Journal of Chemical Technology and Biotechnology J Chem Technol Biotechnol 82:1099–1106 (2007)

A concise synthesis of single-enantiomerβ-lactams and β-amino acids usingRhodococcus globerulusMichael Lloyd,∗ Richard Lloyd, Philip Keene and Andrew OsborneDowpharma, Chirotech Technology Ltd, Unit 162, Cambridge Science Park, Milton Road, Cambridge CB4 0GH, UK

Abstract

BACKGROUND: Pharmaceutical companies continue to evaluate β-amino acids and β-lactams in a range of drugcandidates. The development of a highly efficient and selective bioresolution of cyclic β-lactam substrates couldyield enantiopure lactams and β-amino acids with medicinal potential. The aim of this work was to discover anddevelop a biocatalyst capable of selectively hydrolysing β-lactam substrates.

RESULTS: Screening of our in-house culture collection led to the discovery of a microorganism, Rhodococcusgloberulus (NCIMB 41042) with β-lactamase activity. Whole-cell bioresolutions of the β-lactams 1–4 weresuccessfully carried out and in all cases enantiomeric excesses of the residual lactam and amino acid product werefound to be greater than 98%. For one example, the bioresolution was optimised to operate at 60 g L−1 substrateconcentration with a 20% wt/wt cell paste loading.

CONCLUSIONS: A microorganism, Rhodococcus globerulus (NCIMB 41042), capable of selectively hydrolysinga range of cyclic β-lactams, has been discovered. A scalable whole-cell bioresolution process has been developed,leading to the synthesis of multigram quantities of enantiomerically pure β-lactams and β-amino acids. 2007 Society of Chemical Industry

Keywords: β-lactamase; bioresolution; Rhodococcus globerulus; whole cell; β-amino acid

INTRODUCTIONOver the last few years, pharmaceutical companieshave shown increased interest in the potentialbiological activity of β-amino acids and β-lactamsand their utility in synthetic chemistry.1–6 Single-enantiomer forms of these compounds are potentiallyuseful as chiral synthons in pharmaceutical drugresearch. In the past, they have been evaluatedas antifungal1 and antimalarial agents2 in additionto being utilised in drug candidates targeted atthrombosis3 and cancer treatments.4 More recently,cyclic β-amino acids have been utilised in matrixmetalloproteinase (MMP) inhibitors.5,6 Such enzymeshave attracted considerable interest as they havebeen implicated in physiological and pathologicalconditions ranging from tumour growth to rheumatoidarthritis and cirrhosis of the liver.

Academic groups have also shown considerableinterest in the conformational changes resulting fromincorporation of β-amino acids into peptides.7–10

In addition, β-peptides frequently exhibit retardedmetabolism and are more resistant to proteaseenzymes. The insertion of a cyclic β-amino acidresidue into a peptide chain can modify and potentiallyimprove its pharmacological activity.

The development of a highly efficient and selectivebioresolution of cyclic β-lactam substrates couldyield enantiopure lactams and β-amino acids withmedicinal potential. Early attempts have involvedenzymatic desymmetrisation of meso-diesters followedby conversion to a β-amino acid and then formationof the lactam ring.11 The classical resolution of aring-opened precursor using cinchonidine has alsobeen employed.12 A number of methods describingthe enzymatic production of enantiomerically pureβ-lactams and cyclic β-amino acids from racemicβ-lactams have been reported.13–21 These initiallyinvolved the derivatisation of the β-lactam usingparaformaldehyde and lipase-catalysed resolution ofthe N-hydroxymethyl-β-lactam.14 More recently,work carried out by Forro and Fulop has shownthat β-lactams can be resolved by lipase-catalysedenantioselective hydrolysis in organic solvents.15,16

The products from these resolutions have been furtherelaborated into potentially useful chiral synthons.22

Evans et al. have reported the whole-cell bioresolu-tion of β-lactams using a β-lactamase from Rhodoccocusequi18 but the biotransformation was found to be anextremely slow process and not viable for operationon a commercial scale. Work within our laboratories

∗ Correspondence to: Michael Lloyd, Dowpharma, Chirotech Technology Ltd, A Subsidiary of The Dow Chemical Company, Unit 162, Cambridge SciencePark, Milton Road, Cambridge CB4 0GH, UKE-mail: mlloyd2@dow.com(Received 25 January 2007; revised version received 26 April 2007; accepted 1 May 2007)Published online 6 July 2007; DOI: 10.1002/jctb.1734

2007 Society of Chemical Industry. J Chem Technol Biotechnol 0268–2575/2007/$30.00

M Lloyd et al.

was carried out with a view towards developing animproved whole cell bioresolution of β-lactams and,to date, only partial data from this work has beendisclosed.19–21

In this paper we report the development of awhole-cell bioresolution using a strain of Rhodococcusgloberulus (NCIMB 41042) that can be utilisedfor the production of multigram quantities ofenantiomerically pure β-lactams and cyclic β-aminoacids. The microorganism was isolated from our in-house culture collection by screening for appropriateβ-lactamase activity. A series of racemic β-lactamswere synthesised using the procedure reported byMorriconi and Crawford23 and evaluated as substratesfor this microorganism.

EXPERIMENTALAll reagents and solvents were from commerciallyavailable sources and used as received. Nuclearmagnetic resonance (NMR) spectra were obtainedon a Bruker (Coventry, UK) Avance 400 spectrometeroperating at 400 MHz for proton (1H). Chemical shifts(δ) are reported in ppm using Me4Si or residual non-deuterated solvent as reference. Coupling constants(J) are measured in Hz.

Gas chromatographic (GC) enantiomeric excess(e.e.) data of the resolved β-lactams were obtainedusing a PerkinElmer (Beaconsfield, UK) Autosystemgas chromatograph fitted with a Varian (Oxford, UK)Chirasil-Dex CB column (25 m × 0.25 mm). Sampleswere derivatised using trifluoroacetic anhydride andanalysed using the following oven conditions: 80 ◦Cfor 2 min then ramp to 150 ◦C at 5 ◦C min−1 and holdfor 5 min; helium 20 psi.

High-performance liquid chromatographic (HPLC)e.e. data for the resolved β-amino acids were obtainedusing a Gilson (Luton, UK) HPLC system fitted witha Crownpak CR column (150 mm × 4.6 mm), usingHClO4 pH 1 mobile phase with a 0.5 mL min−1 flowrate and a refractive index detector at 0 ◦C.

Optical rotation data were obtained using aPerkinElmer Polarimeter 341 instrument.

Procedures for racemate synthesis23

Preparation of (±)-7-aza-bicyclo[4.2.0]oct-4-en-8-one,(±)-1To a stirred solution of chlorosulfonyl isocyanate(7.4 mL, 85 mmol) in dichloromethane (150 mL) wasslowly added a solution of 1,3-cyclohexadiene (10 mL,105 mmol) in dichloromethane (35 mL). After theaddition was complete, the reaction mixture wasstirred at room temperature for a further 10 min.The reaction mixture was slowly dosed into a stirredsolution of 25 wt% sodium sulfite solution (100 mL)and dichloromethane (50 mL) and the pH of themixture was maintained between 7 and 8 by additionof 1 mol L−1 sodium hydroxide solution. Stirringwas continued for a further 20 min, the two phaseswere separated and the organic phase was washed

with saturated brine solution (200 mL) and driedover magnesium sulfate. Solvent was removed underreduced pressure to yield the title compound as apale yellow solid (7.3 g, 70%). 1H NMR (CDCl3):6.19–6.10 (1H, m), 6.07 (1H, br s), 5.98–5.90(1H, m), 4.03 (1H, t, J = 5), 3.55–3.48 (1H, m),2.16–2.02 (3H, m), 1.68–1.55 (1H, m).

Preparation of (±)-3-aza-tricyclo[4.2.1.02,5]non-7-en-4-one, (±)-2To a stirred solution of chlorosulfonyl isocyanate(49 mL, 0.56 mol) in dichloromethane (1050 mL)was slowly added a solution of bicyclo[2.2.1]hepta-2,5-diene (70 mL, 0.65 mol) in dichloromethane(280 mL). After the addition was complete, thereaction mixture was stirred at room temperature fora further 20 min. The reaction mixture was slowlydosed into a stirred solution of 25 wt% sodium sulfitesolution (700 mL) and dichloromethane (250 mL) andthe pH of the mixture was maintained between 7 and8 by addition of 5 mol L−1 sodium hydroxide solution.Stirring was continued for a further 20 min, the twophases were separated and the organic phase waswashed with saturated brine solution (1000 mL) anddried over magnesium sulfate. Solvent was removedunder reduced pressure to yield the title compoundas a beige solid (54.4 g, 72%). 1H NMR (CDCl3):6.30–6.19 (1H, m), 6.18–6.00 (2H, m), 3.53–3.44(1H, m), 3.10–2.98 (1H, m), 2.96–2.91 (1H, br s),2.91–2.82 (1H, br s), 1.81 (1H, d, J = 10), 1.65 (1H,d, J = 10).

Preparation of (±)-6-Aza-bicyclo[3.2.0]heptan-7-one,(±)-3To a solution of cyclopentene (75.1 g, 1.10 mol)in dichloromethane (150 mL) at 0–5 ◦C was doseda solution of chlorosulfonyl isocyanate (148.55 g,1.05 mol) in dichloromethane (50 mL), maintainingthe temperature below 10 ◦C. The mixture was stirredat 0–5 ◦C for 1 h and then at 40 ◦C for a further 20 huntil complete conversion was achieved. The mixturewas re-cooled to 0–5 ◦C and cold water (∼100 mL)was dosed into the reaction until effervescence ceased.The reaction mixture was then dosed into a pre-cooledsuspension of sodium sulfite (66.8 g, 0.525 mol) inwater (200 mL), maintaining the temperature below25 ◦C and pH in the range 5–7 by concurrent additionof 5 mol L−1 aqueous sodium hydroxide. The mixturewas stirred at 0–5 ◦C for 1 h, then at pH 7 the layerswere separated, and the aqueous layer was extractedwith dichloromethane (2 × 300 mL). The combineddichloromethane layers were evaporated in vacuo toyield the title compound as an off-white solid (81.1 g,73%). 1H NMR (d6-DMSO): 7.55 (br s, 1H), 3.89 (t,J = 4, 1H), 3.39–3.30 (m, 1H), 1.81–1.65 (m, 3H),1.65–1.48 (m, 1H), 1.41–1.21 (m, 2H).

Preparation of (±)-7-aza-bicyclo[4.2.0]octan-8-one,(±)-4To a solution of chlorosulfonyl isocyanate (66.4 g,0.47 mol) in dichloromethane (100 mL) at 0–5 ◦C was

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dosed a solution of cyclohexene (50 mL, 0.49 mol)in dichloromethane (25 mL). The temperature wasmaintained below 5 ◦C throughout the addition, whichtook approximately 45 min. The mixture was thenstirred at 0–5 ◦C for 1 h, then at 40 ◦C for a further20 h. The mixture was re-cooled to 0–5 ◦C and coldwater (∼30 mL) added until effervescence ceased.This reaction mixture was dosed into a pre-cooledsuspension of sodium sulfite (30.8 g, 0.25 mol) inwater (250 mL) maintaining temperature below 5 ◦Cand pH in the range 5–7 by concurrent addition of5 mol L−1 sodium hydroxide solution. The mixturewas stirred at 0–5 ◦C for 1 h, then ethyl acetate(250 mL) was added and phases were separated.The combined organics were washed with saturatedbrine solution (250 mL) and dried over magnesiumsulfate. Solvent was removed under reduced pressureto yield yellow oil (36.02 g, 61%). This materialwas then crystallised by mixing with 20 mL of 1:1methyl tert-butyl ether: heptane on ice to yield thetitle compound as a yellow crystalline solid (25.29 g,43%). 1H NMR (CDCl3): 6.06 (1H, br s), 3.87–3.81(1H, m), 3.27–3.18 (1H, m), 1.98–1.82 (1H, m),1.81–1.57 (5H, m), 1.56–1.39 (1H, m).

Preparation of (±)-7-aza-bicyclo[4.2.0]oct-3-en-8-one,(±)-5To a solution of 1,4-cyclohexadiene (10 mL,105 mmol) in dichloromethane (30 mL) was slowlyadded a solution of chlorosulfonyl isocyanate (7.4 mL,85 mmol) in dichloromethane (50 mL). The reactionmixture was then stirred at 45 ◦C for 48 h before beingallowed to cool to room temperature and added drop-wise to a stirred solution of 25 wt% sodium sulfitesolution. After stirring for a further 15 min, the mix-ture was extracted with ethyl acetate (2 × 50 mL). Thecombined organics were dried over magnesium sulfateand concentrated under reduced pressure to yield thetitle compound as a yellow solid (2.51 g, 20%). 1HNMR (CDCl3): 5.99 (1H, br s), 5.92–5.82 (1H, m),5.80–5.66 (1H, m), 4.04–3.94 (1H, m), 3.42–3.32(1H, m), 2.51–2.40 (1H, m), 2.40–2.25 (1H, m),2.24–2.04 (1H, m).

Fermentation of Rhodoccocus globerulus (NCIMB41042)A thawed glycerol stock of NCIMB 41 042 (1 mL)was used to inoculate a single seed flask (250 mLTryptone Soya Broth (Oxoid CM129) per 2 LErlenmeyer flask). This culture was incubated at25 ◦C, 250 rpm in a temperature-controlled shaker(New Brunswick G-25 with 25 mm (1 inch) throw)for 22 h. The fermentation was carried out in aB Braun Biotech Biostat (Sartorius, Epsom, UK)C-DCU fermenter containing 20 L fermentationmedium. The fermentation medium contained, perlitre: 20 g yeast extract (Oxoid L21), 8 g KH2PO4,7 g K2HPO4, 1 g MgSO4.7H2O, 1 g (NH4)2SO4,1.0 mL trace elements solution, 1.0 mL polypropyleneglycol and 10 g glucose. The trace elements solution

contained, per litre: 250 mL concentrated HCl, 3.6 gCaCl2.2H2O, 2.0 g ZnO, 0.85 g CuCl2.2H2O, 2.0 gMnCl2.4H2O, 5.4 g FeCl3.6H2O, 2.4 g CoCl2.2H2O,4.8 g Na2MoO4.2H2O and 0.3 g H3BO3. The seedflask (inoculum) was added to the fermenter andthe culture was grown at 25 ◦C, 200 rpm and pHcontrolled between 6.9 and 7.1 with air flow at10 mL min−1 for 25 h. The fermenter was harvestedby continuous flow centrifugation (Beckman J-25 centrifuge; Beckman, High Wycombe, UK) at15 000 × g, 4 ◦C with a flow rate of 205 mL min−1

yielding 308.4 g net wet weight of cells, which werestored at −20 ◦C.

Procedures for small-scale bioresolutionGram-scale resolution of7-aza-bicyclo[4.2.0]oct-4-en-8-one, (±)-1To a solution of (±)-1 (5 g) in 50 mmol L−1 phosphatebuffer pH 7.0 (160 mL) was added Rhodococcusgloberulus cell paste (5 g). The resultant mixture wasstirred at 37 ◦C for 24 h, after which time the reactionmixture was filtered through celite and extracted with3 × 100 mL ethyl acetate. The combined organicswere dried over MgSO4 and solvent was removedunder reduced pressure to yield 1a (2.03 g, 40%; 98%e.e. (chiral GC)), an off-white solid. [α]D

25 = −105.7(c 0.01, MeOH); 1H NMR (CDCl3): 6.19–6.10 (1H,m), 6.07 (1H, br s), 5.98–5.90 (1H, m), 4.03 (1H,t, J = 5), 3.55–3.48 (1H, m), 2.16–2.02 (3H, m),1.68–1.55 (1H, m).

The aqueous phase was concentrated to drynessand the resulting residue was slurried in hot methanol(40 mL). Insoluble material was removed by filtrationand the filtrate was concentrated to dryness andrecrystallised from 10 mL water and 10 mL methanolto yield 1b (2.5 g, 44%; 99.3% e.e. (chiral HPLC)), anoff-white solid. [α]D

25 = −97.9 (c 1, H2O); 1H NMR(D2O); 6.16–6.04 (1H, m), 5.77–5.66 (1H, m), 3.96(1H, br s), 2.77–2.65 (1H, m), 2.24–2.06 (2H, m),2.02–1.90 (1H, m), 1.90–1.74 (1H, m).

Gram-scale resolution of3-aza-tricyclo[4.2.1.02,5]non-7-en-4-one, (±)-2To a suspension of Rhodococcus globerulus cell paste(11.3 g) in 50 mmol L−1 phosphate buffer pH 7.0(300 mL) was added (±)-2 (10.6 g). The resultantmixture was stirred at 37 ◦C for 18 h, after whichtime the reaction mixture was filtered through celiteand extracted with ethyl acetate (3 × 150 mL). Thecombined organic extracts were washed with saturatedbrine solution (50 mL) and dried over MgSO4.Removal of solvent under reduced pressure yielded2a (5.30 g, 50%; 99% e.e. (chiral GC)), a white solid.[α]D

25 = −124 (c 1.223, CDCl3); 1H NMR (CDCl3):6.30–6.19 (m, 1H), 6.18–6.00 (m, 2H), 3.53–3.44(m, 1H), 3.10–2.98 (m, 1H), 2.96–2.91 (br s, 1H),2.91–2.82 (br s, 1H), 1.81 (d, J = 10, 1H), 1.65 (d,J = 10, 1H).

The aqueous phase was concentrated to a volume ofapproximately 40 mL and amino acid was precipitated

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by addition of 400 mL acetone, recovered by filtrationand dried in vacuo to yield 8.17 g of an off-white solid. Material was slurried in hot methanol(75 mL), allowed to cool and filtered. The filtratewas concentrated under reduced pressure to yield 2b(4.18 g, 35%; 99.4% e.e. (chiral HPLC)), an off-whitesolid. [α]D

25 = +13.8 (c 0.989, H2O); 1H NMR (d4-MeOH): 6.37 (dd, J = 3 and 5.4, 1H), 6.15 (dd,J = 3 and 5.4, 1H), 3.16–3.07 (m, 2H), 2.95 (br s,1H), 2.38 (dd, J = 1.6 and 7.6, 1H), 1.95 (d, J = 10,1H), 1.55 (dd, J = 9.2 and 1.6, 1H).

Gram-scale resolution of6-aza-bicyclo[3.2.0]heptan-7-one, (±)-3To a suspension of Rhodococcus globerulus cell paste(13.3 g) in 50 mmol L−1 phosphate buffer pH 7.0(430 mL) was added (±)-3 (13 g). The resultantmixture was stirred at 37 ◦C for 7 h, after whichtime the mixture was filtered through celite and thefiltrate was extracted with 3 × 300 mL ethyl acetate.The organic extracts were combined, washed withsaturated brine solution (100 mL) and dried overMgSO4. Solvent was removed under reduced pressureto yield 3a (38 g, 34%; 99.3% e.e. (chiral GC)),an off-white solid. [α]D

25 = −27.5 (c 0.01, MeOH);1H NMR (CDCl3): 5.95 (1H, br s), 4.05 (1H, t,J = 4 Hz), 3.50 (1H, dt, J = 8 and 2.6 Hz), 2.09–1.94(1H, m), 1.91–1.69 (3H, m), 1.53–1.28 (2H, m).

The aqueous phase was concentrated to dryness,slurried in hot methanol, filtered and evaporated todryness. The residue was then recrystallised fromwater/methanol and dried in vacuo to yield 3b(4.215 g, 28%; 99% e.e. (chiral HPLC)), an off-whitesolid. [α]D

25 = +10.8 (c 1, H2O); 1H NMR (D2O):3.62–3.52 (1H, m), 2.77–2.65 (1H, m), 2.02–1.85(2H, m), 1.76–1.48 (4H, m).

Gram-scale resolution of7-aza-bicyclo[4.2.0]octan-8-one, (±)-4To a suspension of Rhodococcus globerulus cell paste(13.3 g) in 50 mmol L−1 phosphate buffer pH 7.0(290 mL) was added (±)-4 (8.8 g). The resultantmixture was stirred at 37 ◦C for 5 h, after whichtime the mixture was filtered through celite and thefiltrate was extracted with 3 × 300 mL ethyl acetate.The organic extracts were combined, washed withsaturated brine solution (50 mL), dried over MgSO4.Removal of solvent under reduced pressure yielded 4a(3.30 g, 38%; 99% e.e. (chiral GC)), an-off white solid.[α]D

25 = +2.1 (c 0.52, CHCl3); 1H NMR (CDCl3):6.06 (1H, br s), 3.87–3.81 (1H, m), 3.27–3.18(1H, m), 1.98–1.82 (1H, m), 1.81–1.57 (5H, m),1.56–1.39 (2H, m).

The aqueous phase was concentrated to dryness,slurried in hot methanol, filtered and evaporated todryness. The residue was then recrystallised fromwater/methanol and dried in vacuo yielding 4b (3.13 g,31%; 99% e.e. (chiral HPLC)), a white solid. [α]D

25 =+20.1 (c 1, H2O); 1H NMR (D4-MeOH): 3.49–3.38

1. CSI, CH2Cl2

2. Na2SO3

1. CSI, CH2Cl2

2. Na2SO3

NH

O

NH(±)-2

O

(±)-1

n

1. CSI, CH2Cl2

2. Na2SO3n

NH

O

n=1 (±)-3n=2 (±)-4

NH

O

1. CSI, CH2Cl2

2. Na2SO3

(±)-5

Scheme 1. Synthesis of racemic β-lactams.

(1H, m), 3.02–2.91 (1H, m), 2.23–2.07 (1H, m),1.97–1.67 (4H, m), 1.65–1.35 (3H, m).

Scaled-up synthesis of(1S, 2R)-(+)-2-aminocyclopentanecarboxylic acid, 3bTo a suspension of Rhodococcus globerulus cell paste(16 g) in 50 mmol L−1 potassium phosphate buffer,pH 7.0 (2.3 L) in a stirred vessel at 37 ◦C was added(±)-3 (80 g, 0.72 mol). The mixture was stirred at37 ◦C for 5 days, after which time the assay indicatedconversion had reached 50%. The mixture was filteredthrough kieselguhr (40 g) to remove cell debris andconcentrated in vacuo to ∼180 g weight. The aqueousmixture was then warmed to ∼50 ◦C, stirred vigorouslyand 1.6 L acetone was added to precipitate the productas a solid contaminated with inorganic salts. The solidwas recovered by filtration and washed on the filterwith further acetone (3 × 100 mL). The solid was thenstirred in refluxing methanol (650 mL) for 90 min,filtered hot and washed on the filter with methanol(2 × 20 mL). The filtrate was concentrated in vacuoto ∼130 g weight and the resultant slurry reheated to60 ◦C and then cooled slowly to 5 ◦C. After cooling, theproduct was collected by filtration and dried to yield3b (24 g, 26%; 99% e.e. (chiral HPLC)) as a whitesolid. [α]D

25 = +10.8 (c 1, H2O); 1H NMR (D2O):3.62–3.52 (m; 1H), 2.77–2.65 (m; 1H), 2.02–1.85(m; 2H), 1.76–1.48 (m; 4H).

RESULTS AND DISCUSSIONScreeningIn order to screen for a suitable microorganism,the β-lactams 7-aza-bicyclo[4.2.0]oct-4-en-8-one (1)and 3-aza-tricyclo[4.2.1.02,5]non-7-en-4-one (2) wereprepared by cycloaddition of chlorosulfonyl iso-cyanate (CSI) and either 1,3-cyclohexadiene or 2,5-norbornadiene23 (Scheme 1). As reported in theliterature,16,23 the cycloadditions were regioselective,producing single-regioisomer forms of 1 and 2.

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Synthesis of β-lactams and β-amino acids using Rhodococcus globerulus

Over 400 microbial strains taken from our in-house culture collection were screened for hydrolysisactivity against these substrates. Glycerol stocks ofeach microbial strain were used to inoculate 1.0 mLTryptone Soya Broth per well in 2.2 mL 96-well plates.These were then shaken at 25 ◦C on a HeidolphTitramax 1000 incubator at maximum rpm for 45 h.The cells were then harvested by centrifugation at1000 × g, 4 ◦C, for 10 min and the cell pastes werestored at −20 ◦C.

For screening purposes, cell pastes of the 96-wellculture plates were resuspended in 0.5 mL of 20 gL−1 substrate in 0.1 mol L−1 KH2PO4 pH 7 andthen shaken at 25 ◦C on a Heidolph (Schwabach,Germany) Titramax 1000 incubator at maximum rpmfor 40–66 h. Reactions were halted by diluting 1 in 10in a 1:1 mix of methanol:10 mmol L−1 KH2PO4 pH7. The resulting mixtures were then assayed by HPLCusing a 150 mm × 4.6 mm 5 m Hichrom (Theale, UK)KR100 C8 column with a running buffer of a 1:1 mixof methanol:10 mmol L−1 phosphate buffer pH 7.0 ata flow rate of 1.0 mL min−1 with detection at 210 nm.

The screen yielded two hits: Pseudomonas fluorescens(CMC 3060) and Rhodococcus globerulus (NCIMB41042). Results showed that the strain of R. globerulushad reached 43% conversion on lactam 1 and 45%conversion on lactam 2. Further analysis using chiralGC revealed that the lactam substrates had beenhydrolysed enantioselectively. However, analysis of thePseudomonas fluorescens hit indicated that non-selectivehydrolysis of the lactam substrates had taken place. Asa result, work focused on developing a whole-cellbioresolution using R. globerulus.

Scale-upIn order to demonstrate these biotransformations on apreparative scale a larger quantity of biocatalyst (wholecells) was required. The strain was grown up at 20 Lscale in a B Braun Biotech Biostat C-DCU fermenterusing a generic 24 h batch fermentation. The fermenterwas harvested by continuous flow centrifugation usinga Beckman J-25 centrifuge and JCF rotor to produce308 g net wet weight of cells.

Whole-cell bioresolutions of lactams 1 and 2were then undertaken on a 5–10 g scale using oneweight equivalent of wet cell paste (Scheme 2). Thesetransformations were initially carried out in 50 mmolL−1 phosphate buffer pH 7 at 25 ◦C and at asubstrate concentration of 30 g L−1, but subsequentdevelopment work revealed that reaction rates weremore rapid when carried out at 37 ◦C. Cell paste wasremoved by filtration through celite and the filtrate wasextracted with ethyl acetate and concentrated underreduced pressure, yielding 1a of 98% e.e. and 2a of99% e.e. The corresponding amino acids were isolatedfrom the aqueous phase through a combinationof partial concentration, acetone precipitation andrecrystallisation from water/methanol, yielding 1b of99.3% e.e. and 2b of 99.4% e.e. In both casesenantiomeric ratios (E values) well in excess of 1000were determined, which infers the bioresolutions werehighly selective. In addition, isolated yields of theresolved β-lactams and β-amino acids were high, asshown in Table 1.

NH

O

NH

O

NH

O

NH

O HOOC

H2N

R.globerulus+

R.globerulus

pH7, 37 °C

pH7, 37 °C

(±)-1 (1R,6S)-1a (1S,2R)-1b

(±)-2 (1S,2S,3R,4R)-2b(1S,2S,5R,6R)-2a

+

NH2

COOH

NH

O

NH

O

NH

O

NH

O

NH

O

NH2

R.globerulus+

R.globerulus+

R.globerulus

pH7, 37 °C

pH7, 37 °C

pH7, 37 °C

(±)-3 (1R,5S)-3a (1S,2R)-3b

(±)-4 (1S,2R)-4b(1R,6S)-4a

(±)-5

COOH

NH2

COOH

NH

O

NH2+

COOH

(1R,6S)-5a (1S,6R)-5b

Scheme 2. Bioresolution of β-lactams 1–5.

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Table 1. Bioresolution results for substrates 1–5

Reaction time (h) β-Lactam e.e. β-Amino acid e.e. Isolated yield of lactam Isolated yield of amino acid E value

1 24 98% 99.3% 40% 44% >10002 18 99% 99.4% 50% 35% >10003 7 99.3% 99% 34% 28% >10004 5 99% 99% 38% 31% >10005 90 59% – – – –

Substrate specificityHaving successfully demonstrated the preparativescale bioresolutions of 1 and 2 using R. globerulus,we sought to further explore the substrate specificityof the enzyme. With this in mind, the β-lactams3–5 were prepared in an analogous manner to 1 viathe cycloaddition of chlorosulfonyl isocyanate to thecorresponding cycloalkenes and cycloalkadienes.

Bioresolutions involving the β-lactams 3–5 werecarried out on a multigram scale under conditionsdescribed previously. Table 1 summarises the biores-olution results for all substrates and includes isolatedyields for both amino acid and lactam.

The β-lactams 3 and 4 were successfully resolvedusing R. globerulus and in both cases enantiomericexcesses of the residual β-lactam and β-amino acidproduct were found to be greater than 99%. Evanset al. have previously reported that bioresolution of3 using Rhodococcus equi proved unsuccessful.18 Itis interesting to note that under similar reactionconditions the saturated lactams 3 and 4 were resolvedsignificantly quicker than the unsaturated lactams 1and 2.

Despite several attempts at resolving 5, it was foundthat this molecule is not a suitable substrate for theenzyme. Indeed, we subsequently found that by usinga threefold increase in cell loading and reaction timein excess of 90 h a residual lactam e.e. of only 59%was obtained.

Based on the e.e. data obtained from the biores-olutions of β-lactams 1–4, it was predicted that ineach case conversions of approximately 50% shouldhave been reached. However, the results reported inTable 1 show that isolated yields of the bicyclic lac-tams (1a, 3a and 4a) are lower than the tricyclic lactam(2a). The most likely explanation for these lower thanexpected yields is the entrapment of material withinthe cell paste, resulting in loss of product during thecelite filtration step. The β-amino acids 1b–4b wereisolated through a combination of partial concentra-tion of the aqueous phase, acetone precipitation andrecrystallisation from water/methanol. This protractedisolation protocol was required to ensure that aminoacids were isolated essentially free of inorganic salts,but also resulted in lower than expected isolated yields.Further development of the isolation procedure shouldhelp to improve isolated yields.

The substrates 1, 4 and 5 are sterically very similar,yet they exhibit widely differing reactivities. Thissuggests that substrate preference is dictated moreby electrostatic factors rather than sterics.

Optical rotations were measured for compounds1a–4a and the data are presented in Table 2. Bycomparison with literature reported values15,16 it waspossible to assign absolute configurations for the β-lactams 1a–4a and the corresponding amino acids1b–4b. Interestingly, the stereoselectivity observed forR. globerulus-catalysed bioresolutions is the oppositeof that reported by Forro and Fulop for the lipase-catalysed hydrolysis of such substrates in diisopropylether.15,16

Bioresolution developmentCommercial interest in the cyclic amino acid 3b ledto increased interest in the bioresolution of (±)-3 andthe potential for operating the process on larger scale.With this in mind, a series of optimisation studieswere undertaken in order to improve the robustnessand scalability of the process.

Optimisation of substrate concentrationInitial bioresolutions were carried out at substrateconcentrations of 33 g L−1; however, for the processto work efficiently on scale-up we would need toimprove the volume efficiency of the bioresolution.Consequently, a series of experiments were carried outto assess the extent to which substrate concentrationcould be improved.

The results in Table 3 show that the substrateconcentration can be increased to 60 g L−1 withouthaving a major detrimental effect on either reactiontime or e.e. of the residual lactam. However, furtherattempts to improve the substrate loading failed toreach full conversion despite prolonged reaction times.

Optimisation of substrate:cell paste ratioAn improvement in the cell paste loading was alsonecessary for the development of a scalable process.A series of bioresolutions were carried out at variouscell paste loadings while maintaining the substrateconcentration at 33 g L−1.

The results in Table 4 demonstrate that the cellloading can be successfully reduced from 100 wt%

Table 2. Optical rotations for 1a–4a

β-Lactam e.e. [α]D25

(1R, 6S)-1a 98% −105.7(c 0.01, MeOH)(1S, 2S, 5R, 6S)-2a 99% −91.04(c 0.01, MeOH)(1R, 5S)-3a 99.3% −27.5(c 0.01, MeOH)(1R, 6S)-4a 99% +2.1 (c 0.52, CHCl3)

1104 J Chem Technol Biotechnol 82:1099–1106 (2007)DOI: 10.1002/jctb

Synthesis of β-lactams and β-amino acids using Rhodococcus globerulus

Table 3. Optimisation of substrate concentration

Loading(lactam:cell paste)

[Lactam](g L−1)

Reaction time(h)

Lactame.e.

1:1 33 13 >99%1:1 50 20.5 >99%1:1 53.3 24.5 >99%1:1 60 29 >99%1:1 66.6 49 84.3%

to 20 wt%, although extended reaction times areneeded. It is also worth reporting that the reduction incell loading aids product isolation. Such a reductionmeans that sufficient cell paste to resolve 120 kg 6-aza-bicyclo[3.2.0]heptan-7-one could be produced from asingle 1600 L fermentation. The downside is thatat this loading the bioresolution takes approximately4 days compared with a single day at the higher loadinglevel.

Recycling of cell pasteThe operation of a whole-cell biotransformationprocess makes recycling of the biocatalyst an awkwardproposition. The ability to efficiently recycle cellpaste would reduce biocatalyst requirements. Thebioresolution of (±)-3 was carried out with recycles ofR. globerulus cell paste. Reactions were performed witha lactam concentration of ∼33 g L−1 with 5 g cell pasteand 5 g (±)-3. The cell paste from each bioresolutionwas collected by centrifugation, resuspended in freshphosphate buffer and used to resolve further 5 gbatches of the β-lactam substrate.

The results in Table 5 demonstrate that three cyclesof cell paste are possible for the bioresolution of3. Both cycles 1 and 2 were complete within 22 h.However, with bioresolution cycle 3, lactam with∼95% e.e. was only achieved after ∼8 days (168 h).Closer examination of the data collected from cycle3 shows that residual lactam e.e. reaches 80% within3 days and then tails off. It is clear from these resultsthat we could recycle the cell paste (i.e., 2 cycles ofuse). However, collecting the cells by centrifugationis likely to be a processing problem on a large scale.Therefore, we considered removing and recycling thecells using filtration with a celite pad.

Collection of cell paste with a pad of celiteFurther bioresolutions of (±)-3 were performed withrecycles of cell paste (NCIMB 41042). Reactions wereperformed at a lactam concentration of ∼33 g L−1 with

Table 4. Optimisation of cell paste loading

Cell paste loading(wt%)

[Lactam](g L−1)

Reaction time(h)

Lactame.e.

100 33 13 >99%80 33 24.5 >99%41 33 45 >99%20 33 93 96%

Table 5. Recycling of cell paste

CycleLoading

(lactam:cell paste)[Lactam](g L−1)

Reaction time(h)

Lactame.e.

1 1:1 33 22 >99%2 1:1 33 22 >99%3 1:1 33 168 95%

Table 6. Recycling of cell paste on celite

CycleLoading

(lactam:cell paste)[Lactam](g L−1)

Reaction time(h)

Lactame.e.

1 1:1 33 17.5 >99%2 1:1 33 22 >99%3 1:1 33 41.2 23%

5 g cell paste and 5 g lactam. The cell paste (∼5 g)from the bioresolution was collected by filtrationusing a celite pad (5 g). The cells–celite mixture wasresuspended in fresh buffer and used to resolve further5 g batches of (±)3.

The results in Table 6 again demonstrate that atleast two cycles of cell paste can be used in thebioresolution of (±)-3. Both cycles 1 and 2 werecomplete within 22 h. Use of the celite filtrationadditive with cycle 2 did not preclude its use in thebioresolution. However, significant loss in activity isobserved with cycle 3, resulting in a residual lactame.e. of only 23% after 41 h.

CONCLUSIONSScreening of our in-house culture collection fora microorganism capable of selectively hydrolysingβ-lactams led to the identification of a strain ofRhodococcus globerulus (NCIMB 41042). The micro-organism was capable of resolving a series of β-lactamsubstrates, generating good yields of enantiomericallypure β-lactams and β-amino acids. We have alsosuccessfully demonstrated the use of recycled cellpaste in the bioresolution of three batches of (±)-3. Subsequent optimisation has led to the successfuldevelopment of a process that can be operated at 60 gL−1 substrate concentration with a 20% wt/wt cellloading, yielding multigram quantities of enantiopureβ-lactams and β-amino acid products that are availablefor further evaluation.

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