Supporting Information
Wiley-VCH 2013
69451 Weinheim, Germany
Highly Enantioselective Reduction of a-Methylated Nitroalkenes**
Edyta Burda, Tina Reß, Till Winkler, Carolin Giese, Xenia Kostrov, Tobias Huber,
Werner Hummel,* and Harald Grçger*
anie_201301814_sm_miscellaneous_information.pdf
Supporting Information
Highly Enantioselective Reduction of -Methylated Nitroalkenes
Edyta Burda,a Tina Reß,a,b Till Winkler,c Carolin Giese,a,b Xenia Kostrov,a Tobias Huber,a Werner
Hummel,*,c Harald Gröger*,a,b
a
Department of Chemistry and Pharmacy, University of Erlangen-Nürnberg, Henkestr. 42,
91054 Erlangen, Germany
Experimental procedures and analytical data
Table of Content:
1. Fermentation of the ene reductase from Gluconobacter oxydans on a 15L scale
2. Purification of the ene reductase from Gluconobacter oxydans
3. General protocol for the enantioselective reduction of trans-nitroalkenes 5 by means of
an ene reductase from G. oxydans (according to Manuscript Table 1)
4. Protocol for the determination of a potential GDH-mediated reduction of the double bond
5. Protocol for the investigation of the impact of the reaction medium, glucose
dehydrogenase and deactivated ene reductase on the racemization of nitroalkane 5a
6. General protocol for the combination of an in situ-synthesis of the nitroalkene 5b with a
subsequent enantioselective enzymatic reduction (corresponding to Manuscript Figure
4)
7. Spectrophotometric determination of enzyme activity of the ene reductase from G.
oxydans
8. Analytical data of the nitroalkane products (R)-1 (according to Table 1)
9. Chiral HPLC-chromatograms of racemic nitroalkanes 1
10. Chiral GC-chromatogram of racemic 1b
11. 1H NMR-Spectra of nitroalkanes 1
12. References
1
1. Fermentation of the ene reductase from Gluconobacter oxydans on a 15L scale
For the expression of the ene reductase from Gluconobacter oxydans chemically competent E.
coli Bl21(DE3) cells were transformed with the expression plasmid pGOX.[1] The fermentation
was performed on a 15 liter scale as a fed/batch process in a 40 liter bioreactor (Infors). The
components of the batch media are listed subsequently: 0.2 g/L NH4Cl, 2 g/L (NH4)2SO4, 13 g/L
KH2PO4, 10 g/L
K2HPO4, 6 g/L NaH2PO4xH2O, 3 g/L yeast extract, 2 g/L glucose, 1 g/L
MgSO4x7H2O, 0.25% vitamine solution (0.1 g/L riboflavine, 10 g/L thiamine, 0.5 g/L nicotinic
acid, 0.5 g/L pyridoxine, 0.5 g/L Ca-phanthotenate, 0.001 g/L biotin, 0.002 g/L folic acid, 0.01 g/L
cyanocabalamin), 0.16% salt solution (10 g/L CaCl2x2H2O, 0.5 g/L ZnSO4x7H2O, 0.25 g/L
CuCl2x2H2O, 2.5 g/L MnSO4xH2O, 1.75 g/L CoCl2x6H2O, 0.125 g/L H3BO3, 2.5 g/L AlCl3x6H2O,
0.5 g/L Na2MoO4x2H2O, 10 g/L FeSO4x7H2O), 0.1g/L thiamine in distilled water. In addition a
fed-media was used, which consists of the same components, but differs in the following
concentrations: 18 g/L yeast extract, 600 g/L glucose, 10 g/L MgSO4x7H2O, 1 g/L thiamine. After
the sterilisation of the reactor including the batch media the high cell density fermentation was
started by adding 1 % (v/v) over-night culture. For the optimum growth of the recombinant E. coli
glucose limited conditions were used. The addition of the fed-media was carried out at 30 °C
and H3PO4 was used to keep the pH value constant at pH 7. The airflow was controlled by the
speed of the stirring and addition of oxygen. The heterologous protein expression was induced
after 27 h with IPTG with a final concentration of 2 mM. The temperature, pH and airflow was
controlled automatically. After an incubation time of 41 h, the cells were harvested by
centrifugation. Using this method 2.5 kg of cells were obtained with an activity of 100 U/g cells.
2. Purification of the ene reductase from Gluconobacter oxydans
The expression of the ene reductase as N-terminal hexahistidine-tagged fusion protein enables
the purification via immobilized metal affinity chromatography IMAC (Ni-NTA, Qiagen). The
purification was performed with an Äkta purifier system (GE-Healthcare). The following buffers
were used for purification:
buffer A
buffer B
50 mM NaH2PO4,
50 mM NaH2PO4,
300 mM NaCl,
300 mM NaCl,
20 mM imidazole,
250 mM imidazole,
pH 8
pH 8
2
For the disruption process a 25% cell suspension was prepared with buffer A. The cells were
disrupted by pulsed sonication (Branson Sonifier, 2 x 5 min. 40 % power, 50 % pulse) on ice.
Insoluble cell components were separated from the crude extract by a subsequent centrifugation
step (30 min., 18.000 g). The column was equilibrated with buffer A. After loading of the column
a linear gradient (0-100 % buffer B in 100 min) was used to elute non-bound proteins. The ene
reductase elutes by means of a solution of 110 mM of imidazole. The purified enzyme was
transferred via a desalting step (Amicon ultrafiltration cell, 10 kDa membrane, 4 bar pressure) in
a 15 mM Na+/K+-phosphate buffer (pH 7) and stored over night at -20 °C. Afterwards the protein
was lyophilized and the purity of the ene reductase was analyzed by a SDS-PAGE gel. The
enzyme activity was determined as described in ref. [1], and the protein concentration was
determined by means of a method of Bradford as described in ref. [2]. The results on the
purification of the ene reductase from G. oxydans is summarized in the following Figure 1 and
Table 1.
1
2
3
97 kDa
64 kDa
51 kDa
39 kDa
28 kDa
Figure 1. Purification of the ene reductase of G. oxydans by IMAC chromatography. lane 1:
Fermentas See Blue 2 Standard; lane 2: crude extract; lane 3: purified enzyme after IMACchromatography (IMAC: immobilized metal ion affinity chromatography) and desalting step
Table 1. Summary of the purification of the recombinant ene reductase from G. oxydans
Purification step
Total activity
Spezific
Purification
(U)
activity (U/mg)
factor
Crude extract
1512
0.98
1
100
Ni-NTA and
1088
1.86
1.9
72
desalting
3
yield (%)
3. General protocol for the enantioselective reduction of trans-nitroalkenes 5 by means of
an ene reductase from G. oxydans (according to Manuscript Table 1):
The reaction was carried out in a total volume of 100 mL. The corresponding trans-1-aryl-2nitropropene (5, 1.0 mmol, 1.0 eq.) was emulsified stepwise or suspended in a Na+/K+phosphate buffer (pH 6, 10 mM; prepared from an aqueous solution of KH2PO4 (155 mM),
Na2HPO4 (25 mM) with subsequent adjustment of the pH by means of NaOH and dilution with
H2O) via ultrasound. The emulsion or suspension was cooled to 9°C under stirring, and Dglucose (2.0 mmol, 2.0 eq.), NADP+ (0.1 mmol, 0.1 eq.) and glucose dehydrogenase (GDH,
commercially available from Amano Enzymes Inc., Japan, CAS: 9028-53-9, 2.0 mg, specific
activity: 80 U/mg) were added. Subsequently, a purified ene reductase from G. oxydans (21.4
mg, protein content 52%, specific activity 3.6 U/mg referring to trans-hexenal, 40 U) were added
and the reaction mixture was stirred for 5h at 9°C. The pH value was adjusted to pH 6 by
continuous titration with 0.2 M NaOH. The mixture was treated with ultrasound for 10 min,
glucose dehydrogenase (GDH, 2.0 mg, specific activity 80 U/mg) was added. Then, the reaction
mixture was stirred for further 5h at 9°C at constant pH 6. After a reaction time of 10h, the
mixture was extracted with CH2Cl2 and the combined organic phases were dried over MgSO4.
After evaporation of the solvent at 30°C under vacuo, the pale yellow crude product was
analyzed by 1H-NMR-spectroscopy for the determination of the overall conversion as well as
conversion to product 1 (with an accuracy of ±5% being in general assumed for 1H-NMRspectroscopy; see also ref. [3]; the conversion to product 1 is defined as the amount of product 1
obtained relative to the amount of substrate 5 consumed) and by chiral HPLC for the
determination of the enantiomeric excess (ee-value; with an accuracy of ±1% ee according to
own chromatograms of racemates with complete separation of the signals; see below; the
absolute configuration of the major enantiomers of the obtained products 1 was assigned by
comparing the directions of the determined optical rotations with the direction of the reported
optical rotation (see ref. [4]) for product (S)-1g). The conversion related to the formation of the
Nef-product as a side-product was also determined by 1H-NMR-spectroscopy and corresponds
to the difference between the overall conversion and the conversion to product 1. The crude
products 1b and 1c were purified by filtration over SiO2 (eluent: cyclohexane:dichloromethane
(1:1 (v/v)) for 1b, dichloromethane for 1c).
4
4. Protocol for the determination of a potential GDH-mediated reduction of the double
bond
The reaction was carried out in principle as described in the section “3. General protocol for the
enantioselective reduction of trans-nitroalkenes 5 by means of an ene reductase from G.
oxydans (according to Table 1)”, but without adding the ene reductase. As a substrate 1-chloro3-(2-nitroprop-1-en-1-yl)benzene was chosen, and as an enzyme component only the glucose
dehydrogenase was used. The resulting crude product was analyzed by 1H-NMR-spectroscopy
and a conversion of 0% was determined.
5. Protocol for the investigation of the impact of the reaction medium, glucose
dehydrogenase and deactivated ene reductase on the racemization of nitroalkane 1a
For determining the influence of the reaction parameters “reaction medium (buffer)”, “glucose
dehydrogenase” and “ene reductase (in inactivated form)” on the racemization of enantiomerically enriched nitroalkanes 1, the following experiments were carried out:
Under identical reaction conditions as described in the section “3. General protocol for the
enantioselective reduction of trans-nitroalkenes 5 by means of an ene reductase from G.
oxydans (according to Table 1)”, a mixture of 1-chloro-3-(2-nitroprop-1-en-1-yl)benzene (5a) and
1-chloro-3-(2-nitro-propyl)benzene (1a) was used as substrate. Thus, 60 mg of this mixture
(corresponding to a simulated “conversion” of 85%; 93%ee) were emulsified via ultrasound in 25
mL of a Na+/K+-phosphate buffer (pH 6, 10 mM; prepared from an aqueous solution of KH2PO4
(155 mM), Na2HPO4 (25 mM) with subsequent adjustment of the pH by means of NaOH and
dilution with H2O), the corresponding additive was added (10 U GOx-ER (heat-denaturated at
70°C for 20 min) or 0.5 mg (40 U) GDH) and the resulting mixture was stirred at 9°C. After 5h,
the mixture was treated with ultrasound and in when using GDH, a second amount of GDH was
added after this ultrasound step. The pH value was adjusted to pH 6 by means of a continuous
titration with a solution of 0.2 M of NaOH. After a complete reaction time of 10h, the mixture was
extracted with methylene chloride the organic phases were dried over MgSO4. After evaporation
of the solvent, the crude product was analyzed by 1H NMR-spectroscopy and chiral HPLC (for
the accuracy of these methods, see general protocol related to Table 1, which is described
above). In all experiments, the 5a/1a ratio remained constant the ee-values were found to be
unchanged with 93% (Figure 2).
5
100
93
93
93
93
90
substrate mixture
80
70
buffer (pH6, 10 mM)
ee [%]
60
50
40
buffer (pH 6, 10 mM), 2x0.5 mg GHD
30
20
buffer (pH 6, 10 mM), Gox-ER (heatdenaturated)
10
0
Figure 2. Impact of buffer, GDH and inactive ene reductase on racemization of nitroalkanes 1
6. General protocol for the combination of an in situ-synthesis of the nitroalkene 5b with a
subsequent enantioselective enzymatic reduction (corresponding to Manuscript Figure 4)
Step (A) For the organocatalytic Henry reaction with subsequent dehydration, at first L-lysine
(0.15 mmol, 0.3 eq.) was mixed with 500 µl of ethanol (96%). After addition of 3-bromobenzaldehyde (0.5 mmol, 1.0 eq.) and nitroethane (3.8 mmol, 7.6 eq.) the reaction mixture was
stirred for 24 h at room temperature. For quantification of the conversion an aliquot of the
reaction mixture was prepared for GC-analysis. For that purpose L-lysine was separated from
the reaction mixture by filtration, ethanol was evaporated and the remaining yellow oil was
dissolved in ethyl acetate and dried with Na2SO4. After filtration, the conversion was determined
by GC chromatography.
Step (B) The subsequent enzyme catalyzed reduction of the C-C double bond was carried out in
a reaction volume of 1 mL. At first, an amount of the reaction mixture from Step (A),
corresponding to a nitroalkene concentration of 10 mM (5b, 0.01 mmol, 1.0 eq.), was emulsified
in a citric acid/Na2HPO4 buffer (pH 5, 50 mM) by sonication without prior purification. The
emulsion was cooled to 8°C, and mixed with D-glucose (0,1 mmol, 10.0 eq.), NADP+ (0.0005
mmol, 0.05 eq.) and glucose dehydrogenase (GDH, 20 U). Subsequently, the purified ene
reductase from Gluconobacter oxydans (3.0 U referring to trans-hexenal) was added. After a
reaction time of 7 h the extraction was performed using MTBE and the resulting organic phase
was dried over Na2SO4 and analyzed by chiral GC chromatography.
6
7. Spectrophotometric determination of enzyme activity of the ene reductase from G.
oxydans
The enzyme activity of the ene reductase from G. oxydans was determined by means of a
spectrophotometric method described in ref. [1].
8. Analytical data of the nitroalkane products (R)-1 (according to Table 1)
(R)-1-Chloro-3-(2-nitropropyl)benzene ((R)-1a)
1
H-NMR (500 MHz, CDCl3): δ(ppm) = 1.57 (3H, d, J = 6.5 Hz, H-C3), 2.99 (1H, dd, J = 14.1 Hz,
6.5 Hz, H-C1), 3.30 (1H, dd, J 0 14.1 Hz, 7.7 Hz, H-C1), 4.77 (1H, m, H-C2), 7.04-7.26 (4H, m,
H-aromatic)
The spectroscopic data are in accordance with literature data reported in ref. [5].
HPLC (AD-H, CO2:hexanes:i-propanol 90:9:1, 0.8 mL/min, 220 nm, 20°C): 94%ee (R)
tr(1) = 16.1 min
tr(2) = 17.5 min
25
[α]D (CHCl3, c = 2.77 g/L) = -45°
Overall conversion: 96%
Conversion to product (R)-1a: 95%
(Conversion to Nef-product: 1%)
(R)-1-Bromo-3-(2-nitropropyl)benzene ((R)-1b)
1
H-NMR (500 MHz, CDCl3): δ(ppm) = 1.56 (3H, d, J = 6.7 Hz, H-C3), 2.98 (1H, dd, J = 6.5 Hz,
14.1 Hz, H-C1), 3.30 (1H, dd, J = 7.7 Hz, 14.1 Hz, H-C1), 4.76 (1H, m, H-C2), 7.09-7.42 (4H, m,
H-aromatic)
13
C-NMR (100 MHz, CDCl3): δ(ppm) = 18.89 (C3), 40.56 (C1), 84.04 (C2), 122.81 (C6), 127.60
(C5), 130.38 (C7), 130.65 (C8), 132.03 (C9), 137.72 (C4)
MS (EI): m/z = 245 [M+•(81Br)], 243 [M+•(79Br)], 199 [M+• - NO2 (81Br)], 198 [M+• - HNO2 (81Br)], 197
[M+• - NO2 (79Br)], 196 [M+• - HNO2 (79Br)], 171 [M+• - C2H4NO2 (81Br)], 169 [M+• - C2H4NO2 (79Br)]
7
~
IR  (cm-1): 3059 (H-C-aromatic), 1544 (NO2), 1360 (NO2) 78
EA (C9H10BrNO2):
calculated: C: 44.29%, H: 4.13%, N: 5.74%
found: C: 43.95%, H: 4.09%, N: 5.57%
HPLC (OJ-H, hexanes:i-propanol 90:10, 0.8 mL/min, 220 nm, 25°C): 95%ee (R)
tr(1)= 17.5 min
tr(2)= 18.1 min
[α]D25 (CHCl3, c = 4.06 g/L) = -53°
Overall conversion: >99%
Conversion to product (R)-1b: >99%
(Conversion to Nef-product: <1%)
Yield of 1b: 84% (205 mg; 0.840 mmol)
(R)-1-Iodo-3-(2-nitropropyl)benzene ((R)-1c)
1
H-NMR (500 MHz, CDCl3): δ(ppm) = 1.56 (3H, d, J = 6.8 Hz, H-C3), 2.95 (1H, dd, J = 14.1 Hz,
6.6 Hz, H-C1), 3.27 (1H, dd, J = 14.1 Hz, 7.7 Hz, H-C1), 4.75 (1H, m, H-C2), 7.04-7.62 (4H, m,
H-aromatic)
13
C-NMR (100 MHz, CDCl3): δ(ppm) = 18.8 (C3), 40.4 (C1), 84.0 (C2), 94.7 (C8), 128.3 (C5),
130.6 (C6), 136.7 (C7), 137.9, 138.0 (C4, C9)
MS (EI): m/z = 291 (M+•), 244 (M+•-HNO2), 217 (M+•-C2H4NO2), 117 (M+•-HNO2-I)
~
IR  (cm-1): 1543 (NO2), 1359 (NO2)
EA: C9H10INO2
calculated: C: 37.14%, H: 3.46%, N: 4.81%
found: C: 36.57%, H: 3.49%, N: 4.62%
HPLC (OJ-H, hexanes:i-propanol 90:10, 0.8 mL/min, 25°C, 220 nm): 93%ee (R)
tr(1) = 20.5 min
tr(2) = 21.6 min
[α]D25 (CHCl3, c = 1.525 g/L) = -15°
Overall conversion: 99%
Conversion to product (R)-1c: 99%
(Conversion to Nef-product: ≤1%)
8
Yield of 1c: 84% (227 mg; 0.840 mmol)
(R)-1-Methoxy-3-(2-nitropropyl)benzene ((R)-1d)
1
H-NMR (500 MHz, CDCl3): δ(ppm) = 1.55 (3H, d, J = 6.7 Hz, H-C3), 2.98 (1H, dd, J = 13.9 Hz,
6.9 Hz, H-C1), 3.31 (1H, dd, J = 13.9 Hz, 7.4 Hz, H-C1), 3.79 (3H, s, H-C4), 4.78 (1H, q, J = 6.9
Hz, H-C2), 6.70-7.25 (4H, m, H-aromatic).
The spectroscopic data are in accordance with literature data reported in ref. [5].
HPLC (OJ-H+IB, CO2:hexanes:i-propanol 90:9:1, 0.8 mL/min, 35°C, 220nm): 93%ee (R)
tr(1) = 39.4 min
tr(2) = 41.0 min
25
[α]D (CHCl3, c = 1.42 g/L) = -49°
Overall conversion: 95%
Conversion to product (R)-1d: 93%
(Conversion to Nef-product: 2%)
(R)-1-Bromo-4-(2-nitropropyl)benzene ((R)-1e)
1
H-NMR (500 MHz, CDCl3): δ(ppm) = 1.55 (3H, d, J = 6.7 Hz, H-C3), 2.98 (1H, dd, J = 14.2 Hz,
6.4 Hz, H-C1), 3.27 (1H, dd, J = 14.2 Hz, 7.8 Hz, H-C1), 4.75 (1H, m, H-C2), 7.03-7.45 (4H, m,
H-aromatic)
The spectroscopic data are in accordance with literature data reported in ref. [6].
HPLC (OD-H, CO2:hexanes:i-propanol 90:9:1, 0.8 mL/min, 30°C, 220 nm): 81%ee (R)
tr(1) = 23.3 min
tr(2) = 25.0 min
[α]D25 (CHCl3, c = 1.62 g/L) = -33°
Overall conversion: 31%
Conversion to product (R)-1e: 27%
(Conversion to Nef-product: 4%)
9
(R)-1-Methoxy-4-(2-nitropropyl)benzene ((R)-1f)
1
H-NMR (500 MHz, CDCl3): δ(ppm) = 1.53 (3H, d, J = 6.6 Hz, H-C3), 2.96 (1H, dd, J = 14.1 Hz,
6.7 Hz, H-C1), 3.25 (1H, dd, J = 14.1 Hz, 7.5 Hz, H-C1), 3.79 (3H, s, H-C4), 4.73 (1H, m, H-C2),
6.83-7.09 (4H, m, H-aromatic)
The spectroscopic data are in accordance with literature data reported in ref. [5].
HPLC (OD-H, CO2:hexanes:i-propanol 90:9:1, 0.8 mL/min, 20°C, 220nm): 66%ee (R)
tr(1) = 23.3 min
tr(2) = 27.1 min
25
[α]D (CHCl3, c = 1.16 g/L) = -25°
Overall conversion: 53%
Conversion to product (R)-1f: 30%
(Conversion to Nef-product: 23%)
(R)-(2-Nitropropyl)benzene ((R)-1g)
1
H-NMR (500 MHz, CDCl3): δ(ppm) = 1.55 (3H, d, J = 6.6 Hz, H-C3), 3.01 (1H, dd, J = 14.0 Hz,
6.9 Hz, H-C1), 3.33 (1H, dd, J = 14.0 Hz, 7.4 Hz, H-C1), 4.78 (1H, m, H-C2), 7.16-7.34 (4H, m,
H-aromatic)
The spectroscopic data are in accordance with literature data reported in ref. [5].
HPLC (OJ-H, hexanes:i-propanol 90:10, 0.8 mL/min, 25°C, 230nm): 90%ee (R)
tr(1) = 13.9 min
tr(2) = 15.2 min
25
[α]D (CHCl3, c = 1.75 g/L) = -29°
Overall conversion: 76%
Conversion to product (R)-1g: 71%
(Conversion to Nef-product: 5%)
10
9. Chiral HPLC- chromatograms of racemic nitroalkanes 1
11
12
13
14
10. Chiral GC-chromatogram of racemic nitroalkane 1b
GC (FS-Hydrodex-
-Nagel, carrier gas H2, 60 °C for 5 min., 5
°C/min to 150 °C for 110 min, 60 kPa, FID)
tr(1)= 70.8 min
tr(2)= 73.3 min
15
11. 1H NMR-Spectra of nitroalkanes 1
16
17
18
12. References
[1]
N. Richter, H. Gröger, W. Hummel, Appl. Micribiol. Biotechnol. 2011, 89, 79-89.
[2]
M. M. Bradford, Anal. Biochem. 1976, 72, 248-254.
[3]
M. D. Bruch, NMR Spectroscopy Techniques, Practical Spectroscopy Series, vol. 21, 2.
ed., Marcel Dekker, New York, 1996, p. 241.
[4]
A. Fryszkowska, K. Fisher, J. M. Gardiner, G. M. Stevens; J. Org. Chem. 2008, 73, 42954298.
[5]
Y. Kawai, Y. Inaba, N. Tokitoh, Tetrahedron: Asymmetry 2001, 12, 309-318.
[6]
N. M. Goudgaon, P. P. Wadgaonkar, G. W. Kabalka, Synth. Commun. 1989, 19, 805-811.
19