Identification and Implementation of Biocatalytic Transformations in

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Identification and Implementation of Biocatalytic Transformations in Route Discovery: Synthesis of Chiral 1,3-Substituted Cyclohexanone Building Blocks Timin Hadi, Alba Diaz-Rodriguez, Diluar Khan, James P Morrison, Justin M Kaplan, Kathleen T Gallagher, Markus Schober, Michael R Webb, Kristin Brown, Douglas Fuerst, Radka Snajdrova, and Gheorghe-Doru Roiban Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.8b00139 • Publication Date (Web): 22 Jun 2018 Downloaded from http://pubs.acs.org on June 25, 2018

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Organic Process Research & Development

Identification and Implementation of Biocatalytic Transformations in Route Discovery: Synthesis of Chiral 1,3-Substituted Cyclohexanone Building Blocks Timin Hadi,a Alba Diaz-Rodriguez,b Diluar Khan,b James P. Morrison,a Justin M. Kaplan,c Kathleen T. Gallagher,a Markus Schober,d Michael R. Webb,b Kristin K. Brown,e Douglas Fuerst,a Radka Snajdrova,b, † Gheorghe-Doru Roiban*d a

Advanced Manufacturing Technologies, GlaxoSmithKline, 709 Swedeland Road, King of

Prussia, Pennsylvania 19406, United States. b

API Chemistry, GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road,

Stevenage SG1 2NY, United Kingdom. c

API Chemistry, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, 19406, PA, United

States. d

Advanced Manufacturing Technologies, GlaxoSmithKline, Medicines Research Centre,

Gunnels Wood Road, Stevenage SG1 2NY, United Kingdom. Email: [email protected]

ACS Paragon Plus Environment

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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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Molecular Design, Computational and Modeling Sciences, GlaxoSmithKline, 1250 S.

Collegeville Road, Collegeville, Pennsylvania 19426, United States.

Abstract

Several

biocatalytic

approaches

for

the

preparation

of

optically

pure

methyl

3-

oxocyclohexanecarboxylates (S)-, (R)-1 and 3-oxocyclohexanecarbonitriles (S)-, (R)-2 have been successfully demonstrated.

Screening of reaction-focused enzyme collections was used to

identify initial hits using three enzymatic strategies. Reaction optimization and scale-up enabled the production of chiral intermediates for route scouting efforts on scales of up to 100 g. The enzymes applied in these processes (lipases, enoate reductases and nitrilases) have been shown to be robust catalysts for drug manufacturing and represent a green alternative to conventional methods to access these chiral cyclohexanone building blocks.

Keywords: Biocatalysis, Enoate Reductases, Nitrilases, Lipases, Active Pharmaceutical Ingredients. 1. INTRODUCTION The use of biocatalysis in the synthesis of pharmaceutically-relevant intermediates has been an increasingly valuable methodology in the chemist’s synthetic toolbox.1-3 Enzyme-catalyzed reactions allow for potentially greener processes by increasing atom efficiency, simplifying chemical routes and reducing pollution and cost of goods.1,

4, 5

They can especially offer

significant advantages in the production of chiral intermediates where the regio- and stereoselectivity of the enzyme can be harnessed to generate enantiopure products as an alternative to conventional chiral auxiliaries or inorganic catalysts.6 The increasing number of


2

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characterized and, in many cases, commercially available collections of biocatalysts has enabled hundreds of unique enzyme variants to be screened for a desired transformation in a rapid fashion.7 Despite some of the advantages that biocatalysis has to offer, widespread adoption in process development for active pharmaceutical ingredients (APIs) has yet to occur. Frequently cited concerns include: the inability to provide biocatalysts on a multigram scale affordably and promptly, lack of access to enzyme screening collections, and difficulties encountered during downstream processing of biocatalytic reactions.8 We have been actively working to overcome these limitations by increasing access to “ready-to-use” enzyme panels generated from unpurified bacterial cell lysates for common chemical transformations, thereby expanding the biocatalytic toolbox available to synthetic chemists.9 In addition, we have invested in infrastructure improvements that facilitate the progression of an enzyme hit in a microtiter plate to gram-scale process development within a matter of weeks. These improvements have enabled the evaluation of enzymatic reactions in synthetic route scouting in a manner that can be directly compared to more traditional chemical processes. Cyclohexanes with different substitution patterns and absolute configurations can be found in a number of pharmaceutically relevant compounds including Tacrolimus (Prograf),10 Desvenlafaxine,11 Tramadol,12 peroxisome proliferator-activated receptor ligands,13 soluble epoxide inhibitor,14 Oxaliplatin,15 and Gabapentin16 (Fig. 1).


3


Page 4 of 36

Fig. 1 Selected API structures containing cyclohexane motif. During

recent

API

route

scouting

at

GSK,

optically

pure

methyl

3-

oxocyclohexanecarboxylates ((S)-, (R)-1) and 3-oxocyclohexanecarbonitriles ((S)-, (R)-2) were identified as key chiral building blocks. The ester and nitrile groups offered different reactive handles towards the synthesis of more complex structures.


4

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Herein we describe efforts to access the cyclohexanone derivatives (S)- and (R)-methyl 3oxocyclohexanecarboxylate (1) and (S)- and (R)-3-oxocyclohexanecarbonitrile (2) on an industrially-relevant scale using three different enzymatic strategies: a) kinetic resolution of rac1 using lipases, b) enantioselective reduction of prochiral enones using enoate reductases (EREDs) and c) kinetic resolution of rac-2 using nitrilases. Although both enantiomers of 1 and 2 were desired for route scouting, the (S)-enantiomers of the nitrile and methyl ester were ultimately prioritized as they contained the appropriate configuration for downstream chemistry. The aim of this article is also to encourage chemists to consider enzymatic approaches by first intent and to provide several easy-to-use protocols for the preparation of chiral cyclohexanone derivatives. 2. RESULTS AND DISCUSSION Few

reports

describe

the

preparation

of

enantiomerically

enriched

methyl

3-

oxocyclohexanecarboxylate (1) and 3-oxocyclohexanecarbonitrile (2). (R)-1 has been prepared using asymmetric phase transfer catalysis and expensive ruthenium catalysts but result in low product enantioselectivity (47% ee).17 The synthesis of (S)-2 has been reported utilising a catalytic conjugate addition of cyanide to enones at low temperatures, resulting in products with moderate enantiomeric excess (81% ee).18 In the biocatalysis space, syntheses of enantiopure compounds 1 and 2 have been described starting from the corresponding enones using enoate reductases (EREDs)19, cyclohexenes.21,

22

20

or through enzymatic cascades starting from the corresponding

Although there have been a number of published examples of ERED-

mediated catalysis over the last few years reporting high product enantioselectivity (>99%),23-25 EREDs have yet to gain widespread adoption in the field of industrial biocatalysis. Many studies


5


Page 6 of 36

have been limited to milligram scale syntheses26-29 with only a few reporting enzymatic reactions at gram scales.30-33 Several biocatalytic strategies were considered in order to access chirally-enriched compounds 1 and 2 (Scheme 1). Two resolution-based strategies using lipases and nitrilases were explored as well as an ERED-based asymmetric synthesis using a chiral reduction. For the synthesis of chirally-enriched 1, a lipase-mediated resolution of a racemic mixture of 1 was investigated while an analogous nitrilase-mediated resolution of racemic 2 was also pursued. An alternative strategy that was investigated for the syntheses of both 1 and 2 employed a set of enoate reductases to catalyze the chiral reduction of the unsaturated cyclohexenones methyl 3oxocyclohex-1-enecarboxylate (3) and 3-oxocyclohex-1-enecarbonitrile (4).

Scheme 1. Synthetic strategies to access chiral cyclohexanones 1 and 2 2.1 Preparation of (R)- and (S)-methyl 3-oxocyclohexanecarboxylate (1) 2.1.1 Lipase-mediated resolution of racemic methyl 3-oxocyclohexanecarboxylate (rac-1) Lipases are robust and readily available catalysts that have been widely utilized in organic syntheses.34,

35

Due to their widespread use, collections of commercially available lipases are


6

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easy to access on small scales for screening and on larger scales for process development. We envisioned employing a lipase to kinetically resolve rac-1 to access enantiopure (S)- or (R)-1. Although rac-1 is expensive to purchase, it can be made cheaply from methyl 3hydroxybenzoate in two steps36 making this a potentially viable route even though the theoretical yield is only 50%. In order to achieve proof of concept for this biocatalytic approach, various lipases were screened for their activity and selectivity on rac-1 (Table 1), in the hopes of identifying unique enzymes able to resolve both (R)-1 and (S)-1.37 Three enzymes that were tested displayed low selectivity for hydrolysis of the (R)-enantiomer (Table 1 entries 3, 5-6), with lipase CES L-1 giving the best ee for remaining (S)-1 (26% ee) (E= 6). A few lipases showed promising selectivity for the hydrolysis of (S)-1, with Novocor ADL producing (R)-1 with >99.5% ee (Table 1, entry 1, E= 18).

Table 1. Screening results using commercial lipases in the resolution of rac-1

Entry

Lipase a

Conv (%) b

ee (%) 1 c

1

Novocor ADL

63

>99.5 (R)

2

ENZA-06

15

10 (R)

3

ENZA-04

22

8 (S)

4

ENZA-43

57

74 (R)

5

CES L-1

28

26 (S)


7


Page 8 of 36

6

CES L-2

10

3 (S)

7

CES L-4

33

26 (R)

a

Entries 1-7 represent selected hits from several commercial lipases. Reaction conditions: rac-

1 (10 mg) in DMSO (50 µL), potassium phosphate buffer 100 mM (1 mL), pH 7.0, 50% wt enzyme, 15°C, 22 h with orbital shaking at 1000 rpm. b Conversion was determined by HPLC. c Absolute configuration determined after comparison with authentic samples. A 13 g reaction was performed using Novocor ADL to generate (R)-1. The product ee at this scale decreased to 88%, with an isolated yield of 38%. Although ee and yield could be improved through reaction optimization and further process development, the moderate product ee observed on scale-up and lack of a complementary lipase that would enable resolution of the (S)enantiomer led us to draw our attention to an ERED-mediated approach. 2.1.2 ERED-catalyzed reduction of methyl 3-oxocyclohex-1-enecarboxylate (3) An initial screen for the stereoselective reduction of 3 was carried out using an internal collection of ERED enzymes. Reactions were screened under aqueous conditions in the presence of a glucose dehydrogenase (GDH) cofactor recycling system. A number of ERED enzymes displayed conversion of the starting material 3 as well as high product ee for either enantiomer of compound 1 (Table 2). For many enzyme variants, conversion to the desired product was accompanied by the production of the carbonyl reduction products of both 1 and 3, as well as the disproportionation reaction product27 methyl 3-hydroxybenzoate (Fig. 2). Formation of carbonyl reduction products was more pronounced in the case of less active ERED variants. In addition, negative controls (produced from cells harboring plasmids lacking the ERED gene) displayed a background conversion of 3 preferentially to (R)-1, suggesting the presence of endogenous ERED-like activity in the E. coli expression strain. This endogenous background activity is a


8

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possible cause for the lower enantioselectivity of the C26G-YqjM variant under these screening conditions. C26G-YqjM was reported to give (S)-1 in ee >99% when assayed as a purified enzyme preparation.19 The observed ee of the identical variant screened in our hands as a crude bacterial cell lysate was found to be 96%.

Table 2. ERED-catalyzed reduction of 3 → 1

Entry

ERED a

Conv (%) b, c

ee (%) 1 d

1

wt-YqjM

85

92 (R) e

2

C26D-YqjM

80

>99 (R)

3

C26G-YqjM

91

96 (S)

4

ERED-B3

85

98 (S)

5

ERED-C5

80

99 (S)

6

ERED-C12

59

97 (R)

7

ERED-D3

81

98 (S)

8

ERED-E8

72

78 (R)

9

ERED-E11

64

50 (R)

10

ERED-F7

67

61 (R)

11

ERED-G3

78

99 (S)

12

ERED-G5

77

99 (S)


9


13

ERED-H6

80

99 (S)

14

ERED-207

99

99 S)

15

Negative control f

99% ee) for the (S)-2, while a single enzyme variant catalyzed the formation of (R)-2 in 46% yield and 97% ee (Table 4, entry 12). Interestingly, the two YqjM variants C26D-YqjM and C26G-YqjM, which gave high conversion and ee with 3 (Table 2, entries 2,3), gave comparably poor results for the reduction of 4, with < 20% conversion and low product ee values (Table 4, entries 1-2).

Table 4. Screening results using GSK-ERED collection for 3-oxocyclohex-1-enecarbonitrile (4).

Entry

ERED a

% Conv b,c

ee (%) 2 c,d

1

C26D-YqjM

15

64 (R)

2

C26G-YqjM

13

38 (S)

3

ERED-A1

95

>99 (S)

4

ERED-A2

98

>99 (S)

5

ERED-A9

94

>99 (S)

6

ERED-A10

96

>99 (S)

7

ERED-A12

99

>99 (S)


15


Page 16 of 36

8

ERED-B1

99

>99 (S)

9

ERED-B7

98

>99 (S)

10

ERED-D1

97

>99 (S)

11

ERED-G7

99

>99 (S)

12

ERED-H7

46

97 (R)

a

Entries 3-12 represent selected hits obtained from the Codexis ERED Screening plate 1.

b

Reaction conditions: a) entries 1-2: enone 4, 5 g/L, whole cells; potassium phosphate buffer 100 mM, pH 7.4, entries 3-12 enone 4, 24 g/L, clarified lysate, (Codexis plate). c Conversion and ee values measured by GC.

d

Absolute configuration was determined after comparison with

authentic standards.

Additional scouting experiments identified ERED-B1 as the most active variant. ERED-B1 was over-expressed and produced as a lyophilized powder for reaction scale up. An initial 1 g reaction using 10% wt of ERED-B1 produced (S)-2 in 80% isolated yield with excellent ee (99.5%). The reaction was then scaled up to 40 g using 10% wt lyophilized enzyme loading at a substrate concentration of 18.2 g/L. GC monitoring showed that the reaction was complete after just 1 h and (S)-2 was isolated in 87% yield with 99.5% ee after extractive work-up. 2.2.2 Nitrilase-mediated resolution of racemic 3-oxocyclohexanecarbonitrile (rac-2) During the course of the ERED studies, beside the lack of a commercial supplier, additionally it was found that substrate 4 appeared less stable than 2 during distillative purification at higher temperatures. Instability of 4 posed a significant risk that could limit the applicability of the ERED-catalyzed approach in a manufacturing process. To mitigate this risk, we decided to


16

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investigate the use of nitrilases to resolve a racemic mixture of the easily available nitrile, rac2.53-55 Although this resolution only has a theoretical yield of 50%, extractive separation of the desired nitrile from the undesired carboxylic acid (5) should be straight forward leading to isolated yields close to the theoretical maximum. A panel of nitrilases comprised of a set of wild-type enzymes identified from literature reports56 and through protein homology modelling was screened.

Initial screening was

performed using chiral GC analysis to identify nitrilases that were selective for hydrolysis of either (S)-2 or (R)-2. Nitrilase NIT-E6 allowed for the resolution of (R)-2 (up to 38 %ee), while other nitrilases had modest to very good selectivity for the resolution of (S)-2. A representative set of selective nitrilases is summarized in Table 5, with NIT-G6 (97% (S)-2 ee, entry 4) showing the most promising selectivity for the resolution of (S)-2.

Table 5. Nitrilase mediated resolution of racemic 3-oxocyclohexanecarbonitrile (2).

Entry

Nitrilase a, b

% Conv c

ee (%) 2 c, d

1

NIT-D9

23

3 (S)

2

NIT-E6

31

38 (R)

3

NIT-E8

28

11 (S)

4

NIT-G6

51

97 (S)

5

NIT-H6

21

13 (S)


17


a

Entries 1-5 are aquired in house wild-type nitrilases.

b

Page 18 of 36

Reaction conditions: 2, 5 g/L,

potassium phosphate buffer 100 mM pH 7.0, 30 °C, 800 rpm, 20 h. c Conversion and ee were determined by chiral GC.d Absolute configuration was determined after comparison with authentic samples.

Nitrilase G6 was selected for further development work and expressed as a crude clarified cell lysate and used for reaction scale-up and optimization. An achiral GC method was used to monitor formation of the hydrolysis product 5 in conjunction with the chiral GC method for analysis of remaining compound 2. Initial reactions performed on a 0.5 and 2.6 g scale of rac-2, generated (S)-2 in 99.4 and 98.4% ee, respectively (Table 6, entries 1-2). pH adjustment of the reaction mixture and extractive separation of the acid product from the desired nitrile proceeded efficiently, giving isolated yields of 36% and 42%, respectively. A 5 g reaction was then conducted in a jacketed reactor using clarified lysate at 5% wt lysate loading and 26.5 g/L substrate loading. The reaction proceeded to completion after 3h and afforded (S)-2 in 27% yield (99% ee, Table 6, entry 3).

Table 6. Scale up experiments for the preparation of (S)-2 starting from racemic 3oxocyclohexanecarbonitrile (2) using NIT-G6. Entry

rac-2 (g)

Isolated yield 2, (%)

ee (%), 2

1

0.5

36

>99 (S)

2

2.6

42

98 (S)

3

5.0

27

99 (S)

4

20.0

28

> 99 (S)


18

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a

Reaction conditions: 2, entries 1-3: 26.5g/L; entry 4: 50g/L; potassium phosphate buffer 200

mM, pH 7.0, 30 min, 30 °C.

To evaluate NIT-G6 at a higher substrate loading (50 g/L), a 20 g scale reaction was performed and promisingly showed similar results in terms of yield and selectivity to the previous reactions (Table 6, entry 4). This unoptimized process using NIT-G6 was deemed suitable for producing (S)-2 and provided an alternative to the ERED-catalyzed reduction of 4.

Additional enzyme and process

optimization is likely to improve reaction profiles and increase isolated yield. CONCLUSIONS Several biocatalytic approaches have been used on multigram scales to access chiral 3substituted cyclohexanones under mild reaction conditions. These approaches represent a useful alternative to traditional chemical methods and produced the desired compounds with high isolated yields and ee. Three different enzyme type (lipases, enoate reductases and nitrilases) were screened from internal enzyme collections. The majority of these enzymes were produced as crude cell lysates, removing the need for laborious purification and making them ideal catalysts from a cost-of-goods standpoint. Proof of concept experiments on gram scales were conducted rapidly to generate material of sufficient quality (ee, purity) and quantity for downstream chemistry applications. Further reaction optimization and process development allowed for the successful demonstration of these enzymatic reactions on a scale of up to 100 g in a rapid fashion, highlighting the robustness and scalability of these biocatalytic processes. When comparing these strategies it is important to note that the ERED-mediated reduction can produce the desired product in 100% yield, while the theoretical yield of a lipase- or nitrilase-


19


Page 20 of 36

mediated resolution would be limited to 50%. This makes the ERED-mediated reduction an attractive strategy for synthesizing chiral cyclohexanone building blocks. However, resolution approaches were also pursued as factors including alternative methods of substrate synthesis, biocatalyst availability, substrate instability and downstream processing challenges came to light during our work. We have demonstrated that enzymatic approaches can be rapidly (1-3 months) evaluated and implemented in chemical route development on scales that truly compete with other more traditional synthetic approaches. Biocatalysis is an increasingly valuable tool for chemists in the pursuit of cheaper, greener, and more efficient routes for API manufacture. 4. EXPERIMENTAL Biology The genes encoding EREDs and nitrilases were codon optimised for expression in E. coli BL21(DE3), synthesised, and cloned into pET28a at GenScript. Transformation, shake flask enzyme expression and 50 L fermentation trials were performed as described previously.39 Escherichia coli BOU730 cells [an E. coli strain derived from BL21(DE3) that contains a copy of GDH gene from B. megaterium under the control of T7 promoter]57 were used during this study for the wt-YqjM, C26D and C26G variants. Plasmids containing wt-YqjM and mutants C26D and C26G were received from Reetz’s group and were published previously.19 ERED plate 1 and ERED-207 were acquired from Codexis. The nitrilase panel was designed and produced in-house, and is comprised of gene sequences encoding putative nitrilases assembled using an extensive homology search. Sequences of enzyme NIT-D9, E6, E8, G6 and H6 are given in supporting information. Lipases were acquired from Amano, and Enzagen. GDH-CDX-


20

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901 was acquired from Codexis, NADP+ was bought from Bontac Bio-Engineering and used without further purification. Chemistry Rac-2 was either acquired from FCH group (Ukraine) or prepared in house (see supporting information), while rac-1, enone 3, and the other chemical reagents were purchased from the following commercial sources: Manchester Organics, Acros Organics, Sigma Aldrich and Alfa Aesar. Enone 4 was prepared in house according to the procedure described in the supplementary information. All chemicals and solvents were used without any additional purification. NMR spectra were recorded on a Bruker Advance 400 (1H-NMR 400 MHz,

13

C-NMR 101 MHz)

spectrometer using TMS as internal standard (δ=0). Details on plate-based screening as well as analytical methods are given in the supporting information. Scale-up reactions were performed in conical centrifuge tubes (50 mL), and controlled laboratory reactors (CLR) (volumes from 0.25 - 5L). Small amounts of cell paste were lysed using a Fisher Scientific sonicator model FB705 equipped with a specific probe depending on the lysis volume (up to 100 mL). Higher amounts of cell paste were disrupted using a microfluidiser M110Y from analytikLtd Cambridge. Lyophilisation was performed using a BPS VirTris SP Scientific Advantage Pro lyophiliser. For the enzyme fermentation a 50 L (35L working volume) fermenter was used as previously described.39 Preparation of (R)-methyl 3-oxocyclohexanecarboxylate (1) using C26D-YqjM ERED (5 g scale) ERED C26D-YqjM was expressed as previously reported.19 Cell pellets were stored in the freezer at -20°C. To a 500 mL Erlenmeyer flask potassium phosphate buffer was added (KH2PO4 / K2HPO4, 250 mL, pH 8.0, 100 mM). D-glucose (7.5 g, 1.25 equiv), GDH-CDX-901 (0.2 g) and


21


Page 22 of 36

NADP+ (0.256 g, 0.01 equiv) were then added. Reaction was started by addition of methyl 3oxocyclohex-1-enecarboxylate (3) (5.0 g, 32.4 mmol final conc 17.7 g/L) and ERED C26DYqjM (32 g, cell wet mass) and shaken in an Kühner shaker at 200 rpm and 30°C. Reaction samples were extracted with ethyl acetate and monitored by GC. After full conversion was reached, the reaction mixture was extracted with ethyl acetate (2 x 250 mL). The organic extracts were combined and dried over Na2SO4 before being concentrated in vacuo to afford (R)-1 as a pale yellow oil (3.0 g, 60% yield, 77% purity by GC, 99.4 ee%). 1H-NMR (400 MHz, CDCl3) δ ppm 3.7 (s, 3 H) 2.8 (m, 1 H) 2.6 (m, 2 H) 2.4 - 2.3 (m, 2 H) 2.2 - 2.0 (m, 2 H) 1.9 - 1.7 (m, 2 H). 13

C-NMR (101 MHz, CDCl3) δ ppm 209.1 (1 C) 174.0 (1 C) 51.9 (1 C) 43.0 (1 C) 42.9 (1 C)

40.8 (1 C) 27.6 (1 C) 24.3 (1 C). Preparation of (S)-methyl 3-oxocyclohexanecarboxylate (1) using C26G-YqjM ERED (100 g scale) To a controlled laboratory reactor (5 L) potassium phosphate buffer (KH2PO4 / K2HPO4, 2.0 L, 200 mM, pH 7.0) was charged and the temperature was adjusted to 30°C. D-glucose (175 g, 1.28 equiv) and NADP+ (0.5 g) were then added to the vessel and agitation adjusted to 200 rpm. At this point pH monitoring indicated a value of 7.0. When all components dissolved after 5 min, frozen whole cells (180 g) harbouring ERED variant C26G-YqjM in cells coexpressing GDH for cofactor regeneration57 were added and the mixture was stirred for 30 min during which time all the cells resuspended with no lumps being observed. The reaction was started by the addition of methyl 3-oxocyclohex-1-enecarboxylate (3) (100 g, 649 mmol, final conc 34.5 g/L). Additional potassium phosphate buffer (KH2PO4 / K2HPO4, 0.3 L, 200 mM, pH 7.0) was added to rinse the CLR. The reaction mixture was then stirred at 30°C and 200 rpm for 50 min at which point GDH-CDX-901 (0.2 g) and additional frozen cell paste (53 g) were added. After 1 h 30 min


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further amounts of NADP+ (1.0 g) and cell paste (70 g) were added and the reaction was allowed to stir for additional 30 min, after which GC analysis showed complete conversion. The solution pH was maintained during the course of reaction between pH 6.7 - 8.0 by addition of NaOH (5 M). The crude reaction mixture (2.9 L) was then divided in two batches. The first batch (1L) was quenched with acetonitrile and the second batch (1.9 L) with isopropanol (IPA) / isopropyl acetate (iPrOAc) 1:1 v/v. Both work-up approaches resulted in no emulsion formation, acetonitrile delivering a thin interface in between the organic and aqueous layer in comparison with IPA/ iPrOAc extraction where no interface was formed. a) Acetonitrile work-up 1L of reaction mixture was quenched with acetonitrile (1 L) and stirred for 30 min at 444 rpm to ensure proper mixing and then stirring was stopped for 6 min to allow phase separation. The organic layer (0.65 L) was separated and a second acetonitrile extraction (1 L) of the remaining aqueous phase proceeded with no emulsion formation, allowing the recovery of a total of 1.65 L of organic phase. The remaining aqueous phase from the acetonitrile wash was extracted with ethyl acetate (0.5 L) to determine extraction efficiency;mass balance indicated that 98%. The reaction mixture was quenched by adjusting the pH (7.1) to 8.1 by addition of NaOH (5 M) and subsequently adding EtOAc (400 mL). The mixture was stirred for 10 min (200 rpm), then left without stirring for 30 min to allow separation of the organic and aqueous layers. A partial emulsion was observed and this was partially broken by filtration through cotton wool. A second extraction of the aqueous layer with EtOAc (400 mL) resulted in no further emulsion formation. The organic layers were combined and washed with brine twice (300 mL) followed by a saturated potassium bicarbonate wash (200 mL). The combined organic layers were dried over MgSO4 and subsequently concentrated in vacuo to give (S)-2 as a yellow oil (5.6 g, 28% yield, 99% ee). 1H and

13

C spectra of the product were found to be identical with the spectra reported

for the ERED catalysed reduction of enone 4.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI:


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NMR spectra and GC chromatograms (file type, PDF) AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. ORCID Gheorghe-Doru Roiban: 0000-0002-5006-3240 Present Addresses † Current address: Global Discovery Chemistry, Novartis Institute of Biomedical Research, Novartis Pharma AG, Lichtstrasse, Basel CH-4056 Basel, Switzerland. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT This research was supported by the Innovative Medicines Initiative (IMI) joint undertaking project CHEM21 under grant agreement no. 115360, resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/20072013) and EFPIA companies in kind contribution. We thank Prof. Manfred Reetz and Dr. Adriana Ilie for providing ERED variants C96D-YqjM and C96G-YqjM. REFERENCES


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Identification and Implementation of Biocatalytic Transformations in

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