of 111
www.sciencemag.org/content/
354/
6315/
1048/suppl/DC1
Supplementary
Material
s for
Directed evolution of cytochrome c for carbon
–silicon bond formation:
Bringing silicon to life
S. B. Jennifer Kan, Russell D. Lewis, Kai Chen, Frances H. Arnold*
*Corresponding author. E
-mail: frances@cheme.caltech.edu
Published 25 November 2016,
Science
354
, 1048
(2016)
DOI:
10.1126/science.
aah6219
This PDF file includes:
I. Materials and Methods
2
II. General Procedures
3-6
III. Supporting Tables S1 to S7 and
Figs. S1 to S6
7-19
IV. Substrate Synthesis and Characterization
20-
27
V. Synthesis and Characterization of Authentic Organosilicon Products
28-
33
VI. Preparative
-Scale Whole
-Cell Biocatalytic Reaction
34
VII. GC Standard Curves for Organosilicon Products
35-
41
III. Chiral SFC Traces for Racemic and Enzymatically Synthesized
Organosilicon Products
42-
61
IX. References
62-
66
X. NMR Spectra
67-
111
2
I. Materials and Methods
Unless
otherwise noted, all chemicals and reagents for chemical reactions wer
e
obtained from commercial suppliers (Acros,
Arch Bioscience, Fisher Scientific,
Sigma
-
Aldrich, TCI America) and used without further purification. The following proteins
were all purchased from Sigma
-
Aldrich: bovine serum albumin (BSA), cytochrome
c
(from
bovine, equine heart and
S. cerevisiae
), peroxidase II (from horseradish), and
catalase (from
C. glutamicum
). Silica gel chromatography purifications were carried out
using AMD Silica Gel 60, 230
-
400 mesh.
1
H and
13
C NMR spectra were recorded on
a
Bruker
Prodigy 400 MHz instrument and are internally referenced to
the
residual solvent
peak (chloroform).
29
Si NMR spectra were record
ed on the same instrument and
referenced to tetramethoxysilane (
δ
78.9 ppm).
Data for
1
H NMR are reported in the
conventional form: chemical shift (
δ
ppm), multiplicity (s
=
singlet, d
=
doublet, t
=
triplet, q
=
quartet,
hept = heptet,
m
=
multiplet, br
=
broad
, app = appears as
), coupling
constant (Hz), integration.
Data for
13
C
and
29
Si
are reported in terms of chemical shift (
δ
ppm). High
-
resolution mass spectra were obtained with a JEOL JMS
-
600H High
Resolution Mass Spectrometer at the California Institute of Technology Mass Spectral
Facility.
Sonication was perfo
rmed using a Qsonica Q500 sonicator. Chemical r
eactions
were monitored using thin layer chromatography (Merck 60 silica gel plates)
and
a UV
-
lamp for visualization. Gas chromatography (GC) analyses were carried out using a
Shimadzu GC
-
17A gas chromatograph
, a FID detector, and J&W HP
-
5 (30 m x 0.32 mm,
0.25
μ
m film
; 90 °C hold 1 min, 90 to
110
°C
at 15 °C
/min, 110 to 280 °C at 60 °C/min,
280 °C hold 1 min, 6.2 min total
).
Analytical chiral
supercritical fluid chromatography
(
SFC
)
was performed with a JACSO
2000 series instrument
using
i
-
PrOH and
supercritical CO
2
as the mobile phase, with visualization at 210 nm.
The following chiral
columns were used:
Daicel
Chiralpak IC,
Chiralpak
AD
-
H
,
or C
hiralcel OD
-
H (4.6 mm x
25 cm).
Plasmid
pET22 was used as a clonin
g vector,
and
cloning was performed using
Gibson
assembly (
31
). The cytochrome
c
maturation plasmid pEC86 (
32
) was used as
part of a two
-
plasmid system to express prokaryotic cytochrome
c
proteins
.
Cells were
grown using Luria
-
Bertani medium or HyperBroth
(AthenaES) with 100
μ
g/
mL
ampicillin and 20
μ
g/
mL chloramphenicol (LB
amp/chlor
or HB
amp/chlor
). Cells without the
pEC86 plasmid were grown with 100
μ
g/
mL ampicillin (LB
amp
or HB
amp
).
Primer
sequen
ces are available upon request.
Electrocompetent
Escherichia
coli
cells were
prepared following the protocol of Sambrook
et al.
(
33
). T5 exonuclease, Phusion
polymerase, and
Taq
ligase were purchased from New England Biolabs (NEB, Ipswich,
MA).
M9
-
N minimal medi
um
(
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
was 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
.
3
II. General Procedures
(A)
Plasmid construction.
All variants described in this paper were cloned and
expressed using the pET22
(
b
)+
vector (Novagen).
The gene encoding
Rma
cyt
c
(UNIPROT ID B3FQS5) was obtained as a single
gBlock (IDT), codon
-
optimized for
E.
coli
, and clone
d using Gibson
a
ssembly
(
31
)
into pET22(b)+ (Novagen) between
restriction sites
Nde
I and
Xho
I
in frame with an
N
-
terminal pelB lead
er sequence (to
ensure periplas
mic
localization and proper maturation; MKYLLPTAAAGLLLLAAQPA
MA) and a
C
-
terminal 6xHis
-
tag. T
his plasmid was co
-
transformed with the cytochrome
c
maturation plasmid pEC86
(
32
)
into
E. cloni
®
EXPRESS BL21(DE3) cells
(Lucigen).
(B) Cytochrome
c
expression and purification.
Purified cytochrome
c
proteins
were prepared as follows. One liter HB
amp/c
hlor
in a 4 L flask was inoculated with
an
overnight culture (20 mL, LB
amp/chlor
) of recombinant
E. cloni
®
EXPRESS BL21(DE3)
cells containing a pET22(b)+ plasmid encoding the cytochrome
c
variant, and the pEC86
plasmid. The culture was shaken at 37 °C and
200 rpm (no humidity control) until the
OD
600
was 0.7 (approximately 3 hours). The culture was placed on ice for 30 minutes, and
isopropyl
β
-
D
-
1
-
thiogalactopyranoside (
IPTG
)
and 5
-
aminolevulinic acid (ALA)
were
added to final concentrations of 20
μ
M and 20
0
μ
M respectively.
The incubator temperature was reduced to 20 °C, and the culture was allowed to
shake for 20 hours at 200 rpm. Cells were harvested by centrifugation (4 °C, 15 min,
4,000xg), and the cell pellet was stored at
20 °C until further use (at
least 24 hours). The
cell pellet was resuspended in buffer containing 100 mM NaCl, 20 mM imidazole, and 20
mM Tris
-
HCl buffer (pH 7.5 at 25 °C) and cells were lysed by sonication (2 minutes, 2
seconds on, 2 seconds off, 40% duty cycle; Qsonica Q500 sonicat
or). Cell lysate was
placed in a 75 °C heat bath for 10 minutes, and cell debris was removed by centrifugation
for 20 min (5000xg, 4 °C). Supernatant was sterile filtered through a 0.45
μ
m cellulose
acetate filter and purified using a 1 mL Ni
-
NTA column (H
isTrap HP, GE Healthcare,
Piscataway, NJ) using an AKTA purifier FPLC system (GE healthcare). The cytochrome
c
protein was eluted from the column by running a gradient from 20 to 500 mM
imidazole over 10 column volumes.
The purity of t
he
collected
cytochro
me
c
fractions
was analyz
ed using sodium
dodecyl sulfate
-
polyacrylamide gel electrophoresis (SDS
-
PAGE).
Pure fractions were
pooled and
concentrated using a 3 kDa molecular weight cut
-
off centrifugal filter and
dialyzed overnight into 0.05 M phosphate buffe
r (pH = 7.5) using 3 kDa molecular
weight cut
-
off dialysis tubing. The dialyzed protein was concentrated again, flash
-
frozen
on dry ice, and stored at
20 °C.
The concentration of cytochrome
c
was determined in triplicate using the ferrous
assay described
in section (E)
.
(C) P450 and globin expression and purification.
Purified P450s and globins
were prepared differently from the cytochrome
c
proteins, and described as follows. One
liter HB
amp
in a 4 L flask
was inoculated with an overnight culture (20
mL, LB
amp
) of
recombinant
E. cloni
®
EXPRESS BL21(DE3) cells containing a pET22(b)+ plasmid
encoding the P450 or globin variant. The culture was shaken at 37 °C and 200 rpm (no
humidity control) until the OD
600
was 0.7 (approximately 3 hours). The culture w
as
4
placed on ice for 30 minutes, and IPTG and 5
-
ALA were added to final concentrations of
0.5
mM and 1 mM
,
respectively.
The incubator temperature was reduced to 20 °C, and
the culture was allowed to shake for 20 hours at 200 rpm.
Cells were harvested by
c
entrifugation (4 °
C, 15 min, 4,000xg), and the cell pellet was stored at
20 °C until
further use (at least 24 hours). The cell pellet was resuspended in buffer containing 100
mM NaCl, 20 mM imidazole, and 20 mM Tris
-
HCl buffer (pH 7.5 at 25 °C). Hemin (30
mg/mL, 0.1 M NaOH
; Frontier Scientific) was added to the resuspended cells such that 1
mg of hemin was added for every 1 gram of cell pellet. Cells were lysed by sonication (2
minutes, 1 seconds on, 2 seconds off, 40% duty cycle; Qsonica Q500 sonicator). Cell
debris was re
moved by centrifugation for 20 min (27,000xg, 4 °C). Supernatant was
sterile filtered through a 0.45
μ
m cellulose acetate filter, and purified using a 1 mL Ni
-
NTA column (HisTrap HP, GE Healthcare, Piscataway, NJ) using an AKTA purifier
FPLC system (GE healthcare). The P450 and globin proteins were eluted from the
column by running a gradient from 20 to 5
00 mM imidazole over 10 column volumes.
The purity of the collected protein fractions was analyzed using SDS
-
PAGE. Pure
fractions were pooled
and concentrated using a 10 kDa molecular weight cut
-
off
centrifugal filter and buffer
-
exchanged with 0.1 M phosp
ha
te buffer (pH = 8.0). The
purified protein was flash
-
frozen on dry ice and stored at
20 °C.
P450 and globin
concentrations were
determined in triplicate using the hemochrome
assay described
in section (D)
.
(D) Hemochrome assay.
A solution of sodium di
thionite
(10 mg/mL)
was
prepared in M9
-
N
buffer
.
Separately, a solution of
1 M NaOH (0.4 mL)
was mixed with
pyridine (1 mL), followed by centrifugation (10,000xg, 30 seconds) to separate the
excess aqueous layer gave a pyridine
-
NaOH solution. To a cuvette
containing 700
μ
L
protein solution (purified protein or heat
-
treated lysate) in M9
-
N
buffer
, 50
μ
L of
dithionite solution and 250
μ
L pyridine
-
NaOH solution were added. The cuvette was
sealed with Parafilm, and the UV
-
Vis spectrum was recorded immediately.
Cytochrome
c
concentration was determined using
ε
550
-
535
= 22.1 mM
-
1
cm
-
1
(
34
). Protein concentrations
determined by the hemochrome assay were in agreement with that determined by the
bicinchoninic acid (BCA) assay (Thermo Fisher) using bovine serum albumin
(BSA) for
standard curve preparation.
(E) Ferrous assay.
To a cuvette containing 700
μ
L protein solution in M9
-
N
buffer
was added 50
μ
L of dithionite solution (10 mg/mL in M9
-
N
buffer
). The cuvette
was sealed with Parafilm, and the UV
-
Vis spectrum was
recorded immediately.
The
absorbance value for the peak at 550 nm was recorded, and background absorbance at
600 nm was subtracted.
Using the protein concentration as determined by the
hemochrome
assay
, ferrous
ε
550
-
600
was determined to be 27 mM
-
1
cm
-
1
fo
r wild
-
type
Rma
cyt
c
, and 21 mM
-
1
cm
-
1
for
Rma
V75T M100D M103E
(see calibration curves shown on
the following page)
.
Concentrations of
Rma
M100D and V75T M100D were determined
using the extinction coefficient calculated for V75T M100D M103E
.
5
(F
)
L
ibrary construction.
Cytochrome
c
site
-
saturation
mutagenesis
libraries were
generated usi
ng a modified version of the 22
-
codon site
-
saturation method (
35
).
For each
site
-
saturation library, oligonucleotides were ordered such that the coding strand
conta
ined the degenerate codon
NDT, VHG or
TGG.
The reverse complements of th
ese
primers were also ordered.
The three forward primers were mixed together in a 12:9:1
ratio, (NDT:VHG:TGG) and the three reverse
primers were mixed similarly.
Two PCRs
were performe
d, pairing the mixture of forward primers with a pET22
(
b
)+
internal
reverse primer, and the mixture of reverse primers with a p
ET22b internal forward
primer.
The two PCR products were gel purified, ligated together using Gibson assembly
(
3
1
)
, and transform
ed into
E. cloni
®
EXPRESS BL21(DE3) cells.
Primer sequences are
available upon request.
(G
)
Enzyme library
screening
.
Single colonies
were picked with toothpicks off of
LB
amp/chlor
agar plates, a
nd grown in deep
-
well (2 mL) 96
-
well plate
s
containing LB
a
mp/chlor
(400
μ
L)
at 37
°C, 250 rpm shaking, and 80
% relative humidity overnight.
After 16
hours, 30
μ
L aliquots of these overnight cultures were
transferred
to
deep
-
well 96
-
well
plate
s
containing HB
amp/chlor
(1 mL)
using a 12
-
channel EDP3
-
Plus 5
-
50
μ
L pipette
(Rainin).
Glycerol stocks
of
the libraries were
prepared by mixing cells in LB
amp/chlor
(100
μ
L)
with 50% v/v glycerol
(100
μ
L)
. Glycerol stocks were stored at
78 °C in 96
-
well
micro
plates.
Growth plates were allowed to shake for 3 hours at 37
°C, 250 rpm shaking,
and 80% rela
tive humidity. T
he plates were
then placed on ice for
30 min. Cultures were
6
induced by adding 10
μ
L of a solution, prepared in sterile
deionized
water, containing 2
mM isopropyl
β
-
D
-
1
-
thiogalactopyranoside (IPTG) and 20 m
M ALA.
The incubator
t
emperature was reduced to 20
°C, and the
induced
cultures were allowed to shake for 20
hours (250 rpm, no humidity control).
Cells were pelleted (4,000xg,
5
min, 4 °C) and
resuspended in 50
0
μ
L M9
-
N
buffer
. For cell lysis, plates were placed in a 75
°C
wat
er
bath for 10 min
, followed by centrifugation
(4,000xg, 5 min,
4 °C
)
to remove cell debris.
The re
sulting heat
-
treated lysates (34
0
μ
L) were then transferred
to
deep
-
well plates for
biocatalytic
reactions.
In an anaerobic chamber
, to deep
-
well plates of h
eat
-
treated lysates
were added Na
2
S
2
O
4
(40
μ
L per well,
100 mM
in dH
2
O), PhMe
2
SiH (10
μ
L per well, 400
mM in MeCN) and Me
-
EDA (10
μ
L per well, 400 mM in MeCN). The plates were sealed
with aluminum sealing tape
, removed from the anaerobic chamber, and shake
n at 400
rpm for 1.5 h. After quenching with cyclohexane (1 mL),
internal standard was added (20
μ
L
of
20 mM
methyl 2
-
phenylacetate
in cyclohexane) and
the reaction mixtures were
pipetted up and down to thoroughly mix the organic and aqueous layers. The pl
ates were
centrifuged (4,000xg, 5 min) and the organic layer (400
μ
L
) was transferred to
shallow
-
well 96
-
well plates for SFC analysis.
Hits from library screening were confirmed by
sma
ll
-
scale biocatalytic reactions, which were analyzed by GC and SFC for a
ccurate
determination of turnovers and enantioselectivities.
(H)
Protein lysate preparation.
Protein lysates for biocatalytic reactions were
prepared as follow:
E. coli
cells expressing
Rma
cyt
c
variant
were pelleted (4,000xg, 5
min, 4 °C), resuspended
in
M9
-
N
buffer and adjusted to the appropriate OD
600
. The
whole
-
cell solution was heat
-
treated (75 °C for 10 min) then centrifuged
(
1
4,000xg, 10
min, 4 °C) to remove cell debris. The
supernatant was sterile filtered through a 0.45
μ
m
cellulose acetate filter into a 6 mL crimp vial
, crimp sealed, and the head space of the
crimp vial was degassed by bubbling argon through for at least 10 min. The concentration
of cytochrome
c
protein lysate was determined using the ferrous assay descr
ibed
in
section (E)
.
Using this protocol, t
he
protein concentrations we typically observed for
OD
600
= 15 lysates are
in the 8
-
15
μ
M range for wild
-
type
Rma
cyt
c
and 2
-
10
μ
M for
other
Rma
cyt
c
variants
.
(I) Small
-
scale biocatalytic reaction.
In an an
aerobic chamber
, protein lysate (340
μ
L) in a 2 mL crimp vial was added
40
μ
L
Na
2
S
2
O
4
(100 mM in dH
2
O),
10
μ
L
PhMe
2
SiH
(400
or 800
mM in MeCN) and
10
μ
L
Me
-
EDA (400 mM in MeCN). The
vial was crimp
sealed
, removed from the anaerobic chamber,
and shaken at 4
00 rpm
at room temperature
for the stated reaction time
. A
t the end of the reaction, the crimp vial was opened and the
reaction was quenched with cyclohexane (1 mL). I
nternal standard was added (20
μ
L of
20 mM
2
-
phenylethanol
in cycloh
exane) and the reacti
on mixture was
transferred to a
microcentrifuge tube, vortexed (10 seconds, 3 times)
, then
centrifuged (
1
4,000xg, 5
min)
to completely separate the organic and aqueous layers (the vortex
-
centrifugation step was
repeated if complete phase separation was not
achieved)
. The organic layer (75
0
μ
L) was
removed for GC and
SFC analysis.
All biocatalytic reactions were performed in triplicate
unless otherwise stated.
The total turnover numbers (TTNs) reported are
calculated
with
respect to the
protein catalyst
and represent the total number o
f tur
novers
that is possible
to
obtain from the catalyst under the stated reaction conditions.
7
III. Supporting Tables
and Figures
Table S1.
Summary of
known
catalytic systems for enantioselective carbene insertion
into
silicon
hydrogen
bond
s
Chiral catalytic
system
Ref
Reaction condition
Substrate
scope
TTN
%
ee
Copper
A
(
15
)
CH
2
Cl
2
, rt, 18 h
4
17 to 25
17 to 98
B
(
36
)
C
6
H
6
, 0 °C, 13.5 h
1
8 to 10
29 to 78
C
(
14
)
CH
2
Cl
2
, −60
to 0
°C, 2
-
12 h
24
3 to 19
12 to 99
B
(
37
)
CH
2
Cl
2
, −40 to 0 °C, 48
-
72 h
8
5 to 9
49 to 88
B
(
38
)
CH
2
Cl
2
, −40 to −10 °C
9
5 to 8
40 to 84
Iridium
D
(
13
)
CH
2
Cl
2
, −78 or −30 °C, 24 h
15
24 to 50
94 to
99
E
(
39
)
CH
2
Cl
2
, −78 °C, 24 h
7
75 to 94
72 to 91
Rhodium
F
(
12
)
CH
2
Cl
2
, rt to 40 °C, 6
-
12 h
34
9 to 30
77 t
o 99
G
(
40
)
CH
2
Cl
2
, −78 °C or rt, 3
-
12 h
5
8 to 45
20 to 63
H
(
36
)
C
5
H
12
, −78 to −75 °C, 24 h
1
24 to 70
48 to 97
I
(
11
)
CF
3
CH
2
OH, −35 °C
10
51 to 97
20 to 99
H
(
41
)
CH
2
Cl
2
, −78 °C then rt, 23 h
6
22 to 54
77 to 94
various
Rh(II)
-
carboxylate
(
42
)
(
43
)
CH
2
Cl
2
, rt
or −78 °C
, 0.5
to
24
h to overnight
6
<1 to 23
6 to 76
J
(
44
)
CH
2
Cl
2
, −78 °C to rt, 23 h
2
35 to 40
38 to 58
H
(
45
)
CH
2
Cl
2
, −78 °C, 1 to 1.5 h
4
34 to 43
35 to 72
H
(
46
)
C
5
H
12
, −78 °C, 24 h
5
13 to 19
75 to 95
various Rh(II)
-
carboxyla
te
(
47
)
CH
2
Cl
2
, rt or reflux
1
8 to 17
6 to 47
C
atalytic systems that could
yield
enantiopure products are highlighted in blue. r
t = room
temperature
.
In cases where
the reaction times were
not
documented
in the original
literature, this information is n
ot shown in the table
above
.
Rh
Ph
Ar
O
O
R
R
P
O
Rh
Rh
O
4
N
N
Ar
Ar
Cu
B
N
N
Ar
Cu
Ar
C
A
O
N
N
O
Ar
Ar
Cu
N
N
O
O
R
R
Ir
L
D
N
N
N
N
Ir
Me
L
E
F
G
Rh
2
(peptide)
2
I
N
O
MeO
2
C
R
R
Rh
Rh
4
J
O
Rh
Rh
O
H
4
H
R
1
R
2
8
Table S2.
Summary of known catalytic systems that accept
α
-
alkyl diazo compounds as
substrates for enantioselective carbene insertion into silicon
hydrogen bonds
α
-
Alkyl diazo compounds are
challenging substrates for
intermolecular
carbene
-
transfer
chemistry
due t
o their propensity to undergo
competing
intramolecular
β
-
hydride
migration (
48
,
13
).
As a result, only a subset of catalytic systems shown in Table S1
have
been reported to accommodate these substrates, as summarized in the table below:
Chiral catalytic
s
ystem
Ref
Reaction condition
Substrate
scope
TTN
%
ee
Copper
A
(
15
)
CH
2
Cl
2
, rt, 18 h
4
17 to 25
17 to 98
C
(
14
)
CH
2
Cl
2
, −40 °C, 2
-
12 h
2
3 to 12
12 to 35
Iridium
D
(
13
)
CH
2
Cl
2
, −78 °C, 24 h
8
24 to 44
94 to 99
Rhodium
F
(
12
)
CH
2
Cl
2
, rt to 40 °C, 6
-
12 h
2
11 to 15
70 to 77
I
(
11
)
CF
3
CH
2
OH, −35 °C
1
n.d.
§
64
H
(
41
)
CH
2
Cl
2
, −78 °C then rt, 23 h
5
22 to 54
77 to 94
See Table S1 for chemical structures of chiral catalysts.
rt = room temperature.
§
The
amount of product formed was
not reported in the original literature.
Table S3
.
Summary of known catalytic systems for
reaction between
phenyldimethylsilane
and Me
-
EDA
via
enantioselective carbene insert
ion into
silicon
hydrogen bond
Chiral catalytic
system
Ref
Reaction conditi
on
TTN
%
ee
Copper
C
(
14
)
CH
2
Cl
2
, −40 °C, 2
-
12 h
12
35
Iridium
D
(
13
)
CH
2
Cl
2
, −78 °C, 24 h
43
97
Rhodium
F
(
12
)
CH
2
Cl
2
, rt to 40 °C, 6
-
12 h
15
77
See Table S1 for chemical structures of chiral catalysts.
rt = room temperature.
Si
H
Ph
+
O
Et
O
N
2
Catalyst
O
Et
O
H
Si
Ph
3
1
2
9
Table S4
.
Preliminary experiments with heme and purified heme proteins
Catalyst
TTN
%
ee
Controls
None
0
-
Hemin
4
±
1
0
Hemin + BSA
1
± 1
0
P450s
BM3 P450 T268A (
22
)
44
± 15
0
BM3 P450 T268A C400H (
49
)
45
± 3
<5
BM3 P450 CIS I263F C400S T
438S
(5
0
)
24
± 9
<5
BM3 P450 F87A T268A C400S (
51
)
40
± 15
<5
BM3 P450 Hstar H92N H100N (
52
)
46
± 7
0
Myoglobins
Sperm whale Mb
12
± 1
0
Sperm whale Mb H64V V68A (
53
)
17
± 1
0
Cytochromes
c
Horse heart cyt
c
31
± 10
<5
Bovine heart cyt
c
54
± 2
6
S. cerevisiae
cyt
c
11
± 1
<5
R. marinus
cyt
c
34
± 10
97
H. thermophilus
cyt
c
8
± 2
16
R. globiformis
cyt
c
4
± 1
<5
Others
Horse radish peroxidase
0
-
C. glutamicum
catalase
0
-
Heme proteins that are available
commercially or in our labor
atory inventory were
screened to
identify the most
enantio
selective protein variant as starting point for directed
evolution.
Experiments with heme proteins were performed using 10
μ
M purified heme
protein, 10 mM silane, 10 mM diazo ester, 10 mM Na
2
S
2
O
4
, 5 vol% MeCN, M9
-
N buffer
at room temperature under anaerobic conditions for 1.5 h. Experiments with hemin were
performed using
100
μ
M hemin. Experiments with hemin and BSA were performed
using 100
μ
M hemin in the presence of BSA (0.75 mg/mL). Reactions
were
performed in
triplicate.
TTNs reported are the average of three experiments.
Within instrument
detection limit, v
ariability i
n %
ee
was not observed.
Unreacted starting materials were
observed at the end of all reactions and
no
attempt
was made to
optimize these reactions.
Si
H
Ph
+
O
Et
O
N
2
Catalyst
M9-N
buffer
(pH
7.4),
Na
2
S
2
O
4
room
temperature,
1.5
hours
O
Et
O
H
Si
Ph
10
Table S5
.
Carbon
silicon bond formation catalyzed by
Rma
cyt
c
variants
Rma
cyt
c
TTN
WT
44
± 27
M
100D
549
± 24
V75T
150
± 48
M103E
70
± 21
V75T M100D
892
± 20
V75T M100E
154
± 37
M100D M103E
520
± 88
V75T M100D M103E
1518
± 51
Experiments were performed using lysates of
E. coli
expressing
Rma
cyt
c
variant (OD
600
= 15; heat
-
treated at 75 °C f
or 10 min), 10 mM silane, 10 mM diazo ester, 10 mM
Na
2
S
2
O
4
, 5 vol% MeCN, M9
-
N buffer at room temperature under anaerobic conditions
for 1.5 h. Reactions
were
performed in triplicate.
TTNs reported are the average of three
experiments.
Si
H
Ph
+
O
Et
O
N
2
Rma
cyt
c
variant
M9-N
buffer
(pH
7.4),
Na
2
S
2
O
4
room
temperature,
1.5
hours
O
Et
O
H
Si
Ph