1
Supporting Information
Stereoselective Enzymatic Synthesis of Heteroatom
-
Substituted Cyclopropanes
Oliver F. Brandenberg
1
, Christopher K. Prier
1,†
, Kai Chen
1
,
Anders M. Knight
2
,
Zachary
Wu
1
,
Frances H. Arnold
1*
1
Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200
East California Boulevard, Pasadena, California 91125, USA.
2.
Division of
Biology and Bioengineering
, California Institute of
Technology, 1200 East
California Boulevard, Pasadena, California 91125, USA.
†
Present address:
Merck Research Laboratories, Merck & Co., P.O. Box 2000, Rahway,
New Jersey 07065, USA
.
*
Corresponding author: Frances H
.
Arnold
frances@cheme.caltech.edu
Supporting Information content:
I
Experimental Procedures
p.
2
II
Supplementary Tables
p.
11
III
Supplementary Figures
p.
1
6
IV
Characterization of reaction products
p.
2
4
V
NMR spectra
p.
39
V
I
Nucleotide and amino acid sequences of P411 variants
p.
5
3
VI
I
Supplementary References
p.
5
8
2
I
.
Experimental Procedures
General
Unless otherwise noted, all chemicals and reagents were obtained from commercial
suppliers (Sigma
-
Aldrich, VWR, Alfa Aesar) and used without further purification. Silica gel
chromatography was carried out using AMD Silica Gel 60, 230
-
400 mesh.
Synthetic r
eactions
were monitored using thin layer chromatography (Merck 60 gel plates) using a UV
-
lamp for
visualization.
1
H and
13
C NMR spectra were recorded on a Varian Inova 300 MHz or 500 MHz,
or Bruker Prodigy 400 MHz instrument, in CDCl
3
and are internally referenced to the resi
dual
solvent peak.
Data for
1
H NMR are reported as follows: chemical shift (
δ
ppm), multiplicity (s =
singlet, d = doublet, t = triplet, q = quartet, p = pentet, m = multiplet, dd = doublet of doublets, dt
= double
t of triplets, ddd = doublet of doublet of doublets), coupling constant (Hz), integration.
High
-
resolution mass spectra were obtained at the California Institute of Technology Mass
Spectr
ometry
Facility.
Chromatography.
Analytical high
-
performance liquid
chromatography (HPLC) was carried out
using an Agilent 1200 series instrument and a
n Agilent Eclipse XDB
-
C18 column
(4.6
x
1
50
mm,
5 μm) with water and acetonitrile as the mobile phase
s
.
Exemplary HPLC runs for
N
-
vinyl
phthalimide cyclopropanation are show
n in Figure
S
7
.
For quantitative HPLC analysis of
N
-
vinylphthalimide cyclopropanation reaction products, c
alibration curves
using ethyl benzoate as
internal standard were generated (Figure
S
8
)
. The identity of the
reaction
products was confirmed
by HPLC co
-
injections of reaction mixtures with chemically synthesized authentic products,
and
by NMR analysis of products isolated from reactions performed on preparative scale.
C
yclopropanation product
enantiomers
were separated us
ing
chiral
SFC with isopropanol and
liquid CO
2
as the mobile phase, or chiral
HPLC
with
isopropanol and
hexane
as the mobile phase.
Product enantiomers were separated using Chiral
pak
IA, IC
,
or OJ
-
H
column
s
(4.6 x
2
50 mm, 5
μm) from Chiral Technologies
Inc.
Cloning and site
-
saturation
mutagenesis.
pET22b(+)
with the pelB leader sequence removed
was used as a cloning
and expression vector for all constructs
described in this study.
Site
-
saturation libraries were generated employing the
22c
-
trick method
1
.
The
resulting
PCR products
were gel purified, digested w
ith DpnI, repaired using the method of Gibson
2
, and used to directly
transform
E. coli
strain BL21(DE3).
3
Determination of P411 concentration.
The
concentration of P411 enzymes in whole cell
experiments was determined from ferrous carbon monoxide binding diffe
rence spectra using the
previously reported extinction coefficient for serine
-
ligated enzymes (
ε
= 103,000 M
–
1
cm
–
1
)
3
.
The
concentration of purified P411 enzymes was determined by quantifying the amount of free hemin
using the pyridine/hemochrome assay
using
the
corresponding
exti
nction coefficient (
ε
= 191,500
M
–
1
cm
–
1
)
4
.
Expression and
small
-
scale
N
-
vinylphthalimide
cyclopropanation
bioconversions using
whole cells.
E. coli
BL21(DE3) cells transformed with plasmid encoding P411 variants were
grown overnight in
3
mL Luria
-
Bertani medium with 0.1 mg/mL
ampicillin (LB
amp
) at
30 ºC and
250 rpm
.
P
reculture
s
w
ere centrifuged (3000 g, 3 minutes), the supernatant
was
discarded,
and
cell
pellets
were
resuspended in 3 mL fresh LB
amp
and
used to inoculate 4
7
mL of Hyperbroth medium
(prepared from AthenaES© powder, 0.1 mg/mL
ampicilli
n
) in a 125 mL Erlenmeyer flask.
C
ulture
s
w
ere
incu
bated at 37 ºC, 230 rpm for 2 h, typically reaching an OD
600
=
1.5
.
Cultures
were then cooled on ice (20 min) and induced with 0.5 mM
isopropyl β
-
D
-
1
-
thiogalactopyranoside
(
IPTG
)
and 1.0 mM 5
-
aminolevulinic acid
(ALA)
(final concentrations). Expression was
conducted at
22 ºC
, 130 rpm, for 16
-
18 h. Cultures were then centrifuged (
3000
g,
5
min, 4 ºC)
and the pellets
were
resuspended to OD
600
= 30 in M9
-
N
minimal medium (47.7 mM Na
2
H
PO
4
,
22.0 mM KH
2
PO
4
, 8.6 mM NaCl, 2.0 mM MgSO
4
, and 0.1 mM CaCl
2
, pH
6.8
).
To determine
P411 expression level
s
, a
liquots of the cell suspension (4
mL) were
lysed by sonication (Qsonica
Q500 sonicator),
and
the lysate
was
cleared by centrifugation (13,000 g, 10 min, 4
º
C) and
subjected to the carbon monoxide binding assay specified above
.
Small
-
scale
cy
c
lopropanation reaction
s
were set up in 2 mL crimp vials at 500 μL reaction volume.
P411
-
expressing cells
at OD
600
= 30 i
n M9
-
N were degassed by sparging with argon in sealed 6
mL crimp vials for at least
3
0 minutes. Separately,
a
glucose
solution
(
500
mM in M9
-
N) was
degassed by sparging with argon for at least ten minutes.
Following argon sparging, cells, glucose
solution
,
and substrate solutions were
transferred into an anaerobic chamber.
The c
ells (
440 or
465
μL) were added to
2 mL crimp
vials, followed by glucose
solution
(
10 μL), olefin
(
12.5 or
25
μL of a DMSO stock), and
EDA
(
12.5 or 25 μL of an EtOH
stock). Final concentrations were
typically
5 to 30 mM olefin
,
10 to 60
mM
EDA
,
10
mM glucose
, and 5 or 10% cosolvent.
The
4
vials were sealed, removed from the anaerobic chamber, and shaken at room temperature,
5
0
0 rpm
for 16
–
20
h. The reactions were quench
ed by adding acetonitrile (
1
.5
mL
)
supplemented with
internal standard (
ethyl benzoate, to 0.5 mM final concentration
). This mixture was transferred to
a
2 mL
microcentrifuge tube and centrifuged at 1
3
,000
g
for 10 minutes.
The
cleared
supernatant
was tran
sferred to a vial and analyzed by HPLC.
Site
-
saturation library
sc
reening in 96
-
well plate format.
Single colonies of
E. coli
cells
transformed with site
-
saturation
libraries were
picked with sterile toothpicks and
cultured in
96
-
well deep
-
well plates in
LB
amp
(300 μL/well) at 37 ºC, 2
5
0 rpm
,
overnight.
In a fresh 96
-
well deep
-
well plate, h
yperbroth medium (
950
μL/well, 0.1 mg/mL
ampicillin) was inoculated with the
preculture
s
(50
μ
L/well) and incubated at 37 ºC, 2
5
0 rpm, for
2.5
h. The plates were cooled on ice
for
2
0 minutes and then induced with 0.5 mM IPTG and 1.0 mM
ALA
(final concentrations).
Expression was conducted at 20 ºC,
20
0 rpm for 2
0
h.
The cells were pelleted (3,000
g, 5 min
),
the supernatant discarded
,
and the 96
-
w
ell
plate
s were
transferred to an anaerobic chamber. In the
anaerobic chamber,
cell pellets were resuspended in
argon
-
sparged reaction buffer (
20
mM glucose
in M9
-
N,
47
0 μL/well),
and stocks of
olefin
(1
5
μ
L/well, in DMSO) and
EDA
(1
5
μL/well, in
EtOH
)
wer
e added
.
Substrate concentration varied in the course of directed evolution between 10
and 30 mM for both EDA and
N
-
vinylphthalimide.
In
later screening round
s
,
cells were
resuspended in 440 μL
M9
-
N/glucose buffer
followed by addition of 30 μL of each subs
trate
stock,
in order to achieve higher co
-
solvent concentrations and thus aid with
N
-
vinylphthalimide
solubility at higher substrate concentrations
.
The plate
s
w
ere
sealed with aluminum foil, removed
from the anaerobic chamber, and shaken at
5
0 rpm. After
overnight incubation
, the seal was
removed and acetonitrile (
5
00 μL/well)
supplemented with
internal standard (
ethyl benzoate, to
0.5 mM final concentration
) w
as
added. The
plates were
re
sealed, briefly mixed by vortexing, and
incubated for 30
min to 1
h
at room temperature
. T
he plate
s
w
ere
then
centrifuged (5,000
g, 10
min), and the
cleared
supernatant was filtered through an AcroPrep 96
-
well filter plate (0.2
μ
m
cutoff
) into a shallow
-
well plate for HPLC analysis.
Protein purification.
E. co
li
BL21(DE3) cells
freshly
transformed with plasmid encoding P411
variants were grown overnight in 25 mL LB
amp
(3
0
ºC, 250 rpm).
Hyperbroth medium (
500
mL,
0.1 mg/mL
ampicillin) in a
2.8
L flask was inoculated with 2
0
mL of the preculture and inc
ubated
5
at
37 ºC, 230 rpm for 2
h (
to
OD
600
ca. 1.
5
). Cultures were then cooled
in an
ice
-
water bath for 2
0
min and induced with 0.5 mM IPTG and 1.0 mM
ALA
(final concentrations). Expression was
conducted at 2
2
ºC, 1
4
0 rpm, for 16
-
20 h. Cultur
es were then centrifuged
(5,000
g,
10
min, 4 ºC)
and the cell pellets
were
frozen at
–
20 ºC. For
protein
purification, frozen cells from two such
cultures were resuspended in buffer A (25 mM tris, 20 mM imidazole, 100 mM NaCl, pH 7.5, 4
mL/g of cell wet weight), loaded with hemin
(1 mg/gram wet cell weight) and lysed by sonication.
To pellet insoluble material, ly
sates were centrifuged (20,000
g, 20 min, 4 ºC). P
411 proteins
were
purified
from the lysate
using a nickel NTA column (1 mL HisTrap HP, GE Healthcare,
Piscataway, NJ) using an AKTAxpress purifier FPLC system (GE healthcare). P411 enzymes were
eluted on a linear gradient from 100% buffer A to 100% buffer B
(25 mM tris, 300 mM imidazole,
100 mM NaC
l, pH 7.5)
over 10 column volumes.
Fractions containing eluted protein were pooled
and subjected to three rounds of buffer exchange to storage buffer (25 mM Tris
-
HCl, 25 mM NaCl,
pH 7.5) using centrifugal spin filters (10 kDa molecular weight cut
-
off, Amic
on Ultra, Merck
Millipore).
Subsequently, the concentrate
d
protein was aliquoted, flash
-
frozen on powdered
dry
ice, and stored at
–
80 ºC.
Protein concentrations were determined via
the pyridine/hemochrome
assay specified above.
Cyclopropanation reactions
using purified protein.
Portions of
M9
-
N
buffer
(2
9
0 μL) and
NADPH (40 μL,
5
0 mM
in M9
-
N
), or multiples thereof, were combined in a 6 mL crimp vial and
degassed by sparging with argon for at least 30 minutes.
Purified protein solutions were adjusted
to 25
μM in M9
-
N.
After degass
ing was complete, the M9
-
N/NADPH solution and
purified protein
stocks
were brought into the anaerobic chamber.
Reactions were set up on a 400 μL scale in 2 mL
crimp vials: first, 330 μL of the M9
-
N/NADPH solution were added per via
l, followed by 40 μ
L
of purified protein solution.
Next, 20 μL of
N
-
vinylphthalimide stock solution (100 mM in DMSO)
were added, followed by 10 μL of EDA stock solution (
200 mM in
EtOH
). Final concentrations
were typically 5 mM
N
-
vinylphthalimide
, 5 mM
EDA
,
5
mM NADPH
,
and
2.
5 μM P411
, with
7.5% co
-
solvent
. The vials were sealed, removed from the anaerobic chamber, and shaken at room
temperature, 40 rpm for
1 to
6
h. The reactions were quenched by adding acetonitrile (400 μL)
supplemented with
internal stan
dard (
ethyl benzoate, to 0.5 mM final concentration
). This mixture
was transferred to a microcentrifuge tube and centrifuged at 14,000 rpm for 10 minutes. The
supernatant was transferred to a vial and analyzed by HPLC.
6
Determination of initial rates.
(i)
With purified protein:
Reactions were set up as described above
(
Cyclopropanation reactions using purified protein
).
R
eactions were quenched in 1
-
minute
intervals (starting 2 min after the start of the reaction) by addition of 400 μL acetonitrile containing
internal standard, followed by vigorous mixing. The samples were then worked up and analyzed
as described above.
(ii
)
With whole cells: Reactions were set up as described above (
Expression
and small
-
scale N
-
vinylphthalimide cyclopropanation bioconversions using whole cells
).
R
eactions were quenched in 1
-
minute intervals (starting 2 min after the start of the reaction) b
y
addition of 500 μL acetonitrile, followed by vigorous mixing.
The vials were removed from the
anaerobic chamber and 1 mL of acetonitrile supplemented with internal standard (ethyl benzoate,
to 0.5 mM final concentration) was added. Further sample work
-
up
and
HPLC
analysis was
performed as described above.
Preparative scale reactions.
E. coli
BL21(DE3) cells freshly transformed with plasmid encoding
P411 variants were grown overnight in 25 mL LB
amp
(30 ºC, 250 rpm).
Hyperbroth medium (500
mL, 0.1 mg/mL
ampicillin) in a 2.8 L flask was inoculated with 20 mL of the preculture and
incubated at 37 ºC, 230 rpm for 2 h (
to
OD
600
ca. 1.5). Cultures were then cooled in an ice
-
water
bath for 20 min and induced with 0.5 mM IPTG and 1.0 mM
ALA
(final concentrations
).
Expression was cond
ucted at 22 ºC, 140 rpm, for 16
-
20 h. Culture
s were then centrifuged (5,000
g,
5 min, 4 ºC) and the cell pellets were
resuspended to OD
600
=
5
0 in M9
-
N
buffer supplemented
with 20 mM glucose (typically yielding ca. 110 mL of cell susp
ension)
. Aliquots of the cell
suspension (4 mL) were used to determine the P411 expression level after
cell
lysis by sonicat
ion.
A
liqu
o
ts of t
he cell suspension
(23 mL)
w
ere
then transferred to
50
mL Erlenmeyer flask
s
and
degassed by sparging with argon for at least
3
0 minutes. The reaction flask
s
w
ere
then transfe
rred
into an anaerobic chamber and olefin stock solution
(
1
mL, in DMSO) and
EDA
(
1
mL, in
EtOH
)
were added
. Final concentrations were typically
5 to 20
mM
ol
efin and
10 to 40
mM
EDA
,
with
8
% co
-
solvent
. The flask
s were
sealed with parafilm, removed from the anaerobic chamber, and
shaken at room temperature, 1
0
0 rpm for
18
h. The reaction
s were
quenched by adding acetonitrile
(
25
mL) and then centrifuged (4,000
g, 10 min). The supernatant was concentrated
in vacuo
to
remove acetonitrile
and extracted with EtOAc (3 x 25 mL). The organic layers were washed with
brine (20 mL), dried over MgSO
4
, filtered, concentrated, and purified by
silica gel
chromatography.
7
Assi
gnment of absolute configuration
.
To assign the absolute configuration of the
trans
-
configured
N
-
vinylphthalimide cyclopropanation product (
ethyl
trans
-
2
-
(1,3
-
dioxoisoindolin
-
2
-
yl)cyclopropane
-
1
-
carboxylate
,
3b
)
, purified product from preparative
-
scale
enzymatic reactions
with P411
-
VAC
trans
was subjected to crystallization and X
-
ray structure determination
(Table S
1
)
.
Low
-
temperature diffraction data (
-
and
-
scans) were collected on a Bruker AXS D8 VENTURE
KAPPA diffractometer coupled to a PHOTON 100 CMOS detector with Cu
K
radiation (
=
1.54178 Å) from an I
μ
S micro
-
source. The structure was solved by direct methods using SHELXS
and refined against
F
2
on al
l data by full
-
matrix least squares with SHELXL
-
2016
5
using
established refinement techniques. All non
-
hydrogen atoms were refined anisotropically. All
hydrogen atoms were included into the model at geometrically calculated positions and refined
using a riding model. Th
e isotropic displacement parameters of all hydrogen atoms were fixed to
1.2 times the
U
value of the atoms they are linked to (1.5 times for methyl groups).
Ethyl
trans
-
2
-
(1,3
-
dioxoisoindolin
-
2
-
yl)cyclopropane
-
1
-
carboxylate
3b
crystallizes in the orthorhom
bic space
group
P
2
1
2
1
2
1
with one molecule in the asymmetric unit.
The absolute configuration was assigned
as
trans
-
(1
S
,
2
S
)
(Figure S1)
.
The absolute configurations of other cyclopropanes formed by
P411
-
VAC
trans
were assigned by analogy.
Crystallographic coordinates and structure factors
for
e
thyl
trans
-
2
-
(1,3
-
dioxoisoindolin
-
2
-
yl)cyclopropane
-
1
-
carboxylate
3b
have been deposited with the
Cambridge Crystallographic Data Centre (https:// www.ccdc.cam.ac.uk/) under reference number
1815565
.
A
ttempts to crystallize the
cis
-
configured
N
-
vinylphthalimide cyclopropanation product were
unsuccessful; furthermore, attempts to crystallize alternative
cis
-
configured products derived from
P411
-
VAC
cis
(Table 1) were unsuccessful as well. We therefore d
etermined the selectivity of
P411
-
VAC
cis
in the cyclopropanation reaction of styrene with EDA (Figure S
2
), as previously
described
6
. To this end, whole
-
cell reactions with
E. col
i
cells expressing P411
-
VAC
cis
at
OD
600
=30 were
performed
with 10 mM styrene and 10 mM EDA, following the protocol described
above (
Expression and small
-
scale N
-
vinylphthalimide cyclopropanation bioconversions using
whole cells
). Reactions were extracted with cyclohexane and analyzed by GC using a Cyclosil
-
B
column (30m x 0.32 mm x 0.25 μm),
oven temperature = 100
°C
for
5 min, 1
°C per min to 135°C,
135
°C
for
10 min, 10
°C per min to 200
°C, 200
°C
for
5
min
6
. Using a racemic product standard
for comparison
and a previously developed assay
6
, the major styrene cyclopropanation product
8
isomer derived with P411
-
VAC
cis
was thereby assigned as (1
S
,
2
R
), and we assigned the absolute
configuration of other
cyclopropanes formed by
P411
-
VAC
cis
(or its single point mutation
derivatives)
by
analogy
. Of note, we also included P411
-
VAC
trans
in this experiment; the major
styrene cyclopropanation product isomer
obtained with this variant is the (1
S
,
2
S
) isomer, in
agreement with the absolute configuration obtained by crystal structure determination
(Figures S1,
S2)
.
Figure S1
:
Crystal structure of
ethyl
trans
-
2
-
(1,3
-
dioxoisoindolin
-
2
-
yl)cyclopropane
-
1
-
carboxylate, 3b.
The absolute configuration of
3b
was assigned as (1
S
,
2
S
).
9
Table S1
. Crystal data and structure refinement for
ethyl (1S,
2S)
-
2
-
(1,3
-
dioxoisoindolin
-
2
-
yl)
cyclopropane
-
1
-
carboxylate (
3b
).
Empirical formula
C
14
H
13
NO
4
Formula weight
259.25
Temperature
100 K
Wavelength
1.54178 Å
Crystal system
Orthorhombic
Space group
P2
1
2
1
2
1
Unit cell dimensions
a = 6.6275(5) Å,
α
= 90°
b = 8.0167(7) Å,
β
= 90°
c = 22.6419(19) Å,
γ
= 90°
Volume
1202.98(17) Å
3
Z
4
Density (calculated)
1.431 Mg/m
3
Absorption coefficient
0.883 mm
-
1
F(000)
544
Crystal size
0.350 x 0.300 x 0.250 mm
3
Theta range for data collection
3.905 to 74.466°
Index ranges
-
8<=h<=8,
-
10<=k<=10,
-
28<=l<=25
Reflections collected
13445
Independent reflections
2442 [R(int) = 0.0319]
Completeness to theta = 67.679°
99.8 %
Absorption correction
Semi
-
empirical from equivalents
Max. and min. transmission
0.7538 and 0.6780
Refinement method
Full
-
matrix
least
-
squares on F
2
Data / restraints / parameters
2442 / 0 / 173
Goodness
-
of
-
fit on F
2
1.154
Final R indices [I>2sigma(I)]
R1 = 0.0334, wR2 = 0.0889
R indices (all data)
R1 = 0.0336, wR2 = 0.0892
Absolute structure parameter
0.07(5)
Extinction
coefficient
n/a
Largest diff. peak and hole
0.218 and
-
0.329 e.Å
-
3