Biocatalytic preparation of a chiral synthon for a vasopeptidase inhibitor: enzymatic conversion of N2-[N-Phenylmethoxy)carbonyl] L-homocysteinyl]- L-lysine (1- > 1′)-disulfide to [4S-(4I,7I,10aJ)] 1-octahydro-5-oxo-4-[phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b] [1,3]thiazepine-7-carboxylic a...

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Biocatalytic preparation of a chiral synthon for a vasopeptidase inhibitor: enzymatic conversion of N2-[N-Phenylmethoxy)carbonyl] L-homocysteinyl]- L-lysine (1- ⬎ 1⬘)-disulfide to [4S-(4I,7I,10aJ)] 1-octahydro-5-oxo-4-[phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b] [1,3]thiazepine-7-carboxylic acid methyl ester by a novel L-lysine ⑀-aminotransferase Ramesh N. Patel,* Amit Banerjee, Venkata B. Nanduri, Steven L. Goldberg, Robert M. Johnston, Ronald L. Hanson, Clyde G. McNamee, David B. Brzozowski, Thomas P. Tully, Raphael Y. Ko, Thomas L. LaPorte, Dana L. Cazzulino, Shankar Swaminathan, Chien-Kuang Chen, Larry W. Parker, John J. Venit Department of Microbial Technology and Process Development, Process Research & Development, Bristol-Myers Squibb Pharmaceutical Research Institute, P.O. Box 191, New Brunswick, NJ 08903, USA Received 14 September 1999; received in revised form 28 March 2000; accepted 3 April 2000

Abstract [4S-(4I,7I,10aJ)]1-Octahydro-5-oxo-4-[phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b] [1,3]thiazepine-7-carboxylic acid methyl ester (BMS-199541-01) is a key chiral intermediate for the synthesis of Omapatrilat (BMS-186716), a new vasopeptidease inhibitor under development. By using a selective enrichment culture technique we have isolated a strain of Sphingomonas paucimobilis SC 16113, which contains a novel L-lysine ⑀-aminotransferase. This enzyme catalyzed the oxidation of the ⑀-amino group of lysine in the dipeptide dimer N2-[N[phenyl-methoxy)-carbonyl] L-homocysteinyl] L-lysine)1,1-disulphide (BMS-201391-01) to produce BMS-199541-01. The aminotransferase reaction required ␣-ketoglutarate as the amino acceptor. Glutamate formed during this reaction was recycled back to ␣-ketoglutarate by glutamate oxidase from Streptomyces noursei SC 6007. Fermentation processes were developed for growth of S. paucimobilis SC 16113 and S. noursei SC 6007 for the production of L-lysine ⑀-amino transferase and glutamate oxidase, respectively. L-lysine ⑀-aminotransferase was purified to homogeneity and N-terminal and internal peptides sequences of the purified protein were determined. The mol wt of L-lysine ⑀-aminotransferase is 81 000 Da and subunit size is 40 000 Da. L-lysine ⑀-aminotransferase gene (lat gene) from S. paucimobilis SC 16113 was cloned and overexpressed in Escherichia coli. Glutamate oxidase was purified to homogeneity from S. noursei SC 6003. The mol wt of glutamate oxidase is 125 000 Da and subunit size is 60 000 Da. The glutamate oxiadase gene from S. noursei SC 6003 was cloned and expressed in Streptomyces lividans. The biotransformation process was developed for the conversion of BMS201391-01 to BMS-199541-01 by using L-lysine ⑀-aminotransferase expressed in E. coli. In the biotransformation process, for conversion of BMS-201391-01 (CBZ protecting group) to BMS-199541-01, a reaction yield of 65–70 M% was obtained depending upon reaction conditions used in the process. Phenylacetyl or phenoxyacetyl protected analogues of BMS-201391-01 also served as substrates for L-lysine ⑀-aminotransferase giving reaction yields of 70 M% for the corresponding BMS-199541-01 analogs. Two other dipeptides N-[N[(phenylmethoxy)carbonyl]-L-methionyl]-L-lysine (BMS-203528) and N,2-[S-acetyl-N-[(phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine (BMS-204556) were also substrates for L-lysine ⑀-aminotransferase. N-␣-protected (CBZ or BOC)-L-lysine were also oxidized by L-lysine ⑀-aminotransferase. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Chiral synthons; L-lysine ⑀-aminotransferase

* Corresponding author. Tel.: ⫹001-732-519-2026; fax: ⫹011-732-519-1166. E-mail address: [email protected] (R.N. Patel). 0141-0229/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 1 4 1 - 0 2 2 9 ( 0 0 ) 0 0 2 3 3 - 7

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377

Scheme 1.

1. Introduction Recently much attention has been focused on the interaction of small molecules with biological macromolecules. The search for selective enzyme inhibitors and receptor agonists or antagonists is one of the keys for target-oriented research in the pharmaceutical industry. Increasing understanding of the mechanism of drug interaction on a molecular level has led to the wide awareness of the importance of chirality as the key to the efficacy of many drug products. It is now known that in many cases only 1 enantiomer of a drug substance is required for efficacy and the other enantiomer is either inactive or exhibits considerably reduced activity. Chiral synthons can be prepared by asymmetric synthesis by either chemical or biocatalytic processes by using microbial cells or enzymes derived therefrom. The advantages of microbial or enzyme catalyzed reactions over chemical reactions are that they are enantioselective, and can be carried out at ambient temperature and atmospheric pressure. This minimizes problems of isomerization, racemization, epimerization, and rearrangement that may occur during chemical processes. Biocatalytic processes are generally carried out in aqueous solution and avoid the use of environmentally harmful chemicals used in chemical processes and solvent waste disposal. Furthermore, microbial cells or enzymes derived therefrom can be immobilized and reused many cycles. A number of review articles [1– 8] have been published on the use of enzymes in organic synthesis. This report describes enzymatic preparation of [4S-

(4I,7I,10aJ)]1-Octahydro-5-oxo-4-[phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b] [1,3]thiazepine-7-carboxylic acid methyl ester (BMS-199541-01), a key chiral building block used for synthesis of omapatrilat, a vasopeptidase inhibitor now in clinical trials [9]. Our goal was to prepare the compound by a simpler, more convenient route using an intermediate derived from L-lysine as a readily available starting material. An enzymatic process was developed for the oxidation of the ⑀-amino group of lysine in the dipeptide dimer N2-[N[phenyl- methoxy)-carbonyl] L-homocysteinyl] L-lysine)1,1-disulphide (BMS-201391-01) to produce BMS-199541-01 (Scheme 1) by using L-lysine ⑀-aminotransferase from Sphingomonas paucimobilis SC 16113. This enzyme was overexpressed in Escherichia coli and a biotransformation process was developed using the recombinant enzyme. The aminotransferase reaction required ␣-ketoglutarate as the amino acceptor. Glutamate formed during this reaction was recycled back to ␣-ketoglutarate by glutamate oxidase from Streptomyces noursei SC 6007.

2. Materials and methods 2.1. Materials Substrates and authentic product standards were synthesized by the Chemical Process Research and Development Department, Bristol-Myers Squibb Pharmaceutical Research Institute (New Brunswick, NJ, USA). The physicochemical properties including spectral characteristics (1H

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Scheme 2.

nuclear magnetic resonance [NMR], 13C NMR, mass spectra) were in full accord for all these compounds [9 –11]. Starting substrate BMS-201391-01 was prepared as described below. The amino functionality in homocystine was protected with CBZ-Cl under aqueous alkaline conditions to afford CBZ-protected homocystine in ⬎85% yield. The acid group was activated as the hydroxysuccinimide ester by sing N-hydroxy succinimide and DCC. The active ester was coupled with ⑀-N-BOC lysine in aqueous DME under basic conditions to afford bis-protected BMS-201391-01 in ⬎70% yield. Finally, removal of the BOC group by using formic acid furnished the desired peptide, BMS-201391-01, in ⬎80% yield (Scheme 2). 1H NMR data in ppm (solvent D2O at 60°C): 7.2 (5 H), 4.8 –5 (2 H), 4.2 (1 H), 4.1 (1 H), 2.8 (2 H), 2.6 (2 H), 1.1–2.1 (8 H). M ⫹ H ⫽ 793. By using similar procedure the phenylacetate-protected and phenoxyacetate-protected BMS-201391 analogues were prepared. 2.2. Microorganisms Microorganisms were obtained from the American Type Culture Collection (Rockville, MD, USA) and from our culture collection in the Microbiology Department of the Bristol-Myers Squibb Pharmaceutical Research Institute. Microorganisms were stored at ⫺90°C in vials. 2.3. Selective techniques for isolation of microorganisms A selective culture technique was used to isolate microorganisms able to utilize N-␣-CBZ-L-lysine as sole source of nitrogen. Soil samples were collected from various sites in New Jersey. About a gram of soil samples suspended in 5 ml of water, mixed thoroughly and samples were allowed to settle. The supernatant solutions from various samples were inoculated in a medium containing 2% glucose, 1%

N-␣-CBZ-L-lysine, 0.2% KH2PO4, 0.2% K2HPO4, 0.01% MgSO4, 0.001% FeSO4, 0.001% ZnSO4 (adjusted to pH 7) before sterilization. After 4 days of growth when medium became turbid, cultures were transferred to the above medium containing 1.5% agar contained in Petri plates. From this enrichment culture techniques eight different types of colonies were isolated for further studies for oxidation of ⑀-amino group of L-lysine in substrate BMS-201391-01. One culture Z-2 later identified as a S. paucimobilis and designated SC 16113 was used for further studies. 2.4. Growth of S. paucimobilis One vial (containing 1 ml of culture in medium A) was used to inoculate 100 ml of medium A containing 1.5% peptone, 0.01% yeast extract, 0.2% KH2PO4, 0.2% K2HPO4, 0.01% MgSO4, and 0.2% NaCl. Cultures were grown at 28°C and 280 rpm for 48 –72 h on a rotary shaker. One hundred ml of this culture was transferred to 1 l of medium A. Cultures were grown in 4 l flasks at 28°C and 250 rpm for 48 h on a rotary shaker. Cultures were harvested by centrifugation at 18 000 ⫻ g for 15 min, and cells were recovered and stored at ⫺70°C until used. S. paucimobilis SC 16113 were grown in 700 l fermenters containing 500 l of medium A containing 0.025% SAG and 0.025% Dow Corning antifoam (Midland, MI, USA). Growth consisted of two inoculum development stages and one fermentation stage. Inoculum development consisted of F1 and F2 stages. In the F1 stage, 1 ml culture of S. paucimobilis SC 16113 was inoculated into 100 ml of medium B containing 1% glucose, 0.1% KH2PO4, 0.1% K2HPO4, 0.05% MgSO4, 0.05% yeast extract, 0.05% (NH4)2SO4, 0.05% NaCl, and 0.01% CaCl2. The growth was carried out in 500 ml flasks at 28°C and 250 rpm for 24 h. In the F2 stage, 100 ml of F1 stage culture of organism

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was inoculated into 1 l of medium B in a 4 l flask and incubated at 28°C and 250 rpm for 48 h. A fermentor containing medium A was inoculated with 4 l of F2 stage inoculum and grown at 28°C and 220 rpm agitation with 250 L/min. During fermentation, cells were periodically harvested by centrifugation from 200 ml of culture broth. Cell extracts were prepared to assay for enzyme activity. The specific activity (mg of product formed/h/g of protein) was determined. 2.5. Preparation of cell extract of S. paucimobilis SC 16113

379

bile phase solvent A containing 0.1% trifluoroacetic acid (TFA) in water and solvent B containing 0.1% TFA in 70% acetonitrile, 30% water. The following gradients of solvent A and B were used for the separation of BMS-201391-01 and BMS-199541-01: 0 min, 100% A; 0 –15 min, 50% B;15–25 min, 100% B; 25–26 min, 0% B; and 26 –30 min, 0% B. The flow rate was 1 ml/min. The column temperature was ambient, and the detection wavelength was 215 nm. Under these conditions, the retention time for BMS201391-01 was 17.8 min, monomer of BMS-201391-01 was 15.3 min, and BMS-199541-01 was 20.1 min. 2.8. Purification of L-lysine ⑀-aminotransferase

Preparation of cell extracts were carried out at 4 –7°C. Cells were washed with 25 mM potassium phosphate buffer pH 8 (buffer A) and washed cells (2 g) were suspended in 10 ml of buffer A containing 10 mM Na-ethylene diaminetetracaetic acid (EDTA). To the cell suspensions, 0.1 ml of 100 mM phenylmethylsulfonyl fluoride (PMSF) solution in isopropanol, and 0.1 ml of 0.5 M dithiothreitol (DTT) were added. Cell suspensions (20% wt/vol, wet cells) were passed through a French Press at 15 000 psi pressure and disintegrated cells were centrifuged at 25 000 ⫻ g for 30 min at 4°C. The supernatant solution obtained after centrifugation is referred to as cell extract. Cell suspensions of more than 100 ml volumes were disintegrated with a Microfluidizer (Microfluidics) at 12 000 psi (two passages) and disintegrated cells were centrifuged at 25 000 ⫻ g for 30 min to obtain cell extract. Protein in cell extracts was estimated by Bio-Rad (Richmond, CA, USA) protein reagent by using bovine serum albumin as a standard. The assay mixture contained 1–10 ␮l of enzyme fraction, 0.8 ml of water, and 0.2 ml of Bio-Rad reagent. After mixing, the absorbance of solution was measured at 595 nm. 2.6. Assay for L-lysine ⑀-aminotransferase The reaction mixture in 5 ml contained 2.7 ml of cell extract (protein 3.5 mg/ml), 30 ␮l of 10 mM pyridoxal phosphate, 1.95 ml of dipeptide dimer (BMS-201391-01) solution (50 mg/ml stock solution), and 75 ␮l of sodium ␣-ketoglutarate (80 mg/ml stock solution). The reaction mixture was incubated at 30°C and 100 rpm. At 1, 4, 8, and 16 h, 0.95 ml of samples were taken and 0.05 ml of trichloroacetic acid (TCA, 100% w/v) was added. After 1 h incubation with TCA at room temperature, 1 ml of acetonitrile was added to the solution. Samples were analyzed for BMS201391-01 and BMS-199541-01 concentration by high-performance liquid chromatography (HPLC). 2.7. HPLC analysis for BMS-201391-01 and BMS-199541-01 HPLC analysis was performed by using a Hewlett-Packard 1090 instrument (Palo Alto, CA, USA) with a Vydac C-18 reverse-phase column (Hesperia, CA, USA). The mo-

Preparation of cell extract from 200 g of washed cells (S. paucimobilis SC 16113) was carried out as described above. The cell extract was loaded onto a Whatman DE-52 column (Tewksbury, MA, USA; 400 ml packed bed) equilibrated with buffer A. The column was washed with 400 ml of buffer A and then with 400 ml of buffer A containing 0.2 M NaCl. Enzyme activity was eluted with a 2-l gradient of buffer A containing NaCl from 0.2– 0.6 M. Fractions of 20 ml were collected. Fractions containing the highest specific activity were pooled. The pooled fraction from the DE-52 column was adjusted to 1 M ammonium sulfate (132 g/l ammonium sulfate added) and loaded on to a Pharmacia fast-flow Phenyl Sepharose威 column (Piscataway, NJ, USA; 150 ml bed volume) equilibrated with buffer A containing 132 g/l ammonium sulfate (1 M ammonium sulfate). The column was washed with 150 ml of buffer A containing 1 M ammonium sulfate and then with buffer A containing 0.25 M ammonium sulfate. The enzyme activity was eluted with a 400 ml gradient of buffer A containing ammonium sulfate from 0.25– 0 M ammonium sulfate. Fractions of 20 ml were collected. The most active fractions (fractions containing ⬎18 000 specific activity) were pooled and concentrated by ultrafiltration to 4 ml by using an Amicon YM-10 membrane (Beverly, MA, USA). The concentrated fraction (4 ml) from the Amicon step was loaded on to a Sephacryl S-200威 column (450 ml) equilibrated with buffer A. Fractions of 10 ml were collected and activity were measured. 2.9. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) The active fractions from the Sephacryl S-200威 column were evaluated by SDS-PAGE as described in the PhastSystem威 procedure by Pharmacia [12], by using the homogeneous 12.5% Phastgel. The enzyme samples were added to a buffer containing 10 mM Tris-HCl, 1 mM EDTA, pH 8, 2.5% SDS, and 5% ␤-mercaptoethanol. The mixture was heated at 100°C for 5 min and bromophenol blue was added to 0.01%. Gels were stained with silver stain and destained in 10% acetic acid solution. Marker with standard mol wt contained phosphorylase ␤ (94 000), bovine serum albumin

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Scheme 3.

(67 000), ovalbumin (43 000), carbonic anhydrase (30 000), soybean trypsin inhibitor (20 100), and ␣-lactalbumin (14 400). 2.10. Determination of mol wt The mol wt of L-lysine ⑀-aminotransferase was determined by size exclusion chromatography by using a Pharmacia Superose column威 (15 ⫻ 1 cm). The column was equilibrated with buffer A. The aminotransferase (Sephacryl S-200 fraction) was applied to the column and eluted with the buffer A at a flow rate of 0.4 ml/min. Fractions of 1 ml were collected. A standard protein mixture containing thryglobulin (6 690 000 mol wt), ferritin (440 000 mol wt), human IgG (150 000 mol wt), human transferrin (810 000 mol wt), ovalbumin (430 000 mol wt), and myoglobin (17 600 mol wt) was also applied to the column and eluted with buffer A. The mol wt of glutamate oxidase was determined by the same procedure. 2.11. Growth of S. noursei SC 6007 One vial of culture was used to inoculate 100 ml of medium C containing 3% toasted nutrisoy and 1% Maltrin M180. Cultures were grown at 28°C and 280 rpm for 24 h on a rotary shaker. Forty-five ml of this culture was transferred to 1 l of medium C. Cultures were grown in 4 l flask at 28°C and 280 rpm for 22 h on a rotary shaker. Cultures were harvested by centrifugation at 180 000 ⫻ g for 15 min, cells were discarded and the supernatant solution containing glutamate oxidase activity was adjusted to pH 7.2 with 1 M KH2PO4 and assayed for activity.

S. noursei SC 6007 was grown in 380 l fermentors containing 250 l of medium C containing 0.025% SAG and 0.025% Dow Corning antifoam. A fermentor was inoculated with 1 l of inoculum and grown at 28°C and 220 rpm agitation with 250 L/min aeration. During fermentation, cells were periodically removed by centrifugation from 200 ml of culture broth and the supernatant solution was assayed for glutamate oxidase activity. The fermentation broth was cooled to 10°C at the time of harvest, and cells were removed by centrifugation through a Sharples centrifuge (Alfa Laval, PA, USA). The supernatant solution containing glutamate oxidase activity was collected. 2.12. Glutamate oxidase assay Glutamate oxidase catalyzes the conversion of L-glutamate to ␣-ketoglutarate. During this reaction, hydrogen peroxide is generated. Hydrogen peroxide formed by the oxidase reaction is coupled to the oxidation of the colorless dye o-dianisidine by horseradish peroxidase present in the reaction mixture producing a red color (Scheme 3). The formation of red product was measured spectrophotometrically at 460 nm. The reaction mixture in 1 ml contained 0.05 ml of 1 M potassium phosphate buffer, pH 7, 0.04 ml of 50 mM L-glutamate, 0.002 ml of 15 000 U/ml of horseradish peroxidase, 0.05 ml of 1.585 mg/ml o-dianisidine, 0.8 ml of water, and 0.05 ml of enzyme. The reaction was started by the addition of glutamate and the change in absorbance at 460 nm was monitored for 5 min at 0.5 min interval. U/ml ⫽ A460/min/11.3 ⫻ ␮l of enzyme. One unit is defined as 1 ␮mol of hydrogen peroxide formed/min. Protein was determined with Bio-Rad protein reagent by using bovine serum albumin as a standard as described earlier.

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2.13. HPLC Analysis for glutamate and ␣-ketoglutarate HPLC analysis was performed with a Hewlett-Packard (HP) 1090 instrument with a Bio-Rad Aminex HPX-87X (300 ⫻ 7.8 mm) column. The mobile phase was 0.008 N H2SO4 containing 1% acetonitrile for separation of glutamic acid and and ␣-ketoglutarate. The flow rate was 0.6 ml/min. The column temperature was 50°C and the detection wavelength was 210 nm. Under these conditions, the retention time for glutamic acid was 6.4 min and for ␣-ketoglutarate was 10.6 min. 2.14. Purification of glutamate oxidase from S. noursei SC 6007 Filtrate obtained from fermentation broth after removal of cells was used for the purification of extracellular glutamate oxidase. To 3.1 l of filtrate at 4°C, solid ammonium sulfate (165 g/L) was added with continuous stirring. The solution was allowed to sit at 4°C for 30 min, and the precipitate was centrifuged out at 17 000 ⫻ g for 30 min. The supernatant solution was applied to a Pharmacia fastflow Phenyl Sepharose威 column (500 ml bed volume) equilibrated with 50 mM potassium phosphate buffer, pH 7.4, containing 132 g/l ammonium sulfate (1 M ammonium sulfate), 10% glycerol, 1 mM DTT, and 1 ␮M FAD (buffer B). The column was washed with 500 ml of buffer B. The enzyme activity was eluted with a 2-l gradient of buffer B containing 1– 0 M ammonium sulfate. Fractions of 20 ml were collected. The active fractions containing ⬎19 specific activity) were pooled and concentrated by ultrafiltration to 4 ml by using an Amicon YM-10 membrane. The salt was removed by diafiltration by using a YM-10 membrane. The concentrated fractions were applied to a Whatman CM-52 column (50 ml packed bed) equilibrated with buffer C (50 mM sodium acetate buffer, pH 5.7, containing 10% glycerol, 1 mM DTT, and 1 ␮mol FAD). The column was washed with 50 ml of buffer C and eluted with a 200 ml of buffer C containing NaCl from 0 –1 M gradient. Fractions of 10 ml were collected. Fractions containing the highest specific activity were pooled. The active fractions from the CM cellulose column were evaluated by SDS-PAGE as described earlier. The mol wt of purified glutamate oxidase was determined by Pharmacia size-exclusion chromatography by using a Superose-12 column威 (15 ⫻ 1 cm).

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of 0.1 M phosphate buffer, pH 8, containing 5 mM DTT, 1 mM PMSF, and 5 mM EDTA. The homogenized cellsuspension was then passed twice through a Microfluidzer at 7500 psi pressure to prepare cell-free extracts. The chamber and the cell-suspension were chilled to 4°C prior to the cell disruption. The disintegrated cells were centrifuged at 20 000 ⫻ g for 1 h to remove the particulates. To 2 l of cell-free extract, 10 ml each of 10 mM pyridoxal phosphate, and ␣-ketoglutarate (80 mg/ml) were added. The reaction mixture was mixed gently with an overhead Teflon agitator (200 rpm). To the stirred reaction mixture, 60 ml of BMS201391-01 solution (50 mg/ml) was added. The reaction was carried out at ambient temperature. Aliquots were taken out every 30 min to measure the amount of BMS-199541-01 formed. After 1.75 h of reaction, 200 ml of TCA solution (100% v/v) was added to lower the pH of the solution to 3.1. To the reaction mixture 1.8 l of acetonitrile was added and mixed gently for 15 min, then let stand at room temperature for 2 h. The quenched reaction mixture was then centrifuged at 20 000 ⫻ g for 1 h at 4°C to remove precipitated proteins. BMS-199541-01 was isolated from the aqueous/organic supernatant. Assay of the resulting mixture indicated a yield of 0.336 g of BMS-199541-01. A 425 ml portion of the solution (34 mg of BMS-199541-01) was concentrated in vacuo to 228 ml, pH 1.57, and the product extracted into EtOAc. Extraction back into water at pH 9.8 (NaOH) followed by extraction at pH 3.5 (H3PO4) into EtOAc and washing with several portions of 0.5 M sodium phosphate buffer, pH 3.5, gave 56 mg of crude product on removal of the solvent. Chromatography on a 2.6 ml column of silica gel 60 (EM Science, 40⬃63 ␮m), eluting with dichloromethane-MeOH, 19 : 1, and monitoring the effluent by thin-layer chromatography (silica gel, dichloromethane-MeOH, 9 : 1, Rf 0.40), gave 34.1 mg of crystalline solid that was recrystallized from 0.25 ml of EtOAc to give 16 mg of BMS-199541-01, m.p. 141⬃144°C. 1H NMR (400 MHz, CDCl3) ␦ 7.34 (5H, m, H-2⬘, 3⬘, 4⬘, 5⬘, 6⬘), 6.20 (1H, d, J 6 Hz, NH), 5.35 (1H, m, H-5), 5.17 (1H, m, H-2), 5.10 (2H, ABq, H-12), 4.91 (1H, m, H-9), 3.23 (1H, m, H-4), 2.92 (1H, m, H-4), 2.42 (1H, m, H-3), 2.32 (1H, m, H-3), 2.03 (2H, m, H-6) and 1.62 (4H, m, H-7, H-8). 13C NMR (100 MHz, CDCl3) ␦ 174.1 (C-10), 173.6 (C-1), 155.5 (C-11), 136.3 (C-1⬘), 128.5 (2C, C-3⬘, 5⬘), 128.1 (C-4⬘), 127.9 (2C, C-2⬘, 6⬘), 66.9 (C-12), 58.9 (C-5), 52.6 (C-2), 51.2 (C-9), 33.1 (C-3), 31.1 (C-6), 31.0 (C-4), 24.9 (C-8), 16.8 (C-7). HRMS: Calcd for C18H23N2O5S 379.1327; found: 379.1327.

2.15. Preparative-scale biotransformation of BMS-201391-01 by L-lysine ⑀-aminotransferase from S. paucimobilis SC 16113

2.16. Cloning and expression of L-lysine ⑀-Aminotransferase from S. paucimobilis SC 16113

About 700 g of S. paucimobilis SC 16113 cells were suspended in 3.5 l of 10 mM phosphate buffer, pH 8, 5 mM EDTA and homogenized with a Tekmar laboratory homogenizer (Cincinnati, OH, USA). These cells were then centrifuged at 4°C for 30 min at 18 000 ⫻ g. The supernatant solution was discarded. The cells were resuspended in 3.5 l

The L-lysine ⑀-aminotransferase protein (LAT) was purified from S. paucimobilis SC 16113 and its amino terminal and internal peptide sequences were determined. A mixed oligonucleotide based on the amino terminal sequence was synthesized. The downstream (antisense) primer based on a conserved amino acid sequence found in other aminotrans-

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ferases was (Tyr-Gly Asn-Pro-Leu-Ala), with a corresponding oligomer was synthesized. A polymerase chain reaction (PCR) with Tth polymerase was performed to obtain a 850-bp fragment. This fragment was isolated and cloned into plasmid vector pCRII. The sequence of the insert was determined which demonstrated strong homology to bacterial aminotransferases found in GenBank database. An internal peptide sequence of 19 amino acids obtained from a tryptic peptide of purified aminotransferase protein from S. paucimobilis SC 16113 was also located. These data indicated that the PCR fragment amplified was in fact representative of part of the bona fide LAT enzyme. The 850 bp PCR fragment was used as a hybridization probe to identify the entire LAT gene. S. paucimobilis SC 16113 chromosomal DNA was purified and partially digested with restriction endonuclease Sau3A1. Fragments of 6 –10 kilobases (kb) were extracted from an agarose gel following electrophoresis and ligated to BamHI-cleaved plasmid vector pZerol. The DNA was transformed into E. coli TOP 10 F⬘ cells by electroporation and selected on LB medium containing the antiobiotic Zeocin. Transformants were then transferred onto nylon filters and lysed in situ. After hybridization with a 32P-labeled PCR fragment, several strongly hybridizing colonies were seen. They were picked from the master plate and grown in liquid medium for pDNA isolation. One colony contained a plasmid that contained an insert of ⬃6.3 kb and was thus named pLAT6.3. The entire aminotransferase gene of pLAT6.3 was sequenced. A typical Gram-negative promoter and ribosome binding site followed 9 bases later by an initiation codon ATG (Methionine) was found. The size of the coding region of the gene is 1221 bp. Based on our computer analysis of the gene, this region should encode 398 amino acids with a mol wt of 42 457 Da. We identified additional runs of amino acids that matched that obtained from internal peptide sequencing of the purified LAT protein. This information confirmed that the entire lat gene was present on pLAT6.3 and that it encoded the same protein isolated from S. paucimobilis SC 16113. The PCR was used to precisely amplify the lat gene, which also contained restriction sites at both ends for cloning into expression plasmids. For digestion and ligation into pKK223-3, the lat gene was ampified with EcoRI and BamHI sites at the 5⬘ and 3⬘ ends. Similarly, NdeI and XbaI sites were added to the 5⬘ and 3⬘ ends, respectively, for ligation into pAL781. Both the amplified fragment and the vector DNAs were cleaved with the appropriate enzymes and ligated together. The ligation samples were electroporated into E. coli strains TOP 10 F⬘ (pKK223-3-LAT) or GI724 (pAL781-LAT/ A). The presence of the lat gene in the recombinant plasmids was confirmed with PCR and restriction digests. 2.17. Growth of E. coli in shake-flask cultures TOP 10 F⬘(pKK223-3-LAT) was grown in LB medium (tryptone, 1%; yeast extract, 0.5%; NaCl, 1%) containing 100 mg/ml of ampicillin. At an OD600 of ⬃1, the tac

promoter controlling expression was induced with 100 mM isopropylthiogalactoside. Samples were taken 1, 2, and 3 h post-induction and analyzed for aminotransferase activity and used in the bioconversion of BMS-201391-01 to BMS199541-01. GI724(pAL781-LAT) was grown for 18 –20 h in MRM [0.6% Na2HPO4, 0.3% KH2PO4, 0.125% (NH4)2SO4, 2% Casamino acids (Bacto grade), 1.0% glycerol, 1 mM MgSO4]. The culture was then inoculated into MIM medium at a starting OD600 of 0.20 [MIM contained 0.6% Na2HPO4, 0.3% KH2PO4, 0.125% (NH4)2SO4, 0.2% Casamino acids (Bacto grade), 0.5% glucose, and 1 mM MgSO4]. Ampicillin was added to all media at a final concentration of 100 ␮g/ml. At an OD600 of ⬃0.5, Ltryptophan from a filter-sterilized 10 mg/ml solution was added to a final concentration of 100 mg/ml. Samples were removed at 3, 6, and 22 h postinduction for aminotransferase activity and used in the bioconversion of BMS201391-01 to BMS-199541-01. Expression with the tryptophan-inducible promoter was better than with the tac promoter so the former strain was investigated in more detail. A kanamycin-resistant version of the above plasmid was also created. Plasmid pET9b was excised by digestion with restriction endonucleases AlwNI and EcoRI. A 1171 bp fragment containing the KnR gene was purified and the ends made blunt-ended by treatment with Klenow DNA polymerase plus all four deoxyribonucleotides. The modified fragment was ligated into pAL781LAT/A, which had been digested with SspI. After electroporation into GI724, kanamycin-resistant colonies were picked and verified for the presence of both the KnR and lat genes. This plasmid was named pAL781-LAT/K. 2.18. Production of aminotransferase in E. coli G1724 The process described is for the growth of E. coli GI724(pAL781-LAT/A) in 20 l fermentors. E. coli cultures were first grown in a 4 l flask containing 1 l medium (MT3 medium) for 24 h. Two 1 ml vials were inoculated into each 4 l flask (two are needed for a 16 l fermentor) containing 1 l of MT3 medium containing 1% NZ amine A, 2% yeastamine, 2% glycerol, 0.6% Na2HPO4, 0.3 KH2PO4, 0.0246% MgSO4 䡠 7H2O, 0.125% (NH2)SO4, and 0.005% kanamycin. Each flask was incubated at 30°C for 24 h at 250 rpm. 24 h grown cultures were inoculated into a 25 l fermentor containing 16 l of modified MT3 medium containing 1% NZ amine A, 2% yeastamine, 4% glycerol, 0.6% Na2HPO4, 0.3 NaH2PO4, 0.125% (NH2)SO4, and 0.05% polypropylene glycol adjusted to pH 7. Before inoculation, a solution containing magnesium sulfate and ampicillin were added aseptically (after filter sterilization) to the tank to a final concentration of 1 mM and 100 mg/L, respectively. Fermentation was carried out at 37°C, and at 250 rpm agitation for 6 – 8 h. The optical density of properly diluted samples was measured at 600 nm on a spectrophotometer. Cells were recovered by centrifugation. A portion (15 g) of

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centrifuged wet cell paste was suspended in 100 ml of 100 mM phosphate buffer, pH 8, 10 mM Na EDTA and homogenous cell suspensions were prepared. To the cell suspensions, 0.1 ml of 0.1 M PMSF solution in isopropanol and 0.1 ml of 0.5 M DTT solution were added. Cell extracts were prepared as described earlier and assayed for aminotransferase activity by conversion of BMS-201391-01 to BMS199541-01. Unit of activity was defined as ␮mole of BMS199541-01 formed/ml/min. 2.19. Cloning and expression of glutamate oxidase from S. noursei SC 6007 The glutamate oxidase (GOX) enzyme from S. nourseii SC 6007 was purified to homogeneity. The N-terminal sequence as well as three internal amino acid sequences were determined and used to design degenerate oligonucleotide primers to amplify a portion of the gox gene by using PCR. This reaction produced a strongly amplified 265 bp band. DNA sequencing of the fragment indicated a single openreading frame encoding 88 amino acids. BLAST analysis of this amino acid sequence indicated high homology to a variety of amino acid oxidases, suggesting that the PCR fragment contained a portion of the gox gene. The 265-bp fragment was used as a probe to detect the gox gene within a plasmid-based library of S. noursei SC 6007 chromosomal DNA. Positives were isolated and the hybridizing region was found to reside on a 9000 bp KpnI restriction fragment. The regions around the area of probe hybridization were sequenced and found to contain a single open reading frame. Translation of the cloned DNA indicated a mature protein of 60 926 Da, agreeing well with the mol wt of the purified S. noursei SC 6007 glutamate oxidase (⬃58 000 Da). BLAST analysis of the translated DNA sequence revealed extremely strong homology to a variety of bacterial and fungal amino acid oxidases. The N-terminal and the three internal amino acid sequences derived from the purified S. noursei SC6007 glutamate oxidase were found within the cloned gene. Attempts to express the SC6007 glutamate oxidase by using standard E. coli vectors and strains were unsuccessful. As an alternative, the S. noursei SC6007 glutamate oxidase was expressed in S. lividans. The S. noursei SC6007 glutamate oxidase, including its native promoter sequence, was inserted into an S. lividans expression vector. Untransformed S. lividans does not have a native glutamate oxidase activity, whereas S. lividans transformed with the GOX expression plasmid clearly demonstrated glutamate oxidase activity. 2.20. Preparative-scale Biotransformation of BMS-201391-01 by L-lysine aminotransferase from E. coli GI724[pal781-LAT] Four different batches at substrate input of 3, 5, 12, and 22 g were carried out. A typical procedure used for a 22 g batch is described below. E. coli GI724[pal781-LAT] wet cells (75 g) were suspended in 500 ml of 100 mM phosphate

383

buffer, pH 7.8, containing 5 mM DTT, 1 mM PMSF, and 5 mM EDTA and homogenized. The homogenized cell-suspensions were then passed twice through Microfluidzer at 7500 psi pressure to prepare cell-free extracts. The disintegrated cells was centrifuged at 20 000 ⫻ g for 25 min to remove the debris. In a 5 l bioreactor containing 22 g of substrate (BMS201391-01), 2.5 l of 100 mM phosphate buffer, pH 7.8, containing 5 mM EDTA was added and gradually the pH was adjusted to 12 by slow addition of 5 N NaOH while stirring (200 rpm) at room temperature. The pH was maintained at 12 until all solids were dissolved. The pH was then readjusted to 7.8 with slow addition of concentrated phosphoric acid. The temperature of the bioreactor was adjusted to 30°C and 6.75 g of DTT, 19 g of ␣-ketoglutarate (disodium salt) and 100 mg of pyridoxal phosphate were added to the reactor. An additional 1.4 l of 100 mM phosphate buffer containing 5 mM EDTA was then added to the bioreactor. The reaction was started by addition of 100 ml of cell-extracts of E. coli GI724[pal781-LAT] containing 2200 U. Glutamate oxidase solution (2000 U) was added at 30 min, 1 h, 1.5 h and 3.5 h. The reaction was carried out at 30°C, pH 7.8, and 200 rpm. Substrate and product concentrations were determined by HPLC assay. After 4 h of reaction, 600 ml of TCA solution (100% v/v) was added to decrease the pH to 3.1. To the reaction mixture 5 l of acetonitrile was added and mixed gently for 15 min, then let stand at room temperature for 2 h. The product BMS199541 was isolated as described earlier. By using the same procedure other dipeptides and N-␣-protected (CBZ or BOC) L-lysine were evaluated as substrate.

3. Results A selective culture technique was used to isolate microorganisms able to utilize N-␣-CBZ-L-lysine as the sole source of nitrogen. By using this enrichment culture techniques, eight different types of colonies were isolated. Cultures were grown in shake-flasks and cell extracts prepared from cell-suspensions were evaluated for oxidation of the ⑀-amino group of L-lysine in substrate BMS-201391-01. Product BMS-199541-01 formation (0.05– 0.35 mg/ml) was observed with four cultures. One of the cultures Z-2, later identified as a S. paucimobilis SC 16113 contained high activity. In a 10 ml reactor with 2 mg/ml substrate input, the product BMS-199541-01 was obtained at 0.35 mg/ml. Lower mass balance and reaction yield were obtained due to hydrolysis of dipeptide by cell extracts. S. paucimobilis SC 16113 was grown in a 700 l fermentor containing 500 l of medium. During fermentation, cells were harvested from 200 ml broth by centrifugation. Cells were suspended in buffer, cell extract were prepared and evaluated for conversion of BMS-201391-01 to BMS199541-01. Culture grown for 48 – 60 h gave higher [183– 220] specific activity compared to cells harvested at 24 or 72 h (126 –140 specific activity). A specific activity (mg of

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Table 1 Purification of L-lysine ␧-aminotransferase from Spingomonas paucimobilis SC 16113 Step

Volume (m L)

Protein (mg)

Activity (units)

Sp 䡠 activity (mg/h/g)

Recovery (%)

Purification (fold)

Cell extracts DE-52 Phenyl sepharose Sephacryl S-200

800 150 120 40

2544 57 5.04 1.32

400 219 93 53

157 1462 18472 36,666

100 55 23.2 13.2

1 9.3 117 254

Purification was carried out as described in Section 2. Specific activity was expressed as the mg of BMS-199541-01 formed per h per g of protein. Concentration of BMS-199541-01 was determined by HPLC.

BMS-199541-01 formed/h/g of protein in cell extract) of 220 was obtained from culture grown for 60 h. A preparative batch for biotransformation of BMS201391-01 to BMS-199541-01 by using 2 l of cell extract of S. paucimobilis SC 16113 was conducted as described in Section 2. Substrate was used at a concentration of 1.5 g/l. A reaction yield of 10% (0.33 g of BMS-199541-01) was obtained after 1.75 h biotransformation process. The product was isolated and identified by 1H-NMR, 13C-NMR, and mass analysis. Due to the low activity of L-lysine ⑀-aminotransferase in S. paucimobilis SC 16113, we decided to purify the enzyme, determine its sequence and overexpress the protein in a suitable host. The L-lysine ⑀-aminotransferase was purified 354 fold with a specific activity (mg product formed/h/g of protein) of 36 600. Results are as shown in Table 1. The purified enzyme after Sephacryl S-200 column chromatography showed a single protein band on SDS-PAGE by using a silver stain. The mol wt of the enzyme as determined by gel-filtration techniques is 81 000 Da and subunit size as determined SDS-PAGE is 40 000 Da indicating that the L-lysine ⑀-aminotransferase is a dimeric protein. The Nterminal and internal peptides sequence (generated by Lyspeptidase treatment) of purified L-lysine aminotransferase were determined. Results are as shown in Table 2. The purified L-lysine ⑀-aminotransferase was evaluated Table 2 N-terminal and internal peptide sequence of purified L-lysine ␧aminotransferase from Sphingomonas paucimobilis SC 16113 N-terminal: Ser-Ile-Thr-Pro-Leu-Met-Pro-Val-Tyr-Pro-Arg-Arg-Asp-ValArg Peptide 1: Ile-Ile-Ser-Phe-Asp-Asn-Ala-Phe-His-Gly-Arg-Thr-Leu-GlyThr-Ile-Ser-AlaPeptide 2: Ile-Ser-Ala-Gly-Ala-Arg-Ser-Phe-Ala-Asp-Trp-Lys Peptide 3: Ala-Val-Ile-Asp-Glu-Gln-Gly-Leu-Leu-Leu-Ile-Leu-Asp-GluVal-GlnN-terminal and internal peptide sequence of purified glutamate oxidase from Streptomyces noursei SC 6007 N-terminal: Thr-Gly-Gly-Thr-Ala-Glu-Ala-Arg-Gly-Val-Ala-Leu-AlaAla-Arg Peptide 1: Lys-Asn-Asp-Ala-Thr-His-Ile-Gly-Asp-Thr-Pro-Gln-Lys Peptide 2: Lys-Ile-Trp-Tyr-Gln-Ala-Asn-Leu-Asp-Gln-Asp-Ile-Ala-PheGlu-Asn-Arg

for cofactor requirements. The enzyme required ␣-ketoglutarate as amino acceptor. NAD or NADP was not required as a cofactor indicating that enzyme is an aminotransferase and not a dehydrogenase. A reaction yield of 70 M% was obtained for BMS-199541-01 with the complete system. Glutamate oxidase was required to recycle glutamate back to ␣-ketoglutarate. In the absence of glutamate oxidase, a 35 M% reaction yield of BMS-199541-01 was obtained. The L-lysine ⑀-aminotransferase was overexpressed in E. coli strain GI724(pAL781-LAT). The production of enzyme in a 25 l fermentor was conducted as described in Section 2. Enzyme activity of 2000 –2425 U/l (unit is defined as ␮mol BMS-199541-01 formed/ml/min) of broth was obtained after 16 h of fermentation. Screening of microbial cultures led to the identification of S. noursei SC 6007 as a source of extracellular glutamate oxidase. S. noursei SC 6007 were grown in 380-l fermentors containing 250 l of medium A as described in Section 2. Glutamate oxidase activity started after 12 h of growth in a fermentor and reached 750 U/l at harvest (30 h). At the end of the fermentation, fermentation broth was cooled to 8°C and cells were removed by centrifugation. Starting from the extracellular filtrate recovered after removal of cells from fermentation broth, the glutamate oxidase was purified 260-fold with a specific activity (U/mg of protein) of 54 (Table 3). The purified enzyme after CM-cellulose column chromatography showed a single protein band on SDS-PAGE by using a silver stain. The mol wt of the enzyme as determined by gel-filtration techniques is 125 000 Da and subunit size as determined SDS-PAGE is 60 000 Da. The amino terminal and internal peptide sequence (Table 2) of purified glutamate oxidase were determined to synthesize oligonucleotide probes for cloning and overexpression of the enzyme. Attempts to express the S. noursei SC6007 glutamate oxidase using standard E. coli vectors and strains were unsuccessful. As an alternative, the SC6007 glutamate oxidase was expressed in Streptomyces lividans. The S. noursei SC6007 glutamate oxidase, including its native promoter sequence, was inserted into an S. lividans expression vector. Untransformed S. lividans does not have a native glutamate oxidase activity, while S. lividans transformed with the GOX expression plasmid clearly demonstrated glutamate oxidase activity. SDS-PAGE analysis of the transformed S.

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385

Table 3 Purification of glutamate oxidase from Streptomyces noursei SC 6007 Sp 䡠 activity (␮mole/min/mg)

Step

Volume (m L)

Protein (mg)

Activity (units)

Filtrate Ammonium sulfate (30% sat. supernatant) Phenyl sepharose CM cellulose

3145 3920

9435 960

1981 1575

0.21 1.64

1230 528

19.07 54

500 40

64.5 8.2

Recovery (%)

Purification (fold)

100 79

1 7.8

62.3 26.6

90.8 260

Purification was carried out as described in Section 2. Specific activity was expressed as the ␮moles of product formed per min mg of protein. Concentration of BMS-199541-01 was determined by HPLC.

lividans revealed a protein band not seen in an untransformed strain. This band was of same mol wt as the GOX protein purified from S. noursei SC 6007, indicating that the glutamate oxidase activity present in the transformed strain arose from expression of the heterologous gene. About 0.4 U/ml of activity was detected from the S. lividans culture, indicating that the enzyme was expressed at low level. Further research is required to overexpressed this protein. Biotransformation of BMS-201391-01 to BMS199541-01 was carried out by using L-lysine ⑀-aminotransferase from E. coli GI724[pal781-LAT] in the presence of ␣-ketoglutarate and DTT (required to reduce the dipeptide dimer to monomer). Glutamate produced during the reaction was recycled to ␣-ketoglutarate by partially purified glutamate oxidase (7 U/ml) from S. noursei SC 6007. Four different batches were carried out as described in Section 2. Reaction yields of 65– 67 M% were obtained as shown in Table 4. The kinetics of reaction for batch 40457 are as shown in Fig. 1. Two new dipeptides N-[N[(phenylmethoxy)carbonyl]-Lmethionyl]-L-lysine (BMS-203528-01) and N,2-[S-acetylN-[(phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine (BMS-204556) were evaluated as substrates for L-lysine aminotransferase by cell-free extracts of E. coli GI724[pal781-LAT] in the presence of ␣-ketoglutarate. As no product markers were available, the formation of new compounds from the enzymic reaction was investigated by LC-MS. The data indicates the formation of a new compound with mol wt. of 392, which was assigned the tentative structure 1. The ⑀-NH2 group of BMS-203528-01 was oxidized and in the presence of TCA the aldehyde was cyclized to the enamide with a loss of water (Scheme 4).

When BMS-204556 was treated with cell-free extracts of E. coli GI724[pal781-LAT] and ␣-ketoglutarate, several new components were observed by LC-MS. The component with mol wt 420.5 was assigned structure 2, formed by the oxidation of the ⑀-NH2 group of BMS-204556 and subsequent dehydration to produce the the cyclic enamide, the component with mol wt. 397 was proposed as desacetyl BMS-204556 3. The desacetyl BMS-204556 was then oxidized by the enzyme to BMS-199541-01, mol wt. of 378, as shown in Scheme 5. To reduce the cost of producing two enzymes, the transamination reactions were carried out in the absence of glutamate oxidase and higher levels of ␣-ketoglutarate. The reaction yield in the absence of glutamate oxidase averaged about 33–35 M%. At 40 mg/ml of ␣-ketoglutarate (10 ⫻ increase in concentration) and at 40°C, the reaction yield increased to 70 M% equivalent to that in the presence of glutamate oxidase. Phenylacetyl or phenoxyacetyl protected analogues of BMS-201391-01 (Scheme 1) also served as substrates for L-lysine ⑀-aminotransferase giving reaction yield of 70 M% for the corresponding BMS-199541-01 analogs. N␣-t-butoxycarbonyl- L-lysine and N-␣-carbobenzoxy-L-lysine were also oxidized by L-lysine aminotransferase from E. coli GI724[pal781-LAT]. The chiral compounds (S)-3,4-dihydro-1,2(2H)-pyridinedicarboxylic acid, 1-(phenylmethyl)ester (BMS-202665) and (S)-3,4-dihydro1,2(2H)-pyridinedicarboxylic acid, 1,1-dimethylethyl ester (BMS-264406) were obtained as products for oxidation of N-␣-CBZ-L-lysine and N-␣-BOC-L-lysine, respectively (Scheme 6). A reaction yield of 80 – 85 M% was obtained for each product. In the enzymatic reaction to convert BMS-201391-01 to

Table 4 Biotransformation of BMS-201391-01 to BMS-199541-01 by L-lysine ␧-aminotransferase from Escherichia coli Gl724[pal781-LAT] Experiment batch number

BMS-201391-01 input (g)

BMS-201391-01 remaining (g)

BMS-199541-01 (g)

BMS-199541-01 (M% Yield)

40455 40456 40457 40458

3 5 12 22

0.83 1.35 4.3 4.7

1.9 2.92 7.5 14.4

66.5 65 70 67

Reactions were carried out as described in Section 2 using cell extracts of Escherichia coli G1724[pal781-LAT] in presence of dithiothreitol, pyridoxal phosphate, and partially purified glutamate oxidase from Streptomyces noursei SC 6007.

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Fig. 1. Kinetics of enzymatic conversion of BMS-201391 to BMS-199541 by L-lysine ⑀-aminotransferase from E. coli GI724[pal781-LAT]

BMS-199541-01, we used DTT to cleave the disulfide bond of dipeptide dimer BMS-201391 to produce the dipeptide monomer, which is the substrate for the Llysine ⑀-aminotransferase. It was observed, that tributylphosphine (an inexpensive compound) was as effective as DTT for the dipeptide dimer to monomer conversion. In the presence of 10 mM tributylphosphine, 3.5 mg/ml of BMS-201391-01, 40 mg/ml ␣-ketoglutarate and 0.1 U of transaminase, 69 M% yield of BMS199541-01 was obtained. To terminate the L-lysine ⑀-aminotransferase reaction during conversion of BMS-201391-01 to BMS-199541-01, generally 10% v/v TCA was used. After some optimization studies, the amount of TCA required to terminate the reaction was reduced to 5% v/v without loss in yields of BMS-

199541-01. It was also observed that methanesulfonic acid is equally effective as TCA, giving 70 M% yield of BMS199541-01.

4. Discussion In this report, we have described the preparation of a key chiral synthon [4S-(4I,7I,10aJ)]1-octahydro-5-oxo-4-[phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b] [1,3]thiazepine-7-carboxylic acid methyl ester (BMS-199541-01), used for synthesis of Omapatrilat, a vasopeptidase inhibitor. An L-lysine ⑀-aminotransferase-producing culture S. paucimobilis SC 16113 was isolated by enrichment culture tech-

Scheme 4.

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387

Scheme 5.

niques and taxonomically identified. The enzyme was purified, characterized, and overexpressed in E. coli GI724[pal781-LAT]. The enzyme required ␣-ketoglutarate as an acceptor molecule for the ⑀-amino group of L-lysine in a dipeptide dimer (BMS-201391-01). The actual substrate is a dipeptide monomer that was prepared by treatment of BMS-201391-01 with DTT or tributylphosphine. The glutamate formed during this reaction was recycled back to ␣-ketoglutarate by glutamate oxidase from S. noursei SC 6007. By increasing the concentration of ␣-ketoglutarate by 10-fold during the reaction, the recycling of glutamate by using glutamate oxidase was not required to obtain the same yield. Various other L-lysine containing dipeptides and N-

CBZ- or N-BOC-L-lysine were substrates for L-lysine ⑀-aminotransferase. Aminotransferases have been extensively used in the synthesis of L-amino acids from the corresponding ␣-keto acids [13,14]. L-lysine-␣-ketoglutarate aminotransferase from Flavobacterium fuscum was reported by Soda et al. [15], who demonstrated that the product of L-lysine oxidation is 1-piperideine-6-carboxylic acid. In this aminotransferase reaction, the ⑀-amino group of L-lysine is transferred to ␣-ketoglutarate to yield glutamate and ␣-aminoadipate-␦-semialdehyde, which is immediately converted into the intramolecular dehydrated compound 1-piperideine-6-carboxylic acid. Soda and Misono [16] reported that L-lysine ␣-ketoglutarate aminotransferase

Scheme 6.

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(116 000 mol wt) contained two molecules of pyridoxal phosphate as bound prosthetic group. Hammer and Bode [17] purified L-lysine ␣-ketoglutarate aminotransferase from Candida utilis and reported that it is a dimeric protein of 83 000 Da. L-lysine aminotransferase from S. paucimobilis SC 16113 reported in this paper is a dimeric protein of 80 000 Da. In Streptomyces lactamdurans, the precursor of the ␣-aminoadipoyl side-chain of cephamycin C, is L-lysine. Studies with cell extracts demonstrated that an L-lysine ⑀-aminotransferase is involved in the conversion of L-lysine to L-␣-aminoadipic acid [18]. L-lysine ⑀-aminotransferase has also been demonstrated from Penicillus chrysogenum where the oxidation of L-lysine by this enzyme leads to formation of L-␣-aminoadipic acid involved in the synthesis of penicillin [19.]. In this paper, we have for the first time reported the oxidation of the ⑀-amino group of L-lysine in various dipeptide substrates and N-␣-protected lysine by L-lysine ⑀-aminotransferase and its use in the preparation of key chiral compounds for the synthesis of drug substance. L-lysine ⑀-dehydrogenases have been reported from Agrobacterium tumefaciens [20 –22] and Candida albicans [23]. This enzyme catalyzes the NAD⫹ or NADP⫹-dependent conversion of L-lysine to ␣-aminoadipate-␦-semialdehyde. Lysyl oxidase has been studied extensively from calf aortas [24,25]. Lysyl oxidase initiates covalent cross-linking in collagen and elastin by catalyzing the oxidative deamination of specific lysine residues to peptidyl-␣-aminoadipic␦-semialdehyde, which is the precursor to the various crosslinkages found in these proteins [26 –28]. The enzyme also deaminates a variety of aliphatic monoamines and diamines, synthetic elastin-like polypeptides, and lysine in histone H1 and other proteins with basic isoelectric points [27]. L-lysyl oxidase was reported to contain copper, pyridoxal phosphate, and perhaps pyrroloquinoline quinone cofactors [26 –28]. Glutamate oxidases have been reported from Streptomyces sp. X-119-629 –30 Streptomyces violascens [31], and Streptomyces endus [32]. Glutamate oxidase from Streptomyces sp. X-119 has been purified. The enzyme contained 2 mol of FAD and had a mol wt of about 140 000 containing three nonidentical subunits of 44 000, 16 000, and 9 000 Da [29]. The purified glutamate oxidase from Streptomyces violascens has a mol wt of 62 000 and consists of a single polypeptide as determined by SDSPAGE. The purified glutamate oxidase from S. noursei SC 6003 reported in this paper has a mol wt of 125 000 Da and subunit size of 60 000 Da.

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