Biocatalysis in Drug Development â Highlights of the Recent Patent

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Biocatalysis in Drug Development – Highlights of the Recent Patent Literature David L Hughes Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.8b00232 • Publication Date (Web): 10 Aug 2018 Downloaded from http://pubs.acs.org on August 10, 2018

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Organic Process Research & Development

Biocatalysis in Drug Development – Highlights of the Recent Patent Literature David L. Hughes Cidara Therapeutics, Inc., 6310 Nancy Ridge Dr., Suite 101, San Diego, California 92121, United States E-mail: [email protected]

1 ACS Paragon Plus Environment

Organic Process Research & Development 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 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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GRAPHICAL ABSTRACT:

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.

KEY WORDS: biocatalysis, transaminase, ketoreductase, nitrilase, imine reductase, dynamic reductive kinetic resolution, sulfur oxidation, hydroxylation


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The 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 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 so than other technologies in process chemistry, collaboration between the specialty companies and large pharma, and between academia and industry, have been a key driver for 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 – the modification of enzymes to accept non-natural substrates and to be tolerant of conditions outside natural conditions, such as organic co-solvents, high substrate concentrations, higher temperatures and wider pH ranges; 4. Metabolic engineering – modifying the metabolism of an organism by altering genetic and regulatory processes to produce or increase production of a desired molecule; 3 ACS Paragon Plus Environment


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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. 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,4 and Lalonde.5 In addition, Chemical Reviews devoted its first issue in 2018 to biocatalysis and 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 includes an outstanding review on industrial implementation of biocatalysis by Sheldon.9 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.


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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 have become more common and at least one transaminase process has been introduced into commercial manufacturing, Merck’s sitagliptin.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 will be unstable and may be present in only small quantities; (2) 5 ACS Paragon Plus Environment


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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 keto-reductase 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 ketoacids as substrates. For these reasons, 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 ketoacids as their natural substrate, 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 only included primary amines as products. The first example of an enzyme-catalyzed asymmetric imine reduction was reported by Vaijayanthi and Chadha in 2008 using whole cells of the fungal species Candida parapsilosis for the asymmetric reduction of water-stable aryl imines, with yields ranging from 55-80% and ee’s of the R-enantiomer ranging from 95 to 99%.14 In 2010 Mitsukura and co-workers reported 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


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dihydro-beta-carboline imines to chiral amines using various yeast species with ee’s ranging from 50-97%.17 Since these early reports, imine reductases have been characterized from several microbial species, suggesting these enzymes are perhaps more widespread in Nature than originally anticipated. Based on analyses of protein databases, Fadermrecht and co-workers have built a database that currently includes 1400 sequences that could be potential IRED enzymes.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 Given 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.

22 18

13 12 11

5 1

0

1

2

1

2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018



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Figure 1. Publications and Patents on Imine Reductase Enzymes for Asymmetric Synthesis through April 2018

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 application disclosed enzyme-catalyzed reactions of pyruvate with several unnatural amines (EtNH2, 2-PrNH2, and n-BuNH2) as well as the unnatural substrate, cyclohexanone, with L-norvaline. The application only provided screening information with no details on conversions, yields, or enantioselectivity. Scheme 2. Opine Dehydrogenase Catalyzes the Reductive Amination of Pyruvate with LNorvaline

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 information regarding substrate scope and no conversions, yields, nor enantioselectivities are included. The claims are focused only on new enzyme sequences.


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Other than the Codexis patents, during the past five years, only one other patent application on 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 IP.com Journal is a disclosure from Pfizer on the use of IREDs for the preparation of the anti-depressant (S,S)-sertraline.23 An enzyme from the salttolerant bacteria Myxococcus fulvus was isolated, sub-cloned and expressed in E. 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 these companies are not interested in IRED’s. 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



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development, 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, ErnstMoritz-Arndt-Universität Greifswald and Albert-Ludwigs-Universität Freiburg, have published three papers on development of IREDs. In the first paper, the team used bioinformatics tools to mine protein databases to identify 23 potential IRED’s from several bacterial sources.24 These enzymes were expressed in E. coli, isolated and purified to >90%, 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 absolute stereochemistry. Both substrates afforded product with >98% ee.24 In the second paper, the group screened 33 IRED’s 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 (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 GDH’s from multiple sources had IRED activity with conversions of >50% and ee’s up to 99%.26


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In a contribution from the laboratories of Turner at Manchester and process chemists at Astrazeneca, reductions of several cyclic 5-, 6-, and 7-membered imines were carried out with an R-selective IRED from Streptomyces sp. GF3587, originally identified by Mitsukura15b as having (R)-selectivity for reduction of 5-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 previously reported IREDs. A one gram preparative scale reduction of 2-n-propyl-1- piperideine was carried out (Table 1, entry 1) to afford product in 90% yield and >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. IRED enzymes were identified that catalyzed 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, 1-N-Cbz-3-pyrrolidinone was reacted with methylamine (5 equiv) to afford the reductive amination product in 18% yield and 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 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 for



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allowing binding of larger substrates in the binding pocket, improving catalyst performance by two orders of magnitude. Table 1. Imine Reductase and Reductive Aminase Enzyme Reactions Carried out on Preparative Scale Entry

Substrate

Product

Reaction Scale

Reference

and Outcome 1

1 g scale, 90%

27

yield, >98% ee 2

4-5 mg scale, 8-

22

99% ee, both enantiomers accessible, 9196% yields 3

NHMe

O

150 mg scale,

25

98% CH3

CH3

conversion, 98% de 4

400 mg scale,

25

62% yield, 96% ee 5

600 mg scale,

24,25

77% yield,


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>99% ee 6

98% ee, 82%

24

yield 7

16 mg scale,

26

92% ee 8

225 mg, 18%

28

yield, 94% ee

In summary, over the past decade, considerable progress has been made toward the development of IRED enzymes for reductive amination of ketones. The creation of a database18a of potential IRED enzymes, containing >1400 protein structures, 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 IRED enzymes for further protein engineering.

1.2 Ketoreductase (Carbonyl Reductase) Enzymes for Asymmetric Ketone Reductions Ketoreductase enzymes, also referred to as carbonyl reductases, (KRED, CRED) reduce ketones and aldehydes to their corresponding alcohols using NAD(P)H as co-factor and a terminal reductant that is generally either glucose, formate, or 2-propanol (Scheme 4). Scheme 4. Enzymatic Carbonyl Reduction using Glucose for Cofactor Recycle



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Reactions are often carried out with 2-PrOH as co-solvent, which also serves as the terminal reductant (oxidation to acetone). KRED reductions have a long history with the use of yeast for reductions that date back at least a century.29 However, yeast reductions were difficult to implement due the numerous enzymes present with opposing selectivity that often resulted in low enantio-enrichment.30 In addition, the need for high yeast loadings and dilute substrate conditions often resulted in difficult workups and tedious product purification.31 Isolated KREDs became commercially available in the mid 1990’s30 and have now become a mature “tool” in the biocatalysis toolkit, with off-the-shelf R- and S-selective screening kits and the availability at scale of enzymes for use in commercial processes.32,33 Current research on ketoreductases is focused on incremental but important advancements, such as (1) widening the scope of substrates; (2) improving activity; (3) minimizing the loading of the expensive co-factor (nicotinamide adenine dinucleotide phosphate, NADP); (4) increasing overall robustness by using higher substrate concentrations, a wider temperature range, and improving compatibility with organic solvents; (5) gaining efficiency through immobilization; and (6) applying KREDs for dynamic kinetic resolutions to set two contiguous chiral centers. This section is divided into 3


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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. Table 2. Aryl Ketone Reductions Using Ketoreductase Enzymes Entry

Product

Conditions

Comments

Patent Owner (Reference)

1

2

pH 7.0, 30 oC, no

Hindered substrate

co-solvent; highly

unreactive with

concentrated:

natural KRED’s.

initial loading of

94% yield, >99.8%

400 g/L.

ee.

130 g/L substrate;

91% yield as

50% 2-PrOH co-

crystalline HCl salt,

solvent; NADP

ee>99% on 50 g

0.05 g/L; 30 oC;

scale

Codexis (34)

Codexis (35)

pH 7



3

4

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20 g/L substrate;

Medicinal Chemistry Merck (36)

50/50 2-PrOH/aq.

example, 50 g scale,

phosphate buffer;

product isolated as

pH 7; 1.0 g/L

oxirane after in situ

NADP; 1.0 g/L

cyclization with

KRED; 28 oC

Et3N

125 g/L substrate;

100 g scale. Biphasic Roche (37)

3:1 phosphate

solvent mixture.

buffer: n-heptane;

Product crystallized

30 oC; pH 7.2;

from reaction

0.125 g/L NAD;

mixture in 97%

1.25 g/L KRED;

yield; 99.9% ee;

1.25 g/L GDH

2.6% epoxide byproduct.

5

250 g/L substrate;

100 g scale; product

3:1 phosphate

crystallized in 96%

buffer: 2-PrOH;

yield, >99% ee,

30 oC; pH 6.5-7.7;

1.2% epoxide by-

NADP 0.5 g/L;

product.

Roche (37)

KRED 1.25 g/L


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6

2:1 phosphate

Med Chem example;

buffer:2-PrOH, 30

650 mg scale

Merck (38)

o

C, pH 7.0

7

135 g/L substrate,

Intermediate for

Hangzhou

7:3 tris buffer: 2-

Brilinta; 1.35 kg

Meiyi

PrOH; pH 7; 30

scale; 80% yield,

Biotechnology

o

99.5% ee, 99.2%

(39)

C; 7.5 g/L KRED

purity.

8

75 g/L;

30 kg scale,

water/heptane/PEG 100% assay yield; 6000 bi-phasic

Genentech (40)

99.5% ee.

solvent mixture; pH 6.5-7.0; 29-31 o

C; 0.8 g/L for

GDH, NAD, and KRED

9

220 g/L substrate;

Intermediate for

Enzymeworks

pH 7; 0.7 g/L

duloxetine; 2 g scale, (41)

KRED and GDH;

very low



0.05 g/L NADP

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concentration of NADP, 95% isolated yield

10

100 g/L; 60% 2-

(S)-(+)-

PrOH co-solvent;

licarbazepine; 96%

1 g/L KRED; 55

yield, 99.9% ee

Codexis (42)

o

C

Highlights from the table entries: 1. High substrate loading >100 g/L is achieved in most of the examples (entries, 1, 2, 4, 5, 7, 9, 10). 2. Use of 2-PrOH as co-solvent ranging from 25-60% level (entries 2, 3, 5, 6, 7, 10). 3. Use of bi-phasic conditions with n-heptane (entries 4 and 8). 4. Use of PEG 6000 as co-solvent (entry 8). 5. Two examples from Medicinal Chemistry groups, both from Merck (entries 3 and 6). 6. Back-integration of biocatalysis into commercial products: 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


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Table 3 provides a summary of examples of aliphatic ketone reductions from recent patents and patent applications.

Table 3. Aliphatic Ketone Reductions Using Ketoreductase Enzymes Entry Product

Conditions

Comments


1

195 g/L substrate;

Atazanavir

400 mL

intermediate,

triethanolamine

90 g scale, 81%

buffer, pH 9.0; 2-

yield of

PrOH (9 % v/v),

oxirane,

0.65 g/L NAD;

>99.9% de.

Codexis (43)

1.3 g/L KRED, 45 o

C

2

25 g/L substrate;

Medicinal

2-PrOH (10%

Chemistry

v/v); pH 7.05; 1.0

example; 55%

g/L NADP; 4 g/L

yield after

KRED; 30 oC

crystallization

Pfizer (44)



3

Biphasic

Simeprevir

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Janssen (45)

toluene/aq. buffer; intermediate, 20 oC

1.2 kg scale, >98.5% de

4

8 g/L substrate;

Alios

5:1 aq.

Biopharma

buffer:MTBE; 30

(46)

o

C; pH 7.5

5

250 g/L substrate,

Ibrutinib

Syncozymes

5:3 buffer:2-

intermediate; 4

(47)

PrOH; 19 g/L

kg scale, 97%

KRED; pH 6.3

isolated yield, 99.9% ee

6

17 g/L substrate;

2 kg scale;

Kailaiying &

0.1 g/L NAD; 8

99.5% ee; 82%

Asymchem

g/L KRED; pH

yield

(48)

biphasic1:1 2-

5 g scale, 85%

Enzymeworks

MeTHF:buffer,;

yield

(49)

7.0 7

pH 6.5; 100 g/L; 1 g/L KRED; 0.4 g/L NAD; 25 oC


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Highlights from Table 3: 1. Entry 1 (atazanavir), entry 3 (simeprevir) and entries 5 and 6 (ibrutinib) are examples of back integration using ketoredutases for the preparation of intermediates for approved drugs. 2. Entries 3, 4 and 7 use biphasic solvent systems (toluene, MTBE, and 2methyltetrahydrofuran). All three substrates are hydrophobic with very low water solubility. 3. Entry 2 is a contribution from Medicinal Chemistry (Pfizer).

1.2.3

Dynamic Reductive Kinetic Resolution (DYRKR)

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 DYRKR using an example disclosed by Codexis in a 2009 patent application for the preparation of chiral alcohol 4, useful as an intermediate for the synthesis of β-lactam antibiotics.51 In this process, the KRED enzyme only reduces 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 rapid), then the racemic starting material can be converted into a single product, diastereomer 4. Scheme 5. Dynamic Reductive Kinetic Resolution Using a KRED Enzyme



O

O

OH OMe

NH O

OMe

KRED k1 (fast)

Ph

O

OMe NH O

Ph

Ph 5

4 krac

O

+

NH

3S

O

OH

O

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X OH

O

O

OH

O

KRED OMe

OMe NH O

k2 (slow)

Ph 3R

+

OMe

NH O

Ph 6

NH O

Ph 7

As noted in the Huisman review article,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 have achieved a highly productive process with an initial substrate concentration of 350 g/L, ee > 99% and dr >99:1 (Table 4, entry 1).53 Table 4. Recent Dynamic Reductive Kinetic Resolution (DYRKR) Examples Entry Ketone Precursor

Product

Conditions



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1

25 g scale, 350

Codexis

g/L, pH 7.2, 37

(51,53)

o

C, 1 g/L

KRED, 0.14 g/L NADP, >95% yield 2

120 g scale, 36

Merck (54)

g/L, 30-35 oC, DMSO cosolvent, >99% ee, >99:1 dr, 91% yield 3

1:1 buffer (pH

Merck &

10):2-PrOH, 50

Codexis (55)

g/L substrate, 0.1 g/L NADP, 1 g/L KRED, 45 o

C

4

100 g/L, pH 6.5,

Suzhou Lead

30 oC, 0.1 g/L

Biotechnology

NADP, 2 g/L

Co. (58)

GDH, 2 g/L g



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KRED, dr >98:2, ee>99%.

5

100 g/L, pH 6.5,

Suzhou Lead

30 oC, 3 g/L

Biotechnology

KRED, 2 g/L

Co. (58)

GDH, 0.1 g/L NADP, ee >99%, dr >99:1.

With keto-ester substrate 8 (Table 4, entry 2), Merck carried out the DYRKR with a KRED obtained from Codexis and used DMSO as co-solvent, likely required to provide some 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 DYRKR on substrate 10 (Table 4, entry 3).55 Noteworthy about this substrate is that it is far less acidic than the doubly-activated ketoesters that have been the standard substrates for enzymatic DYRKRs. In the Applegate and Berkowitz review,50 the authors 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 in water of 10.7.56 Compound 10 is well outside this range, having an aq. pKa near 17.57 To achieve racemization of this compound for the DYRKR, the enzyme was engineered to be stable at pH 10, a temperature of 45 oC, and a solvent mixture of 50% 2-PrOH. No yield, ee, or 24 ACS Paragon Plus Environment

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dr are provided for the example provided although dr’s >100 were reported for screening experiments. The patent application also describes non-covalent immobilization of the KRED enzyme and the co-factor 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 reaction to be conducted in 90% 2-PrOH/10% water using a strong organic base (1,4-diazabicyclooctane, DABCO) and a temperature of 50 oC to facilitate the racemization. Conversion and stereospecificity were similar to 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 dr’s ranged from 2:1 to 99:1. Using the best enzyme from the survey, the DYRKR of the methyl analog 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 dr>99:1 and ee>99%. A different enzyme was required for the dichloro analog 14. The DYRKR for this substrate was also carried out on a 50 g scale to afford chloramphenicol (15) with dr>99:1 and ee>99%. The DYRKR reactions of both substrates were carried out at a pH of 6.5 and a



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temperature of 30 oC, conditions that would not be expected to afford rapid racemization of the substrates. The enzyme may also be serving 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 ketoacids to amino acids. The substrate scope for transaminases has been expanded beyond natural ketoacids 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 for 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 Scheme 6. The Conversion of Ketones to Amines via Transaminase Enzymes

A summary of transamination reactions reported in recently published patents and patent applications is presented in Table 5.


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Table 5. Enantio- and Diastero-selective Transaminase Reactions Entry Product

Conditions


1

Infinity: 10% MeOH,

Infinity (61),

vibrio fluvialis enzyme,

Codexis (62)

pH 7.5, L-alanine as amine donor. Codexis: 20 g/L substrate, 0.5 g/L PLP, 1M 2-PrNH2, 25% DMSO, 2 g/L transaminase, pH 7.0.

2

2.5 g/L substrate, 2 g/L

Merck (63)

enzyme, 10% DMSO, 40 o

C, pH 7, 10 g/L 2-

PrNH2 3

NH2

2.5 g/L substrate, 2 g/L

Merck (63)

N

enzyme, 10% DMSO, 40 EtO

N 18

o

C, pH 7, 10 g/L 2-

PrNH2



4

30 g/L substrate, 0.2 g/L

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Novartis (64)

PLP, 10% PEG 200, 1.6 g/L enzyme, 50 oC, pH 7

5

70 g/L substrate, pH

Pfizer (65)

7.25, 45 oC

6

Zhejiang: 1.1M

Zhejiang

substrate, pH 7.3, 40 oC;

University (67),

Agrimetis: amino acid

AgriMetis (68)

oxidase and transaminase enzymes immobilized 7

pH 8, 25-35 oC

Embio (69)

8

3-enzyme process

Forschungszentrum

starting from

Jülich (70)

benzaldehyde and acetaldehyde or pyruvic


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acid DKR, pH 9, 35 oC, 20%

9

Enzymeworks (71)

DMSO

Both Infinity61 and Codexis62 describe 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 4-step original approach that required reduction of the ketone to 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 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, achieving >95% conversion and 99% dr on a 200 mg scale with a substrate concentration of 20 g/L.62 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 five gram scale.63 Alternate routes to similar compounds within the patent application required use of the Ellman auxiliary to install the desired amine stereochemistry in 3-steps. Cipargamin is a drug candidate being studied by Novartis for the treatment of malaria. Transaminase chemistry was reported on a 30 g scale to produce the cipargamin indole intermediate 19 from the corresponding ketone (Table 5, entry 4). The solvent mixture included



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10% PEG200 and a reaction temperature of 50 oC. The indole 19 was crystallized as (+)-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% and an ee of 99.8%. 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. Scheme 7. Transaminase Chemistry to Prepare Pregabalin

Glufosinate is a naturally occurring herbicide produced by soil bacteria.66 Glufosinate 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 3-enzyme process (Scheme 8).67 In the primary step, ketoester 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 αketoglutamic acid back to glutamic acid using ammonium formate as the amine source. This transformation requires NADH, which is recycled with glucose using glucose dehydrogenase (GDH) as the third enzyme in the overall process. With the optimized conditions, the substrate concentration was 1.1M and final product had an ee of 99.9%. 30 ACS Paragon Plus Environment

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Scheme 8. Transaminase Process to Prepare (S)-Glufosinate

A patent application from AgriMetis has published that 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 ketoacid 20, leaving the (S)enantiomer untouched. The ketoacid is then converted to (S)-glufosinate using a transaminase enzyme and 2-PrNH2 as the terminal amine source. Both the amino acid oxidase and transaminase enzymes were immobilized together on porous glass beads and were reused for 15 cycles, retaining >50% activity. An example was described on a 100g scale. Scheme 9. Two-Enzyme Process to Convert Racemic Glufosinate to (S)-Glufosinate



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A patent application from Embio discloses a transaminase approach to (1R,2S)-norephedrine from 1-(R)-hydroxy-1-phenylacetone at pH 8, 25-35 oC. The product was isolated as an oxalate salt with a dr >99% (Table 5, entry 7).69 A patent application from Forschungszentrum Jülich 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 (S)-selective lyase is used to form the carbon-carbon bond to prepare 1-(S)-hydroxy-1-phenylacetone with an ee of 70%. 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 product with high ee. This step is followed by the transaminase reaction which selectively generates the (1S,2S)-product with an ee of 97%. Scheme 10. Three-Enzyme Route to Prepare (1S,2S)-Norpseudoephedrine


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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 oC, conditions which 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. Scheme 11. Transaminase DKR to Form a Fused Ring System

Scientists from Evonik have disclosed conversion of primary alcohols to amines using a dual enzyme system including an alcohol dehydrogenase to oxidize the alcohol to the corresponding aldehyde followed by a transaminase for conversion to the amine.72 1-Hexanol, 1-octanol, 133 ACS Paragon Plus Environment


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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-hexyl amine. Conversion of 1,8-octanediol to the corresponding diamine was also successful when 10-20% DME was used as co-solvent. BASF has described an alcohol to chiral amine transformation using a combination of an alcohol dehydrogenase (ADH) and transaminase (Scheme 12).73 Either enantiomer of the starting alcohol (23R and 23S) can be oxidized depending on the type of ADH used (Prelog vs antiPrelog). NAD+/NADH is used as the co-factor 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 one mL scale. Scheme 12. Conversion of a Chiral Alcohol to Chiral Amine with Retention or Inversion of Stereochemistry

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 34 ACS Paragon Plus Environment

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was carried out on 50 mg of substrate, proceeded to 34% conversion, and afforded ten milligrams of isolated product 26. Larger quantities were made by a synthetic route which already incorporated the hydroxyl group in an early intermediate.74 Scheme 13. Selective Enzymatic Oxidation of Nilotinib

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 (S)pipecolic acid. Ascorbic acid is co-factor which 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 activity toward hydroxylation of (S)pipecolic acid and improve regioselectivity. The early variants gave poor conversions and furnished nearly equal amounts of hydroxylation at the 3- and 5-positions. After several rounds of optimization, enzymes were generated capable of >95% conversion at a substrate loading of 30 g/L using 6 g/L enzyme, generating less than 1% of the 3-hydroxy isomer. In addition, using a dual enzyme system (lysine cyclodeaminase and proline hydroxylase), the conversion of L-lysine to (2S,5S)-hydroxypipecolic acid was demonstrated on a 16 g scale. 35 ACS Paragon Plus Environment


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

2.2 Sulfur Oxidation As noted in a review article from Osborne and Milczek, enantioseletive 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, over-oxidation to the sulfone, formation of reactive hydrogen peroxide that can cause degradation, and development of a compatible cofactor regeneration system with a second enzyme.77 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 co-factors 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 ketoreductase (KRED) enzyme is also required to oxidize NADP back to NADPH. In one example (Scheme 15), 2-PrOH was used as the terminal oxidant. 36 ACS Paragon Plus Environment

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

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 oC. The CHMO enzyme contains 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 6M HCl, which resulted in precipitation of the product with quantitative yield and 99.85% ee.78 Codexis has also engineered a CHMO enzyme for the synthesis of esomeprazole, the (S)enantiomer of the proton pump inhibitor omeprazole, via oxidation of the sulfide to sulfoxide (Scheme 16).81,82 Successive rounds of enzyme screening were conducted to generate enzymes with improved activity that were stable to cosolvents, high pH, and the other enzymes present (KRED, catalase), while affording high conversions and ee with minimal over-oxidation.



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Scheme 16. Asymmetric Sulfur Oxidation to Produce Esomeprazole

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 oC for 48 h, with 4% 2-PrOH. Catalase enzyme (50,000 units/L) was also added to reduce hydrogen peroxide to water to prevent product degradation. After work up, esomeprazole was crystallized from MIBK/n-heptane in 87% yield, 99% chemical purity, and 99.8% ee.81,82 A number of other pyrazole analogs 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 oC and a substrate concentration of 150 g/L to afford esomeprazole in 97% yield and 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)praziquantel.84 An early step in the process is an enzymatic dynamic kinetic deracemizationresolution of tetrahydroquinoline 32 using recombinant D-amino acid oxidase (DAOO) (Scheme 17). In the presence of oxygen, this enzyme oxidizes the (S)-enantiomer of the starting material 38 ACS Paragon Plus Environment

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to imine 34, which is then reduced back to racemic starting material using borane—t-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 Scheme 17. Enzymatic Dynamic Kinetic Deracemization-Resolution with D-Amino Acid Oxidase

NH DAAO, air NH

o

35 C, pH 8.3

33 CO2H +

CO2H 32

BH3 t-BuNH2

N 34 CO2H

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 resolution of nitrile rac-35 on a 200 g scale at pH 7 or 9 and 20-25 oC (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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Zhejiang University of Technology and Zhejiang Chiral Medicine Chemicals Co. reported a route to a pregabalin intermediate that relies on a nitrilase resolution of bis-nitrile rac-37 (Scheme 19).86 Only the primary nitrile was hydrolyzed, affording the acid 38 with 99.5% ee on a 1.5 g scale. Optimum pH was 7.5-10 and optimum temperature range was 30-35 oC. Scheme 19. Nitrilase Resolution of a Pregabalin Intermediate

Scientists at Les Laboratories Servier report using a nitrilase for resolution of an intermediate for ivabradine (Scheme 20).87 The hydrolysis was described on 500 mg scale of rac-39 in 5% DMSO at pH 7. 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. Scheme 20. Nitrilase Resolution of an Ivabradine Intermediate 40 ACS Paragon Plus Environment

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MeO MeO MeO

Nitrilase rac-39

CN

MeO 40 MeO

CO2H

+

DBU MeO

CN (R)-39

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 three years. Biocatalysis has become an integral technology for the design and development of efficient processes for the pharmaceutical and fine chemical industry. 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. Abbreviations ADH, alcohol dehydrogenase; CHMO, cyclohexanone monooxygenase; CSA, camphorsulfonic acid; CRED, carbonyl reducatase enzyme; DAAO, D-amino acid oxidase enzyme; DABCO, 1,441 ACS Paragon Plus Environment


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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 enzyme; KRED, ketone reductase enzyme; MeTHF, 2-methyltetrahydrofuran; MIBK, methyl iso-butyl ketone; MTBE, methyl tert-butyl ether; NADP, nicotinamide adenine dinucleotide phosphate; PEG, polyethylene glycol; PLP, pyridoxal 5’-phosphate.

References 1) Hughes, D. L., Asymmetric Organocatalysis in Drug Development—Highlights of Recent Patent Literature, Org. Process Res. Dev. 2018, 22, 574–584. 2) Hughes, D. L., Highlights of the Recent U.S. Patent Literature: Focus on Biocatalysis, Org. Process Res. Dev. 2016, 20, 700–706. 3) Rosenthal, K.; Lutz, S. Recent Developments and Challenges of Biocatalysis Processes in the Pharmaceutical Industry, Curr. Opinion Green and Sustainable Chem. 2018, 11, 58-64. 4) Li, G.; Wang, J.-b.; Reetz, M. T. Biocatalysts for the Pharmaceutical Industry Created by Structure-guided Directed Evolution of Stereoselective Enzymes, Bioorg. Med. Chem. 2018, 26, 1241-1251. 5) Lalonde, J. Highly Engineered Biocatalyts for Efficient Small Molecule Pharmaceutical Synthesis, Curr. Opinion Biotech. 2016, 42, 152-158.


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6) Kelly, S. A.; Pohle, S.; Wharry, S.; Mix, S.; Allen, C. C. R.; Moody, T. S.; Gilmore, B. F. Application of ω-Transaminases in the Pharmaceutical Industry, Chem. Rev. 2018, 118, 349-367. 7) Latham, J.; Brandenburger, E.; Shepherd, S. A.; Menon, B. R. K.; Micklefield, J., Development of Halogenase Enzymes for Use in Synthesis, Chem. Rev. 2018, 118, 232-269.

8) Parmeggiani, F.; Weise, N. J.; Ahmed, S. T.; Turner, N. J. Synthetic and Therapeutic Applications of Ammonia-lyases and Aminomutases, Chem. Rev. 2018, 118, 73-118. 9) Sheldon, R. A.; Woodley, J. M. Role of Biocatalysis in Sustainable Chemistry, Chem. Rev. 2018, 118, 801-838. 10) Turner, N. J.; Humphreys, L. Biocatalysis in Organic Synthesis: The Retrosynthetic Approach, London: Royal Society of Chemistry, 2018. 11) http://www.homepages.ucl.ac.uk/~ucbepad/RSCBiocatpdf/David%20Rozzell%20presentation% 20RSC.pdf; accessed June 11, 2018. 12) Savile, C. K.; Janey, J. M.; Mundorff, E. C.; Moore, J. C.; Tam, S.; Jarvis, W. R.; Colbeck, J. C.; Krebber, A.; Fleitz, F. J.; Brands, J.; et al. Biocatalytic Asymmetric Synthesis of Chiral Amines from Ketones Applied to Sitagliptin Manufacture. Science 2010, 329, 305−309. 13) Rozzell, J. D., Jr. Method for Reductive Amination of a Ketone Using A Mutated Enzyme. U.S. Patent 7,202,070 B2, Apr 10, 2007. 14) Vaijayanthi, T.; Chadha, A. Asymmetric Reduction of Aryl Imines using Candida parapsilosis ATCC 7330, Tetrahedron: Asymmetry, 2008, 19, 93-96.



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15) (a) Mitsukura, K.; Suzuki, M.; Tada, K.; Yoshida, T.; Nagasawa, T. Asymmetric Synthesis of Chiral Cyclic Amine from Cyclic Imine by Bacterial Whole-cell Catalyst of Enantioselective Imine Reductase, Org. Biomol. Chem. 2010, 8, 4533–4535. (b) Mitsukura, K.; Suzuki, M.; Shinoda, S.; Kuramoto, T.; Yoshida, T.; Nagasawa, T. Purification and Characterization of a Novel (R)-Imine Reductase from Streptomyces sp. GF3587, Biosci. Biotechnol. Biochem. 2011, 75, 1778–1782. 16) (a) Nagasawa, T.; Yoshida, T.; Ishida, K.; Yamamoto, H.; Kimoto, N. Process for Production of Optically Active Amine Derivative, U.S. Patent Application 2011/0287494 A1, Nov 24, 2011. (b) Nagasawa, T.; Yoshida, T.; Ishida, K.; Yamamoto, H.; Kimoto, N. Process for Production of Optically Active Amine Derivative, U.S. Patent Application 2011/0262977 A1, Oct 27, 2011. 17) Espinoza‐Moraga, M.; Petta, T.; Vasquez‐Vasquez, M.; Laurie, V. F. ; Moraes, L. A. B.; Santos, L. S. Bioreduction of Beta-carboline Imines to Amines Employing Saccharomyces bayanus, Tetrahedron: Asymmetry 2010, 21, 1988–1992. 18) (a) Fademrecht, S.; Scheller, P. N.; Nestl, B. M.; Hauer, B.; Pleiss, J. Identification of Imine Reductase-specific Sequence Motifs, Proteins Struct. Funct. Bioinforma. 2016, 84, 600– 610. (b) Scheller, P. N.; Fadermrecht, 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 a very young field, many reviews have already been written on imine reductase enzymes. (a) Mitsukura, K.; Yoshida, T. Imine Reductases for Chiral Amine Synthesis, in Future Directions in Biocatalysis, 2nd Ed, (ed., Matsuda, T), New York: Elsevier, 2017, Chapter 5, 97-117. (b) Mangas-Sanchez, J.; France, S. P.; Montgomery, S. 44 ACS Paragon Plus Environment

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Ketone and Amine Compounds, U.S. Patent 9,932,613, Apr 3, 2018. (b) ibid., U.S. Patent 9,828,614, Nov 28, 2017. (c) ibid., U.S. Patent 9,803,224, Oct 31, 2017. (d) ibid., U.S. Patent Application 2017/0268028 A1, Sep 21, 2017. (e) ibid., U.S. Patent Application 2017/0022527 A1, Jan 26, 2017. (f) ibid., U.S. Patent Application 2016/0168545 A1, Jun 16, 2016. (g) ibid., U.S. Patent Application 2016/0040140 A1, Feb 11, 2016. (h) Agard, N. J.; Alvizo, O.; Mayo, M. A.; Minor, S. N.; Riggins, J. N.; Moore, J. C. Engineered Imine Reductases and Methods for the Reductive Amination of Ketone and Amine Compounds, U.S. Patent 9,822,346, Nov 21,2017. (i) Ibid., U.S Patent Application 2018/0030419 A1, Feb 1, 2018. (j) ibid., U.S. Patent Application 2015/0132807 A1, May 14, 2015. (k) 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. Polynucleotides encoding engineered imine reductases, U.S. Patent 9,695,451, Jul 4, 2017. (l) ibid., U.S. Patent 9,487,760 B2, Nov 8, 2016. (m) ibid., U.S. Patent 9,296,993 B2, Mar 29, 2016. (n) U.S. Patent 9,193,957 B2, Nov 24, 2015. (o) ibid., U.S. Patent Application, 2018/0066292 A1, Mar 8, 2018. (p) Alvizo, O.; Mayo, M. A.; Moore, J. C., Engineered Imine Reductases and Methods for the Reductive Amination of Ketone and Amine Compounds, U.S. Patent Application 2017/0355966, Dec 14, 2017. 22) (a) Qu, X.; Zhu J. Synthesis of (S)-1-Aryl-1,2,3,4-tetrahydroisoquinoline in Imine Reductases and Mutants Thereof, Chinese Patent Application CN 107384885, Nov 24, 2017. (b) Zhu, J.; Tan, H.; Yang, L.; Dai, Z.; Zhu, L.; Ma, H.; Deng, Z.; Tian, Z.; Qu, X. Enantioselective Synthesis of 1-Aryl-Substituted Tetrahydroisoquinolines Employing Imine Reductase, ACS Catal. 2017, 7, 7003-7007.


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23) Anonymous, Imine Reductase Mediated Synthesis of (S,S)-Sertraline, IP.com Journal, Dec 11, 2017, Vol. 12B, Disclosure Number IPCOM000251929D, pages 1-3. https://priorart.ip.com/IPCOM/000251929; Accessed, Apr 19, 2018. 24) Wetzl, D.; Berrera, M.; Sandon, N.; Fishlock, D.; Ebeling, M.; Müller, M.; Hanlon, S.; Wirz, B.; Iding, H. Expanding the Imine Reductase Toolbox by Exploring the Bacterial Protein‐Sequence Space, ChemBioChem 2015, 16, 1749-1756. 25) Wetzl, D.; Gand, M.; Ross, A.; Muller, H.; Matzel, P.; Hanlon, S. P.; Muller, M.; Wirz, B.; Hohne, M.; Iding, H. Asymmetric Reductive Amination of Ketones Catalyzed by Imine Reductases, ChemCatChem 2016, 8, 2023-2026. 26) Roth, S.; Präg, A.; Wechsler, C.; Marolt, M.; Ferlaino, S.; Lüdeke, S.; Sandon, N.; Wetzl, D.; Iding, H.; Wirz, B.; Muller, M., Extended Catalytic Scope of a Well‐Known Enzyme: Asymmetric Reduction of Iminium Substrates by Glucose Dehydrogenase, ChemBioChem 2017, 8, 1703-1706. 27) Hussain, S.; Leipold, F.; Man, H.; Wells, E.; France, S. P.; Mulholland, K. R.; Grogan, G.; Turner, N. J. An (R)-Imine Reductase Biocatalyst for the Asymmetric Reduction of Cyclic Imines, ChemCatChem 2015, 7, 579-583. 28) France, S. P.; Howard, R. M.; Steflik, J.; Weise, N. J.; Mangas-Sanchez, J.; Montgomery, S. L.; Crook, R.; Kumar, R.; Turner, N. J., Identification of Novel Bacterial Members of the Imine Reductase Enzyme Family that Perform Reductive Amination, ChemCatChem 2018, 10, 510-514. 29) Early yeast reductions: (a) Neuberg, C.; Kerb, E., Phytochemische Reduktionen. XV. Die Uberfuhrung von Acetaldol in Optisch Aktives Butylen-glykol durch Hefe Nebst Anhang: Darstellung, Reaktionen und Krystallisierte Abkommlinge des Aldols. Biochem. Z. 1918,



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92, 96-110. (b) Farber, E.; Nord, F. F.; Neuberg, C. Die Phytochemische Reduktion des Acetols za Optisch-aktivem Propylenglykol. Nebst Bemerkungen und Versuchen zur Frage des Auftretens Sowie der Verarbeitung von Racemkorpern bei Tier und Pflanze, Biochem. Z. 1920, 112, 313. (c) Levene, P. A.; Walti, A.; Haller, H. L. The Configurational Relationship of Dextro-Methylethyl Carbinol to Dextro-Lactic Acid. J. Biol. Chem. 1927, 71, 465-469. (d) Levene, P. A.; Walti, A.Phytochemical Reductions. The Configurations of Glycols Obtained by Reduction with Fermenting Yeast, J. Biol. Chem. 1931, 94, 361-366. (e) Lemieux, R. U.; Giguere, J. Biochemistry of the Ustilaginales: IV. The Configurations of Some α-Hydroxyaicds and the Bioreduction of α-Ketoacids, Can. J. Chem. 1951, 29, 678– 690. 30) De Gonzalo, G.; Lavandera, I.; Gotor, V. Recent Advances in Biocatalysis Applied to Organic Synthesis, In Catalytic Methods in Asymmetric Synthesis: Advanced Materials, Techniques; Gruttadauria, M.; Giacalone, F., Eds.; John Wiley and Sons: Hoboken, NJ, 2011, Chapter 12, pp. 491-529. 31) A good example of a straightforward process involving a yeast reduction is described by Seebach and coworkers. Seebach, D.; Sutter, M. A.; Weber, R. H.; Züger, M. F. Yeast Reduction of Ethyl Acetoacetate: (S)-(+)-Ethyl 3-Hydroxybutanoate [Butanoic acid, 3hydroxy-, ethyl ester, (S)], Org. Synth. 1985, 63, 1-9. http://orgsyn.org/demo.aspx?prep=cv7p0215, accessed May 4, 2018. 32) (a) Reviews on the early implementation of ketoreductases for industrial applications: (a) Huisman, G. W.; Liang, J.; Krebber, A., Practical chiral alcohol manufacture using ketoreductases, Curr. Opinion Chem. Biol. 2009, 14, 1–8. (b) Moore, J. C.; Pollard, D. J.; Kosjek, B.; Devine, P. N., Advances in the enzymatic reduction of ketones. Acc. Chem. Res. 48 ACS Paragon Plus Environment

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34) Liang, J.; Jenne, S. J.; Mundorff, E.; Ching, C.; Gruber, J. M.; Krebber, A.; Huisman, G. W. Ketoreductase Polypeptides for the Reduction of Acetophenones. U.S. Patent Application 2017/0067032 A1, March 9, 2017. 35) (a) Alviso, O.; Collier, S. J.; Hennemann, H.-G. J.; Oh, S. H.; Zha, W. Ketoreductase for the Preparation of Phenylephrine. U.S. Patent 9,834,758 B2, December 5, 2017. (b) Alviso, O.; Collier, S. J.; Hennemann, H.-G. J.; Oh, S. H.; Zha, W. Ketoreductase for the Preparation of Phenylephrine. U.S. Patent 9,102,959 B2, Aug 11, 2015. (c) Alviso, O.; Collier, S. J.; Hennemann, H.-G. J.; Oh, S. H.; Zha, W. Ketoreductase for the Preparation of Phenylephrine. U.S. Patent Application 2018/0066239 A1, Mar 8, 2018. 36) Pasternak, A.; De Jesus, R. K.; Ding, F.-x.; Dong, S.; Frie, J.; Gu, X.; Jiang, J.; Shahripour, A.; Pio, B.; Tang, H.; Walsh, S.; Inhibitors of the Renal Outer Medullary Potassium Channel. U.S. Patent Application 2016/0002259 A1, Jan 7, 2016. 49 ACS Paragon Plus Environment


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37) Trussardi, R.; Iding, H. Process for the Preparation of Chiral 2-Aryl Morpholines. U.S. Patent Application 2016/0289718 A1, Oct 6, 2016. 38) Berger, R.; Blizzard, T. A.; Campbell, B. T.; Chen, H. Y.; Debenham, J. S.; Dewnani, S. V.; Dubois, B.; Gude, C.; Guo, Z. Z.; Harper, B.; Hu, Z.; Lin, S.; Liu, P.; Wang, M.; Ujjainwalla, F.; Xu, J.; Xu, L.; Zhang, R. Isoquinoline Derivatives as MGAT2 Inhibitors. U.S. Patent Application 2018/0009796 A1, Jan 11, 2018. 39) Zhou, S.; Lai, D.; Wang, R.; Zhang, S.; Chen, Z. Method for Preparing Chiral 2-Chloro-2,3difluorophenethyl Alcohol. Chinese Patent Application CN 106520849, Mar 22, 2017. 40) Lin, J.; Chestakova, A.; Gu, W.; Iding, H.; Li, J.; Xin, L.; Meier, P.; Sha, C.; Stults, J.; Wang, Y.; Zhang, H.; Zhang, J.; Zhang, T. Process for the Manufacturing of Medicaments. U.S. Patent Application 2017/0022183 A1, Jan 26, 2017. 41) Fu, M.; Zhang, T. Engineered Ketoreductase Polypeptide and Method for Preparing (S)-3Dimethylamino-1-thiophen-2-yl)-1-propanol by Using Same. Chinese Patent Application CN 106011096, Oct 12, 2016. 42) (a) Gohel, A.; Smith, D. J.; Wong, B.; Sukumaran, J.; Yeo, W. L.; Collier, S. J.; Novick, S. Biocatalytic Process for Preparing Eslicarbazepine and Analogs Thereof, U.S. Patent Application 2017/0159028 A1, Jun 8, 2017. (b) Modukuru, N. K.; Sukumaran, J.; Collier, S. J.; Chan, Z. S.; Gohel, A.; Huisman, G. W.; Keledjian, R.; Narayanaswamy, K.; Novick, S. J.; Palanivel, S. M.; Smith, D.; Wei, Z.; Wong, B.; Yeo, W. L.; Entwistle, D. A. Development of a Practical, Biocatalytic Reduction for the Manufacture of (S)Licarbazepine Using an Evolved Ketoreductase, Org. Process Res. Dev. 2014, 18, 810–815.


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43) Bong, Y. K.; Vogel, M.; Collier, S. J.; Mitchell, V.; Mavinahalli, J. Ketoreductase-mediated Stereoselective Route to Alpha Chloroalcohols, U.S. Patent Application 2016/0160187 A1, Jun 9, 2016. (b) Bong, Y. K.; Vogel, M.; Collier, S. J.; Mitchell, V.; Mavinahalli, J. Polypeptides for a Ketoreductase-mediated Stereoselective Route to Alpha Chloroalcohols. U.S. Patent 8,796,002 B2, Aug 5, 2014. 44) Pettersson, M. Y.; Johnson, D. S.; Subramanyam, C.; O’Donnell, J.; am Ende, W.; Green, M. E.; Patel, N. C.; Stiff, C. M.; Tran, T. P.; Kauffman, G. W.; Stepan, A. F.; Verhoest, P. R. Novel Pyrido1,2-A]pyrazines and Their Use in the Treatment of Neurodegenerative and Neurological Disorders, U.S. Patent Application 2016/0024088 A1, Jan 28, 2016. 45) Depre, D. P. M.; Ormerod, D. J.; Horvath, A.; Moody, T. S.; Brossat, M.; Riant, O.; Vriamont, N.; Lemaire, S. F. E.; Hermant, S. N. J. Processes and Intermediates for Preparing a Macrocyclic Protease Inhibitor of HCV, U.S. Patent Application 2018/0093943 A1, Apr 5, 2018. 46) Welch, M. H.; Beigelman, L.; Hendricks, R. T.; Horvath, A.; Brown, G.; Ganji, P.; Mix, S. Method of Preparing Aza-Pyridone Compounds, U.S. Patent Application 2017/0260189, Sep 14, 2017. 47) Zhu, W.; Wang, B.; Wu, H. Enzymatic Preparation Method of (S)-N-tert-Butoxycarbonyl-3hydroxypiperidine, Chinese Patent Application CN 106520856, Mar 22, 2017. 48) Hong, H.; Gage, J.; Lu, J.; Li, Y.; Zhang, N.; He, X.; Gao, J.; Shi, B. Enzymatic Catalysis Preparation Method of Chiral Alcohol, Chinese Patent Application CN 106497996, Mar 15, 2017.



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49) Zhang, Y. Method for Preparing Dehydroepiandrosterone Through Chemical-Enzyme Method, Chinese Patent Application CN 106086148. Nov 9, 2016. 50) Recent review: Applegate, G. A.; Berkowitz, D. B. Exploiting Enzymatic Dynamic Reductive Kinetic Resolution (DYRKR) in Stereocontrolled Synthesis, Adv. Synth. Catal. 2015, 357, 1619–1632. 51) Campopiano, O.; Mundorff, E.; Borup, B.; Voladri, R., Ketoreductase Polypeptides for the Production of Azetidinone. PCT Int. Patent Application WO2009046153, Apr 9, 2009. 52) Huisman, G. W.; Liang, J.; Krebber, A. Practical Chiral Alcohol Manufacture using Ketoreductase, Curr. Opinion Chem. Biol. 2010, 14, 122–129. 53) Campopiano, O.; Mundorff, E.; Borup, B.; Voladri, R. Ketoreductase Polypeptides for the Production of Azetidinones. U.S. Patent 8,980,606 B2, Mar 17, 2015. (b) ibid., U.S. Patent Application 2017/0313992 A1, Nov 2, 2017. 54) Xu, F.; Chung, J. Y.; Moore, J. C.; Liu, Z.; Yoshikawa, N.; Hoerrner, R. S.; Lee, J.; Royzen, M.; Cleator, E.; Gibson, A. G.; Dunn, R.; Maloney, K. M.; Alam, M.; Goodyear, A.; Lynch, J.; Yasuda, N.; Devine, P. N., Asymmetric Synthesis of cis-2,5-Disubstituted Pyrrolidine, the Core Scaffold of β3-AR Agonists, Org Lett. 2013, 15, 1342-1345. 55) Xu, F.; Liu, Z.; Desmond, R.; Park, J.; Kalinin, A.; Kosjek, B.; Strotman, H.; Li, H.; Moncecchi, J. Process the Preparing Beta 3 Agonists and Intermediates. U.S. Patent Application 2017/0145014 A1, May 25, 2017. (b) Cabirol, F. L.; Chen, H.; Gohel, A.; Collier, S.; Smith, D. J.; Kosjek, B.; Janey, J. Biocatalysts for the Preparation of Hydroxy Substituted Carbamates. U.S. Patent Application 2018/0010100 A1, Jan 11, 2018. (c) ibid., U.S. Patent Application 2016/0319247 A1, Nov 3, 2016. (d) ibid., U.S. Patent Application 52 ACS Paragon Plus Environment

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2015/0344850 A1, Dec 3, 2015. (e) ibid., U.S. Patent Application 2014/0322769 A1, Oct 30, 2014. (f) ibid., U.S. Patent 9,803,178 B2, Oct 31, 2017. (g) ibid., U.S. Patent 9,422,531 B2, Aug 23, 2016. (h) ibid., U.S. Patent 9,139,819 B2, Sep 22, 2015. 56) Ethyl acetoacetate pKa: https://en.wikipedia.org/wiki/Ethyl_acetoacetate; accessed May 8, 2018. 57) Based on an aq. pKa of acetophenone of 18.4 (Guthrie, J. P.; Cossar, J.; Klym, A. pKa Values for Substituted Acetophenones: Values Determined by Study of Rates of Halogenation, Can. J. Chem. 1987, 65, 2154-2159) and the estimated lowering of the pKa of about 1 unit due to the NH-Boc group (in DMSO solution, an alpha –NMe2 group lowers the pKa of acetophenone by 1.1 unit; PhCOMe = 24.7; PhCOCH2NMe2 = 23.7). https://www.chem.wisc.edu/areas/reich/pkatable/; accessed May 8, 2018. 58) Xie, X.; Huang, X.; Zhang, J.; Zhang, R. Preparation Method of Chloramphenicol Compounds. Chinese Patent Application CN 106566851, Nov 11, 2016. 59) Based on aq. pKa of 16.65 for 4-nitroacetophenone (ref 57). 60) Several recent reviews have been published on transaminase enzymes with a focus on industrial use: (a) reference 6. (b) Slabu, I.; Galman, J. L.; Lloyd, R. C.; Turner, N. J., Discovery, Engineering, and Synthetic Application of Tranaminase Biocatalysts, ACS Catal. 2017, 7, 8263-8284. (c) Ferrandi, E. E.; Monti, D., Amine Transaminases in Chiral Amines Synthesis: Recent Advances and Challenges, World J. Microbiol. Biotech. 2018, 34, article 13. (d) Guo, F.; Berglund, P. Transaminase Biocatalysis: Optimization and Application. Green Chem. 2017, 19, 333−360. (e) Fuchs, M.; Farnberger, J. E.; Kroutil, W., The Industrial Age of Biocatalytic Transamination. Eur. J. Org. Chem. 2015, 6965−6982. 53 ACS Paragon Plus Environment


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61) Austad, B. C.; Bhadoor, A.; Belani, J. D.; Janardanannair, S.; Wallerstein, S. L.; Keaney, G. F.; White, P. L.; Johannes, C. W. Enzymatic Transamination of Cyclopamine Analogs, U.S. Patent 9,879, 293 B2, Jan 30, 2018. 62) Tang, W. L.; Hsieh, H.; Pham, S.; Smith, D.; Collier, S. J., U.S. Patent Application 2017/0022484 A1, Jan 26, 2017. (b) ibid, U.S. Patent 9,902,943 B2, Feb 27, 2018. 63) Berger, R.; Blizzard, T. A.; Campbell, B. T.; Chen, H. Y.; Debenham, J. S.; Dewnani, S. V.; Dubois, B.; Gude, C.; Guo, Z. Z.; Harper, B.; Hu, Z.; Lin, S.; Liu, P.; Wang, M.; Ujjainwalla, F.; Xu, J.; Xu, L.; Zhang, R., Isoquinoline Derivatives as MGAT2 Inhibitors, U.S. Patent Application 2018/0009796 A1, Jan 11, 2018. 64) Crowe, M.; Foulkers, M.; Francese, G.; Grimler, D.; Kuesters, E.; Laumen, K.; Li, Y.; Lin, C.; Nazor, J.; Ruch, T.; Smith, D.; Song, S.; Teng, S., Chemical Processes for Preparing Spiroindolones and Intermediates Thereof, U.S. Patent 9,598,712 B2, Mar 21, 2017. 65) Debarge, S.; Erdman, D. T.; Karmilowicz, J.; Kumar, R.; O’Neill, P. M. Process and Intermediates for the Preparation of Pregabalin, U.S. Patent Application 2016/0024540 A1, Jan 28, 2016. 66) https://en.wikipedia.org/wiki/Glufosinate 67) Yang, L.; Zhou, H.; Meng, L.; Yin, X.; Xu, G.; Wu, J. Production Method of LPhosphinothricin, Chinese Patent Application CN106916857, Jul 4, 2017. 68) Green, B. M.; Gradley, M. L. Methods for Making L-Glufosinate, U.S. Patent Application 2018/0030487 A1, Feb 1, 2018.


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