Asymmetric bioreduction of a keto ester to its corresponding (S)-hydroxy ester by Microbacterium sp. MB 5614

JOURNAL OFFERMENTATION ANDBIOENGINEERING Vol. 81, No. 6, 530-533. 1996

Asymmetric Bioreduction of a Keto Ester to Its Corresponding (S’)-Hydroxy Ester by Microbacterium sp. MB 5614 CHRISTOPHER

ROBERGE, ANTHONY KING, VICTOR PECORE, RANDOLPH GREASHAM, AND MICHEL CHARTRAIN*

Merck Research Laboratories, R Y BOY-105, PO Box 2000, Rah way, NJ 07065, USA Received 5 February 1996IAccepted 9 April 1996

A bioprocess employing Microbacterium sp. MA 5614 was developed for the production of the MK-0476(s)hydroxy ester by asymmetric bioreductlon of the corresponding keto ester. The cysteinyl leukotrlene 1 receptor antagonist MK-0476 [3-[(lS)-[3~)-[2-(7-chloroquinolinyl)ethenyl]phenyll-3-(ace~lphenyl)-propyl~~ol]-2(~methylpropanoic acid] is currently undergoing clinical evaluation for the treatment of asthma. Optimization of the bioconversion medium composition and the tlme of keto ester addition, as well as the development of a keto ester feeding strategy yielded improvements ln the bioreduction rate and final (S)-hydroxy ester titer, by 20- and 25fold respectively. This asymmetric bioreduction process supported the production of preparative quantities of (S)-hydroxy ester with an enantiomeric excess greater than 95%. [Key words: biotransformation,

asymmetric

bioreduction,

biocatalysis,

MK-0476,bioprocess]

final hydroxy ester titer were improved by 25 and 20fold respectively.

Recent studies demonstrating that the enantiomers of racemic pharmaceutical drugs can exhibit different pharmokinetic and bioavailability properties have prompted the use of only the active form of the drug (1, 2). Consequently, the manufacture of optically pure new drug candidates is now becoming standard practice throughout the pharmaceutical industry (3). The synthesis of optically active molecules is generally achieved through classical chemical methods employing novel chiral catalysts and reagents. However, for economic reasons or due to the lack of availability of a suitable chiral catalyst, the use of biocatalysis, employing either isolated enzymes or whole microbial cells, has greatly increased during the past years (4-7). The asymmetric bioreduction of prochiral carbonyls, which are important precursors to a large number of pharmaceutical drugs, is one of the best-established fields of biocatalysis (8-11). Asymmetric bioreductions are usually catalyzed by baker’s yeast, the prominent catalyst used so far, but also by many other yeast, fungal, and bacterial species (8-10, 12, 13). MK-0476,[3-[(1S)-[3(E)-[2-(7-chloroquinolinyl)ethenyl] phenyl] - 3- (acetylphenyl) - propylthiol] -2(s) - methylpropanoic acid] is a very potent cysteinyl leukotriene 1 receptor antagonist that is under clinical investigation for the treatment of asthma (14). MK-0476 presents a chiral hydroxy group (15), and a practical approach to building this chiral center is through the asymmetric reduction of a keto ester precursor (Fig. 1). This particular chiral synthesis appeared to be an excellent candidate for an asymmetric bioreduction approach. Extensive screening studies undertaken at Merck and Co. by Drs. Shtiee and Chen identified Microbacterium sp. MB 5614 (Heimbuch, B. M. et al., Annu. Meet. Sot. Indus. Microbial., Boston, 1994) as a suitable biocatalyst, yielding the desired (S)-hydroxy ester. This report presents the development studies that yielded a process supporting the production of preparative quantities of (S)-hydroxy ester with an enantiomeric excess greater than 95%. During the course of these studies, the bioreduction rate and the

MATERIALS

AND METHODS

Bioconversion procedures Cells of Microbacterium sp. MB 5614 preserved in 25% glycerol in a cryovial (1.5 ml) at -70°C were thawed at room temperature and used to inoculate a 250-ml Erlenmeyer flask containing 50ml of KE seed medium [KE seed medium contained per liter: dextrin lO.Og, ardamine pH (Champlain Industries, Clifton, NJ, USA) 5.Og, NZ amine type E (Sheffield, Norwich, NY, USA) 5.Og, beef extract (Difco, Detroit, MI, USA) 3.0 g, dextrose l.Og, K2HP04 0.37 g, MgS04-7Hz0 0.05 g, deionized water to bring the total volume to 1.0 I, and NaOH to bring the pH to 7.11. CaC03 (0.5 g/l) was added prior to sterilization. The culture was incubated for 24 h on an orbital shaker at 220rpm and 28°C. A lo-ml aliquot of this first stage seed culture was used to inoculate a 2.0-l Erlenmeyer flask containing 500ml of KE seed medium (second seed stage). The 2-1 flask was incubated for 24 h on an orbital shaker at 200rpm and 28°C. A lo-ml aliquot of the 24 h second stage seed was

Keto ester

Hydroxy ester FIG. 1. Asymmetric reduction of the MK-0476 keto ester to the corresponding Q-hydroxy ester.

* Corresponding author. 530

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VOL. 81, 1996

used to inoculate a 2-l Erlenmeyer flask containing 5OOml of biotransformation medium (BT medium) [BT medium contained per liter: dextrose 20 g, N-morpholino ethanesulfonic acid (MES) 9.8 g, hysoy peptone 5 g, yeast extract 5 g/l, NaCl 5 g/l, deionized water to bring the total volume to 1 .O I, and NaOH to bring the pH to 7.01. An amount of 25 mg of keto ester [(E)-2-[-3[-3[2(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-oxypropyl] benzoic acid methyl ester] (15) dissolved in 1 ml of DMSO was added to the culture immediately after inoculation. The culture was incubated on an orbital shaker at 200rpm and 28”C, and the progress of the biotransformation was periodically monitored by sampling the flasks. Analytical procedures Keto ester and the desired hydroxy ester were extracted from the fermentation broth by adding two volumes of ethyl acetate and shaking for 20min. After breaking the resulting emulsion by centrifugation at 2,500rpm in a Beckman TJ6 table top model (Beckman Instruments, Fullerton, CA, USA), the extracts were dried under nitrogen and resuspended in acetonitrile. The concentrations of keto ester and hydroxy ester were determined with a Rainin HPLC system (Rainin Instrument, Woburn, MA, USA) and a Zorbax RX-C8 column (MacMod Analytical, Chadds Ford, PA, USA). The elution was performed with a gradient of acidified (0.1% H3P04) acetonitrile (10 to 90%) and acidified (0.1% H3P0,+) water (90 to 10%) delivered at a flow rate of 1.5 ml/min over 25 min. Detection was performed at 254nm, and under these conditions hydroxy ester and keto ester eluted after 4.8 and 9.4min respectively. The enantiomeric excess of the (S)-hydroxy ester was determined using a normal phase HPLC system as previously described (15). Biomass was determined by measuring the light absorbance at 660 nm (ODm) of culture samples with a diode array Hewlett Packard spectrophotometer model 8451 A (Hewlett Packard). RESULTS AND DISCUSSION Initial growth and bioconversion kinetics The bioreduction of the MK-0476 keto ester to the corresponding (S)-hydroxy ester was first evaluated at the 21 shaker flask scale employing the BT medium. A keto ester solution in DMSO (10.0 ml) was added immediately after inoculation to each flask to give a final keto ester concentration of 50mg/l. Typical growth and glucose consumption kinetics of Microbacterium sp. MB 5614 are presented in Fig. 2A. Microbacterium sp. MB 5614 in BT cultivation medium grows very slowly, requiring five days to achieve maximum biomass production (OD = 12,&, and the glucose consumption kinetics observed were correspondingly slow. A typical keto ester bioreduction to hydroxy ester time course in BT medium is presented in Fig. 2B. A final hydroxy ester titer of 25 mg/l, representing a conversion yield of about 50%, was achieved after 150 h of cultivation (conversion rate of 5 mg/f/d). These data show that keto ester reduction occurred during active growth, and suggested a close link between cell growth and bioreduction activity. The enantiomeric excess of the (S)-hydroxy ester produced under these conditions was evaluated as greater than 95%. Substrate addition timing Timing the substrate ad-

531 ‘25

60 50

0

50

150

100

200

Time /h)

FIG. 2.

Growth and bioreduction performances of Microbactesp. was cultivated in BT medium (see Materials and Methods for composition) in a 2-l shake flask. Growth performances were monitored by measuring the optical density at 660 nm. Symbols: 0, ODW; 0, glucose (g/f). (B) Kinetics of the asymmetric bioreduction of the MK-0476 keto ester to the corresponding (S)-hydroxy ester. Keto ester and hydroxy ester concentrations were measured by HPLC as described in the Materials and Method section. Symbols: 0 , keto ester; 0, hydroxy ester. rium sp. (A) Microbacterium

dition in a way that correlates with the metabolic stage of the biocatalyst has been reported to have great influence on the outcome of several asymmetric bioreduction processes (16, 17). In this study, we tested the effect of adding the keto ester at 0, 24, 48, 72 or 96 h postinoculation. Data were collected from each experiment until hydroxy ester production ceased. The data summarized in Fig. 3 show that a maximum hydroxy ester titer was achieved when adding the keto ester between 48 and 72 h post inoculation. However, these data also show that maximum bioreduction rate was achieved when adding the keto ester to a 120-h old culture. In order to take advantage of both observations, a substrate addition 40

40

s 3oc

-30 s

5 al 5

g

$ g 20-

-20

g R =

= .P ;ii E b

-10

‘;; s 3

lo-

m Ol

I

0

50

Post-inoculation substrate

I

100

I

0

150

addition time(h)

FIG. 3. Effect of Microbacterium sp. culture age on MK-0476 keto ester bioreduction rate and yields. MK-0476 keto ester was added to cultures of Microbacterium sp. at different times post inoculation (from 0 to 120 h). Daily samples were analyzed by HPLC and used to determine the production rate and the final concentration of (S)hydroxy ester. Symbols: 0 , final hydroxy ester titer; 0, biotransformation rate.

532

J. FERMENT.BIOENG.,

ROBERGE ET AL.

TABLE 1.

Effect of medium nitrogen source on bioconversion process

Nitrogen source Hysoy peptone Sheftone B Sheftone S Sheftone D Acid hydrolyzed peptone Casamino acids Sodium glutamate Ammonium chloride

ODW after 5-d cultivation

Hydroxy ester (mg/f) after 3-d bioconversion

10.4 5.5 10.9 6.8 6.4 1.1 6.0 1.4

41.1 12.6 5.4 7.9 43.2 8.6 42.4 25.7

performed at a compromise time of 72 h post inoculation was selected for the remainder of these studies. Growth performance of Microbacterium sp. (growth rate and biomass production) were unaffected by the timing of the keto ester addition, which suggested a lack of keto ester toxicity at the concentrations used. A maximum hydroxy ester titer of 35 mg/l (conversion yield of 70X), and a bioconversion rate of 18 mg/l/d were achieved under these selected conditions. Correlation of these data with the time course data presented in Fig. 2A, suggests that the optimum time for keto ester addition is when the culture has reached its late growth phase. A large part of the initial glucose is still present at that stage and is readily available to support the regeneration of the required NAD(P)H co-factor (12, 18). The effects of Cultivation medium improvement the concentrations of the three major components in BT medium (dextrose, hysoy peptone, and yeast extract) on the bioreduction process were evaluated. A statistically designed Box Behnken factorial experiment (21) was used to test various levels of these ingredients (concentrations ranging from 5 to 20 g/l). Statistical analyses of the results indicated that higher concentrations of glucose had a positive effect whereas higher concentrations of hysoy peptone inhibited both biomass and hydroxy ester production. The effect of the third ingredient, yeast extract, was found to be negligible. Consequently, alternative sources of nitrogen were investigated, and the results of this screening are presented in Table 1. Acid hydrolyzed peptone and monosodium glutamate proved to be capable of supporting biomass and hydroxy ester production levels similar to those observed when using hysoy peptone. Monosodium glutamate was selected for the replacement of hysoy peptone because it resulted in a more defined production medium. A second Box Behnken experiment was used to study the composition of this new medium by examining glucose concentrations in the higher range from 20 to 50 g/l, monosodium glutamate concentrations in the TABLE 2.

Medium

Effect of medium enhancements on bioconversion process ODssoafter 5-d cultivation

SC 4.8 SG+O.l g/l FeC13.6Hz0 9.1 SGfO.5 g/l FeC1s.6H20 11.3 9.0 SC + 2% trace solution #2 SC + 3 g/l beef extract 7.2 SC + 5 g/l ardamine pH 11.1 SG+2 g/l yeast nitrogen base 8.7

Hydroxy ester (mg/[) after 7-d bioconversion 36.4 116.1 171.5 84.6 85.6 109.2 127.7

Yeast extract = 5

5,

I

I

I

I

I

I

I

15 20

25

30 35 40 45 50 Glucose (g/l) Yeast extract = 5

55

15 20

25

30 35 40 45 50 55 Glucose (g/1)

FIG. 4. Effect of glucose and glutamate on biomass production and asymmetric bioreduction of Microbacterium sp. (A) The effects of glucose and sodium glutamate concentrations on Microbacterium sp. biomass production were determined using a Box Behnken statistical design. The contours show the biomass (OD%a) achieved when using the glucose and sodium glutamate concentrations listed on the axes. The contours presented here were obtained for a fixed yeast extract concentration of 5 g/l. (B) The effects of glucose and sodium glutamate concentrations on Microbacterium sp. asymmetric bioreduction activity were determined using a Box Behnken statistical design. The contours show the maximum hydroxy ester concentration achieved when using the glucose and sodium glutamate concentrations listed on the axes. The contours presented here were obtained for a fixed yeast extract concentration of 5 g/l.

range from 10 to 30 g/l, and yeast extract concentrations in the range from 2 to 8 g/l. The results, given in Fig. 4A and 4B, show that a medium made up of 3Og/l of glucose, 2Og/f of monosodium glutamate, and 5 g/l of yeast extract, supported both high biomass and high hydroxy ester production. In a further effort to improve both biomass and bioconversion yields, we tested the addition of several organic and inorganic nutrients to this new monosodium glutamate-based medium (SG medium). In order to fully evaluate the potential of these additions, the initial keto ester charge was increased from 0.05 g/l to 0.125 g/l. The effects of these additions on both biomass production and bioconversion activity are presented in Table 2. These data clearly show that the most significant increases in productivity were achieved when employing a medium containing 0.5 g/l FeC& . 6H20. Under these conditions, biomass production was doubled while the final hydroxy ester titer was increased five-fold when com-

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ASYMMETRIC BIOREDUCTION

500 g

400

5 .Z= h

300

5 e s

200 100 0 100

150

200

250

300

350

400

Time (h)

FIG. 5. Optimized asymmetric bioreduction time course. The bioreduction kinetics presented in this figure were obtained when using the sodium glutamate medium supplemented with 0.5 g/l of FeCl,. 6Hz0. The initial addition of MK-0476 keto ester (250mg/f) was made 120 h after inoculation (time at which late log phase was reached for this new medium). Two subsequent additions of MK0476 keto ester (250 mg each) were made 200 and 230 h post-inoculation. Symbols: 0, hydroxy ester; 0, keto ester.

pared to the original process, which suggested that the addition of FeC& .6Hz0 not only increased biocatalyst concentration but also increased the biocatalyst specific activity. Substrate feeding Finally, we investigated the effect of multiple keto ester additions on final bioreduction rate and hydroxy ester titer. An additional two keto ester feedings of 250mg/l each were made after 200 and 230 h of cultivation respectively. Under these conditions, a bioreduction rate of about 100 mg/I/d yielding a final hydroxy ester titer of 500mg/l was achieved after about 280 h of cultivation (Fig. 5). The biotransformation yield observed was about 67% and the enantiomeric excess of the (S)-hydroxy ester produced was measured to be greater than 95%. It is interesting to note that for this particular asymmetric bioreduction, the enantiomeric excess of the (S)-hydroxy ester produced was not affected by the metabolic stage of the biocatalysts or by the cultivation conditions employed in these studies (16, 17, 20-22). When compared with the initial process, a 25-fold increase in the final (5’)-hydroxy ester titer and a 20-fold increase in the rate of (S)-hydroxy ester were achieved when employing these optimal conditions. The asymmetric bioreduction process developed as a result of these studies supported the production of highly optical pure (S)-hydroxy ester at the preparative scale. REFERENCES 1. Ariens, E.: Nonchiral, homochiral and composite chiral drugs. Trends Biochem. Sci., 14, 68-76 (1993). 2. Rauws, A. and Groen, K.: Current regulatory (draft) guidance on chiral medicinal products: Canada, EEC, Japan, United States. Chirality, 6, 72-75 (1994). 3. Stinson, S.: Chiral drugs. Chem. Eng. News, Sept. 38-72 (1994).

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