OF ARMODAFINIL

The present invention relates to non-naturally occurring polypeptides useful for preparing armodafinil, polynucleotides encoding the polypeptides, and methods of using the polypeptides. The non-naturally occurring polypeptides of the present invention are effective in carrying out biocatalytic conversion of the (i) 2-(benzhydrylsulfinyl)acetamide to (−)-2-[(R)-(diphenylmethyl)sulfinyl]acetamide (armodafinil), or (ii) benzhydryl-thioacetic acid to (R)-2-(benzhydrylsulfinyl)acetic acid, which is a pivotal intermediate in the synthesis of armodafinil, in enantiomeric excess. 1. A non-naturally occurring polynucleotide encoding a non-naturally occurring polypeptide having cyclohexanone monooxygenase (CHMO) activity wherein the amino acid sequence of the polypeptide has at least 90% sequence identity to SEQ ID NO: 2 , and one or more amino acid substitutions compared to the naturally occurring polypeptide at one or more positions corresponding to positions in SEQ ID NO: 2 , selected from the group consisting of 280 , 322 , 325 , 426 , 430 , 432 , 435 , and 532.3. The non-naturally occurring polynucleotide encoding the non-naturally occurring polypeptide of claim 1 , wherein said non-naturally occurring polypeptide further comprises one or more amino acid substitutions relative to SEQ ID NO: 2 claim 1 , wherein the polypeptide comprises an alanine claim 1 , glutamic acid claim 1 , glycine claim 1 , isoleucine claim 1 , lysine claim 1 , proline claim 1 , serine claim 1 , threonine claim 1 , or valine at a position corresponding to position 246 of SEQ ID NO: 2.5. The non-naturally occurring polynucleotide encoding the non-naturally occurring polypeptide of claim 1 , wherein said non-naturally occurring polypeptide further comprises one or more amino acid differences relative to SEQ ID NO: 2 claim 1 , wherein said polypeptide further comprises one or more substitutions corresponding to substitutions in SEQ ID NO: 2 selected from the group consisting of a glycine at position ...

BIOCATALYSTS FOR THE PREPARATION OF HYDROXY SUBSTITUTED CARBAMATES

The present disclosure relates to engineered ketoreductase polypeptides for the preparation of hydroxyl substituted carbamate compounds, and polynucleotides, vectors, host cells, and methods of making and using the ketoreductase polypeptides. 2. The engineered ketoreductase polypeptide of claim 1 , wherein the amino acid sequence of said ketoreductase polypeptide comprises the substitution X206F/L and one or more residue differences as compared to SEQ ID NO:4 selected from: X7S; X17M; X17Q; X17R; X23V; X27L; X29G; X40R; X60I; X64V; X71P; X87L; X94A; X94P; X94S; X95M; X105G; X113I; X122A; X127R; X131S; X144V; X145L; X147I; X147L; X147Q; X150Y; X152G; X153G; X157C; X173L; X196M; X198S; X208R; X216R; X221S; X243S; X245I; X249F; X249G; and X249Y.3. The engineered ketoreductase polypeptide of claim 1 , wherein the amino acid sequence of said ketoreductase polypeptide comprises X206F/L claim 1 , and at least one or more residue differences as compared to SEQ ID NO:4 selected from: X17Q/R/M; X40R; X64V; X94P; X96L/Y; X144V; X147Q/I/L; X157C; X195A/G; X196M; and X199H.4. The engineered ketoreductase polypeptide of claim 1 , wherein the ketoreductase polypeptide is capable of converting the substrate compound (2) to the product compound (1) with at least 10 fold the activity of the reference polypeptide of SEQ ID NO:4 claim 1 , wherein the amino acid sequence comprises the substitution X206F/L claim 1 , and one or more residue differences as compared to SEQ ID NO:4 selected from: X40R; X60I; X71P; X94P; X94A; X95M; X96L; X96Y; X127R; X144V; X1451; X150Y; X152G; X153G; X157C; X195A; X195G; X196M; X198S; X199H; X216R claim 1 , X245I claim 1 , X245F; X249Y; and X249F.5. The engineered ketoreductase polypeptide of claim 1 , wherein the ketoreductase polypeptide has increased thermal stability as compared to the reference polypeptide of SEQ ID NO:4 or 32 claim 1 , wherein the amino acid sequence comprises the substitution X206F/L claim 1 , and one or more residue differences as ...

Enzymatic Route For The Preparation Of Chiral Gamma-Aryl-Beta-Aminobutyric Acid Derivatives

The present invention relates to a process for preparing chiral γ-aryl-β-aminobutyric acid compounds and derivatives thereof. 2. The process according to claim 1 , wherein in step (iii) the compound of Formula (R)-II is obtained by acidifying the aqueous medium and extracting the compound of Formula (R)-II from acidic aqueous medium with a water immiscible solvent claim 1 , optionally subsequently removing the water immiscible solvent.3. The process according to claim 1 , wherein Ar is halo substituted phenyl.4. The process according to claim 1 , wherein W is phenyl claim 1 , and/or wherein R is H.11. The compound according to claim 10 , wherein W is phenyl and Y is H.13. Use of a compound as set forth in in a process for preparing a gliptin compound.15. The process according to claim 14 , wherein said gliptin compound or a pharmaceutically acceptable salt thereof is combined with another pharmaceutically active ingredient claim 14 , either within the same pharmaceutical composition or another pharmaceutical composition claim 14 , wherein said another pharmaceutically active ingredient is selected from the group consisting of insulin sensitizers claim 14 , insulin claim 14 , insulin mimetics claim 14 , sulfonylureas claim 14 , (α-glucosidase inhibitors claim 14 , glucagon receptor antagonists claim 14 , GLP-1 claim 14 , GLP-1 analogues claim 14 , GLP-1 mimetics claim 14 , GLP-1 receptor agonists claim 14 , GIP claim 14 , GIP mimetics claim 14 , PACAP claim 14 , PACAP mimetics claim 14 , PACAP receptor agonists claim 14 , cholesterol lowering agents claim 14 , PPAR-δ agonists claim 14 , anti-obesity compounds claim 14 , ileal bile acid transporter inhibitors claim 14 , agents intended for use in inflammatory conditions claim 14 , antihypertensive agents claim 14 , glucokinase activators (GKAs) claim 14 , inhibitors of 11(-hydroxysteroid dehydrogenase type 1 claim 14 , inhibitors of cholesteryl ester transfer protein (CETP) and inhibitors of fructose 1 claim 14 ,6- ...

Methods for Making L-Glufosinate

Methods for the production of L-glufosinate (also known as phosphinothricin or (S)-2-amino-4-(hydroxy(methyl)phosphonoyl)butanoic acid) are provided. The methods comprise a two-step process. The first step involves the oxidative deamination of D-glufosinate to PPO (2-oxo-4-(hydroxy(methyl)phosphinoyl)butyric acid). The second step involves the specific amination of PPO to L-glufosinate, using an amine group from one or more amine donors. By combining these two reactions, the proportion of L-glufosinate in a mixture of L-glufosinate and D-glufosinate can be substantially increased. 1. A method for making L-glufosinate , comprising:reacting D-glufosinate with a D-amino acid oxidase (DAAO) enzyme to form PPO (2-oxo-4-(hydroxy(methyl)phosphinoyl)butyric acid);aminating the PPO to L-glufosinate by a transaminase (TA) enzyme, using an amine group from one or more amine donors,wherein at least 70% of the D-glufosinate is converted to L-glufosinate; andsubjecting the composition to further purification methods selected from ion exchange, extraction, salt formation, crystallization, and filtration.2. The method of claim 1 , wherein the one or more amine donors is selected from the group consisting of glutamate and L-glutamate.3. The method of claim 1 , wherein the D-glufosinate is originally present in a racemic mixture of D- and L-glufosinate or salts thereof.4. A D-amino acid oxidase (DAAO) enzyme comprising one or more mutations at positions 54 claim 1 , 56 claim 1 , 58 claim 1 , 213 claim 1 , and 238 using SEQ ID NO: 2 as a reference sequence.5. The DAAO enzyme of claim 4 , wherein said mutation at position 54 is selected from the group consisting of N54C claim 4 , N54L claim 4 , N54T claim 4 , and N54V.6. The DAAO enzyme of claim 5 , wherein said mutation at position 56 is T56M.7. The DAAO enzyme of claim 4 , wherein said mutation at position 58 is selected from the group consisting of F58A claim 4 , F58G claim 4 , F58H claim 4 , F58K claim 4 , F58N claim 4 , F58Q claim ...

PROCESSES USING AMINO ACID DEHYDROGENASES AND KETOREDUCTASE-BASED COFACTOR REGENERATING SYSTEM

The present disclosure relates to the use of an amino acid dehydrogenase in combination with a cofactor regenerating system comprising a ketoreductase. In particular embodiments, the process can be used to prepare L-tert-leucine using a leucine dehydrogenase. 2. The process of claim 1 , wherein R is a substituted or unsubstituted —(C-C)alkyl claim 1 , —(C-C)alkenyl claim 1 , —(C-C)alkynyl claim 1 , —(C-C)cycloalkyl claim 1 , heterocycloalkyl claim 1 , aryl claim 1 , or heteroaryl.3. The process of claim 1 , wherein the lower secondary alcohol is isopropanol and the ketone is acetone.4. The process of claim 1 , further comprising the step of removing the lower secondary alcohol formed from the lower alkyl ketone from the reaction medium.5. The process of claim 1 , wherein the reaction medium is at a pH of about 8.5 to about 10.6. The process of claim 5 , wherein the reaction medium is at a pH of about 8.5 to about 9.0.7. The process of claim 1 , wherein the reaction medium is at a temperature of about 25° C. to about 45° C.8. The process of claim 7 , wherein the reaction medium is at a temperature of about 35° C. to about 40° C.9. The process of claim 1 , wherein the secondary alcohol is present in at least 1.5 fold stoichiometric excess of substrate.10. The process of claim 1 , wherein the ketoreductase is capable of recycling cofactor by converting isopropanol (IPA) to acetone in a reaction medium of 3 to 20% IPA at a pH of about 9.0 to 10.5 with an activity at least 2.0-fold greater than a reference ketoreductase encoded by the polynucleotide of SEQ ID NO: 1.11. The process of claim 1 , wherein the activity of the ketoreductase with compound of formula I is less than 2% claim 1 , 1% claim 1 , 0.5% claim 1 , 0.1% claim 1 , 0.05% claim 1 , or 0.01% of the activity of the amino acid dehydrogenase with the compound of formula I claim 1 , or optionally no activity with the compound of formula I.12. The process of claim 1 , wherein the compound of formula IIa is 3 claim ...

KETOREDUCTASE POLYPEPTIDES

The present disclosure provides engineered ketoreductase enzymes having improved properties as compared to a naturally occurring wild-type ketoreductase enzyme including the capability of reducing 5-((4S)-2-oxo-4-phenyl (1,3-oxazolidin-3-yl))-1-(4-fluorophenyl) pentane-1,5-dione to (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one. Also provided are polynucleotides encoding the engineered ketoreductase enzymes, host cells capable of expressing the engineered ketoreductase enzymes, and methods of using the engineered ketoreductase enzymes to synthesize the intermediate (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one in a process for making Ezetimibe. 1. An engineered polypeptide having ketoreductase activity , wherein said engineered polypeptide comprises a region with an amino acid sequence having at least 90% sequence identity to residues 90 to 211 of SEQ ID NO: 130 , comprising a substitution at position X153 , and further comprising one or more of the following features selected from:residue corresponding to X94 is asparagine, glycine, serine, or a polar residue;residue corresponding to X95 is an aliphatic residue;residue corresponding to X96 is glutamine, asparagine, or threonine;residue corresponding to X101 is an acidic, non-polar, or a polar residue;residue corresponding to X105 is an acidic or non-polar residue;residue corresponding to X108 is a hydrophilic, polar or constrained residue;residue corresponding to X111 is a non-polar or aliphatic residue;residue corresponding to X112 is an acidic or polar residue;residue corresponding to X113 is a non-polar or aliphatic residue;residue corresponding to X127 is a basic residue;residue corresponding to X147 is a non-polar, aliphatic, aromatic, or hydrophobic residue;residue corresponding to X152 is a non-polar, basic, or hydrophilic residue;residue corresponding to X157 is a polar residue;residue corresponding to X163 is a non-polar or aliphatic ...

BIOCATALYSTS AND METHODS FOR THE SYNTHESIS OF ARMODAFINIL

The present invention relates to non-naturally occurring polypeptides useful for preparing armodafinil, polynucleotides encoding the polypeptides, and methods of using the polypeptides. The non-naturally occurring polypeptides of the present invention are effective in carrying out biocatalytic conversion of the (i) 2-(benzhydrylsulfinyl)acetamide to (−)-2-[(R)-(diphenylmethyl)sulfinyl]acetamide (armodafinil), or (ii) benzhydryl-thioacetic acid to (R)-2-(benzhydrylsulfinyl)acetic acid, which is a pivotal intermediate in the synthesis of armodafinil, in enantiomeric excess. 1. A non-naturally occurring polynucleotide encoding a non-naturally occurring polypeptide having cyclohexanone monooxygenase (CHMO) activity , wherein the amino acid sequence of the polypeptide has at least 90% sequence identity to SEQ ID NO:136 , and one or more amino acid substitutions at one or more positions corresponding to positions in SEQ ID NO: 136 , selected from the group consisting of 37 , 277 , 278 , 280 , 281 , 326 , 432 , 433 , 435 , and 490.3. The non-naturally occurring polynucleotide encoding the non-naturally occurring polypeptide of claim 1 , wherein said non-naturally occurring polypeptide further comprises one or more amino acid substitutions relative to SEQ ID NO: 136 claim 1 , wherein the polypeptide comprises an alanine claim 1 , glutamic acid claim 1 , glycine claim 1 , isoleucine claim 1 , lysine claim 1 , proline claim 1 , serine claim 1 , threonine claim 1 , or valine at a position corresponding to position 246 of SEQ ID NO:136.4. The non-naturally occurring polynucleotide encoding the non-naturally occurring polypeptide of claim 1 , wherein said non-naturally occurring polypeptide is further capable of converting the acid substrate of compound (1b) to the R-enantiomer compound (2b) in at least 50% enantiomeric excess.5. The non-naturally occurring polynucleotide encoding the non-naturally occurring polypeptide of claim 1 , wherein said non-naturally occurring ...

TRANSAMINASE POLYPEPTIDES

The present disclosure provides engineered transaminase enzymes having improved properties as compared to a naturally occurring wild-type transaminase enzyme. Also provided are polynucleotides encoding the engineered transaminase enzymes, host cells capable of expressing the engineered transaminase enzymes, and methods of using the engineered transaminase enzymes to synthesize a variety of chiral compounds. 1. An engineered transaminase polypeptide , wherein said transaminase polypeptide comprises a polypeptide sequence at least 80% identical to SEQ ID NO: 2 , and wherein the transaminase polypeptide comprises a substitution at one or more positions selected from the group consisting of X57 , X211 , X294 , X324 , and X391.2. The engineered transaminase polypeptide of claim 1 , wherein said polypeptide has at least about 10% residual activity in the conversion of pyruvate to L-alanine in presence of amino donor isopropylamine after treatment of the polypeptide at 50° C. for 23 h.3. The engineered transaminase polypeptide of claim 1 , wherein the transaminase polypeptide further comprises at least one additional substitution at a residue position selected from: X4 claim 1 , X12 claim 1 , X21 claim 1 , X157 claim 1 , X208 claim 1 , X253 claim 1 , X272 claim 1 , X302 claim 1 , X316 claim 1 , X398 claim 1 , X418 claim 1 , X420 claim 1 , X431 claim 1 , X444 claim 1 , and X446.4. The engineered transaminase polypeptide of claim 3 , wherein the transaminase polypeptide sequence comprises the substitution X233L and at least one further substitution selected from: X4 is R claim 3 , Q claim 3 , or L; X12 is K; X21 is N; X45 is H; X86 is Y; X157 is T; X177 is L; X208 is I; X211 is K; X253 is M; X272 is A; X294 is V; X302 is K; X316 is K; X324 is G; X391 is A; X398 is R; X418 is V; X420 is N; X431 is D; X444 is V; and X446 is V.5. The engineered transaminase polypeptide of claim 1 , wherein the transaminase polypeptide has increased transaminase activity as compared to SEQ ID NO ...

TRANSAMINASE POLYPEPTIDES

The present disclosure provides engineered transaminase enzymes having improved properties as compared to a naturally occurring wild-type transaminase enzyme. Also provided are polynucleotides encoding the engineered transaminase enzymes, host cells capable of expressing the engineered transaminase enzymes, and methods of using the engineered transaminase enzymes to synthesize a variety of chiral compounds. 1. An engineered transaminase polypeptide , wherein said transaminase polypeptide comprises a polypeptide sequence at least 80% identical to SEQ ID NO:2 , and wherein the transaminase polypeptide comprises a substitution at position 177.2. The engineered transaminase polypeptide of claim 1 , wherein said polypeptide has at least about 10% residual activity in the conversion of pyruvate to L-alanine in presence of amino donor isopropylamine after treatment of the polypeptide at 50° C. for 23 h.3. The engineered transaminase polypeptide of claim 1 , wherein the transaminase polypeptide further comprises at least one additional substitution at a residue position selected from: X4 claim 1 , X9 claim 1 , X12 claim 1 , X21 claim 1 , X45 claim 1 , X86 claim 1 , X157 claim 1 , X177 claim 1 , X208 claim 1 , X211 claim 1 , X253 claim 1 , X272 claim 1 , X294 claim 1 , X302 claim 1 , X316 claim 1 , X324 claim 1 , X391 claim 1 , X398 claim 1 , X418 claim 1 , X420 claim 1 , X431 claim 1 , X444 claim 1 , and X446.4. The engineered transaminase polypeptide of claim 3 , wherein the transaminase polypeptide sequence comprises the substitution X177L and at least one further substitution selected from: X4 is R claim 3 , Q claim 3 , or L; X12 is K; X21 is N; X45 is H; X86 is Y; X157 is T; X208 is I; X211 is K; X253 is M; X272 is A; X294 is V; X302 is K; X316 is K; X324 is G; X391 is A; X398 is R; X418 is V; X420 is N; X431 is D; X444 is V; and X446 is V.5. The engineered transaminase polypeptide of claim 4 , wherein the transaminase polypeptide sequence further comprises at least one ...

PROCESS FOR THE ENZYMATIC REGENERATION OF REDOX COFACTORS

A process for the enzymatic regeneration of the redox cofactors NAD/NADH and NADP/NADPH in a one-pot reaction, wherein, as a result of at least two further enzymatically catalyzed redox reactions proceeding in the same reaction batch (product-forming reactions), one of the two redox cofactors accumulates in its reduced form and, respectively, the other one in its oxidized form, characterized in that a) in the regeneration reaction which reconverts the reduced cofactor into its original oxidized form, oxygen or a compound of general formula RC(O)COOH is reduced, and b) in the regeneration reaction which reconverts the oxidized cofactor into its original reduced form, a compound of general formula RCH(OH)Ris oxidized and wherein R, Rand Rin the compounds have different meanings. 1a) in the regeneration reaction which reconverts the reduced cofactor into its original oxidized form, oxygen or a compound of general formula. A process for the enzymatic regeneration of the redox cofactors NAD/NADH and/or, in particular and, NADP/NADPH in a one-pot reaction, wherein, as a result of at least two further enzymatically catalyzed redox reactions proceeding in the same reaction batch (product-forming reactions), one of the two redox cofactors accumulates in its reduced form and, respectively, the other one in its oxidized form, characterized in that{'sub': 1', '1', '4', '1', '4, 'wherein Rrepresents a linear-chain or branched (C-C)-alkyl group or a (C-C)-carboxy alkyl group, is reduced, and'}{'sub': 4', '8, 'b) in the regeneration reaction which reconverts the oxidized cofactor into its original reduced form, a (C-C)-cycloalcanol or a compound of general formula'}{'sub': 2', '3', '1', '6', '1', '6', '6', '12', '1', '4', '3', '8, 'wherein Rand Rare independently selected from the group consisting of H, (C-C) alkyl, wherein alkyl is linear-chain or branched, (C-C) alkenyl, wherein alkenyl is linear-chain or branched and comprises one to three double bonds, aryl, C-Caryl, carboxyl, ...

Ketoreductase polypeptides and polynucleotides

The present invention provides engineered ketoreductase and phosphite dehydrogenase enzymes having improved properties as compared to a naturally occurring wild-type ketoreductase and phosphite dehydrogenase enzymes, as well as polynucleotides encoding the engineered ketoreductase and phosphite dehydrogenase enzymes, host cells capable of expressing the engineered ketoreductase and phosphite dehydrogenase enzymes, and methods of using the engineered ketoreductase and phosphite dehydrogenase enzymes to synthesize a chiral catalyst used in the synthesis of antiviral compounds, such as nucleoside inhibitors. The present invention further provides methods of using the engineered enzymes to deracemize a chiral alcohol in a one-pot, multi-enzyme system.

METHOD FOR PRODUCING PYRROLE DERIVATIVE, AND INTERMEDIATE THEREOF

The present invention provides a method for producing an atropisomer of a pyrrole derivative having excellent mineralocorticoid receptor antagonistic activity, and an intermediate thereof. A method for producing an atropisomer of a pyrrole derivative using a compound represented by (B) [wherein Rrepresents a C1-C4 alkyl group, and Rrepresents a 2-hydroxyethyl group or a carboxymethyl group] as a production intermediate. 5. The method according to claim 3 , wherein the enzyme is a lipase.6. The method according to claim 3 , wherein the enzyme is an immobilized lipase.7. The method according to claim 3 , wherein the solvent is an organic solvent.10. The method according to claim 8 , wherein the optically active amine is (R)-(+)-1-(1-naphthyl)ethylamine.12. A method for producing ethyl (S)-1-(2hydroxyethyl)-4-methyl-5-[2-(trifluoromethyl)phenyl]-1H-pyrrole-3-carboxylate claim 8 , comprising the following steps of:(i) obtaining an optically active amine salt of (S)-2-[4-ethoxycarbonyl-3-methyl-2-[2-(trifluoromethyl)phenyl]-1H-pyrrol-1-yl]acetic acid by resolving (RS)-2-[4-ethoxycarbonyl-3-methyl-2-[2-(trifluoromethyl)phenyl]-1H-pyrrol-1-yl]acetic acid in a solvent using an optically active amine;(ii) removing the optically active amine under an acidic condition; and thereafter(iii) performing reduction using a reducing agent.13. The method according to claim 11 , wherein the reducing agent is sodium borohydride.14. The method according to claim 11 , wherein the optically active amine is quinine claim 11 , cinchonine claim 11 , or (R)-1-(1-naphthyl)ethylamine.15. The method according to claim 11 , wherein the optically active amine is cinchonine.16. The method according to claim 11 , wherein the solvent is an organic solvent.18. The production method according to claim 17 , wherein the reagent is a Grignard reagent.20. The method according to claim 4 , wherein the enzyme is a lipase.21. The method according to claim 4 , wherein the enzyme is an immobilized lipase.22. The ...

METHOD FOR PRODUCING PYRROLE DERIVATIVE, AND INTERMEDIATE THEREOF

The present invention provides a method for producing an atropisomer of a pyrrole derivative having excellent mineralocorticoid receptor antagonistic activity, and an intermediate thereof. A method for producing an atropisomer of a pyrrole derivative using a compound represented by (B) [wherein Rrepresents a C1-C4 alkyl group, and Rrepresents a 2-hydroxyethyl group or a carboxymethyl group] as a production intermediate. 2. A compound according to claim 1 , wherein Ris an ethyl group.3. A compound according to claim 1 , wherein Ris a 2-hydroxyethyl group.4. A compound according to claim 1 , wherein Ris a carboxymethyl group. This application is a division of U.S. application Ser. No. 15/699,954, filed Sep. 8, 2017, which is a division of U.S. application Ser. No. 15/043,260, filed Feb. 12, 2016, which is a continuation of International Application No. PCT/JP2014/072332, filed Aug. 26, 2014, which claims priority from Japanese Application No. 2013-175172, filed Aug. 27, 2013. Each application is incorporated herein by reference in its entirety.The present invention relates to a method for producing an atropisomer of a pyrrole derivative having excellent mineralocorticoid receptor antagonistic activity, and a production intermediate thereof.A mineralocorticoid receptor (MR) (aldosterone receptor) is known to play an important role in regulating electrolyte balance and blood pressure in the body, and MR antagonists having a steroidal structure such as spironolactone and eplerenone are known to be useful for the treatment of hypertension and heart failure.1-(2-Hydroxyethyl)-4-methyl-N-[4-(methylsulfonyl)-phenyl]-5-[2-(trifluoromethyl) phenyl]-1H-pyrrole-3-carboxamide, which is a pyrrole derivative, is disclosed in PTL 1. Further, an atropisomer thereof is disclosed in PTL 2 and is known to be useful for the treatment of hypertension, diabetic nephropathy, and the like.PTL 1: WO 2006/012642 (US Patent Application No. US 2008-0234270)PTL 2: WO 2008/126831 (US Patent ...

SOLID FORMS OF A THIENOPYRIMIDINEDIONE ACC INHIBITOR AND METHODS FOR PRODUCTION THEREOF

The present invention provides solid forms of compounds useful as inhibitors of Acetyl CoA Carboxylase (ACC), compositions thereof, methods of producing the same, and methods of using the same in the treatment of ACC-mediated diseases. 116.-. (canceled)18. The process of claim 17 , wherein Ris bromo.1940.-. (canceled)4244.-. (canceled) This application is a continuation of U.S. application Ser. No. 15/446,873, filed Mar. 1, 2017, which claims the benefit under 35 U.S.C. § 119(e) to U.S. Application No. 62/302,755, filed Mar. 2, 2016, and U.S. Application No. 62/303,237, filed Mar. 3, 2016, each of which are incorporated herein by reference.Obesity is a health crisis of epic proportions. The health burden of obesity, measured by quality-adjusted life-years lost per adult, has surpassed that of smoking to become the most serious, preventable cause of death. In the U.S., about 34% of adults have obesity, up from 31% in 1999 and about 15% in the years 1960 through 1980. Obesity increases the rate of mortality from all causes for both men and women at all ages and in all racial and ethnic groups. Obesity also leads to social stigmatization and discrimination, which decreases quality of life dramatically. The chronic diseases that result from obesity cost the U.S. economy more than $150 billion in weight-related medical bills each year. Furthermore, about half of the obese population, and 25% of the general population, have metabolic syndrome, a condition associated with abdominal obesity, hypertension, increased plasma triglycerides, decreased HDL cholesterol, and insulin resistance, which increases the risk for type-2 diabetes (T2DM), stroke and coronary heart disease (Harwood, 9: 267, 2005).Diet and exercise, even when used in conjunction with the current pharmacotherapy, do not provide sustainable weight loss needed for long-term health benefit. Currently, only a few anti-obesity drugs are approved in the U.S., the fat absorption inhibitor orlistat (Xenical®), the 5- ...

Systems and methods for microbial production

Methods to produce microbial biomass are provided. The methods involve the inoculation of a liquid growth medium including a controlled substrate with a microbial inoculum and incubation under conditions that are suitable for the production of biomass. The biomass and/or the liquid growth medium may be used to produce valuable products including food and feed products.

MUTANTS OF HYDANTOINASE

The present invention relates to it hydantoinase having an amino acid sequence selected from (i) or (ii), with (i) amino acid sequence selected from SEQ ID NO: 6-20 and SEQ ID NO: 73-119 (ii) amino acid sequence wherein in the amino acid sequence of SEQ ID NO: 6-20 and SEQ ID NO: 73-119, 1 to 75 amino acid residues have been substituted, deleted, inserted and/or added, and wherein further the catalytic activity of the hydantoinase is higher by a factor of at least 1.2 than the catalytic activity of the hydantoinase having amino acid sequence SEQ ID NO: 1. The present invention further relates to a process for preparing amino acids, wherein said hydantoinase is used. 15-. (canceled)6. An isolated polynucleotide encoding an hydantoinase , wherein the catalytic activity of the hydantoinase encoded by the isolated polynucleotide is higher by a factor of at least 1.2 than the catalytic activity of the hydantoinase having the amino acid sequence SEQ ID NO: 1 , and wherein the catalytic activity of an hydantoinase is the catalytic activity of the hydantoinase with respect to transformation of a 5-substituted D-hydantoin compound to the corresponding D-carbamoylamino acid.7. The isolated polynucleotide of claim 6 , wherein the isolated polynucleotide comprises a nucleotide sequence encoding an hydantoinase comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 6 claim 6 , SEQ ID NO: 7 claim 6 , SEQ ID NO: 8 claim 6 , SEQ ID NO: 9 claim 6 , SEQ ID NO: 10 claim 6 , SEQ ID NO: 11 claim 6 , SEQ ID NO: 12 claim 6 , SEQ ID NO: 13 claim 6 , SEQ ID NO: 14 claim 6 , SEQ ID NO: 15 claim 6 , SEQ ID NO: 16 claim 6 , SEQ ID NO: 17 claim 6 , SEQ ID NO: 18 claim 6 , SEQ ID NO: 19 claim 6 , SEQ ID NO: 20 claim 6 , SEQ ID NO: 73 claim 6 , SEQ ID NO: 74 claim 6 , SEQ ID NO: 75 claim 6 , SEQ ID NO: 76 claim 6 , SEQ ID NO: 77 claim 6 , SEQ ID NO: 78 claim 6 , SEQ ID NO: 79 claim 6 , SEQ ID NO: 80 claim 6 , SEQ ID NO: 81 claim 6 , SEQ ID NO: 82 claim 6 , SEQ ID NO: ...

NOVEL HYDROLASE PROTEIN

It is an object of the present invention to provide a novel hydrolase, which is used when dialkyl 2-vinylcyclopropane-1,1-dicarboxylate is hydrolyzed with an enzyme, so as to efficiently obtain (1S,2S)-1-alkoxycarbonyl-2-vinylcyclopropanecarboxylic acid that is useful as an intermediate for synthesizing therapeutic agents for hepatitis C. According to the present invention, there is provided a hydrolase protein, which consists of the amino acid sequence shown in any one of SEQ ID NOS. 2 to 5 and which has activity of catalyzing, at higher selectivity than the protein consisting of the amino acid sequence shown in SEQ ID NO. 1, a reaction of producing (1S,2S)-1-ethoxycarbonyl-2-vinylcyclopropanecarboxylic acid from diethyl 2-vinylcyclopropane- 1,1 -dicarboxylate. 2. A method for producing (1S ,2S)-1-alkoxycarbonyl-2-vinylcyclopropanecarboxylic acid , which comprises contacting dialkyl 2-vinylcyclopropane-1 ,1-dicarboxylate with a hydrolase protein to produce (1S ,2S)-1-alkoxycarbonyl-2-vinylcyclopropanecarboxylic acid , wherein the hydrolase protein consists of an amino acid sequence shown in any one of SEQ ID NOs: 2 to 5. This application is a Divisional of U.S. patent application Ser. No. 13/813,047, which is a National Stage of International Application No. PCT/JP2011/069680, filed Aug. 31, 2011, which claims priority to Japanese Application No. 2010-194630, filed Aug. 31, 2010. The disclosures of each of U.S. patent application Ser. No. 13/813,047 and PCT/JP2011/069680 are expressly incorporated by reference herein in their entireties.The present invention relates to a novel hydrolase protein that can be used in the production of (1S,2S)-1-alkoxycarbonyl-2-vinylcyclopropanecarboxylic acid, and a use thereof.(1S,2S)-1-alkoxycarbonyl-2-vinylcyclopropanecarboxylic acid is an intermediate that is useful for the production of various types of HCV NS3 protease inhibitors that are currently under development as therapeutic agents for hepatitis C. Non Patent Document 1 ...

Novel amidase

An object of the present invention is to provide a means for producing an optically active tropic acid that is a compound useful as a synthetic raw material or an intermediate for pharmaceutical products and the like. The present invention provides a novel polypeptide having activity to (R)-selectively hydrolyze a racemic tropic acid amide, DNA encoding the polypeptide, a vector containing the DNA, a transformant prepared by transformation with the vector, and a method for producing an optically active carboxylic acid amide and an optically active carboxylic acid using them.

PROCESS FOR PREPARING (CYCLOPENTYL[d]PYRIMIDIN-4-YL)PIPERAZINE COMPOUNDS

The present disclosure relates to processes for preparing (cyclopentyl[d]pyrimidin-4-yl)piperazine compounds, and more particularly relates to processes for preparing (R)-4-(5-methyl-7-oxo-6,7-dihydro-5H-cyclopenta[d] pyrimidin-4-yl)piperazine and N-protected derivatives thereof, which may be used as an intermediate in the synthesis of Ipatasertib (i.e., (S)-2-(4-chlorophenyl)-1-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-(isopropylamino)-propan-1-one). The present disclosure additionally relates to various compounds that are intermediates employed in these processes. 3. The process of claim 2 , wherein Y is bromo.4. The process of or claim 2 , wherein Lv is selected from hydrogen and a halogen claim 2 , and Ris selected from acetyl claim 2 , trifluoroacetyl claim 2 , phthalimidyl claim 2 , benzyl claim 2 , triphenylmethyl claim 2 , benzylidenyl claim 2 , p-toluenesulfonyl claim 2 , p-methoxybenzyl claim 2 , tertbutyloxycarbonyl claim 2 , 9-fluorenylmethyloxycarbonyl and carbobenzyloxy.5. The process of any one of - claim 2 , wherein the compound of Formula IV claim 2 , or salt thereof claim 2 , is contacted with from about 1 molar equivalent to about 1.5 molar equivalents of the piperazine compound.6. The process of any one of - claim 2 , wherein the compound of Formula IV claim 2 , or salt thereof claim 2 , is contacted with the piperazine compound at room temperature.8. The process of claim 7 , wherein each X is chloro.9. The process of claim 7 , wherein each X is hydroxyl.10. The process of any one of - claim 7 , wherein the brominating agent is selected from bromine claim 7 , bromotrimethylsilane claim 7 , phosphorus oxybromide claim 7 , N-bromosuccinimide claim 7 , and phosphorus tribromide.11. The process of any one of - claim 7 , wherein the compound of Formula V claim 7 , or salt thereof claim 7 , is contacted with from about 2 molar equivalents to about 7 molar equivalents of the brominating agent.12. The ...

TRANSAMINASE POLYPEPTIDES

The present disclosure provides engineered transaminase enzymes having improved properties as compared to a naturally occurring wild-type transaminase enzyme. Also provided are polynucleotides encoding the engineered transaminase enzymes, host cells capable of expressing the engineered transaminase enzymes, and methods of using the engineered transaminase enzymes to synthesize a variety of chiral compounds. 1. An engineered transaminase polypeptide , wherein said transaminase polypeptide comprises a polypeptide sequence at least 80% identical to SEQ ID NO:2 , and wherein the transaminase polypeptide comprises a substitution at position 233.2. The engineered transaminase polypeptide of claim 1 , wherein said polypeptide has at least about 10% residual activity in the conversion of pyruvate to L-alanine in presence of amino donor isopropylamine after treatment of the polypeptide at 50° C. for 23 h.3. The engineered transaminase polypeptide of claim 1 , wherein the transaminase polypeptide further comprises at least one additional substitution at a residue position selected from: X4 claim 1 , X9 claim 1 , X12 claim 1 , X21 claim 1 , X45 claim 1 , X86 claim 1 , X157 claim 1 , X177 claim 1 , X208 claim 1 , X211 claim 1 , X253 claim 1 , X272 claim 1 , X294 claim 1 , X302 claim 1 , X316 claim 1 , X324 claim 1 , X391 claim 1 , X398 claim 1 , X418 claim 1 , X420 claim 1 , X431 claim 1 , X444 claim 1 , and X446.4. The engineered transaminase polypeptide of claim 3 , wherein the transaminase polypeptide sequence comprises the substitution X233L and at least one further substitution selected from: X4 is R claim 3 , Q claim 3 , or L; X12 is K; X21 is N; X45 is H; X86 is Y; X157 is T; X177 is L; X208 is I; X211 is K; X253 is M; X272 is A; X294 is V; X302 is K; X316 is K; X324 is G; X391 is A; X398 is R; X418 is V; X420 is N; X431 is D; X444 is V; and X446 is V.5. The engineered transaminase polypeptide of claim 4 , wherein the transaminase polypeptide sequence further comprises ...

Solid forms of a thienopyrimidinedione acc inhibitor and methods for production thereof

The present invention provides solid forms of compounds useful as inhibitors of Acetyl CoA Carboxylase (ACC), compositions thereof, methods of producing the same, and methods of using the same in the treatment of ACC-mediated diseases.

PROCESSES USING AMINO ACID DEHYDROGENASES AND KETOREDUCTASE-BASED COFACTOR REGENERATING SYSTEM

The present disclosure relates to the use of an amino acid dehydrogenase in combination with a cofactor regenerating system comprising a ketoreductase. In particular embodiments, the process can be used to prepare L-tert-leucine using a leucine dehydrogenase. 4Bacillus, Clostridium, Corynebacterium, Geobacillus, Natronobacterium, Synechocystis, Thermoactinomyces, Thermos, ThermomicrobiumCarderia.. The process of claim 3 , wherein the L-amino acid dehydrogenase is from claim 3 , or5. The process of claim 4 , wherein the L-amino acid dehydrogenase is selected from L-alanine dehydrogenase claim 4 , L-aspartate dehydrogenase claim 4 , L-erythro-3 claim 4 ,5-diaminohexanoate dehydrogenase claim 4 , L-leucine dehydrogenase claim 4 , L-glutamate dehydrogenase claim 4 , lysine dehydrogenase claim 4 , L-phenylalanine dehydrogenase claim 4 , L-serine dehydrogenase. L-valine dehydrogenase claim 4 , L-2 claim 4 ,4-diaminopentanoate dehydrogenase claim 4 , L-glutamate synthase claim 4 , L-diaminopimelate dehydrogenase claim 4 , L-N-methylalanine dehydrogenase claim 4 , L-lysine 6-dehydrogenase claim 4 , and L-tryptophan dehydrogenase.7Halobacterium, Methanosarcina, Pseudomonas, Pyrobaculum, Salmonella, CorynebacteriumEscherichia.. The process of claim 6 , wherein the D-amino acid dehydrogenase is from claim 6 , and8Pseudomonas aeruginosa, Pseudomonas fluorescens, Pyrobaculum islandicum, Salmonella typhimurium, Corynebacterium glutamicumEscherichia coli.. The process of claim 7 , wherein the D-amino acid dehydrogenase is from claim 7 , and9. The process of claim 8 , wherein the D-amino acid dehydrogenase is selected from D-alanine dehydrogenase claim 8 , D-threonine dehydrogenase claim 8 , and D-proline dehydrogenase.10. The process of which is carried out in a cell free system.11. The process of claim 1 , wherein the amino acid dehydrogenase is present as a crude extract.12. The process of claim 1 , wherein the amino acid dehydrogenase is substantially purified.13. The process ...

A PROCESS OF CHIRAL RESOLUTION OF CYCLIC AND ACYCLIC ACETATES TO ENANTIOMERICALLY PURE (R)--ALCOHOLS

The patent discloses herein a process for the chiral resolution of racemic cyclic and acyclic acetates to obtain (R)-alcohol. Further, it discloses the resolution of racemic cyclic and acyclic acetates to obtain enantiomerically pure (R)-(−)-alcohol as single enantiomer through fungal catalyzed deacylation in single fermentation, wherein fungal strains are 1. A process for the chiral resolution of racemic cyclic and acyclic acetate to obtain enantiomerically pure (R)-Alcohol as single enantiomer comprising the steps of;i) incubating fungus for 24-48 hrs at temperature ranging between 28° C. to 30° C. in a media;ii) adding substrates selected from cyclic and acyclic acetates in media of step (i) and incubating further for 6 hrs to 3 days at temperature ranging between 28° C. to 30° C. to obtain enantiomerically pure (R)-Alcohol.2F. proliferatum.. The process according to claim 1 , wherein the fungal strains is3F. proliferatum. The process according to claim 1 , wherein spores and mycelia of fungal strains were used.4. The process according to claim 1 , wherein the acyclic acetate is selected from the group consisting of 2-Heptyl acetate claim 1 , lavandulyl acetate and 2-Hexyl acetate.5. The process according to claim 1 , wherein the cyclic acetate is selected from the group consisting of 1-Phenylethyl acetate claim 1 , 1-Phenylpropyl acetate.6. The process according to claim 1 , wherein yield of the enantiomerically pure (R)-Alcohol as single enantiomer is in the range of 95-99.9%.7. The process according to claim 1 , wherein enantiomerically pure (R)-Alcohol is selected from the group consisting of R-lavandulol claim 1 , R-2-Hexnol claim 1 , R-2-Heptanol claim 1 , R-1-Phenyl ethanol and R-1-Phenyl propanol. The present invention relates to a process of chiral resolution of cyclic and acyclic acetates to obtain (R)-Alcohols using whole cell microorganisms.The present invention further relates to resolution of racemic cyclic and acyclic acetates to obtain ...

Protein complex capable of catalyzing asymmetric oxidation reaction and method for producing same

Provided are: a protein complex capable of selectively and asymmetrically oxidizing an enantiomer of a secondary alcohol without adding a coenzyme and having an asymmetric oxidation activity in a water-soluble solvent system in the presence of oxygen; a method for producing the same; and a method for coating the protein complex with a high molecular weight compound. The method for producing the protein complex includes: (1) enclosing a crude water-soluble protein in a gel, air-oxidizing the gel, and eluting the protein complex into an aqueous solution; and (2) applying gravity to concentrate and precipitate the protein complex, redissolving the precipitate in an aqueous glycine sodium hydroxide solution of about 0.5 mM and allowing the same to homogeneously coexist with a high molecular weight compound, and re-precipitating the solution and dehydrating and drying the same to yield a protein complex coated with a high molecular weight compound.

METHOD FOR PREPARING (R)-PRAZIQUANTEL

The invention relates to a new method for preparing (R)-praziquantel. In the invention, by taking advantage of the high stereo selectivity, site selectivity and region selectivity of an enzyme, an intermediate of a pure optical and chiral (R)-praziquantel are obtained by means of the dynamic kinetic resolution of an enantiomer from the synthesized racemate or derivatives thereof, and the (R)-praziquantel is obtained by using various conventional and mature organic chemical reactions with higher yield. The method of the invention has the potential advantages of easily available raw materials, low cost, environmentally safer process and convenience for large-scale production. Also, the purity of the end product can be more than 98%. By adopting the invention, the quality of the product is improved and a basis for developing high quality of active pharmaceutical ingredients and formulations is established, and thus the pending industrial problem of purifying praziquantel over 30 years becomes solvable. 2. The method for preparing (R)-praziquantel as claimed in claim 1 , wherein R is selected from the group consisting of methyl claim 1 , ethyl claim 1 , isopropyl and tert-butyl.3Aspergillus niger, Candida rugosa, Candida cylindracea, Rhizomucor miehei, Candida Antarctica, Pseudomonas cepacia, Pseudomonas fluorescens, Thermomyces lanuginose, Bacillus subtilis, Fusarium solani pisi, AlcaligenesRhizopus niveus, Mucor javanicusRhizopus oryzaeThermomyces lanuginose, Fusarium solani pisi, Bacillus subtilis, Pseudomonas cepaciaPseudomonas fluorescens.. The method for preparing (R)-praziquantel as claimed in claim 1 , wherein the lipase is selected from one or more microbial lipases derived from the group consisting of sp claim 1 , and claim 1 , and the lipases derived from and4. The method for preparing (R)-praziquantel as claimed in claim 1 , wherein the step (2) comprises:adding the racemic compound 3e, an ionic liquid, a solvent and an optional organic base to a sealed ...

Method for producing optically active amine compounds by deracemization

Disclosed are methods for producing optically active amino acids and amines. According to the methods, α-keto acids are generated as reaction intermediates, and as a result, ω-transaminase-catalyzed kinetic resolution of racemic amino acids or amines as racemic amine compounds enables the production of optically active amine compounds without the need to use expensive α-keto acids as starting materials. Therefore, the optically active amine compounds are produced at greatly reduced costs. In addition, the optically active amine compounds have high enantiomeric excess.

PROCESS FOR THE ENZYMATIC REGENERATION OF REDOX COFACTORS

A process for the enzymatic regeneration of the redox cofactors NAD/NADH and NADP/NADPH in a one-pot reaction, wherein, as a result of at least two further enzymatically catalyzed redox reactions proceeding in the same reaction batch (product-forming reactions), one of the two redox cofactors accumulates in its reduced form and, respectively, the other one in its oxidized form, characterized in that a) in the regeneration reaction which reconverts the reduced cofactor into its original oxidized form, oxygen or a compound of general formula RC(O)COOH is reduced, and b) in the regeneration reaction which reconverts the oxidized cofactor into its original reduced form, a compound of general formula RCH(OH)Ris oxidized and wherein R, Rand Rin the compounds have different meanings. 3. A process according to claim 1 , wherein Rand Rindependently of each other are selected from the group consisting of H claim 1 , (C-C) alkyl claim 1 , wherein alkyl is linear-chain or branched claim 1 , (C-C) alkenyl claim 1 , wherein alkenyl is linear-chain or branched and comprises one to three double bonds claim 1 , aryl claim 1 , C-Caryl claim 1 , carboxyl claim 1 , or (C-C) carboxy alkyl.4. A process according to claim 1 , characterized in that oxidation reaction(s) and reduction reaction(s) take place on the same substrate (molecular backbone).5. A process according to claim 1 , characterized in that oxidation reaction(s) and reduction reaction(s) proceed chronologically parallel.6. A process according to claim 1 , characterized in that claim 1 , in the regeneration reaction which reconverts the oxidized cofactor into its original reduced form claim 1 , 2-propanol is oxidized to acetone by means of an alcohol dehydrogenase.7. A process according to claim 1 , characterized in that claim 1 , in the regeneration reaction which reconverts the reduced cofactor into its original oxidized form claim 1 , pyruvate is reduced to lactate by means of a lactate dehydrogenase.8. A process according ...

OMEGA-TRANSAMINASE MUTANTS WITH ACTIVITY IMPROVEMENTS TOWARD KETONES AND METHODS FOR PRODUCING OPTICALLY PURE AMINES

Provided is an omega-transaminase mutant with improved activity using a point mutation in an active site of an omega-transaminase. Specifically, provided is a method for improving activity and extending a substrate spectrum of omega-transaminase by introducing a point mutation into a wild-type omega-transaminase rendered by replacing tryptophan at position 58 with the other amino acid. 1. An omega-transaminase mutant comprising a point mutation of a wild-type omega-transaminase represented by an amino acid sequence of SEQ ID NO: 1 , the point mutation is rendered by replacing tryptophan at position 58 with the otheramino acid.2. The omega-transaminase mutant according to claim 1 , wherein the other amino acid is an sterically less demanding amino acid having a lower molecular weight than that of an inherent amino acid of the wild-type omega-transaminase.3. The omega-transaminase mutant according to claim 2 , wherein the sterically less demanding amino acid is a neutral amino acid or an anionic amino acid.4. The omega-transaminase mutant according to claim 3 , wherein the neural amino acid is a hydrophobic amino acid or a polar uncharged amino acid.5. The omega-transaminase mutant according to claim 4 , wherein the hydrophobic amino acid comprises any one hydrophobic amino acid selected from the group consisting of alanine claim 4 , valine claim 4 , leucine claim 4 , isoleucine claim 4 , proline claim 4 , glycine claim 4 , phenylalanine and methionine.6. The omega-transaminase mutant according to claim 4 , wherein the polar uncharged amino acid comprises any one amino acid selected from the group consisting of serine claim 4 , threonine claim 4 , cysteine claim 4 , glutamine claim 4 , asparagine and tyrosine.7. The omega-transaminase mutant according to claim 4 , wherein the anionic amino acid comprises any one amino acid selected from the group consisting of aspartic acid and glutamic acid.8. The omega-transaminase mutant according to claim 3 , wherein the omega- ...

PROCESSES USING AMINO ACID DEHYDROGENASES AND KETOREDUCTASE-BASED COFACTOR REGENERATING SYSTEM

The present disclosure relates to the use of an amino acid dehydrogenase in combination with a cofactor regenerating system comprising a ketoreductase. In particular embodiments, the process can be used to prepare L-tert-leucine using a leucine dehydrogenase. 4Bacillus, Clostridium, Corynebacterium, Geobacillus, Natronobacterium, Synechocystis, Thermoactinomyces, Thermos, ThermomicrobiumCarderia.. The process of claim 3 , wherein the D-amino acid dehydrogenase is from claim 3 , or6Halobacterium, Methanosarcina, Pseudomonas, Pyrobaculum, Salmonella, CorynebacteriumEscherichia.. The process of claim 5 , wherein the D-amino acid dehydrogenase is from claim 5 , and7Pseudomonas aeruginosa, Pseudomonas fluorescens, Pyrobaculum islandicum, Salmonella typhimurium, Corynebacterium glutamicumEscherichia coli.. The process of claim 6 , wherein the D-amino acid dehydrogenase is from claim 6 , and8. The process of claim 7 , wherein the D-amino acid dehydrogenase is selected from D-alanine dehydrogenase claim 7 , D-threonine dehydrogenase claim 7 , and D-proline dehydrogenase.9. The process of which is carried out in a cell free system.10. The process of claim 1 , wherein the amino acid dehydrogenase is present as a crude extract.11. The process of claim 1 , wherein the amino acid dehydrogenase is substantially purified.12. The process of claim 1 , wherein the ketoreductase is a wild type ketoreductase or an engineered ketoreductase.13Lactobacillus, Candida, NovosphingobiumSaccharomyces.. The process of claim 12 , wherein the ketoreductase is from claim 12 , or14LactobacillusLactobacillus kefir, Lactobacillus brevisLactobacillus minor.. The process of claim 13 , wherein the ketoreductase of is from claim 13 , or15CandidaCandida magnoliae.. The process of claim 14 , wherein the ketoreductase of is from16SaccharomycesSaccharomyces cerevisiae.. The process of claim 14 , wherein the ketoreductase of is from17NovosphingobiumNovosphingobium aromaticivorans.. The process of claim 14 , ...

METHOD FOR THE PREPARATION OF CHIRAL ALPHA HALOALKANOIC ACIDS

What is described herein relates to a method of selectively hydrolyzing an enantiomer of an alpha haloalkanoic acid according to formula I employing a polypeptide having dehalogenase activity comprising an amino acid sequence as set forth in SEQ ID NO. 1 or SEQ ID NO. 4 or a sequence with at least 80% sequence identity to either of said sequences and to the use of said method. 2. Method according to claim 1 , wherein the ratio of racemate of the alpha haloalkanoic acid according to Formula I to biomass of whole cells comprising the polypeptide having dehalogenase activity comprising an amino acid sequence as set forth in SEQ ID NO. 1 or SEQ ID NO. 4 or a sequence with at least 80% sequence identity to either of said sequences is between 2:1 to 15:1 claim 1 , optionally between 3:1 to 10:1 optionally 4:1.3. Method according to claim 1 , wherein the halogen of said alpha haloalkanoic acid of formula I is bromide or chloride.4. Method according to claim 1 , wherein moiety R of said alpha haloalkanoic acid of formula I is chosen from the group consisting of ethyl claim 1 , butyl claim 1 , 2-methyl-propyl and methyl-cyclopropyl.5. Method according to for selective hydrolysis of the S-enantiomer of said alpha haloalkanoic acid claim 1 , wherein the enantiomeric excess of the R-enantiomer is between 90.0 and 99.9.7. Method according to for selective hydrolysis of the S-enantiomer of said alpha haloalkanoic acid claim 5 , wherein the enantiomeric excess of the R-enantiomer is between 90.0 and 99.9.8. Method according to claim 1 , wherein said alkyl chain is branched at carbon atoms γ or δ claim 1 , and wherein the carbon atoms following the branch at carbon atoms γ or δ are cyclic. It is well known that there is sometimes a marked difference in the effects of enantiomers of the same chemical substance in biological systems such as organisms. In drugs, for example, often only one of a drug's enantiomers is responsible for the desired physiologic effects, while the other ...

METHOD FOR PRODUCING D-FORM OR L-FORM AMINO ACID DERIVATIVE HAVING THIOL GROUP

The object of the present invention is to provide a method of efficiently manufacturing an optically active D- and/or L-form amino acid possessing a thiol group in the side chain by a simple method. The present invention provides a method of manufacturing an amino acid derivative possessing a thiol group in the side chain, characterized in manufacturing an intermediate composition comprising D- and L-forms of an amino acid derivative possessing a thiol group at the β-position, reacting a hydrolase selective for D- or L-amino acids, and separating the hydrolyzed D- or L-amino acid derivative, as well as an intermediate thereof. 2. The method according to claim 1 , which is a method of manufacturing a non-natural D- or L-amino acid derivative possessing a protected or non-protected thiol group and substituent Rat the β-position claim 1 , wherein Rrefers to the substituent moiety bound to the β carbon atom among side chain substituents that configure amino acids claim 1 , except when it is a hydrogen atom claim 1 , andsaid step (I) comprises the following step (P) before said reaction (A):{'sup': '1', '(P) manufacturing an amino acid derivative possessing substituent Rand a leaving group L on the β carbon atom, and'}said reaction (A) is carried out simultaneously with a reaction of detaching said leaving group L from the β carbon atom of the amino acid derivative.3. The method according to claim 2 , wherein{'sup': '1', 'substituent Ris an aromatic substituent, and'}said step (P) comprises the following step (P-1):{'sup': '1', '(P-1) introducing a leaving group L at the β carbon atom of an amino acid derivative possessing substituent Ron the β carbon atom.'}4. The method according to claim 2 , whereinsaid step (P) comprises the following step (P-2):{'sup': '1', '(P-2) reacting glycine with an aldehyde compound represented by RCHO.'}5. The method according to claim 1 , whereinsaid step (B) comprises converting the amino group bound to the α carbon atom of said amino acid ...

TRANSAMINASE POLYPEPTIDES

The present disclosure provides engineered transaminase enzymes having improved properties as compared to a naturally occurring wild-type transaminase enzyme. Also provided are polynucleotides encoding the engineered transaminase enzymes, host cells capable of expressing the engineered transaminase enzymes, and methods of using the engineered transaminase enzymes to synthesize a variety of chiral compounds. 1. An engineered transaminase polypeptide , wherein said transaminase polypeptide comprises a polypeptide sequence at least 80% identical to SEQ ID NO:2 , and wherein the transaminase polypeptide comprises a substitution at position 417.2. The engineered transaminase polypeptide of claim 1 , wherein said polypeptide has at least about 10% residual activity in the conversion of pyruvate to L-alanine in presence of amino donor isopropylamine after treatment of the polypeptide at 50° C. for 23 h.3. The engineered transaminase polypeptide of claim 1 , wherein the transaminase polypeptide further comprises at least one additional substitution at a residue position selected from: X4 claim 1 , X9 claim 1 , X12 claim 1 , X21 claim 1 , X45 claim 1 , X86 claim 1 , X157 claim 1 , X177 claim 1 , X208 claim 1 , X211 claim 1 , X253 claim 1 , X272 claim 1 , X294 claim 1 , X302 claim 1 , X316 claim 1 , X324 claim 1 , X391 claim 1 , X398 claim 1 , X418 claim 1 , X420 claim 1 , X431 claim 1 , X444 claim 1 , and X446.4. The engineered transaminase polypeptide of claim 3 , wherein the transaminase polypeptide sequence comprises the substitution X417C and at least one further substitution selected from: X4 is R claim 3 , Q claim 3 , or L; X12 is K; X21 is N; X45 is H; X86 is Y; X157 is T; X177 is L; X208 is I; X211 is K; X253 is M; X272 is A; X294 is V; X302 is K; X316 is K; X324 is G; X391 is A; X398 is R; X418 is V; X420 is N; X431 is D; X444 is V; and X446 is V.5. The engineered transaminase polypeptide of claim 4 , wherein the transaminase polypeptide sequence further comprises ...

PROTEIN COMPLEX CAPABLE OF CATALYZING ASYMMETRIC OXIDATION REACTION AND METHOD FOR PRODUCING SAME

Provided are: a protein complex capable of selectively and asymmetrically oxidizing an enantiomer of a secondary alcohol without adding a coenzyme and having an asymmetric oxidation activity in a water-soluble solvent system in the presence of oxygen; a method for producing the same; and a method for coating the protein complex with a high molecular weight compound. The method for producing the protein complex includes: (1) enclosing a crude water-soluble protein in a gel, air-oxidizing the gel, and eluting the protein complex into an aqueous solution; and (2) applying gravity to concentrate and precipitate the protein complex, redissolving the precipitate in an aqueous glycine sodium hydroxide solution of about 0.5 mM and allowing the same to homogeneously coexist with a high molecular weight compound, and re-precipitating the solution and dehydrating and drying the same to yield a protein complex coated with a high molecular weight compound. 18-. (canceled)9. An iron-containing protein complex derived from an animal or plant , which has reactivity with hydrogen peroxide , even in the absence of NAD(H) and/or NADP(H).10. The protein complex according to claim 9 , which has a function attributable to a cell organelle having asymmetric oxidation activity and/or catalase activity.11. The protein complex according to claim 10 , wherein the cell organelle is a mitochondrion claim 10 , and the function is derived from iron-oxygen-dependent electron transfer-based oxidation activity of PQQ-dehydrogenase and cytochrome c oxidase present as membrane protein.12. The protein complex according to claim 10 , wherein the cell organelle is a chloroplast or endoplasmic reticulum claim 10 , and the function is derived from tissue in which catalase activity is localized that exhibits reactivity with hydrogen peroxide.13. The protein complex according to claim 10 , wherein the cell organelle is present as a membrane protein claim 10 , and contains two or more enzymes of any of PQQ- ...

KETOREDUCTASE POLYPEPTIDES

The present disclosure provides engineered ketoreductase enzymes having improved properties as compared to a naturally occurring wild-type ketoreductase enzyme including the capability of reducing 5-((4S)-2-oxo-4-phenyl (1,3-oxazolidin-3-yl))-1-(4-fluorophenyl) pentane-1,5-dione to (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one. Also provided are polynucleotides encoding the engineered ketoreductase enzymes, host cells capable of expressing the engineered ketoreductase enzymes, and methods of using the engineered ketoreductase enzymes to synthesize the intermediate (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one in a process for making Ezetimibe. 1. An engineered polypeptide having ketoreductase activity , wherein said engineered polypeptide comprises a region with an amino acid sequence having at least 90% sequence identity to residues 90 to 211 of SEQ ID NO: 130 , comprising a substitution at position X190 , and further comprising one or more of the following features selected from:residue corresponding to X94 is asparagine, glycine, serine, or a polar residue;residue corresponding to X95 is an aliphatic residue;residue corresponding to X96 is glutamine, asparagine, or threonine;residue corresponding to X101 is an acidic, non-polar, or a polar residue;residue corresponding to X105 is an acidic or non-polar residue;residue corresponding to X108 is a hydrophilic, polar or constrained residue;residue corresponding to X111 is a non-polar or aliphatic residue;residue corresponding to X112 is an acidic or polar residue;residue corresponding to X113 is a non-polar or aliphatic residue;residue corresponding to X117 is a non-polar or a polar residue;residue corresponding to X127 is a basic residue;residue corresponding to X147 is a non-polar, aliphatic, aromatic, or hydrophobic residue;residue corresponding to X152 is a non-polar, basic, or hydrophilic residue;residue corresponding to X157 is a polar ...

Biocatalysts for the preparation of hydroxy substituted carbamates

The present disclosure relates to engineered ketoreductase polypeptides for the preparation of hydroxyl substituted carbamate compounds, and polynucleotides, vectors, host cells, and methods of making and using the ketoreductase polypeptides.

Process for the preparation of pregabalin

The present invention provides an improved process for the preparation of a compound of formula (I), which comprises the steps of: formula (I), (a) reacting isovaleraldehyde of formula (II) and alkyl cyanoacetate of formula (III) optionally in presence of salts of weak acid and weak base or weak base in a suitable solvent to get 2-cyano-5-methyl-hex-2-enoic acid alkyl ester of formula (IV); (b) reacting 2-cyano-5-methyl-hex-2-enoic acid alkyl ester of formula (IV) with a suitable cyanide source in water or in an organic solvent or mixture thereof to get 2-isobutylsuccinonitrile of formula (V); (c) obtaining optionally 2-isobutylsuccinonitrile of formula (V) by reacting isovaleraldehyde of formula (II) and alkyl cyanoacetate of formula (III) in presence of suitable cyanide source in water or in an organic solvent or mixture thereof in single step; (d) converting 2-isobutylsuccinonitrile of formula pa (V) to racemic 3-cyano-5-methyl-hexanoic acid or salt thereof of formula (VI) with a genetically modified nitrilase enzyme (Nit pt 9N_56_2) in water or optionally with an organic co-solvent at appropriate pH and temperature; (e) converting racemic 3-cyano-5-methyl-hexanoic acid or salt thereof of formula (VI) to racemic alkyl 3-cyano-5-methyl-hexanoate of formula (VII) by treatment with alcohol (R3OH) and acidic catalyst or alkyl halide (R3X) in presence of a base in a suitable solvent or a mixture of solvents thereof; (f) obtaining (S)-alkyl 3-cyano-5-methyl-hexanoate of formula (VIII) and (R)-3-cyano-5-methyl-hexanoic acid or salt thereof of formula (X) by enzymatic enantioselective hydrolysis in water or organic solvent or a mixture thereof from racemic alkyl 3-cyano-5-methyl-hexanoate of formula (VII); (g) obtaining optionally the compound of formula (VII) by racemizing unwanted (R)-3-cyano-5-methyl-hexanoic acid or salt thereof of formula (X) or substantially enriched (R)-3-cyano-5-methyl-hexanoic acid salt thereof of formula (X) in presence of a base in organic ...

PROCESS FOR THE ENZYMATIC REGENERATION OF REDOX COFACTORS

A process for the enzymatic regeneration of the redox cofactors NAD/NADH and NADP/NADPH in a one-pot reaction, wherein, as a result of at least two further enzymatically catalyzed redox reactions proceeding in the same reaction batch (product-forming reactions), one of the two redox cofactors accumulates in its reduced form and, respectively, the other one in its oxidized form, characterized in that a) in the regeneration reaction which reconverts the reduced cofactor into its original oxidized form, oxygen or a compound of general formula RC(O)COOH is reduced, and b) in the regeneration reaction which reconverts the oxidized cofactor into its original reduced form, a compound of general formula RCH(OH)Ris oxidized and wherein R, Rand Rin the compounds have different meanings. 1. A process for the enzymatic regeneration of the redox cofactors NAD/NADH and NADP/NADPH in a one-pot reaction , wherein , as a result of at least two further enzymatically catalysed redox reactions proceeding in the same reaction batch (product-forming reactions) , the redox cofactor NAD/NADH accumulates in its reduced form as NADH and the redox cofactor NADP/NADPH accumulates in its oxidized form as NADP , wherein:a) in the regeneration reaction which reconverts NADH into its original oxidized form, oxygen or pyruvate is reduced by means of an NADH oxidase or a lactate dehydrogenase, and{'sup': '+', 'b) in the regeneration reaction which reconverts NADP into its original reduced form, 2-propanol or malate is oxidized by means of an alcohol dehydrogenase or a malate dehydrogenase.'}2. The process according to claim 1 , wherein oxidation reaction(s) and reduction reaction(s) take place on the same substrate (molecular backbone).3. The process according to claim 1 , wherein oxidation reaction(s) and reduction reaction(s) proceed chronologically parallel.4. The process according to claim 1 , wherein claim 1 , in the regeneration reaction which reconverts NADP+ to NADPH claim 1 , 2-propanol is ...

NITRILASES, NUCLEIC ACIDS ENCODING THEM AND METHODS FOR MAKING AND USING THEM

The invention relates to nitrilases and to nucleic acids encoding the nitrilases. In addition methods of designing new nitrilases and method of use thereof are also provided. The nitrilases have increased activity and stability at increased pH and temperature. 113-. (canceled)14. An isolated , synthetic or recombinant nucleic acid , encoding a polypeptide having nitrilase activity , comprising:(a) a nucleic acid having a sequence at least 85% identical to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383 or 385; or(b) nucleic acid encoding a polypeptide comprising consecutive amino acids having a sequence at least 85% identical to SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218 ...

PROTEIN COMPLEX CAPABLE OF CATALYZING ASYMMETRIC OXIDATION REACTION AND METHOD FOR PRODUCING SAME

Provided are: a protein complex capable of selectively and asymmetrically oxidizing an enantiomer of a secondary alcohol without adding a coenzyme and having an asymmetric oxidation activity in a water-soluble solvent system in the presence of oxygen; a method for producing the same; and a method for coating the protein complex with a high molecular weight compound. The method for producing the protein complex includes: (1) enclosing a crude water-soluble protein in a gel, air-oxidizing the gel, and eluting the protein complex into an aqueous solution; and (2) applying gravity to concentrate and precipitate the protein complex, redissolving the precipitate in an aqueous glycine sodium hydroxide solution of about 0.5 mM and allowing the same to homogeneously coexist with a high molecular weight compound, and re-precipitating the solution and dehydrating and drying the same to yield a protein complex coated with a high molecular weight compound. 121-. (canceled)22. A process for producing an optically active alcohol , comprising: selectively obtaining one enantiomer of a racemic alcohol by allowing an iron-containing protein complex derived from an animal or plant , which has reactivity with hydrogen peroxide even in the absence of NAD(H) and/or NADP(H) , to act on a substrate in the form of the racemic alcohol.23. The process for producing an optically active alcohol according to claim 22 , wherein the other enantiomer not involved in the reaction is selectively obtained by selectively asymmetrically oxidizing the one enantiomer of the racemic alcohol to a ketone.24. The process for producing an optically active alcohol according to claim 23 , wherein the protein complex is coated with a polymer compound and water is used for the solvent in the reaction system.25. The process for producing an optically active alcohol according to claim 24 , wherein the protein complex is subjected to chemical modification treatment claim 24 , and glycine-NaOH buffer (50 mM claim 24 , ...

BIOCATALYSTS AND METHODS FOR THE SYNTHESIS OF ARMODAFINIL

The present invention relates to non-naturally occurring polypeptides useful for preparing armodafinil, polynucleotides encoding the polypeptides, and methods of using the polypeptides. The non-naturally occurring polypeptides of the present invention are effective in carrying out biocatalytic conversion of the (i) 2-(benzhydrylsulfinyl)acetamide to (−)-2-[(R)-(diphenylmethyl)sulfinyl]acetamide (armodafinil), or (ii) benzhydryl-thioacetic acid to (R)-2-(benzhydrylsulfinyl)acetic acid, which is a pivotal intermediate in the synthesis of armodafinil, in enantiomeric excess. 1. A non-naturally occurring polynucleotide encoding a non-naturally occurring polypeptide having cyclohexanone monooxygenase (CHMO) activity wherein the amino acid sequence of the polypeptide has at least 90% sequence identity to SEQ ID NO: 2 , and one or more amino acid substitutions at one or more positions corresponding to positions in SEQ ID NO: 2 , selected from the group consisting of 75 , 79 , 82 , 99 , 110 , 166 , 172 , 208 , 216 , 273 , 324 , 364 , 395 , 412 , 491 , 503 , and 504.3. The non-naturally occurring polynucleotide encoding the non-naturally occurring polypeptide of claim 1 , wherein said non-naturally occurring polypeptide further comprises one or more amino acid substitutions relative to SEQ ID NO: 2 claim 1 , wherein the polypeptide comprises an alanine claim 1 , glutamic acid claim 1 , glycine claim 1 , isoleucine claim 1 , lysine claim 1 , proline claim 1 , serine claim 1 , threonine claim 1 , or valine at a position corresponding to position 246 of SEQ ID NO:2.5. The non-naturally occurring polynucleotide encoding the non-naturally occurring polypeptide of claim 1 , wherein said non-naturally occurring polypeptide further comprises one or more amino acid differences relative to SEQ ID NO: 2 claim 1 , wherein said polypeptide further comprises one or more substitutions corresponding to substitutions in SEQ ID NO: 2 selected from the group consisting of a glycine at position ...

KETOREDUCTASE POLYPEPTIDES FOR THE SYNTHESIS OF CHIRAL COMPOUNDS

The present disclosure provides engineered ketoreductase enzymes having improved properties as compared to a naturally occurring wild-type ketoreductase enzyme. Also provided are polynucleotides encoding the engineered ketoreductase enzymes, host cells capable of expressing the engineered ketoreductase enzymes, and methods of using the engineered ketoreductase enzymes to synthesize a variety of chiral compounds. 1. An engineered polynucleotide encoding an engineered polypeptide comprising an amino acid sequence with at least 90% sequence identity to SEQ ID NO:4 and an arginine or lysine at position X249 as compared to SEQ ID NO:4 , and wherein said polypeptide has ketoreductase activity with at least 2-fold greater selectivity to the chiral compound 2a relative to chiral compound 2c as compared to SEQ ID NO:4.2. The engineered polynucleotide encoding the engineered polypeptide of claim 1 , wherein said amino acid sequence further comprises at least one of the following:the residue corresponding to X68 is a non-polar, or aliphatic residue;the residue corresponding to X102 is an acidic residue;the residue corresponding to X110 is an acidic, or aromatic residue;the residue corresponding to X114 is a non-polar residue;the residue corresponding to X135 is a basic residue;the residue corresponding to X144 is a non-polar, or aromatic residue;the residue corresponding to X147 is a non-polar, or aliphatic residue;the residue corresponding to X149 is a polar residue;the residue corresponding to X153 is a polar, or aromatic residue;the residue corresponding to X158 is a non-polar, or aliphatic residue;the residue corresponding to X175 is a polar residue;the residue corresponding to X190 is an aromatic, aliphatic, non-polar, or polar residue;the residue corresponding to X196 is an aromatic, polar, basic, non-polar, or aliphatic residue;the residue corresponding to X197 is an aromatic residue;the residue corresponding to X198 is a non-polar, polar residue;the residue ...

Ketoreductase polypeptides

The present disclosure provides engineered ketoreductase enzymes having improved properties as compared to a naturally occurring wild-type ketoreductase enzyme including the capability of reducing 5-((4S)-2-oxo-4-phenyl (1,3-oxazolidin-3-yl))-1-(4-fluorophenyl) pentane-1,5-dione to (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one. Also provided are polynucleotides encoding the engineered ketoreductase enzymes, host cells capable of expressing the engineered ketoreductase enzymes, and methods of using the engineered ketoreductase enzymes to synthesize the intermediate (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one in a process for making Ezetimibe.

METHOD FOR PRODUCING PYRROLE DERIVATIVE, AND INTERMEDIATE THEREOF

The present invention provides a method for producing an atropisomer of a pyrrole derivative having excellent mineralocorticoid receptor antagonistic activity, and an intermediate thereof. A method for producing an atropisomer of a pyrrole derivative using a compound represented by (B) [wherein Rrepresents a C1-C4 alkyl group, and Rrepresents a 2-hydroxyethyl group or a carboxymethyl group] as a production intermediate. 2. The method according to claim 1 , wherein the reagent is a Grignard reagent.3. The method according to claim 1 , wherein the Grignard reagent is selected from ethylmagnesium bromide claim 1 , ethylmagnesium chloride claim 1 , isopropylmagnesium chloride claim 1 , methylmagnesium bromide claim 1 , and phenylmagnesium bromide.4. The method according to claim 1 , wherein the Grignard reagent is ethylmagnesium bromide.5. The method according to claim 2 , wherein the reaction solvent is tetrahydrofuran.6. The method according to claim 2 , wherein reacting ethyl (S)-1-(2-hydroxyethyl)-4-methyl-5-[2-(trifluoromethyl)phenyl]-1H-pyrrole-3-carboxylate with the Grignard reagent is carried out at a reaction temperature from room temperature to 150° C.7. The method according to claim 6 , wherein the reaction temperature is from 60° C. to 100° C.8. The method according to claim 2 , wherein reacting ethyl (S)-1-(2-hydroxyethyl)-4-methyl-5-[2-(trifluoromethyl)phenyl]-1H-pyrrole-3-carboxylate with the Grignard reagent is carried at a reaction time from 0.5 to 5 hours.9. The method according to claim 8 , wherein the reaction time is from 0.5 to 2 hours.10. The method according to claim 1 , wherein the reagent is a metal alkoxide.11. The method according to claim 1 , wherein the metal alkoxide is selected from potassium t-butoxide claim 1 , sodium t-butoxide claim 1 , sodium methoxide claim 1 , and potassium ethoxide.12. The method according to claim 10 , wherein the reaction solvent is selected from tetrahydrofuran claim 10 , toluene claim 10 , dimethylsulfoxide ...

NEW PROCESS FOR EARLY SACUBITRIL INTERMEDIATES

The invention relates to a new enantioselective process for producing useful intermediates for the manufacture of NEP inhibitors or prodrugs thereof, in particular NEP inhibitors comprising a γ-amino-δ-biphenyl-α-methylalkanoic acid, or acid ester, backbone. 2. A process according to claim 1 , wherein the amine donor is an achiral amine donor selected from the group consisting of achiral C-C-alkylamine claim 1 , achiral C-C-cycloalkylamine claim 1 , achiral C-C-aryl-C-C-alkylamine claim 1 , achiral C-Calkyldiamine claim 1 , achiral amino-C-C-alkanoic acid claim 1 , and achiral C-C-aryl-di(C-C-alkylamine).3. A process according to claim 2 , wherein the achiral amine donor is isopropylamine (2-aminopropane).14. A process according to any one of to claim 2 , wherein Ra is a nitrogen protecting group in each formula where it is present it is selected from C-C-alkyl claim 2 , which is unsubstituted or mono- claim 2 , di- or tri-substituted by tri-C-C-alkylsilylC-C-alkoxy claim 2 , C-C-aryl claim 2 , or a heterocyclic group being a mono- claim 2 , bi- or tricyclic ring system with 5 to 14 ring atoms and 1 to 4 heteroatoms independently selected from N claim 2 , O claim 2 , S claim 2 , S(O) or S(O) claim 2 , wherein the aryl ring or the heterocyclic group is unsubstituted or substituted by one claim 2 , two or three residues claim 2 , selected from the group consisting of C-C-alkyl claim 2 , hydroxyl claim 2 , C-Calkoxy claim 2 , C-C-alkanoyl-oxy claim 2 , halogen claim 2 , nitro claim 2 , cyano claim 2 , and CF;{'sub': 6', '10', '1', '2', '1', '10', '1', '6', '6', '10', '1', '6', '6', '10', '1', '6, 'C-C-aryl-C-C-alkoxycarbonyl; C-C-alkenyloxycarbonyl; C-C-alkylcarbonyl; C-C-arylcarbonyl; C-C-alkoxycarbonyl; C-C-aryl-C-C-alkoxycarbonyl; allyl; cinnamyl; sulfonyl;'}sulfenyl; succinimidyl, and silyl,{'sub': 1', '7', '6', '10', '1', '4, 'wherein each silyl group is a SiR11R12R13 group, wherein R11, R12 and R13 are, independently of each other, C-C-alkyl, C-C-aryl or phenyl-C ...

METHYLOPILA SP. AND USE THEREOF IN SELECTIVE RESOLUTION PREPARATION OF (S)-A-ETHYL-2-OXO-1-PYRROLIDINEACETATE

sp. and use thereof in the selective resolution preparation of (S)-α-ethyl-2-oxo-1-pyrrolidineacetate. sp. that produces enzymes is subjected to cell immobilization, and is then applied to the biological resolution of a racemate (R,S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid ethyl ester to prepare high optically pure (S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid ethyl ester, which is further subjected to a hydrolysis reaction to obtain (S)-α-ethyl-2-oxo-1-pyrrolidineacetate. The present invention achieves a high conversion yield up to 50.0% or more, a good stereoselectivity, and an enantiomeric excess value e.e.(%) of (S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid ethyl ester not less than 99.5; the catalytic efficiency is high; the concentration of the racemic substrate in the resolution reaction is up to 500 g/L, the reaction time does not exceed 15 h, the number of reuse times of the immobilized cells is not lower than 35. 1MethylopilaMethylopila. A sp. , which is classified and named as a cxzy-L013 strain of sp. , deposited in China Center for Type Culture Collection on September 18 , 2016 under the conservation number CCTCC NO. M2016494.2MethylopilaMethylopilaMethylopila. Use of sp. in the selective resolution preparation of (S)-α-ethyl-2-oxo-1-pyrrolidineacetate , wherein the sp. is a sp. cxzy-L013 strain deposited in China Center for Type Culture Collection on Sep. 18 , 2016 under the conservation number CCTCC NO. M2016494.3Methylopila. The use of sp. in the selective resolution preparation of (S)-α-ethyl-2-oxo-1-pyrrolidineacetate according to claim 2 , comprising the following three steps:{'i': 'Methylopila', '(1) treating a bacterial solution of sp. by a cell immobilization method to obtain an immobilized bacterial agent containing an immobilized resolution enzyme;'}(2) with (R,S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid ethyl ester as a substrate, adding a certain amount of water and the immobilized bacterial agent for a resolution reaction to obtain (S)-α-ethyl-2- ...

BIOCATALYSTS FOR THE PREPARATION OF HYDROXY SUBSTITUTED CARBAMATES

The present disclosure relates to engineered ketoreductase polypeptides for the preparation of hydroxyl substituted carbamate compounds, and polynucleotides, vectors, host cells, and methods of making and using the ketoreductase polypeptides. 2. The engineered polynucleotide encoding the engineered ketoreductase polypeptide of claim 1 , wherein the amino acid sequence of said ketoreductase polypeptide comprises the substitution X40R claim 1 , and one or more residue differences as compared to SEQ ID NO:4 selected from: X7S; X17M; X17Q; X17R; X23V; X27L; X29G; X60I; X64V; X71P; X87L; X94A; X94P; X94S; X95M; X105G; X113I; X122A; X127R; X131S; X144V; X145L; X147I; X147L; X147Q; X150Y; X152G; X153G; X157C; X173L; X196M; X198S; X208R; X216R; X221S; X243S; X245I; X249F; X249G; and X249Y.3. The engineered polynucleotide encoding the engineered ketoreductase polypeptide of claim 1 , wherein the amino acid sequence of said ketoreductase polypeptide comprises X40R claim 1 , and at least one or more residue differences as compared to SEQ ID NO:4 selected from: X17Q/R/M; X64V; X94P; X96L/Y; X144V; X147Q/I/L; X157C; X195A/G; X196M; X199H; and X206L/F.4. The engineered polynucleotide encoding the engineered ketoreductase polypeptide of claim 1 , wherein the ketoreductase polypeptide is capable of converting the substrate compound (2) to the product compound (1) with at least 10 fold the activity of the reference polypeptide of SEQ ID NO:4 claim 1 , wherein the amino acid sequence comprises the substitution X40R claim 1 , and one or more residue differences as compared to SEQ ID NO:4 selected from: X60I; X71P; X94P; X94A; X95M; X96L; X96Y; X127R; X144V; X1451; X150Y; X152G; X153G; X157C; X195A; X195G; X196M; X198S; X199H; X206F/L claim 1 , X216R claim 1 , X245I claim 1 , X245F; X249Y; and X249F.5. The engineered polynucleotide encoding the engineered ketoreductase polypeptide of claim 1 , wherein the ketoreductase polypeptide has increased thermal stability as compared to the ...

BIOCATALYSTS AND METHODS FOR THE SYNTHESIS OF ARMODAFINIL

The present invention relates to non-naturally occurring polypeptides useful for preparing armodafinil, polynucleotides encoding the polypeptides, and methods of using the polypeptides. The non-naturally occurring polypeptides of the present invention are effective in carrying out biocatalytic conversion of the (i) 2-(benzhydrylsulfinyl)acetamide to (−)-2-[(R)-(diphenylmethyl)sulfinyl]acetamide (armodafinil), or (ii) benzhydryl-thioacetic acid to (R)-2-(benzhydrylsulfinyl)acetic acid, which is a pivotal intermediate in the synthesis of armodafinil, in enantiomeric excess. 1. A non-naturally occurring polypeptide having cyclohexanone monooxygenase (CHMO) activity wherein the amino acid sequence of the polypeptide has at least 90% sequence identity to SEQ ID NO: 2 , and one or more amino acid substitutions at one or more positions in SEQ ID NO: 2 , selected from the group consisting of 75 , 79 , 82 , 99 , 110 , 166 , 172 , 208 , 216 , 273 , 324 , 364 , 395 , 412 , 491 , 503 , and 504.3. The non-naturally occurring polypeptide of claim 1 , wherein said non-naturally occurring polypeptide further comprises one or more amino acid substitutions relative to SEQ ID NO:2, wherein the polypeptide comprises an alanine, glutamic acid, glycine, isoleucine, lysine, proline, serine, threonine, or valine at a position corresponding to position 246 of SEQ ID NO:2.4. The non-naturally occurring polypeptide of claim 1 , wherein said non-naturally occurring further is capable of converting the acid substrate of compound (1b) to the R-enantiomer compound (2b) in at least 50% enantiomeric excess.5. The non-naturally occurring polypeptide of claim 1 , wherein said non-naturally occurring polypeptide further comprises one or more amino acid differences relative to SEQ ID NO: 2 claim 1 , wherein said polypeptide further comprises one or more substitutions selected from the group consisting of a glycine at position 143 claim 1 , glycine at position 278 claim 1 , an arginine at position 326 ...

METHODS FOR MAKING L-GLUFOSINATE

Methods for the production of L-glufosinate (also known as phosphinothricin or (S)-2-amino-4-(hydroxy(methyl)phosphonoyl)butanoic acid) are provided. The methods comprise a two-step process. The first step involves the oxidative deamination of D-glufosinate to PPO (2-oxo-4-(hydroxy(methyl)phosphinoyl)butyric acid). The second step involves the specific amination of PPO to L-glufosinate, using an amine group from one or more amine donors. By combining these two reactions, the proportion of L-glufosinate in a mixture of L-glufosinate and D-glufosinate can be substantially increased. 1. A herbicidal formulation comprising an L-glufosinate composition wherein said L-glufosinate composition is prepared by reacting D-glufosinate with a D-amino acid oxidase (DAAO) enzyme to form PPO (2-oxo-4-(hydroxy(methyl)phosphinoyl)butyric acid); andaminating the PPO to L-glufosinate by a transaminase (TA) enzyme, using an amine group from one or more amine donors,wherein at least 70% of the D-glufosinate is converted to L-glufosinate, andwherein said formulation further comprises at least one adjuvant.2. The formulation of claim 1 , wherein said adjuvant is selected from a surfactant claim 1 , a solvent claim 1 , a polysaccharide humectant claim 1 , and a diluent.3. The formulation of claim 2 , wherein said surfactant is sodium alkyl ether sulfate.4. The formulation of claim 2 , wherein said solvent is selected from at least one of 1-methoxy-2-propanol claim 2 , dipropylene glycol claim 2 , and ethylene glycol.5. The formulation of claim 2 , wherein said polysaccharide humectant is selected from at least one of alkyl polysaccharides claim 2 , pentoses claim 2 , high fructose corn syrup claim 2 , sorbitol claim 2 , and molasses.6. The formulation of claim 2 , wherein said diluent is an aqueous component.7. The formulation of claim 2 , wherein said diluent is water.8. The formulation of claim 1 , wherein said L-glufosinate composition comprises L-glufosinate ammonium in an amount from ...

Processes for the Preparation of Dasotraline and Intermediates Thereof

The present invention provides processes for the preparation of Dasotraline, as well as intermediates useful in the preparation thereof. In particular, processes are provided for the enzymatic transamination of a compound of Formula (2) to afford Dasotraline.

Stereoselective transaminase, gene encoding said protein and use thereof

According to the present invention, a novel protein capable of catalyzing transamination stereoselectively and a gene encoding said protein can be provided.

Preparation of 3,3,3-tri:fluoro-2-hydroxy-2-methyl propionic acid,

Preparation of (S)- or (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acids of formulae (I) and (II) comprises: (a) reacting a trifluoroacetic acid ester of formula (III) with a mineral acid to give trifluoroacetone of formula (IV); (b) reacting (III) with a cyanide to give propionic acid nitrile of formula (V); (c) reacting (V) with a 1-4C alcohol to give a propionic acid ester of formula (VI); (d) converting (VI) with an esterase or a lipase to give (II), which can then be isolated, or (e) bio-transforming (II) to give a compound of formula (VII) followed by hydrolysation to give (I). R = 1-4C alkyl group. Also claimed is reaction of (VII) with (VIII) to give (IX).

Enzymatic process for the enantioselective reduction of keto compounds

The invention relates to an enzymatic method for the enantioselective reduction of organic keto compounds to the corresponding chiral hydroxy compounds, an alcohol dehydrogenase from Lactobacillus minor and a method for the enantioselective production of (S)-hydroxy compounds from a racemate.

Ketoreductase polypeptides and uses thereof

The present disclosure provides engineered ketoreductase enzymes having improved properties as compared to a naturally occurring wild-type ketoreductase enzyme including the capability of stereo specifically reducing (R)-2-methylpentanal to (R)-2-methylpentanol. Also provided are polynucleotides encoding the engineered ketoreductase enzymes, host cells capable of expressing the engineered ketoreductase enzymes, and methods of using the engineered ketoreductase enzymes to produce (R)-2-methylpentanol and related compounds.

Ketoreductase polypeptides for the stereoselective production of (4s)-3-[(5s)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one

The present disclosure provides engineered ketoreductase enzymes having improved properties as compared to a naturally occurring wild-type ketoreductase enzyme including the capability of reducing 5-((4S)-2-oxo-4-phenyl(1,3-oxazolidin-3-yl))-1-(4-fluorophenyl)pentane-1,5-dione to (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one. Also provided are polynucleotides encoding the engineered ketoreductase enzymes, host cells capable of expressing the engineered ketoreductase enzymes, and methods of using the engineered ketoreductase enzymes to synthesize the intermediate (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one in a process for making Ezetimibe.

Patent DE3920570C2

Phosphinothricin-resistance gene

The phosphinothricin (PTC)-resistance gene has been isolated from the genome of Streptomyces viridochromogenes DSM 40736, and a large part of it has been sequenced. The sequenced fragment confers PTT resistance in Streptomyces lividans and in E. coli and is suitable for producing PTC-resistant plants, but also as resistance marker.

Process for enzymatic racemate separation of pantolacton

(R)-(-)-pantolactone can be prepared in high optical purity by enzymatic racemate resolution of racemic pantolactone in the presence of a vinyl ester and lipase from Pseudomonas and from pig pancreas.

2－氨基－4－甲膦酰丁酸抗性基因

通过在绿色产色链霉菌DSM4112中选择2-氨基-4-甲膦酰-丙氨酰-丙氨酸(PTT)抗性而产生PTT抗性的选择体。用BamHI酶切这些选择体的总DNA得到携有Phosphinothricin(PTC)抗性基因的DNA片段，克隆4.0Kb大小的片段并选择PTT抗性。此基因适用于生产PTC抗性的植物，以及作为抗性标记用于L型外消旋PTC的选择性N-乙酰化作用。