S
0
Supporting
Information
Enzymatic Lactone
-
Carbene C‒H Ins
ertion to Build
Contiguous
Chi
ral Centers
Andrew Z. Zhou, Kai Chen,
*
Frances H. Arnold
*
a
Division of Chemistry and Chemical Engineering 210
-
41, California Institute of
Technology, Pasadena, CA 91125, USA.
*
To whom correspondence should be addressed. E
-
mail:
chenkaikc1990@gmail.com
and
frances@cheme.caltech.edu
This PDF file includes:
I.
General Procedures
S
1
-
S
3
II.
Directed Evolution
of P411
-
C10
for
Lactone
-
Carbene C
‒H Insertion
S
4
-
S
6
III.
Screening of
C10
Lineage for Activity on Different Substrates
S
7
-
S
1
2
IV
.
Preparation
and Characterization of
β
-
A
mino
L
actone
P
roducts
S
1
3
-
S
1
9
V.
Analysis
of
Enzymatic
Lactone
-
Carbene C
‒H Insertion
S
20
-
S
4
8
V
I
.
Sequence Information
S
4
9
-
S
5
8
VI
I
.
NMR Spectra
S
5
9
-
S
7
8
V
I
II.
References
S
7
9
S
1
I.
G
e
neral 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.
1
H and
13
C
NMR spectra were
taken using a
Bruker Prodigy 400 MHz
instrument and are internally referenced to the residual solvent peak (chloroform)
. 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 =
doublet of triplets, ddd = doublet of doublet of doublets), coupling constant (Hz),
integration. Sonication was performed using a Qsonica Q500 sonicator. High
-
resolution
mass spectra we
re obtained at the California Institute of Technology Mass Spectral
Facility. Synthetic reactions were monitored using thin layer chromatography (Merck 60
gel plates) using a UV
-
lamp for visualization.
Chromatography.
Analytical
reversed
-
phase
high
-
performance liquid
chromatography (HPLC) was carried out using an Agilent 1200 series instrument and a
Kromasil
C18 column (4.6
×
50 mm, 5 μm)
with water and acetonitrile as the mobile phase
and visualization at 2
3
0 nm for library screening
.
Analytica
l normal
-
phase HPLC was
carried out using an Agilent 1200 series instrument and chiral columns
Chiralpak
IC
/IA/IB/OJ
-
H/OD
-
H
(4.6 mm
×
25 cm)
with
n
-
hexane and isopropanol as the mobile
phase and visualization at
230 or
2
54
nm for chiral separation.
Cloning and site
-
saturation
mutagenesis.
Vector
pET22b(+)
containing a C
-
terminal 6x
-
His tag
was used
for
cloning and expression
of
all enzymes described in this
study. Site
-
saturation
mutagenesis was performed using a modified Qu
ikChange
TM
mutagenesis pro
tocol
.
1
Primer sequences are available upon request.
The PCR products
were digested with
Dpn
I, gel purified, and
fragments were assembled
using Gibson Mix
.
2
The
Gibson assembly products
w
ere
used to directly transform
E
scherichia
coli
strain
BL21
E. cloni
®
(Lucigen).
Cells were grown using Luria
-
Bertani medium
(LB)
or
Hyper
b
roth (AthenaES)
(HB)
with
0.1 m
g/mL ampicillin (LB
amp
or HB
amp
).
Electrocompetent
E. coli
cells were prepared following the protocol of Sambrook
et al
.
3
T5 exonuclease, Phusion polymeras
e, and Taq ligase were purchased from New England
Biolabs (NEB, Ipswich, MA). M9
-
N minimal medium (abbreviated as M9
-
N buffer; pH
7.4) was used as a buffering system for whole cells, lysates, and purified proteins, unless
otherwise specified. M9
-
N buffer w
as used without a carbon source; it contains 47.7 mM
Na
2
HPO
4
, 22.0 mM KH
2
PO
4
, 8.6 mM NaCl, 2.0 mM MgSO
4
, and 0.1 mM CaCl
2
.
Determination of
hemeprotein
co
ncentration.
1.
Preparation of cell lysate
: Aliquots of ~3 mL OD
600
= 60 cells were prepared in
15
-
mL conical tubes, which were then placed on wet ice. Cells were lysed by sonication
following the
program
below: sonication for 4 min, 1 second on
-
1 second off, 35%
amplitude. The sonicated samples were then transferred to two Eppendorf tubes, and then
c
entrifuged down (14,000 rpm, 15 min, 4 ºC). The supernatants
(
~
2.5
mL
)
were then
collected
to
a
5
-
mL glass vial for analysis.
2.
Hemechrome assay for protein concentration measurement
:
A solution of
S
2
NaOH/pyridine was prepared by mixing 1 mL of NaOH aqueous
solution (1 M), 2 mL of
water and 2 mL of pyridine. To 4.5 mL of NaOH/pyridine solution, 22.5 μL of K
3
Fe(CN)
6
aqueous solution
(0.1 M) w
ere
added to make
solution 1
. A
background solution
was
prepared by mixing 500 μL M9
-
N and 500 μL of the NaOH/pyridine solution, which was
used for UV background subtraction. When measuring samples
with
a UV
spectrometer
, a
spectrum of a mixed solution (oxidized spectrum) with 500 μL cell lysate + 500 μL
s
olution 1
was taken at the wavelength range 380 nm to 650 nm. Subsequently, 5 μL of
dithionite solution (0.5 M in 0.1 M NaOH solution) w
ere
added to the same sample and
mixed by pipetting; a spectrum of this solution (reduced spectrum) was taken at 380 nm
to
650 nm. The protein concentration was calculated using the extinction coefficient and
dilution factor (2
X
dilution in volume): ε_[55
7
reduced
‒ 540
oxidized
] = 23.98 mM
‒
1
cm
‒
1
.
4
Expression
of
P411
proteins
.
E. coli
BL21
E. cloni
®
cells carrying a plasmid
encoding a
P411
variant were grown overnight in 5 mL LB
amp
(
37 ºC, 2
2
0 rpm). The pre
-
culture was used to inoculate 45 mL of HB
amp
in a 125
-
mL Erlenmeyer flask; this culture
was incubated at 37 ºC, 2
2
0 rpm for 2 h
and 15 min
. Cultu
res were then cooled on ice (
4
0
min), and expression was induced with 0.5 mM
i
sopropyl β
-
D
-
1
-
thiogalactopyranoside
(
IPTG
)
and 1.0 mM 5
-
aminolevulinic acid (final concentrations). Expression was
conducted at room temperature (2
4
or 22
ºC), at 1
4
0
(or 150)
rpm, for
20
h
(± 20 min)
.
Cultures were then centrifuged (
4
,
5
00
×
g,
5
min, 4 ºC), and the pellets were resuspended
to an OD
600
of
6
0 in M9
-
N
buffer
. Aliquots of the cell suspension (
3
mL) were used to
determine
protein concentration
after lysis by sonicat
ion.
The expression
level
in
OD
600
=
6
0
lysates is typically in the range
of
3
‒
1
3
μM for
the
P411
-
C10
variant
s
.
Biotransformations.
All the biocatalytic reactions were set up in
an anae
robic
chamber
(oxygen level: < 40 ppm)
. Resuspended cells
(340 μL
, diluted to
a given
OD
600
with M9
-
N minimal buffer
) were added to 2 mL vials, followed by
D
-
glucose (40 μL, 250
mM in M9
-
N),
aniline derivatives
(10 μL of an EtOH stock
, 400
or 480
mM), and
α
-
diazo
-
γ
-
lactone
(
LAD
,
10 μL of an EtOH stock
, 400
or 480
mM
). Final concentrations were
typically
1
0.0
or 12.0
mM
aniline derivative
,
1
0.0
or 12.0
mM
LAD
, and 25 mM
D
-
glucose; final reaction volume was 400 μL. The vials were sealed,
shaken in
s
ide the
anaerobic chamber
at room temperature
for a set time
(600 rpm
)
.
After the reaction w
as
completed
and
the vials
removed
from the
anae
robic chamber
, internal standard 1,3,5
-
trimethoxybenzene
(1,3,5
-
TMOB)
,
p
-
methyl anisole
(pMe
-
anisole)
,
ethyl 2
-
phenylacetate
(PhEA)
,
or allyl phenyl ether
(AllylOPh)
(20 μL of 20 mM stock solution in
acetonitrile)
was added followed by acetonitrile
(
0.58
mL). The mixture was transferred to a 1.
7
-
mL
Eppendorf
tube, and then subjected to vortex
ing
(15 s × 3) and centrifugation (14,000 rpm,
5 min
, 4 °C
).
A sample of the
s
upernatant
(
0.8 mL
)
was
transferred to a vial
for
reverse
-
phase HPLC
analysis.
Reaction screening in 96
-
well plate format.
Libraries (s
ingle
site
-
saturation libraries
generated employing the “22c
-
trick” method
1
or collections of heme protein variants
) were
screened
in
96
-
well plate
s
.
E. coli
libraries
for P411 variants
were cultured in LB
amp
(3
5
0 μL/well) at 37 ºC, 2
5
0
rpm and 80% relative humidity overnight.
HB
amp
(
95
0 μL/well) was inoculated with the
pre
-
culture (50 μL/well) and incubated at 37 ºC, 2
3
0 rpm, 80% humidity for
2
h
and 45
S
3
min
. The plates were co
oled on ice for 30 minutes, and
expression was induced with
0.5
mM IPTG and 1.0 mM 5
-
aminolevulinic acid (final concentrations). Expression was
conducted at 2
2
ºC and
2
20
rpm for
20
h.
The cells were pelleted (
4,5
00
×
g
, 5 min, 4 ºC) and resuspended
with M9
-
N
buffer
(
340
μL/well)
and
D
-
glucose solution (40 μL/well
, in M9
-
N)
. The 96
-
well plate was then
transferred to an anaerobic chamber. In the anaerobic chamber,
aniline derivative
(10
μL/well, 400 mM in EtOH) and
LAD
(10 μL/well, 400 mM in EtOH)
were added
.
The
plate was sealed with aluminum foil
and shaken
inside
the anaerobic chamber
(600 rpm)
.
After
24
h,
the plate was moved out of the anaerobic chamber. T
he seal was removed
and acetonitrile (580 μL/well) and internal standard
(
1,3,5
-
trimethoxybenzene
,
p
-
methyl
anisole,
ethyl 2
-
phenylacetate
or allyl pheny
l ether
;
20 mM in acetonitrile
;
20 μL/well)
were added. The plate was tightly sealed with a reusable silicone mat
,
vortex
ed
(15 s × 3)
and centrifug
ed
(4,500
×
g, 5 min).
T
he supernatant
(200 μL/well)
was filtered through an
AcroPrep 96
-
well filter plate (0.2 μm) into a shallow
-
well plate for
reversed
-
phase
HPLC
analysis
(
C18
Kromasil
column, MeCN:H
2
O
50
:
5
0
, 1.2 mL/min flow,
230 or
2
54
nm
)
.