In the format provided by the authors and unedited.
1
Supplementary Information
Enantioselective, intermolecular benzylic C–H amination catalysed by an engineered iron-
haem enzyme
Christopher K. Prier,
†
Ruijie K. Zhang,
†
Andrew R. Buller, Sabine Brinkmann-Chen, and
Frances H. Arnold*
Division of Chemistry and Chemical Engineering, California Institute of Technology,
1200 East
California Boulevard, MC 210-41, Pasadena, CA 91125, United States
Email: frances@cheme.caltech.edu
This PDF includes:
I. Experimental Procedures
2–6
II. Supplementary Tables 1–11 and Figure 1
7–21
III. Characterization of reaction products and HPLC calibration curves
22–32
IV. Determination of enantioselectivity and assignment of absolute stereochemistry
33–41
V. Preparative scale
whole-cell biocatalytic reactions
41–42
VI. Tosyl group removal
43–44
VII. Determination of initial rates
45–46
VIII. Kinetic isotope effect
47
IX. Sequence of cytochrome P411
CHA
48–49
X. Protein crystallization and structure analysis
50–54
XI. Docking simulations
55–58
XII. Supplementary References
59–61
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
SUPPLEMENTARY INFORMATION
DOI: 10.1038/NCHEM.2783
NATURE CHEMISTRY
|
www.nature.com/naturechemistry
1
2
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.
1
H and
13
C
N
MR
spectra were recorded on a Varian Inova
300 MHz or
500 MHz
, or Bruker Prodigy 400 MHz
instrument
,
in
CDCl
3
and are referenced
to residual protio solvent signals
.
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
u
si
ng a Qsonica Q500
sonicator
.
High
-
resolution mass spectra were obtained at the California
Institute of Technology Mass Spectral Facility.
Synthetic reactions were monitored using thin
layer
chromatograph
y (Merck 60 gel plates) using a
UV
-
lamp for visualization.
Substrates were
p
urchased
from commercial suppliers.
Tosyl azide was prepared according to de Nanteuil and
Waser.
1
Chromatography
.
Analytical
high
-
performance liquid chromat
ography (HPLC) was carried out
using an Agilent 1200
series
instrument and a Kromasil 100 C18
column (4.6 x 50 mm, 5 μm)
with
water
and acetonitrile as the mobile phase.
Semi
-
preparative
HPLC was performed
using an
Agilent XDB
-
C18 column (9.4 x 250 mm, 5 μm) with
water and acetonitrile as the mobile phase.
A
nalytical chiral HPLC was
conducted using a supercritical fluid chromatography (SFC) system
with isopropanol and liquid C
O
2
as the mobile phase.
Product enantiomers were separated using
a
Chiral
pak
AS
column
(4.6 x 150 mm, 5 μm)
from Chiral Technologies
Inc.
Cloning and site
-
directed mutagenesis
.
pET22b(+) was used as a cloning and expression
vector for all enzymes described in this study. Site
-
directed mutagenesis was performed using a
modified QuikChange
TM
mutagenesis
protocol.
2
The PCR products were d
igested with
Dpn
I,
gel
purified,
and the ga
ps were
repaired using Gibson Mix
TM
.
3
The ligation mixture was
used to
directly transform
E. coli
strain BL21
E. cloni
(Lucigen
).
3
Determination
of P411
concentration.
The
c
oncentration of
P411 enzymes
in whole cell
experiments
was determined from ferrous carbon monoxide binding
difference spectra using
the
previously
reported extinction coefficient
for serine
-
ligated enzymes (
ε
= 103,000
M
–
1
c
m
–
1
).
4
The c
oncentration of purified P411 enzymes was
determined by quantifying the amount of free
hemin present u
sing the pyridine/hemochrome assay
and the published extinction coefficient (
ε
=
191,500
M
–
1
cm
–
1
)
.
5
E
xpression and
amination bioconversions
using whole cells.
E. coli
BL21
E. cloni
cells
carrying a
plasmid encoding
a
P411 variant
were grown overnight
in 5 mL
Luria
-
Bertani
medium
with 0.1 mg/
m
L
ampicillin (
LB
amp
,
37
º
C, 250
rpm). The preculture was used to
inoculate 45 mL of Hyperbroth
(HB)
medium (prepare
d from AthenaES© powder, 0.1 mg/
mL
ampicillin) in a 125
-
mL Erlenmeyer flask; this cu
lture was incubated at 37 º
C, 23
0 rpm for 2 h.
C
ultures were
then
cooled on ice
(20 min)
,
an
d
expression was
induced with 0.5 mM IPTG and
1.0
mM 5
-
aminolevulinic acid (final concentration
s
). Expression was c
onducted at room
temperature
(23 ºC)
,
at
130 rpm, for 16
–
18
h. C
ultures were then
centrif
uged (
2,600 x g, 10 min,
4 º
C
)
,
and
the pellets
were
resuspended to
an
O
D
600
of
30 in M9
-
N
minimal media (no nitrogen)
.
Aliquots of the cell suspension (4 mL) were
used to determine the P411 expression level after
lysis
by
sonication
.
For amination bioconversions, the cells
(
OD
600
of
30 in M9
-
N
media)
were
degas
sed by
sparging with argon in
sealed 6
-
mL crimp vial
s
for at least 40
min
u
t
es. Separately,
a
glucose
solution
(250 mM
in
M9
-
N
) was degassed by sparging with argon for at least
10
minutes.
A
n
oxygen
depletion system (2
0 μL
of a stock solution
containing 14,000 U/mL catalase and
1,000
U/mL glucose
oxidase
in 0.1 M KPi
,
pH
8.0)
was added to 2
-
mL crimp vials
. All solutions were
uncapped and transferred into an anaerobic chamber. Resuspended cells (320 μL) were added to
the vial
s
, followed by glucose (40 μL
, 250 mM in M9
-
N
),
alkane
(10 μL of a
DMSO
stock
), a
nd
tosyl azide (10 μL of a
DMSO
stock
).
Final concentrations were
typically 2.5
–
5
.0
mM
alkane
,
5
.0
mM tosyl azide, and 25 mM glucose
; final reaction volume was 400
μL
.
The vial
s were
sealed
, removed from the anaerobic chamber
,
and
shaken
at
room temperature
and
40 rpm
for
16
–
20
h.
Reactions
under aerobic conditions were performed by
combining resuspended cells
(32
0
μ
L), glucose
(40 μL
, 250 mM in M9
-
N
),
oxygen depletion system
(20
μ
L, or
20
μ
L
M9
-
N),
alkane
(10 μL, 200 mM in DMSO), and tosyl
azide (10 μL, 200 mM in DMSO) on the benchtop,
4
witho
ut any argon sparging
(final reaction volume 400
μL
).
The reactions
were que
nched by
addi
tion of
acetonitrile (40
0 μL) and internal standard (10
μ
L of a
D
MSO stock).
T
his
mixture
was then transferred to a microc
entrifuge tube and centrifuged at
20
,000
x g
for 10 minutes. The
supernatant
w
as transferred to a
vial and analyzed by HPLC.
Reaction
screening in 96
-
well plate format.
Site
-
satura
tion libraries were generated
employing
the “22c
-
trick”
method.
2
E. coli
libraries were culture
d
in
LB
amp
(
3
00 μL/well)
at 37 ºC, 220 rpm
and 80% relative humidity
overnight
.
Hyperbroth
medium
(
1000 μL/well, 0.1 mg/mL
ampicillin
)
was inoculated with the preculture (50
μ
L/well) and
incubated at 37
º
C
,
22
0 rpm, 80%
humidity
for 3 h
. The plates were cooled on ice for 30 minut
es
,
and then
expression was
induced
with 0.5
mM IPTG and
1
.0
mM
5
-
aminolevulinic acid (
final concentration
s)
. Expression was conducted
at 20
ºC
and
120 rpm for 24
h. The cells we
re pelleted (3,
000
x
g, 5
min, 4
º
C
)
and resuspended
in t
he oxygen depletion system (20
μL/well). T
he 96
-
well plate was
then
transferred to an
anaerobic chamber.
In the
anaerobic chamber,
argon
-
sparged reaction buffer (
50
mM glucose in
M9
-
N, 300 μL/
well) was added
,
followed by
the
alkane
(10
μ
L
/
well, 200 mM in
DMSO
)
and
tosyl
azide (
10 μL/
well, 200 mM in DMSO
). The plate was sealed with
an
aluminum
foil
,
removed from the anaerobic chamber, and shaken at 40 rpm.
After 16
–
20
h, the seal was
removed and acetonitrile
(
4
00 μL
/well)
and
internal
standard (
10
μ
L
/
well o
f a DMSO sto
ck
)
were added.
The wells were mixed, the
plate was centrifuged (5,
000
x g, 10 min
)
, and the
supernatant
was
filtered through an AcroPrep
96
-
well
filter
plate
(0.2
μ
m)
into a
shallow
-
well
plate for
HPLC
analysis
.
Protein purification.
E. coli
BL21
E. cloni
cells
carrying a
plasmid encoding
a
P411 variant
were grown overnight in 25 mL LB
amp
(37
º
C
, 250
rpm).
Hyperbroth
medium
(
630
mL,
0.1
mg/mL
ampicillin
) in a 1
-
L flask
was i
noculated with
25
mL of the pre
culture
and
incubated
at
37
º
C
and
23
0 rpm
for 2.5 h
(
to
OD
600
ca. 1.8).
Cultures were then cooled on ice (30 min) and
induced with 0.5 mM
IPTG
and 1
.0
mM 5
-
aminolevulinic acid (final concentrations)
.
Expression
was conducted at
23 ºC
, 130 rpm, for
1
6
–
20
h.
Cultures were then centrifuged
(
5,000 x g,
8
min,
4 ºC) and
the cell pellet
s
frozen at
–
20
º
C.
For purification, f
rozen
cells
from two such cultures
were resuspended in buffer A (25 mM tris, 20 mM imidazole
,
1
00 mM NaCl, pH 7.5, 4 mL/g of
cell wet weight)
,
loaded with hemin
(1 mg/
gram
wet cell weight
)
and pow
dered DNase
I
,
and
5
lysed by sonication. To pellet
cell debris
, lysates wer
e cen
trifuged (
20
,000 x g,
20
min,
4
º
C
)
.
Proteins were expressed in a construct containing a 6x
-
His tag and purified using a nickel NTA
column (1 mL Hi
sTrap H
P, GE Healthcare, Piscataway, NJ) using an
AKTA or
AKTAxpress
purifier
FPLC system (GE healthcare). P411 enzymes were eluted
with
a linear gradient from
100% buffer A
to 100
% b
uffer B
(25 mM tris, 300 mM imidazole,
100 mM NaCl, pH 7.5)
over
10 column
volumes.
Proteins used for crystallography were subjected to an additional ion
-
exchange
purification step. For these proteins,
fractions were pooled and subjected to three exchanges of
anion exchange buffer
A
(25 mM tris
-
HCl, pH 7.5)
using centrif
ugal spi
n filters (
10 kDa
molecular weight cut
-
off,
Amicon Ultra, Merck Millipore
)
.
Next
, the protein was loaded onto an
anion exchange Q Sepharose column (HiTrap
TM
Q HP, GE
Healthcare)
and purified
using an
AKTA
or
AKTA
express
purifier FPLC system
(GE healthcare).
The enzyme was eluted from the
Q
-
column by running a gradient from 0 to 0.5 M NaCl over 10 column volumes. Fractions
containing the enzyme were pooled, concentrated, and exchanged into storage buffer (25 mM
tris
-
HCl, 25 mM NaCl, pH 7.5)
a
s before
.
Subsequently, the concentrate
d
protein was aliquoted,
flash
-
frozen on powdered dry ice, and stored at
–
80
ºC
.
Protein concentration
s were determined
via Bradford assay with a bovine serum albumin standard curve.
Proteins used for other purpose
s were eluted from the Ni
-
NTA column as described
above, pooled, concentrated, and subjected to
three exchanges of phosphate buffer (0.1 M KPi
,
pH 8.0)
using centrifugal filters
(
10 kDa molecular weight cut
-
off, Amicon Ultra, Merck
Millipore)
to remove excess salt and imidazole
. Concentrated proteins were
aliquoted, flash
-
frozen on powdered dry ice, and stored
at
–
8
0
or
–
20
ºC.
Amination bioconversions using purified protein.
Aliquots
of phosphate buffer
(26
0 μL 0.1 M
KPi, pH
8.0)
and NADPH
(40 μL, 100 mM), or multiples thereof, were combined in a 6
-
mL
crimp vial
and degassed by sparging with argon for at least 30 minutes. Separately,
a
glucose
solution (250 mM in 0.1 M KPi
,
pH 8.0) was also degassed in the same manner
.
C
rimp vials
(2
mL)
wer
e each charged with the oxygen depletion system (
20 μL of a stock solution
containing
14,000 U/mL catalase and
1,000
U/mL glucose oxidase in 0.1 M KPi, pH 8.0)
. After degassing
was complete, all solutions, 2
-
mL crimp vials, and purified protein
(100
μM in
0.1 M KPi
,
pH
8.0
), kept on ice, were brought into the anaerobic chamber.
Glucose solution (40
μL
),
NADPH
6
solution (300
μL
), and purified protein (20
μL
of 100 μM stock solution) were added to each 2
-
mL vi
al. Reaction vials
were then charged with alkane
(1
0
μ
L, 20
0 mM in DMSO) and
tosyl
azide (10
μ
L, 200 mM in DMSO)
. Final concentrations were
typically
5 mM
alkane
, 5 mM tosyl
azide, 10 mM NADPH, 25 mM glucose,
and 5
μM P411
; final reaction volume was 400
μL
.
The
vials were sealed, removed from the anaerobic
chamber, and shaken at room temperature
and
40
rpm for
16
–
20 h. The reactions
were que
nched by addi
tion of
acetonitrile (40
0 μL) and internal
standard (10
μ
L of a
D
MSO stock). T
his
mixture was then transferred to a microcentrifuge tube
and centrifuged at
20
,000
x g
for 10 minutes
. The
supernatant
w
as transferred to a
vial and
analyzed by HPLC
. Sodium dithionite (5 mM) was used as the reductant instead of NADPH for
reactions with hemin and myoglobin.
7
Supplementary
Table
1
.
C
–
H a
mination
of 4
-
ethylanisole
with
variants of
cytochrome
P450
BM3
.
a
V
ariant
M
utations relative to wild
-
ty
pe
P450
BM3
Y
ield
pET22b(+)
vector
N/A
N.D.
P450
BM3
None
N.D.
P411
BM3
C400S
N.D.
P411
BM3
T268A
T268A, C400S
N.D.
P411
BM3
-
CIS
T438S ("P")
V78A, F87V, P142S,
T175I, A184V,
S226R, H236Q, E252G, T268A, A290V,
L353V, I366V, C400S, T438S, E442K
N.D.
P411
BM3
-
CIS
T438S A268T
V78A, F87V, P142S, T175I, A184V,
S226R, H236Q, E252G, A290V, L353V,
I366V, C400S, T438S, E442K
N.D.
P411
BM3
-
H2
-
5
-
F10
L75A, V78A, F87V, P142S,
T175I,
A184V, S226R, H236Q, E252G, I263A,
T268A, A290V, L353V, I366V, C400S,
L437A, E442K
N.D.
P411
BM3
-
H2
-
A
-
10
L75A, V78A, F87V, P142S, T175I,
L181A, A184V, S226R, H236Q, E252G,
T268A, A290V, L353V, I366V, C400S,
E442K
N.D.
P411
BM3
-
H2
-
4
-
D4
L75A, V78A, F
87V, P142S, T175I,
M177A, L181A, A184V, S226R, H236Q,
E252G, T268A, A290V, L353V, I366V,
N.D.
Me
NHTs
Me
TsN
3
P450
BM3
variant
whole
cells
MeO
MeO
8
C400S, L437A, E442K
P411
BM3
T268A F87A
b
F87A, T268A, C400S
N.D.
P
-
I263F
b
V78A, F87V, P142S, T175I, A184V,
S226R, H236Q, E252G, I263F, T268A,
A290V, L353V,
I366V, C400S, T438S,
E442K
<1% yield
P
-
I263F A268T
P
-
I263F A268T
N.D.
P
-
I263F A328V
P
-
I263F A328V
<1% yield
P
-
I263F A328V L437V
c
P
-
I263F A328V L437V
N.D.
P
-
I263F V87A
P
-
I263F V87A
2% yield
P
-
I263F V87A A328V
P
-
I263F V87A A328V
2% yield
P
-
I263F V87A
A328V A82L
P
-
I263F V87A A328V A82L
4% yield
P
-
5
P
-
I263F V87A A328V A268G A82I
14% yield
P
-
4 A82L A78V F263M
d
P
-
4 A82L A78V F263M
55% yield,
99% ee
P
-
4 A82L A78V F263Y
d
P
-
4 A82L A78V F263Y
68% yield,
61% ee
P
-
4 A82L
A78V F263L
(heme domain only)
P
-
4 A82L
A78V F263L
(heme domain only)
<1% yield
a
Reactions performed in
whole cells
at OD
6
00
= 30
with 5 mM
of
each substrate
;
results are the
average of duplicate reactions.
N.D. = none
detected.
b
Variants
identified for regioselective
intramolecular
C
–
H
amination.
6
c
Variant identified for aziridination
of styrenes
.
7
d
Other
active
variants identified in the course of evolution of cytochrome P411
CHA
.
9
Supplementary
Table
2
.
C
–
H a
mination
of 4
-
ethylanisole
performed with
hemin
or heme
-
containing proteins
.
a
C
atalyst
Y
ield
hemin (25
μ
M)
b
N.D.
hemin (25
μ
M) + imidazole (1 mM)
b
N.D.
hemin (25
μ
M) + bovine serum albumin (10
μ
M)
b
N.D.
Myoglobin (
Mb,
equine heart
, 10
μ
M
)
b
N.D.
Mb
H64V V68A
(sperm whale
, 10
μ
M
)
b
,
c
N.D.
Rhodothermus marinus
cytochrome c
d
N.D.
Hydrogenobacter
thermophilus
cytochrome c
d
N.D.
Rhodopila
globiformis
cytochrome c
d
N.D.
Rhodothermus marinus
cytochrome c V75T M100D M103E
d,e
N.D.
a
Reactions
pe
r
formed
with
5
mM
4
-
ethylanisole
and 5 mM tosyl azide;
results are the average of
duplicate reactions.
N.D. = none
detected.
b
Performed
in vitro
with 5 mM sodium
d
ithionite
.
c
Variant identified
for
intramolecular C
–
H amination
.
8
d
Performed
in whole cells
at OD
600
= 30
.
e
Variant identified for
carbene Si
–
H insertion
.
9
Me
NHTs
Me
TsN
3
catalyst
MeO
MeO
10
Supplementary
Table
3.
Mutations
present in cytochrome P450
BM3
variants used in this work.
Variant
Mutations relative to wild
-
type P450
BM3
P
-
4
V78A, F87A, P142S, T175I, A184V, S226R, H236Q, E252G,
I263F,
T268G, A290V, A328V, L353V, I366V, C400S, T438S, E442K
P
-
4 A82L
V78A, A82L, F87A, P142S, T175I, A184V, S226R, H236Q, E252G,
I263F, T268G, A290V, A328V, L353V, I366V, C400S, T438S,
E442K
P
-
4 A82L A78V
A82L, F87A, P142S, T175I, A184V, S226R, H236Q,
E252G, I263F,
T268G, A290V, A328V, L353V, I366V, C400S, T438S, E442K
P
-
4 A82L A78V
F263L
A82L, F87A, P142S, T175I, A184V, S226R, H236Q, E252G, I263L,
T268G, A290V, A328V, L353V, I366V, C400S, T438S, E442K
P
-
4 A82L A78V
F263L E267D
(P411
CHA
)
A82L, F87A, P
142S, T175I, A184V, S226R, H236Q, E252G, I263L,
E267D, T268G, A290V, A328V, L353V, I366V, C400S, T438S,
E442K
11
Supplementary
Table
4
.
Summary of directed evolution for
intermolecular
C
–
H amination.
a
Generation
Parent
enzyme
Site
-
saturation
libraries evaluated
Screening
substrate
Mutation
identified
1
P
-
4 A82L
A78X, L181X,
F263X, T327X
4
-
ethylanisole
A78V
2
P
-
4 A82L A78V
F263X, T327X
4
-
ethylanisole
F263L
3
a
P
-
4 A82L A78V
F263L
A74X, A264X,
F393X, G402
X
4
-
ethylanisole
none
3
b
P
-
4 A82L
A78V
F263L
R47X, S142X,
V184X, G252X,
E267X
4
-
ethyltoluene
E267D
4
P
-
4 A82L A78V
F263L E267D
(P411
CHA
)
R47X, Y51X
4
-
ethyltoluene
none
a
Some resid
ues were saturated more than once, in different parent variants.
A74, L181, F263, A264, E267, T327: Distal
face of heme, active site residues
F393, G402: Axial face of heme
A78, S142, V184, G252: Already mutated in
P
-
4
relative to wild
type
P450
BM3
R47, Y51: Polar residues that
interact with
the carboxylate of the native
fatty acid
substrates
12
Supplementary
Table 5
.
Intermolecular C
–
H amination
data presented in Figure 3
.
V
ariant
S
ubstrate
[P411]
Y
ield
ee
TO
N
P
-
4
4
-
ethylanisole
1.8
μ
M
11 ± 1%
14% (
S
)
31
0
P
-
4 A82L
4
-
ethylanisole
2.4
μ
M
51 ± 3%
77% (
R
)
1000
P
-
4 A82L
A78V
4
-
ethylanisole
2.7
μ
M
66 ± 2%
80% (
R
)
1200
P
-
4 A82L
A78V
F263L
4
-
ethylanisole
N.A.
a
66 ± 2%
>99% (
R
)
N.A.
a
P411
CHA
4
-
ethylanisole
3.2
μ
M
66 ± 3%
>99% (
R
)
1000
P
-
4
4
-
ethyltoluene
1.8
μ
M
2.0 ± 0.3%
N.A.
58
P
-
4 A82L
4
-
ethyltoluene
2.4
μ
M
11 ± 1%
98% ee (
R
)
220
P
-
4 A82L
A78V
4
-
ethyltoluene
2.7
μ
M
29 ± 1%
>99% ee (
R
)
530
P
-
4 A82L
A78V
F263L
4
-
ethyltoluene
N.A.
a
32 ± 1%
>99% ee
(
R
)
N.A.
a
P411
CHA
4
-
ethyltoluene
3.2
μ
M
34 ± 3%
>99% ee
(
R
)
530
P
-
4
ethylbenzene
1.8
μ
M
0.50 ± 0.01%
N.A.
15
P
-
4 A82L
ethylbenzene
2.4
μ
M
2.2 ± 0.1%
N.A.
46
P
-
4 A82L
A78V
ethylbenzene
2.7
μ
M
6.5 ± 0.4%
92% ee (
R
)
120
P
-
4 A82L
A78V
F263L
ethylbenzene
N.A.
a
6.7 ± 0.4%
>99% ee
(
R
)
N.A.
a
P411
CHA
ethylbenzene
3.2
μ
M
15 ± 1%
>99% ee
(
R
)
240
a
Protein concentration could not be accurately determined;
variant is poorly
behaved in carbon
monoxide
-
binding assay.
13
Supplementary
Table
6
.
Activity
of P411
variants
toward C
–
H amination of
indan, tetralin, 1
-
bromo
-
4
-
ethylbenzene, and 4
-
propylanisole
. Reactions
were
performed
as in Table 2
(2.5 mM
alkane, 5
.0
mM tosyl azide)
.
S
ubstrate
P
roduct
P
-
4 A82L
A78V F263L
P411
CHA
indan
N
-
t
osyl
-
1
-
aminoindane
72% yield, 90% ee
80% yield, 96% ee
tetralin
N
-
t
osyl
-
1
-
aminotetralin
49% yield, 81% ee
76% yield, 95% ee
1
-
bromo
-
4
-
ethylbenzene
N
-
t
osyl
-
1
-
(
p
-
bromophenyl
)ethyl
amine
13% yield, >99% ee
19% yield, >99% ee
4
-
propylanisole
N
-
t
osyl
-
1
-
(
p
-
methoxyphenyl)propy
lamine
15% yield, >99% ee
16% yield, >99% ee
14
Supplementary
Table 7
.
C
–
H amination r
eactions
performed
under aerobic conditions.
a
V
ariant
C
onditions
Y
ield
P
-
4 A82L A78V
anaerobic, full system
66%
P
-
4 A82L A78V
aerobic, full system
13%
P
-
4 A82L A78V
aerobic, no glucose oxidase/catalase
7
.5
%
P
-
4 A82L A78V F263L
anaerobic, full system
66%
P
-
4 A82L A78V F263L
aerobic, full system
20
%
P
-
4 A82L A78V
F263L
aerobic, no glucose oxidase/catalase
7.6
%
P411
CHA
anaerobic, full system
66
%
P411
CHA
aerobic, full system
15%
P411
CHA
aerobic, no glucose oxidase/catalase
9.5
%
a
Reactions
performed as in Figure 3
(5 mM 4
-
ethylanisole, 5 mM tosyl azide). For reactions
without the glucose oxidase/catalase mixture, 20
μ
L of M9
-
N
media
was added instead.
Me
NHTs
Me
TsN
3
MeO
MeO
P411
variant
whole
cells
15
Supplementary
Table 8
.
C
–
H amination of 4
-
ethylanisole performed with purified P411
variants.
a
C
atalyst
Y
ield, turnovers, ee
P
-
4 (5
μ
M)
2.2% yield, 22 TON
, 4% ee
(S
)
P
-
4 A82L (5
μ
M)
9.7% yield, 97
TON, 80
% ee
(
R
)
P
-
4 A82L A78V F263L (5
μ
M)
19% yield, 190 TON, >99% ee
(
R
)
P411
CHA
(5
μ
M)
15% yield, 150 TON, >99% ee
(
R
)
P411
CHA
(20
μ
M)
47% yield, 120 TON,
>99% ee
(
R
)
P
-
4 A82L A78V F263L (heme domain only, 10
μ
M)
b
1.4% yield, 7 TON
a
Reactions performed
with
10 mM NADPH,
5 mM 4
-
ethylanisole
, and 5 mM tosyl azide;
results
are the average of
duplicate reactions.
b
Performed with 5 mM sodium dithionite
instead of
NADPH.
Supplementary
Table 9
.
Reaction mass
balances; r
epresentative
reactions showing amount of
p
-
toluenesulfonamide
(TsNH
2
,
5
)
formed
.
Reactions
are those
in Table 2
(
performed with
2.5
mM alkane, 5 mM tosyl azide)
.
Table 2
entry
S
ubstrate
C
–
H amination
product
p
-
toluene
-
sulfonamide
T
otal
of
nitrogen
-
containing
products
1
4
-
ethylanisole
2.14 mM
2.74 mM
4.88 mM
2
4
-
ethyltoluene
1.33 mM
3.51 mM
4.84 mM
5
ethylbenzene
0.39 mM
4.63 mM
5.01 mM
Me
NHTs
Me
TsN
3
MeO
MeO
P411
variant
in
vitro
16
Supplementary Table 10.
Enantio
s
e
lective
C
–
H amination of
isochroman
with
cytochrome
P411
CHA
followed by post
-
reaction racemization
.
a
Entry
C
onditions
ee
1
1 hour reaction time
65%
2
2 hour reaction time
65%
3
4 hour reaction time
65%
4
6 hour reaction time
65
%
5
20 hour reaction time
65%
6
20 hour reaction time, followed by treatment with silica
racemic
a
Reactions
performed as in Table 2, using
whole
E.
coli
cells overexpressing P411
CHA
at OD
600
=
30
(~3
μ
M enzyme)
,
with 2.5 mM alkane and
5
mM tosyl azide.
Identical small scale reactions
(400
μ
L)
were set up in parallel. After the indicated
time, c
rude reaction mixtures were extracted
with
1:1
cyclohexane
:
ethyl acetate,
concentrated
to dryness, and redissolved in 50% isopropanol
in cyclohexane. Silica treatm
ent was performed by allowing the crude reaction mixture (in
cyclohexane/ethyl acetate) to rest on a silica pipette column (approx. 1 hour) followed by elution
with ethyl acetate. Samples were analyzed by SFC chromatography
using Chiralpak AS column
(35
% i
sopropanol).
O
TsN
3
P411
CHA
whole
cells
O
NHTs
cytochrome
17
Representative traces
.
Racemic standa
rd
,
N
-
tosyl
-
isochroman
-
1
-
amine
:
Supplementary Table 10,
Entry 5, with P411
CHA
(65% ee):
Supplementary Table 10,
Entry 6, with P411
CHA
post silica treatment (racemic):