Biocatalytic Racemization of Sec-Alcohols and -Hydroxyketones Using Lyophilized Microbial

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Biocatalytic Racemization of sec-Alcohols and-Hydroxyketones
Using Lyophilized Microbial Cells.

Electronic Supporting Information.

Bettina M. Nestla,b, Constance V. Vossa, Anne Bodlennera,b, Ursula Ellmer-Schaumbergera,b, Wolfgang Kroutila, Kurt Fabera[*]

a Department of Chemistry, Organic and Bioorganic Chemistry, University of Graz, Heinrichstrasse 28, A-8010 Graz, Austria.

b Research Centre Applied Biocatalysis, Petersgasse 14, Graz, Austria.

Media composition

The following components of the standard-complex medium were sterilized in four separate groups: Group I: Yeast extract (10 g L-1, Oxoid L21), bacteriological peptone (10 g L-1, Oxoid L37). Group II: glucose (10 g L-1, Fluka 49150). Group III: NaCl (2 g L-1, Roth 9265.1), MgSO4*7H2O (0.15 g L-1, Fluka 63140). Group IV: NaH2PO4 (1.3 g L-1, Fluka 71496), KH2PO4 (4.4 g L-1, Merck 5101).

In case of the SNLH-medium for fungi, the following components were sterilized: Glucose (30 g L-1), asparagine monohydrate (4.5 g L-1, Fluka 11160), KH2PO4 (1.5 g L-1, Fluka 60220), MgSO4*7H2O (1.03 g L-1, Fluka 63140), trace element solution SL4 (1 mL L-1), yeast extract (3 g L-1, Oxoid L21). The medium was adjusted to pH 6 with NaOH solution. The trace element solution contained the following components: FeCl3*6H2O (80 mg L-1, Fluka 44944), ZnSO4*7H2O (90 mg L-1, Aldrich 22,137-6), MnSO4*H2O (30 mg L-1, Fluka 63554), CuSO4*5H2O (5 mg L-1, Aldrich 46,193-0), EDTA or Triplex III (0.4 g L-1, Aldrich 43178,8).

For medium DSMZ #186 the following components were sterilized: Yeast extract (3 g L-1, Oxoid L21), malt extract (3 g L-1, Oxoid L39), peptone (5 g L-1, Oxoid L37), glucose (10 g L-1, Fluka 49150).

For the tomato juice medium, distilled water was added to tomato juice (200 mL L-1) and CaCO3 (3 g L-1, Merck 102066) to give a final volume of 1L, adjusted to pH 7.2 and sterilized.

For the PEP medium the following components were sterilized: Glucose (10 g L-1, Fluka 49150), malt extract (5 g L-1, Oxoid L39), KH2PO4 (2 g L-1, Fluka 60220), yeast extract (2 g L-1, Oxoid L21), peptone (2 g L-1, Oxoid L37).

For the soja glucose medium the following components were sterilized: Glucose (20 g L-1, Fluka 49150), KH2PO4 (5 g L-1, Fluka 60220), sodium chloride (5 g L-1, Roth 9265.1), soja peptone (5 g L-1, Oxoid L44), yeast extract (5 g L-1, Oxoid L21).

For medium DSMZ #90 the following components were sterilized: Malt extract (30 g L-1, Oxoid L39), soja peptone (3 g L-1, Oxoid L44).

For the Luria broth medium the following components were sterilized: Luria broth (15.5 g L-1, Sigma L-3522), KH2PO4 (1.4 g L-1, Fluka 60220), K2HPO4 (4.4 g L-1, Merck 5101).

For medium DSMZ #393 the following components were sterilized: Yeast extract (10 g L-1, Oxoid L21), peptone (20 g L-1, Oxoid L37), glucose (20 g L-1, Fluka 49150). The pH was adjusted to 6.5.

The following components of the medium DSMZ #11 were sterilized in five separated groups: Group I: Pepticase (10 g/L, Sigma), bacteriological peptone (10 g/L, Oxoid), yeast extract (5 g/L, Oxoid). Group II: Glucose (20 g/L, Fluka). Group III: Tween 80 (polyoxyethylene-sorbitan-monooleate, 1 g/L, Aldrich). Group IV: K2HPO4 (2 g/L, Merck). Group V: Na-acetate*3H2O (8.3 g/L, Fluka), (NH4)2-citrate (2 g/L, Fluka), MgSO4*7H2O (0.20 g/L, Fluka), MnSO4 (0.05 g/L, Fluka).

Media for Active Strains

Rhodococcus ruber DSM 44541, Rhodococcus erythropolis FCC 173, Rhodococcus ruber DSM 44540, Rhodococcus ruber FCC 169, Rhodococcus ruber DSM 44539, Rhodococcus sp. NCIMB 11216, Rhodococcus sp. R312 CBS 717.73, Rhodococcus erythropolis DSM 312, Rhodococcus equi IFO 3730, Rhodococcus ruber DSM 43338, Agrobacterium tumefaciens FCC 148, Streptomyces caeruleus DSM 40008, Alcaligenes faecalis DSM 13975, Bacillus megaterium DSM 32, Nocardia G FCC 146 and Nocardia H FCC 147 were grown on standard-complex medium. Rhizopus oryzae CBS 906, Beauveria bassiana DSM 1344, Penicillium simplicissimum FCC 072 and Botrytis cinerea DSM 877 were grown on SNLH-medium for fungi. Candida parapsilosis DSM 70125 was grown on medium DSMZ #186, Helminthosporium sp. NRRL 4671 on tomato-juice medium, Syncephalastrum racemosum ATCC 18192 on PEP-medium, Aspergillus niger DSM 821 on soja-glucose medium, Geotrichum candidum DSM 6401 on medium DSMZ #90, Pseudomonas putida ATCC 47054 on Luria broth medium and Kluyveromyces lactis DSM 3795 on medium DSMZ #393. Lactobacillus paracasei DSM 20207 was grown on medium #11 as suggested by DSMZ.

Strain Maintenance

Bacteria, fungi and yeast were maintained in frozen stock solutions and long-term storage of lyophilized cells was at +4°C.

Preparation of Lyophilized Cells

Strains were grown in flask cultures with shaking at 30°C (Rhodococcus ruber DSM 44541, Rhodococcus erythropolis FCC 173, Rhodococcus ruber DSM 44540, Rhodococcus ruber FCC 169, Rhodococcus ruber DSM 44539, Rhodococcus sp. NCIMB 11216, Rhodococcus sp. R312 CBS 717.73, Rhodococcus erythropolis DSM 312, Rhodococcus equi IFO 3730, Rhodococcus ruber DSM 43338, Agrobacterium tumefaciens FCC 148, Bacillus megaterium DSM 32, Kluyveromyces lactis DSM 3795, Aspergillus niger DSM 821, Nocardia G FCC 146 and Nocardia H FCC 147, Lactobacillus paracasei DSM 20207), at 24°C (Beauveria bassiana DSM 1344), at 25°C (Candida parapsilosis DSM 70125, Helminthosporium sp. NRRL 4671, Rhizopus oryzae CBS 906, Syncephalastrum racemosum ATCC 18192, Geotrichum candidum DSM 6401, Botrytis cinerea DSM 877), at 26°C (Penicillium simplicissimum FCC 072), at 28°C (Streptomyces caeruleus DSM 40088, Pseudomonas putida ATCC 47054) and at 37°C (Alcaligenes faecalis DSM 13975). After transfer from agar plates, the microorganisms were grown for 3d (Rhodococcus ruber DSM 44541, Rhodococcus erythropolis FCC 173, Rhodococcus ruber DSM 44540, Rhodococcus ruber FCC 169, Rhodococcus ruber DSM 44539, Rhodococcus sp. NCIMB 11216, Rhodococcus sp. R312 CBS 717.73, Rhodococcus erythropolis DSM 312, Rhodococcus equi IFO 3730, Rhodococcus ruber DSM 43338, Agrobacterium tumefaciens FCC 148, Streptomyces caeruleus DSM 40088, Alcaligenes faecalis DSM 13975, Bacillus megaterium DSM 32, Nocardia G FCC 146 and Nocardia H FCC 147, Penicillium simplicissimum FCC 072, Candida parapsilosis DSM 70125, Pseudomonas putida ATCC 47054, Kluyveromyces lactis DSM 3795, Lactobacillus paracasei DSM 20207) and 5d (Rhizopus oryzae CBS 906, Beauveria bassiana DSM 1344, Botrytis cinerea DSM 877, Helminthosporium sp. NRRL 4671, Syncephalastrum racemosum ATCC 18192, Aspergillus niger DSM 821, Geotrichum candidum DSM 6401), respectively. Then the cells were harvested by centrifugation (18000 x g), washed twice with buffer, lyophilized, and stored at +4°C. Cells were washed using the following buffers: bacteria (sodium/potassium phosphate buffer 50 mM, pH 7.5), fungi (TRIS buffer 10 mM, pH 7.5) and Lactobacillus sp. (BIS-TRIS buffer, 50 mM, 10-2 M MgCl2, pH 6).

Cell Disruption

Cell disruption was carried out using a digital ultrasonifier (Branson, 250 W). For cell breakage a portion of wet cells of Lactobacillus paracasei DSM 20207, which was obtained from 0.33 L of culture (about 2.5 g wet cell paste) was suspended in 6 mL BIS-TRIS buffer (50 mM, 10-2 M MgCl2, pH6). The cells were broken by treatment with energy for 20 min (1 sec pulse followed by 2 sec rest period for cooling) with an amplitude of 30% (corresponding to 60 W). During the cell disruption the suspension was externally cooled with ice. Afterwards the crude cell lyzate was centrifuged at +4°C and 18.000 rpm (38.000xg) for 30 min for the removal of cell debris.

Experimental and Analytic Procedures

General

NMR spectra were recorded in CDCl3 using a Bruker AMX 360 at 360 (1H) and 90 (13C) MHz. Chemical shifts are reported relative to TMS ( 0.00) and coupling constants (J) are given in Hz. TLC plates were run on silica gel Merck 60 (F254) and compounds were visualized by spraying with Mo-reagent [(NH4)6Mo7O24*4H2O (100 g/L), Ce(SO4)2*4H2O (4 g/L) in H2SO4 (10%)]. The degree of conversion (expressed as % of racemization, 100% corresponding to the racemate) and enantiomeric excess were determined via GC or HPLC on a chiral stationary phase. GC analyses were carried out on a Varian 3900 gas chromatograph equipped with a FID detector using a Chrompack Chirasil-DEX CB column (Varian, 25 m x 0.32 mm x 0.25 m, 1.0 bar H2).HPLC analyses were carried out on a Shimadzu HPLC system equipped with DGU-20A5 degasser, LC 20AD liquid chromatograph, SIL 20AC autosampler, CBM 20A communications bus module, SPD M20A diode array detector and CTO 20AC column oven using a Chiralpak AD column (0.46 x 25 cm, DAICEL) and Chiralpak OD-H column (0.46 x 25 cm, DAICEL). Melting points were obtained on a Gallenkamp melting point apparatus MFB-595 in open capillary tubes. Optical rotation values ([]D20) were measured on a Perkin-Elmer polarimeter 341 at 589 nm (Na-line) in a 1 dm cuvette and are given in units of 10 deg cm2 g-1.

(R)-1-Hydroxy-1-phenyl-2-propanone (5)was kindly provided by BASF AG (Ludwigshafen). The sample was purified by flash chromatography (petroleum ether/ethyl acetate 9:1) to give (R)-5 as white crystals. []D20 –103.4 (c 0.2, EtOH); (S)-5 []D20 +164.7 (c 2.2, EtOH) (Taka et al. 2002); mp 95°C, 1H-NMR (360 MHz, CDCl3)  = 2.08 (1H, s, OH), 2.53 (3H, s, CH3), 5.10 (1H. s, CH-OH), 7.32-7.53 (3H, m, aryl), 7.93 (2H, d, J = 7.7, aryl); 13C-NMR (90 MHz, CDCl3)  = 22.3, 69.3, 128.7, 128.9, 129.0, 134.0, 202.4.

Synthesis of (R)-2-hydroxyindan-1-one (R)-6

rac-2-Acetoxy-indan-1-one was obtained from 1-indanone using the following procedure adapted from literature (Demir et al. 2004). A solution of 1-indanone (1.3g, 10 mmol) and Mn(OAc)3*2H2O (3.3 g, 12.5 mmol) in benzene-AcOH (10:1, 110 mL) was stirred under reflux using a Dean-Stark apparatus during which the dark brown color of Mn(OAc)3*2H2O gradually disappeared. After the starting material was consumed, the reaction mixture was diluted with diethyl ether and washed with brine. The resulting organic phase was directly filtered through a pad of silica, dried over sodium sulfate and concentrated under vacuum. The residue was purified by flash chromatography (petroleum ether/ethyl acetate 9:1) to give rac-2-acetoxyindan-1-one as yellow oil (1.3 g, 93%). 1H-NMR (360 MHz, CDCl3)  = 1.11 (3H, t, J = 4.9, CH2-CH3), 2.74 (2H, t, J = 6.0, CH2), 3.16 (2H. q, J = 5.6, CH2-CH3), 5.43 (1H, t, J = 4.9, CH-OH), 7.37-7.49 (2H, m, aryl), 7.59 (1H, t, J = 7.3, aryl), 7.77 (1H, d, J = 7.7, aryl); 13C-NMR (90 MHz, CDCl3)  = 20.7, 36.2, 74.1, 123.8, 126.7, 127.3, 128.2, 134.6, 137.1, 155.2, 207.3.

(R)-2-Hydroxy-indan-1-one (R)-6 was obtained from rac-2-acetoxyindan-1-one using the following procedure adapted from literature (Kajiro et al. 1998). rac-2-Acetoxyindan-1-one (1.3 g, 7.4 mmol) was dissolved in phosphate buffer (400 mL, 100 mM, pH 7) and CH3CN (100 mL). Pseudomonas sp. lipase (Amano PS, 130 mg) was added and the mixture was vigorously stirred for 48 h at 30°C. The immobilized enzyme was removed by filtration, products were extracted with ethyl acetate, dried over sodium sulfate and the solvent was evaporated under reduced pressure. Flash chromatography (petroleum ether/ethyl acetate 5:1) yielded (R)-6 as yellow oil (56 mg, 86%, e.e. >99%). []D20 +2.4 (c 1.0, CHCl3); []D25 +57.1 (c 1, MeOH) (Kajiro et al. 1999); 1H-NMR (360 MHz, CDCl3)  = 2.18 (1H, s, OH), 3.67 (2H, q, J = 8.0 Hz, CH2), 5.43 (1H, q, J = 4.9, CH-OH), 7.27-7.47 (2H, m, aryl), 7.62 (1H, t, J = 6.6, aryl), 7.80 (1H, d, J = 7.7, aryl); 13C-NMR (90 MHz, CDCl3)  = 33.4, 74.1, 124.5, 126.6, 128.2, 134.5, 135.9, 150.4, 200.6.

Table 1. GC-analyses using a chiral stationary phase

Substrate / Compound / Conditions / Retention Times [min]

(R)a

/ (S) a / Ketone
1 / / [b] / 2.52 / 2.16 / (1a) 1.36
2 / / [b] / 2.42 / 2.12 / (2a) 1.34
3 / / [b] / 4.36 / 3.11 / (3a) 3.09

a Analysed as corresponding acetate ester; b conditions: Column Chirasil-DEX CB, temperature program (start temperature [°C]/ holding time [min]/ heating rate [°C min-1]/ plateau temperature [°C]/ holding time [min]/ heating rate [°C min-1]/ final temperature [°C]/ holding time [min]):110/1/2.5/120/10/160/1.

Table 2. HPLC-analyses using a chiral stationary phase

Substrate / Compound / Conditions / Retention Times [min]

(R)

/ (S) / Diketone
4 / / [a] / 21.8 / 20.1 / (4a) 10.4
5 / / [b] / 17.7 / 19.3 / (5a) 10.4
6 / / [c] / 20.8 / 22.6 / (6a) 8.1

a Conditions: Column Chiralpak AD, 0.46 x 25 cm, n-heptane/i-propanol (90/10), flow 1.0 mL min-1; 18 °C. b Conditions: Column Chiralpak OD-H, 0.46 x 25 cm, n-heptane/i-propanol (90/10), flow 0.5 mL min-1; 18 °C. c Conditions: Column Chiralpak AD, 0.46 x 25 cm, n-heptane/i-propanol (90/10), flow 0.5 mL min-1; 18 °C.

References

Demir AS, Reis Oe, Idgir AC (2004) Enzyme-catalyzed hydroxymethylation of aromatic aldehydes with formaldehyde. Synthesis of hydroxyacetophenones and (S)-benzoins. Tetrahedron 60: 3427-3432

Kajiro H, Mitamura S, Mori A, Hiyama T (1998) Enantioselective synthesis of 2-hydroxy-1-indanone, a key precursor of enantiomerically pure 1-amino-2-indanol. Tetrahedron Asymmetry 9: 907-910

Kajiro H, Mitamura S, Mori A, Hiyama T (1999) A practical synthesis of (1S,2R)-1-amino-2-indanol, a key component of an HIV protease inhibitor, indinavir. Bull Chem Soc Jpn 72: 1093-1100

Taka H, Fujita K, Oishi A, Taguchi Y (2002) Asymmetric nucleophilic addition reactions of aldehydes with optically active dithioacetals and their application to optically active -hydroxy ketone synthesis. Heterocycles 57: 1487-1493

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