Biocatalysis in Drug DevelopmentâHighlights of the Recent Patent

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Cite This: Org. Process Res. Dev. XXXX, XXX, XXX−XXX

Biocatalysis in Drug DevelopmentHighlights of the Recent Patent Literature David L. Hughes*

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Cidara Therapeutics, Inc., 6310 Nancy Ridge Drive, Suite 101, San Diego, California 92121, United States ABSTRACT: The recent patent literature on biocatalysis is reviewed, with a focus on significant advances in enzymatic catalysis involving ketoreductases, transaminases, hydroxylases, sulfur oxidation, and nitrilase resolutions and a progress report on the emerging area of imine reductases highlighting collaborations between academia and pharmaceutical companies. KEYWORDS: biocatalysis, transaminase, ketoreductase, nitrilase, imine reductase, dynamic reductive kinetic resolution, sulfur oxidation, hydroxylation

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These techniques provide access to new and more productive enzymes that can be tuned for a wide variety of unnatural substrates and lay the groundwork for creating biocatalysts for reactions that are currently unknown or rare in nature. A number of reviews on the use of biocatalysis in the pharmaceutical industry have been published recently. Of note to the pharmaceutical industry are reviews on the opportunities and challenges for implementing biocatalysis in the pharmaceutical industry by Rosenthal and Lutz,3 Reetz and coworkers,4 and Lalonde.5 In addition, Chemical Reviews devoted its first issue in 2018 to biocatalysis, and it includes in-depth overviews of transaminases,6 halogenases,7 and ammonialyases.8 Sustainable chemistry is the theme of the second issue of Chemical Reviews in 2018, and it includes an outstanding review by Sheldon and Woodley9 on industrial implementation of biocatalysis. Finally, Turner and Humphreys have authored a comprehensive book on integrating biocatalysis into organic synthesis.10 In a 2009 presentation, David Rozzell, one of the early pioneers of biocatalysis, asserted that “to truly be considered as a mainstream technology, biocatalysis must be a first-line option, not an alternative that is tried after everything else has failed.”11 Nearly a decade later, it is safe to conclude that biocatalysis has matured into a core technology within industry and is poised to realize even greater impact in the future. This review is organized into the following sections: (1) reductions, including imine reductases, ketoreductases, and transaminases; (2) oxidations, including hydroxylation and asymmetric sulfide to sulfoxide oxidations; and (3) hydrolytic resolutions using nitrilases.

he current article is a continuation of a series of thematic reviews on the recent patent literature, focusing primarily on chemistry that has not been published in peer-reviewed articles.1 The theme of the current review is biocatalysis, with the intent to cover the patent literature since our previous review that was published in early 2016.2 Biocatalysis has become an area of core expertise in most process chemistry departments in large pharma and fine chemical companies. In addition, a number of smaller companies have emerged with biocatalysis and enzyme technology as a core business. Perhaps more than other technologies in process chemistry, collaborations between the specialty companies and large pharma and between academia and industry have been key drivers of the rapid advance of the industrial use of biocatalysis. These collaborations bring together diverse expertise and perspectives that foster new discoveries and focused development for industrial implementation. Recent advances in biocatalysis are being driven in part by the following developments in adjacent technologies: 1. metagenomicsthe analysis of genetic material from the environment to identify new enzymes from the vast diversity provided by nature; 2. bioinformaticsthe ability to use computing power to mine large protein databases to identify those proteins that are most likely to be useful enzymes for a particular transformation; 3. protein engineeringmodification of enzymes to accept non-natural substrates and to be tolerant of conditions outside of natural conditions, such as organic cosolvents, high substrate concentrations, higher temperatures, and wider pH ranges; 4. metabolic engineeringmodification of the metabolism of an organism by altering genetic and regulatory processes to produce or increase production of a desired molecule; 5. high-throughput screening and analysisthe ability to quickly screen vast numbers of reactions to determine the best enzymes and conditions for a desired transformation. © XXXX American Chemical Society

1. REDUCTIONS 1.1. Imine Reductase and Reductive Aminase Enzymes. Over the past decade, the study and development of transaminase enzymes has progressed to the point that a variety of isolated transaminase enzymes are now readily available for routine screening. Implementation of transaminase reactions at scale has become more common, and at Received: July 16, 2018 Published: August 10, 2018 A

DOI: 10.1021/acs.oprd.8b00232 Org. Process Res. Dev. XXXX, XXX, XXX−XXX

Organic Process Research & Development

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carboline imines to chiral amines using various yeast species with ee’s ranging from 50 to 97%.17 Since these early reports, imine reductases from several microbial species have been characterized, suggesting that these enzymes are perhaps more widespread in nature than originally anticipated. On the basis of analyses of protein databases, Fademrecht and co-workers have built a database that currently includes 1400 sequences that could be potential IREDs.18 This database provides a rich starting point for researchers to identify and expand the use of IREDs as well as a starting point for protein engineering. The growth of journal publications and patents that incorporate the term “imine reductase” has increased significantly over the past 5 years, as shown in Figure 1.19 In

least one transaminase process, Merck’s sitagliptin, has been introduced into commercial manufacturing.12 A key limitation of transaminases is that only primary amines can be generated from ketone starting materials. To address this limitation, an emerging focus of biocatalysis is the development of imine reductase enzymes (IREDs), which are NAD(P)H-dependent oxidoreductases that reduce imines to amines and therefore can generate substituted amines. More powerfully, a subset of imine reductases, the reductive aminases, can catalyze both imine formation and imine reduction to permit conversion of ketones into substituted amines (Scheme 1). Scheme 1. Pathway for Reductive Aminase Transformation

Enzyme-catalyzed reductive aminations face a number of challenges: (1) in an aqueous environment, many imines are unstable and may be present in only small quantities; (2) binding both the amine and ketone in the enzyme active site, such that reduction of the resulting imine can occur before hydrolysis, may be possible but requires binding of three molecules simultaneously in the active siteNADPH, the amine, and the ketone; (3) the enzyme must be selective for imine reduction and must not have ketoreductase activity to avoid reduction of the ketone precursor. In addition, since most amines in nature are produced by transamination rather than by reduction of imines, the pool of native imine reductase enzymes was initially thought to be limited to a small number of enzymes having keto acids as substrates. For these reasons, the development of imine reductases and amine reductases was quite limited until very recently. Early work on reductive aminases was carried out by Rozzell at Biocatalytics, who in 2007 disclosed the conversion of prochiral ketones to chiral amines using ammonia and amine dehydrogenase enzymes.13 Reductive aminase enzymes, which have keto acids as their natural substrates, were engineered to operate on non-natural ketones, expanding the scope of this class of enzyme. However, ammonia was still the only amine donor that was viable, resulting in a scope that included only primary amines as products. The first example of an enzyme-catalyzed asymmetric imine reduction was reported in 2008 by Vaijayanthi and Chadha,14 who used whole cells of the fungal species Candida parapsilosis for the asymmetric reduction of water-stable aryl imines, with yields ranging from 55 to 80% and ee’s of the R enantiomer ranging from 95 to 99%. In 2010, Mitsukura and co-workers reported the reduction of 2-methyl-1-pyrroline using whole cells of Streptomyces species, obtaining high ee’s of either enantiomer depending on the strain used.15 This work also appeared in two patent applications assigned to Daicel Corporation, although neither patent was issued in the U.S.16 In 2010, Espinoza-Moraga and co-workers reported the biocatalytic reduction of 14 water-stable dihydro-β-

Figure 1. Publications and patents on imine reductase enzymes for asymmetric synthesis through April 2018.

view of the short time from the initial interest in the development of imine reductases, it is not surprising that no scalable processes have yet been reported in the literature or patents. As noted in our previous review,2 the majority of the patent activity regarding imine reductases has come from the laboratories of Codexis. A 2013 patent application assigned to Codexis disclosed the company’s initial efforts on the development of viable imine reductases.20 The starting enzyme for this work was opine dehydrogenase, a naturally occurring enzyme that catalyzes the reductive amination of pyruvate with L-norvaline to produce amine 1 (Scheme 2). The patent Scheme 2. Opine Dehydrogenase Catalyzes the Reductive Amination of Pyruvate with L-Norvaline

application disclosed enzyme-catalyzed reactions of pyruvate with several unnatural amines (EtNH2, 2-PrNH2, and nBuNH2) as well as the reaction of the unnatural substrate cyclohexanone with L-norvaline. The application provided only screening information with no details on conversions, yields, or enantioselectivity. Since then, eight additional applications from Codexis have been published, as well as eight granted patents.21 However, these patents and patent applications have provided no new B



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(Table 1, entry 7) to afford product with 92% ee. In these experiments, glucose dehydrogenase (GDH) was used to regenerate NADPH by oxidation of D-glucose to gluconolactone, which is irreversibly hydrolyzed to gluconic acid. In control experiments without IRED present, the team surprisingly found that GDHs from multiple sources had IRED activity with conversions of >50% and ee’s up to 99%.26 In a contribution from the laboratories of Turner at Manchester and process chemists at Astrazeneca, reductions of several five-, six-, and seven-membered cyclic imines were carried out with an R-selective IRED from Streptomyces sp. GF3587, originally identified by Mitsukura15b as having R selectivity for reduction of five-membered cyclic imines.27 The gene for this IRED was overexpressed in E. coli to generate a whole-cell biocatalyst with broad substrate scope and activity that was orders of magnitude greater than those of previously reported IREDs. A 1 g preparative-scale reduction of 2-npropyl-1-piperideine was carried out (Table 1, entry 1) to afford product in 90% yield with >98% ee. The researchers also noted that this enzyme had no activity for ketone reduction, which sets the stage for intermolecular reductive aminations. Turner’s group at Manchester is also collaborating with Pfizer on the use of IREDs for reductive amination. IREDs were identified that catalyze the reaction of simple ketones with ammonia, methylamine, benzylamine, allylamine, pyrrolidine, and aniline.28 This is one of the first reported examples of IREDs capable of accepting a more diverse set of amines. In the only asymmetric preparative-scale experiment reported, 1N-Cbz-3-pyrrolidinone was reacted with methylamine (5 equiv) to afford the reductive amination product in 18% yield with 94% ee (Table 1, entry 8). Wuhan University and Abiochem have teamed up to develop IREDs capable of handling more sterically congested imine substrates.22 Starting from a known IRED (IR71) in the Imine Reductase Engineering Database,18a 95 novel IREDs and five known IREDs were synthesized and expressed in E. coli. Of these, four enzymes were found to be active for the enantioselective reduction of a range of 1-aryl dihydroisoquinolines with high conversion (Table 1, entry 2). Replacing a tryptophan amino acid at position 191 with alanine was found to be critical to allow binding of larger substrates in the binding pocket, improving the catalyst performance by 2 orders of magnitude. In summary, over the past decade, considerable progress has been made toward the development of IREDs for reductive amination of ketones. The creation of a database of potential IREDs containing >1400 protein structures18a has shown that such enzymes are not as rare in nature as originally thought. The proteins from this database are proving highly valuable in providing a source of IREDs for further protein engineering. 1.2. Ketoreductase (Carbonyl Reductase) Enzymes for Asymmetric Ketone Reductions. Ketoreductase enzymes (KREDs), also called carbonyl reductases (CREDs), reduce ketones and aldehydes to their corresponding alcohols using NAD(P)H as a cofactor and a terminal reductant that is generally either glucose, formate, or 2-propanol (Scheme 4). Reactions are often carried out with 2-PrOH as a cosolvent, which also serves as the terminal reductant (oxidation to acetone). KRED reductions have a long history, with the use of yeast for reductions dating back at least a century.29 However, yeast reductions were difficult to implement because of the numerous enzymes present with opposing selectivity that often resulted in low enantioenrichment.30 In addition, the need for

information regarding substrate scope, and no conversions, yields, or enantioselectivities are included. The claims are focused only on new enzyme sequences. Other than the Codexis patents, during the past 5 years only one other patent application on the use of IREDs for asymmetric synthesis has been filed, from Wuhan University,22a and the content in this application has also been reported in a journal publication.22b This work is discussed further below. One interesting short publication in the IP.com Prior Art Database is a disclosure from Pfizer on the use of IREDs for the preparation of the antidepressant (S,S)-sertraline.23 An enzyme from the salt-tolerant bacteria Myxococcus fulvus was isolated, subcloned, and expressed in Escherichia coli to provide an IRED enzyme capable of carrying out a diastereoselective imine reduction of 2 to afford (S,S)-sertraline (Scheme 3). Scheme 3. Diastereoselective Imine Reduction To Furnish (S,S)-Sertraline

The lack of patent applications by pharmaceutical and fine chemical companies does not imply that these companies are not interested in IREDs. Many large pharma companies are actively pursuing IRED technology via collaborations with academia and are reporting results via journal publications instead of patents. Recent publications from four such collaborations are highlighted below. Table 1 summarizes IRED-mediated imine reductions or reductive aminations that have been reported on preparative scale (4 mg to 1 g). At this early stage of development, the substrate scope is still limited to relatively simple ketones and amines, and no larger-scale processes have been reported. Process chemists at Roche, in collaboration with teams at two German universities, Ernst-Moritz-Arndt-Universität Greifswald and Albert-Ludwigs-Universität Freiburg, have published three papers on the development of IREDs. In the first paper, the team used bioinformatics tools to mine protein databases to identify 23 potential IREDs from several bacterial sources.24 These enzymes were expressed in E. coli, isolated, purified to >90%, and then screened for imine reductase activity. All 23 enzymes provided some IRED activity. Two preparative-scale experiments (Table 1, entries 5 and 6) were carried out to provide enough product to establish the absolute stereochemistry. Both substrates afforded product with >98% ee.24 In the second paper, the group screened 33 IREDs for reductive amination of simple ketones with ammonia, methylamine, and 1-butylamine.25 The preparative-scale experiments (Table 1, entries 3−5) were carried out on 150−600 mg of ketone with purified enzyme preparations, providing ee’s or de’s of 96 to 99%. In the third paper, iminium ions were also shown to be amenable to asymmetric reduction by IREDs to form tertiary amines.26 One preparative-scale run (16 mg) was carried out C



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Table 1. Imine Reductase and Reductive Aminase Enzyme Reactions Carried Out on Preparative Scale

Isolated KREDs became commercially available in the mid 1990s30 and have now become a mature “tool” in the biocatalysis toolkit, with off-the-shelf R- and S-selective screening kits and the availability of enzymes at scale for use in commercial processes.32,33 Current research on ketoreductases is focused on incremental but important advancements, such as (1) widening the substrate scope; (2) improving the activity; (3) minimizing the loading of the expensive cofactor (nicotinamide adenine dinucleotide phosphate, NADP); (4) increasing the overall robustness by using higher substrate concentrations and a wider temperature range and improving the compatibility with organic solvents; (5) gaining efficiency through immobilization; and (6) applying KREDs for dynamic kinetic resolutions (DKRs) to set two contiguous chiral

Scheme 4. Enzymatic Carbonyl Reduction Using Glucose for Cofactor Recycle

high yeast loadings and dilute substrate conditions often resulted in difficult workups and tedious product purification.31 D



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Table 2. Aryl Ketone Reductions Using Ketoreductases

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Table 2. continued

centers. This section is divided into three parts: (a) aryl ketone reductions, (b) aliphatic ketone reductions, and (c) dynamic reductive kinetic resolutions. 1.2.1. Reduction of Aryl Ketones. Table 2 provides a summary of examples of aryl ketone reductions from recent patents and patent applications. Highlights from the table entries include the following:

1. use of high substrate loadings (>100 g/L) in most of the examples (entries, 1, 2, 4, 5, 7, 9, and 10); 2. use of 2-PrOH as a cosolvent ranging from the 25% level to the 60% level (entries 2, 3, 5, 6, 7, and 10); 3. use of biphasic conditions with n-heptane (entries 4 and 8); 4. use of PEG 6000 as a cosolvent (entry 8); F



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5. two examples from medicinal chemistry groups, both from Merck (entries 3 and 6); 6. back-integration of biocatalysis into commercial products, including phenylephrine (entry 2), Brilinta (entry 7), duloxetine (entry 9), and (S)-licarbazepine (entry 10)all four are highly productive processes with low KRED, NADP, and GDH loadings. 1.2.2. Aliphatic Ketone Reductions. Table 3 provides a summary of examples of aliphatic ketone reductions from recent patents and patent applications. Highlights from the table entries include the following: 1. Entries 1 (atazanavir), 3 (simeprevir), and 5 and 6 (ibrutinib) are examples of back-integration using ketoreductases for the preparation of intermediates for approved drugs. 2. Entries 3, 4, and 7 use biphasic solvent systems (toluene, MTBE, and 2-methyltetrahydrofuran). All three substrates are hydrophobic with very low water solubility. 3. Entry 2 is a contribution from a medicinal chemistry group (Pfizer). 1.2.3. Dynamic Reductive Kinetic Resolution. When a prochiral ketone has an adjacent chiral center, a selective ketone reduction accompanied by a dynamic kinetic resolution of the adjacent center can permit setting of two stereocenters.50 Scheme 5 outlines the elements of an enzymatic dynamic reductive kinetic resolution (DYRKR) using an example disclosed by Codexis in a 2009 patent application for the preparation of chiral alcohol 4, which is useful as an intermediate for the synthesis of β-lactam antibiotics.51 In this process, the KRED reduces only the S substrate 3S and is selective for reduction of the ketone to the (2S,3R) isomer 4 (k1 ≫ k2). If the reaction conditions can be tuned appropriately such that the R substrate 3R can epimerize (krac is large), then the racemic starting material can be converted into a single product, diastereomer 4. As noted in the review by Huisman,52 the early enzymes developed by Codexis provided good selectivity for reducing the ketone, but considerable enzyme engineering was required to evolve an enzyme that could set both stereocenters with good activity and high selectivity. Codexis has continued to patent additional enzymes for this transformation and has achieved a highly productive process with an initial substrate concentration of 350 g/L, >99% ee, and >99:1 dr (Table 4, entry 1).53 With keto ester substrate 8 (Table 4, entry 2), Merck carried out the DYRKR with a KRED obtained from Codexis and used DMSO as a cosolvent, which was likely required to increase the solubility of the substrate. Alcohol 9 was prepared with >99:1 dr and >99% ee.54 In an alternate approach to the same final target molecule, Merck and Codexis have disclosed a DYRKR on substrate 10 (Table 4, entry 3).55 Noteworthy about this substrate is that it is far less acidic than the doubly activated keto esters that have been the standard substrates for enzymatic DYRKRs. In their review, Applegate and Berkowitz50 note that most substrates for enzymatic DYRKRs have pKa’s in the 7.5 to 12.5 range. As an example, ethyl acetoacetate has a pKa of 10.7 in water.56 Compound 10 is well outside this range, having an aqueous pKa near 17.57 To achieve racemization of this compound for the DYRKR, the enzyme was engineered to be stable at pH 10 and a temperature of 45 °C in a solvent mixture of 50% 2PrOH. No yield, ee, or dr data are given for the example

Table 3. Aliphatic Ketone Reductions Using Ketoreductases

provided, although dr’s > 100 were reported for screening experiments. The patent application also describes non-covalent immobilization of the KRED and the cofactor NADP on a polymethacrylate resin simply by stirring the enzyme and NADP in a buffer with the resin present.55 The enzyme was also immobilized with an apolar covalent resin on porous beads (ChiralVision). Immobilization on the beads allowed for the DYRKR to be conducted in 90% 2-PrOH/10% water using G



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Scheme 5. Dynamic Reductive Kinetic Resolution Using a KRED

Table 4. Recent Dynamic Reductive Kinetic Resolution (DYRKR) Examples

the strong organic base 1,4-diazabicyclooctane (DABCO) and a temperature of 50 °C to facilitate the racemization. The conversion and stereospecificity were similar to those for the reaction in solution, with 98% conversion, 99% ee, and 100:1 dr. The immobilized enzyme was recycled for nine rounds with no loss of specificity. After filtration of the immobilized enzyme, phosphoric acid was added to crystallize the DABCO phosphate salt, which was filtered away to afford the product 11 as a solution in 2-PrOH. Suzhou Lead Biotechnology Co has disclosed DYRKRs on two substrates for the preparation of chloramphenicol (15) and the chloramphenicol intermediate 13 (Table 4, entries 4 and 5).58 Similar to the Merck/Codexis example in entry 3, these substrates are weakly acidic with an estimated pKa in water of about 16.59 Six enzymes were surveyed for the two substrates. Most gave excellent ee’s, but the dr’s ranged from 2:1 to 99:1. Using the best enzyme from the survey, the DYRKR of the methyl analogue 12 was carried out on a 50 g scale. After hydrolysis of the amide, the (R,R) free amine product was isolated in 87% yield after crystallization with >99:1 dr and >99% ee. A different enzyme was required for the dichloro analogue 14. The DYRKR for this substrate was also carried out on a 50 g scale to afford 15 with >99:1 dr and >99% ee. The DYRKRs of both substrates were carried out at pH 6.5 and 30 °C, conditions that would not be expected to afford rapid racemization of the substrates. The enzyme may also serve as a racemization catalyst for these substrates. 1.3. Transaminase Reactions. Transaminase enzymes catalyze the formation of primary amines from ketones and aldehydes via transfer of an amine group from an amine donor via pyridoxal 5′-phosphate (Scheme 6). The primary natural function of transaminases is to convert keto acids to amino acids. The substrate scope for transaminases has been expanded beyond natural keto acids through protein engineering of known transaminase enzymes, through searches for new enzymes in the environment, and from structure-based mining of the vast protein database.60 The pioneering work of Merck and Codexis on the development of an engineered transaminase enzyme for the manufacture of sitagliptin has stimulated the expanded use of transaminase chemistry in the pharmaceutical and fine chemical industry.12 A summary of transamination reactions reported in recently published patents and patent applications is presented in Table 5. Both Infinity61 and Codexis62 have described transamination chemistry to install the amine of 16, an intermediate for the

synthesis of drug candidate IPI-926 (Table 5, entry 1). The one-step transamination was developed to replace the four-step original approach that required reduction of the ketone to the alcohol, activation as a mesylate, displacement with azide, and reduction to the amine. The lack of water solubility of the large molecule presented a significant challenge for the biotransformation. The best conditions from Infinity involved the use of 10% MeOH and a pH of 7.5, which afforded 40% conversion on a 14 mg scale.61 Codexis employed a solvent system that included 25% DMSO and achieved >95% conversion and 99% dr on a 200 mg scale with a substrate concentration of 20 g/ L.62 H



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A patent application from Embio discloses a transaminase approach to (1R,2S)-norephedrine from 1-(R)-hydroxy-1phenylacetone at pH 8, 25−35 °C. The product was isolated as an oxalate salt with >99% dr (Table 5, entry 7).69 A patent application from Forschungszentrum Jü lich discloses a three-enzyme process to prepare (1S,2S)norpseudoephedrine starting from benzaldehyde and acetaldehyde or pyruvic acid (Scheme 10).70 In the first step, an Sselective lyase is used to form the carbon−carbon bond to prepare 1-(S)-hydroxy-1-phenylacetone with 70% ee. To upgrade the ee, a benzaldehyde lyase is added to convert the undesired R enantiomer back to starting materials, which are then recycled through the lyase process to ultimately generate the product with high ee. This step is followed by the transaminase reaction, which selectively generates the (1S,2S) product with 97% ee. Enzymeworks has disclosed in a Chinese patent application a dynamic kinetic resolution to set two stereocenters in an approach to a key fragment of moxifloxacin (Scheme 11).71 The transaminase reaction of ketone 21 is carried out at pH 9 in 20% DMSO at 35 °C, conditions that allow racemization of the starting material since only one enantiomer reacts. The amine intermediate readily cyclizes to the fused ring system 22 under the reaction conditions. The chemistry is described on a 50 g scale to provide 99.8% conversion, 99.9% ee, 99.9% de, and 95% yield. Scientists from Evonik have disclosed the conversion of primary alcohols to amines using a dual enzyme system including an alcohol dehydrogenase (ADH) to oxidize the alcohol to the corresponding aldehyde followed by a transaminase for conversion of the aldehyde to the amine.72 1-Hexanol, 1-octanol, 1-decanol, and 1-dodecanol were used as substrates in screening experiments on a 5 mg scale, but only 1-hexanol gave complete conversion to 1-hexylamine. Conversion of 1,8-octanediol to the corresponding diamine was also successful when 10−20% DME was used as cosolvent. BASF has described the transformation of an alcohol to a chiral amine using a combination of an ADH and a transaminase (Scheme 12).73 Either enantiomer of the starting alcohol (23R or 23S) can be oxidized depending on the type of ADH used (Prelog vs anti-Prelog). NAD+/NADH is used as the cofactor for both enzymes. The resulting phenylacetone (24) is then reacted with an R-selective transaminase using ammonium ion as the amine source to afford chiral amine 25 with >99% ee. Examples were described only on a 1 mL scale.

Scheme 6. Conversion of Ketones to Amines via Transaminase Enzymes

Compounds 17 and 18 (Table 5, entries 2 and 3) are intermediates for MGAT2 inhibitors from the medicinal chemistry group at Merck, with transaminase examples described on a 5 g scale.63 Alternate routes to similar compounds within the patent application required use of the Ellman auxiliary to install the desired amine stereochemistry in three steps. Cipargamin is a drug candidate being studied by Novartis for the treatment of malaria. Transaminase chemistry on a 30 g scale to produce cipargamin indole intermediate 19 from the corresponding ketone was reported (Table 5, entry 4). The solvent mixture included 10% PEG 200 and a reaction temperature of 50 °C. Indole 19 was crystallized as the(+)CSA salt in 84.5% yield. Pfizer scientists have devised a route to pregabalin that uses transaminase chemistry in the final step (Scheme 7 and Table 5, entry 5). The chemistry was demonstrated on a 2.6 g scale, affording a yield of 61% with 99.8% ee. Since the stereochemistry is set adjacent to the aldehyde, this is an example of a dynamic kinetic resolution, wherein only the S enantiomer of the aldehyde reacts and the R enantiomer is racemized as the reaction proceeds. Glufosinate is a naturally occurring herbicide produced by soil bacteria.66 It is sold as a racemate, although the S isomer is the active species. Zhejiang University has applied for a Chinese patent of a route to (S)-glufosinate using a threeenzyme process (Scheme 8).67 In the primary step, keto ester 20 is converted to the amino acid (S)-glufosinate using a transaminase enzyme. Glutamic acid is used as the amine source and is converted to α-ketoglutamic acid during the process. Glutamate dehydrogenase (GLDH) is included in the mixture to convert α-ketoglutamic acid back to glutamic acid using ammonium formate as the amine source. This transformation requires NADH, which is recycled with glucose using GDH as the third enzyme in the overall process. Under the optimized conditions, the substrate concentration was 1.1 M and the final product had 99.9% ee. A patent application from AgriMetis describes a two-enzyme process to generate the S isomer of glufosinate starting from the racemate (Scheme 9).68 In the first step, a D-amino acid oxidase is employed to oxidize the R enantiomer to keto acid 20, leaving the S enantiomer untouched. The keto acid is then converted to (S)-glufosinate using a transaminase enzyme and 2-PrNH2 as the terminal amine source. The amino acid oxidase and transaminase were immobilized together on porous glass beads and reused for 15 cycles, retaining >50% activity. An example performed on a 100 g scale was described.

2. OXIDATION 2.1. Hydroxylation. Selective enzymatic hydroxylation of active molecules has emerged as a method to generate new leads for drug discovery programs. In a Codexis patent application, the cancer drug nilotinib was selectively hydroxylated at one of the two aromatic methyl groups (Scheme 13). The reaction was carried out on 50 mg of substrate, proceeded to 34% conversion, and afforded 10 mg of isolated product 26. Larger quantities were made by a synthetic route that already incorporated the hydroxyl group in an early intermediate.74 Codexis has disclosed a selective hydroxylation of (S)pipecolic acid to provide (2S,5S)-hydroxypipecolic acid using engineered proline hydroxylase enzymes (Scheme 14).75 The reaction involves Fe2+ catalysis of the oxidative decarboxylation of α-ketoglutaric acid to succinic acid and CO2, which generates a reactive iron(IV)−oxo species that hydroxylates I



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Table 5. Enantio- and Diasteroselective Transaminase Reactions

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Table 5. continued

Scheme 7. Transaminase Chemistry To Prepare Pregabalin

Scheme 10. Three-Enzyme Route To Prepare (1S,2S)Norpseudoephedrine

Scheme 8. Transaminase Process To Prepare (S)Glufosinate

Scheme 11. Transaminase DKR To Form a Fused Ring System

Scheme 12. Conversion of a Chiral Alcohol to a Chiral Amine with Retention or Inversion of Stereochemistry

Scheme 9. Two-Enzyme Process To Convert Racemic Glufosinate to (S)-Glufosinate

(S)-pipecolic acid. Ascorbic acid is a cofactor that presumably serves to reduce oxidized iron species back to Fe2+.76 Starting with the wild-type cis-4-proline hydroxylase enzyme from Sinorhizobium meliloti, enzymes were progressively engineered to increase the activity toward hydroxylation of (S)-pipecolic acid and improve the regioselectivity. The early K



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Scheme 13. Selective Enzymatic Oxidation of Nilotinib

NADPH and flavin adenine dinucleotide (FAD), which are both bound to the enzyme. The catalytic cycle involves NAD(P)H reduction of FAD, which then reacts with oxygen to form a C4a-(hydro)peroxyflavin intermediate, which is the active oxygenating agent.80 A KRED is also required to oxidize NADP back to NADPH. In one example (Scheme 15), 2PrOH was used as the terminal oxidant. In the largest-scale example provided in the patents and patent applications (15 g of carboxylic acid 28), the conversion was carried out with a substrate loading of 100 g/L in a solvent composition consisting of 85% buffer at pH 8.5, 5% 2-PrOH, and 10% PEG 200 at 35 °C. The CHMO contained FAD, so no additional FAD was required. After a reaction time of 48 h, the sulfoxide was isolated by acidifying the mixture to pH 3 with 6 M HCl, which resulted in precipitation of the product in quantitative yield with 99.85% ee.78 Codexis has also engineered a CHMO for the synthesis of esomeprazole, the S enantiomer of the proton pump inhibitor omeprazole, via oxidation of the sulfide to the sulfoxide (Scheme 16).81,82 Successive rounds of enzyme screening were

Scheme 14. Selective Hydroxylation of (S)-Pipecolic Acid

variants gave poor conversions and furnished nearly equal amounts of hydroxylation at the 3- and 5-positions. Several rounds of optimization generated enzymes capable of >95% conversion at a substrate loading of 30 g/L using 6 g/L enzyme, generating less than a 1% yield of the 3-hydroxy isomer. In addition, the conversion of L-lysine to (2S,5S)hydroxypipecolic acid using a dual enzyme system (lysine cyclodeaminase and proline hydroxylase) was demonstrated on a 16 g scale. 2.2. Sulfur Oxidation. As noted in a review by Osborne and Milczek,77 enantioselective sulfide to sulfoxide oxidations that are useful for chemical synthesis at scale require balancing a large number of factors, including poor solubility of the substrate, overoxidation to the sulfone, formation of reactive hydrogen peroxide that can cause degradation, and the development of a compatible cofactor regeneration system with a second enzyme. Codexis has developed an enantioselective sulfur oxidation to prepare armodafinil (29) or its carboxylic acid precursor 30 (Scheme 15).78 Armodafinil is the single enantiomer of the racemic drug modafinil, a treatment for sleep disorders. Codexis initiated work with a cyclohexanone monooxygenase (CHMO) from the bacteria Acinetobacter sp., which had been previously reported to oxidize thioethers to sulfoxides with S selectivity.79 This enzyme uses oxygen and the cofactors

Scheme 16. Asymmetric Sulfur Oxidation To Produce Esomeprazole

conducted to generate enzymes with improved activity that were stable toward cosolvents, high pH, and the other enzymes present (KRED, catalase) while affording high conversions and ee with minimal overoxidation. On a 30 g scale, the reaction was carried out with a substrate concentration of 50 g/L in pH 9.0 buffer at 25 °C for 48 h with 4% 2-PrOH. Catalase (50 000 units/L) was also added to reduce hydrogen peroxide to water to prevent product degradation. After workup, esomeprazole was crystallized from MIBK/n-heptane in 87% yield with 99% chemical purity and 99.8% ee.81,82 A number of other pyrazole analogues were also oxidized selectively, providing either the R or S enantiomer depending on enzyme used, although these examples were described only on a screening scale.81 A Chinese patent application from Qingdao University of Science and Technology describes an immobilized whole cell oxidation for the production of esomeprazole.83 One example was described on a 5000 L reactor scale. The reaction was carried out at pH 8.5 in a biphasic system with toluene at 25 °C and a substrate concentration of 150 g/L to afford esomeprazole in 97% yield with 99.5% ee.83 2.3. D-Amino Acid Oxidase. Tongli Biomedical has been granted a patent for a process for the preparation of (R)-

Scheme 15. Enzymatic Cycle for the Enantioselective Sulfur Oxidation in the Synthesis of Armodafinil

L



Review

praziquantel.84 An early step in the process is an enzymatic dynamic kinetic deracemization−resolution of tetrahydroquinoline 32 using recombinant D-amino acid oxidase (DAOO) (Scheme 17). In the presence of oxygen, this enzyme oxidizes

Scheme 19. Nitrilase Resolution of a Pregabalin Intermediate

Scheme 17. Enzymatic Dynamic Kinetic Deracemization− Resolution with D-Amino Acid Oxidase

Scheme 20. Nitrilase Resolution of an Ivabradine Intermediate

the S enantiomer of the starting material to imine 34, which is then reduced back to racemic starting material using borane tert-butylamine complex. Catalase is added to the reaction mixture to reduce hydrogen peroxide, the reduction product of oxygen, to water. The cycle leads to complete conversion to the R enantiomer 33 with 92.5% recovery and 99.3% ee.84

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SUMMARY In this review, we have highlighted biocatalysis employed for the synthesis of compounds of pharmaceutical interest described in the patent literature over the past 3 years. Biocatalysis has become an integral technology for the design and development of efficient processes for the pharmaceutical and fine chemical industries. Through protein engineering, enzymes can be tailored for a particular substrate, allowing for the development of highly selective and efficient biocatalytic reactions on an industrial scale. Advances in adjacent technologies, such as metagenomics, genetic engineering, and bioinformatics, are laying the groundwork for the identification and development of new enzymes to enable a wider scope of unnatural substrates for reactions that are either rare or unknown in nature and for completely new reactions. Genetic engineering and synthetic biology are giving rise to the next generation of biocatalysis, the creation of modified organisms in which several enzymatic steps can be organized within a cell to synthesize complex molecules starting from basic fermentation starting materials.

3. HYDROLYSIS 3.1. Nitrilase Resolutions. Resolution of nitriles can be accomplished by selective hydrolysis of one enantiomer of a racemic compound to its carboxylic acid, which can then be separated from the unreacted nitrile by acid−base extraction. Genentech/Hoffman-La Roche reported the resolution of nitrile rac-35 on a 200 g scale at pH 7 or 9 and 20−25 °C (Scheme 18).85 After 55% conversion, the ee of the remaining nitrile (S)-35 was 99.7%. After an extractive workup, the resolved nitrile (S)-35 was obtained in 45% yield. Scheme 18. Nitrilase Resolution of rac-35

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Zhejiang University of Technology and Zhejiang Chiral Medicine Chemicals Co. reported a route to a pregabalin intermediate that relies on a nitrilase resolution of bisnitrile rac-37 (Scheme 19).86 Only the primary nitrile was hydrolyzed, affording acid 38 with 99.5% ee on a 1.5 g scale. The optimum pH was 7.5−10, and the optimum temperature range was 30−35 °C. Scientists at Les Laboratories Servier reported the use of a nitrilase for resolution of an intermediate for ivabradine (Scheme 20).87 The hydrolysis of rac-39 on a 500 mg scale in 5% DMSO at pH 7 was described. The unreacted nitrile (R)39 was recovered in 36% yield (96% ee) while the yield of the isolated acid 40 was 39% with 96% ee. The unreacted (R)-39 could be racemized in a separate step using DBU. While the application includes a claim for an in situ racemization, no example was provided.

David L. Hughes: 0000-0001-5880-8529 Notes

The author declares no competing financial interest.

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ABBREVIATIONS ADH, alcohol dehydrogenase; CHMO, cyclohexanone monooxygenase; CSA, camphorsulfonic acid; CRED, carbonyl reductase enzyme; DAAO, D-amino acid oxidase enzyme; DABCO, 1,4-diazabicyclooctane; de, diastereomeric excess; DKR, dynamic kinetic resolution; DMSO, dimethyl sulfoxide; dr, diastereomeric ratio; DYRKR, dynamic reductive kinetic resolution; ee, enantiomeric excess; er, enantiomeric ratio; FAD, flavin adenine dinucleotide; GDH, glucose dehydrogenase; GLDH, glutamate dehydrogenase; IRED, imine reductase; KRED, ketone reductase; MeTHF, 2-methyltetrahydrofuran; M



Review

Fademrecht, S.; Hofelzer, S.; Pleiss, J.; Leipold, F.; Turner, N. J.; Nestl, B. M.; Hauer, B. Enzyme Toolbox: Novel Enantiocomplementary Imine Reductases. ChemBioChem 2014, 15, 2201−2204. (19) (a) Although it is a very young field, many reviews have already been written on imine reductase enzymes. See: (a) Mitsukura, K.; Yoshida, T. Imine Reductases for Chiral Amine Synthesis. In Future Directions in Biocatalysis, 2nd ed.; Matsuda, T., Ed.; Elsevier: New York, 2017; Chapter 5, pp 97−117. (b) Mangas-Sanchez, J.; France, S. P.; Montgomery, S. L.; Aleku, A. G.; Man, H.; Sharma, M.; Ramsden, J. I.; Grogan, G.; Turner, N. J. Imine Reductases (IREDs). Curr. Opin. Chem. Biol. 2017, 37, 19−25. (c) Lenz, M.; Borlinghaus, N.; Weinmann, L.; Nestl, B. M. Recent Advances in Imine ReductaseCatalyzed Reactions. World J. Microbiol. Biotechnol. 2017, 33, 199. (d) Grogan, G. Synthesis of Chiral Amines Using Redox Biocatalysis. Curr. Opin. Chem. Biol. 2018, 43, 15−22. (e) Leipold, F.; Hussain, S.; France, S. P.; Turner, N. J. Imine Reductases. In Science of Synthesis, Biocatalysis in Organic Synthesis; Faber, K., Fessner, W.-D., Turner, N. J., Eds.; Thieme: Stuttgart, Germany, 2015; Vol. 2, pp 359−381. (f) Kohls, H.; Steffen-Munsberg, F.; Höhne, M. Recent Achievements in Developing the Biocatalytic Toolbox for Chiral Amine Synthesis. Curr. Opin. Chem. Biol. 2014, 19, 180−192. (g) Sharma, M.; MangasSanchez, J.; Turner, N. J.; Grogan, G. NAD(P)H-Dependent Dehydrogenases for the Asymmetric Reductive Amination of Ketones: Structure, Mechanism, Evolution and Application. Adv. Synth. Catal. 2017, 359, 2011−2025. (h) Schrittwieser, J. H.; Velikogne, S.; Kroutil, W. Biocatalytic Imine Reduction and Reductive Amination of Ketones. Adv. Synth. Catal. 2015, 357, 1655−1685. (20) Chen, H.; Moore, J. C.; Collier, S. J.; Smith, D.; Nazor, J.; Hughes, G.; Janey, J.; Huisman, G. W.; Novick, S. J.; Agard, N. J.; Alvizo, O.; Cope, G. A.; Yeo, W. L.; Minor, S. N.; Sukumaran, J.; Ng, S. Engineered Imine Reductases and Methods for the Reductive Amination of Ketone and Amine Compounds. PCT Int. Patent Application WO 2013/170050A1, Nov 14, 2013. (21) (a) Chen, H.; Collier, S. J.; Nazor, J.; Sukumaran, J.; Smith, D.; Moore, J. C.; Hughes, G.; Janey, J.; Huisman, G. W.; Novick, S. J.; Agard, N. J.; Alvizo, O.; Cope, G. A.; Yeo, W. L.; Minor, S. N. Engineered Imine Reductases and Methods for the Reductive Amination of Ketone and Amine Compounds. U.S. Patent 9,932,613, April 3, 2018. (b) Chen, H.; Collier, S. J.; Nazor, J.; Sukumaran, J.; Smith, D.; Moore, J. C.; Hughes, G.; Janey, J.; Huisman, G. W.; Novick, S. J.; Agard, N. J.; Alvizo, O.; Cope, G. A.; Yeo, W. L.; Minor, S. N. Engineered Imine Reductases and Methods for the Reductive Amination of Ketone and Amine Compounds. U.S. Patent 9,828,614, Nov 28, 2017. (c) Chen, H.; Collier, S. J.; Nazor, J.; Sukumaran, J.; Smith, D.; Moore, J. C.; Hughes, G.; Janey, J.; Huisman, G. W.; Novick, S. J.; Agard, N. J.; Alvizo, O.; Cope, G. A.; Yeo, W. L.; Minor, S. N. Engineered Imine Reductases and Methods for the Reductive Amination of Ketone and Amine Compounds. U.S. Patent 9,803,224, Oct 31, 2017. (d) Chen, H.; Collier, S. J.; Nazor, J.; Sukumaran, J.; Smith, D.; Moore, J. C.; Hughes, G.; Janey, J.; Huisman, G. W.; Novick, S. J.; Agard, N. J.; Alvizo, O.; Cope, G. A.; Yeo, W. L.; Minor, S. N. Engineered Imine Reductases and Methods for the Reductive Amination of Ketone and Amine Compounds. U.S. Patent Application 2017/0268028 A1, Sept 21, 2017. (e) Chen, H.; Collier, S. J.; Nazor, J.; Sukumaran, J.; Smith, D.; Moore, J. C.; Hughes, G.; Janey, J.; Huisman, G. W.; Novick, S. J.; Agard, N. J.; Alvizo, O.; Cope, G. A.; Yeo, W. L.; Minor, S. N. Engineered Imine Reductases and Methods for the Reductive Amination of Ketone and Amine Compounds. U.S. Patent Application 2017/0022527 A1, Jan 26, 2017. (f) Chen, H.; Collier, S. J.; Nazor, J.; Sukumaran, J.; Smith, D.; Moore, J. C.; Hughes, G.; Janey, J.; Huisman, G. W.; Novick, S. J.; Agard, N. J.; Alvizo, O.; Cope, G. A.; Yeo, W. L.; Minor, S. N. Engineered Imine Reductases and Methods for the Reductive Amination of Ketone and Amine Compounds. U.S. Patent Application 2016/0168545 A1, June 16, 2016. (g) Chen, H.; Collier, S. J.; Nazor, J.; Sukumaran, J.; Smith, D.; Moore, J. C.; Hughes, G.; Janey, J.; Huisman, G. W.; Novick, S. J.; Agard, N. J.; Alvizo, O.; Cope, G. A.; Yeo, W. L.; Minor, S. N. Engineered Imine Reductases and Methods for the Reductive Amination of Ketone and

MIBK, methyl isobutyl ketone; MTBE, methyl tert-butyl ether; NADP, nicotinamide adenine dinucleotide phosphate; PEG, poly(ethylene glycol); PLP, pyridoxal 5′-phosphate

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boxylic Acid and Application in the Synthesis of Ivabradine and Salts Thereof. U.S. Patent 9,476,071 B2, Oct 25, 2016.

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