Journal of the American Chemical Society
Article
unless otherwise stated. All dry reactions were performed under argon
or nitrogen gas in flame-dried glassware with magnetic stirring by
using standard gastight syringes, cannula, and septa. 1H and 13C NMR
spectra were measured on a Bruker DPX-500 spectrometer (operating
addition of 20 μL of diazoketone (from 100 mM stock solution in
ethanol). The reaction mixture was stirred for 16 h at room
temperature inside an anaerobic chamber. The reactions were
prepared for chiral SFC analysis by adding 20 μL of internal standard
(50 mM benzodioxole in ethanol) to the reaction mixture, followed
by extraction with 400 μL of dichloromethane. The TON for the
whole-cell reactions was calculated based on Mb concentration in the
reaction mixture as measured via UV−vis spectroscopy (ε410 = 156
mM−1 cm−1) after cell lysis.
General Procedure for Synthesis of Diazoketones 2 and
6a−6n (Procedure A). The diazoketone reagents were prepared
from the corresponding carboxylic acids according to the following
procedure. To a flame-dried round-bottom flask, carboxylic acid (1.0
mmol) was dissolved in DCM (1.0 mL) under argon with a drop of
dimethylformamide as catalyst. After dropwise addition of thionyl
chloride (1.5 equiv), the reaction was stirred for 2 h at room
temperature. Solvent was removed in vacuo and used in the next step
without further purification. Synthesis of diazoketones from acetyl
chlorides was performed using a diazomethane generator (Aldrich)
with System 45 compatible connection. Caution: diazomethane is a
toxic and explosive gas, and it must be handled with great caution at all
times! In the outer tube of the diazomethane generator, acetyl chloride
(0.3 mmol) was dissolved in Et2O (3.0 mL), and in the inner tube,
diazald (5.7 equiv) was suspended in carbitol (1.0 mL). Once the
reaction mixture was immersed in an ice bath, aqueous KOH (37%,
1.5 mL) was injected dropwise via a syringe into the inner tube. After
stirring for 3 h at 0 °C, silicic acid (0.15 g) was added to the inner
tube to quench any unreacted diazomethane. Solvent in the outer
tube was removed, and the reaction mixture was subjected to column
chromatography to yield diazoketones 2 and 6a−6n. See the
General Procedure for Preparative-Scale Enzymatic Syn-
thesis of Cyclopropanation Products 3, 5a−5k, 7a−7n, and
8a−8c (Procedure B). To a round-bottom flask, 18 mL of 20 μM
myoglobin in argon-purged sodium borate buffer (50 mM, pH 9.0)
and 70 mg of sodium dithionite were added under argon. The
reaction was initiated by addition of 1 mL of 400 mM alkene solution
in ethanol (20 mM final concentration), followed by addition 1 mL of
100 mM diazoketone solution in ethanol (5 mM final concentration).
The reaction was stirred for 16 h at room temperature under argon.
After extraction with DCM (3 × 50 mL), organic layers were
combined and dried over NaSO4. The solvent was removed in vacuo,
and the reaction mixture was subjected to column chromatography to
yield cyclopropanes 3, 5a−5k, 7a−7n, and 8a−8c. See the Supporting
Racemic Standards. Analytical amounts of racemic products
were prepared from 400 μL scale cyclopropanation reactions with 1
mM Fe(TPP)Cl (10 mol %), 20 mM olefin, 10 mM diazoketone, and
10 mM sodium dithionite in toluene:H2O:EtOH (8:1:1). In a typical
procedure, a mixture containing 40 μL of sodium dithionite (100 mM
stock solution in distilled H2O) and 300 μL of toluene was degassed
by sparging with argon for 5 min in a sealed vial. A separate vial
containing 20 μL of Fe(TPP)Cl (20 mM stock solution in toluene)
was carefully degassed in a similar manner. The two solutions were
then mixed together via a cannula. Reactions were initiated by
addition of 20 μL of olefin (400 mM stock solution in ethanol),
followed by the addition of 20 μL of diazoketone (200 mM stock
solution in ethanol) with a syringe, and the reaction mixture was
stirred for 16 h at room temperature under positive argon pressure.
The product was isolated via preparative TLC (hexanes:EtOAc 9:1),
extracted with dichloromethane, and analyzed by chiral SFC, GC, or
1
at 500 MHz for H and 125 MHz for 13C) or a Bruker DPX-400
(operating at 400 MHz for 1H and 105 MHz for 13C). 19F was
measured on a Bruker DPX-400 (operating at 375 MHz). TMS was
used as the internal standard (0 ppm) for 1H NMR, CDCl3 was used
as the internal standard (77.0 ppm) for 13C NMR, and
trifluorotoluene served as the internal standard (−63 ppm) for 13F
NMR. Silica gel chromatography purifications were performed by
using AMD Silica Gel 60 230−400 mesh. Preparative thin layer
chromatography was performed on TLC plates (1 mm thickness,
Sigma-Aldrich).
Protein Expression and Purification. Wild-type sperm whale
myoglobin and the engineered Mb variants were cloned and expressed
in E. coli C41(DE3) cells as described previously.36 Briefly, cells were
grown in terrific broth (TB) medium (ampicillin, 100 mg L−1) at 37
°C (200 rpm) until OD600 reached 1.0−1.2. Cells were then induced
with 0.25 mM β-D-1-thiogalactopyranoside (IPTG) and 0.3 mM δ-
aminolevulinic acid (ALA). After induction, cultures were shaken at
180 rpm and 27 °C and harvested after 18−20 h by centrifugation at
4000 rpm at 4 °C. After cell lysis by sonication, the proteins were
purified by Ni-affinity chromatography. The lysate was transferred to a
Ni-NTA column equilibrated with Ni-NTA Lysis Buffer (50 mM KPi,
250 mM, NaCl, 10 mM imidazole, pH 8.0). The resin was washed
with 50 mL of Ni-NTA Lysis Buffer and then 50 mL of Ni-NTA Wash
Buffer (50 mM KPi, 250 mM, NaCl, 20 mM imidazole, pH 8.0).
Proteins were eluted with Ni-NTA Elution Buffer (50 mM KPi, 250
mM, NaCl, 250 mM histidine, pH 7.0). After elution, the proteins
were buffer exchanged against 50 mM KPi buffer (pH 7.0) by using
10 kDa Centricon filters. Protein concentration in ferric form was
determined by using ε410 = 156 mM−1 cm−1 as the extinction
coefficient.
Enzymatic Reactions with Purified Protein. Substrate screen-
ing reactions were performed at a 400 μL scale by using 20 μM
myoglobin, 20 mM styrene, 5 mM diazoketone, and 10 mM sodium
dithionite inside an anaerobic chamber. In a typical procedure, a
buffered solution containing the myoglobin variant was carefully
charged to a vial inside an anaerobic chamber. After the catalyst
solution was diluted with sodium borate buffer (50 mM, pH 9.0), 20
μL of sodium dithionite solution (200 mM stock solution in 50 mM
pH 9 sodium borate buffer) was added. Reactions were initiated by
addition of 20 μL of styrene (from 400 mM stock solution in
ethanol), followed by the addition of 20 μL of diazoketone (from 100
mM stock solution in ethanol). The reaction mixture was stirred for
16 h at room temperature inside an anaerobic chamber. The reaction
mixtures were added with 20 μL of internal standard (50 mM
benzodioxole in ethanol), followed by extraction with 400 μL of
dichloromethane. For determination of product yield, diastereomeric
excess, and enantiomeric excess, the extraction solutions were
analyzed by chiral SFC, chiral GC, or chiral HPLC as described in
performed in a similar manner by using 10 μM enzyme, 10 mM
styrene 1, 2.5 mM diazoketone 2, 10 mM Na2S2O4 in 50 mM sodium
borate buffer (pH 9.0) with 10% EtOH, room temperature, and an
anaerobic atmosphere. The reactions were quenched with 3 M HCl at
the different time points, followed by extraction and chiral SFC
analysis as described above.
Reactions with Whole Cells. Whole cell experiments were
performed at a 400 μL scale by using 370 μL of E. coli cells expressing
Mb(H64V,V68A), 20 mM styrene derivative, and 5 mM diazoketone
inside an anaerobic chamber. In a typical procedure, cells suspended
in potassium phosphate buffer (50 mM, pH 7.2) with 10% glycerol
were charged to a vial inside an anaerobic chamber. After the catalyst
solution was diluted with potassium phosphate buffer (50 mM, pH
7.2) with 10% glycerol, reactions were initiated by addition of 20 μL
of styrene (from 400 mM stock solution in ethanol), followed by the
ASSOCIATED CONTENT
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* Supporting Information
The Supporting Information is available free of charge at
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J. Am. Chem. Soc. 2021, 143, 2221−2231