Preparation of β-hydroxy-α-amino Acid Using Recombinant d

Aug 4, 2015 - The chiral β-hydroxy-α-amino acid, (2R,3S)-2-amino-3-hydroxy-3-(pyridin-4-yl)-propanoic acid, is a key intermediate in the synthesis o...
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Preparation of β‑hydroxy-α-amino Acid Using Recombinant D‑Threonine Aldolase Steven L. Goldberg,* Animesh Goswami,* Zhiwei Guo, Yeung Chan, Ehrlic T. Lo, Andrew Lee, Vu Chi Truc, Kenneth J. Natalie, Chao Hang, Lucius T. Rossano, and Michael A. Schmidt Chemical Development, Bristol-Myers Squibb, One Squibb Drive, New Brunswick, New Jersey 08903, United States ABSTRACT: The chiral β-hydroxy-α-amino acid, (2R,3S)-2-amino-3-hydroxy-3-(pyridin-4-yl)-propanoic acid, is a key intermediate in the synthesis of the API (2R,3S)-2-amino-3-hydroxy-3-(pyridin-4-yl)-1-(pyrrolidin-1-yl)propan-1-one, a developmental drug candidate. Two D-threonine aldolase enzymes were identified to catalyze the aldol addition of glycine and pyridine 4-carboxaldehyde for the synthesis of the β-hydroxy-α-amino acid. The two D-threonine aldolase enzymes have similar properties. Efficient recombinant E. coli fermentation processes were developed for producing the enzymes. The stabilities of the enzymes were significantly improved by addition of divalent cations. An unexpected and beneficial finding was that the βhydroxy-α-amino acid aldol addition product directly crystallized out from the reaction mixture in high purity and high diastereoand enantioselectivity, contributing also to high yield and allowing easy isolation, processing, and downstream utilization. The temperature, pH, and amounts of reactants and enzyme were optimized to minimize reaction time and enzyme and raw material usage and maximize amino acid formation. Efficient D-threonine aldolase-catalyzed synthesis and recovery of the β-hydroxy-αamino acid at the 100 L scale was demonstrated leading to a highly efficient and environmentally friendly process for the production of the API.

1. INTRODUCTION

2. RESULTS AND DISCUSSION 2.1. Expression and Characterization of Recombinant D-Threonine Aldolases. Isolation and characterization of Dthreonine aldolases and their respective gene sequences from Alcaligenes (Achromobacter) xylosoxidans IFO 12699 and Arthrobacter sp. DK-38 were carried out by Liu.7,8 The Dthreonine aldolase enzymes from Alcaligenes (Achromobacter) xylosoxidans IFO 12699 (AXDTA) and Arthrobacter sp. DK-38 (ARDTA) are both monomeric proteins of nearly identical size, containing 377 and 379 amino acids, respectively, with 54% amino acid homology. They have nearly identical cofactor requirements and substrate ranges and can carry out aldol addition of glycine with many different aldehydes with similar specific activities. Though there is no report of aldol addition between glycine and pyridine-4-carboxaldehyde catalyzed by these enzymes, given their broad aldehyde substrate range, we reasoned that they should be able to catalyze such a reaction. Synthetic versions of both D-threonine aldolase genes optimized for E. coli codon usage were prepared and cloned into expression vectors pBMS20049 and pET30a. After overnight growth in shake flasks in the presence of IPTG, three of the strains were noticeably yellow in color, suggesting sequestration of the cofactor pyridoxal 5′-phosphate by overproduction of D-TA enzyme, which became more apparent upon lysis of the cells. SDS-PAGE indicated overexpression of a protein band of the expected molecular weight of D-TA (42 kDa) in all samples except BL21Gold(pBMS2004-AXDTA), where there was apparently no expression of the recombinant protein. Examination of soluble and insoluble fractions indicated that the overexpressed protein was located exclusively

The active pharmaceutical ingredient of the drug development candidate, (2R,3S)-2-amino-3-hydroxy3-(pyridin-4-yl)-1-(pyrrolidin-1-yl)propan-1-one 2, was initially synthesized by a conventional chemical process using classical resolution for the separation of desired isomer.1,2 The initial synthesis also used toxic ethyl isocyanoacetate. Since a theoretical maximum of 50% possesses the correct stereochemistry in the racemic synthesis, the initial process had low yield and generated significant waste. A more productive and environmentally friendly synthesis of 2 was sought. The API 2 is the pyrolidine amide of the chiral β-hydroxy-αamino acid 1, (2R,3S)-2-amino-3-hydroxy-3-(pyridin-4-yl)propanoic acid. The chiral β-hydroxy-α-amino acid 1 resembles the structure of D-threonine and could be synthesized by a Dthreonine aldolase (D-TA)-catalyzed condensation of pyridine4-carboxaldehyde 3 and glycine 4. The use of threonine aldolases to catalyze C−C bond formation and create nonnatural β-hydroxy-α-amino acids has been well-documented.3−5 Several different aliphatic and aromatic aldehydes were successfully used as acceptor with glycine as donor in the condensation. This article describes the development of an efficient, environmentally friendly process for the production of βhydroxy-α-amino acid 1 by a recombinant D-threonine aldolasecatalyzed aldol addition of glycine and pyridine 4-carboxaldehyde. More details regarding the initial synthesis of 1 and 2 and the chemical conversion of the enzymatically produced chiral βhydroxy-α-amino acid 1 to the API 2 are presented in an accompanying article.6 © XXXX American Chemical Society

Received: June 12, 2015

A

DOI: 10.1021/acs.oprd.5b00191 Org. Process Res. Dev. XXXX, XXX, XXX−XXX

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results were obtained for both ARDTA and AXDTA recombinant enzymes. Isolated solid precipitate, obtained in 70% yield, was exclusively the desired diastereomer (2R,3S) 1. The clear solution left after removal of solid contained relatively higher proportion of undesired hydroxy amino acid diastereomer (2R,3R) with the ratio of 88.8:11.2 for the desired to undesired isomer. The reaction was scaled up using 20 mL of pyridine 4carboxaldehyde as limiting reagent at 4 °C, and after 24 h 34 g of the hydroxy amino acid 1 was obtained as a white solid. NMR analysis showed the product to be a dihydrate. The yield was 70% as is. To establish the identity of the hydroxy amino acid 1 obtained by D-TA catalyzed aldol addition, it was converted to the drug substance 2 as shown in Scheme 2. The amino group of 1 was first protected by Boc derivatization to 5; the Boc acid 5 was then condensed with pyrrolidine 6 to the amide 7, and finally Boc deprotection gave the drug substance 2. HPLC indicated the 2 obtained had 100% enantiomeric excess (ee) and 97.5% diastereomeric excess (de), thus establishing the stereochemistry of 1. The reactions of Scheme 2 were carried out later in larger scale and will be described in the accompanying publication.6 It has also been demonstrated that the slight loss of de (from 100% for 1 to 97.5% for 2) seen here occurred during the chemical steps of the first small-scale synthesis. Later, large scale reactions confirmed that hydroxy amino acid 1 obtained from D-TA catalyzed reaction is almost exclusively one diastereomer (99.1% de) and one enantiomer (99.8% ee). X-ray crystallography also confirmed the absolute configuration of the β-hydroxy-α-amino acid 1 to be 2R,3S. Enzyme catalyzed aldol additions are reversible and generally require large excess of one reagent to drive the reaction to the desired direction. A previous report11 described obtaining yields and de of 79% and 98%, respectively, of D-synphenylserine (2R,3S) upon condensation of glycine and benzaldehyde catalyzed by A. xylosoxidans D-threonine aldolase. However, low temperatures (5 °C) and a 10:1 ratio of glycine to aldehyde were required to minimize the reverse (cleavage) reaction. The unexpected propensity of the desired isomer of 1 to crystallize out of the solution pushed the reaction in the desired direction in high yield and suggested that a highly efficient and environmentally friendly process could be attained through further development. 2.3. Substitution of Glycine in Condensation Reaction. In order to synthesize the API 2 directly using the D-TA catalyzed aldol addition, reaction of pyridine-4-carboxaldehyde 3 with pyrrolidine amide of glycine 8 (Scheme 3) was tried with both enzymes. Unfortunately, there was no reaction with either ARDTA or AXDTA even at a 10:1 molar ratio of 3 and 8. In addition, attempts to carry out D-TA catalyzed aldol addition reaction between 3 and glycine amide 9 or Boc-glycine 10 in order to shorten the synthetic sequence for 2 were unsuccessful. The two D-TA enzymes work with glycine only but not substituted glycine (e.g., alanine 12) as donor. This result is in agreement with literature13−15 reports that the D-TA from Alcaligenes (Achromobacter) xylosoxidans and Arthrobacter sp. can only accept glycine as donor. One Dthreonine aldolase from Pseudomonas sp. capable of accepting D-enantiomers of alanine, serine, and/or cysteine has recently been reported.12−14 2.4. Optimization of Reaction Conditions. D-TAs have a reported pH optimum of 8.0−9.5.11 Reactions (1 mL) were conducted as described in Materials and Methods using

in the cytoplasm. These results were verified by performing an activity assay measuring the ability of the enzyme to convert Dthreonine into glycine and acetaldehyde, as shown in Table 1: Table 1. D-Threonine Aldolase Activities of Shake Flask Cultures bacterial strain

IPTG added

activity (U/mL)

BL21Gold (pBMS2004-ARDTA) BL21Gold (pBMS2004-ARDTA) BL21(DE3) (pET30a-ARDTA) BL21(DE3) (pET30a-ARDTA) BL21Gold (pBMS2004-AXDTA) BL21Gold (pBMS2004-AXDTA) BL21(DE3) (pET30a-AXDTA) BL21(DE3) (pET30a-AXDTA)

50 μM 1 mM 50 μM 1 mM 50 μM 1 mM 50 μM 1 mM

32.33 159.83 172.34 62.26 0.62 0.57 97.90 60.90

It was apparent that only background activity was present in extracts of BL21Gold (pBMS2004-AXDTA): Nearly identical activities were observed if an extract derived from the negative control strain BL21Gold (pBMS2004) was used or if Lthreonine was used in place of D-threonine. While both genes could be efficiently expressed using the T7 promoter of pET30a, only ARDTA could be synthesized from the tac promoter of pBMS2004. We have not investigated the reason for these results. Kudla et al.10 showed that 5′ end of a gene might influence mRNA folding and protein expression levels which may explain the lack of expression for pBMS2004 and AXDTA. 2.2. Aldol Addition Reactions. Sample of the active extracts were used to determine if they could catalyze the condensation of pyridine 4-carboxaldehyde and glycine to form the intermediate 1: Scheme 1. Aldol Addition Using D-Threonine Aldolase

Steinreiber et al.5 and Fesko et al.11 suggested conducting similar reactions at 4 °C to favor the condensation reaction and to improve the diastereoselectivity. The reactions (1 mL volume) were carried out with 1 M glycine, 100 mM pyridine 4-carboxaldehyde, and 1 U/mL of ARDTA or AXDTA enzyme at pH 8.0 at 4 °C for overnight (18 h). A heavy crystalline precipitate had formed after the overnight incubation. The result of a typical experiment is shown in Table 2. The total suspension showed a high preference (97.1%) for the desired isomer 1 with only 2.9% of the undesired diastereomer. Similar Table 2. HPLC Analysis of Condensation Reaction Using Enzyme Solution Containing Recombinant AXDTA suspension supernatant isolated solid

desired isomer 1 %

undesired diasteromer %

97.1 88.8 100.0

2.9 11.2 NDa

a

ND = not detected at sensitivity of HPLC system used. HPLC area percent was directly used to determine concentration without correction. B

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Scheme 2. Synthesis of API 2 Using 1 Obtained from D-TA Catalyzed Reaction

mM potassium phosphate buffer was used for all subsequent studies and scale-up activities. The optimum pH is close to the isoelectric point of 1, which facilitates its precipitation and drives the reaction to completion. Reactions were initially performed at 4 °C as this temperature was reported to improve diastereoselectivity.5 The first series of experiments to increase volumetric productivity was limited to a maximum pyridine 4-carboxaldehyde 3 concentration of 3% using the initial 10:1 glycine− pyridine 4-carboxaldehyde molar ratio due to precipitation of the reactants at higher than 3% concentrations of 3 at 4 °C. Given the propensity of 1 to crystallize out from the reaction mixture, we suspected that the glycine−pyridine 4-carboxaldehyde ratio could be lowered while still favoring the condensation reaction. Consequently, later experiments with concentration of 3 greater than 3% were carried out at higher temperatures (room temperature of 22−25 °C) to improve the solubility of the reactants. The effect of supplying additional amounts of the cofactors pyridoxal 5′-phosphate and MnCl2· 4H2O to increase product formation was also investigated. Results are summarized in Figure 1. The results clearly indicate that (1) raising the temperature from 4 °C to room temperature (22−25 °C) significantly improved the yield of 1; (2) glycine−pyridine 4-carboxaldehyde ratio could be lowered to 2:1 while maintaining yield of 1; and (3) higher pyridine 4-carboxaldehyde concentrations also required an increase in the concentration of cofactors to obtain the higher yield of desired product 1. HPLC analysis of crystallized 1 under optimized conditions revealed no change in the high quality of the material formed. The product 1 formation at 3, 4, and 5% pyridine 4carboxaldehyde 3 were investigated in detail, along with the effect of varying temperature (23 °C, 30 °C, and 37 °C), and the results are shown in Figure 2. A reaction temperature of 30 °C gave the highest formation of 1 at all pyridine 4-carboxaldehyde concentrations with 23 °C giving similar yield. The yield was definitely lower at 37 °C. It is preferable to avoid heating above 30 °C; therefore, the reactions were henceforth carried out between 23 and 30 °C. Later experiments revealed that the yield of 1 at 5% pyridine 4carboxaldehyde could be increased to that seen at 3% and 4% by raising both cofactor concentrations to 0.5 mM. The maximum aldehyde 3 concentrations was kept at 5%, since at greater than 5% of 3 the reaction mixture turned into a distinct

Scheme 3. Condensation of Pyridine 4-Carboxaldehyde with Alternative Compounds

dilutions of 1 M Tris-HCl stock solutions at pH 8.0, 8.5, 9.0, and 9.5 to 50 mM. After 5 h at 4 °C, the entire reaction mix was diluted 1:100 in 10% acetonitrile and dissolved before filtering and HPLC analysis. Results are presented in Table 3 with AXDTA enzyme; results with ARDTA were essentially identical: A pH of 8.0 was found to be the optimum for the yield and diastereoselectivity of the desired product 1. A follow-up experiment substituted 50 mM potassium phosphate buffer at pH 8.0 for Tris-HCl with no significant change in formation of 1. Since potassium phosphate is less expensive than Tris, 50 Table 3. Effect of pH on Efficiency of Aldol Addition pH of Tris buffer

area % 1

area % undesired isomer

7.0 7.5 8.0 8.5 9.0 9.5

56.30 80.30 84.69 81.58 76.72 74.50

0.97 2.36 2.27 2.45 2.35 2.48 C

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Figure 1. Varying glycine:pyridine 4-carboxaldehyde (gly:P4C) ratio, temperature of reaction (RT: room temperature), and cofactor (CF) concentration vs 1 formation. All reactions (1 mL) were carried out at pH 8.0 with 3% pyridine 4-carboxaldehyde. “1X CF” (cofactor) = 50 nmol each pyridoxal 5′-phosphate and MnCl2·4H2O.

Figure 3. Enzyme activity vs 1 formation. P4C = pyridine 4carboxaldehyde. Figure 2. Varying pyridine 4-carboxaldehyde (P4C) concentration and temperature vs 1 area %.

presence of divalent cations was critical for thermal stability of the D-TA enzymes at higher than ambient temperatures for a short time (15 min).14 The effect of divalent cations to extend the storage stability of D-TA in cold (4 °C) was explored. The results are for AXDTA are shown in Table 4 (results using ARDTA were essentially identical): Divalent transition metal cations (cobalt and nickel) improved the stability significantly. Ni2+ was the best and retained almost all activity on storage at 4 °C for a week. Although MnCl2 was required to catalyze the condensation

orange color, an unidentified precipitate was formed, and the formation of 1 was greatly inhibited (99.95% >99.95% >99.95%

1 after drying to remove water.

2.7. Scale up of D-Threonine Aldolase Catalyzed Condensation Reaction. Based on the above optimization experiments, the aldol addition was scaled up using a 100 L reaction vessel with AXDTA enzyme as described in Section 4.9d. A summary of three batches prepared using this protocol is presented in Table 5. There was no apparent difference between the recombinant ARDTA and AXDTA enzymes in their ability to catalyze condensation of glycine and pyridine 4-carboxaldehyde to make 1. We were able to exploit the propensity of 1 to crystallize out of solution to optimize the reaction to make it more cost- and process efficient. Low temperatures (4−10 °C) and a large molar (10-fold) excess of glycine are normally required to obtain amino acids with high enantio- and diastereoselectivity.11 Baer et al. describe the use of two L-threonine aldolases to prepare β-hydroxy L-α-amino acids.15 They achieved high yields and excellent enantioselectivity of the desired substituted hydroxy amino acids, but due to thermodynamic limitations the maximum diasteromeric ratio that could be obtained was 82:21 (syn/anti). A glycine−aldehyde molar ratio of 8:1 was necessary to achieve these results, and the maximum aldehyde loading was 250 mM. In our study, we speculate that precipitation of the amino acid shifted the equilibrium of the reaction sufficiently to obviate the need for the extreme conditions previously used. We were able to obtain very pure material despite increasing the temperature of the reaction to 25−30 °C and reducing the glycine:pyridine 4-carboxaldehyde ratio to 2:1. Volumetric productivity was improved by increasing the concentration of the reactants 5-fold (equivalent to 500 mM aldehyde). The crystalline material was isolated on a large scale by simple pad filtration as dihydrate of 1. It was used to prepare drug substance 2 with the very high e.e. (>99%) and d.e. (99.8%), of the aldol product 1. 2.8. Absence of Residual Protein in the Aldol Product. The residual protein coming from a process which utilizes biocatalysis is usually well below the acceptable impurity level and therefore not an issue.16 Current guidelines suggest that residual protein in a sample of drug substance or API be present at no greater than 0.05%.17 At the maximum solubility of 1 (1% w/v in water), this would be equivalent to 0.0005 mg/ mL. Known amounts of protein from a lysate of BL21(DE3) (pET30a-AXDTA) were mixed with a solution of 1 to provide internal standards for polyacrylamide gel electrophoresis. The protein solution (0.0001 mg/mL) and protein solution added to compound 1 at 0.01% clearly showed the protein bands, while proteins were not visible in the solution of 1. Thus, compound 1 produced by the biocatalytic process was shown to contain at least less than 0.01% of protein, well below the current guidelines for residual protein levels in the API.

purity. Due to the propensity of 1 to crystallize out during the course of the reaction, the reaction could be carried out at higher temperatures and with significantly reduced glycine− pyrdine 4-carboxaldehyde ratio than anticipated based on previous reports. Moreover, the concentrations of the reactants could be increased without solubility issues, leading to greatly improved volumetric productivity. The process described was demonstrated at a 100 L scale with no significant issues using D-TA obtained from extracts of recombinant E. coli produced by an optimized fermentation protocol. An accompanying communication from our group6 describes the chemical conversion of the enzymatically produced chiral βhydroxy-α-amino acid 1 to the API 2 resulting in a highly efficient and environmentally friendly synthesis of the API.

4. EXPERIMENTAL SECTION 4.1. Chemicals and Materials. Glycine was obtained from TCI (Tokyo, Japan). Pyridine 4-carboxaldehyde was purchased from Alfa Aesar, Ward Hill, MA. D-Threonine was obtained from Sigma-Aldrich, St. Louis, MO. All other chemicals were of reagent grade. 4.2. Bacterial Strains, Plasmids, and Culture Conditions. The amino acid sequences encoding D-threonine aldolase (D-TA) of Alcaligenes (Achromobacter) xylosoxidans IFO 126997 and Arthrobacter sp. DK-388 were obtained from PubMed under accession numbers BAA86032.1 and O82872.1, respectively. The sequences were used to prepare synthetic genes with optimized E. coli codon usage (GeneArt, Regensburg, Germany). Electrocompetent E. coli DH10B [FmcrA Δ(mrr-hsdRMS-mcrBC) ø80dlacZΔM15 ΔlacX74 deoR recA1 endA1 araD139 Δ(ara, leu)7697 galU galK lambda- rpsL nupG] was used for routine cloning and obtained from Life Technologies, Grand Island, NY. Chemically competent E. coli BL21Gold [F− ompT hsdSB(rB−, mB−) gal dcm and BL21(DE3) Gold [F− ompT hsdSB(rB−, mB−) gal dcm (DE3) (Agilent Technologies, Inc., Santa Clara, CA) were used as hosts for expression of D-threonine aldolase. pBMS2004 is a derivative of a proprietary expression vector with a tac promoter.9 T7 expression vector pET30a was purchased from EMD Millipore Biosciences, Billerica, MA. Luria−Bertani (LB) agar powder medium was purchased from Life Technologies (Grand Island, NY) and used for growth of recombinant E. coli by supplementation with 50 μg/mL filter sterilized kanamycin sulfate after autoclaving. Shake flask liquid cultures were conducted using MT5-M2/ Kan medium (40 g/L glycerol, 20 g/L Quest Pea hydrolysate, 18.5 g/L Bacto Yeastolate, 6 g/L Na2HPO4, 1.25 g/L (NH4)2SO4 containing 50 mg/mL filter sterilized kanamycin sulfate). After overnight growth at 30 °C, 250 rpm in MT5M2/Kan the optical density at 600 nm (OD600) was determined and used to inoculate fresh medium to a starting OD of ca. 0.3 and incubated as described. At OD 0.8−1.0, expression of recombinant D-threonine aldolase was induced by addition of filter sterilized solution of isopropylthio-β-Dgalactoside (IPTG) at appropriate concentrations. Cells were

3. CONCLUSION Recombinant D-threonine aldolases (D-TAs) from Alcaligenes (Achromobacter) xylosoxidans and Arthrobacter sp. were used to catalyze formation of the chiral intermediate β-hydroxy-αamino acid 1 with very high enantio- and diastereometric F

DOI: 10.1021/acs.oprd.5b00191 Org. Process Res. Dev. XXXX, XXX, XXX−XXX

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250 mM IPTG solution was added to a final concentration 0.2 mM. The flow rate was reduced to 2/3 of that at induction and fermentation continued until the entire 2 L of feed was consumed (ca. 45 h postinoculation). Fermentation broths were centifuged at 4 °C to collect the cells containing the D-TA enzyme. For fermentation at 15 L scale, the above media and inoculation conditions were scaled up proportionately from the 2 L experiments based on an 8 L starting volume in a Sartorius Biostat C vessel with three Rushton-type impellers set at 500 rpm. Airflow was 12−15 L/min and pressure 10 psig. Oxygen concentration was maintained at 30% or higher as needed by increasing tip speed. Feeding continued until the entire 6 L feed was consumed (ca. 20−24 h after initiation of feeding program), whereupon the tank batch temperature was reduced to 5 °C and agitation reduced to 400 rpm. Cells were pelleted in a CARR Pilot centrifuge operated at 15 325 rpm (20 000 × g). The cell paste was transferred to polypropylene containers and the wet cell weight determined before processing or storage at −70 °C. 4.7. Preparation of D-Threonine Aldolase Enzyme Solutions from Recombinant E. coli Cells. a. Small-Scale Lysates. The cells were resuspended (frozen cells were thawed to room temperature before suspension) in 50 mM potassium phosphate buffer, pH 8.0 at 1 mL/0.2 g wet cell weight (20% suspension). A portion of 1 mL of the cell suspension was placed into a Lysing Matrix “B” tube and disrupted using the FastPrep unit at 6.0 m/s, 2 × 20 s (MP Biomedicals, Solon, OH). The lysates were centrifuged at 18 000 × g for 4 min (“soluble” fraction). The samples had a distinct yellow color due to the binding of pyridoxal 5′-phosphate, which is a cofactor for this enzyme. b. Preparation of Microfluidized Extract from Cell Paste. Pelleted cells were suspended at 20% (w/v) in 50 mM potassium phosphate buffer, pH 8.0. They were disrupted by two passages of the suspension through a model 110F microfluidizer at 12 000−15 000 psig (Microfluidics, Newton, MA). Debris was pelleted by centrifugation for 30 min at 17 700 × g and the supernatant assayed for enzyme activity. c. Preparation of Microfluidized Extract from Cell Paste for Large Scale Reaction. For large-scale preparation of 1, clarification was accomplished by passage of the extract through a Sartopore 2 XLG MidiCaps membrane (Sartorius AG, Bohemia, NY). d. Preparation of a Microfluidized Extract from Whole Broth. After termination of fermentation, cell broth was held at 4 °C until use. Microfluidization was carried out with this material as described in Section 2.7b. e. Lyophilization of Cell Lysates. Twenty-five mL of supernatant from microfluidized BL21(DE3) (pET30aAXDTA) cells (see Section 2.7b) was pipetted into a 150 cm diameter Pyrex crystallizing dish. The dish was covered with Saran Wrap and placed into a −75 °C freezer overnight. The dish was covered with a sheet of Whatman 1 filter and transferred to a Virtis Genesis freeze-drying chamber set to maintain a vacuum of 100 mbar at room temperature. Lyophilization was terminated after 16 h and the flaky material transferred to a polypropylene container. 4.8. D-TA Activity Assay. D-threonine aldolase (D-TA) activity was determined as described by Fesko et al.11 Acetaldehyde produced by the action of D-TA on D-threonine is reduced by yeast alcohol dehydrogenase in the presence of

harvested by centrifugation of the culture 18−22 h postinduction. 4.3. General Recombinant DNA Techniques. Restriction enzymes were purchased from Life Technologies. The FastLink DNA ligase kit was obtained from Epicenter Biotechnologies, Madison, WI. Both materials were used according to the manufacturer’s protocols. Transformation of E. coli with plasmid DNA by electroporation was performed under standard conditions using a BioRad Gene Pulser II system (BioRad, Hercules, CA). Chemically competent cells were transformed following the manufacturer’s recommendations. Polymerase chain reaction (PCR) was performed using the GoTaq Green Master Mix (Promega, Madison, WI) and appropriate template and primers. 4.4. Sodium Docecyl Sulfate−Polyacrylamide Gel Electrophoresis. Sodium docecyl sulfate−polyacrylamide (SDS/PAGE) gel electrophoresis was performed in a 10% NuPAGE Bis-Tris polyacrylamide gel with NuPAGE MOPS running buffer (Life Technologies). Proteins were visualized using the Simply Blue colloidal Coomassie Blue dye solution (Life Technologies). 4.5. Cloning of D-TA Genes into Expression Vectors. The two synthetic D-TA genes were prepared by the vendor (GeneArt) in plasmid pMK with specified NdeI and KpnI restriction sites at the 5′ and 3′ ends of the genes, respectively. A portion of 2 μg of plasmid DNA was digested with 10 U each NdeI and KpnI in 20 μL final volume for 1 h at 37 °C. The 1134 or 1140 base pair (bp) DNA fragments containing D-TA coding regions from Alcaligenes (AXDTA) or Arthrobacter (ARDTA), respectively, were excised following electrophoresis on a 1.0% agarose gel and purified using a MinElute column and buffers (Qiagen, Chatsworth, CA). The isolated DNAs were ligated to NdeI-KpnI-digested plasmids pBMS2004 and pET30a and transformed into DH10B. Colonies were screened for plasmids containing an insert of the correct size by PCR using plasmid-based primers flanking the putative insert. The plasmids were confirmed to contain the desired genes by DNA sequencing (GeneWiz, South Plainfield, NJ). 4.6. Production of Recombinant E. coli Cells in Fermentor. Seed medium consisting of 10 g/L soy protein (HySoy, Quest), 5 g/L yeast extract (Yeastolate), 5 g/L NaCl, and 50 mg/mLfilter sterilized kanamycin was used to prepare the initial inoculum from frozen vials of the recombinant strain. The culture was grown in a shake flask at 30 °C, 250 rpm for 24 h. A portion of 2 L of sterile production medium (2.2 g/L glucose monohydrate, 5.0 g/L Yeastolate, 1.7 g/L (NH4)2SO4, 1.0 g/L citric acid, 7.0 g/L K2HPO4, 0.03 g/L FeSO4·7H2O, 0.4 g/L UCON antifoam (Dow Corning, Midland, MI), 2.3 g/L MgSO4·7H2O, and 50 mg/mL kanamycin sulfate (the latter two ingredients added as a combined filter-sterilized solution postautoclaving) in a 5 L Biostat B fermentation vessel (Sartorius Stedim Biotech, Bohemia, NY) was prepared and adjusted to pH 7.2 with 10 N NaOH. The seed culture was used to inoculate the production medium to a starting OD600 = 0.2. Growth in the Biostat was carried out under the following conditions: 30 °C, impeller at 1000 rpm, and airflow at 3.0 L/ min. When the OD reached ca. 1.0 feeding of a sterile medium consisting of 220 g/L glucose, 100 g/L Yeastolate, and 50 mg/ L filter sterilized kanamycin sulfate by means of a programmable peristaltic pump was initiated. The starting feed rate was 15 mL/h with an increase of 2.5 mL/h/h. pH was controlled at 6.8 with 28% NH4OH. Twenty-four hours after inoculation, the OD of the culture was 55−60, where upon a filter sterilized, G

DOI: 10.1021/acs.oprd.5b00191 Org. Process Res. Dev. XXXX, XXX, XXX−XXX

Organic Process Research & Development

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through a 20 μM polypropylene cloth and dried in a Nutschetype Aurora filter dryer at 40 °C for 24 h. 4.10. Analysis of Amino Acid Diasteromers. For small scale reactions, diasteromers of 1 were analyzed on a Shimadzu LC-10AT high-pressure liquid chromatography (HPLC) system using a Primesep 100 (5 μm, 4.6 × 150 mm) column. Chromatography conditions: Ambient temperature; injection vol 10 μL; flow rate 1 mL/min; detection 257 nm. Eluent: 20% acetonitrile/0.05% H2SO4. Isocratic, 15 min. Retention times: 1 9.5 min; 1 diasteromer 10.6 min. During development work, it was necessary to analyze various fractions of the reaction mixtures containing precipitated 1 (e.g., reaction mixture with suspended crystalline material, supernatant after removing precipitated 1, and the precipitate with liquid removed). In these instances, the entire suspension was dissolved in 100 vol 10% acetonitrile prior to HPLC analysis. The suspension was also centrifuged for 1 min, 18 000 × g and the clear supernatant added to 100 vol 10% acetonitrile for HPLC. Finally, the suspension was centrifuged for 1 min at 18 000 × g and the supernatant removed. The pellet was washed with 1 mL 100% acetonitrile, dried in a SpeedVac for 5 min with heat, and dissolved in 100 vol of 10% acetonitrile for HPLC. All samples were filtered through a 0.2 μM nylon filter prior to HPLC analyses. A different method was used to analyze formation and quality of 1 prepared in the 100 L reactor. Samples were diluted 1:10 in 90:10 H2O−acetonitrile prior to injection onto the Primesep 100 column. Chromatography conditions: Ambient temperature; injection vol 5 μL; flow rate 1 mL/min; detection 257 nm. Eluent A, water; eluent B, acetonitrile; eluent C, 1% TFA in water. A gradient program over 35 min was used to resolve 1 and its diastereomer: 0 min, 90% A/5% B/5% C; 20 min, 75% A/15% B/10% C; 25 min, 20% A/60% B/20% C; 26 min, 20% A/60% B/20% C; 26.1 min, 90% A/5% B/5% C; 30 min, 90% A/5% B/5% C. Retention times: 1 25.6 min; 1 diastereomer 25.9 min. 4.11. Assay for Protein in Preparations of 1. Isolated 1 prepared in a 100 L reactor was dissolved in deionized water at its solubility limit (1% w/v). Total protein concentration of a 20% (w/v) lysate of BL21Gold(DE3) (pET30a-AXDTA) cells was determined using the BioRad Protein Dye concentrate vs a standard curve of known concentrations of bovine serum albumin. The amount of protein is present in the lysate was 8.5 mg/mL. Therefore, 1 mL of the 1% solution of 1 was mixed with 6, 3, and 1.2 μL of a 1:10 dilution of the lysate to prepare samples of containing 0.05, 0.25, and 0.10% protein, respectively. In addition, samples with protein dilutions alone were prepared. A portion of 20 μL of each sample as well as the solution of 1 with no additions were analyzed by polyacrylamide gel electrophroresis and stained (see Section 4.4).

NADH. The oxidation of NADH is measured by decrease in A340 nm. A 10× master mix composed of 0.5 M D-threonine (0.596 g/ 10 mL), 0.5 mM pyridoxal 5′-phosphate (0.00124 g/10 mL), 0.5 mM MnCl2·4H2O (0.001 g/10 mL), 2 mM NADH (0.014 g/10 mL), and yeast alcohol dehydrogenase (15 mg/10 mL @ 200 U/mg) was prepared. The reaction is composed of 400 μL of deionized water, 500 μL of 0.1 M potassium phosphate buffer pH 8.0, and 100 μL of the master mix. A dilution of 10 μL lysate to 990 μL deionized water was prepared immediately before use. The reaction was initiated by addition of 10 μL enzyme solution to the assay mix. Activity/mL was calculated by the formula (ΔA340 × dilution factor)/(6.2 × t[min] × volume extract used [mL]). The typical assay time is 2 min. One unit of enzyme catalyzes the formation of 1 μmol of acetaldehyde (= 1 μmol of NADH oxidized) per minute at room temperature under the above conditions. 4.9. Synthesis of 1. a. First Demonstration of D-TA Catalyzed Reaction. The reactions (1 mL volume) were performed as suggested by Steinreiber5 and Fesko11 using 1 M glycine and 100 mM of pyridine 4-carboxaldehyde and 1 U/mL of ARDTA or AXDTA enzyme. Reactions were placed on a rotating platform at 4 °C and left overnight (18 h). A heavy crystalline precipitate had formed after the overnight incubation. HPLC analysis of the dissolved precipitate indicated one peak with a retention time identical to the standard (chemically prepared 1). b. Initial Small Reaction and Product Isolation for Characterization. To 583.5 mL of deionized water in a 1 L beaker, 33.5 mL of 1 M potassium phosphate solution (pH 8.0), 20 mL of pyridine 4-carboxaldehyde (limiting reagent), 30 g of glycine, 26 mg of pyridoxal 5′-phosphate, and 12 mg of MnCl2·4H2O were added with stirring. The beaker was placed in a cold room (4 °C) and the solution mixed gently on a stir plate for 24 h. The precipitate was captured by vacuum filtration through filter paper using a Buchner funnel, washed twice with 100 mL of n-heptane, and dried in vacuo for 20 h at 40 °C. A sample of 34 g of white solid was obtained out of a theoretical 36 g. NMR analysis confirmed the structure of the product 1 as a dihydrate. The yield was determined to be 70%. c. Small-Scale Reactions under Optimum Conditions. Optimized reaction conditions for formation of 1 were: 5% v/v (0.46 M) pyridine 4-carboxaldehyde, a 2.2-fold molar excess (1.0 M) glycine, 0.5 mM pyridoxal 5′-phosphate, 0.5 mM MnCl2·4H2O, in 50 mM potassium phosphate buffer, pH 8.0, and enzyme solution containing 175 U D-TA per mL of pyridine 4-carboxaldehyde. The sample was maintained at 25− 30 °C with gentle agitation. Crystalline 1 began to form after 45 min to 1.5 h, and the reaction was terminated after 20−24 h. The precipitate was isolated as described in Section 4.9b. d. Synthesis of 1 in kg Scale. The optimized reaction conditions were used as a basis for preparation of 1 at the 100 L scale. For this process, a 50 mM phosphate buffer pH 8.0 solution was prepared and the temperature of the solution maintained at 25 °C with agitation. 1.9 eq glycine, 0.00047 eq pyridoxal 5′-phosphate, 0.00029 eq MnCl2·4H2O, and 4 vol % pyridine 4-carboxaldehyde was charged to the reactor. Condensation was initiated by addition of enzyme solution containing 175 U/mL D-threonine aldolase per mL pyridine 4carboxaldehyde to the reactor. At 5.5 h a heavy slurry was observed in the reactor. The reaction was considered complete when the mother liquor contained