Supplementary information
Table of Contents
1 Equipment and analytical methods...2
1.1 Thin layer chromatography (TLC)...2
1.2 Optical rotation measurements...2
1.3 Nuclear magnetic resonance (NMR) and infrared (IR) spectroscopy...2
1.4 High-performance liquid chromatography (HPLC)...2
1.5 Enantiomeric ratio (E) calculation...2
1.6 Thermostated multicolumn continuous-flow reactor system and filled columns...2
2 Methods...3
2.1 Synthesis of the racemic N-Boc-phenylalanine thioethyl ester (rac-1)...3
2.2 Synthesis of racemic N-Boc-phenylalanine benzylamide (rac-2)...3
2.3 Kinetic resolution of racemic N-Boc-phenylalanine thioethyl ester (rac-1) in batch modes...4
2.3.1 Kinetic resolution of racemic N-Boc-phenylalanine thioethyl ester (rac-1) by the freshly immobilized Alcalase biocatalysts in batch mode...4
2.3.2 Kinetic resolution of racemic N-Boc-phenylalanine thioethyl ester (rac-1) by the one year long stored Alcalase biocatalysts in batch mode...4
2.4 Kinetic resolution of the racemic N-Boc-phenylalanine thioethyl ester (rac-1) in continuous-flow bioreactors...5
2.4.1 Kinetic resolution of the racemic N-Boc-phenylalanine thioethyl ester (rac-1) in continuous-flow bioreactors at various temperatures...5
2.4.2 Operational stability of the Alc-Dv250-Et and Alc-Dv250-MePhe biocatalysts in kinetic resolution of racemic N-Boc-phenylalanine thioethyl ester (rac-1) in continuous-flow bioreactors...6
2.5 Racemization tests with N-Boc protected (R)-phenylalanine thioethyl ester [(R)-1] in continuous-flow reactor...6
2.6 Calculation of the theoretical, technical and kinetics controlled limits of the DKR of rac-1 in a continuous-flow system...6
References...9
1 Equipment and analytical methods
1.1 Thin layer chromatography (TLC)
TLC was carried out using Kieselgel 60 F254 (Merck) sheets. Spots were visualized under UV
light (254 nm and 365 nm) or by treatment with 5% ethanolic phosphomolybdic acid solution and heating of the dried plates.
1.2 Optical rotation measurements
Optical rotations were taken on a Perkin-Elmer 241 polarimeter that was calibrated by measuring the specific optical rotations of both enantiomers of menthol, [α]D values were
given in units of 10−1 deg cm2 g−1.
1.3 Nuclear magnetic resonance (NMR) and infrared (IR) spectroscopy
The NMR spectra were recorded in CDCl3 on a Bruker DRX-300 spectrometer operating at
300 MHz for 1H and 75 MHz for 13C, and signals are given in ppm on the δ scale. Infrared
spectra were recorded on a Bruker ALPHA FT-IR spectrometer and wavenumbers of bands are listed in cm−1.
1.4 High-performance liquid chromatography (HPLC)
Reactions were analyzed by HPLC on a Hewlett Packard 1090 Series II equipment equipped with a Chiralpack® IB column [Daicel, 150×2.1 mm, 5 μm, cellulose
tris(3,5-dimethylphenyl)carbamate] using hexane/isopropanol 98:2 (v/v) as eluent at a flow rate of 0.25 mL min−1 at 25°C and diode array detection (DAAD) at λ=220 nm.
1.5 Enantiomeric ratio (E) calculation
The conversion (c) and enantiomeric excess (ee) were derived from the chromatogram (HPLC) of the samples taken at various time in the course of the reactions of rac-1 with benzylamine catalyzed by the immobilized Alcalase preparations.
Enantiomeric ratio (E) was calculated from conversion (c) and enantiomeric excess of the product (eeP) using the equation E = ln[1 − c(1+eeP)]/ln[1 − c(1 − eeP)].[1] For simplicity, E
values calculated in the range of 100–200 were given as ›100, those in the range of 200–500 as ›200 and above 500 as »200.
In Figure 2 of the main article the solid curves and filled markers indicate data which arose from a precise integration of chromatograms in which both enantiomers of the product [(S)-2 and (R)-2] were clearly visible. When signals for the minor enantiomer [(R)-2] were indistinguishable from noise, the dashed lines indicate values which are the possible minimum values of ee and E (calculated assuming the presence of the minor enantiomer [(R)-2] equal to the noise level).
1.6 Thermostated multicolumn continuous-flow reactor system and filled columns
Continuous-flow kinetic resolutions were performed in a laboratory flow reactor system (Figure S1). The system comprised an isocratic HPLC pump (Knauer K-120) attached to stainless steel columns (ThalesNano CatCart™) filled with the immobilized Alcalase
biocatalysts placed in an in-house made thermostated aluminum metal block multicolumn holder with precise temperature control. Before use, the columns were washed with tert-amyl alcohol (0.5 mL min−1, 20 min).
The immobilized Alcalase biocatalysts were filled into stainless steel CatCartTM columns
(stainless steel, inner diameter: 4 mm; total length: 70 mm; packed length: 65 mm; inner volume: 0.816 mL) according to the process of ThalesNano Inc. For the continuous-flow enzymatic applications both ends of the columns were sealed with PTFE sealing and silver metal filter membranes (Sterlitech Silver Membrane Filter from Sigma–Aldrich, Z623237, pore size 0.45 µm; pure metallic silver, 99.97% with no extractable or detectable contaminants).
Figure S1. A multicolumn continuous-flow reactor block (A). Scheme of the KR of N-Boc-phenylalanine thioethyl ester (rac-1) in the continuous-flow reactor (B)
2 Methods
2.1 Synthesis of the racemic N-Boc-phenylalanine thioethyl ester (rac-1) The reaction was carried out as described earlier.[2]
White crystals, yield: 74% (20.97 mmol, 6.49 g), mp: 78.5–79.1°C (lit.[2]: 79–80°C), HPLC
(tr): 3.47 min, 1H NMR (CDCl3): 1.25 (3H, t, J=7.5 Hz, CH3), 1.42 (9H, s, 3×CH3), 2.89 (2H, q, J=7.5 Hz, CH2), 2.98–3.22 (2H, m, CH2), 4.60–4.70 (1H, m, NH), 4.89–4.97 (1H, m, CH) 7.13–7.41 (5H, m, 5×CH), 13C NMR (CDCl 3): 14.65, 23.53, 28.47 (3×CH3), 35.51, 61.16, 80.44, 127.23, 128.77 (2×CH), 129.56 (2×CH), 136.00, 155.19, 201.34, IR (KBr): 3363, 2971, 2931, 2120, 1688, 1509, 1365, 1251, 1162, 1017.
To a stirred solution of N-Boc-Phe (5.7 mmol, 1.50 g) in acetonitrile (100 mL) N,N’-dicyclohexylcarbodiimide (11.3 mmol, 2.35 g), hydroxybenzotriazole (5.7 mmol, 0.77 g) and benzylamine (5.7 mmol, 0.61 g) were added at room temperature under argon atmosphere. After completion of the reaction (monitored by TLC, eluent: hexane–ethyl acetate 3:1 + 1% CH3COOH) ethyl acetate (100 mL) and 1 M HCl (25 mL) were added and the mixture was
stirred vigorously for further 45 min. The precipitated dicyclohexyl urea (DCU) was filtered off, then the filtrate was washed with 15% HCl solution (50 mL). The organic phase was dried on MgSO4 and the solvents were removed by vacuum rotary evaporator. The crude
product was purified by filtering through on a thin layer of silica gel (eluent: hexane–ethyl acetate 3:1) to yield the desired amide (rac-2) as white crystals (0.46 g, 1.29 mmol).
Yield: 23% (1.29 mmol, 0.46 g), mp: 135.7–136.0°C, HPLC (tr): 6.18 min for (R)-2, 7.15 min
for (S)-2, 1H NMR (CDCl 3): 1.40 (9H, s, 3×CH3), 3.09 (2H, d, J=7 Hz, CH2), 4.31–4.40 (3H, m, CH2, CH), 5.15 (1H, s, NH), 6.23 (1H, s, NH), 7.08–7.30 (10H, m, 10×CH), 13C NMR (CDCl3): 28.45 (3×CH3), 38.87, 43.64, 55.92, 79.82, 127.10, 127.63, 127.85 (2×CH), 128.79 (2×CH), 128.88 (2×CH), 129.54 (2×CH), 136.88, 137.90, IR (KBr): 3322, 3261, 2901, 1664, 1532, 1364, 1236, 1022, 714.
2.3 Kinetic resolution of racemic N-Boc-phenylalanine thioethyl ester (rac-1) in batch modes
The kinetic resolution of N-Boc-phenylalanine thioethyl ester (rac-1) with benzylamine (Scheme S1) was performed on analytical scale to characterize the biocatalytic performance (Table S1) and storability (Figure 1 in the main article, MA) of the grafted silica gel based Alcalase biocatalysts.
Scheme S1. Kinetic resolution of racemic thioethyl ester (rac-1) in batch mode
2.3.1 Kinetic resolution of racemic N-Boc-phenylalanine thioethyl ester (rac-1) by the freshly immobilized Alcalase biocatalysts in batch mode
To a solution of rac-N-Boc-Phe-SEt (rac-1, 0.048 mmol, 15 mg) and benzylamine (0.058 mmol, 6.34 μL) in tert-butyl alcohol or tert-amyl alcohol (2 mL) in an amber screw cap vial, the immobilized Alcalase preparation (20 mg) was added and the resulted reaction mixture was shaken (1000 rpm) in the closed vial at 30°C for 24 h. Samples (60 μL, diluted with hexane/isopropanol 98:2 to 500 μL) were taken directly from the reaction mixture after 1, 2, 4, 6 and 24 h and analyzed by HPLC. Results for the freshly immobilized Alcalase biocatalysts are shown in Table S1.
2.3.2 Kinetic resolution of racemic N-Boc-phenylalanine thioethyl ester (rac-1) by the one year long stored Alcalase biocatalysts in batch mode
Samples of the adsorbed Alcalase biocatalyst (150 mg of each preparation) were stored in an amber screw cap vial at 4°C under argon for 12 months. After this storing period, the Alcalase biocatalysts were tested in the kinetic resolution of racemic N-Boc-phenylalanine thioethyl ester (rac-1) as described in section 2.3.1. The results are shown in Figure 1 of the MA.
Table S1. Kinetic resolution of racemic thioethyl ester (rac-1) in batch mode with the immobilized Alcalase preparations (tert-amyl alcohol, 30°C, 24 h)
Entry Alcalase preparationa) rbatch
[mmol g−1 h−1] E [-] c [%] ee(S)-2 [%] 1 Alc-Dv250 45.9 ›100 33.9 97 2 Alc-Dv250-Et 55.7 ›200 41.3 98 3 Alc-Dv250-DiMe 55.7 ›100 41.1 98 4 Alc-Dv250-MePhe 55.0 ›100 40.9 98 5 Alc-Dv250-Dodec 54.9 ›100 40.8 98 6 Alc-Dv250-ODec 51.8 ›100 38.5 98 7 Alc-Dv250-Hex 51.6 ›100 37.8 98 8 Alc-Dv250-CyEt 47.2 ›100 35.3 98 9 Alc-Dv250-Pr 58.6 ›100 43.8 97 10 Alc-Dv250-DiPhe 58.7 ›100 43.6 97 11 Alc-Dv250-Phe 55.4 ›100 40.9 97 12 Alc-Dv250-iBu 54.7 ›100 40.6 97 13 Alc-Dv250-AmPr 53.4 ›100 40.0 97 14 Alc-Dv250-CHexMe 52.5 ›100 39.0 97 15 Alc-Dv250-Vin 52.1 ›100 38.7 97 16 Alc-Dv250-AmEtAmPr 52.0 ›100 38.4 97 17 Alc-Dv250-Me 50.2 ›100 37.3 97 18 Alc-Dv250-AmEtAmPrMe 48.1 ›100 36.0 97 19 Alc-Dv250-GlyOPr 43.9 ›100 32.6 97 20 Alc-Dv250-Dec 42.9 ›100 32.1 97 21 Alc-Dv250-Oct 26.4 78 19.6 97 22 Alc-Dv250-ClPr 56.0 ›100 41.6 96 23 Alc-Dv250-MerPr 55.8 ›100 41.4 96 24 Alc-Dv250-pFOct 54.1 ›100 40.7 96
a) Grafting functions on the silica gel supports are listed in Experimental Section of the MA.
2.4 Kinetic resolution of the racemic N-Boc-phenylalanine thioethyl ester (rac-1) in continuous-flow bioreactors
2.4.1 Kinetic resolution of the racemic N-Boc-phenylalanine thioethyl ester (rac-1) in continuous-flow bioreactors at various temperatures
Separate CatCartTM columns were packed with the following enzyme preparations (filling
weights in mg): Alc-Dv250-Et (215.4), Alc-Dv250-Dodec (225.1), Alc-Dv250-DiMe (213.3) Alc-Dv250-MePhe (231.7).
A solution of rac-N-Boc-Phe-SEt (rac-1, 5 mg mL−1) and benzylamine (1.2 equiv.) in
tert-amyl alcohol was pumped through the enzyme-packed column thermostated at various temperatures at a flow rate of 0.2 mL min−1. Experiments were run in 10°C steps at
temperatures between 0–100°C. Samples (sample size: 200 μL, diluted with hexane/isopropanol 98:2 to 500 μL) were collected after stationary operation has been established (30 min after changing conditions) and analyzed by HPLC. Results for KRs of
rac-1 are shown in Figure 2 of the MA.
The product (S)-2 isolated from the combined solutions of the KRs (Section 2.4) was used in the racemization tests (Section 2.5).
2.4.2 Operational stability of the Alc-Dv250-Et and Alc-Dv250-MePhe biocatalysts in kinetic resolution of racemic N-Boc-phenylalanine thioethyl ester (rac-1) in continuous-flow bioreactors
Separate CatCartTM columns were packed with the following enzyme preparations (filling
weights in mg): Alc-Dv250-Et (214.9), Alc-Dv250-MePhe (237.8).
Kinetic resolutions of rac-N-Boc-Phe-SEt (rac-1) by Alc-Dv250-Et and Alc-Dv250-MePhe were carried out for 120 h at 50°C in continuous-flow bioreactors as described in section 2.4.1. The results are shown in Figure 3 of the MA.
2.5 Racemization tests with N-Boc protected (R)-phenylalanine thioethyl ester [(R)-1] in continuous-flow reactor
A solution of (S)-N-Boc-Phe-NHBn and (R)-N-Boc-Phe-SEt [(S)-2:(R)-1≈1:1, 5 mg mL−1],
1,8-diazabicycloundec-7-ene (DBU, 3 equiv.) and benzylamine (1.2 equiv.) in tert-amyl alcohol was pumped through the CatCartTM column, filled with Dv250-Et (215 mg),
thermostated at various temperatures (0–150°C) at a flow rate of 0.2 mL min−1. Samples
(sample size: 200 μL, diluted with hexane/isopropanol 98:2 to 500 μL) were collected after stationary operation has been established (45 min after changing conditions) and analyzed by HPLC. Results for the racemization test are shown in Figure 4 of the MA.
2.6 Calculation of the theoretical, technical and kinetics controlled limits of the DKR of rac-1 in a continuous-flow system
The theoretical limits of conversion (c) and ee(S)-2 at the outlet of each units of the system
performing DKR of rac-1 were calculated assuming fully selective kinetic resolution (KR) and complete racemization (Rac) steps (Table S2).
The technical limits of conversion (c) and ee(S)-2 at the outlet of each units of the system
performing DKR of rac-1 were calculated assuming units achieving the performance of the single column KR (c= 45.9% and ee(S)-2= 99.5%) and the single column Rac (ee(R)-1= 4%) in
each corresponding units (Table S3).
The kinetics controlled limits of conversion (c) and ee(S)-2 at the outlet of each units of the
system performing DKR of rac-1 were calculated assuming decrease of c in the subsequent KR steps proportional with the decrease of the (S)-1 concentration but retaining selectivity (ee(S)-2= 99.5%) and reaching ee(R)-1= 4% in all racemization steps (Table S4).
Table S2. Calculated values of the theoretical limits of conversion and enantiomeric excess of the product (R)-2 at the outlet of each units of the continuous-flow system performing DKR of rac-1 [KR (6×) and Rac (5×) in alternating cascade]. (S)-2 [%] (R)-2 [%] (S)-1 [%] (R)-1 [%] ee(R)-1 [%] rac-1 [%] cunit [%] ctotal [%] Start 0.0 0.0 50.0 50.0 0.0 100.0 0.0 KR 1 50.0 0.0 0.0 50.0 100.0 50.0 50.0 Rac 1 50.0 0.0 25.0 25.0 0.0 50.0 KR 2 75.0 0.0 0.0 25.0 100.0 50.0 75.0 Rac 2 75.0 0.0 12.5 12.5 0.0 25.0 KR 3 87.5 0.0 0.0 12.5 100.0 50.0 87.5 Rac 3 87.5 0.0 6.3 6.3 0.0 12.5 KR 4 93.8 0.0 0.0 6.3 100.0 50.0 93.8 Rac 4 93.8 0.0 3.1 3.1 0.0 6.3 KR 5 96.9 0.0 0.0 3.1 100.0 50.0 96.9 Rac 5 96.9 0.0 1.6 1.6 0.0 3.1 KR 6 98.4 0.0 0.0 1.6 100.0 50.0 98.4
Table S3. Calculated values of the technical limits of conversion and enantiomeric excess of the product (R)-2 at the outlet of each units of the continuous-flow system performing DKR of rac-1 [KR (6×) and Rac (5×) in alternating cascade] assuming units achieving the performance of the single column KR [(rac-1, 5 mg mL−1 and
benzylamine, 1.2 equiv. in tert-amyl alcohol at 50°C, 0.2 mL min−1 in an Alc-Dv250-Et-packed reactor): c=
45.9% and ee(S)-2= 99.5%] and the single column Rac [(R)-1, 3 mg mL−1 with DBU (3 equiv.), (S)-2, 2.5 mg mL−1 tert-amyl alcohol, 150°C, 0.2 mL min−1 in a Dv250-Et-packed reactor): ee
(R)-1= 4%] in each corresponding units.
(S)-2 [%] (R)-2 [%] (S)-1 [%] (R)-1 [%] ee(R)-1 [%] rac-1 [%] cunit [%] ctotal [%] Start 0.0 0.0 50.0 50.0 0.0 100.0 0.0 KR 1 45.8 0.1 4.2 49.9 84.4 45.9 45.9 Rac 1 45.8 0.1 26.0 28.1 4.0 51.9 KR 2 69.6 0.2 2.2 28.1 85.5 45.9 69.7 Rac 2 69.6 0.2 14.5 15.7 4.0 29.1 KR 3 82.9 0.2 1.2 15.7 85.5 45.9 83.1 Rac 3 82.9 0.2 8.1 8.8 4.0 16.2 KR 4 90.3 0.2 0.7 8.8 85.5 45.9 90.5 Rac 4 90.3 0.2 4.5 4.9 4.0 9.1 KR 5 94.5 0.2 0.4 4.9 85.5 45.9 94.7 Rac 5 94.5 0.2 2.5 2.8 4.0 5.1 KR 6 96.8 0.2 0.2 2.7 85.5 45.9 97.0
Table S4. Calculated values of the kinetics controlled limits of conversion and enantiomeric excess of the product (R)-2 at the outlet of each units of the continuous-flow system performing DKR of rac-1 [KR (6×) and Rac (5×) in alternating cascade] assuming decrease of c in the KR steps (rac-1, 5 mg mL−1 and benzylamine, 1.2
equiv. in tert-amyl alcohol at 50°C, 0.2 mL min−1 in an Alc-Dv250-Et-packed reactor) proportional with the
decrease of the (S)-1 concentration but retaining selectivity [ee(S)-2= 99.5%] and reaching ee(R)-1= 4% in all
racemization steps [(R)-1, 3 mg mL−1 with DBU (3 equiv.), (S)-2, 2.5 mg mL−1 tert-amyl alcohol, 150°C,
0.2 mL min−1 in a Dv250-Et-packed reactor].
(S)-2 [%] (R)-2 [%] (S)-1 [%] (R)-1 [%] ee(R)-1 [%] rac-1 [%] cunit [%] ctotal [%] Start 0.0 0.0 50.0 50.0 0.0 100.0 0.0 KR 1 45.8 0.1 4.2 49.9 84.4 45.9 45.9 Rac 1 45.8 0.1 26.0 28.1 4.0 51.9 KR 2 58.1 0.1 13.6 28.1 34.7 23.8 58.3 Rac 2 58.1 0.1 20.0 21.7 4.0 40.1 KR 3 65.5 0.2 12.7 21.7 26.2 18.4 65.6 Rac 3 65.5 0.2 16.5 17.9 4.0 33.0 KR 4 70.5 0.2 11.5 17.9 21.6 15.1 70.6 Rac 4 70.5 0.2 14.1 15.3 4.0 28.2 KR 5 74.1 0.2 10.5 15.3 18.7 12.9 74.3 Rac 5 74.1 0.2 12.3 13.4 4.0 24.7 KR 6 76.9 0.2 9.6 13.4 16.6 11.3 77.1 References
1[] C. S. Chen, Y. Fujimoto, G. Girdaukas, C. J. Sih, J. Am. Chem. Soc. 1982, 104, 7294–7299. 2[] D. Tessaro, L. Cerioli, S. Servi, F. Viani, P. D’Arrigo, Adv. Synth. Catal. 2011, 353, 2333–2338.
