Technical Considerations for Scale-Up of Imine Reductase Catalyzed

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Technical Considerations for Scale-Up of Imine Reductase Catalyzed Reductive Amination: A Case Study Amin Bornadel, Serena Bisagni, Ahir Pushpanath, Sarah Louise Montgomery, Nicholas J Turner, and Beatriz Dominguez Org. Process Res. Dev., Just Accepted Manuscript • Publication Date (Web): 16 May 2019 Downloaded from http://pubs.acs.org on May 16, 2019

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Organic Process Research & Development

Technical Considerations for Scale-Up of Imine Reductase Catalyzed Reductive Amination: A Case Study

Amin Bornadel,† ,* Serena Bisagni,† Ahir Pushpanath,† Sarah L. Montgomery,‡ Nicholas J. Turner,‡ and Beatriz Dominguez† ,*

† Johnson Matthey Plc., 28 Cambridge Science Park, Milton Road, Cambridge CB4 0FP, UK ‡ School of Chemistry, University of Manchester, Manchester Institute of Biotechnology, 131 Princess Street, Manchester M1 7DN, UK

* E-mail: [email protected]; [email protected]

ACS Paragon Plus Environment

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Abstract Graphic


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Abstract

Imine reductases (IREDs) have attracted increasing attention as novel biocatalysts for the synthesis of various cyclic and acyclic amines. Herein, a number of guidelines and considerations towards the development and scale-up of IRED catalyzed reactions have been determined based on the reductive amination of cyclohexanone (1) with cyclopropylamine (2). A Design of Experiments (DoE) strategy has been followed to study the different reaction parameters, facilitating resourceefficient and informative screening. Enzyme stability was identified to be the limiting factor. By moving from batch to fed-batch it was possible to double the concentration of the substrate and turnover number (TON). Kinetic studies revealed that IRED-33 was the best enzyme for the reaction with respect to both activity and stability. Under the optimal reaction conditions, it was possible to react 1 and 2 at 750 mM concentration and reach 100% conversion to the desired amine (>90% isolated yield) in the space of 8 h. Hence, excellent volumetric productivity of 12.9 g L-1 h-1 and TON above 48000 were achieved.

Keywords IRED catalyzed reaction, reductive amination, enzyme kinetics, fed-batch reaction, biocatalytic process development.


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Amines are high profile building blocks owing to increasing demand for these moieties in the production

of

pharmaceuticals,

agrochemicals,

and

fine

chemicals.1-3

Biocatalyzed

transformations such as transaminase- and amine dehydrogenase catalyzed routes for the transformation of ketones into chiral amines have been known for several years, allowing the use of renewable catalysts and mild reaction conditions to obtain optically pure primary amines.4-6 In the past few years, a growing interest has surged in NAD(P)H-dependent imine reductases for the reduction of cyclic imines7-9 and the reductive amination of aldehydes and ketones to offer both primary and secondary amines.10-12 In the latter case, these enzymes have been interchangeably called "reductive aminases". Enantioselective (S)- and (R)-IREDs from Streptomyces sp. were used for the asymmetric reduction of prochiral imines.7-9 In a report published in 2015, (R)-IRED was shown to have high activity and enantioselectivity towards a range of cyclic imines and iminium ions.9 Preparative scale synthesis of the alkaloid (R)-coniine was demonstrated at 25 mM imine concentration, which resulted in 90% yield and 99% ee.9 Reductive aminase (RedAm) from Aspergillus oryzae was reported to catalyze the reductive amination of a spectrum of carbonyl compounds with primary and secondary amines to give >98% conversion and ee.10 The demonstration at preparative scale was performed with purified RedAm (0.1 mg/mL) and 50 mM ketone to achieve space time yield (STY) of 3.7 g L-1 d-1. In a more recent paper by France and coworkers, IRED catalyzed reductive aminations were studied through screening a diverse library of IREDs against a panel of ketones and amines, to show the potential of this type of transformation for broader applications.11


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At the same time, work performed in Kroutil’s lab showed the potential of IREDs in terms of substrate scope and enantioselectivity through sequence-based in-silico IRED discovery for the reduction of cyclic imines.12 100 mM substrate concentration was observed to be the threshold, after which the conversion and productivity dropped, highlighting the sensitivity of these enzymes to higher substrate concentrations. Furthermore, IRED catalyzed reductive amination has been coupled with alcohol dehydrogenase (ADH) in cascade fashion to enable biocatalytic hydrogen borrowing through direct alkylation of amines with primary and secondary alcohols.13 Very recently, in situ reductive amination of aldehydes has been studied through cascade reactions coupling RedAm with primary alcohol oxidase or carboxylic acid reductase.14 Considering the importance of this area of biocatalysis as a rapidly growing field and its significant impact on facilitating the sustainable synthesis of amines for industrial applications, it is crucial to set practical guidelines and technical considerations applicable to the development and intensification of such a reaction system. Whilst most of the work reported focuses on the discovery of new IREDs, and their enzyme engineering, characterization, and mechanistic studies, more emphasis on process development is needed to understand and overcome the limitations of these enzymes for industrial applications. This work aims to enable the scale-up and intensification of the reactions catalyzed by IREDs to facilitate the application of these enzymes in industrially relevant transformations. The reductive amination reaction between cyclohexanone (1) and cyclopropylamine (2) was used in this study (Scheme 1). The product (3) and similar amines derived from prochiral ketones, which are currently synthesized using hazardous hydrides,15,16 are applied to the production of benzamide derivatives (Scheme 1). The latter is used for the treatment of diabetes and obesity by inhibiting


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the activity of 11-β- hydroxysteroid dehydrogenases (type 1) to control the cortisol concentration in the liver and adipose tissue.15-17 The IRED catalyzed route can serve as a less hazardous, enantioand stereoselective alternative to conventional methods.10,18

Scheme 1. General reaction scheme for reductive amination catalyzed by imine reductase (IRED) or reductive aminase (RedAm) (top); model reaction with cyclohexanone (1) and cyclopropylamine (2) catalyzed by IRED to form the secondary amine product (3), coupled with glucose dehydrogenase (GDH) for the in situ cofactor regeneration (bottom left); the application of 3 for the synthesis of benzamide derivatives (bottom right) for the treatment of diabetes and obesity by inhibiting the activity of 11-β-hydroxysteroid dehydrogenases (type 1).

Robotic, high-throughput technologies enable development of flexible, robust, and rapid screening methods for hit identification and optimization of enzymes.19 Access to a broad and varied enzyme library is a stepping stone to explore and access reductive amination for a wide range of ketone


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and amine pairs. In addition to the size and diversity of the IRED library, experimental conditions such as temperature, pH, and amine to ketone ratio are usually very important factors influencing the outcome of the screening experiments. Typical reaction conditions usually applied to these types of studies include 5-20 mM ketone, 1-10 molar equivalent amine, 1-10 mg/mL enzyme, 5% co-solvent DMSO utilized at pH 7 to 9, and 30-37 °C.10,13,18

Figure 1. A circular cladogram including approximately 1400 sequences, currently part of the IRED database (https://ired.biocatnet.de)20, and other notable literature sequences. Johnson Matthey’s IREDs are shown in green, Pfizer’s in blue, Roche’s in yellow and GSK’s in orange.


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After the initial screening of Johnson Matthey’s IRED collection for the reductive amination of 1 and 2, enzymes IRED-17, -18, -33, and -69 were selected for further investigation. High similarity between these enzymes and characterized literature sequences was observed (Figure 1). For example, IRED-18 shares a 70.9% sequence similarity (based on the BLOSUM62 substitution matrix) to the IRED from S. kanamyceticus21 while IRED-33 shares a 74.2% sequence similarity to AspRedAm10 (Table S1). IRED catalyzed reductive amination is a multi-parameter reaction system comprising two substrates, two co-substrates, two enzymes, co-solvent, buffer, pH, and temperature. Given the complexity of the IRED catalyzed reductive amination systems, it is useful to perform an initial screen of the reaction parameters. Design of Experiments (DoE) is a known and useful approach for structured and efficient analysis of multi-variable systems.22,23 To screen the aforementioned parameters and identify those exhibiting a dominant effect on the reaction outcome, we applied a customized factorial design at three levels (Table S2). The design’s center point was performed in triplicate to measure the experimental error. Detailed experimental procedures have been made available in the Supporting Information (SI). The results summarized in Table S3 were analyzed by the statistical software JMP® (v. 14.1.0). The data collected (Table S3) was investigated using principle component analysis (Figure S1) to visualize the data points’ distribution, detect any outliers, and study the significance and correlations of the variables. Further analyses through fitting least squares models were also attempted to confirm the important variables with the highest effects on the responses. The most significant parameters were identified to be the concentration of ketone 1 and amine 2, reaction temperature, and IRED loading, followed by the concentration of DMSO. The concentration of


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NADP+, GDH, and glucose, on the other hand, were observed to have less impact on the reaction’s outcome. Furthermore, the optimum reaction conditions revealed by the DoE studies fell in a window of operation involving 20-100 mM 1, 1.1-2.0 molar equivalent 2, 1.0 mg/mL (or 3-5% w/w of 1 and 2) IRED, 0.5-1.0 mg/mL GDH-101, 0.5-1.0 mM NADP+, 1.1-2.0 molar equivalent D-glucose, 25-35°C reaction temperature, and 0-5% (v/v) DMSO. More specifically, the sweet spot was the outcome of the DoE experiment 16 (Table 2S – Exp. 16) ran under 100 mM 1, 2 molar equivalent 2, 1 mg/mL IRED-69, 1 mg/mL GDH-101, 1 mM NADP+, 2 molar equivalent glucose, 5% DMSO, and 25°C. The implications of choice of buffer and pH on the reaction were also studied. Potassium phosphate and Tris-HCl buffers were compared. Both buffers provided full conversion under the pH range 6.5-8.0 for potassium phosphate and 7.5-9.0 for Tris-HCl (Table 4S). The effect of pH on IRED stability, however, can be quite significant24 as extreme pH conditions can destabilize proteins by influencing the electrostatic interactions.25 Higher pH, in the range of 9 or more, was observed (data not shown) to be detrimental to the IRED stability and led to its irreversible deactivation. Nevertheless, higher pH can also improve the reaction due to improving the imine formation as a result of promoting the amine’s deprotonation, especially when less nucleophilic aromatic amines are used.10,19,26 The process of intensifying a laboratory scale reaction into an industrial process benefits from early scale-up studies. The outcomes and information obtained from preliminary scale-up experiments can be implemented into designing subsequent scale-up trials, as well as new smallscale studies. The first attempt to scale up the reaction was made by performing it in 25 mL batch at 200 mM 1 (20 g/L) under controlled temperature and pH, as shown in Table 1 (Entry 1). After 16 hours, conversion was about 87% and yield about 83% (167 mM 3). The conversion and yield


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were calculated based on the consumption of 1 and generation of 3, respectively, where the difference between the two was due to the presence of the imine intermediate 4 (Table S5). These values did not change after an additional 8 hours, suggesting catalyst deactivation. The turnover number (TON), calculated as the ratio of the final concentration of 3 over the concentration of IRED, was 15261. Further intensification of the reaction was achieved by running it at the same scale (25 mL) using 500 mM 1 (Table 1 – Entry 2). This time, conversion was 7.4% after 4 hours and no product 3 was detected. The conversion increased to 31% after 20 hours, but unfortunately formation of product 3 was not observed. Decreasing the initial concentration of 1 to 400 mM, despite the IRED loading being reduced by half, allowed the conversion to double up to 62% and a minor amount of 3 (0.2%) be formed. These results confirm the findings of the DoE as they verify that a lower concentration of 1 and co-solvent significantly improve both conversion and yield. The presence of higher substrate concentrations (>200 mM 1) led to the accumulation of the imine 4, which in turn may significantly inhibit the IRED,9,10,27 leading to low or no formation of 3. Various strategies are commonly undertaken to overcome substrates’ negative effects on the biocatalyst including inhibition and deactivation. Such strategies involve protein engineering, enzyme immobilization, use of enzyme in whole cells preparation, reaction engineering, medium engineering, or combinations thereof. For the next attempt to run the reaction at 400 mM 1 (25 mL scale), a fed-batch28 reaction setup was used to allow slow feeding 1 and 2 (Table 1 – Entry 4) into the mixture over the course of the reaction. The results obtained demonstrated that the fed-batch strategy was extremely successful and unlocked the potential for scale-up. After 5 h, the yield of 3 was about 25%, which increased to above 84% (337 mM 3) after the feeding was finished (>20 h). Analysis of the reaction after 29 h showed no further progress. The implementation of the fed-


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batch strategy improved the reaction’s efficiency significantly as the concentration of 1 was easily doubled up to 400 mM (under the same conditions applied for the batch at 200 mM) and TON increased by more than two folds to 30777.


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Table 1. Summary of the experiments performed at larger scale. Entry

1

2

Reaction

IRED

IRED

GDH-101

NADP+

Glucose

DMSO

T

Time

Conversion

Imine

Amine

(mM)

(mol. equiv.)

Mode

Type

(mg/mL)

(mg/mL)

(mM)

(mol. equiv.)

(v/v %)

(°C)

(h)

(%)

(mM)

(mM)

1

200

2

Batch

IRED-69

5

0.5

0.5

2

5

30

16

87.1

8

166

24

86.5

5

167

2

500

2

Batch

IRED-69

10

1.0

1.0

1.1

5

30

4

7.4

37

0

20

30.8

154

0

3

400

2

Batch

IRED-69

5

1.0

1.0

1.1

0

30

20

59.5

162

0

24

62.5

249

1

4

400

2

Fed-batch (33 mM + 18.4 mM/h)

IRED-69

5

1.0

1.0

1.1

0

30

22

91.5

29

337

29

93.0

37

335

16

92.9

60

398

24

94.4

31

439

40

99.1

8

485

16

56.5

299

97

24

67.4

379

95

5

500

2


IRED-18

5

1.0

1.0

1.1

0

25

6

750

2


IRED-18 Whole cells

15

1.0

1.0

2

0

25

7

750

2

Fed-batch (100 mM + 100 mM/h)

IRED-33

5

1.0

1.0

1.1

0

25

8

100.0

N.D.

750

8

1500

2

Fed-batch (100 mM + 100 mM/h)

IRED-33

5

1.0

1.0

1.1

0

25

16

69.9

927

122


12

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In-process stability for the IREDs was studied by measuring the residual enzymatic activity after exposure to the reaction conditions for varying time spans. The stability profiles determined by this method were compared to identify the most robust enzyme candidate. The results obtained (Figure S2) showed one enzyme (IRED-18) specifically stood out with respect to its relative activity retained over time. Whilst all four enzymes tested showed very similar activities at the beginning of these experiments, activities of three of them dropped by 50% upon 16 h incubation, while the activity loss for IRED-18 was about 20%. After 24 h incubation, IRED-18 still showed about 8% activity remained, while the other enzymes showed no or trace activity. The longer half-life of IRED-18 was verified by performing the reaction under a higher substrate concentration at larger scale (25 mL) with 500 mM 1, in fed-batch (Table 1 – Entry 5). After feeding completion (16 h), conversion was about 93% and yield about 80% (398 mM 3). These values gradually increased to 99% and 97% (485 mM 3), respectively, by running the reaction for 40 h. Hence, the yield was increased by 44% from 337 to 485 mM 3 by replacing IRED-69 with the same amount of, the more stable, IRED-18. The crude product was subject to a work-up procedure for the isolation of 3 as described in the SI. The product (1.6 g) was isolated as an oil (~90% yield, >98% chemical purity). Amine 3 was purified as the hydrochloride salt (>99% chemical purity) and analyzed by 1H NMR (Figure S4). During the workup, protein precipitates were separated using Celite® 545 to facilitate the liquid-liquid extraction.29 The kinetic properties of the different IREDs were studied to identify the fastest enzyme for achieving higher productivities. Specific activities were determined empirically for various substrate concentrations ranging from 5 to 200 mM 1. The data obtained was used to estimate the apparent kinetic parameters of the IRED samples based on the Michaelis-Menten double substrate equation. As presented in Table 2, IRED-69 was the fastest enzyme followed by IRED-33 with


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kcat values of 6.93 and 5.84 s-1, respectively. However, stability studies (Figure S2) showed IRED69 had the highest deactivation rate among the four enzymes studied. The scale-up results were found to be in line with the kinetic data.

Table 2. Summary of the kinetic studies and apparent parameters estimated. IRED

Vmax

Km

kcat

(U/mg)

(mM)

(s-1)

IRED-17

8.61 ± 0.02

57.16 ± 20.46

4.74 ± 0.01

IRED-18

1.12 ± 0.08

20.22 ± 13.07

0.62 ± 0.04

IRED-33

10.88 ± 0.01

69.79 ± 23.03

5.84 ± 0.01

IRED-69

14.01 ± 0.01

29.60 ± 7.59

6.93 ± 0.00

The application of whole cells containing IRED for the reductive amination has been successfully shown at lower substrate concentrations.30,31 In an attempt to run the reaction catalyzed by whole cells containing IRED, to alleviate the substrate effect on the enzyme, lyophilized whole cells containing IRED-18 were used. The reaction was performed at 25 mL scale using 750 mM 1 (Table 1 – Entry 6). After 16 h the conversion was around 57% and yield 13%. Continuing the reaction for an extra 8 h allowed the conversion to increase by 10% to 67%, whereas the yield remained the same. Furthermore, about 4% (30 mM) cyclohexanol was also formed during this reaction by the E. coli endogenous ketoreductases. This is not an issue, as it can be shifted back towards the ketone whilst the reaction is achieving complete conversion.13,32 Thus, IRED-33 was selected as a better candidate with respect to both activity and stability. Using the latter enzyme and its kinetic data, the reaction using 750 mM 1 was run in fed-batch mode


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under the best conditions identified (Table 1 – Entry 7). The feeding was completed in 6.5 h (100 mM/h 1). Analysis of a sample of the reaction after 8 h showed full conversion of the starting material to amine 3. Therefore, an excellent turnover number of 48279 was achieved by IRED-33. Further increasing the substrate concentration in the reaction to about 150 g/L (1.5 M) 1 did not prove successful. After 16 h, conversion was about 70%, whereas the main product was the imine intermediate and only about 122 mM 3 (8%) was formed. It is postulated that the unsatisfactory results are due to higher concentration of the starting materials used, especially amine 2, accounting for 3 M, and their detrimental effects on the enzymatic activity.

Figure 2. Different steps included for the development of the IRED-catalyzed reductive amination and their impact on the productivity and overall biocatalytic reaction efficiency.


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In conclusion, the present study successfully demonstrates the development and intensification of an IRED catalyzed reaction and highlights various strategies and considerations (Figure 2) for achieving industrially viable processes employing this family of enzymes. Whilst the DoE studies performed on the model reaction enabled identifying the most significant parameters and their optimal levels, the kinetic studies facilitated the selection of a high-performing IRED candidate in terms of both stability and activity. The reductive amination of cyclohexanone 1 with cyclopropylamine 2 was scaled-up and intensified efficiently in fed-batch fashion, completely transforming 750 mM 1 to the desired amine 3 in 8 h. In this reaction the volumetric productivity surpassed 100 g/L per batch (103.5 g/L with respect to the product), STY was enhanced to 12.9 g L-1 h-1 and TON was above 48000 – all features of a sustainable biocatalytic process.33 The detrimental effect of higher substrate concentrations on the IRED was overcome by reaction engineering and employing fed-batch strategy. The rapid growth and diversity seen in the application of IREDs is very promising. The process development tactics demonstrated herein can serve as a guideline to help understand the limitations of IRED catalyzed reactions and promote the industrial application of these enzymes. The limitations addressed at the end of the current development cycle, such as the overall IRED stability or reaction rate, will eventually be overcome by molecular studies, followed by protein engineering and rapid screenings to obtain more active and stable IREDs. Ultimately, the application of IREDs in flow for continuous synthesis would further enhance process productivity and economy.34,35


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Supporting Information Experimental procedures for the following studies are included in the Supporting Information: (I) Design of Experiments, (II) Buffer and pH effects, (III) Stability studies, (IV) Kinetic studies, (V) Scale-up demonstrations in batch and fed-batch, (VI) Product isolation, and (VII) Analytics.

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Technical Considerations for Scale-Up of Imine Reductase Catalyzed

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