(S)-Modified oxynitrilase polypeptide and its use

Abstract

The present disclosure relates to a process for producing chiral β-nitroalcohols. The present invention particularly relates to an (S)-selective oxynitrilase that can enantioselectively catalyze the Henry reaction, in which an aldehyde or ketone compound is converted to the corresponding β-nitroalcohol compound in the presence of a nitroalkane compound and an oxynitrilase.

Description

Technical Field

[0001] Claim of Priority This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 350,047, filed on Jun. 8, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application is electronically filed in ST.26 format and includes a sequence listing which is incorporated herein by reference in its entirety. The name of the said ST.26 copy created on Jun. 7, 2023 is PAT059273-WO-PCT_SL.xml.

[0003] The present disclosure relates to the field of biotechnology, and in particular, to modified oxynitrilase polypeptides and their applications in industrial biocatalysts. In particular, the present disclosure relates to a method for producing chiral β-nitroalcohol compounds, which converts an aldehyde or ketone compound into a corresponding β-nitroalcohol compound in the presence of a nitroalkane compound and a modified oxynitrilase. The present disclosure particularly relates to (S)-selective oxynitrilase that enantioselectively catalyzes the Henry reaction.

Background Art

[0004] Processes using biocatalysts have become very important for the chemical industry. Of particular importance is the use of enzymes in chemical reactions with chiral or prochiral compounds such that one of the two enantiomers preferentially reacts or is formed due to the properties of the biocatalyst.

[0005] An essential requirement for exploiting such favorable properties of enzymes is that a sufficient amount required in an industrial process can be utilized at low cost and that they have sufficiently high reactivity, selectivity and high stability under the realistic conditions of an industrial process.

[0006] β-Nitroalcohol is a precursor of β-aminoalcohol, which is an important chiral building block for synthesizing bioactive compounds. Nitroaldol or Henry reaction is one of the classical named reactions in organic synthesis for C-C bond formation. Due to the possibility of creating up to two new chiral centers, the ability to perform nitroaldol addition enantioselectively and stereoselectively is fundamentally important for synthetic applications. This reaction has been known for over a century (Henry, 1895), but stereospecific protocols using non-enzymatic organic catalysts or chiral metal catalysts have been developed only recently. There are still many drawbacks in the development of these methods. For example, in the case of metal catalysts, there are long reaction times and sometimes extreme reaction conditions, and in the case of organic catalysts, the selectivity may not be sufficient.

[0007] Hydroxynitrile lyase (HNL), often also called oxynitrilase, belongs to the enzyme class of aldehyde lyase (E.C. 4.1.2.X). Originally, HNL catalyzes the reversible stereoselective cleavage of hydroxynitriles into hydrogen cyanide and aldehydes or ketones. This cyanation reaction is utilized by plants to defend against fungi or predators by releasing hydrogen cyanide in cells. Contrary to these original reactions, HNL also catalyzes the stereoselective addition of hydrocyanic acid to aldehydes or ketones to produce enantiopure hydroxynitriles, which are often used as building blocks for various pharmaceuticals and pesticides (Dadashipour & Asano ACS Catalysis 2011 1(9), 1121-1149). In the formation of nitriles, HNL has shown a limited substrate scope with respect to the nature of the electrophilic acceptor, which can be aliphatic and aromatic aldehydes or aliphatic ketone compounds, while only cyanide is accepted as a nucleophile (Liu et al. Front. Bioeng. Biotechnol. 2021 9:653682).

[0008] The HNL derived from Hevea brasiliensis was the first enzyme reported to be able to catalyze the enzymatic nitroaldol (Henry) reaction of aldehyde and nitromethane (Mandana Gruber-Khadjawi et al. Adv. Synth. Catal. 2007, 349, 1445-1450). In recent years, more examples of (R)-selective HNLs that catalyze the Henry reaction and are derived from Acidobacterium capsulatum, Granulicella tundriculata (Bekerle-Bogner et al. ChemCatChem 2016, 8, 2214), or Arabidopsis thaliana (Fuhshuku et al. J. Biotechnol. 2011, 153, 153-159) have been reported. Among these, the latter HNL derived from Arabidopsis thaliana (AtHNL) has been the most widely described (Fuhshuku et al. J. Biotechnol. 2011, 153, 153-159).

[0009] In addition to HNL, it has been reported that TGase (protein-glutamine-glutamyltransferase; EC2.3.2.13) from Streptorerticillium griseoverticillatum catalyzes the Henry reaction.

Summary of the Invention

Problems to be Solved by the Invention

[0010] Therefore, there is a great demand for the development of the asymmetric synthesis of β-nitroalcohols. There is still a need for a novel oxynitrilase that can catalyze the Henry reaction enantioselectively.

Means for Solving the Problems

[0011] The present disclosure provides a series of modified polypeptides having high stereoselectivity, which overcome the above-mentioned drawbacks.

Brief Description of the Drawings

[0012]

Figure 1A

Figure 1B

Figure 2A

Figure 2B

Figure 2C

Figure 2D

Figure 3A

Figure 3B

Figure 4

[0013] Summary of the Disclosure The present disclosure provides a modified polypeptide having high stereoselectivity, high catalytic activity, and good stability, which can asymmetrically synthesize β-nitroalcohol, and in particular, can asymmetrically synthesize (S)-1,1,1-trifluoro-2-methyl-3-nitropropan-2-ol. The present disclosure relates in particular to an (S)-selective oxynitrilase that can enantioselectively catalyze the Henry reaction.

[0014] Surprisingly, the modified polypeptides of the present disclosure are particularly suitable for substrates containing an electron-withdrawing group. As a result, it has been found that by introducing an electron-withdrawing substituent into the substrate of an aldehyde or a ketone, the β-nitroalcohol product can be synthesized in high yield with high stereoselectivity.

[0015] The present disclosure also provides a gene sequence of the modified polypeptide, a recombinant expression vector containing the gene, a modified strain, and an efficient method for its production, as well as a reaction process for asymmetrically synthesizing β-nitroalcohol using the modified polypeptide.

[0016] The modified oxynitrilase polypeptides disclosed herein have good catalytic properties. These modified polypeptides are derived from wild-type oxynitrilase, which has low stereoselectivity for the product, by substitution, insertion, or deletion of many amino acid residues in the directed evolution process. The wild-type oxynitrilase is derived from Baliospermum montanum (BmHNL), consists of 263 amino acids, and has the sequence shown in SEQ ID NO: 606 (also accessible under the UniProt accession number D1MX73). The wild-type oxynitrilase showed low stereoselectivity for the product. In the production of (S)-1,1,1-trifluoro-2-methyl-3-nitropropan-2-ol (i.e., (IA)) by the reaction of 1,1,1-trifluoropropan-2-one with nitromethane using SEQ ID NO: 606, the enantiomeric excess (i.e., ee) of IA was ≤2%.

[0017] In a first aspect, there is provided an oxynitrilase polypeptide that is a polypeptide of the following (a) or (b): (a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 to 604, and 608 to 640; or (b) a polypeptide having oxynitrilase activity, comprising (i) at least 80% sequence identity to one of the polypeptides described in (a), and (ii) one or more amino acid residue substitutions, deletions, additions, or insertions with respect to said one amino acid sequence described in (a).

[0018] In a second aspect, there is provided an oxynitrilase polypeptide capable of coupling 1,1,1-trifluoropropan-2-one with nitromethane to produce (S)-1,1,1-trifluoro-2-methyl-3-nitropropan-2-ol with a stereoselectivity and / or activity higher than the stereoselectivity and / or activity of SEQ ID NO: 606 under suitable reaction conditions.

[0019] In a third aspect, under suitable reaction conditions, 1,1,1-trifluoropropan-2-one is coupled with nitromethane to produce (S)-1,1,1-trifluoro-2-methyl-3-nitropropan-2-ol with an enantiomeric excess of at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. An oxynitrilase polypeptide is provided that includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 606.

[0020] In a further aspect, a polypeptide immobilized on a solid material by a chemical bond or physical adsorption method is provided, the polypeptide being selected from the oxynitrilase polypeptides according to the present disclosure.

[0021] In a further aspect, a polynucleotide encoding the polypeptide of the present disclosure is provided.

[0022] In a further aspect, an expression vector comprising the polynucleotide according to the present disclosure is provided.

[0023] In a further aspect, a host cell comprising the expression vector of the present disclosure is provided, and the host cell is preferably E. coli.

[0024] In a further aspect, a method for preparing an oxynitrilase polypeptide is provided, the method including culturing the host cell according to the present disclosure and obtaining the oxynitrilase polypeptide from the culture.

[0025] In a further aspect, there is provided an oxynitrilase catalyst obtained by culturing a host cell of the present disclosure, wherein the oxynitrilase catalyst comprises a cell or a culture solution containing an oxynitrilase polypeptide, or an article treated therewith, and the article refers to an extract obtained from a culture of transformed cells, an isolated product obtained by isolating or purifying oxynitrilase from the extract, or an immobilized product obtained by immobilizing transformed cells, an extract thereof, or an isolated product of the extract.

[0026] In a further aspect, there is provided a process for the asymmetric synthesis of β-nitroalcohol using an oxynitrilase polypeptide, the process comprising contacting a nitroalkane and an aldehyde or ketone substrate with the oxynitrilase polypeptide to obtain a β-nitroalcohol product.

[0027] In a further aspect, there is provided a process for the asymmetric synthesis of β-nitroalcohol using a modified oxynitrilase polypeptide disclosed herein, the process comprising contacting a nitroalkane and an aldehyde or ketone substrate with the oxynitrilase polypeptide of the present disclosure to obtain a β-nitroalcohol product.

[0028] In a further aspect, a process for the asymmetric synthesis of (S)-1,1,1-trifluoro-2-methyl-3-nitropropan-2-ol (IA):

Chemical formula

[0029] In a further aspect, (S)-3-amino-1,1,1-trifluoro-2-methylpropan-2-ol of formula (IB): [Chemical formula] A process for synthesizing (S)-3-amino-1,1,1-trifluoro-2-methylpropan-2-ol (IB) by contacting (IA) with hydrogen under suitable hydrogenation conditions, wherein (IA) is synthesized by the process according to the present disclosure, is provided.

[0030] In a further aspect, a process for synthesizing (S)-3-amino-6-methoxy-N-(3,3,3-trifluoro-2-hydroxy-2-methylpropyl)-5-(trifluoromethyl)picolinamide of formula (IC), [Chemical formula] A process is provided that includes contacting nitromethane and 1,1,1-trifluoropropan-2-one with the oxynitrilase polypeptide according to the present disclosure to obtain (S)-1,1,1-trifluoro-2-methyl-3-nitropropan-2-ol of formula (IA).

[0031] Detailed Description The present disclosure describes the directed evolution of HNL for obtaining nitroalcohols in excellent yields and enantiomeric excesses even with equimolar ratios of substrates. This is the first description of an HNL that accepts a ketone as a substrate for the Henry reaction and the first S-selective oxynitrilase to catalyze the Henry reaction. This HNL is derived from the organism Baliospermum montanum (BmHNL), and it has not been reported so far that it can catalyze the Henry reaction. A series of modified polypeptides with high S-stereoselectivity are provided.

[0032] These modified polypeptides were developed by directed evolution towards the selection of (S)-1,1,1-trifluoro-2-methyl-3-nitropropan-2-ol, a compound of formula (IA) as defined herein.

[0033] The present disclosure describes a modified polypeptide derived from BmHNL that catalyzes the Henry reaction of trifluoroacetone (1) and nitromethane (2) to produce a nitroalcohol compound (IA). All previously reported oxynitrilases require 10- to 45-fold large excesses of nitro compounds to reach a sufficient conversion rate >50%, resulting in low atom efficiency (<20%) and low economic viability of these processes. However, the newly reported polypeptide can reach a conversion rate >80% and >80% ee under equimolar conditions, or a conversion rate of 100% and >90% ee with 1.2-fold excess substrate nitromethane (2). This is the first report of an HNL that accepts a ketone as a substrate for the Henry reaction and the first reported highly S-selective oxynitrilase that catalyzes the Henry reaction with >80% ee.

[0034] The present disclosure also provides a process for the asymmetric synthesis of β-nitroalcohols using the modified oxynitrilase polypeptide disclosed herein, the process comprising contacting a nitroalkane and an aldehyde or ketone substrate with the oxynitrilase polypeptide of the present disclosure to obtain a β-nitroalcohol product.

[0035] In particular, the present disclosure provides a process for the asymmetric synthesis of (S)-1,1,1-trifluoro-2-methyl-3-nitropropan-2-ol (IA), which stereoselectively produces the desired (S) enantiomer over the (R) enantiomer. Scheme 1

Chemical formula

Chemical formula

[0036] Definitions Unless otherwise clearly defined, technical and scientific terms used in this disclosure have the meanings commonly understood by those of ordinary skill in the art to which this invention pertains. The following references provide many common definitions of terms used in this invention to those of ordinary skill in the art: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings set forth below unless otherwise specified. Abbreviations used for genetically encoded amino acids are conventional and are as follows:

[0037] [Table 1]

[0038] A “-” was used for amino acid deletions and a “*” was used for stop codons. When three-letter abbreviations were used, amino acids were in the L-configuration unless specifically preceded by “L” or “D” or otherwise apparent from the context in which the abbreviation was used, at the α-carbon (Cα ) can be either in the L-configuration or the D-configuration for. For example, while "Ala" represents alanine that does not specify the configuration about the α-carbon, "D-Ala" and "L-Ala" represent D-alanine and L-alanine, respectively.

[0039] When single-letter abbreviations are used, capital letters indicate amino acids with the L-configuration about the α-carbon, and lowercase letters indicate amino acids in the D-configuration about the α-carbon. For example, "A" indicates L-alanine and "a" indicates D-alanine. When a polypeptide sequence is presented as a string of single-letter or three-letter abbreviations (or a mixture thereof), the sequence is presented in the amino (N)-to-carboxy (C) direction according to common convention.

[0040] The abbreviations used for genetically encoding nucleotides are conventional and are as follows: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless specifically indicated, the abbreviated nucleotides can be ribonucleotides or 2'-deoxyribonucleotides. Nucleotides can be specified as ribonucleotides or 2'-deoxyribonucleotides on an individual or aggregate basis. When a nucleic acid sequence is shown as a string of single-letter abbreviations, the sequence is shown in the 5'-to-3' direction according to common convention, and phosphate is not shown.

[0041] "Difference in amino acids" or "residue difference" refers to the difference in amino acid residues at the position of the polypeptide sequence relative to the amino acid residue at the corresponding position in the reference sequence. The position of the amino acid difference is generally referred to herein as "Xn", where n refers to the corresponding position in the reference sequence on which the residue difference is based. For example, "residue difference at position X2 compared to SEQ ID NO:2" refers to the difference in amino acid residues at the polypeptide position corresponding to position 2 of SEQ ID NO:2. Thus, if the reference polypeptide of SEQ ID NO:2 has leucine at position 2, "residue difference at position X2 compared to SEQ ID NO:2" refers to an amino acid substitution of a residue other than leucine at the polypeptide position corresponding to position 2 of SEQ ID NO:2. In most of the examples herein, the difference in a particular amino acid residue at that position is shown as "XnY", where "Xn" is specified at the corresponding position as described above, and "Y" is the one-letter identifier of the amino acid found in the modified polypeptide (i.e., a residue different from the residue in the reference polypeptide). In some examples (e.g., Figure 4), the present disclosure also provides a difference in a particular amino acid indicated by the conventional notation "AnB", where A is the one-letter identifier of the residue in the reference sequence, "n" is the number of the residue position in the reference sequence, and B is the one-letter identifier of the residue substitution in the sequence of the modified polypeptide. In some examples, the modified polypeptides of the present disclosure may include one or more amino acid residue differences relative to the reference sequence, which are shown in a list of the specific positions where there are residue differences relative to the reference sequence. In some embodiments, two or more amino acid residues can be used at a particular residue position of the modified polypeptide, and the various amino acid residues that can be used are separated by " / " (e.g., X38F / X38F). "Deletion" refers to the modification of a polypeptide by removing one or more amino acids from the reference polypeptide. Deletions can include the removal of one or more amino acids, two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids, up to 10% of the total number of amino acids of the enzyme, or up to 20% of the total number of amino acids constituting the reference enzyme, while retaining the enzyme activity of the modified oxynitrilase and / or retaining the improved properties of the modified oxynitrilase.The deletion may include an internal portion and / or a terminal portion of the polypeptide. In various embodiments, the deletion may include a continuous segment or may be discontinuous.

[0042] The term “and / or” means “and” or “or” unless otherwise indicated.

[0043] “Coding sequence” refers to a portion of a nucleic acid (e.g., a gene) that encodes an amino acid sequence of a protein.

[0044] “Comparison window” refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acid residues, wherein a sequence can be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids, and the portion of the sequence in the comparison window can include no more than 20% addition or deletion (i.e., gaps) as compared to the reference sequence (not including additions or deletions) for optimal alignment of the two sequences. The comparison window may be longer than 20 contiguous residues and can optionally include 30, 40, 50, 100 or more residues.

[0045] Unless otherwise specified, the terms “compounds of the present disclosure,” “compounds of the disclosure,” or “compound of the disclosure” refer to compounds of formula (1), (2), (I), (I-i), (II), (III), (IA), (IB), (IB).HCl, (IC), (ID), E1, E3, E4, E5, E6, exemplary compounds, salts thereof, particularly pharmaceutically acceptable salts, hydrates, solvates, prodrugs, and all stereoisomers (including diastereoisomers and enantiomers), rotamers, tautomers, and isotopically labeled compounds (including deuterium substitution), and essentially formed moieties.

[0046] "Conversion" refers to the enzymatic conversion reaction of a substrate to its corresponding product. "Percent conversion" or "conversion" refers to the proportion of the substrate that is changed to the product within a certain time under specific conditions. Therefore, the "enzymatic activity" or "activity" of an oxynitrilase polypeptide can be expressed as the "conversion rate" of the substrate to the product.

[0047] In the context of numbering a given amino acid or polynucleotide sequence, "corresponding to", "reference to", or "compared to" refers to the numbering of the residues of a particular reference when comparing the given amino acid or polynucleotide sequence to a reference sequence. In other words, the residue number or residue position of a given sequence is specified with respect to the reference sequence, rather than by the actual numerical position of the residue within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence such as a modified oxynitrilase can be aligned with a reference sequence by introducing gaps to optimize the residue match between these two sequences. In such cases, although there are gaps, the numbering of the residues of the given amino acid or polynucleotide sequence is done with respect to the aligned reference sequence.

[0048] As used herein, the term "electron-withdrawing substituent" refers to a substituent having a strong electron-withdrawing property. Exemplary electron-withdrawing groups include, but are not limited to, halogen atoms such as fluorine atom, haloalkyl groups such as trifluoromethyl group, carboxyl group, alkoxycarbonyl groups such as methoxycarbonyl group, aryloxycarbonyl groups such as phenoxycarbonyl group, acyl groups such as acetyl group, acyloxy groups such as acetoxy group, cyano group, aryl group, alkenyl group, nitro group, sulfo group, alkanesulfonyl group, alkanesulfinyl group, and alkoxysulfonyl group, and any of the substituents disclosed herein such as alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, and arylalkyl groups containing these electron-withdrawing groups as substituents. Among these, preferred examples are fluorine-containing groups such as fluorine atom and trifluoromethyl group, acyloxy groups such as acetoxy group, cyano group, nitro group, sulfo group, alkylsulfonyl groups such as C1-C6 alkylsulfonyl groups, alkylsulfinyl groups such as C1-C6 alkylsulfinyl groups, and alkoxysulfonyl groups such as C1-C6 alkoxysulfonyl groups.

[0049] The terms "modified oxynitrilase", "modified oxynitrilase polypeptide", "oxynitrilase polypeptide", "improved oxynitrilase polypeptide", and "modified polypeptide" are used interchangeably herein.

[0050] As used herein, "fragment" refers to a polypeptide having deletions at the amino terminus and / or carboxyl terminus, but with the remaining amino acid sequence being the same as the corresponding position in the sequence. Fragments can be at least 10 amino acids in length, at least 20 amino acids in length, at least 50 amino acids in length, or more, and can be up to 70%, 80%, 90%, 95%, 98% and 99% of the full-length oxynitrilase polypeptide.

[0051] "Improved enzyme characteristics" refers to enzyme characteristics that are better or more desirable for a particular purpose compared to a reference oxynitrilase, such as a wild-type oxynitrilase or another improved modified oxynitrilase. The improved enzyme characteristics are exhibited by the modified oxynitrilase polypeptides in the present disclosure. Enzyme characteristics that are expected to be improved include, but are not limited to, enzyme activity (which can be expressed as a percentage of substrate conversion), thermal stability, solvent stability, pH activity profile, cofactor requirements, resistance to inhibitors (e.g., substrate or product inhibition), stereospecificity, and stereoselectivity (including enantioselectivity and diastereoselectivity).

[0052] "Insertion" refers to the modification of a polypeptide by adding one or more amino acids to a reference polypeptide. In some embodiments, the improved modified oxynitrilase includes the insertion of one or more amino acids into a naturally occurring oxynitrilase polypeptide, as well as the insertion of one or more amino acids into other modified oxynitrilase polypeptides. It can be inserted into the internal portion of the polypeptide or at the carboxyl or amino terminus. As used herein, insertion includes fusion proteins known in the art. The insertion can be a continuous segment of amino acids or can be separated by one or more amino acids in a naturally occurring or modified polypeptide.

[0053] "Isolated polypeptide" refers to a polypeptide that is substantially separated from other substances with which it is naturally associated, such as proteins, lipids, and polynucleotides. This term includes polypeptides that have been removed or purified from their natural environment or expression system (e.g., a host cell or in vitro synthesis). The modified oxynitrilase polypeptide may be present intracellularly, in a cell culture medium, or may be prepared in various forms such as a lysate or an isolated preparation. Thus, in some embodiments, the modified oxynitrilase polypeptide can be an isolated polypeptide.

[0054] "Naturally occurring" or "wild-type" refers to the form found in nature. For example, a naturally occurring or wild-type polypeptide or polynucleotide sequence is a sequence that exists in an organism that can be isolated from a natural source and has not been intentionally modified by manual procedures.

[0055] As used herein, "oxynitrilase" or "HNL" refers to a wild-type or modified enzyme having oxynitrilase activity.

[0056] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein.

[0057] The terms "protein", "polypeptide", and "peptide" are used interchangeably herein to denote a polymer of at least two amino acids covalently linked by amide bonds, regardless of length or post-translational modifications (e.g., glycosylation, phosphorylation, lipidation, myristoylation, ubiquitination, etc.). This definition includes D-amino acids and L-amino acids, as well as mixtures of D-amino acids and L-amino acids. Preferably, the amino acids have the L configuration.

[0058] "Recombinant" or "modified" or "not naturally occurring", when used with respect to, for example, a cell, nucleic acid or polypeptide, refers to a material that is otherwise not naturally occurring or is the same as a naturally occurring or native form of the material but has been modified by a method that is produced or derived from synthetic materials and / or by manipulation using recombinant techniques.

[0059] "Reference array" refers to a defined array used as a basis for array comparison. The reference array may be a subset of a larger array, such as a full-length gene or a fragment of a polypeptide sequence. Generally, the reference array is at least 20 nucleotides or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, or a full-length nucleic acid or polypeptide. Since two polynucleotides or polypeptides can each (1) contain sequences that are similar between the two sequences (i.e., part of the complete sequence) and (2) further contain sequences that differ between the two sequences, the comparison of sequences between two (or more) polynucleotides or polypeptides is usually carried out by comparing the sequences of the two polynucleotides or polypeptides over a "comparison window" to identify and compare local regions of sequence similarity. In some embodiments, it is not intended to limit the "reference array" to the wild-type sequence, and it may include engineered or modified sequences. For example, "a reference array having proline at the residue corresponding to X35 based on SEQ ID NO: 2" refers to a reference array in which the corresponding residue at position X35 of SEQ ID NO: 2, which is alanine, has been changed to proline.

[0060] "Sequence identity" and "homology" are used interchangeably herein and refer to a comparison between polynucleotide sequences or polypeptide sequences (generally expressed as a percentage), which is determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide or polypeptide sequence in the comparison window may include additions or deletions (i.e., gaps) as compared to a reference sequence for optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions at which the identical nucleic acid bases or amino acid residues exist in both sequences, or the number of positions at which the nucleic acid bases or amino acid residues are aligned by gaps, obtaining the number of matched positions, dividing the number of the matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Those skilled in the art will understand that there are many established algorithms available for aligning two sequences. Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm by Smith and Waterman (1981) Adv. Appl. Math. 2:482c, by the homology alignment algorithm by Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, by the similarity search method by Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Package), or by visual inspection (generally, see Current Protocols in Molecular Biology, FM Ausubel et al. eds., Current Protocols, a joint venture of Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (1995 Supplement) (Ausubel)).Examples of algorithms suitable for determining percent sequence identity and percent sequence similarity are the BLAST algorithm and the BLAST 2.0 algorithm, described in Altschul et al., 1990, J. Mol. Biol. 215:403-410 and Altschul et al., 1977, Nucleic Acids Res. 3389-3402, respectively. Software for performing BLAST analysis is publicly available from the website of the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short word lengths W in the query sequence that match or satisfy a positive-valued threshold score T when aligned with words of the same length within the database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits serve as seeds for initiating a search for longer HSPs that contain them. The word hits are extended in either direction along each sequence as long as the cumulative alignment score can increase. For nucleotide sequences, the cumulative score is calculated using parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for a non-matching residue; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. The extension of the word hits in each direction is stopped when the cumulative alignment score drops by an amount X from its maximum value achieved; when the cumulative score drops to 0 or below due to the accumulation of one or more negative-score residues in the alignment; or when the end of either sequence is reached. The sensitivity and speed of the alignment are determined by the BLAST algorithm parameters W, T, and X. In the BLASTN program (for nucleotide sequences), a word length (W) of 11, an expectation value (E) of 10, M = 5, and N = -4 are used as defaults, and comparisons of both strands are performed.For the BLASTP program for amino acid sequences, by default, a word length of 3, an expectation value (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA 89:10915) are used. Exemplary determination of sequence alignment and percent sequence identity can be done using the BESTFIT or GAP programs with the default parameters provided in the GCG Wisconsin software package (Accelrys, Madison WI).

[0061] "Solvent stability" refers to an oxynitrilase polypeptide that maintains similar activity (e.g., more than 50% - 80%) after exposure to various solvents (such as ethanol, isopropanol, dimethyl sulfoxide, tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene, butyl acetate, methyl tert-butyl ether, etc.) for a certain period of time (e.g., 0.5 - 24 hours).

[0062] "Stereoisomers" and similar expressions are used interchangeably herein and refer to all isomers arising from differences in the orientation of atoms only in their space. It includes enantiomers and compounds having two or more stereocenters that are not mirror images of each other (i.e., diastereomers).

[0063] "Stereoselectivity" refers to the preferential formation of one stereoisomer over the other in a chemical or enzymatic reaction. Stereoselectivity can be partial, where the formation of one stereoisomer is preferred over the other, or complete when only one stereoisomer is formed. When the stereoisomers are enantiomers, the stereoselectivity is called enantioselectivity. This is often reported as the "enantiomeric excess" (abbreviated as ee). When the stereoisomers are diastereomers, the stereoselectivity is called diastereoselectivity. This is often reported as the "diastereomeric excess" (abbreviated as de). The ratio (usually a percentage) is generally reported in the art as the enantiomeric excess (i.e., ee) derived therefrom according to the following formula: [major enantiomer - minor enantiomer] / [major enantiomer + minor enantiomer].

[0064] "Suitable reaction conditions" refer to the conditions (e.g., catalyst loading, substrate loading, temperature, solvent, etc.) in a reaction system in which a substrate is converted into a desired product.

[0065] In the context of the biocatalytic processes of the present disclosure, "suitable reaction conditions" refer to the conditions (e.g., enzyme loading, substrate loading, cofactor loading, temperature, pH, buffer, cosolvent, etc.) in a biocatalytic reaction system under which the oxynitrilase polypeptide of the present disclosure can convert a substrate into a desired product compound. Examples of "suitable reaction conditions" are provided in the present disclosure and illustrated by the examples.

[0066] "Thermal stability" means an oxynitrilase polypeptide that retains similar activity (e.g., greater than 50%) after exposure to a high temperature (e.g., 30 - 85°C) for a certain period of time (e.g., 0.5 - 24 hours).

[0067] Furthermore, when a term indicating a monovalent radical is used where a divalent radical is appropriate, it is to be construed as indicating each divalent radical, and vice versa. Unless otherwise specified, in all formulas and groups, the conventional definitions of the term "control" and conventional stable valences are assumed and achieved. The articles "a" and "an" refer to one or more (e.g., at least one) of the grammatical referents of the article. By way of example, "an element" means one element or two or more elements.

[0068] The term "substituted" means that a particular group or moiety has one or more suitable substituents, and the substituents can be linked to the particular group or moiety at one or more positions. For example, an aryl substituted with cycloalkyl can indicate that the cycloalkyl is bonded to one atom of the aryl or is fused to the aryl and bonded by sharing two or more common atoms.

[0069] In the groups, radicals, or moieties defined below, the number of carbon atoms is often specified before the group, e.g., C1-C8 alkyl means an alkyl group or radical having 1 to 8 carbon atoms. Generally, for a substituent containing two or more sub-groups, the last-named group is the point of attachment of the group, e.g., "alkylaryl" means a monovalent group of the formula alkyl-aryl- while "arylalkyl" means a monovalent group of the formula aryl-alkyl-. "Halogen" or "halo" means fluorine, chlorine, bromine, or iodine. The term "nitro" shall mean the radical -NO2.

[0070] As used herein, the term "alkyl" refers to a branched or straight-chain saturated hydrocarbon group having, for example, 1 to 50 carbon atoms, such as C1-C3 alkyl, C1-C6 alkyl, C2-C8-alkyl, C3-C8-alkyl, C1-C8-alkyl, C1-C 10 alkyl, C1-C 20 alkyl, C1-C 30 alkyl, C1-C 40 alkyl, C1-C50 It represents alkyl and the like. Representative examples include methyl, ethyl, propyl (e.g., propan-1-yl, propan-2-yl (or isopropyl)), butyl (e.g., 2-methylpropan-2-yl (or tert-butyl), butan-1-yl, butan-2-yl), pentyl (e.g., pentan-1-yl, pentan-2-yl, pentan-3-yl), 2-methylbutan-1-yl, 3-methylbutan-1-yl, hexyl (e.g., hexan-1-yl), heptyl (e.g., heptan-1-yl), octyl (e.g., octan-1-yl), nonyl (e.g., nonan-1-yl), etc. The term "C1-C6 alkyl" should be interpreted as appropriate.

[0071] As used herein, the term "alkenyl" refers to a branched or straight-chain hydrocarbon group having at least one double bond, for example having 2 to 50 carbon atoms and having at least one double bond, such as C2-C3 alkenyl, C2-C6 alkenyl, C2-C7 alkenyl, C2-C8 alkenyl, C3-C5 alkenyl, C1-C 10 alkenyl, C1-C 20 alkenyl, C1-C 30 alkenyl, C1-C 40 alkenyl, C1-C 50 It represents alkenyl and the like. Representative examples include ethenyl (or vinyl), propenyl (e.g., propan-1-enyl, propan-2-enyl), butadienyl (e.g., buta-1,3-dienyl), butenyl (e.g., buta-1-en-1-yl, buta-2-en-1-yl), pentenyl (e.g., penta-1-en-1-yl, penta-2-en-2-yl), hexenyl (e.g., hexa-1-en-2-yl, hexa-2-en-1-yl), 1-ethylpropan-2-enyl, 1,1-(dimethyl)propan-2-enyl, 1-ethylbut-3-enyl, 1,1-(dimethyl)but-2-enyl, etc.

[0072] As used herein, the term "alkynyl" refers to a branched or straight-chain hydrocarbon group having at least one triple bond, for example having from 2 to 50 carbon atoms and having at least one triple bond, such as C2-C3 alkynyl, C2-C6 alkynyl, C2-C7 alkynyl, C2-C8 alkynyl, C3-C5 alkynyl, C1-C 10 alkynyl, C1-C 20 alkynyl, C1-C 30 alkynyl, C1-C 40 alkynyl, C1-C 50 and the like. Representative examples are ethynyl, propynyl (e.g., prop-1-ynyl, prop-2-ynyl), butynyl (e.g., but-1-ynyl, but-2-ynyl), pentynyl (e.g., pent-1-ynyl, pent-2-ynyl), hexynyl (e.g., hex-1-ynyl, hex-2-ynyl), 1-ethylprop-2-ynyl, 1,1-(dimethyl)prop-2-ynyl, 1-ethylbut-3-ynyl, 1,1-(dimethyl)but-2-ynyl, and the like.

[0073] As used herein, the term "aryl" is intended to include monocyclic, bicyclic, or polycyclic carbocyclic aromatic rings. Representative examples include phenyl, naphthyl (e.g., naphthalen-1-yl, naphthalen-2-yl), anthryl (e.g., anthracen-1-yl, anthracen-9-yl), phenanthryl (e.g., phenanthren-1-yl, phenanthren-9-yl), and the like. Aryl is also intended to include monocyclic, bicyclic, or polycyclic carbocyclic aromatic rings substituted with a carbocyclic aromatic ring. Representative examples include biphenyl (e.g., biphenyl-2-yl, biphenyl-3-yl, biphenyl-4-yl), phenylnaphthyl (e.g., 1-phenylnaphthalen-2-yl, 2-phenylnaphthalen-1-yl), and the like. Aryl is also intended to include bicyclic or polycyclic partially saturated carbocyclic rings having at least one unsaturated moiety (e.g., a benzo moiety). Representative examples include indanyl (e.g., indan-1-yl, indan-5-yl), indenyl (e.g., inden-1-yl, inden-5-yl), 1,2,3,4-tetrahydronaphthyl (e.g., 1,2,3,4-tetrahydronaphthalen-1-yl, 1,2,3,4-tetrahydronaphthalen-2-yl, 1,2,3,4-tetrahydronaphthalen-6-yl), 1,2-dihydronaphthyl (e.g., 1,2-dihydronaphthalen-1-yl, 1,2-dihydronaphthalen-4-yl, 1,2-dihydronaphthalen-6-yl), fluorenyl (e.g., fluorene-1-yl, fluorene-4-yl, fluorene-9-yl), and the like. Aryl is also intended to include bicyclic or polycyclic partially saturated carbocyclic aromatic rings containing one or two bridges. Representative examples include benzonorborn-yl (e.g., benzoborn-3-yl, benzoborn-6-yl), 1,4-ethano-1,2,3,4-tetrahydronaphthyl (e.g., 1,4-ethano-1,2,3,4-tetrahydronaphthalen-2-yl, 1,4-ethano-1,2,3,4-tetrahydronaphthalen-10-yl), and the like. Aryl is also intended to include bicyclic or polycyclic partially saturated carbocyclic aromatic rings containing one or more spiro atoms.Representative examples include spiro[cyclopentane-1,1'-indan]-4-yl, spiro[cyclopentane-1,1'indene]-4-yl, spiro[piperidine-4,1'-indan]-1-yl, spiro[piperidine-3,2'-indan]-1-yl, spiro[piperidine-4,2'-indan]-1-yl, spiro[piperidine-4,1'-indan]-3'-yl, spiro[pyrrolidine-3,2'-indan]-1-yl, spiro[pyrrolidine-3,1'-(3',4'-dihydronaphthalene)]-1-yl, spiro[piperidine-3,1'-(3',4'-dihydronaphthalene)]-1-yl, spiro[piperidine-4,1'-(3',4'-dihydronaphthalene)]-1-yl, spiro[imidazolidine-4,2'-indan]-1-yl, spiro[piperidine-4,1'-indene]-1-yl, and the like.

[0074] The term C6-C 14 Aryl should be interpreted as appropriate.

[0075] Preferably, aryl refers to a monocyclic or bicyclic carbocyclic aromatic ring.

[0076] The term "arylalkyl" (e.g., benzyl, phenylethyl, 3-phenylpropyl, 1-naphthylmethyl, 2-(1-naphthyl)ethyl, etc.) represents an aryl group as defined above bonded via an alkyl chain having the indicated number of carbon atoms or a substituted alkyl group as defined above. The term C7-C 20 Arylalkyl should be interpreted as appropriate.

[0077] Preferred examples of aryl include, but are not limited to, phenyl and naphthyl. In one embodiment, aryl is phenyl.

[0078] As used herein, the term "cycloalkyl" means a monocyclic or polycyclic saturated or partially saturated carbocyclic ring, containing, for example, from 3 to 20 carbon atoms, for example, containing from 3 to 18 carbon atoms, and having no delocalized pi electrons (aromaticity) shared between ring carbons. "C3-C 20 cycloalkyl" and "C3-C 10 cycloalkyl" are terms to be construed as appropriate. The term polycyclic includes bridged (e.g., norbornane), fused (e.g., decalin), and spirocyclic cycloalkyls. Preferably, cycloalkyl, for example, "C3-C 20 cycloalkyl" and "C3-C 18 cycloalkyl" are each monocyclic or spirocyclic hydrocarbon groups of 3 to 20 and 3 to 18 carbon atoms, respectively.

[0079] Representative examples are spiro[2.5]octanyl, spiro[4.5]decanyl, cyclopropenyl, cyclopropyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclohexyl, cycloheptanyl, cyclooctanyl, spiro[2.3]hexanyl, spiro[3.3]heptyl, spiro[3.4]octanyl, spiro[3.5]nonanyl, spiro[4.5]decanyl, spiro[5.5]undecanyl, spiro[4.4]nonanyl, bicyclo[2.2.2]octanyl, bicyclo[2.2.2]octenyl, bicyclo[1.1.1]pentanyl, decahydronaphthalenyl, bicyclo[3.3.0]octanyl, adamantyl, norbornanyl, norbornenyl, nortricyclanyl, bicyclo-[3.2.1]octanyl, tricyclo[5.2.1.0 2,6 decanyl, bicyclo[2.2.1]heptyl, and the like.

[0080] As used herein, the term "fluoroalkyl" refers to a straight-chain or branched alkyl group as defined above, wherein some or all of the hydrogen atoms of these groups are replaced by fluorine atoms. The term C1-C6 fluoroalkyl should be construed as appropriate. Examples include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 1-fluoroethyl, 2-fluoroethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, pentafluoroethyl, 3,3,3-trifluoropropan-1-yl, 1,1,1-trifluoropropan-2-yl, heptafluoroisopropyl, 1-fluorobutyl, 2-fluorobutyl, 3-fluorobutyl, 4-fluorobutyl, 4,4,4-trifluorobutyl, fluoro-tert-butyl, and the like.

[0081] As used herein, the term "haloalkyl" refers to an alkyl radical as defined above that is substituted by one or more halo radicals as defined herein. The terms C1-C 20 Haloalkyl and C1-C6 haloalkyl should be construed as appropriate. Examples of haloalkyl include, but are not limited to, trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, 1,1-difluoroethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, 2-fluoropropyl, 1,1,1-trifluoropropyl, 2,2-difluoropropyl, 3,3-difluoropropyl, and 1-fluoromethyl-2-fluoroethyl, 1,3-dibromopropan-2-yl, 3-bromo-2-fluoropropyl, 1,1,2,2-tetrafluoropropyl, and 1,4,4-trifluorobutan-2-yl.

[0082] As used herein, the term "heteroaryl" is intended to include monocyclic heteroaromatic rings containing one or more heteroatoms selected from oxygen, nitrogen and sulfur (O, N and S). Representative examples include pyrrolyl, furanyl, thienyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isothiazolyl, isoxazolyl, triazolyl (e.g., 1,2,4-triazolyl), oxadiazolyl (e.g., 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl), thiadiazolyl (e.g., 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl), tetrazolyl, pyranyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, thiadiazinyl, azepinyl, azecinyl, and the like.

[0083] Heteroaryl is also intended to include bicyclic heteroaromatic rings containing one or more heteroatoms selected from oxygen, nitrogen and sulfur (O, N and S). Representative examples include indolyl, isoindolyl, benzofuranyl, benzothiophenyl, indazolyl, benzopyranyl, benzimidazolyl, benzothiazolyl, benzoisothiazolyl, benzoxazolyl, benzoisoxazolyl, benzoxazinyl, benzotriazolyl, naphthyridinyl, phthalazinyl, pteridinyl, purinyl, quinazolinyl, cinnolinyl, quinolinyl, isoquinolinyl, quinoxalinyl, oxazolopyridinyl, isoxazolopyridinyl, pyrrolopyridinyl, furopyridinyl, thienopyridinyl, imidazolopyridinyl, imidazolopyrimidinyl, pyrazolopyridinyl, pyrazolopyrimidinyl, pyrazolotriazinyl, thiazolopyridinyl, thiazolopyrimidinyl, imidazolothiazolyl, triazolopyridinyl, triazolopyrimidinyl, and the like.

[0084] Heteroaryl is also intended to include polycyclic heterocyclic aromatic rings containing one or more heteroatoms selected from oxygen, nitrogen, and sulfur (O, N, and S). Representative examples include carbazolyl, phenoxazinyl, phenazinyl, acridinyl, phenothiazinyl, carbolinyl, phenanthrolinyl, and the like.

[0085] Heteroaryl is also intended to include partially saturated monocyclic, bicyclic, or polycyclic heterocyclyl containing one or more heteroatoms selected from oxygen, nitrogen, and sulfur (O, N, and S). Representative examples include imidazolinyl, indolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, dihydropyranyl, dihydropyridooxazinyl, dihydrobenzodioxinyl (e.g., 2,3-dihydrobenzo[b][1,4]dioxinyl), benzodioxolyl (e.g., benzo[d][1,3]dioxole), dihydrobenzoxazinyl (e.g., 3,4-dihydro-2H-benzo[b][1,4]oxazine), tetrahydroindazolyl, tetrahydrobenzimidazolyl, tetrahydroimidazo[4,5-c]pyridyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, tetrahydroquinoxalinyl, and the like.

[0086] The heteroaryl ring structure may be substituted with one or more substituents. The substituents may themselves be optionally substituted. The heteroaryl ring may be attached via a carbon atom or a heteroatom.

[0087] The term "5- to 20-membered heteroaryl" should be interpreted as appropriate.

[0088] As used herein, the term "monocyclic heteroaryl" is intended to include the monocyclic heterocyclic aromatic rings defined above.

[0089] As used herein, the term "bicyclic heteroaryl" is intended to include the bicyclic heterocyclic aromatic rings defined above.

[0090] Examples of heteroaryl having 5 to 20 members include indolyl, imidazopyridyl, isoquinolinyl, benzoxazolonyl, pyridinyl, pyrimidinyl, pyridinonyl, benzotriazolyl, pyridazinyl, pyrazolotriazinyl, indazolyl, benzimidazolyl, quinolinyl, triazolyl (e.g., 1,2,4-triazolyl), pyrazolyl, thiazolyl, oxazolyl, isoxazolyl, pyrrolyl, oxadiazolyl (e.g., 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl), imidazolyl, pyrrolopyridinyl, tetrahydroindazolyl, quinoxalinyl, thiadiazolyl (e.g., 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl), pyrazinyl, oxazolopyridinyl, pyrazolopyrimidinyl, benzoxazolyl, indolinyl, isoxazolopyridinyl, dihydropyridooxazinyl, tetrazolyl, dihydrobenzodioxinyl (e.g., 2,3-dihydrobenz[b][1,4]dioxinyl), benzodioxolyl (e.g., benz[d][1,3]dioxole), and dihydrobenzoxazinyl (e.g., 3,4-dihydro-2H-benz[b][1,4]oxazine), but are not limited thereto.

[0091] As used herein, the term "heterocyclyl" refers to a saturated or partially saturated monocyclic or polycyclic ring containing one or more heteroatoms selected from nitrogen, oxygen, sulfur, S(═O), and S(═O)2, and there are no delocalized pi electrons (aromaticity) shared between the carbon or heteroatoms of the ring. The heterocyclyl ring structure may be substituted with one or more substituents. The substituents may themselves be optionally substituted. The heterocyclyl group may be attached via a carbon atom or a heteroatom. The term polycyclic includes bridged, fused, and spirocyclic heterocyclyls.

[0092] Representative examples include aziridinyl (e.g., aziridin-1-yl), azetidinyl (e.g., azetidin-1-yl, azetidin-3-yl), oxetanyl, pyrrolidinyl (e.g., pyrrolidin-1-yl, pyrrolidin-2-yl, pyrrolidin-3-yl), imidazolidinyl (e.g., imidazolidin-1-yl, imidazolidin-2-yl, imidazolidin-4-yl), oxazolidinyl (e.g., oxazolidin-2-yl, oxazolidin-3-yl, oxazolidin-4-yl), thiazolidinyl (e.g., thiazolidin-2-yl, thiazolidin-3-yl, thiazolidin-4-yl), isothiazolidinyl, piperidinyl (e.g., piperidin-1-yl, piperidin-2-yl, piperidin-3-yl, piperidin-4-yl), homopiperidinyl (e.g., homopiperidin-1-yl, homopiperidin-2-yl, homopiperidin-3-yl, homopiperidin-4-yl), piperazinyl (e.g., piperazin-1-yl, piperazin-2-yl), morpholinyl (e.g., morpholin-2-yl, morpholin-3-yl, morpholin-4-yl), thiomorpholinyl (e.g., thiomorpholin-2-yl, thiomorpholin-3-yl, thiomorpholin-4-yl), 1-oxothiomorpholinyl, 1,1-dioxothiomorpholinyl, tetrahydrofuranyl (e.g., tetrahydrofuran-2-yl, tetrahydrofuran-3-yl), tetrahydrothienyl, tetrahydro-1,1-dioxothienyl, tetrahydropyranyl (e.g., 2-tetrahydropyranyl), tetrahydrothiopyranyl (e.g., 2-tetrahydrothiopyranyl), 1,4-dioxanyl, 1,3-dioxanyl, and the like. Heterocyclyl is also intended to represent a saturated 6- to 8-membered bicyclic ring containing one or more heteroatoms selected from nitrogen, oxygen, sulfur, S(═O), and S(═O)2.Representative examples include octahydroindolyl (e.g., octahydroindol-1-yl, octahydroindol-2-yl, octahydroindol-3-yl, octahydroindol-5-yl), decahydroquinolinyl (e.g., decahydroquinolin-1-yl, decahydroquinolin-2-yl, decahydroquinolin-3-yl, decahydroquinolin-4-yl, decahydroquinolin-6-yl), decahydroquinoxalinyl (e.g., decahydroquinoxalin-1-yl, decahydroquinoxalin-2-yl, decahydroquinoxalin-6-yl), and the like. Heterocyclyl is also intended to represent a saturated 6- to 8-membered ring containing one or more heteroatoms selected from nitrogen, oxygen, sulfur, S(═O), and S(═O)2 and having one or two bridges. Representative examples include 3-azabicyclo[3.2.2]nonyl, 2-azabicyclo[2.2.1]heptyl, 3-azabicyclo[3.1.0]hexyl, 2,5-diazabicyclo[2.2.1]heptyl, atropinyl, tropinyl, quinuclidinyl, 1,4-diazabicyclo[2.2.2]octanyl, and the like. Heterocyclyl is also intended to represent a 6- to 8-membered saturated ring containing one or more heteroatoms selected from nitrogen, oxygen, sulfur, S(═O), and S(═O)2 and having one or more spiro atoms.Representative examples include 1,4-dioxaspiro[4.5]decan-2-yl (e.g., 1,4-dioxaspiro[4.5]decan-2-yl, 1,4-dioxaspiro[4.5]decan-7-yl), 1,4-dioxa-8-azaspiro[4.5]decan-2-yl (e.g., 1,4-dioxa-8-azaspiro[4.5]decan-2-yl, 1,4-dioxa-8-azaspiro[4.5]decan-8-yl), 8-azaspiro[4.5]decan-1-yl (e.g., 8-azaspiro[4.5]decan-1-yl, 8-azaspiro[4.5]decan-8-yl), 2-azaspiro[5.5]undecan-2-yl (e.g., 2-azaspiro[5.5]undecan-2-yl), 2,8-diazaspiro[4.5]decan-2-yl (e.g., 2,8-diazaspiro[4.5]decan-2-yl, 2,8-diazaspiro[4.5]decan-8-yl), 2,8-diazaspiro[5.5]undecan-2-yl (e.g., 2,8-diazaspiro[5.5]undecan-2-yl), 1,3,8-triazaspiro[4.5]decan-1-yl (e.g., 1,3,8-triazaspiro[4.5]decan-1-yl, 1,3,8-triazaspiro[4.5]decan-3-yl, 1,3,8-triazaspiro[4.5]decan-8-yl), and the like.

[0093] The term "3- to 14-membered heterocyclyl" should be interpreted as appropriate.

[0094] As used herein, the terms "optional" or "optionally substituted" mean that the described event or situation may or may not occur. For example, "optionally substituted aryl" refers to an aryl group that may or may not be substituted. This description includes both substituted and unsubstituted aryl groups.

[0095] The term "pharmaceutically acceptable" is defined as being free of adverse events and suitable for administration to humans.

[0096] As used herein, the terms "spongy nickel and spongy cobalt" shall refer to nickel and cobalt catalysts having a skeletal structure formed from a nickel or cobalt alloy. Typically, these catalysts are formed from nickel or cobalt alloyed with aluminum, which is then removed. Included within the terms spongy nickel and spongy cobalt are the trade names, well-known Raney nickel and Raney cobalt catalysts.

[0097] Some of the defined terms may appear more than once in the structural formulas, and in such an event, each term shall be defined independently of the other terms.

[0098] The oxynitrilase of the present disclosure In another aspect, the oxynitrilase polypeptide disclosed herein can asymmetrically couple an aldehyde or ketone substrate with a nitroalkane substrate.

[0099] In some embodiments, the modified oxynitrilase polypeptide of the present disclosure can convert 1,1,1-trifluoropropan-2-one and nitromethane to produce (S)-1,1,1-trifluoro-2-methyl-3-nitropropan-2-ol (i.e., (IA)) with at least equivalent or greater stereoselectivity as compared to SEQ ID NO: 606 and / or SEQ ID NO: 2. The modified oxynitrilase polypeptide of the present disclosure can produce a β-nitroalcohol product, such as compound IA, with an enantiomeric excess of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0100] In some embodiments, the modified oxynitrilase polypeptide converts 1,1-trifluoropropan-2-one and nitromethane to produce (S)-1,1,1-trifluoro-2-methyl-3-nitropropan-2-ol (i.e., (IA)) with higher stereoselectivity than the polypeptide of SEQ ID NO: 606 under suitable reaction conditions, such as the conditions disclosed herein. The modified oxynitrilase polypeptide has a sequence identity of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% to the polypeptide of SEQ ID NO: 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, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428,The sequences of 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638 and 640, and an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical. The identity between two amino acid sequences or two nucleotide sequences can be obtained by algorithms commonly used in the art, and can be calculated according to the default parameters by using NCBI Blastp and Blastn software, or by using the Clustal W algorithm (Nucleic Acid Research, 22(22):4673-4680, 1994). For example, when using the Clustal W algorithm, the amino acid sequence identity of the sequence of SEQ ID NO: 4 to SEQ ID NO: 606 is 92.78%.

[0101] The modified oxynitrilase polypeptides represented by SEQ ID NOs: 4 to 604 and 608 to 640 exhibit higher activity and / or stereoselectivity than the polypeptide of SEQ ID NO: 606, as shown in the examples.

[0102] In some embodiments, the modified oxynitrilase polypeptide comprises an amino acid sequence having an insertion of one or more amino acid residues in SEQ ID NO: 2 and having oxynitrilase activity. In any embodiment of the modified oxynitrilase polypeptides of the present disclosure, the insert fragment can include one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, six or more amino acids, eight or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids, and the related functional and / or improved characteristics of the modified oxynitrilase described herein are maintained. The insert fragment can be inserted at the amino terminus or carboxy terminus, or an internal portion of the oxynitrilase polypeptide. In some embodiments, the insert fragment can include 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 amino acid residues. In some embodiments, the number of insertion occurrences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60 or more.

[0103] In some embodiments, the insert fragment may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acid residues. In some embodiments, the modified oxynitrilase polypeptide comprises an amino acid sequence that is different from the sequence of SEQ ID NO:2 at one or more residue positions selected from the following: X2, X12, X28, X29, X32, X39, X50, X55, X64, X105, X111, X147, X152, X154, X160, X185, X196, X203, X208, X209, X232, X233 and X250. In some embodiments, the modified oxynitrilase polypeptide comprises an amino acid sequence comprising at least one of the following features (these features are amino acid residue substitutions relative to the reference sequence of SEQ ID NO:2): VX12I; SX28G; AX29W; NX32T; WX39F; WX39V; QX50E; QX50D; RX55G; LX64A; AX105G; DX111S; EX147K; TX152L; NX160M; TX185R; SX196G; YX203C; QX208R; QX208S; IX209V; SX232G; AX233G; or QX250G.

[0104] In certain embodiments, the modified oxynitrilase polypeptide comprises an amino acid sequence that is different from the sequence of SEQ ID NO:2 at one or more residue positions selected from the following: X39, X105 and X154. In certain embodiments, the modified oxynitrilase polypeptide comprises an amino acid sequence comprising at least one of the following features (these features are amino acid residue substitutions relative to the reference sequence of SEQ ID NO:2): WX39F; WX39V; or AX105G.

[0105] In some embodiments, the modified oxynitrilase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 606 at one or more residue positions selected from: X2, X11, X12, X28, X29, X32, X33, X39, X43, X44, X46, X50, X55, X64, X80, X103, X105, X111, X118, X121, X147, X152, X154, X160, X172, X180, X185, X196, X203, X208, X209, X232, X233, X238, X241, X250 and X263. In some embodiments, the modified oxynitrilase polypeptide comprises an amino acid sequence comprising at least one of the following features, which are amino acid residue substitutions relative to the reference sequence of SEQ ID NO: 606: TX11S; IX12V; SX28G; AX29W; NX32T; AX33V; VX39F, IX43S; DX44N; RX46H; QX50E; QX50D; EX55R; EX55G; LX64A; SX80A; HX103V; AX105G; DX111S; YX118V; FX121Y; EX147K; TX152L; NX160M; LX172R; EX180L; TX185R; SX196G; YX203C; QX208R; QX208S; IX209V; SX232G; AX233G; QX238M; KX241R; QX250G; or AX263S.

[0106] In certain embodiments, the modified oxynitrilase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 606 at one or more residue positions selected from: X2, X105, X111, X154, X160, X185, X209, X232 and X250. In certain embodiments, the modified oxynitrilase polypeptide comprises an amino acid sequence comprising at least one of the following features, which are amino acid residue substitutions relative to the reference sequence of SEQ ID NO: 606: AX105G; DX111S; NX160M; TX185R; IX209V; SX232G; or QX250G.

[0107] In another aspect, the present disclosure provides a polynucleotide comprising a sequence encoding a modified oxynitrilase polypeptide, an expression vector, and a host cell capable of expressing the modified oxynitrilase polypeptide. In some embodiments, the host cell can be a bacterial host cell such as E. coli. The host cell can be used to express and isolate the modified oxynitrilase described herein, or directly in a reaction to convert a substrate to a product.

[0108] In some embodiments, the modified oxynitrilase in the form of whole cells, crude extracts, isolated enzymes, or purified enzymes can be used alone or in an immobilized form such as immobilized on a resin.

[0109] Polynucleotides, control sequences, expression vectors, and host cells that can be used to produce a modified oxynitrilase polypeptide In another aspect, the present disclosure provides a polynucleotide encoding a modified polypeptide having oxynitrilase activity as described herein. The polynucleotide can be ligated to one or more heterologous regulatory sequences that can control gene expression to produce a recombinant polynucleotide capable of expressing the modified polypeptide. An expression construct comprising the heterologous polynucleotide encoding the modified oxynitrilase can be introduced into a suitable host cell to express the corresponding modified oxynitrilase polypeptide.

[0110] As will be apparent to those skilled in the art, the availability of protein sequences and knowledge of the codons corresponding to various amino acids provide an exemplification of all possible polynucleotides encoding a protein sequence of interest. The degeneracy of the genetic code, where the same amino acid is encoded by alternative or synonymous codons, allows for the production of a vast number of polynucleotides, all of which encode the modified oxynitrilase polypeptides disclosed herein. Thus, given a particular amino acid sequence, one of ordinary skill in the art can generate any number of different polynucleotides by modifying one or more codons in a manner that does not change the amino acid sequence of the protein. In this regard, the present disclosure specifically contemplates any possible modification of the polynucleotides that can be made by selecting combinations based on possible codon choices for any of the polypeptides disclosed herein, including the amino acid sequences of the exemplary modified polypeptides recited in Examples 6 - 12 and 19, and any of the polypeptides disclosed as even sequence identifiers of SEQ ID NOs: 4 - 604 and 608 - 640 in the Sequence Listing incorporated by reference.

[0111] In various embodiments, the codons are preferably selected to be compatible with the host cell in which the recombinant protein is to be produced. For example, codons preferred for bacteria are used to express genes in bacteria, codons preferred for yeast are used to express genes in yeast, and codons preferred for mammals are used to express genes in mammalian cells.

[0112] In some embodiments, the polynucleotide encodes a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a reference sequence that is an even sequence identifier of SEQ ID NOs: 4-604 and 608-640. Here, the polypeptide has oxynitrilase activity and has one or more of the improved properties described herein, for example, the ability to convert 1,1,1-trifluoropropan-2-one to compound (IA) with higher stereoselectivity compared to the polypeptides of SEQ ID NOs: 2 and / or 606.

[0113] In some embodiments, the polynucleotide encodes a modified oxynitrilase polypeptide that comprises an amino acid sequence having the above percentage of identity and has one or more amino acid residue differences compared to SEQ ID NO: 606.

[0114] In some embodiments, the present disclosure provides a modified polypeptide having oxynitrilase activity, the modified polypeptide having at least 80% sequence identity to the reference sequence of SEQ ID NO: 2 and comprising a combination of residue differences selected from the following positions: X2, X12, X28, X29, X32, X39, X50, X55, X64, X105, X111, X147, X152, X154, X160, X185, X196, X203, X208, X209, X232, X233, X250.

[0115] In some embodiments, the polynucleotide encoding the modified oxynitrilase polypeptide comprises a sequence having an odd sequence identifier of SEQ ID NOs: 3-603 and 607-639.

[0116] In some embodiments, the polynucleotide encodes the polypeptides described herein, but at the nucleotide level, the polynucleotide has about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polynucleotide that encodes a modified oxynitrilase polypeptide described herein. In some embodiments, the reference polynucleotide is selected from the sequences having odd sequence identifiers of SEQ ID NOs: 3-603 and 607-639.

[0117] The isolated polynucleotide encoding the modified oxynitrilase polypeptide can be engineered in various ways to express the modified polypeptide, including further modification of the sequence by codon optimization to improve expression, insertion into suitable expression elements, with or without additional control sequences, and transformation into a host cell suitable for expression and production of the modified polypeptide.

[0118] Depending on the expression vector, it may be desirable or necessary to engineer the isolated polynucleotide before inserting it into the vector. Techniques for modifying polynucleotides and nucleic acid sequences using recombinant DNA methods are well known in the art. Guidance is provided below: Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press; and Current Protocols in Molecular Biology, Ausubel, F. Eds., Greene Pub. Associates, 1998, 2010 Year update.

[0119] In another aspect, the present disclosure also relates to a recombinant expression vector comprising a polynucleotide encoding a modified oxynitrilase polypeptide or a variant thereof, and one or more expression regulatory regions such as a promoter and a terminator, an origin of replication, etc., depending on the type of host into which they are introduced. Alternatively, the nucleic acid sequence of the present disclosure can be expressed by inserting a nucleic acid sequence or a nucleic acid construct containing the sequence into a suitable expression vector. In constructing an expression vector, the coding sequence is positioned in the vector such that it is linked to a control sequence suitable for expression. A recombinant expression vector can be any vector (e.g., a plasmid or a virus) that can be conveniently used in recombinant DNA procedures and can bring about the expression of a polynucleotide sequence. The vector is generally selected depending on its compatibility with the host cell into which it is introduced. The vector can be a linear or circular plasmid. The expression vector can be an autonomously replicating vector, i.e., a vector whose replication exists as an extrachromosomal entity independent of chromosomal replication such as a plasmid, an episomal element, a minichromosome, or an artificial chromosome. The vector may contain any tool that ensures self-replication. Alternatively, the vector can be a vector that, when introduced into a host cell, is integrated into the genome and replicates with the chromosome into which it is integrated. Further, a single vector or plasmid containing all the DNA to be introduced into the genome of the host cell, or two or more vectors or plasmids can be used.

[0120] Many expression vectors useful in the embodiments of the present disclosure are commercially available. Exemplary expression vectors can be prepared by inserting a polynucleotide encoding a modified oxynitrilase polypeptide into the plasmid pACYC-Duet-1 (Novagen), the pBR322 vector (New England Biolabs), the pUC19 vector (New England Biolabs), or the pET T7 expression vector (Novagen).

[0121] In another aspect, the present disclosure provides a host cell comprising a polynucleotide encoding a modified oxynitrilase polypeptide. The polynucleotide is linked to one or more control sequences for expressing the oxynitrilase polypeptide in the host cell. Host cells for expressing the polypeptide encoded by the expression vector of the present disclosure are well known in the art and include bacterial cells such as E. coli, Streptomyces, and Salmonella typhimurium; fungi (e.g., Saccharomyces cerevisiae or Pichia pastoris); insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS, BHK, 293, and Bowes melanoma cells; and plant cells, but are not limited thereto. An exemplary host cell is E. coli BL21(DE3). The above host cells may be wild-type or cells modified by genome editing such as knockout of the wild-type oxynitrilase gene carried on the genome of the host cell. Suitable media and growth conditions for the above host cells are well known in the art.

[0122] The polynucleotide used to express the modified oxynitrilase can be introduced into cells by various methods known in the art. The techniques include, among others, electroporation, biolistic bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion. Various methods for introducing polynucleotides into cells are known to those skilled in the art.

[0123] Method for Producing a Modified Oxynitrilase Polypeptide The modified oxynitrilase can be obtained by subjecting the polynucleotide encoding the oxynitrilase to mutagenesis and / or directed evolution. Exemplary directed evolution techniques can be found in “Biocatalysis for the Pharmaceutical Industry: Discovery, Development, and Manufacturing” (2009 John Wiley & Sons Asia (Pte) Ltd. ISBN: 978-0-470-82314-9).

[0124] If the sequence of the modified polypeptide is known, the coding polynucleotide can be prepared by standard solid-phase methods according to known synthetic methods. In some embodiments, fragments up to about 100 bases can be synthesized separately and then ligated (e.g., by enzymatic or chemical ligation methods or polymerase-mediated methods) to form any desired contiguous sequence. For example, the polynucleotides and oligonucleotides of the present disclosure can be prepared by chemical synthesis using, for example, the classical phosphoramidite method generally practiced in automated synthesis methods, as described in Beaucage et al., 1981, Tet Lett 22:1859-69, or Matthes et al., 1984, EMBO J. 3:801-05. Oligonucleotides are synthesized, purified, annealed, ligated, and cloned into a suitable vector, for example, in an automated DNA synthesizer. Furthermore, essentially any nucleic acid is available from any of a variety of commercial sources.

[0125] In some embodiments, the disclosure also provides a process for preparing or manufacturing a modified oxynitrilase polypeptide capable of converting 1,1,1-trifluoropropan-2-one to a compound (IA) under suitable reaction conditions, the process comprising culturing a host cell capable of expressing a polynucleotide encoding the modified polypeptide under culture conditions suitable for the expression of the polypeptide. In some embodiments, the process for preparing the polypeptide further comprises isolating the polypeptide. The modified polypeptide is expressed in a suitable cell and can be isolated (or recovered) from the host cell and / or the culture medium using any one or more of well-known techniques for purifying proteins, including, in particular, lysozyme treatment, sonication, filtration, salting out, ultracentrifugation, and chromatography.

[0126] Methods of Using Modified Oxynitrilase and Compounds Prepared Thereby The disclosure also provides a process for preparing a wide range of compounds or their structural analogs using the modified oxynitrilase polypeptides disclosed herein.

[0127] Accordingly, the disclosure provides a process for the asymmetric synthesis of β-nitroalcohols using the modified oxynitrilase polypeptides disclosed herein, the process comprising contacting a nitroalkane and an aldehyde or ketone substrate with the oxynitrilase polypeptide disclosed herein to obtain a β-nitroalcohol product.

[0128] The selection of applicable electrophiles ranges from aromatic to heteroaromatic and includes aliphatic aldehydes and ketones. Depending on the substrate and reaction system, the methods of the disclosure were able to achieve yields of up to at least 90% or enantiomeric excesses greater than 99%.

[0129] In one embodiment, the aldehyde or ketone substrate comprises an electron-withdrawing substituent.

[0130] In one embodiment, the present disclosure also provides a process for asymmetric synthesis of β-nitroalcohols using the modified oxynitrilase polypeptides disclosed herein, wherein the resulting β-nitroalcohol has the formula (I):

Chemical formula

Chemical formula

[0131] In one embodiment, the modified oxynitrilase polypeptide can be used in a process for preparing a β-nitroalcohol compound, such as a compound of structural formula (I) defined above.

[0132] By adding a nitroalkane other than nitromethane to an aldehyde or ketone substrate, two new stereocenters are generated simultaneously. The addition of nitromethane to an aldehyde or ketone substrate generates one new stereocenter.

[0133] Thus, in one embodiment, the β-nitroalcohol product, such as a compound of structural formula (I), is diastereomerically enriched over other diastereomers. In one embodiment, the β-nitroalcohol product is present at a diastereomeric excess of at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

[0134] In one embodiment, the β-nitroalcohol product, such as a compound of structural formula (I), is enantiomerically enriched over other enantiomers. In one embodiment, the β-nitroalcohol product is present at an enantiomeric excess of at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more over other enantiomers.

[0135] In one embodiment, the oxynitrilase polypeptide has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 2, and couples an aldehyde or ketone substrate, such as an aldehyde or ketone substrate of formula (III), with a nitroalkane substrate, such as a nitroalkane substrate of formula (II), such as nitromethane, to form a β-nitroalcohol product, such as a β-nitroalcohol product of formula (I), having a conversion rate and / or high stereoselectivity higher than that of SEQ ID NO: 606.

[0136] As disclosed herein, an oxynitrilase polypeptide useful in the processes of the present disclosure can be characterized by its ability to condense 1,1,1-trifluoropropan-2-one and nitromethane to form (S)-1,1,1-trifluoro-2-methyl-3-nitropropan-2-ol (IA).

[0137] Thus, in any of the embodiments of the processes disclosed herein, the oxynitrilase polypeptide can couple 1,1,1-trifluoropropan-2-one and nitromethane to form (S)-1,1,1-trifluoro-2-methyl-3-nitropropan-2-ol (IA) having a conversion rate and / or high stereoselectivity higher than that of SEQ ID NO: 606, and can perform a process having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 2.

[0138] In one embodiment, the resulting β-nitroalcohol has the formula (I-i):

Chemical formula

Chemical formula

[0139] In any of the embodiments of the process disclosed herein, R 1 and R 2 are each independently selected from H, C1 - C 20 alkyl, C2 - C 20 alkenyl, C2 - C 20 alkynyl, C3 - C 10 cycloalkyl, C6 - C 14 aryl, C7 - C 20Arylalkyl, 3- to 14-membered heterocycloalkyl, and 5- to 20-membered heteroaryl, where C1-C 20 alkyl, C2-C 20 alkenyl, and C2-C 20 alkynyl are each optionally, independently, substituted with 1 to 6 R a ; C3-C 10 cycloalkyl, C6-C 14 aryl, C7-C 20 arylalkyl, 3- to 14-membered heterocycloalkyl, and 5- to 20-membered heteroaryl are each optionally, independently, substituted with 1 to 6 R b ; Each R a is, when it occurs, independently, C3-C 10 cycloalkyl, C6-C 14 aryl, 3- to 14-membered heterocycloalkyl, 5- to 20-membered heteroaryl, halogen, such as F, haloalkyl, such as C1-C 20 haloalkyl, such as -CF3, -CN, -OR c , -NR c R c , -(CH2) n COOR c , -(CH2) n -C(=O)R c , -(CH2) n -C(=O)NR c R c ; Each R b is, when it occurs, independently, halogen, such as F, haloalkyl, such as, C1-C 20 haloalkyl, such as -CF3, -CN, -NO2, -OR c , -NR c R c , -(CH2) n COOR c , -(CH2) n -C(=O)R c , -(CH2) n -C(=O)NR c R c , C1-C 20 alkyl, C2-C 20Alkenyl, or C2-C 20 selected from alkynyl; Each R c , when each occurs, is independently H, C1-C 20 alkyl, C2-C 20 alkenyl, and C2-C 20 alkynyl; and n is 0, 1, 2, 3, 4, 5 or 6, for example 0, 1, 2 or 3.

[0140] In any of the embodiments of the processes disclosed herein, R 1 and R 2 are each independently H, C1-C 20 alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl, C3-C 10 cycloalkyl, C6-C 14 aryl, C7-C 20 arylalkyl, 3- to 14-membered heterocycloalkyl, and 5- to 20-membered heteroaryl, wherein C1-C 20 alkyl, C2-C 20 alkenyl, and C2-C 20 alkynyl are each optionally substituted with 1 to 6 R a ; C3-C 10 cycloalkyl, C6-C 14 aryl, C7-C 20 arylalkyl, 3- to 14-membered heterocycloalkyl, and 5- to 20-membered heteroaryl are each optionally substituted with 1 to 6 R b ; Each R a , when each occurs, is independently C3-C 10 cycloalkyl, C6-C 14 aryl, 3- to 14-membered heterocyclyl, 5- to 20-membered heteroaryl, halogen, for example F, haloalkyl, for example C1-C 20 haloalkyl, for example -CF3, -CN, -OR c , and -NR c R cselected from; each R b when each occurs independently, halogen, such as F, haloalkyl, such as C1-C 20 haloalkyl, such as -CF3, -CN, -NO2, -OR c , -NR c R c , C1-C 20 alkyl, C2-C 20 alkenyl, and C2-C 20 alkynyl; each R c when each occurs independently, H, C1-C 20 alkyl, C2-C 20 alkenyl, and C2-C 20 alkynyl.

[0141] In any of the embodiments of the processes disclosed herein, R 1 and R 2 are each independently selected from H, C1-C 20 alkyl, C3-C 10 cycloalkyl, C6-C 14 aryl, C7-C 20 arylalkyl, 3- to 14-membered heterocyclyl, and 5- to 20-membered heteroaryl, wherein C1-C 20 alkyl is optionally substituted with 1 to 6 R a ; C3-C 10 cycloalkyl, C6-C 14 aryl, C7-C 20 arylalkyl, 3- to 14-membered heterocyclyl, and 5- to 20-membered heteroaryl are each optionally substituted with 1 to 6 R b ; each R a when each occurs independently, is selected from halogen, such as F, C1-C 20 haloalkyl, such as -CF3, -CN, -OR c , and -NR c R c ; each R bWhen each occurs independently, a halogen, such as F, a haloalkyl, such as C1-C 20 haloalkyl, such as -CF3, -CN, -NO2, -OR c , and -NR c R c is selected from; wherein each R c when each occurs independently, is selected from H, and C1-C 20 alkyl.

[0142] In any of the embodiments of the process disclosed herein, R 1 is selected from hydrogen, and C1-C 20 alkyl, wherein the C1-C 20 alkyl is optionally substituted with 1 to 6 R a s, R 2 is C1-C 20 alkyl, wherein the C1-C 20 alkyl is optionally substituted with 1 to 6 R a s, wherein each R a when each occurs independently, is selected from halogen, such as F, and C1-C 20 haloalkyl, such as C1-C 20 fluoroalkyl, such as -CF3.

[0143] In any of the embodiments of the process disclosed herein, at least one of R 1 and R 2 is C1-C 20 fluoroalkyl, such as C1-C6 fluoroalkyl.

[0144] In any of the embodiments of the process disclosed herein, R 1 is selected from hydrogen, and C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted with 1 to 6 Fs, and R 2 is C1-C6 alkyl or phenyl.

[0145] In any of the embodiments of the processes disclosed herein, R 1 is selected from hydrogen and trifluoromethyl, and R2 is methyl or phenyl.

[0146] In any of the embodiments of the processes disclosed herein, R 3 and R 4 are each independently selected from H and C1-C 20 alkyl, where the C1-C 20 alkyl is optionally substituted with 1 to 6 R a ; each R a is independently selected, when each occurs, from C3-C 10 cycloalkyl, C6-C 14 aryl, 3- to 14-membered heterocyclyl, 5- to 20-membered heteroaryl, halogen such as F, haloalkyl such as C1-C 20 haloalkyl such as -CF3, -OR c , and -NR c R c ; each R c is independently selected, when each occurs, from H, C1-C 20 alkyl, C2-C 20 alkenyl, and C2-C 20 alkynyl.

[0147] In any of the embodiments of the processes disclosed herein, R 3 and R 4 are each independently selected from H and C1-C 20 alkyl, where the C1-C 20 alkyl is optionally substituted with 1 to 6 R a ; each R a is independently selected, when each occurs, from halogen such as F, C1-C 20 haloalkyl such as -CF3, -OR c , and -NR c R c ; each R cWhen each occurs independently, H, C1-C 20 alkyl, C2-C 20 alkenyl, and C2-C 20 alkynyl are selected from.

[0148] In any of the embodiments of the processes disclosed herein, R 3 and R 4 each independently is selected from H and C1-C 20 alkyl, where C1-C 20 alkyl is optionally substituted with 1 to 6 R a ; each R a when each occurs independently, is halogen, such as F, C1-C 20 haloalkyl, such as -CF3, -OR c , and -NR c R c is selected from; each R c when each occurs independently, is selected from H, and C1-C 20 alkyl.

[0149] In any of the embodiments of the processes disclosed herein, the substrate is a ketone.

[0150] In any of the embodiments of the processes disclosed herein, the nitroalkane substrate is nitromethane or nitroethane.

[0151] In any of the embodiments of the processes disclosed herein, the ketone substrate is

Chemical formula

[0152] This disclosure also provides a process for asymmetric synthesis of (S)-1,1,1-trifluoro-2-methyl-3-nitropropan-2-ol (IA):

Chemical formula

[0153] As disclosed herein and exemplified in the examples, the present disclosure contemplates a range of suitable reaction conditions that can be used in the processes herein, including but not limited to pH, temperature, buffer, solvent system, substrate loading, product stereoisomers, e.g., mixtures of enantiomers, polypeptide loading, cofactor loading, pressure, and reaction time. Further suitable reaction conditions for carrying out the method of enzymatically converting a substrate compound to a product compound using the modified oxynitrilase polypeptides described herein can be readily optimized by routine experimentation, which includes contacting the modified oxynitrilase polypeptide with the substrate compound under experimental reaction conditions of various concentrations, pH, temperature, and solvent conditions, and detecting the product compound using, for example, the methods described in the examples provided herein, but is not limited thereto.

[0154] As described above, the modified polypeptides having oxynitrilase activity for use in the processes of the present disclosure generally have at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a reference amino acid sequence selected from any one of the even-numbered sequences of SEQ ID NOs: 4 to 604, and 608 to 640.

[0155] The substrate compound in the reaction mixture can be varied, for example, considering the amount of the desired product compound, the effect of substrate concentration on enzyme activity, the stability of the enzyme under the reaction conditions, and the conversion rate of the substrate to the product. In any of the embodiments of the processes disclosed herein, suitable reaction conditions include an aldehyde or ketone substrate, such as a loading amount of formula (III), of at least about 0.5 to about 200 g / L, about 1 to about 200 g / L, about 5 to about 150 g / L, about 10 to about 150 g / L, or about 50 to about 150 g / L. In one embodiment, suitable reaction conditions include an aldehyde or ketone substrate, such as a loading amount of formula (III), of at least about 0.5 g / L, at least about 1 g / L, at least about 5 g / L, at least about 10 g / L, at least about 15 g / L, at least about 20 g / L, at least about 30 g / L, at least about 50 g / L, at least about 75 g / L, at least about 100 g / L or more. The values of the substrate loading amounts provided herein are based on the molecular weight of the aldehyde or ketone substrate, such as formula (III), although equimolar amounts of various hydrates and salts of the aldehyde or ketone substrate, such as formula (III), are also contemplated for use in this process.

[0156] In any of the embodiments of the processes disclosed herein, the modified oxynitrilase polypeptide forms a β-nitroalcohol product compound using an aldehyde or ketone substrate and a nitroalkane compound. In one embodiment, suitable reaction conditions include a nitroalkane present at a loading amount of at least about 1-fold the molar loading amount of the aldehyde or ketone substrate, such as formula (III). In one embodiment, the nitroalkane is present at a loading amount of 2, 3, 4, 5, 6, 7, 8, 9, or 10 times the molar loading amount of the aldehyde or ketone substrate, such as formula (III).

[0157] In any of the embodiments of the processes disclosed herein, suitable reaction conditions include nitroalkanes present at a loading amount of the nitroalkane substrate of 0.5 to about 200 g / L, about 1 to about 200 g / L, about 5 to about 150 g / L, about 10 to about 150 g / L, or about 50 to about 150 g / L. In one embodiment, suitable reaction conditions include a loading amount of the nitroalkane substrate of at least about 0.5 g / L, at least about 1 g / L, at least about 5 g / L, at least about 10 g / L, at least about 15 g / L, at least about 20 g / L, at least about 30 g / L, at least about 50 g / L, at least about 75 g / L, at least about 100 g / L or more.

[0158] In any of the embodiments of the processes disclosed herein, the reaction conditions can include a suitable pH. As noted above, the desired pH or desired pH range can be maintained using an acid or base, a suitable buffer, or a combination of a buffer and an acid or base to be added. The pH of the reaction mixture can be controlled before and / or during the reaction. In some embodiments, suitable reaction conditions include a solution pH of about 4 to about 8, a pH of about 5 to about 7, a pH of about 6 to about 7. In some embodiments, the reaction conditions include a solution pH of about 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8.

[0159] In any of the embodiments of the processes disclosed herein, for example, considering the increase in reaction rate at higher temperatures and the activity of the enzyme over a sufficient reaction time, a temperature suitable for the reaction conditions can be used. Thus, in some embodiments, suitable reaction conditions include a temperature of about 10°C to about 60°C, about 25°C to about 50°C, about 25°C to about 40°C, about 25°C to about 30°C, or about 10°C to about 30°C. In some embodiments, suitable reaction temperatures include about 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, or 60°C. In some embodiments, the temperature during the enzyme reaction can be maintained at a specific temperature throughout the reaction. In some embodiments, the temperature during the enzyme reaction can be adjusted along a temperature profile during the reaction process.

[0160] Processes using modified oxynitrilase are generally carried out in a solvent. However, the process can also be carried out in the absence of a solvent.

[0161] Suitable solvents include water, aqueous buffers, organic solvents, and / or cosolvent systems (generally including aqueous and organic solvents). The organic solvent can be any organic solvent, preferably one that does not interfere with or inhibit the enzyme reaction. In certain embodiments, the organic solvent is water-miscible or partially water-miscible. The organic solvent is particularly an aprotic organic solvent.

[0162] The aqueous solution (water or an aqueous cosolvent system) can be pH-buffered or unbuffered. In some embodiments, processes using the modified oxynitrilase polypeptide are generally carried out in a solvent system containing an organic solvent. In some embodiments, processes using the modified oxynitrilase polypeptide are generally carried out in an aqueous cosolvent system containing an organic solvent.

[0163] In any of the embodiments of the processes disclosed herein, the organic solvent is selected from methanol, ethanol, propanol, isopropanol, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), isopropyl acetate, ethyl acetate, butyl acetate, 1-octanol, hexane, heptane, octane, methyl tert-butyl ether (MTBE), toluene, benzene, glycerol, polyethylene glycol, and ionic liquids such as 1-ethyl-4-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium hexafluorophosphate.

[0164] The organic solvent component of the aqueous cosolvent system may be miscible with the aqueous component and provide a single liquid phase, or it may be partially miscible or immiscible with the aqueous component and provide two liquid phases. Exemplary aqueous cosolvent systems include water and one or more organic solvents as defined herein. Generally, the organic solvent component of the aqueous cosolvent system is selected so as not to completely inactivate the oxynitrilase. Suitable cosolvent systems can be readily identified by measuring the enzyme activity of a particular modified oxynitrilase with a substrate of a predetermined purpose in a candidate solvent system using an enzyme activity assay such as those described herein. In any of the embodiments of the processes disclosed herein, suitable reaction conditions include an aqueous cosolvent system containing isopropyl acetate at a concentration of about 1% to about 60% (v / v), about 1% to about 50% (v / v), about 1% to about 40% (v / v), about 2% to about 40% (v / v), about 5% to about 40% (v / v), about 10% to about 40% (v / v), from about 10% to about 30% (v / v), or about 10% to about 20% (v / v). In some embodiments of the present process, suitable reaction conditions include an aqueous cosolvent system containing isopropyl acetate at a concentration of at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% (v / v).

[0165] Suitable reaction conditions can include a combination of reaction parameters that provide for the biocatalytic conversion of a substrate compound to its corresponding product compound. Thus, in some embodiments of the present process, the combination of reaction parameters includes: (a) the substrate loading, e.g., the loading of 1,1,1-trifluoropropan-2-one of about 5 g / L to about 150 g / L; (b) the loading of the nitroalkane, e.g., the loading of nitromethane is about twice the molar amount of the substrate, e.g., 1,1,1-trifluoropropan-2-one; (c) a modified polypeptide concentration of at least about 3 g / L; (d) an aqueous isopropyl acetate concentration of about 1% (v / v) to about 60% (v / v); (e) a pH of about 4.0 to 8.0; and (f) a temperature of about 10°C to 30°C.

[0166] Exemplary reaction conditions include the assay conditions provided in the Examples section.

[0167] In the practice of the reactions described herein, the modified oxynitrilase polypeptide biocatalyst can be added to the reaction mixture in different formulation forms as frozen or lyophilized whole cells (FWC or LWC) transformed with the gene encoding the modified oxynitrilase polypeptide and / or as cell lysates or lyophilized cell lysates of such cells, so-called shake flask powders (SFP) (where cell debris has been removed and / or further purified as fermentation powders (FP)). Whole cells or cell extracts transformed with the gene encoding the modified oxynitrilase, lysates thereof, and isolated enzymes can be used in a wide variety of forms including solids (e.g., lyophilized, spray dried, etc.) or semi-solids (e.g., crude paste). Cell extracts or cell lysates can be partially purified by precipitation (e.g., ammonium sulfate, polyethyleneimine, heat treatment, etc.) followed by a desalting procedure (e.g., ultrafiltration, dialysis, etc.) prior to lyophilization. Any enzyme preparation can be stabilized by crosslinking with a known crosslinking agent such as glutaraldehyde or immobilization on a solid phase material (such as a resin).

[0168] In any of the embodiments of the processes disclosed herein, the reaction is carried out under suitable reaction conditions as described herein, and the modified oxynitrilase polypeptide is immobilized on a solid support such as a membrane, resin, solid support, or other solid phase material. The solid support can be composed of organic polymers such as polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy, polymethacrylate, and polyacrylamide, as well as copolymers and grafts thereof. The solid support can also be an inorganic material such as glass, silica, controlled pore glass (CPG), reversed-phase silica, or a metal such as gold or platinum. The form of the solid support can be in the form of beads, spheres, particles, granules, gels, membranes, or surfaces. The surface can be planar, substantially planar, or non-planar. The solid support can be porous or non-porous and can have swelling or non-swelling properties. The solid support can be configured in the form of wells, depressions, or other containers, vessels, functions, or positions. Examples of solid supports useful for immobilizing the modified oxynitrilase enzyme to carry out the reaction include, but are not limited to, beads or resins such as polymethacrylate, such as polymethacrylate having an epoxy functional group, polymethacrylate having an amino epoxy functional group, polymethacrylate, styrene / DVB copolymer, or polymethacrylate having an octadecyl functional group.

[0169] In certain embodiments, the solid support is beads or resin comprising polymethacrylate.

[0170] Exemplary solid supports include, but are not limited to, chitosan beads, Eupergit C, IB-150, IB-350, IB-C435, IB-A369, IB-A161, IB-A171, IBS500, IB-S861, SEPABEADS (Mitsubishi), for example, Sepabeads EC-EP, Sepabeads EC-HFA, Sepabeads EC-HG, Sepabeads EC-BU, Sepabeads EC-OD, Sepabeads EC-CM, Sepabeads EC-IDA, Sepabeads EC-EA, Sepabeads EC-HA, Sepabeads EC-QA, Sepabeads EXE, Sepabeads EXA, Dilbeads-TA, Amberzyme Oxirane, Amberlite XAD-7HP, Amberlite FPA98Cl, Amberlite IRA958Cl, Amberlite IRA67, Amberlite FPA90Cl, Amberlite FPA40Cl, Amberlite XAD18, Accurel EP100, ECR8206F / 5730, ECR8206 / 5803, ECR8206M / 5749, ReliZyme EP403, ReliZyme EP113, Lewatit VP OC 1600, Diaion WA20, Diaion WA21J, Diaion WA30, Dowex 66, Diaion HPA-25L, Lewatit VP OC 1064 MD PH, Lewatit VP OC 1163, Lifetech ECR8304F, Lifetech ECR8309F, Lifetech ECR8315F, Lifetech ECR8204F, Lifetech ECR8285, Lifetech ECR1090M, Lifetech ECR1030M, Lifetech ECR8806M, Chromalite (MAM2 / F) D6591, Chromalite MIDA / M, Chromalite MIDA / M / Fe, Chromalite MIDA / M / Co, Chromalite MIDA / M / Ni, Chromalite MIDA / M / Cu, and Chromalite MIDA / M / Zn.

[0171] In any of the embodiments of the processes disclosed herein in which the modified polypeptide is expressed in the form of a secreted polypeptide, a culture medium containing the secreted polypeptide can be used in the processes herein.

[0172] In any of the embodiments of the processes disclosed herein, solid reactants (e.g., enzymes, salts, etc.) can be provided to the reactants in various forms, including powders (e.g., lyophilized, spray-dried, etc.), solutions, emulsions, suspensions, etc. The reactants can be readily lyophilized or spray-dried using methods and equipment known to those skilled in the art. For example, a protein solution can be aliquoted, frozen at -80 °C, then placed into a pre-cooled lyophilization chamber, and thereafter, a vacuum can be applied.

[0173] In any of the embodiments of the processes disclosed herein, the order of addition of the reactants is not critical. The reactants can be added simultaneously together to a solvent (e.g., a single-phase solvent, a two-phase aqueous co-solvent system, etc.), or some of the reactants can be added separately, or some can be added together at different times. For example, oxynitrilase and the substrate can be added to the solvent first. When using an aqueous co-solvent system, to improve the mixing efficiency, oxynitrilase can be added to the aqueous phase first and mixed. Then, an organic phase, such as isopropyl acetate, can be added and mixed, and thereafter, the substrate can be added. Alternatively, the substrate can be premixed in the organic phase and then before adding to the aqueous phase.

[0174] The method of performing an enzymatic reaction may further include a step of isolating the product of the enzymatic reaction. In particular, this step is carried out after completion of the enzymatic reaction. The product is separated, in particular, from one or more of the reaction mixture, in particular from substantially all other components. For example, the product is separated from the remaining substrate, by-products, enzyme, and / or organic solvent. Isolation of the product can be achieved by means and techniques known in the art, including, for example, evaporation of the solvent, aggregation or crystallization, and filtration, phase separation, chromatographic separation, etc.

[0175] The modified oxynitrilase polypeptides that can be used in any of the embodiments of the processes disclosed herein have the sequences of SEQ ID NO: 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, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498,It can include one or more sequences selected from amino acid sequences corresponding to 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, or 640.

[0176] In any of the embodiments of the processes disclosed herein, the β-nitroalcohol product, for example of formula (IA), is obtained with an enantiomeric excess (e.e.) of at least 55% ee, for example at least 65% ee, for example at least 75%, for example at least 80%, for example at least 90% ee, for example at least 95% ee, for example at least 99%.

[0177] In any of the embodiments of the processes disclosed herein, the β-nitroalcohol compound, for example of formula (IA), is obtained with a conversion rate of at least 10%, for example at least 20%, for example at least 30%, for example at least 40%, for example at least 50%, more preferably at least 75%, at least 85%, at least 90%, for example 95%.

[0178] The present disclosure also relates to (S)-3-amino-1,1,1-trifluoro-2-methylpropan-2-ol of formula (IB):

Chemical formula

[0179] The hydrogen source can be selected from gaseous hydrogen (H2), a hydrogen donor (transfer hydrogenation, e.g., formic acid or its salt), a hydride reagent (LiAlH4), and the like.

[0180] Suitable hydrogenation reaction conditions include the presence of hydrogen gas, a transition metal catalyst, and a suitable solvent. In one embodiment, the transition metal catalyst is selected from sponge metals such as sponge nickel, sponge cobalt, Pd / C, Pt / C, Pt2, Rh / Al2O3, and Pd / BaSO4. In particular, the catalyst is sponge nickel. In one embodiment, the transition metal catalyst is present in a loading amount of at least 2 wt%, such as at least 5 wt%, at least 10 wt%. The sponge metal or Raney metal catalyst that can be used in the reaction contains, as a promoter, 0.1 to 10 wt%, such as about 0.5 to 3 wt% of molybdenum based on the weight of the catalyst. Sponge nickel promoted with molybdenum is a preferred catalyst, but sponge cobalt or Raney cobalt is also sufficiently suitable. These catalysts can be used in the reaction medium in a loading amount of at least 2 wt%, such as at least 5 wt%, at least 10 wt%. In one embodiment, the transition metal catalyst is a metal-promoted sponge nickel, such as molybdenum-promoted sponge nickel. In one embodiment, the transition metal catalyst is unpromoted sponge nickel. In one embodiment, the solvent is selected from water, ethanol, methanol, n-propanol, isopropanol, ethyl acetate, isopropyl acetate, butyl acetate, tert-butyl methyl ether, and tetrahydrofuran. In one embodiment, the reaction is carried out at about 20 to 60 °C, such as 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C. In one embodiment, the reaction is carried out at a pressure of about 0.1 to 40 bar. In one embodiment, the compound of formula (IA) is present at a concentration of at least 2 wt%, such as 2, 4, 8, 10 wt%. In one embodiment, the hydrogenation reaction is carried out in a batch or flow mode.

[0181] In a further embodiment, the above process for manufacturing (IB) further comprises the step of converting the compound of formula (IB) into an acid salt. In a further embodiment, the process comprises an additional step of crystallizing the acid salt. Preferably, the acid salt is the HCl salt. The crystallization step increases the enantiomeric purity of compound (IB). Advantageously, compound (IB) having an ee of about 92% before isolation increased to about 98% after isolation of the crystalline compound (IB) HCl salt.

[0182] The present disclosure also provides a process for synthesizing (S)-3-amino-6-methoxy-N-(3,3,3-trifluoro-2-hydroxy-2-methylpropyl)-5-(trifluoromethyl)picolinamide of formula (IC)

Chemical formula

[0183] In certain embodiments, the process further comprises contacting the compound of (IA) with hydrogen under suitable hydrogenation conditions to obtain (S)-3-amino-1,1,1-trifluoro-2-methylpropan-2-ol (IB). In a further embodiment, the process further comprises the step of converting the compound of formula (IB) into an acid salt. Preferably, the acid salt is the HCl salt (compound (IB)·HCl). In a further embodiment, the process comprises an additional step of crystallizing the acid salt.

[0184] The additional steps necessary to synthesize the compound of formula (IC) from formula (IB) or (IB). HCl can be used according to the disclosure of European Patent No. 3555048 B1, for example, according to Scheme 5 and

[0028] and the examples in that specification.

[0185] Alternatively, this process may further comprise coupling a compound of formula (IB)·HCl or crystalline (IB)·HCl with a compound of formula (E6) in the presence of a coupling reagent under suitable reaction conditions to

Chem.

[0186] Suitable reaction conditions can be those commonly used in the amidation of carboxylic acids, as known in the art. These include, for example, in the presence of a suitable base such as Et3N, DIEA, DMAP or pyridine, in a suitable solvent such as THF, DCM or toluene, at a suitable temperature such as 0 °C to 120 °C, with a suitable coupling reagent such as tetramethyl orthosilicate (TMOS), N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC), dicyclohexylcarbodiimide (DCC), 1,1’-carbonyldiimidazole (CDI). In certain embodiments, the coupling reagent is tetramethyl orthosilicate. In certain embodiments, suitable reaction conditions include Et3N, toluene, and heating the reaction to about 110 °C.

[0187] The complete procedure for the synthesis of compound (IC) is described in Scheme 2 below. Scheme 2

Chem.

[0188] In a further embodiment, a process is provided for preparing a compound (IC) of formula (I)

Chem.

[0189] The various features and embodiments of the present disclosure are illustrated in the following representative examples, which are for illustrative purposes only and are not intended to be limiting.

Example

[0190] The following examples (including experiments and achieved results) are provided for illustrative purposes only and should not be construed as limiting the invention.

[0191] In the following examples, the following abbreviations are applied: ppm (parts per million); M (mole); mM (millimole); uM and μM (micromole); nM (nanomole); mol (mole); gm and g (gram); mg (milligram); ug and μg (microgram); L and l (liter); ml and mL (milliliter); cm (centimeter); mm (millimeter); um and μM (micrometer); sec. (second); min(s) (minute); h(s) and hr(s) (hour); U (unit); MW (molecular weight); rpm (revolutions per minute); psi and PSI (pound per square inch); °C (Celsius temperature); RT and rt (room temperature); OD600 (optical density at 600 nm); CAM and cam (chloramphenicol); IPAc (isopropyl acetate), DMSO (dimethyl sulfoxide); FP (fermentation powder); FWC (frozen whole cell), LWC (lyophilized whole cell); PMBS (polymyxin B sulfate); IPTG (isopropyl β-D-1-thiogalactopyranoside); LB (lysogeny broth); NM (nitromethane), MeOH (methanol), MTBE (methyl-tert-butyl ether), TB (terrific broth; 12 g / L bacto-tryptone, 24 g / L yeast extract 4 mL / L glycerol, 65 mM potassium phosphate, pH 7.0, 1 mM MgSO4); PLP (5'-pyridoxal phosphate), TEoA (triethanolamine buffer), HEPES zwitterionic buffer; 4-(2-hydroxyethyl)-piperazineethanesulfonic acid); SFP (shaken flask powder); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); E. coli W3110 (a commonly used laboratory E. coli strain, available from Coli Genetic Stock Center [CGSC], New Haven, CT); HTP (high throughput); HPLC (high performance liquid chromatography); GC (gas chromatography), MS (mass spectrometer), RF (Rapid Fire), FIOP (improvement rate relative to positive control); Microfluidics (Microfluidics, Corp., Westwood, MA); Sigma-Aldrich (Sigma-Aldrich, St.Louis, MO); Difco (Difco Laboratories, BD Diagnostic Systems, Detroit, MI); Agilent (Agilent Technologies, Inc., Santa Clara, CA); Corning (Corning, Inc., Palo Alto, CA); Dow Corning (Dow Corning, Corp., Midland, MI); and Gene Oracle (Gene Oracle, Inc., Mountain View, CA).

[0192] Throughout the examples, “T100%conv” corresponds to the conversion calculated for each single sample expressed as a percentage with respect to the theoretical 100% conversion value. The amount of nitroaldol product generated in each single sample was quantified by HPLC using an external standard of nitroaldol product of known concentration. The conversion rate of each single sample was obtained by dividing the amount calculated for the generated nitroaldol product by the theoretical amount of nitroaldol product if 100% conversion was achieved, then multiplying the resulting value by 100 to express the result of the conversion rate as a percentage.

[0193] Example 1 Production of the Modified Polypeptide in pCK110900 The polynucleotide (SEQ ID NO: 1) encoding the polypeptide (SEQ ID NO: 2) having oxynitrilase activity was cloned into the pCK110900 vector system (see, for example, FIG. 3 of U.S. Patent Application Publication App. No. 2006 / 0195947A1, which is incorporated herein by reference in its entirety), and then expressed in E. coli W3110fhuA under the control of the lac promoter. The expression vector also contained the P15a origin of replication and the chloramphenicol (CAM) resistance gene. This polynucleotide and the related polypeptide were the product of six rounds of directed evolution starting from (S)-hydroxynitrile lyase found in Baliospermum montanum (UniProt D1MX73).

[0194] Example 2 Preparation of Cell Pellets E. coli W3110 fhuA cells were transformed with the pCK110900 plasmid containing the gene encoding oxynitrilase. The transformed cells were plated on a lysogeny broth (LB) agar plate containing 1% glucose and 30 μg / mL CAM and grown overnight at 37°C. Subsequently, single colonies were picked into a 96-well format and grown in 190 μL of LB medium containing 1% glucose and 30 μg / mL CAM at 30°C, 200 rpm, and 85% humidity. After overnight growth, 20 μL of the grown culture was transferred to a deep-well plate containing 380 μL of terrific broth (TB) medium containing 30 μg / mL of CAM. The culture was grown at 30°C, 250 rpm, and 85% humidity for approximately 2.5 hours. When the optical density (OD 600 ) of the culture reached 0.4 - 0.8, the expression of the oxynitrilase gene was induced by adding isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 1 mM. After induction, growth was continued at 30°C, 250 rpm, and 85% humidity for 18 - 20 hours. The cells were harvested by centrifugation at 4,000 rpm and 4°C for 10 minutes, and then the supernatant was discarded. The cell pellet was stored at -80°C until ready for use.

[0195] Example 3 Preparation of Lysates and Clarified Lysates Before performing the assay, the cell pellet was thawed and resuspended in 300 μL of lysis buffer (containing 1 g / L lysozyme, 0.5 g / L PMBS, and 0.1 μL / mL or 0.2 U / ml of commercially available DNAse (New England BioLabs, M0303L) in 0.1 M citrate buffer, pH 6.0). The plate was shaken and stirred at medium speed for 2.5 hours at room temperature using a microtiter plate shaker. The plate was then centrifuged at 4,000 rpm for 10 minutes at 4°C, and the clarified supernatant was used for the HTP assay reaction for activity determination as described in the following examples.

[0196] Example 4 Preparation of Frozen Whole Cells (FWC), Lyophilized Whole Cells (LWC), Shake Flask Powder (SFP), and Fermentation Powder (FP) Using the shake flask procedure, a modified oxynitrilase polypeptide shake flask powder (SFP) can be produced, which is useful for use in secondary screening assays and / or the biocatalytic processes described herein. The shake flask powder preparation of the enzyme provides a more concentrated preparation of the modified enzyme compared to the cell lysate used in the HTP assay. To initiate the culture, a single colony from the plate or a glycerol stock of E. coli containing the plasmid encoding the modified polypeptide of interest was inoculated into 25 mL of LB supplemented with 30 μg / mL of CAM and 1% glucose in a 250 ml baffled shake flask. The culture was grown overnight (16 - 20 hours and OD 600 > 3.8) with shaking at 250 rpm in a 37°C incubator. A 1 L shake flask containing 250 mL of TB medium containing 30 μg / mL of CAM was inoculated with 5 mL of the overnight grown culture. The 250 mL culture was incubated at 30°C, 250 rpm, OD 600The mixture was incubated for 3 - 3.5 hours until it reached 0.6 - 0.8. The expression of the oxynitrilase gene was induced by adding IPTG to a final concentration of 1 mM, and the growth was continued for an additional 18 - 20 hours. The cells were harvested by transferring the culture into centrifuge bottles and then centrifuged at 7,000 rpm for 5 minutes at 4°C. The supernatant was discarded, and the remaining cell pellet was either lysed or, in some embodiments, stored at -80°C as frozen whole cells (FWC) until ready for use. For lysis, the cell pellet was resuspended in 30 mL of 50 mM citrate buffer at pH 6.0 and lysed using a LM20 MICROFLUIDIZER® processor system (Microfluidics). Cell debris was removed by centrifugation at 14,000 rpm for 30 minutes at 4°C. The clarified lysate was recovered, frozen at -80°C, and then lyophilized using standard methods known in the art. Lyophilization of the frozen clarified lysate yielded a dried shake flask powder (SFP), and lyophilization of the FWC yielded a dried lyophilized whole cell (LWC), both of which contained the crude modified oxynitrilase polypeptide. To obtain larger amounts of enzyme required for large-scale reactions as in Example 12, the cells were fermented in a bioreactor using standard methods known in the art. Compared to the SFP, this fermented powder (FP) contained a higher concentration of the modified oxynitrilase polypeptide due to its lower salt content.

[0197] Example 5 Analytical Methods for Activity and Selectivity Evaluation The improvement of the activity of the modified oxynitrilase was analyzed by high performance liquid chromatography (HPLC) using the method described in Table 5.1, or by RapidFire® mass spectrometry (RF-MS) using the method described in Table 5.2. To analyze the conversion of compounds (1) and (2) to compound (IA), an HPLC method using UV detection was developed. Only the nitromethane substrate (2) and the nitroalcohol product (IA) could be detected, and (IA) could not be distinguished from the (R)-enantiomer of (IA). In Examples 9, 10, and 11, RF-MS was used for the rapid quantification of the product (IA). The corresponding method parameters are described in Table 5.2.

[0198]

Table 2

[0199]

Table 3

[0200] The selectivity of the selected samples for the desired (S)-(IA) or the undesired (R)-(IA) was analyzed by gas chromatography (GC) using the method described in Table 5.3.

[0201]

Table 4

[0202] In Example 20, LC-MS was used to detect the products 1,1,1-trifluoro-2-methyl-3-nitrobutan-2-ol (4) and 1,1,1-trifluoro-3-nitro-2-phenylpropan-2-ol (8). The method parameters are described in Table 5.4.

[0203]

Table 5

[0204] In Example 20, the product 1-nitro-2-phenylpropan-2-ol (6) was detected using GC-MS / FID. The method parameters are described in Table 5.5.

[0205]

Table 6

[0206] The methods provided herein are used for the analysis of variants generated using the present invention. However, since there are other suitable methods known in the art that are applicable to the analysis of variants provided herein and / or generated using the methods provided herein, the present invention is not intended to be limited to the methods described herein.

[0207] Example 6 Round 7 Evolution and Screening of Modified Polypeptides Derived from SEQ ID NO: 2 to Improve the Production of Compound (IA) Modified polynucleotides (SEQ ID NO: 1) encoding polypeptides having oxynitrilase activity of SEQ ID NO: 2 were used to generate the modified polypeptides of Table 6-1. These polypeptides showed an improvement in oxynitrilase activity under desired conditions, for example, an improvement in the formation of nitroalcohol compounds (IA) generated in situ from the substrates trifluoroacetone and nitromethane (compounds (1) and (2), respectively), as compared to the starting polypeptide. Some polypeptides showing an improvement in product formation of the nitroalcohol product (IA) as compared to the starting polypeptide are shown in Table 6-1. Modified polypeptides having the amino acid sequences of even-numbered sequence identifiers were generated from the "backbone" amino acid sequence of SEQ ID NO: 2, together with the analysis methods described in Table 5-1, as described below.

[0208] Directed evolution was initiated with the polynucleotide set forth in SEQ ID NO: 1. A library of modified polypeptides was generated using various well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences), and screened using the HTP assay and analysis methods described below to measure the ability of the polypeptides to produce compound (IA).

[0209] The enzyme assay was performed in 96-well deep-well (total volume 1.1 mL) plates with a total reaction volume of 100 μL per well. The reaction mixture contained 91 v / v% undiluted oxynitrilase lysate prepared as described in Example 3, 0.6 M trifluoroacetone (compound (1)), 0.6 M nitromethane (compound (2)), and 0.1 M citrate buffer (pH 5.5). The reaction plates were heat-sealed and shaken at 150 rpm and 22 °C.

[0210] After incubation overnight (about 22 h), 300 μL / well of MTBE was added to the reaction plates. The quenched reaction plates were sealed, mixed well, and centrifuged at 4,000 rpm for 5 min. Then, a 150 μL aliquot of the upper organic phase was removed from each well and added to a shallow-well 96-well plate. For selectivity determination, the plates were sealed and analyzed by chiral GC using the analytical method described in Table 5-3. For activity analysis by HPLC, 20 μL / well of the upper organic phase of the quenched reaction plates was removed and added to a shallow-well 96-well plate containing 180 μL of MeOH. The plates were sealed and mixed well. These samples were then analyzed by HPLC to determine the activity of the enzyme variants using the analytical method described in Table 5-1. The selected oxynitrilase variants that showed faster product formation of (IA) compared to SEQ ID NO: 2 are shown in Table 6.1.

[0211]

Table 7

[0212] Example 7 Round 8 Evolution and Screening of Modified Polypeptides Derived from SEQ ID NO: 48 to Improve the Production of Compound (IA) Using the polynucleotide from SEQ ID NO: 47 of Example 6 that encodes the most active polypeptide having the oxynitrilase activity of SEQ ID NO: 48, the modified polypeptides in Table 7-1 were generated. These polypeptides showed an improvement in oxynitrilase activity under desired conditions, for example, an improvement in the formation of the nitroalcohol compound (IA) generated in situ from the substrate trifluoroacetone and nitromethane (compounds (1) and (2), respectively), as compared to the starting polypeptide. Table 7-1 shows several polypeptides that showed an improvement in the product formation of the nitroalcohol product (IA) as compared to the starting polypeptide. Modified polypeptides having the amino acid sequences of even-numbered sequence identifiers were generated from the "skeleton" amino acid sequence of SEQ ID NO: 48, together with the analytical methods described in Table 5-1, as described below.

[0213] Directed evolution was initiated with the polynucleotide described in SEQ ID NO: 47. A library of modified polypeptides was prepared using various well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences), and screened using the HTP assay and analytical methods described below that measure the ability of the polypeptide to produce compound (IA).

[0214] The enzyme assay was performed in a 96-well deep well (total volume 1.1 mL) plate with a total reaction volume of 100 μL per well. The reaction mixture contained 20 v / v% of the undiluted oxynitrilase lysate prepared as described in Example 3, 0.62 M trifluoroacetone (compound (1)), 0.63 M nitromethane (compound (2)), and citrate buffer (pH 5.5). The reaction plate was heat-sealed and shaken at 150 rpm and 22 °C.

[0215] After incubating overnight (about 22 hours), 600 μL / well of MeOH was added to the reaction plate. The quenched reaction plate was sealed, mixed well, and centrifuged at 4,000 rpm for 15 minutes. Subsequently, 20 μL / well of the quenched reaction plate was taken out and added to a 96-well plate with shallow wells containing 180 μL of MeOH. The plate was sealed and mixed thoroughly. Then, these samples were analyzed by HPLC to determine the activity of the enzyme variants using the analytical method described in Table 5-1. The selected oxynitrilase variants showing faster product formation of (IA) compared to SEQ ID NO: 48 are shown in Table 7.1. After confirming the selectivity of the most active polypeptide SEQ ID NO: 74 for the (IA) product by chiral GC analysis using the analytical method described in Table 5-3, the evolution method was continued as described in Example 8. All variants showed >75% S-selectivity (>50% ee) under the given reaction conditions.

[0216]

Table 8

[0217]

Table 9

[0218] Example 8 Round 9 Evolution and Screening of Modified Polypeptides Derived from SEQ ID NO: 74 to Improve the Production of Compound (IA) Using the polynucleotide from SEQ ID NO: 73 of Example 7 that encodes the most active polypeptide having the oxynitrilase activity of SEQ ID NO: 74, the modified polypeptides in Table 8-1 were generated. These polypeptides showed an improvement in oxynitrilase activity under desired conditions, for example, an improvement in the formation of the nitroalcohol compound (IA) generated in situ from the substrate trifluoroacetone and nitromethane (compounds (1) and (2), respectively), compared to the starting polypeptide. Table 8-1 shows several polypeptides that showed an improvement in product formation of the nitroalcohol product (IA) compared to the starting polypeptide. Modified polypeptides having the amino acid sequences of even-numbered sequence identifiers were generated from the "scaffold" amino acid sequence of SEQ ID NO: 74, together with the analytical methods described in Table 8-1, as described below.

[0219] Directed evolution was initiated with the polynucleotide described in SEQ ID NO: 73. A library of modified polypeptides was prepared using various well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using the HTP assay and analytical methods described below to measure the ability of the polypeptides to produce compound (IA).

[0220] The enzyme assay was performed in a 96-well deep well (total volume 1.1 mL) plate with a total reaction volume of 100 μL per well. The reaction mixture contained 15 v / v% of the undiluted oxynitrilase lysate prepared as described in Example 3, 0.62 M trifluoroacetone (compound (1)), 0.63 M nitromethane (compound (2)), and citrate buffer (pH 6.0). The reaction plate was heat-sealed and shaken at 150 rpm and 22 °C.

[0221] After incubating overnight (about 22 hours), 300 μL / well of MeOH was added to the reaction plate. The quenched reaction plate was sealed, mixed well, and centrifuged at 4,000 rpm for 15 minutes. Subsequently, 20 μL / well of the quenched reaction plate was taken out and added to a 96-well plate with shallow wells containing 180 μL of MeOH. The plate was sealed and mixed thoroughly. Then, these samples were analyzed by HPLC to determine the activity of the enzyme variant using the analytical method described in Table 5-1. The selected oxynitrilase variants showing faster product formation of (IA) compared to SEQ ID NO: 74 are shown in Table 8.1. Note that the oxynitrilase polypeptide having SEQ ID NO: 142 is rationally constructed. It combines the beneficial mutations of polypeptide SEQ ID NO: 124 and SEQ ID NO: 140. After confirming the high selectivity for the (IA) product of the most active polypeptide SEQ ID NO: 124, 140, and 142 by chiral GC analysis using the analytical method described in Table 5-3, the evolution method was continued as described in Example 9. All variants showed >80% S-selectivity (>60% ee) under the given reaction conditions.

[0222]

Table 10

[0223]

Table 11

[0224] Example 9 Round 10 Evolution and Screening of Modified Polypeptides Derived from SEQ ID NO: 142 to Improve the Production of Compound (IA) Using the polynucleotide from SEQ ID NO: 141 of Example 8 that encodes the most active polypeptide having oxynitrilase activity of SEQ ID NO: 142, the modified polypeptides of Table 9-1 were generated. These polypeptides showed an improvement in oxynitrilase activity under desired conditions, for example, an improvement in the formation of the nitroalcohol compound (IA) generated in situ from the substrate trifluoroacetone and nitromethane (compounds (1) and (2), respectively), compared to the starting polypeptide. Some polypeptides that showed an improvement in product formation of the nitroalcohol product (IA) compared to the starting polypeptide are shown in Table 8-1. Modified polypeptides having the amino acid sequences of even-numbered sequence identifiers were generated from the "scaffold" amino acid sequence of SEQ ID NO: 74, together with the analytical methods described in Table 8-1, as described below.

[0225] Directed evolution was initiated with the polynucleotide described in SEQ ID NO: 141. A library of modified polypeptides was generated using various well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using the HTP assay and analytical methods described below to measure the ability of the polypeptides to produce compound (IA).

[0226] The enzyme assay was performed in a 96-well deep well (total volume 1.1 mL) plate with a total reaction volume of 100 μL per well. The reaction mixture contained 10 v / v% of the undiluted oxynitrilase lysate prepared as described in Example 3, 0.6 M trifluoroacetone (compound (1)), 0.9 M nitromethane (compound (2)), and citrate buffer (pH 6.0). The reaction plate was heat-sealed and shaken at 150 rpm and 22 °C.

[0227] After incubating overnight (about 22 hours), 700 μL / well of MeOH was added to the reaction plate. The quenched reaction plate was sealed, mixed well, and centrifuged at 4,000 rpm for 15 minutes. Subsequently, 20 μL / well of the quenched reaction plate was taken out and added to a 96-well plate with shallow wells containing 180 μL of MeOH. The plate was sealed and mixed thoroughly. Then, these samples were diluted 1:400 with MeOH and analyzed by RF-MS to determine the activity of the enzyme variant using the analytical method described in Table 5-2. The selected oxynitrilase variants showing faster product formation of (IA) compared to SEQ ID NO: 142 are shown in Table 9.1. After confirming the high selectivity of the most active polypeptide SEQ ID NO: 156 for the (IA) product by chiral GC analysis using the analytical method described in Table 5-3, the evolution method was continued as described in Example 10. All variants showed >90% S-selectivity (>80% ee) under the given reaction conditions.

[0228]

Table 12

[0229]

Table 13

[0230] Example 10 Round 11 Evolution and Screening of Modified Polypeptides Derived from SEQ ID NO: 156 for Improved Stability in Isopropyl Acetate and Production of Compound (IA) Using the polynucleotide from SEQ ID NO: 155 of Example 9 that encodes the most active polypeptide having oxynitrilase activity of SEQ ID NO: 156, the modified polypeptides of Table 10-1 were generated. These polypeptides showed an improvement in oxynitrilase activity under the desired conditions, for example, an improvement in the formation of the nitroalcohol compound (IA) generated in situ from the substrate trifluoroacetone and nitromethane (compounds (1) and (2), respectively), compared to the starting polypeptide. Table 10-1 shows several polypeptides that showed an improvement in product formation of the nitroalcohol product (IA) compared to the starting polypeptide. Modified polypeptides having the amino acid sequences of the even-numbered sequence identifiers were generated from the "scaffold" amino acid sequence of SEQ ID NO: 156, together with the analytical methods described in Table 8-3, as described below.

[0231] Directed evolution was initiated with the polynucleotide described in SEQ ID NO: 155. A library of modified polypeptides was generated using various well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using the HTP assay and analytical methods described below that measure the ability of the polypeptide to produce compound (IA).

[0232] The enzyme assay was performed in a 96-well deep well (total volume 1.1 mL) plate with a total reaction volume of 100 μL per well. The reaction mixture contained 5 v / v% of the undiluted oxynitrilase lysate prepared as described in Example 3, 0.6 M trifluoroacetone (compound (1)), 0.9 M nitromethane (compound (2)), and 30% isopropyl acetate (in citrate buffer (pH 6.0)). The reaction plate was heat-sealed and shaken at 150 rpm and 22 °C.

[0233] After incubation overnight (about 22 hours), 700 μL / well of MeOH was added to the reaction plate. The quenched reaction plate was sealed, mixed well, and centrifuged at 4,000 rpm for 15 minutes.

[0234] Subsequently, 20 μL / well of the quenched reaction plate was taken out and added to a 96-well plate with shallow wells containing 180 μL of MeOH. The plate was sealed and mixed well. Then, these samples were diluted 1:400 with MeOH and analyzed by RF-MS to determine the activity of the enzyme variants using the analysis method described in Table 5-2. The selected oxynitrilase variants showing faster product formation of (IA) compared to SEQ ID NO: 156 are shown in Table 10.1. After confirming the high selectivity of the most active polypeptide SEQ ID NO: 234 for the (IA) product by chiral GC analysis using the analysis method described in Table 5-3, the evolution method was continued as described in Example 11. All variants showed >92% S-selectivity (>84% ee) under the given reaction conditions.

[0235]

Table 14

[0236]

Table 15

[0237] Example 11 Round 12 Evolution and Screening of Modified Polypeptides Derived from SEQ ID NO: 234 to Improve the Production of Compound (IA) Using the polynucleotide from SEQ ID NO: 233 of Example 10 encoding the most active polypeptide having oxynitrilase activity of SEQ ID NO: 234, the modified polypeptides in Table 11-1 were generated.

[0238] These polypeptides showed an improvement in oxynitrilase activity under desired conditions, for example, an improvement in the formation of the nitroalcohol compound (IA) generated in situ from the substrate trifluoroacetone and nitromethane (compounds (1) and (2), respectively), compared to the starting polypeptide. Table 11-1 shows several polypeptides that showed an improvement in the product formation of the nitroalcohol product (IA) compared to the starting polypeptide. Modified polypeptides having the amino acid sequences of even-numbered sequence identifiers were generated from the "scaffold" amino acid sequence of SEQ ID NO: 234, together with the analytical methods described in Table 8-3, as described below.

[0239] Directed evolution was initiated with the polynucleotide described in SEQ ID NO: 233. A library of modified polypeptides was prepared using various well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using the HTP assay and analytical methods described below to measure the ability of the polypeptides to produce compound (IA).

[0240] The enzyme assay was performed in 96-well deep-well (total volume 1.1 mL) plates with a total reaction volume of 100 μL per well. The reaction mixture contained 2.5 v / v% of the undiluted oxynitrilase lysate prepared as described in Example 3, 0.75 M trifluoroacetone (compound (1)), 0.9 M nitromethane (compound (2)), and 30% isopropyl acetate in citrate buffer (pH 6.0). The reaction plates were heat-sealed and shaken at 150 rpm and 22 °C.

[0241] After incubation overnight (about 22 hours), 650 μL / well of MeOH was added to the reaction plates. The quenched reaction plates were sealed, mixed well, and centrifuged at 4,000 rpm for 15 minutes.

[0242] Subsequently, 15 μL / well of the quenched reaction plate was taken out and added to a 96-well plate with shallow wells containing 185 μL of MeOH. The plate was sealed and mixed well. Then, these samples were diluted 1:400 with MeOH and analyzed by RF-MS to determine the activity of the enzyme variants using the analytical method described in Table 5-2. The selected oxynitrilase variants showing faster product formation of (IA) compared to SEQ ID NO: 234 are shown in Table 11.1. After confirming the high selectivity of the most active polypeptide SEQ ID NO: 410 for the (IA) product by chiral GC analysis using the analytical method described in Table 5-3, the evolution method was continued as described in Example 12. All variants showed >92% S-selectivity (>84% ee) under the given reaction conditions.

[0243]

Table 16

[0244]

Table 17

[0245]

Table 18

[0246] Example 12 Round 13 Evolution and Screening of Modified Polypeptides Derived from SEQ ID NO: 410 to Improve the Production of Compound (IA) Using the polynucleotide from SEQ ID NO: 409 of Example 11 that encodes the most active polypeptide having the oxynitrilase activity of SEQ ID NO: 410, the modified polypeptides of Table 12-1 were generated. These polypeptides showed an improvement in oxynitrilase activity under desired conditions, for example, an improvement in the formation of the nitroalcohol compound (IA) generated in situ from the substrate trifluoroacetone and nitromethane (compounds (1) and (2), respectively), compared to the starting polypeptide. Some of the polypeptides that showed an improvement in product formation of the nitroalcohol product (IA) compared to the starting polypeptide are shown in Table 12-1. Modified polypeptides having the amino acid sequences of even-numbered sequence identifiers were generated from the "skeleton" amino acid sequence of SEQ ID NO: 410, together with the analytical methods described in Table 8-3, as described below.

[0247] Directed evolution was initiated with the polynucleotide described in SEQ ID NO: 409. A library of modified polypeptides was generated using various well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences), and screened using the HTP assay and analytical methods described below that measure the ability of the polypeptide to generate compound (IA).

[0248] The enzyme assay was performed in 96-well deep-well (total volume 1.1 mL) plates with a total reaction volume of 100 μL per well. The reaction mixture contained 5 v / v% of the undiluted oxynitrilase lysate prepared as described in Example 3, 0.75 M trifluoroacetone (compound (1)), 0.9 M nitromethane (compound (2)), and 30% isopropyl acetate (in citrate buffer (pH 6.0)). The reaction plates were heat-sealed and shaken at 150 rpm and 22 °C.

[0249] After incubation overnight (about 22 hours), 650 μL / well of MeOH was added to the reaction plates. The quenched reaction plates were sealed, mixed well, and centrifuged at 4,000 rpm for 15 minutes.

[0250] Subsequently, 15 μL / well of the quenched reaction plate was taken out and added to a 96-well plate with shallow wells containing 185 μL of MeOH. The plate was sealed and mixed thoroughly. Then, these samples were analyzed by HPLC to determine the activity of the enzyme variants using the analytical method described in Table 5-1. The selected oxynitrilase variants showing faster product formation of (IA) compared to SEQ ID NO: 410 are shown in Table 12.1. After confirming the high S-selectivity (>95% for the (IA) product of the most active polypeptide SEQ ID NO: 414, >90% ee) by chiral GC analysis using the analytical method described in Table 5-3, the evolution method was continued as described in Example 13. All other mutants also showed high S-selectivity of >94% (>88% ee) under the given reaction conditions.

[0251]

Table 19

[0252]

Table 20

[0253]

Table 21

[0254]

Table 22

[0255] Example 13 Comparison of Catalytic Activity and Selectivity of Wild-Type Polypeptide SEQ ID NO: 606 and Modified Polypeptide SEQ ID NO: 2 The polynucleotide SEQ ID NO: 605 encoding the wild-type (S)-hydroxynitrile lyase from Baliospermum montanum with accession number 606, Uniprot ID: D1MX73, and the modified polynucleotide SEQ ID NO: 1 encoding the starting enzyme of Example 6 with SEQ ID NO: 2 were both used in the production of SFP as described in Example 4.

[0256] The catalytic activity and selectivity for converting the substrates trifluoroacetone (1) and nitromethane (2) into the desired nitroalcohol compound (IA) were carried out in a 96-well deep well plate (total volume 1.1 mL) with a total reaction volume of 100 μL per well. The reactants were in 100 mM citrate buffer at pH 5.5 and contained 0 g / L, 0.049 g / L, 0.098 g / L, 0.195 g / L, 0.391 g / L, 0.781 g / L, 1.563 g / L, 3.125 g / L, 6.25 g / L, 12.5 g / L, 25 g / L, 50 g / L of SFP, 0.623 M (70 g / L) of trifluoroacetone (1), and 0.631 M (39 g / L) of nitromethane (2) prepared as described in Example 3. Each reaction was performed in duplicate. Two reaction plates were set up in parallel, one for chiral GC analysis and the other for achiral LC analysis. Both reaction plates were heat-sealed and shaken at 150 rpm and 22 °C. After incubating overnight (about 22 hours), 450 μL / well of MTBE was added to the GC reaction plate and 650 μL / well of MeOH was added to the LC reaction plate. The quenched reaction plates were sealed, mixed well, and centrifuged at 4,000 rpm for 15 minutes.

[0257] Subsequently, 15 μL / well of the quenched LC reaction plate was taken out and added to a 96-well plate with shallow wells containing 185 μL of MeOH. The plate was sealed and mixed well. Then, these samples were analyzed by HPLC to determine the activity of the enzyme variants using the analytical method described in Table 5-1.

[0258] For selectivity and enantiomeric excess (ee) analysis, 150 μL / well of the upper organic phase of the quenched GC reaction plate was removed and added to a shallow-well 96-well plate. This plate was then analyzed by chiral GC using the analytical method described in Table 5-3. Figure 1 clearly shows higher selectivity and activity of SEQ ID NO: 2 compared to the wild-type polypeptide of SEQ ID NO: 606.

[0259] The comparative data in Figures 1A and 1B show the conversion rates and enantiomeric excesses of trifluoroacetone (1) and nitromethane (2) to the nitroalcohol compound (IA) by the wild-type (S)-hydroxynitrile lyase from Baliospermum montanum, Uniprot ID: D1MX73, having SEQ ID NO: 606, compared to those by the modified polypeptide SEQ ID NO: 2. The experiments were performed in duplicate. The reaction mixture contained 0.623 M (70 g / L) trifluoroacetone (1) and 0.631 M (39 g / L) nitromethane (2) in 100 mM citrate buffer at pH 5.5, and the SFP catalyst concentration was varied from 0 to 50 g / L. The modified polypeptide having SEQ ID NO: 2 showed higher selectivity and activity than the wild-type polypeptide of SEQ ID NO: 606. Since the selective enzyme reaction always competes with the non-selective chemical reaction in parallel, the actual ee resulting from the selectivity (>90%) of the oxynitrilase of SEQ ID NO: 606 is at most 25%. Therefore, the evolutionary campaign described in the present disclosure was designed to further enhance the activity of the enzyme while maintaining high selectivity.

[0260] In the follow-up experiment, the best enzyme of each evolutionary round was used to convert the substrate trifluoroacetone (1) and nitromethane (2) into the desired nitroalcohol compound (IA). Unless otherwise specified, the experimental procedures were the same as those shown in FIGS. 1A and 1B for comparison with the wild-type sequence. Enzyme SFP was prepared as described in Example 3 and used at the following concentrations of SFP: 0 g / L, 0.049 g / L, 0.098 g / L, 0.195 g / L, 0.391 g / L, 0.781 g / L, 1.563 g / L, 3.125 g / L, 6.25 g / L, 12.5 g / L. The following two different conditions were investigated: (1) the initial screening conditions of Examples 6 and 7 in 100 mM citrate buffer at pH 5.5 with 0.623 M (70 g / L) trifluoroacetone (1) and 0.631 M (39 g / L) nitromethane (2) (see FIGS. 2A, 2B), and (2) the final screening conditions of Examples 11, 12 and 13 in 100 mM citrate buffer at pH 6.0 with 0.75 M (84 g / L) trifluoroacetone (1), 0.9 M (55 g / L) nitromethane (2) and 30% isopropyl acetate (see FIGS. 2C, 2D). FIGS. 2A and 2C show the conversion rate, while FIGS. 2B and 2D show the enantiomeric excess of the most active polypeptide of each round for the conversion of trifluoroacetone (1) and nitromethane (2) into the nitroalcohol compound (IA). As shown in FIG. 2, the ranking of the various enzyme variants is in good agreement with the progress of each round under both conditions. Oxynitrilase having SEQ ID NO: 414 shows the highest selectivity and conversion rate under both conditions. However, under the first condition, SEQ ID NO: 414 with a SFP loading of 12.5 g / L reached a conversion rate of 91% and ee of 87% (see FIGS. 2A, 2B), while under the improved second reaction condition, it reached a conversion rate of 100% and ee of 93% with a loading of only 6.25 g / L (see FIGS. 2C, 2D). Then, the enzyme having SEQ ID NO: 414 was used in a large-scale reaction (see Example 14).

[0261] Example 14 Production of Nitro Alcohol Compound (IA) from Substrates Trifluoroacetone (1) and Nitromethane (2) Using Modified Polypeptide SEQ ID NO: 414 as a Catalyst [Chemical formula] The polypeptide of SEQ ID NO: 414, which showed the highest oxynitrilase activity in Example 12, was produced as a fermentation powder and used to convert the substrates trifluoroacetone (1) and nitromethane (2) into the nitroalcohol compound (IA). 0.504 g of the polypeptide of SEQ ID NO: 414 was dissolved in sodium citrate buffer (4.0 mL, 0.1 M, pH 6.0 ± 0.5) and loaded into a reactor pre-cooled to 10°C. A 10°C solution of trifluoroacetone (12.13 g, 108.3 mmol) dissolved in sodium citrate buffer (27.8 mL, 0.1 M, pH 6.0) and additional sodium citrate buffer (78.6 mL, 0.1 M, pH 6.0) were charged into the reactor. After setting the internal temperature and stirring to 22°C and 350 rpm, nitromethane (13.2 g, 11.7 mL, 216.5 mmol) and then isopropyl acetate (10.2 g, 11.7 mL) were added. Isopropyl acetate (21.1 g, 24.3 mL) was added and the mixture was stirred at 22°C for 22 hours. CellFlock 40 (6.0 g) and isopropyl acetate (5.0 g, 5.7 mL) were added to the emulsion and the mixture was stirred at 22°C for 2 hours. The resulting suspension was filtered to obtain a clear two-phase mixture. The aqueous layer was separated and extracted with isopropyl acetate. The organic layers were combined and concentrated at a jacket temperature of 50°C and 180 - 200 mbar. A small amount of isopropyl acetate was added to the resulting distillation residue and it was concentrated again. This procedure was repeated once more to obtain a solution of the desired concentration of nitroalcohol in isopropyl acetate (91.7 g solution containing 16.84 g of nitroalcohol, yield 90.8%, e.r. = 96:4). 1 H NMR (DMSO-d6): 6.96 (s, OH), 4.92 (d, 1H), 4.82 (d, 1H), 1.48 (s, 3H)

[0262] Example 15 Production of Nitro Alcohol Compound (IA) from Substrates Trifluoroacetone (1) and Nitromethane (2) Using Whole Cells Containing Modified Polypeptide of SEQ ID NO: 254 The polynucleotide from Example 10 having SEQ ID NO: 233, which encodes the most active polypeptide from Example 10 having oxynitrilase activity with SEQ ID NO: 234, was produced as three different enzyme formulations as described in Example 4, such as SFP, FWC or LWC. In all three formulations, the nitroalcohol compound (IA) could be successfully formed, and the nitroalcohol compound was generated in situ from the substrates trifluoroacetone and nitromethane (compounds (1) and (2), respectively).

[0263] The enzyme assay was performed in 96-well deep well (total volume 1.1 mL) plates with a total reaction volume of 100 μL per well. The reaction contained one of three oxynitrilase formulations (SFP, LWC or FWC), and 0.62 M trifluoroacetone (compound (1)), 0.98 M nitromethane (compound (2)), 30% isopropyl acetate (in citrate buffer, pH 6.0). The reaction plates were heat sealed and shaken at 150 rpm and 22 °C.

[0264] After incubation overnight (about 22 h), 300 μL / well of MTBE was added to the reaction plates. The quenched reaction plates were sealed, mixed well, and centrifuged at 4,000 rpm for 5 min. Then, a 150 μL aliquot of the upper organic phase was removed from each well and added to a shallow well 96-well plate. For selectivity determination, the plates were sealed and analyzed by chiral GC using the analytical method described in Table 5-3. For activity analysis by HPLC, 20 μL / well of the upper organic phase of the quenched reaction plates was removed and added to a shallow well 96-well plate containing 180 μL of MeOH. The plates were sealed and mixed well. These samples were then analyzed by HPLC to determine the activity of the enzyme variants using the analytical method described in Table 5-1.

[0265] Catalysts of three formulations made from the same amount of cells normalized to a SFP loading of 20 g / l, using SFP, FWC or LWC, after 20 h reaction time, showed 50% conversion to (IA) for the SFP formulation, 55% conversion for the LWC formulation and 65% conversion for the FWC. All samples showed 93% S-selectivity.

[0266] Example 16 Immobilization of Modified Polypeptide of SEQ ID NO: 410 The fermented powder of the polypeptide of SEQ ID NO: 410 having oxynitrilase activity was immobilized on a solid methoxy methacrylate support. This makes the filtration and reuse of the polypeptide faster and thus the process options for the nitroaldol reaction from substrates (1) and (2) to product (IA) more flexible. As a proof of concept, the polypeptide of SEQ ID NO: 410 was cross-linked to an epoxy resin (ECR8204F) or an amino-functionalized methacrylate resin (ECR8304F) obtained from Purolite Inc. For this, 6 g of each resin was used and all washing steps were carried out at a ratio of 1:4 (resin: washing solution). First, the resin was washed with 1 M (for ECR8204F) and 50 mM (for ECR8304F) sodium citrate buffer (pH 6.0) of the immobilization buffer. To cross-link to the amino-functionalized methacrylate resin (ECR8304F), it was incubated with a 1% glutaraldehyde solution for 2 h and the activated resin was washed 3 times with the immobilization buffer. Thereafter, both resins were incubated overnight in the immobilization buffer with a 15 mg / mL solution of the lyophilized polypeptide of SEQ ID NO: 410. Both types of resins (ECR8304F and ECR8204F) were washed once with 20 mM sodium citrate buffer (pH 6.0) and twice with 0.5 M brine. Thereafter, both resins were washed 3 times with 50 mM sodium citrate buffer (pH 6.0).

[0267] Subsequently, these resins were used under the same reaction conditions as in Example 12, and 170 mg of the immobilized enzyme was incubated in 1 ml of 0.1 M citrate buffer (pH 6) containing 0.85 M (1), 1.02 M (2), and 20% - 88% concentrations of isopropyl acetate (IPAc) at 22°C for 22 hours. After incubating overnight, 650 μL / well of MeOH was added to the reaction plate. The quenched reaction plate was sealed, mixed well, and centrifuged at 4,000 rpm for 15 minutes. Subsequently, 15 μL / well of the quenched reaction plate was taken out and added to a shallow well 96-well plate containing 185 μL of MeOH. The plate was sealed and mixed thoroughly. Then, these samples were analyzed by HPLC to determine the activity of the enzyme variant using the analytical method described in Table 5-1. Figure 3 shows the activity (see Figure 3A) and selectivity (see Figure 3B) of the free enzyme compared to the enzyme immobilized on an amino carrier or an epoxy carrier. As shown in Figure 3, the amino resin showed performance superior to that of the epoxy resin in terms of conversion rate (see Figure 3A) and immobilization yield, but the selectivity was equivalent in all cases (see Figure 3B). A high isopropyl acetate (IPAc) concentration (in the range of 20% - 88%) was applied to the screening. Note that the substrate solution itself had 12% by volume, and for samples with 88% by volume of IPAc, IPAc was water-saturated prior to the reaction.

[0268] The conditions for the amino resin were further optimized, and the best conditions were used for the large-scale immobilization reaction. For this purpose, 30 g of ECR8304F obtained from Purolite Inc. was used, and all washing steps were carried out at a ratio of 1:4 (resin: washing solution). The resin was first washed with 50 mM sodium citrate (pH 6.0) immobilization buffer. Cross-linking incubation was carried out with a 1% glutaraldehyde solution for 2 hours, and the activated resin was washed 3 times with the immobilization buffer. Thereafter, both resins were incubated overnight in the immobilization buffer with a 20 mg / mL solution of the freeze-dried polypeptide of SEQ ID NO: 410. Both resins were washed once with 20 mM sodium citrate buffer (pH 6.0), twice with 0.5 M brine, and twice with the buffer. This procedure resulted in an immobilization yield of 70 - 80% and a total recovered enzyme activity of approximately 30%. The 30 g of immobilized polypeptide (SEQ ID NO: 410) thus produced was incubated in 180 ml of 0.1 M citrate buffer pH 6 containing 0.61 M (1), 1.22 M (2) and 30% IPAc at 22 °C for 22 hours to obtain a yield of 80% and ee 92% (S-selectivity 96%). This immobilized enzyme was reused twice by filtering the reaction mixture, and no significant loss of reaction yield and selectivity was observed. The second reuse was carried out after a total storage time of 25 days at 4 °C.

[0269] Example 17 Hydrogenation of the Compound of Formula (IA)

Chem.

[0270] HCl (1.25 M in MeOH, 2.0 eq) was added and stirred at room temperature for 6 hours. The resulting solution was filtered and then concentrated at a jacket temperature of 40 °C and 60 mbar. A small amount of isopropyl acetate was added to the resulting distillation residue and concentrated again. This procedure was repeated two more times to obtain the HCl salt of the amino alcohol free of methanol. Acetonitrile was added and the resulting solution was heated to a jacket temperature of 35 °C, and then a suspension of the resulting seed material in n-heptane ((IB)·HCl 1 part and n-heptane 33 parts) was slowly added. Additional n-heptane was added and the resulting mixture was slowly cooled to a jacket temperature of 5 °C. Stirring was continued for 18 hours. The suspension was filtered and the filter cake was washed with some n-heptane. After drying the filter cake under vacuum, it was dissolved in acetonitrile, heated to a jacket temperature of 35 °C, n-heptane was added, and then a suspension of the resulting seed material consisting of 1 part of (IB)·HCl and 33 parts of n-heptane was added. Additional n-heptane was added and the resulting suspension was cooled to a jacket temperature of -1 °C. Stirring was continued for 18 hours. Then, the resulting suspension was filtered and the filter cake was washed with n-heptane. The filter cake was dried under vacuum at room temperature to obtain the desired product, namely crystalline (IB)·HCl (6.1 g, yield 61%, e.r. 99:1). 1 H NMR (DMSO-d6): 8.32 (br s, NH3 + ), 6.84 (br s, OH), 2.99 (s, 2H), 1.39 (s, 3H). 1313C NMR (DMSO-d6): 125.8 (CF3), 70.4 (C-CF3), 42.2 (CH2), 18.6 (CH3).

[0271] Flow Procedure A stainless steel cartridge with an inner diameter of 9.5 mm and a length of 90 mm was filled with a pre-thoroughly mixed mixture of water-wet sponge-nickel (14.5 g) and inactive silicon carbide (9.58 g). The cartridge is equipped with inlets for liquid and gas and contains a 5 μm frit to prevent the solid material from exiting the cartridge outlet. At the start, the cartridge loaded with the catalyst was placed in a water-ice bath and flushed with more than 12 column volumes of a 1:4 v / v mixture of methanol and isopropyl acetate. Subsequently, the liquid flow was switched to a 0.25 M solution of (IA) in a 1:4 v / v mixture of methanol and isopropyl acetate. The feed was also pre-cooled to 0 - 5 °C in an ice bath. The feed was supplied to the cartridge at a flow rate of 3 mL / min by a gear pump. Hydrogen gas was generated by the electrolysis of water, which was supplied at a flow rate of 99.0 mL / min by a mass flow controller, and the entire apparatus was pressurized to 10 bar using a back pressure regulator. Considering the presence of hydrogen gas, an apparent residence time of 31 seconds was calculated. Each time the cartridge was passed through, a conversion to the product of 50 - 60 A% was achieved, at which time the solution containing the product was returned to the feed bottle and the cartridge was passed through again until a total of 3 passes were made.

[0272] For the 180-minute experiment, a total of 17.92 g of starting material was used in a volume of 500 mL of solvent (4.0 m%). The conversion rate to the product after 3 passes was 98 A%, and in the post-treatment of the liquid fraction of the product to the hydrochloride (by the standard crystallization method using HCl / methanol as described above), an isolation yield of 75% was observed with 99 A% HPLC quality.

[0273] Example 18 Coupling Process for Synthesizing Compound (IC) [Chemical formula] E6 (41.4 g, 152 mmol) and crystallization (IB)·HCl (30 g, 167 mmol) were charged into a reactor, followed by the addition of 410 ml of toluene. While stirring, triethylamine (38 g, 380 mmol) and then tetramethyl orthosilicate (TMOS) (46 g, 304 mmol) were added. The reaction mixture was heated to 110 °C and methanol formed by the reaction was slowly distilled off while stirring for 3 - 16 hours. The completion of the reaction was monitored by HPLC. An aqueous NaOH solution (330 ml, 2 mol) was added and the mixture was stirred at 50 °C for 2 hours. The aqueous phase was separated and discarded. At 50 °C, an aqueous HCl solution (330 ml, 10%) was added and the aqueous phase was discarded. After washing with water, the organic solution was dried and concentrated by distillation. (IC) was crystallized by adding 240 ml of n-heptane and cooling to 0 °C. After drying at 40 °C under vacuum, compound (IC) was obtained in a yield of 80% (44 g). 1 1H-NMR (DMSO-d6): δ ppm 8.29 (m, 1H), 7.67 (s, 1H), 6.68 (br, 2H), 6.28 (s, 1H), 3.92 (s, 3H), 3.69 - 3.43 (m, 2H), 1.25 (s, 3H). 13 13C-NMR (DMSO-d6): δ ppm 18.95, 42.2, 53.5, 72.6 - 71.9 (m), 116.5 - 115.6 (m), 124.9 - 121.0 (2C, m), 126.3, 128.3 (m), 140.9, 148.5, 166.3.

[0274] Example 19 Round 14 Evolution and Screening of Modified Polypeptides Derived from SEQ ID NO: 606 to Improve the Production of Compound (IA) Using the polynucleotide from SEQ ID NO: 413 of Example 12 that encodes the most active polypeptide having the oxynitrilase activity of SEQ ID NO: 414, the modified polypeptides in Table 19-1 were generated. These polypeptides showed improvement in oxynitrilase activity, for example, improvement in heat resistance and improvement in the formation of the nitroalcohol compound (IA) generated in situ from the substrate trifluoroacetone and nitromethane (compounds (1) and (2), respectively), compared to the starting polypeptide, under the desired conditions. Table 19-1 shows several polypeptides that showed improvement in the product formation of the nitroalcohol product (IA) compared to the starting polypeptide. Modified polypeptides having the amino acid sequences of the even-numbered sequence identifiers were generated from the "skeleton" amino acid sequence of SEQ ID NO: 414, together with the analytical methods described herein, as described below.

[0275] Directed evolution was initiated with the polynucleotide described in SEQ ID NO: 413. A library of modified polypeptides was prepared using various well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences), and screened using the HTP assay and analytical methods described below that measure the ability of the polypeptide to produce compound (IA).

[0276] The enzyme assay was performed in a 96-well deep well (total volume 1.1 mL) plate with a total reaction volume of 100 μL per well. The reaction mixture was prepared as described in Example 3 and then pre-incubated for 1 hour at 4 °C (described as "no heat incubation" in Table 19-1) or 52 °C (described as "with heat incubation" in Table 19-1) containing 5 v / v% of undiluted oxynitrilase lysate. All other conditions were unchanged from the previous round of evolution: 0.75 M trifluoroacetone (compound (1)), 0.9 M nitromethane (compound (2)), 30% isopropyl acetate in citrate buffer (pH 6.0). The reaction plate was heat-sealed and shaken at 150 rpm and 22 °C.

[0277] After incubating overnight (about 22 hours), 650 μL / well of MeOH was added to the reaction plate. The quenched reaction plate was sealed, mixed well, and centrifuged at 4,000 rpm for 15 minutes.

[0278] Subsequently, 15 μL / well of the quenched reaction plate was taken out and added to a 96-well plate with shallow wells containing 185 μL of MeOH. The plate was sealed and mixed thoroughly. These samples were then analyzed by HPLC to determine the activity of the enzyme variants using the analytical method described in Table 5-1. The selected oxynitrilase variants showing faster product formation of (IA) compared to SEQ ID NO: 414 are shown in Table 19.1. The >95% high S selectivity (>90% ee) for the (IA) product of the most active polypeptides of SEQ ID NOs: 610 and 620 was confirmed by chiral GC analysis using the analytical method described in Table 5-3.

[0279]

Table 23

[0280] Example 20 Substrate Scope of the Evolved Oxynitrilase

Chem.

[0281] In the first experimental setup, for Reactions 1, 2 and 3, the activities of the oxynitrilase variants were examined. The enzyme assay was performed in a 96-well deep well plate (total volume 2 mL) with a total reaction volume of 400 μL per well. The reaction mixture contained 0.1 M sodium citrate buffer (pH 6.0), 10% by volume of iPrOAc, and, for Reaction 1, 1 g / L (9 mM) of trifluoroacetone (1) and 11 g / L (180 mM) of nitromethane (2); for Reaction 2, 1 g / L (9 mM) trifluoroacetone (1) and 13.5 g / L (180 mM) of nitroethane (3); for Reaction 3, 1.1 g / L (9 mM) of acetophenone (5) and 11 g / L (180 mM) of nitromethane (2). The reaction plate was heat sealed and shaken at 200 rpm and 22 °C.

[0282] After incubating overnight (about 22 hours), 1.2 mL / well of MeOH was added to the reaction plate. The quenched reaction plate was sealed, mixed well, and centrifuged at 4,000 rpm for 10 minutes. Subsequently, 200 μL / well of the quenched reaction was transferred to a shallow well 96-well plate for HPLC-MS analysis, and the activity of the enzyme variant was determined using the analytical method described in Table 5.4. The relative product peak areas observed in Reaction Settings 1, 2, and 3 are shown in Table 20-1. The masses of the products (IA) and (4) could be confirmed by LC-MS. In Reaction 3, no product formation of (6) was observed by LC-MS using the analytical method described in Table 5.4 and GC-MS / FID using the analytical method described in Table 5.5, and no enzyme activity was shown when using acetophenone as the substrate. The results of Reaction 2 showed an increase in the substrate acceptance of the evolved oxynitrilase with nitroethane (3), while no activity was detected with the wild-type enzyme of SEQ ID NO: 606. After the first activity was detected with SEQ ID NO: 2, an increase in enzyme productivity of >25-fold was achieved in subsequent evolution rounds, as can be seen in the comparison with SEQ ID NO: 2 with less evolution at 100 g / L, which resulted in less product formation than 4 g / L of SEQ ID NO: 414.

[0283] To obtain the results shown in Table 20-1 for Reaction 4, the enzyme assay was performed in a 96-well deep well plate (total volume 1.1 mL) with a total reaction volume of 200 μL per well. The reaction mixture contained 0.1 M sodium citrate buffer (pH 6.0), 20% by volume of iPrOAc, and 5.2 g / L (30 mM) of 2,2,2-trifluoro-1-phenylene-1-one (7) and 36.6 g / L (600 mM) of nitromethane (2). The reaction plate was heat-sealed and shaken at 200 rpm and 22 °C.

[0284] After incubating overnight (about 22 hours), 0.6 mL / well of MeOH was added to the reaction plate. The quenched reaction plate was sealed, mixed well, and centrifuged at 4,000 rpm for 10 minutes. Subsequently, 100 μL / well of the quenched reaction was transferred to a shallow-well 96-well plate containing 100 μL of MeOH per well. The samples were then analyzed by LC-MS to determine the activity of the enzyme variants using the analytical method described in Table 5.4. The results of Reaction 4 show an increase in the conversion rate to product (8) with the evolved oxynitrilase. The most evolved oxynitrilase with SEQ ID NO: 414 showed the highest product formation among all substrate combinations tested.

[0285]

Table 24

[0286]

Table 25

Claims

1. A modified oxynitrilase polypeptide having at least 80% sequence identity with SEQ ID NO: 606, wherein 1,1,1-trifluoropropan-2-one can be coupled with nitromethane under suitable reaction conditions to produce (S)-1,1,1-trifluoro-2-methyl-3-nitropropan-2-ol in an enantiomer excess of at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.

2. The oxynitrilase polypeptide according to claim 1, wherein the preferred reaction conditions include about 5 g / L to about 150 g / L of 1,1,1-trifluoropropan-2-one, a loading amount of nitromethane about twice the molar amount of 1,1,1-trifluoropropan-2-one, at least 3 g / L of oxynitrilase polypeptide, isopropyl acetate at a concentration of about 20% (v / v) to about 60% (v / v), a pH of about 4.0 to 8.0, and a temperature of about 10°C to 30°C.

3. A polypeptide immobilized on a solid material by chemical bonding or physical adsorption, wherein the polypeptide is a modified oxynitrilase polypeptide according to (a) or (b) below: (a) Sequence numbers 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, 8 4, 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, 15 0, 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 ,214,216,218,220,222,224,226,228,230,232,234,236,238,240,242,244,246,248,250,252,254,256,258,260,262,264,266,268,270,272,274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 3 38, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 40 0, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462 ,464,466,468,470,472,474,476,478,480,482,484,486,488,490,492,494,496,498,500,502,504,506,508,510,512,514,516,518,520,522,524,Polypeptides containing an amino acid sequence selected from the group consisting of 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, and 640; or (b) A polypeptide having oxynitrilase activity, comprising an amino acid sequence having at least 80% sequence identity to one of the polypeptides described in (i)(a), and having one or more amino acid residue substitutions, deletions, additions or insertions to the amino acid sequence described in (ii)(a); This is the oxynitrilase polypeptide.

4. Use of a polynucleotide to provide the polypeptide according to any one of claims 1 to 3.

5. The sequence of the aforementioned polynucleotides is sequence numbers 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 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, 2 69, 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, 3 31, 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, 385, 387, 389, 391, 39 3, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455 ,457,459,461,463,465,467,469,471,473,475,477,479,481,483,485,487,489,491,493,495,497,499,501,503,505,507,509,511,513,515,517,519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581 The use according to claim 4, which is 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, or 639.

6. Use of an expression vector comprising the polynucleotide according to claim 4 or 5 to provide the polypeptide according to any one of claims 1 to 3.

7. The use according to claim 6, comprising a plasmid, cosmid, bacteriophage, or viral vector.

8. A method for preparing an oxynitrilase polypeptide, comprising the steps of culturing a host cell containing the expression vector described in claim 6 or 7, and obtaining an oxynitrilase polypeptide from the culture, wherein the host cell is preferably E. coli.

9. An oxynitrilase catalyst comprising cells or a culture medium containing the oxynitrilase polypeptide described in any one of Claims 1 to 3, or an article treated with the same, wherein the article refers to an extract obtained from the culture of the transformed cells, an isolation product obtained by isolating or purifying oxynitrilase from the extract, or an immobilization product obtained by immobilizing the transformed cells, an extract thereof, or an isolation product of the extract.

10. A process for the asymmetric synthesis of β-nitroalcohols, wherein the process involves using an oxynitrilase polypeptide of (a) or (b) below to deliver a nitroalkane and an aldehyde or ketone substrate: (a) Sequence numbers 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, 8 4, 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, 15 0, 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 ,214,216,218,220,222,224,226,228,230,232,234,236,238,240,242,244,246,248,250,252,254,256,258,260,262,264,266,268,270,272,274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 3 38, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 40 0, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462 ,464,466,468,470,472,474,476,478,480,482,484,486,488,490,492,494,496,498,500,502,504,506,508,510,512,514,516,518,520,522,524,Polypeptides containing an amino acid sequence selected from the group consisting of 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, and 640; or (b) A polypeptide having oxynitrilase activity, comprising an amino acid sequence having at least 80% sequence identity to one of the polypeptides described in (i)(a), and having one or more amino acid residue substitutions, deletions, additions or insertions to the amino acid sequence described in (ii)(a); A process for the asymmetric synthesis of β-nitro alcohols, including a step of contact with [a specific substance].

11. The process according to claim 10, wherein the aldehyde or ketone substrate includes an electron-withdrawing substituent.

12. The resulting β-nitro alcohol is given by formula (I): 【Chemistry 1】 [In the formula, R １ and R ２ are each independently selected from H, alkyl, such as C １ to C ２０ alkyl, alkenyl, such as C ２ to C ２０ alkenyl, alkynyl, such as C ２ to C ２０ alkynyl, cycloalkyl, such as C ３ to C １０ cycloalkyl, aryl, such as C ６ to C １４ aryl, arylalkyl, such as C ７ to C ２０ arylalkyl, heterocyclyl, such as 3- to 14-membered heterocyclyl, and heteroaryl, such as 5- to 20-membered heteroaryl Here, the alkyl, alkenyl, and alkynyl are each optionally selected as one or more R ａ For example, 1 to 6 R ａ Replaced by, The cycloalkyl, aryl, arylalkyl, heterocyclyl, and heteroaryl are each optionally selected as one or more R ｂ For example, 1 to 6 R ｂ Replaced by; R ３ and R ４ Each independently consists of H and an alkyl group, for example, C. １ ~C ２０ Selected from alkyl groups, where the alkyl group is, for example, C １ ~C ２０ The alkyl group is optionally substituted with one or more Ra, for example, 1 to 6 Ra; Each R ａ Each of these, when generated, independently produces a cycloalkyl, for example, C ３ ~C １０ Cycloalkyl, aryl, for example C ６ ~C １４ Aryl, arylalkyl, for example, C ７ ~C ２０ Arylalkyls, heterocyclyls, e.g., 3-14 member heterocyclyls, and heteroaryls, e.g., 5-20 member heteroaryls, halogens, e.g., F, haloalkyls, e.g., C １ ~C ２０ Haloalkyl, e.g., -CF ３ -CN, -OR ｃ , -NR ｃ R ｃ ,-(CH ２ ) ｎ COOR ｃ ,-(CH ２ ) ｎ -C(=O)R ｃ ,-(CH ２ ) ｎ -C(=O)NR ｃ R ｃ Selected from; Each R ｂ When each occurs, it independently produces a halogen, e.g., F, and a haloalkyl, e.g., C. １ ~C ２０ Haloalkyl, e.g., -CF ３ -CN, -NO ２ , -OR ｃ , -NR ｃ R ｃ ,-(CH ２ ) ｎ COOR ｃ ,-(CH ２ ) ｎ -C(=O)R ｃ ,-(CH ２ ) ｎ -C(=O)NR ｃ R ｃ , C １ ~C ２０ Alkyl, C ２ ~C ２０ Alkenyl, or C ２ ~C ２０ Selected from Alkinil; Each R ｃ When each occurs, H and C occur independently. １ ~C ２０ Alkyl, C ２ ~C ２０ Alkenyl and C ２ ~C ２０ Alkinyl (one or more R selected at will) ｂ For example, 1 to 6 R ｂ Selected from (which will be replaced by); and n is 0, 1, 2, 3, 4, 5, or 6, for example, 0, 1, 2, or 3. It has the structure shown; The aforementioned process is R ３ R ４ CHNO ２ The method includes the step of contacting a nitroalkane of formula (II) and an aldehyde or ketone substrate with an oxynitrilase polypeptide according to any one of claims 1 to 3 to obtain a β-nitroalcohol product of formula (I), wherein the aldehyde or ketone substrate is of formula (III) 【Chemistry 2】 The process according to claim 10 or 11, comprising:

13. R ３ and R ４ are each independently selected from H and C １ to C ２０ alkyl, wherein said C １ to C ２０ alkyl is optionally substituted with 1 to 6 R ａ groups, Each R ａ is independently selected, when each occurs, from C ３ to C １０ cycloalkyl, C ６ to C １４ aryl, 3- to 14-membered heterocyclyl, 5- to 20-membered heteroaryl, halogen, for example F, haloalkyl, for example C １ to C ２０ haloalkyl, for example -CF ３ , -OR ｃ , and -NR ｃ R ｃ ; Each R ｃ When each occurs, H and C occur independently. １ ~C ２０ Alkyl, C ２ ~C ２０ Alkenyl and C ２ ~C ２０ The process according to claim 12, selected from alkynyl.

14. R ３ and R ４ H and C are independent of each other. １ ~C ２０ Selected from alkyl, where C １ ~C ２０ The alkyl group can be optionally selected to have 1 to 6 R groups. ａ Replaced by, Each R ａ Each of these occurs independently of halogens, such as F and C. １ ~C ２０ Haloalkyl, e.g., -CF ３ , -OR ｃ , and -NR ｃ R ｃ Selected from; Each R ｃ When each occurs, H and C occur independently. １ ~C ２０ Alkyl, C ２ ~C ２０ Alkenyl and C ２ ~C ２０ The process according to claim 12, selected from alkynyl.

15. R ３ and R ４ H and C are independent of each other. １ ~C ２０ Selected from alkyl, where C １ ~C ２０ The alkyl group can be optionally selected to have 1 to 6 R groups. ａ Replaced by, Each R ａ Each of these occurs independently of halogens, such as F and C. １ ~C ２０ Haloalkyl, e.g., -CF ３ , -OR ｃ , and -NR ｃ R ｃ Selected from; Each R ｃ When each occurs, H and C occur independently. １ ~C ２０ The process according to claim 12, selected from alkyl groups.

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