Biocatalytic process for preparing eslicarbazepine and analogs thereof

Abstract

The present disclosure relates to biocatalysts and its uses for the efficient preparation of eslicarbazepine, eslicarbazepine acetate, and analogs thereof.

Claims

1. A non-naturally occurring polynucleotide encoding a polypeptide capable of converting compound (2c) ##STR00027## to compound (1c) ##STR00028## in enantiomeric excess in presence of NADPH, the ketoreductase polypeptide comprising an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:10 and has at least the following features: X80 is T; X96 is V or R; X145 is L; X153 is T; X190 is P; X196 is L or M; and X226 is V.

2. The non-naturally occurring polynucleotide of claim 1, wherein the encoded polypeptide sequence has at least 93% sequence identity to SEQ ID NO: 36.

3. The non-naturally occurring polynucleotide of claim 1, wherein the encoded polypeptide sequence further comprises one or more of the following features: X17 is H; X29 is T; X43 is R; X71 is P or G; X87 is L; X95 is Y; X131 is C; X173 is L; and X199 is M.

4. The non-naturally occurring polynucleotide of claim 1, wherein the encoded polypeptide sequence further comprises one or more of the following features: X17 is M or H; X29 is T; X40 is R; X43 is R or V; X64 is V; X94 is G or A; X95 is Y or M; X147 is Q or M; X152 is L or A; X157 is C or S; X173 is L; X199 is M; and X200 is P.

5. The non-naturally occurring polynucleotide of claim 1, where the encoded polypeptide sequence further comprises one or more of the following features: X25 is T; X76 is A; X144 is V; X150 is L; X194 is R; X233 is G; and X249 is W or F.

6. The non-naturally occurring polynucleotide of claim 1, wherein the encoded polypeptide sequence comprises at least the following features: X64 is V; X71 is P or G; X80 is T; X87 is L; X94 is G or A; X96 is V or R; X145 is L; X147 is Q or M; X153 is T; X173 is L; X190 is P; X196 is M or L; X199 is M; and X226 is V.

7. The non-naturally occurring polynucleotide of claim 1, wherein the encoded polypeptide sequence further comprises one or more of the following features: X17 is M or H; X29 is T; X40 is R; X43 is R or V; X95 is M or Y; X131 is C; X152 is L or A; and X200 is P.

8. The non-naturally occurring polynucleotide of claim 1, wherein the encoded polypeptide sequence further comprises one or more of the following features: X25 is T; X76 is A; X144 is V; X150 is L; X157 is C or S; X194 is R; X233 is G; and X249 is W or F.

9. The non-naturally occurring polynucleotide of claim 1, wherein the encoded polypeptide sequence comprises at least the following features: X17 is H or M; X25 is T; X29 is T; X40 is R; X43 is R or V; X64 is V; X71 is G or P; X80 is T; X87 is L; X94 is G; X95 is Y or M; X96 is R or V; X131 is C; X145 is L; X147 is Q or M; X152 is A or L; X153 is T; X157 is S or C; X173 is L; X190 is P; X196 is M or L; X199 is M; X200 is P; and X226 is V.

10. The non-naturally occurring polynucleotide of claim 9, wherein the encoded polypeptide sequence further comprises one or more of the following features: X76 is A; X144 is V; X150 is L; X194 is R; X233 is G; and X249 is W or F.

11. The non-naturally occurring polynucleotide of claim 1, wherein the encoded polypeptide sequence comprises a sequence selected from SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 38.

12. A non-naturally occurring polynucleotide that encodes a polypeptide sequence comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO:2 and having one or more residue differences as compared to the reference sequence of SEQ ID NO:2 at residue positions corresponding to X71, X87, and X131, wherein the polypeptide has ketoreductase activity.

13. The non-naturally occurring polynucleotide of claim 12, wherein encoded polypeptide sequence comprises one or more of the following features: X71 is P or G; X87 is L, and X131 is C.

14. The non-naturally occurring polynucleotide of claim 1, wherein the encoded polypeptide sequence is immobilized on a solid support.

15. The non-naturally occurring polynucleotide of claim 12, wherein the encoded polypeptide sequence is immobilized on a solid support.

16. An isolated polynucleotide encoding the polypeptide of claim 12.

17. An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, and 37.

18. An expression vector comprising the polynucleotide of claim 1.

19. An expression vector comprising the polynucleotide of claim 16.

20. An expression vector comprising the polynucleotide of claim 17.

Description

7. EXAMPLES

(1) Various features and embodiments of the disclosure are illustrated in the following representative examples, which are intended to be illustrative, and not limiting.

Example 1

Wild-Type Ketoreductase Gene Acquisition and Construction of Expression Vectors

(2) The wild-type ketoreductase gene from L. kefir (SEQ ID NO: 1) was designed for expression in E. coli using standard codon optimization. (Codon-optimization software is reviewed in e.g., OPTIMIZER: a web server for optimizing the codon usage of DNA sequences, Puigb et al., Nucleic Acids Res. 2007 July; 35(Web Server issue): W126-31. Epub 2007 Apr. 16.) Genes were synthesized using oligonucleotides composed of 42 nucleotides and cloned into expression vector pCK110900 (vector depicted as FIG. 3 in US Patent Application Publication 20060195947, which is hereby incorporated by reference herein) under the control of a lac promoter. The expression vector also contained the P15a origin of replication and the chloramphenicol resistance gene. Resulting plasmids were transformed into E. coli W3110 (fhu) using standard methods. Polynucleotides encoding the engineered ketoreductase polypeptides were also cloned into vector pCK110900 for expression in E. coli W3110.

(3) The engineered ketoreductase polypeptide of SEQ ID NO: 4 was obtained by directed evolution of a codon-optimized gene encoding the wild-type ketoreductase of Lactobacillus kefir (Genbank acc. No. AAP94029.1; GI: 33112056). SEQ ID NO: 4 has 9 amino acid residue differences relative to the WT ketoreductase (A80T, A94G, S96V, E145L, L153T, Y190P, V196L, I226V, and Y249W) of SEQ ID NO:2. The polypeptide of SEQ ID NO: 4 was found to be able to convert compound (2c) to compound (1c) in >99% e.e. and with about 25% conversion rate in 24 h under initial screening conditions (100 g/L compound (2a) substrate; 0.5 g/L NADP, 100 mM TEA, pH 9.0, 1 mM MgSO.sub.4, 40 C.). The polypeptide SEQ ID NO: 4 was used as the starting backbone for subsequent rounds of evolution. Multiple rounds of directed evolution of the gene encoding SEQ ID NO: 4 (i.e., SEQ ID NO: 3) were carried out. Each round used the gene encoding the most improved engineered polypeptide from each round as the parent backbone sequence for the subsequent round of evolution. The resulting engineered ketoreductase polypeptide sequences and specific mutations and relative activities are listed in Table 3.

Example 2

Production of Engineered Polypeptides

(4) The engineered ketoreductase polypeptides of the disclosure were produced in E. coli W3110 as an intracellular protein expressed under the control of the lac promoter. The polypeptide accumulates primarily as a soluble cytosolic active enzyme. A shake-flask procedure is used to generate engineered polypeptide powders that can be used in activity assays or biocatalytic process disclosed herein.

(5) Fermentation for Shake Flask Powders.

(6) A single microbial colony of E. coli containing a plasmid encoding an engineered ketoreductase of interest is inoculated into 50 mL Luria Bertani broth containing 30 g/ml chloramphenicol and 1% glucose. Cells are grown overnight (at least 16 hours) in an incubator at 30 C. with shaking at 250 rpm. The culture is diluted into 250 mL Terrific Broth (12 g/L bacto-tryptone, 24 g/L yeast extract, 4 mL/L glycerol, 65 mM potassium phosphate, pH 7.0, 1 mM MgSO.sub.4) containing 30 g/ml chloramphenicol, in a 1 liter flask to an optical density at 600 nm (0D600) of 0.2 and allowed to grow at 30 C. Expression of the ketoreductase gene is induced by addition of isopropyl--D-thiogalactoside (IPTG) to a final concentration of 1 mM when the OD600 of the culture is 0.6 to 0.8. Incubation is then continued overnight (at least 16 hours). Cells are harvested by centrifugation (5000 rpm, 15 min, 4 C.) and the supernatant discarded.

(7) Production of Ketoreductase Shake-Flask Powders:

(8) The cell pellet is resuspended with an equal volume of cold (4 C.) 100 mM phosphate buffer, pH 9.0 (optionally including 2 mM MgSO.sub.4), and harvested by centrifugation as above. The washed cells are resuspended in two volumes of the cold phosphate buffer and passed through a French Press twice at 12,000 psi while maintained at 4 C. Cell debris is removed by centrifugation (9000 rpm, 45 minutes, 4 C.). The clear lysate supernatant was collected and stored at 20 C. Lyophilization of frozen clear lysate provides a dry shake-flask powder of crude ketoreductase polypeptide. Alternatively, the cell pellet (before or after washing) can be stored at 4 C. or 80 C.

(9) Fermentation for Production Downstream Process (DSP) Powders.

(10) Larger-scale (100-120 g) fermentation of the engineered ketoreductases for production of DSP powders can be carried out as a short batch followed by a fed batch process according to standard bioprocess methods. Briefly, ketoreductase expression is induced by addition of IPTG to a final concentration of 1 mM. Following fermentation, the cells are harvested and resuspended in 100 mM triethanolamine-H.sub.2SO.sub.4 buffer, then mechanically disrupted by homogenization. The cell debris and nucleic acid are flocculated with polyethylenimine (PEI) and the suspension clarified by centrifugation. The resulting clear supernatant is concentrated using a tangential cross-flow ultrafiltration membrane to remove salts and water. The concentrated and partially purified enzyme concentrate can then be dried in a lyophilizer and packaged (e.g., in polyethylene containers).

Example 3

High Throughput (HTP) Assay Protocols

(11) High-Throughput Growth & Expression.

(12) Cells were picked and grown using standard KRED protocol for W3110 with direct induction: (1) Master growth=single colonies picked from agar Q-trays by Q-bot and grown overnight in LB media containing 1% glucose and 30 g/mL chloramphenicol (CAM), 30 C., 200 rpm, 85% humidity. (2) Subculture=20 L of overnight growth transferred to a deep well plate containing 380 L 2YT growth media containing 30 g/mL CAM, 1 mM IPTG, 1 mM MgSO.sub.4, and incubated for 18 h at 30 C., 200 rpm, 85% humidity. Subculture TB media was made up of TB media (380 L/well), 30 g/mL CAM, 1 mM MgSO.sub.4, and 1 mM IPTG. Cell culture was centrifuged at 4000 rpm, 4 C. for 10 min., and the media discarded. Cell pellets were resuspended in 200-400 L lysis buffer (0.1 M triethanolamine (TEA) buffer, pH 9.0, containing 1 mM MgSO.sub.4, 400 g/mL PMBS and 500 g/mL Lysozyme).

(13) HTP Screening Procedure.

(14) Standard HTP reaction assays were carried out on 200 L reaction volume scales in 96-wells deep well plates (reaction assay blocks). The reaction mixtures in each well typically consisted of: 5.0 g/L substrate oxcarbazepine prepared in DMSO or approximately 80 g/L solid substrate oxcarbazepine; 0.05 g/L NADP.sup.+; 70% isopropyl alcohol (IPA); 0, 0.5 or 1% acetone; and 10 L, 20 L or 50 L of clear lysates, as further specified below. Generally, reaction reagents were added using automated HTP robotics, such as Biomek NX. Lysis volumes were fixed at 150 L or 300 L. In later rounds, lysates were diluted 2 or 0.6 after lysis.

(15) Assay Protocols I and II.

(16) Cell lysates were prepared by one of two methods. Cell pellets were lysed with 300 L/well lysis buffer (1.0 mg/mL lysozyme, 0.5 mg/mL PMBS in 0.1M TEA-HCl with 1 mM MgSO.sub.4, pH 9.0) for plates grown using TB medium as subculture media. 150 L/well or 300 L/well lysis buffer was used for plates grown using 2YT medium as subculture media. Lysates were also prepared by lysing cell pellets with 300 L/well lysis buffer for plates grown using TB medium as subculture media and then 2 diluted with 0.1M TEA-HCl with 1 mM MgSO.sub.4 (pH 9.0) buffer.

(17) Plates were then left to shake at speed of 1.5-2.5 g on a titre-plate shaker, room temperature, for 1.5 hrs. Plates containing lysed cells were centrifuged at 4000 rpm for 10 min at 4 C. Plates with lysates were stored at 4 C. if they were not immediately used.

(18) The reaction condition comprised oxcarbazepine, 5 g/L (5% v/v DMSO); NADP.sup.+, 0.05 g/L; IPA 70% v/v; and lysate, 10 L or 20 L. Assay was carried out by adding into reaction assay blocks 10 L/well of 100 g/L warmed substrate in DMSO (freshly prepared), 140 L/well of isopropyl alcohol (IPA), and 40 L/well of 0.25 g/L NADP.sup.+, prepared in 0.1M TEA-HCl with 1 mM MgSO.sub.4 (pH 9.0). Clear lysates (10 L/well) from lysate plates were transferred to their reaction mixtures. Plates were heat sealed and left to shake overnight at 40 C. and 570-575 rpm.

(19) For reactions in which cells were lysed in 150 L/well lysis buffer, 10 L/well of 100 g/L warmed substrate in DMSO, freshly prepared, were added into reaction assay blocks. This was followed by the addition of 30 L/well of 0.33 g/L NADP+ prepared in 0.1M TEA-HCl with 1 mM MgSO.sub.4 (pH 9.0) followed by 140 L/well of isopropyl alcohol (IPA). Clear lysates (20 L/well) from lysate plates were respectively transferred to their reaction mixtures. Plates were heat sealed and left to shake overnight at 40 C. and 570 rpm.

(20) Assay Protocol III.

(21) Lysates were prepared as described for Assay Protocols I and II above. The reaction conditions comprised oxcarbazepine, 75 g/L; NADP.sup.+, 0.05 g/L; IPA, 70% v/v; and lysate, 10 L, 20 L or 50 L. Assays were carried out by dispensing 15 mg substrate solid into reaction assay blocks using solid dose template plate Millipore MACL09625. This was followed by 10 L/well of 1 g/L NADP.sup. prepared in 0.1M TEA-HCl with 1 mM MgSO.sub.4 (pH 9.0). IPA of 140 L/well of was then dispensed into the assay blocks. Clear lysates of 50 L/well from lysate plates were respectively transferred to their reaction mixtures. Plates were heat sealed and left to shake overnight at 45 C. and 575 rpm.

(22) Assay Protocol IV.

(23) Cell pellets were lysed with 300 L/well lysis buffer and then diluted 0.6 with 0.1M TEA-HCl with 1 mM MgSO.sub.4 (pH 9.0) buffer, and worked up as described in Reactions I and II above. The reaction conditions comprised oxcarbazepine, 75 g/L; NADP.sup.+ 0.05 g/L; IPA, 70% v/v; and lysate, 10 L, 20 L or 50 L. Assays were carried out by dispensing 15-20 mg substrate solid into reaction assay blocks using solid dose template plate Millipore MACL09625. IPA was dispensed into the reaction assay blocks at 140 L/well, followed by 50 L/well of NADP 0.2 g/L prepared in 0.1M TEA-HCl with 1 mM MgSO.sub.4 (pH 9.0). Clear lysates of 10 L/well from lysate plates were respectively transferred to their reaction mixtures. Plates were heat sealed and left to shake overnight at 50 C. and 575 rpm.

(24) Assay Protocol V.

(25) Cell pellets were processed as described in Assay Protocol IV. The reaction condition comprised oxcarbazepine, 75 g/L; NADP.sup.+ 0.05 g/L; IPA/Acetone, 70:0.5% v/v; and lysate 10 L. Assays were carried out by dispensing 15-20 mg solid substrate into reaction assay blocks using solid dose template plate Millipore MACL09625, followed by 49 L/well of 0.2 g/L NADP+ prepared in 0.1M TEA-HCl with 1 mM MgSO.sub.4 (pH 9.0). IPA:acetone (99.3:0.7% v/v) of 141 L/well was dispensed into reaction assay blocks containing the substrate to obtain a final concentrations of 70% IPA and 0.5% acetone in the reaction mixtures. Clear lysates of 10 L/well from lysate plates were transferred to the reaction mixtures. Plates were heat sealed and left to shake overnight at 50 C. and 575 rpm.

(26) Assay Protocol VI.

(27) Cell pellets were processed as described in Assay Protocol IV. Reaction conditions comprised oxcarbazepine, 75 g/L; NADP+, 0.05 g/L; IPA, 70% v/v; and lysate, 10 L, 20 L or 50 L. Assays were carried out by dispensing 15-20 mg solid substrate into reaction assay blocks using solid dose template plate Millipore MACL09625, followed by 40 L/well of 0.25 g/L NADP+ prepared in 0.1M TEA-HCl with 1 mM MgSO.sub.4 (pH 9.0). IPA of 140 L/well was dispensed into reaction assay blocks. Clear lysates of 20 L/well from lysate plates were transferred to the reaction mixtures. Plates were heat sealed and left to shake at 57 C. and 575 rpm for the following periods: (a) overnight, i.e., approximately 18 hrs, or (b) 4 hrs.

(28) Assay Protocol VII.

(29) Cell pellets were processed as described in Assay Protocol IV above. Reaction conditions comprised oxcarbazepine, 75 g/L; NADP.sup.+, 0.05 g/L; IPA/Acetone, 70:1% v/v; and lysate, 10 L. Assays were carried out by dispensing 15-20 mg solid substrate into reaction assay blocks using solid dose template plate Millipore MACL09625, followed by 48 L/well of 0.21 g/L NADP.sup.+ prepared in 0.1M TEA-HCl with 1 mM MgSO.sub.4 (pH 9.0). IPA:acetone (98.6:1.4% v/v) of 142 L/well were dispensed into reaction assay blocks containing substrate to obtain the final concentrations of 70% IPA and 1% acetone in the reaction mixtures. Clear lysates of 10 L/well from lysate plates were respectively transferred to their reaction mixtures. Plates were heat sealed and left to shake overnight at 57 C. with a speed of 575 rpm.

(30) Assay Protocol VIII.

(31) Oxcarbazepine, 75 g/L; NADP+, 0.05 g/L; IPA/Acetone, 70:1% v/v; and lysate, 10 L. Assays were carried out by dispensing 15-20 mg solid substrate into reaction assay blocks using solid dose template plate Millipore MACL09625, followed by 48 L/well of 0.21 g/L NADP+ prepared in 0.1M TEA-HCl with 1 mM MgSO.sub.4 (pH 9.0). IPA:acetone (98.6:1.4% v/v) of 142 L/well was dispensed into reaction assay blocks containing substrate to obtain the final concentrations of 70% IPA and 1% acetone in the reaction mixtures. Clear lysates of 10 L/well from lysate plates were transferred to their reaction mixtures. Plates were heat sealed and left to shake at 62 C. and 575 rpm for the following periods: (i) overnight i.e. approximately 18 hrs, or (ii) 6 hrs.

(32) For reaction plates with substrate loaded as homogenous DMSO solution, after designated reaction times, plates were centrifuged at 4000 rpm for 1 min at 25 C. Acetonitrile at 800 L/well were added into the reaction assay mixes. Plates were heat sealed and vigorously shaken for about 15 min at RT on titre-plate shaker (approximately 8 g) and visually inspected to ensure that there was no precipitation of substrates or products. Quenched plates were spun down at 4000 rpm for 10 min at 25 C.

(33) For reaction plates with solid dosed substrate, after designated reaction times, plates were centrifuged at 4000 rpm for 1 min at 25 C. DMSO of 1200 L/well was added into the reaction assay mixes. Plates were heat sealed and vigorously shaken for about 15 min at RT on titre-plate shaker (approximately 8 g). Plates with quenched reactions were visually inspected to ensure that all solid substrate were dissolved. When necessary, plates were shaken again for approximately 15 min until no solid substrate was observed. Quenched reactions were left to shake at 575 rpm for about 10 min at 45 C. to ensure that residual solid substrate in the mixtures was dissolved. Quenched plates were spun down at 4000 rpm for 10 min at 25 C.

(34) HPLC Screening Assay for Reactions with Solid Dosed Substrate.

(35) HTP reaction assays performed using substrate loaded as DMSO solution were prepared for analysis by taking 40 L of quenched reaction mixtures and diluting with 160 L of acetonitrile in Costar 96-wells round bottom plates.

(36) HTP reaction assays performed using solid dosed substrate, i.e., high substrate loading of 75 g/L substrate was prepared for analysis by taking 10 L of quenched reaction mixtures and diluting with 190 L of acetonitrile in Costar 96-wells round bottom plates. The samples were then analyzed by HPLC.

(37) HPLC Protocol 1: HPLC chromatographic analysis used Agilent's Eclipse XDB C-18 column, 4.6150 mm, 5 m diameter column, at a temperature of 25 C., and a flowrate of 1.2 mL/min using a mobile phase of water containing 0.1% acetic acid/acetonitrile, 60/40% v/v, where the acetic acid is prepared in 18.2 M cm-1 milli-Q water. The column detection wavelengths were 210 nm, 230 nm and 254 nm. Retention time for the ketone is 2.1 min, and retention time for the alcohol is 1.5 min, respectively. This protocol was primarily used for HTP screening assays in all rounds.

(38) HPLC Protocol 2: HPLC chromatographic analysis used Agilent's Eclipse XDB C-8 column, 4.6150 mm, 5 m diameter column, at a temperature of 25 C. and a flowrate of 1.5 mL/min using a mobile phase of 10 mM ammonium acetate/acetonitrile, 45/55% v/v. The column detection wavelengths were 210 nm, 230 nm and 254 nm. Retention time for the ketone is 1.3 min and retention time for the alcohol is 1.1 min, respectively. This protocol was used for rapid screenings, e.g., in Round 1 libraries screenings.

Example 4

Biocatalytic Process I for Preparation of Compound (1c) from Compound (2c)25 g Scale Reaction

(39) This example illustrates a first biocatalytic process using an engineered ketoreductase polypeptide of the disclosure to prepare compound (1c) on a 25 g scale. The reaction was carried out in an aqueous co-solvent system of 0.1 M TEA, 1 mM MgSO.sub.4, pH 9.0, 70% IPA, and compound (2a) loading of 50 g/L. The engineered ketoreductase (SEQ ID NO:36) was at a loading of 3 g/L along with co-factor NADPH at 0.1 g/L. The engineered ketoreductase also has secondary alcohol dehydrogenase activity, acting as a recycling system with the IPA to regenerate the oxidized co-factor NADP+ to NADPH through the oxidation of the IPA to acetone. Execution of this procedure as described afforded 23.2 g (93% isolated yield) of crude desired product in one run with 96.2% chemical purity (w/w, HPLC Method 5).

(40) Reaction Protocol.

(41) A 1 L baffled jacketed reactor was charged sequentially with the following: 0.1 M TEA buffer solution (pH 9.0), 100 L; IPA, 350 mL; oxcarbazepine, 25 g charged as a solid. The reactor vessel was equipped with an overhead anchor stirrer. A temperature probe was inserted to check the internal solution temperature and a nitrogen inlet connected to a flow meter. An outlet tube with condenser was also attached. The reaction mixture was stirred at 45 C. (internal temperature) at 200 rpm with a nitrogen flow rate of 1 L/min for 10 min.

(42) The reaction mixture was then charged sequentially with the following: NADP+(50 mg), 1 mL prepared in TEA buffer; ketoreductase polypeptide of SEQ ID NO: 36 (1.5 g of DSP Powder), and 10 mL prepared in TEA buffer. The reaction mixture at the start of the process is a white slurry with an initial pH of about 8. The reaction mixture was stirred at the above conditions for 24 hours. The reaction volume was maintained by the intermittent addition of deionized water (Total vol. added: 410 mL). After 8 hours, the nitrogen flow rate was turned off to prevent any significant overnight evaporation.

(43) The reaction course was followed periodically by taking samples from the reaction mixture, quenching, and analyzing as described in Method 1. Samples were also frequently monitored for acetone content using the procedure described in Method 4. For the purposes of tracking the process, t=0 was set at the time at which the enzyme was added.

(44) After in-process analyses indicated >99% conversion (24 hours), the reaction mixture was taken for subsequent workup and isolation. The solution was allowed to cool to room temperature then drained from the reactor into a 500 mL round-bottom flask. IPA was distilled by rotary evaporation (60 torr, 40 C. bath) until 5-10% IPA remained relative to the start of distillation (IPA concentration determined by Method 4).

(45) The crude product was collected by filtration through a sintered funnel and washed with 20 mL of heptane. The solid was dried for 24 h under vacuum (3-20 mm Hg) at 30 C. Upon drying, 23.2 g (93% yield) of crude product was obtained with a chemical purity of 96.2% (as determined by HPLC Method 5) and >99.9% e.e (as determined by HPLC Method 3). The residual protein content in the crude isolated product measured 0.87% by weight. The level of residual protein was measured using a UV absorbance-based protein quantification assay (SPN-Assay) commercially available from G-Biosciences (Maryland Heights, Mo., USA).

(46) Analytical Methods for Example 3.

(47) Five different methods were used for determining % conversion, purity of compound (1a), enantiomeric purity, IPA and acetone concentration, and % potency.

(48) Method 1In-Process % Conversion.

(49) To prepare a sample for HPLC analysis, 100 L of reaction mixture was quenched with 900 L 1:1 DMSO:MeCN. The sample was centrifuged to remove precipitated enzyme. 10 L of supernatant was added to 990 L MeCN and used for chromatographic analysis.

(50) Method 1 Chromatographic Parameters

(51) TABLE-US-00004 Instrument Agilent 1200 HPLC system Column Agilent Eclipse XDB C18 4.6 150 mm, 5 m Mobile Phase MeCN/0.1% HOAc in water (40/60) Column temperature 25 C. Flow rate 1.2 mL/min Injection volume 10 L UV Wavelength 210 nm Runtime 3.2 min Product 1.8 min Substrate 2.5 min Linearity 0.99 (R.sup.2 at 2-200 ppm substrate and product) RF (Product to substrate) 1.23

(52) The percent (%) conversion was calculated as follows:

(53) $\%\mathrm{Conversion}=\frac{\left[\mathrm{Peak}\mathrm{Area}\mathrm{of}\mathrm{product}\right]}{\left[\mathrm{Peak}\mathrm{Area}\mathrm{of}\mathrm{product}\right]+\left[\mathrm{Peak}\mathrm{Area}\mathrm{of}\mathrm{substrate}\text{*}\mathrm{RF}\right]}*100\%$

(54) Method 2Identification and Impurity Determination by HPLC.

(55) 20.0 mg of isolated eslicarbazepine was weighed into 100 mL volumetric flask and dissolved in approximately 80 mL in MeCN. The mixture was sonicated for 5 min and brought up to the final volume with MeCN.

(56) Method 2 Chromatographic Parameters

(57) TABLE-US-00005 Instrument Agilent 1200 HPLC system Column Agilent Eclipse XDB C18 4.6 150 mm, 5 m Mobile Phase (Premixed) Gradient Eluent A: MeCN Eluent B: 0.1% HOAc in water Time (min) Eluent A (%) Eluent B (%) 0 20 80 12 30 70 18 50 50 20 50 50 22 20 80 Detection Wavelength 230 nm Column Temperature 25 C. Injection Volume 10 L Run time 22 min (Post Time 8 min) Product 6.2 min Substrate 10.7 min Flow rate 1 mL/min

(58) Method 3Enantiomeric Purity of (S)-Licarbazepine.

(59) Samples were prepared by taking 40.0 mg of isolated (S)-licarbazepine weighed into a 100 mL volumetric flask and dissolving in 80 mL of MeCN. The mixture was sonicated for 5 min and brought up to the final volume with MeCN.

(60) Method 3 Chromatographic Parameters

(61) TABLE-US-00006 Instrument Agilent HPLC 1200 system Column Chiralcel OD-H, 4.6 250 mm, 5 m Mobile Phase (premixed) 90% n-Hexane, 10% IPA Flow Rate 2.0 mL/min Detection Wavelength 230 nm Column Temperature 15 C. Injection Volume 10 L Run time 35 min (R)-Eslicarbazepine 18.5 min (S)-Eslicarbazepine 27.3 min

(62) Enantiomeric purity was calculated as follows:

(63) $\%e.e.=\frac{\begin{array}{c}\left[\mathrm{Peak}\mathrm{Area}\mathrm{of}\left(S\right)\text{-}\mathrm{licarbazepine}\right]-\\ \left[\mathrm{Peak}\mathrm{Area}\mathrm{of}\left(R\right)\text{-}\mathrm{licarbazepine}\right]\end{array}}{\begin{array}{c}\left[\mathrm{Peak}\mathrm{Area}\mathrm{of}\left(S\right)\text{-}\mathrm{licarbazepine}\right]+\\ \left[\mathrm{Peak}\mathrm{Area}\mathrm{of}\left(R\right)\text{-}\mathrm{licarbazepine}\right]\end{array}}100$

(64) Method 4Determining Acetone and IPA Concentration (GC).

(65) Samples for GC analysis were prepared by taking 100 L of reaction solution and adding it to 900 L of methanol. Samples were centrifuged for 1-2 min and 200 L of supernatant dispensed into a GC glass vial with insert.

(66) Method 4 Chromatographic Parameters

(67) TABLE-US-00007 Instrument Agilent GC 6890N Column Roticap WAX Capillary, 50 m 250 m (ID) 0.25 m (FT) Gas flow Helium with split ratio 60:1 Inlet Pressure 22.4 psi Column Pressure 6.0 psi Helium flow rate 1.1 mL/min (Constant Pressure Mode) Inlet Temperature 180 C. Detector Temperature 200 C. FID Hydrogen Flow 30 mL/min FID Air Flow 350 mL/min FID Nitrogen Flow 35 mL/min Injection Volume 1 L Run time 6.71 min Acetone Retention Time 3.97 min IPA Retention Time 4.41 min
Temperature Program:

(68) TABLE-US-00008 Hold Time Run Time Oven Ramp C./min Next C. (min) (min) Initial 65 0 0 Ramp 7 105 1 6.71

(69) Method 5Determining (S)-Licarbazepine % Potency.

(70) Samples were prepared by placing 1.0 mg of reference standard into a 5 mL volumetric flask and adding 4 mL MeCN to disperse the solid. The mixture was sonicated for 5 min, then made up to volume with MeCN. After passing through a 0.5 m disc membrane, a sample was injected into the HPLC using the chromatographic conditions specified below. Isolated sample solution was prepared in the same way.

(71) Method 5 Chromatographic Parameters

(72) TABLE-US-00009 Instrument Agilent 1200 HPLC system Column Agilent Eclipse XDB C18 4.6 150 mm, 5 m Mobile Phase (Premixed) MeCN/0.1% HOAc in water (25/75) Detection Wavelength 230 nm Column Temperature 25 C. Injection Volume 10 L Run time 10 min Flow rate 1.0 mL/min % Potency 96.2 LOD <0.2 ppm (S/N~3) LOQ <0.6 ppm (S/N~10)

(73) Percent (%) potency was calculated as follows:

(74) $\%\mathrm{potency}=\frac{\mathrm{Peak}\mathrm{Area}\mathrm{of}\mathrm{Sample}\mathrm{Weight}\mathrm{of}\mathrm{Std}\mathrm{Potency}\mathrm{of}\mathrm{Std}}{\mathrm{Peak}\mathrm{Area}\mathrm{of}\mathrm{Std}\mathrm{Weight}\mathrm{of}\mathrm{Sample}}100$

Example 5

Biocatalytic Process I for Preparation of Compound (1c) from Compound (2c)50 g Scale Reaction

(75) This example illustrates a second biocatalytic process using an engineered ketoreductase polypeptide to prepare compound (1c) on a 50 g scale. The reaction is carried out in an aqueous co-solvent system of 0.1M TEA, 1 mM MgSO.sub.4, pH 10, 60% IPA, and compound (2c) loading of 100 g/L. The engineered ketoreductase (SEQ ID NO: 36) was at a loading of 1 g/L along with co-factor NADPH at 0.1 g/L. This protocol afforded 48 g (96% isolated yield) of crude desired product in one run with 98.7% chemical purity (w/w, as determined by HPLC Method 5), >99.9% enantiomeric excess (as determined by HPLC Method 3) and a protein residue content of <100 ppm.

(76) Biocatalytic Reaction Procedure.

(77) A 1 L jacketed reactor was charged sequentially with the 300 mL of IPA, 190 mL of TEA buffer solution (pH 10.0), and oxcarbazepine 50.0 g (charged as solid under stirring). The reaction vessel was equipped with an overhead stirrer fitted with an anchor shaped stir blade, a temperature probe, a nitrogen inlet connected to a flow meter and an outlet for solvent collection. The reaction mixture was stirred at 200 rpm and heated until an internal temperature of 55 C. was attained. A stock solution of enzyme and NADP, prepared separately in buffer, was charged to the reaction mixture at 55 C. The reaction mixture was stirred at 55 C., 200 rpm under a nitrogen atmosphere with a flow rate of 0.8 liters per minute. The reaction volume was maintained by the intermittent addition of a pre-mixed solution of 60% IPA and 40% buffer (0.1 M TEA with 1 mM MgSO.sub.4, pH 10.0). The reaction course was followed periodically by taking samples from the reaction mixture, quenching, and analyzing as described in Method 1. Samples were also frequently monitored for acetone content using the procedure described in Method 4. For the purposes of tracking the process, t=0 was set at the time at which the enzyme was added. After in-process analyses indicated >99% conversion in 24 h, the reaction mixture was taken for subsequent workup and isolation.

(78) Following complete conversion (>99%), the reaction mixture was drained from the reactor into a 1 L round-bottom flask. IPA was distilled by rotary evaporation (75 torr, 50 C. bath). Upon partial distillation of reaction volume, 100 mL of water was added to the white slurry and the distillation continued to completely remove IPA.

(79) The crude product was collected by filtration through a Buchner funnel and washed with water (100 mL) and heptane (200 mL). The solid was dried for 24 h in a vacuum oven (2 mbar) at 30 C. Upon drying, 48.0 g (96% yield) of crude product was obtained as an off white solid with a chemical purity of 98.7% (as determined by HPLC Method 5) with >99.9% e.e (as determined by HPLC Method 3) and a residual protein content of 80 ppm.

(80) Purification of Compound (1c).

(81) A 10 g suspension of crude product from above in 100 ml methanol was heated to 40 C. (internal temperature) to allow maximum dissolution of product. Celite (2.0 g) was added to the slightly turbid solution, and the mixture was stirred at 40 C. for 15-20 minutes. The slurry with Celite was then filtered through a sintered funnel, and the residue was washed with 20 mL of pre-heated (40 C.) methanol. The clear filtrate obtained was then distilled under reduced pressure to reduce the volume to approximately 30 mL. The thick solution was gradually cooled to 5 C. using an ice-bath. Cold water (50 mL) was added dropwise to the white precipitate, and the resulting slurry was stirred at 5 C. for 30 minutes. The precipitated product was filtered through a sintered funnel, rinsed and washed with 20 mL of water before being dried in a vacuum oven for 16 h (30 C., 2 mbar). This resulted in 9.0 g (90% recovery) of purified product in a single run as a white solid with 99.6% chemical purity (as determined by HPLC Method 5) and a residual protein content of <10 ppm.

(82) Analytical Methods Used in the Process of Example 4:

(83) Chromatographic methods were employed to analyze the products of the ketoreductase mediated conversion of compound (2c) to compound (1c)

(84) Method 1Rapid Conversion Method.

(85) 1 mL of reaction mixture was quenched in 9 mL of pre-mixed solution of 1:1 DMSO:MeCN. The sample was sonicated to completely dissolve any undissolved substrate or product. The sample was then centrifuged to separate the enzyme from the solution. 50 L of supernatant was added to 950 L MeCN and submitted for analysis.

(86) Method 1 Chromatographic Parameters

(87) TABLE-US-00010 Instrument Agilent 1200 HPLC system Column Agilent Eclipse XDB C18 4.6 150 mm, 5 m Mobile Phase MeCN/0.1% AcOH in water (40/60, Isocratic) Column temperature 25 C. Flow rate 1.4 mL/min Injection volume 10 L UV Wavelength 210 nm Runtime 2.7 min Substrate 1.91 min Linearity 0.99 (R2 at 2-200 ppm substrate and product) RF (product to substrate) 1.23

(88) The % conversion was calculated from the chromatogram as follows:

(89) $\%\mathrm{Conversion}=\frac{\left[\mathrm{Peak}\mathrm{Area}\mathrm{of}\mathrm{product}\right]}{\left[\mathrm{Peak}\mathrm{Area}\mathrm{of}\mathrm{product}\right]+\left[\mathrm{Peak}\mathrm{Area}\mathrm{of}\mathrm{substrate}\mathrm{RF}\right]}100\%$

(90) Method 2Purity Method (HPLC).

(91) To prepare the sample for analysis, 20.0 mg of isolated eslicarbazepine was weighed into 100 mL volumetric flask and dissolved in approximately 80 mL in MeCN. The mixture was sonicated for 5 min and topped up to volume with MeCN. Alternatively, the reaction sample prepared in Method 1 above can also be used in this method to determine in-process purity and % conversion.

(92) Method 2 Chromatographic Parameters

(93) TABLE-US-00011 Instrument Agilent 1200 HPLC system Column Agilent Eclipse XDB C18 4.6 150 mm, 5 m Mobile Phase (premixed) Gradient Eluent A: MeCN Eluent B: 0.1% AcOH in water Time (min) Eluent A (%) Eluent B (%) 0 20 80 12 30 70 18 50 50 20 50 50 22 20 80 Detection Wavelength 210 nm Column Temperature 25 C. Injection Volume 10 L Run time 22 min Equilibration time 5 min Product 7.03 min Substrate 11.34 min Flow rate 1.0 mL/min

(94) The % conversion was calculated by the same method described in Method 1 above.

(95) Method 3Chiral Method (HPLC).

(96) To prepare the sample, 40.0 mg of isolated S-licarbazepine was weighed into a 100 mL volumetric flask and dissolved in 80 mL of MeCN. The mixture was sonicated for 5 min and topped up to volume with MeCN.

(97) Method 3 Chromatographic Parameters

(98) TABLE-US-00012 Instrument Agilent 1200 HPLC system Column ChiraDex, LiChroCART 250-4, 5 m Mobile Phase (premixed) 95% Na.sub.2HPO.sub.4 (100 mM; pH 7.0) and 5% MeOH Column temperature 15 C. Flow rate 1.0 mL/min Injection volume 10 L Detection Wavelength 254 nm Runtime 24 min R-licarbazepine 15.4 min S-licarbazepine 18.3 min

(99) $\%e.e.=\frac{\begin{array}{c}\left[\mathrm{Peak}\mathrm{Area}\mathrm{of}S\text{-}\mathrm{licarbazepine}\right]-\\ \left[\mathrm{Peak}\mathrm{Area}\mathrm{of}R\text{-}\mathrm{licarbazepine}\right]\end{array}}{\begin{array}{c}\left[\mathrm{Peak}\mathrm{Area}\mathrm{of}S\text{-}\mathrm{licarbazepine}\right]+\\ \left[\mathrm{Peak}\mathrm{Area}\mathrm{of}R\text{-}\mathrm{licarbazepine}\right]\end{array}}100$

(100) Method 4Residual Solvent Detection Method (GC).

(101) Sample was prepared by diluting 1 mL of reaction mixture with 9 mL of methanol, prior to centrifugation for 1 min. The supernatant was removed for GC analysis.

(102) Method 4 Chromatographic Parameters

(103) TABLE-US-00013 Instrument Agilent GC 6890N Column Roticap WAX Capillary, 50 m 250 m (ID) 0.25 m (FT) Gas flow Helium with split ratio 60:1 Inlet Pressure 22.4 psi Column Pressure 6.0 psi Helium flow rate 1.1 mL/min (Constant Pressure Mode) Inlet Temperature 180 C. Detector Temperature 200 C. FID Hydrogen Flow 30 mL/min FID Air Flow 350 mL/min FID Nitrogen Flow 35 mL/min Injection Volume 1 L Run time 6.7 min Acetone Retention Time 3.9 min IPA Retention Time 4.4 min
Temperature Program

(104) TABLE-US-00014 Hold Time Run Time Oven Ramp C./min Next C. (min) (min) Initial 65 0 0 Ramp 7 105 1 6.71

(105) Method 5Potency Method (HPLC).

(106) 1.0 mg of reference standard eslicarbazepine was dissolved with 4 mL MeCN in a 5 mL volumetric flask. The solution was sonicated for 5 min and made up to volume with MeCN. After filtering the solution through a 0.5 m disc membrane, sample was used for HPLC analysis. Sample with isolated eslicarbazepine was prepared in a similar manner.

(107) Method 5 Chromatographic Parameters

(108) TABLE-US-00015 Instrument Agilent 1200 HPLC system Column Agilent Eclipse XDB C18 4.6 150 mm, 5 m Mobile Phase (Premixed) MeCN/0.1% AcOH in water (25/75) Detection Wavelength 230 nm Column Temperature 25 C. Injection Volume 10 L Run time 10 min Flow rate 1.0 mL/min LOD <0.2 ppm (S/N~3) LOQ <0.6 ppm (S/N~10)

(109) % potency of isolated product is calculated as follows:

(110) $\%\mathrm{potency}=\frac{\begin{array}{c}\mathrm{Peak}\mathrm{Area}\mathrm{of}\mathrm{Sample}\mathrm{Weight}\mathrm{of}\mathrm{Standard}\\ \mathrm{Potency}\mathrm{of}\mathrm{Standard}\end{array}}{\mathrm{Peak}\mathrm{Area}\mathrm{of}\mathrm{Standard}\mathrm{Weight}\mathrm{of}\mathrm{Sample}}100$

(111) All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes.

(112) While various specific embodiments have been illustrated and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the invention(s).