Materials and chemicals
All reagents were purchased from Sigma-Aldrich (Milan, Italy) and/or from VWR International and were used without further purification. All the solvents were of HPLC grade. Analytical Thin Layer Chromatography TLC was performed on silica gel 60 F254 precoated aluminum sheets (0.2 mm layer; Merck, Darmstadt, Germany). Components were detected under an UV lamp (λ 254 nm), by spraying with a vanillin/H2SO4 solution in EtOH [6% (w/v) vanillin + 1% (v/v) H2SO4], followed by heating at about 150 °C. Product purification was accomplished by flash chromatography (silica gel 60, 40–63 mm, Merck).
Prepararion of recombinant E. coli harbouring CYP153A6
The synthetic gene encoding CYP153A6 (BaseClear B.V., Leiden, The Netherlands) operon has been designed and amplified using the following primer:
Forward: 5′-CACCATATGACCGAAATGACCGTGGC-3′.
Reverse: 5′-ATTGCTCGAGTCAATGCTGCGCGGC-3′.
The amplified gene was then cloned into the pET100/D-TOPO® vector (Invitrogen) downstream the EK cleavage site, and correct construct sequence was confirmed by DNA sequencing (Eurofins Biolab Srl). The synthetic gene sequence has been deposited in NCBI database with accession number OM622424. Recombinant BL21(DE3)Star E. coli cells harbouring the pET100-CYP53A6 plasmid were obtained through heat-shock transformation.
Expression of CYP153A6
Expression of the recombinant CYP153A6 operon was performed using BL21(DE3)Star E. coli strain harbouring pET100-CYP53A6 expression vector. Seed cultures were prepared inoculating 0.2 mL of glycerol stock of the recombinant strain in 20 mL of broth with 100 mg mL−1 ampicillin and incubated at 37 °C at 120 rpm in Erlenmeyer flasks for 16 h. The seed cultures were used as inoculum in 1 L baffled flasks containing 200 mL of the selected medium supplemented with ampicillin (100 mg mL−1) to get an initial cells density of 0.1 OD600. The resulting suspensions were incubated at 37 °C and 120 rpm until 0.6–0.8 OD600 (2–4 h), brought to 4 °C for 5 min and induced for 4 h with isopropyl-β-d-thiogalactopyranoside 0.5 mM at 28 °C and 150 rpm. The following liquid media were used: Luria–Bertani (LB: 10 g L−1 bacto-tryptone, 5 g L−1 yeast extract, 10 g L−1 NaCl), Super Broth (SB: 32 g L−1 bacto-tryptone, 20 g L−1 yeast extract and 5 g L−1 NaCl), Terrific Broth (TB: 12 g L−1 bacto-tryptone, 24 g L−1 yeast extract, 8 g L−1 glycerol, 9.4 g L−1 KH2PO4 and 2.2 g L−1 K2HPO4).
Biotransformations
Cell growth was measured both as optical density (OD600nm, Eppendorf BioSpectrometer) than as cell dry weight (mg mL−1) of washed cells coming from a known volume of culture.
The enzymatic activity (Units) was calculated dividing the moles of substrate converted into product by the time unit (min) per weight of biocatalyst (U g−1 dry weight) or reaction volume (U L−1).
Optimization of biotransformations was carried out by varying different parameters of the reactions (pH, temperature, and biomass concentration) in sequential experimental trials selected by Multisimplex® 2.0 software. Cell pellets were recovered by centrifugation at 5000 rpm for 10 min at 4 °C, washed once with 100 mM potassium phosphate buffer pH 7.0 and suspended in different phosphate buffers to get the desired cells density; the suspensions were transferred to flat bottom baffled flasks without exceeding 10–15% of the total volume, and incubated at the desired temperature at 150 rpm. The substrates at different concentrations were added to the suspension and the flasks tightly sealed. To standardize the effect of volatility of 1a on the time course of the reaction, each flask was dedicated to a single analysis, thus avoiding repeated sampling.
Preparative biotransformations were carried out with 50 mgdry cells mL−1 in a total volume of 50 mL of phosphate buffer (100 mM) at pH 8.0 at 28° C and 150 rpm. For GC analysis, proper amounts of the mixture (500 µL) were withdrawn at fixed times, extracted with EtOAc (1:1 volume ratio), dried under nitrogen stream at 4° C, diluted in EtOAc and directly injected. For product purification, the mixture was extracted with EtOAc (3 × reaction volume). The reunited organic phases were dried over anhydrous Na2SO4 and evaporated under vacuum at 4° C. The resulting crude material was purified by flash chromatography (n-hexane/EtOAc, from 5 to 45% EtOAc in n-hexane) to get either a mixture of constitutional isomers (2c + 3c and 2d + 3d) or pure products (2e, 2f, 2k and 2l). The ratio between isomers was determined by GC analysis.
Analyses
GC analyses was performed using a Dani® 86.10 HT gas chromatographer equipped with a flame ionization detector (200 °C, p(H2) 0.8 atm, p(air) 1.5 atm). Chromatographic conditions were as follows: column, DeMePeβCDxPS086 Mega® (25 m × 0.25 mm); injection volume: 1 µL (split (1/50), 230 °C); injection solvent: EtOAc; carrier: H2 (0.6 mL/min). Analyses were performed with the following program: (i) gradient from 80 °C to 110 °C (10 °C/min), (ii) isocratic at 110 °C for 9 min. Data were processed with the EZChrom Elite software. Retention times were reported in minutes. 1H NMR spectra were recorded on a Varian Oxford 300 MHz NMR spectrometer equipped with a VnmrJ software package (Varian Medical Systems, Palo Alto, California, USA) at 300 K, unless stated otherwise. 1H chemical shifts (δ) are given in parts per million and were referenced to the solvent signals (δH 7.26 ppm from tetramethylsilane (TMS) for CDCl3).
Chemical synthesis of substrates
Synthesis of 1e and 1f: LiAlH4 (1.0 M in THF, 4.0 mL, 4.00 mmol, 1.20 equiv) was added dropwise at -78° C to a solution of 1c or 1d (0.52 mL, 3.32 mmol, 1.00 equiv) in dry THF (10 mL) under inert atmosphere. The mixture was stirred while warming to room temperature for 3 h. Water (1 mL), 2 M NaOH (2 mL) were added at 0° C and the suspension was extracted with Et2O (3 × 20 mL). The reunited organic phases were then washed with brine (2 × 10 mL), dried over anhydrous Na2SO4 and evaporated. The resulting crude material was purified by flash column chromatography (cyclohexane-EtOAC, 85:15). Compound 1e was obtained as a colorless oil in quantitative yield (504 mg, 3.31 mmol): 1H NMR (300 MHz, CDCl3): δ 5.53–5.48 (m, 1H, H1), 4.75–4.73 (m, 2H, CH29), 4.24–4.15 (m, 1H, H3), 2.33–2.22 (m, 1H, H4/5/6), 2.21–2.12 (m, 1H, H4/5/6), 2.08–2.02 (m, 1H, H4/5/6), 2.02–1.88 (m, 1H, H4/5/6), 1.76 (dt, J = 4.0, 1.5 Hz, 3H, CH37/10), 1.75 (br t, J = 1.1 Hz, 3H, CH37/10), 1.51 (td, J = 12.1, 9.5 Hz, 1H, H4/5/6, partially covered by H2O) (Elamparuthi et al. 2012). Compound 1f was obtained as a colorless oil in 99% yield (502 mg, 3.30 mmol): 1H NMR (300 MHz, CDCl3): δ 5.53–5.48 (m, 1H, H1), 4.75–4.73 (m, 2H, CH29), 4.24–4.15 (m, 1H, H3), 2.33–2.22 (m, 1H, H4/5/6), 2.21–2.12 (m, 1H, H4/5/6), 2.08–2.02 (m, 1H, H4/5/6), 2.02–1.88 (m, 1H, H4/5/6), 1.76 (dt, J = 4.0, 1.5 Hz, 3H, CH37/10), 1.75 (br t, J = 1.1 Hz, 3H, CH37/10), 1.51 (td, J = 12.1, 9.5 Hz, 1H, H4/5/6, partially covered by H2O) (Elamparuthi et al. 2012).
Synthesis of 1g and 1h: pyridine (151 µL, 1.87 mmol, 1.89 equiv) and acetyl chloride (1.50 equiv) were added at 0 °C to a solution of 1e or 1f (150 mg, 0.99 mmol, 1.00 equiv) in dry CH2Cl2 (2.0 mL) under N2. The mixture was stirred at room temperature for 5 h. The resulting yellow suspension was diluted with CH2Cl2 (30 mL) and washed with sat. NH4Cl (2 × 30 mL), followed by sat. NaHCO3 (2 × 30 mL). The organic phase was dried over anhydrous Na2SO4 and evaporated. The resulting crude was purified by flash column chromatography (n-hexane–EtOAc, 7:3). 1g was obtained as a colorless oil in 43% yield (82 mg, 0.42 mmol); 1h was obtained as a colorless oil in 42% yield (86 mg, 0.41 mmol): 1H NMR (300 MHz, CDCl3): δ 5.61 (dt, J = 5.2, 3.4, 1.7 Hz, 1H, H1), 5.50–5.40 (m, 1H, H3), 4.76–4.66 (m, 2H, CH29), 2.37–2.25 (m, 1H, H4/5/6), 2.24–2.15 (m, 1H, H4/5/6), 2.09–2.03 (m, 4H, CH312, H4/5/6), 2.02–1.88 (m, 1H, H4/5/6), 1.73–1.71 (m, 3H, CH37/10), 1.64 (td, J = 2.5, 1.4 Hz, 3H, CH37/10), 1.49 (ddd, J = 13.0, 11.9, 10.0 Hz, 1H, H4/5/6) (Trost and Schmuff 1985).
Synthesis of 1i and 1j: pyridine (302 µL, 3.74 mmol, 3.78 equiv) and benzoic anhydride (335 mg, 1.48 mmol, 1.50 equiv) were added to a solution of 1e or 1f (150 mg, 0.99 mmol, 1.00 equiv) in EtOAc (2.0 mL) under N2. The mixture was refluxed overnight. The light orange solution was washed with sat. NH4Cl (2 × 30 mL) and sat. NaHCO3 (2 × 30 mL), dried over Na2SO4 and evaporated. The resulting crude was purified by flash column chromatography (n-hexane–EtOAc, 8:2) to get compounds 1i and 1j as colorless oils (1i: 33 mg, 0.13 mmol, 13%; 1j: 38 mg. 0.15 mmol, 15%): 1H NMR (300 MHz, CDCl3): δ 8.10–8.04 (m, 2H, Ph), 7.60–7.53 (m, 1H, Ph), 7.48–7.41 (m, 2H, Ph), 5.76–5.63 (m, 2H, H1, H3), 4.77–4.70 (m, 2H, CH29), 2.46–2.28 (m, 2H, 2 × H4/5/6), 2.22–1.94 (m, 2H, 2 × H4/5/6), 1.74 (br t, 3H, J = 1.1 Hz, CH37/10), 1.71 (tq, J = 2.4, 1.1 Hz, 3H, CH37/10), 1.67–1.61 (m, 1H, H4/5/6) (Correia and DeShong 2001).
7-Hydroxy-(R)-carvone and 10-hydroxy-(R)-carvone (2c + 3c). The product mixture was obtained as an off-white solid in 67% yield. 1H NMR (300 MHz, CDCl3): major isomer (2c): δ 6.97–6.90 (m, 1H, H1), 4.82–4.79 (m, 1H, CH9a), 4.76–4.73 (m, 1H, CH9b), 4.24 (br s, 2H, CH27), 2.77–2.25 (m, 5H, CH24, CH26, H5), 1.74 (s, 3H, CH310); minor isomer (3c): δ 6.97–6.90 (m, 1H, H1, covered by 2c), 5.11–5.08 (m, 1H, CH9a), 4.93–4.91 (m, 1H, CH9b), 4.11 (br s, 2H, CH210), 2.77–2.25 (m, 5H, CH24, CH26, H5, covered by 2c), 1.74 (s, 3H, CH37, covered by 2c) (Lakshmi et al. 2005).
7-Hydroxy-(S)-carvone and 10-hydroxy-(S)-carvone (2d + 3d). The product mixture was obtained as an off-white solid in 70% yield. 1H NMR (300 MHz, CDCl3): major isomer (2d): δ 6.97–6.92 (m, 1H, H1), 4.85–4.80 (m, 1H, CH9a), 4.79–4.75 (m, 1H, CH9b), 4.30–4.23 (br s, 2H, CH27), 2.79–2.27 (m, 5H, CH24, CH26, H5), 1.76 (s, 3H, CH310); minor isomer (3d): δ 6.97–6.92 (m, 1H, H1, covered by 2d), 5.12 (br s, 1H, CH9a), 4.95 (br s, 1H, CH9b), 4.16–4.08 (m, 2H, CH210), 2.79–2.27 (m, 5H, CH24, CH26, H5, covered by 2d), 1.76 (s, 3H, CH37, covered by 2d) (Lakshmi et al. 2005).
(4R,6R)-7-Hydroxycarveol (2e). The product was obtained as an off-white solid in 68% yield. 1H NMR (300 MHz, CDCl3): δ 5.77 (br s, 1H, H1), 4.75 (s, 2H, CH27), 4.57–4.47 (m, 1H, H3), 4.23 (s, 2H, CH27), 2.56 (br s, 2H, 2 × OH), 2.35–2.22 (m, 1H, CH24/CH26/CH5), 2.21–2.09 (m, 2H, CH24/CH26/CH5), 2.09–1.94 (m, 1H, CH24/CH26/CH5), 1.74 (s, 3H, CH310), 1.58 (td, J = 12.1, 10.0 Hz, 1H, CH24/CH26/CH5) (Gimalova et al. 2012).
(4S,6S)-7-Hydroxycarveol (2f). The product was obtained as an off-white solid in 64% yield. 1H NMR (300 MHz, CDCl3): δ 5.76 (br s, 1H, H1), 4.74 (s, 2H, CH27), 4.56–4.45 (m, 1H, H3), 4.21 (s, 2H, CH27), 2.99–2.57 (m, 2H, 2 × OH), 2.34–2.21 (m, 1H, CH24/CH26/CH5), 2.20–2.08 (m, 2H, 2 × CH24/CH26/CH5), 2.07–1.92 (m, 1H, CH24/CH26/CH5), 1.73 (s, 3H, CH310), 1.56 (q, J = 12.0 Hz, 1H, CH24/CH26/CH5) (Gimalova et al. 2012).
δ-3-Caren-10-ol (2k). The product was obtained as an oil in 39% yield. 1H NMR (300 MHz, CDCl3): δ
5.72–5.49 (m, 1H, H4), 3.92 (s, 2H, CH210), 2.55–2.32 (m, 2H, CH22/CH25), 2.08–1.95 (m, 2H, CH22/CH25), 1.63 (s, 1H, OH), 1.11 (s, 3H, CH38/9), 0.82 (s, 3H CH38/9), 0.74–0.81 (m, 1H, H1/6), 0.69 (br t, J = 8.4 Hz, 1H, H1/6) (Frąckowiak et al. 2006).
7-Hydroxy-α-terpineol (2l). The product was obtained as an off-white solid in 65% yield. 1H NMR (300 MHz, CDCl3): δ 5.71 (br s, 1H, H1), 4.07–3.97 (m, 2H, CH27), 2.22–2.02 (m, 3H, CH23/CH24/ CH26/CH5), 2.01–1.94 (m, 1H, CH23/CH24/CH26/CH5), 1.93–1.82 (m, 1H, CH23/CH24/CH26/CH5), 1.61–1.51 (m, 1H, CH23/CH24/CH26/CH5), 1.34–1.24 (m, 1H, CH23/CH24/CH26/CH5), 1.22 (s, 3H, CH39), 1.21 (s, 3H, CH310) (Constantino et al. 2007).