Supporting Information
Asymmetric Enzymatic Synthesis of Allylic Amines: A Sigmatropic
Rearrangement Strategy
Christopher K. Prier, Todd K. Hyster, Christopher C. Farwell, Audrey Huang, and
Frances H. Arnold*
anie_201601056_sm_miscellaneous_information.pdf
1
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
NMR
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, m = multiplet, dd = doublet of
doublets,
dt = doublet of triplets, dq = doublet of quartets,
ddd = doublet of doublet of doublets, td = triplet o
f doublets),
coupling constant (Hz)
,
integration
.
Sonication
was performed u
sing a Qsonica Q500 sonicator
.
High
-
resolution mass
spectra were obtained at
the California Institute of Technology Mass Spectral Facility.
Specific
o
ptical rotations were
measured
using a Jasco P
-
2000 Polarimeter, and
are reported in units of
deg
·
cm
3
·
g
–
1
·
dm
–
1
.
Synthetic reactions
were monitored using thin layer chromatograph
y (Merck
60 gel plates) using a
UV
-
lamp for visualization.
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.
Analytical 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
Chiral
cel
OD
-
H
, Chiralcel OJ
-
H, and Chiralpak
AS
-
H columns
(4.6 x 150 mm, 5 μm)
from
Chiral Technologies Inc.
2
Cloning and site
-
directed
mutagenesis.
pET22b(+) was used as a cloning and expression
vector for all enzymes described in this study. Site
-
directe
d mutagenesis was performed using a
modified QuikChange
TM
mutagenesis protocol. The PCR products were gel purified, digested
with DpnI, repaired using Gibson Mix
TM
, and
used to directly transform
E. coli
strain
BL21(DE3).
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
).
1
The
c
oncentration of purified P411 enzymes was determined by quantifying the amount of free
hemin present using the pyridine/hemochrome assay.
2
E
xpression and
amination
bioconversions using whole cells.
E. coli
BL21(DE3) cells
transformed with plasmid encodin
g P411 variants
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 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 induced with 0.5 mM IPTG and 0.5
mM 5
-
aminolevulinic acid (final concentration
s
). Expression was c
onducted at room temperature, 130
rpm, f
or 16 h. C
ultures were then
centrifuged (
2,600 x g, 10 min, 4 º
C
)
and
the pellets
resuspended to O
D
600
= 30 in M9
-
N. Aliquots of the cell suspension (4 mL) were used to
determine the P411 expression level after lysis
by
sonication
.
For amination
bioconversions, the cells at
OD
600
= 30 in M9
-
N
were
degas
sed by
3
sparging with argon in
sealed 6 mL crimp vial
s
for at least 40
min
u
t
es. Separately, glucose (250
mM
in
M9
-
N
) was degassed by sparging with argon for at least five minutes.
To
2 mL crimp
vial
s
were
added
an
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)
. All solutions were uncapped
and transferred into an anaerobic chamber. Resuspended cells (320 μL) were a
dded to the vial
s
,
followed by glucose (40 μL
, 250 mM in M9
-
N
), sulfide
(10 μL, 200 mM in DMSO), and tosyl
azide (10 μL, 200 mM in DMSO).
Final concentrations were 5 mM sulfide, 5 mM tosyl azide,
and 25 mM glucose.
The vial
s were
sealed, removed from the a
naerobic chamber
,
and
shaken
at
room temperature
, 40 rpm
for 20
h.
Reactions under aerobic conditions were performed by
combining resuspended cells (340
μ
L),
glucose (40 μL
, 250 mM in M9
-
N
), sulfide
(10 μL, 200
mM in DMSO), and tosyl
azide (10 μL, 200 mM in DMSO) on the benchtop, without any argon
sparging.
The reactions
were quenched by adding acetonitrile (450 μL) and internal standard (10
μ
L of a
D
MSO stock).
For reactions proceeding to
>30% conversion (
at
which
point
the
phenylthi
osulfonamide is not completely degraded by cellular reductants), dithiothreitol was
added (20
μ
L, 20 mM DTT in 0.1 M KPi, pH 8.0).
T
his
mixture was then transferred to a
microc
entrifuge tube and centrifuged at 14,000 rpm 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.
3
E. coli
libraries were culture
d
in
LB
amp
(
3
00 μL/well)
at 37 ºC, 220 rpm.
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 minutes
and then
induced
with 0.5 mM IPTG and
1
mM
5
-
aminolevulinic
acid (
final
4
concentration
s)
. Expression was conducted at 20
ºC, 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
sulfide
(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
hours, the seal was removed and acetonitrile
(
4
00 μL
/well)
and
internal
standard (
10
μ
L
/
well
of a DMSO sto
ck
) were added.
The wells were mixed, the
plate was centrifuged (5,
000
x g, 10
mi
n
)
, and the supernatant
was
filtered through an AcroPrep
96
-
well
filter
plate
(0.2
μ
m)
into a
shallow
-
well plate for
HPLC
analysis
.
For
the
screening
of site
-
saturation libraries for activity on sulfide
1
,
we used
batches
of
sulfide
1
favoring the
E
-
alkene
(
3:1 or 4.5:1
E:Z
) for the
first three libraries
(at residues A268,
L437, and S438),
while
for the next set of
libraries (at
residues A78, A82, T260, and P329)
we
performing
screening using
Z
-
1
(>15:1
Z:E
)
.
Protein purification.
E. coli
BL21(
DE3)
cells transformed with plasmid encoding P411 variants
were grown overnight in 25 mL LB
amp
(37
º
C
, 250
rpm).
Hyperbroth
medium
(
470 mL,
0.1
mg/mL
ampicillin
) in a 1 L flask
was i
noculated with
19 mL of the pre
culture
and
incubated
at
37
º
C,
23
0 rpm
for 2.5 h
(OD
600
ca. 1.8).
Cultures were then cooled on ice (30 min) and
induced
with 0.5 mM
IPTG
and 1 mM 5
-
aminolevulinic acid (final concentrations)
.
Expression was
conducted at room temperature, 130 rpm, for 20
–
24 h.
Cultures were then centrifuged
(
5,0
00 x g,
5 min,
4 ºC) and
the cell pellet
s
frozen at
–
20
º
C.
F
rozen
cells were resuspended in buffer A (25
mM tris, 20 mM imidazole
, 200 mM NaCl, pH 7.5, 4 mL/g of cell wet weight)
,
loaded with
300
5
μ
g/mL hemin,
and lysed by sonication. To pellet insoluble material, lysates wer
e cen
trifuged
(
20
,000 x g, 15 min,
4
º
C
)
. Proteins were expressed in a construct containing a 6x
-
His tag and
purified 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
/
0% buffer B (25 mM tris, 300 mM imidazole, 2
00 mM NaCl, pH
7.5) to 100
% buffer B over 10 column volumes. Fractions containing
P411 enzymes were
pooled, concentrated, and subjected to three exchanges of phosphate buffer (0.1 M KPi
,
pH 8.0)
to remove excess salt and imi
dazole. Concentrated proteins were
aliquoted, flash
-
frozen on
powdered dry ice, and stored at
–
20
º
C.
A
mination b
ioconversions using purified protein.
Purified P411
e
nzyme
(
in
0.1 M KPi, pH
8.0
,
30
μ
L)
and
the
oxygen depletion
system
(20
μ
L)
were added
to
2 mL crimp
vial
s
. P
ortions
of phosphate buffer (250
μL
0.1 M KPi, pH
8.0), glucose (40 μL, 250 mM)
, and NADPH (40
μL,
10
0 mM), or multiples thereof, were combined in a
6 mL
crimp vial and degassed by sp
arging
with argon for at least 10
min.
All solutions were uncapped and transferred into an anaerobic
chamber. The buffer/reductant/glucose solution (330 μL) was added
to the reaction vial
s
,
followed by sulfide (10
μ
L, 20
0 mM in DMSO) and tosyl azide (10
μ
L, 200 mM in DMSO).
Final concentrations were
typically 5
mM
sulfide, 5 mM
tosyl azide, 10
mM NADPH, 25 mM
glucose,
and 10 μM P411
.
The vials were
sealed, removed from the anaerobic chamber, and
shaken at room temperature, 40 rpm for 20 h.
The reactions
were quenched by adding
acetonitrile (450 μL) and internal standard (10
μ
L of a
D
MSO stock).
For reactions proceeding
to >10% conversion, dithiothrei
tol was added (20
μ
L, 20 mM DTT in 0.1 M KPi, pH 8.0).
T
his
mixture was then transferred to a microcentrifuge tube and centrifuged at 14,000 rpm for 10
6
minutes
. The
supernatant
w
as transferred to a
vial and analyzed by HPLC
.
Sodium dithionite (5
mM) was used as the reductant for reactions with hemin, myoglobin, and cytochrome
c
.
Myoglobin (equine heart) and cytochrome
c
(bovine heart) were purchased from Sigma
-
Aldrich.
Synthesis o
f s
ubstrates
.
Thioanisole
and
ethyl phenyl su
lfide
were purchased from Sigma
-
Aldrich and used without
further
purification.
Other sulfides were synthesized according to know
n
procedures, and
their
s
pectral data
are in agreement
with
reported valu
es.
4
Tosyl
azide was prepared according to de
Nanteuil and Waser.
5
(
Z
)
-
But
-
2
-
en
-
1
-
yl(phenyl)sulfide
(
Z
-
1
)
was prepared
in two steps via Lindlar hydrogenation of 2
-
butyn
-
1
-
ol according to Balduzzi
et al
.
6
followed by mesylation and displacement with
thiophenol
ate
according to Hiro
i and Makino
.
7
The sulfide was obtained
in 26%
yield from (
Z
)
-
2
-
buten
-
1
-
ol
as a >15:1
Z:E
mixture of alkene isomers:
1
H NMR
(3
00 MHz, CDCl
3
)
δ
7
.40
–
7.24
(m, 4H), 7.22
–
7.14 (m, 1
H), 5
.87
–
5.47 (m, 2H), 3.58 (d
,
J
= 6.6 Hz,
2H), 1.61
–
1.54
(m, 3H)
;
13
C
NMR
(101 MHz, CDCl
3
)
δ
136
.5, 130.0, 128.9,
127.8, 126.3, 125.5, 31.
0,
12.8
;
HRMS (FAB)
exact mass calculated for C
10
H
12
S requires
m/z
164.0660, found 164.0653.
HO
Me
Lindlar
catalyst
H
2
,
MeOH
HO
Me
1)
MsCl,
THF
2)
PhSH,
NaH,
EtOH
S
Me
Ph
7
(
Z
)
-
Pent
-
2
-
en
-
1
-
yl(phenyl)
sulfide
was prepared
as above
from
cis
-
2
-
penten
-
1
-
ol
in
15% yield
,
and obtained as a >15
:1
Z
:
E
mixture
:
1
H NMR
(3
00 MHz, CDCl
3
)
δ
7
.40
–
7.14 (m, 5H), 5.56
–
5.42 (m, 2H),
3.57 (d,
J
=
6.4 Hz, 2H), 2.1
0
–
1.92 (m, 2H), 0.90 (t,
J
= 7.5 Hz, 3H)
;
13
C NMR
(101
MHz, CDCl
3
):
δ
136.5, 135.4, 130.1, 128.9, 126.3, 123.9, 31.3, 20.7,
14
.2;
HRMS (FAB) exact mass calculated for C
1
1
H
1
4
S requires
m/z
178.0816, found 178.0822.
(
Z
)
-
Hex
-
2
-
en
-
1
-
yl(phenyl)
sulfide
was prepared
as above
from
cis
-
2
-
hexen
-
1
-
ol in 53% yield
,
and
o
btained as
a
>15
:1
Z
:
E
mixture
:
1
H NMR
(
3
00 MHz, CDCl
3
):
δ
7.39
–
7.32
(m, 2H), 7.31
–
7.23 (m, 2H), 7.22
–
7.14
(m, 1H), 5.59
–
5.45 (m, 2H), 3.63
–
3.52 (m, 2H), 2.05
–
1.90 (m, 2H), 1.32
(ddt,
J
= 14.3, 7.9, 7.0, 2H), 0.87 (t,
J
= 7.3 Hz, 3H);
13
C NMR (101
MHz, CDCl
3
):
δ
136.6, 133.6, 130.0, 128.9, 126.3
, 124.7, 31.4, 29.4,
22.7, 13.9;
HRMS (FAB) exact mass calculated for C
1
2
H
1
6
S requires
m/z
192.0973, found 192.0964.
Characterization of reaction p
roducts
.
Authentic standards
corresponding to enzymatic
reaction products were prepared
by tosylation of
the corresponding allylic amine or
by nitrene transfer to
the corresponding sulfide according to
the method of Okamura and Bolm
.
8
Phenylthiosulfonamides
were cleaved to the corresponding
sulfonamides by treatment with 0.5 M potassium hydroxide in methanol, according to Murakami
Ph
S
Me
Ph
S
Me
8
et al
.
9
All products are known compounds, and their s
pectral data are in
agreement with
reported
values.
9,10
S
-
Me
thyl
-
S
-
phenyl
-
N
-
(
4
-
methylbenzene
sulfonyl)sulfimide
(
1
1
)
:
1
H NMR
(
4
00 MHz
,
CDCl
3
)
δ
7.77
–
7.67 (m
,
4
H),
7.57
–
7.45 (m, 3
H),
7.20
–
7.14
(
m
,
2 H),
2.84 (s, 3H), 2.35
(s, 3H).
S
-
Ethyl
-
S
-
phenyl
-
N
-
(
4
-
methylbenzene
sulfonyl
)sulfimide
(
1
2
)
:
1
H
NMR
(
3
00 MHz, CDCl
3
)
δ
7.76
–
7.70
(
m
,
2H), 7.69
–
7.62 (m, 2H), 7.55
–
7.43 (m,
3H), 7.19
–
7.10 (m, 2H),
3.12
–
2.90 (m, 2H), 2.33 (s, 3H), 1.17 (t,
J
= 7.3 Hz,
3H).
N
-
A
llyl
-
4
-
methylbenzenesulfonamide
(
1
3
)
:
1
H
NMR (
3
00 MHz,
CDCl
3
)
δ
7.
75 (d,
J
= 8.3 Hz, 2H), 7.34
–
7.
27
(
m
, 2H),
5.71
(ddt,
J
= 17.1, 10.2, 5.8
Hz, 1H), 5.16 (dq,
J
= 17.1, 1.5 Hz, 1H),
5.08 (dq,
J
= 10.2, 1.4 Hz, 1H),
4.73 (t,
J
= 6.2 Hz, 1H),
3.57 (tt
,
J
= 6.0, 1.5 Hz, 2H), 2.4
2 (s, 3H).
N
-
(B
ut
-
3
-
en
-
2
-
yl)
-
4
-
methylbenzenesulfonamide
(
10
)
:
1
H NMR (500 MHz,
CDCl
3
)
δ
7.75
(d,
J
= 8.3 Hz, 2H),
7.30
–
7.26 (m, 2H), 5.63 (ddd,
J
= 17.2,
10.4, 5.8 Hz, 1H),
5.05 (ddd,
J
= 17.1, 1.5, 1.0 Hz, 1H),
4.96 (dt,
J
= 10.4,
1.2 Hz, 1H), 4.76 (d,
J
= 7.7
Hz, 1H), 3.93
–
3.84 (m
, 1H),
2.42 (s, 3H), 1.17
(d,
J
= 6.8 Hz, 3H).
S
Me
Ph
N
Ts
S
Me
Ph
N
Ts
NHTs
Me
HN
Ts
9
N
-
(P
ent
-
1
-
en
-
3
-
yl)
-
4
-
methyl
benzenesulfonamide
(
1
4
)
:
1
H NMR (3
00
MHz,
CDCl
3
)
δ
7
.
74 (d,
J
= 8.3 Hz, 2H), 7.31
–
7.25 (m, 2H), 5.53 (ddd,
J
= 17.0,
10.3, 6.4 Hz, 1H), 5.0
3
–
4.94 (m
,
2
H),
4.49 (d,
J
= 8.0 Hz, 1H),
3.76
–
3.63
(m, 1H), 2.42 (s, 3H),
1.50 (p,
J
= 7.3 Hz, 2H), 0.82 (t,
J
= 7.4 Hz, 3H).
N
-
(H
ex
-
1
-
en
-
3
-
yl)
-
4
-
methylbenzenesulfonamide
(
1
5
)
:
1
H NMR (
4
00 MHz,
CDCl
3
)
δ
7.
74 (d,
J
= 8.3 Hz, 2H),
7.30
–
7.24 (m, 2H), 5.53 (ddd,
J
= 17.0,
10.3, 6.6 Hz, 1H),
5.01
–
4.91 (m, 2H), 4.63 (d,
J
= 7.9 Hz, 1H),
3.80
–
3.70
(m, 1H), 2.41 (s, 3H), 1.48
–
1.39 (m, 2H), 1.34
–
1.18 (m, 2H), 0.82 (t,
J
= 7.3
Hz, 3H).
N
-
(2
-
M
ethylallyl)
-
4
-
methylbenzenesulfonamide
(
1
6
)
:
1
H NMR (
3
00 MHz,
CDCl
3
)
δ
7.
75 (d,
J
= 8.3 Hz, 2H),
7.33
–
7.28 (m, 2H), 4.67
–
4.79 (m, 2H),
4.64 (t,
J
= 6.4 Hz, 1H), 3.47 (d,
J
= 6.5 Hz, 2H), 2.43 (s, 3H), 1.67 (s, 3H).
N
-
(2
-
M
ethylbut
-
3
-
en
-
2
-
yl)
-
4
-
methyl
benzenesulfonamide
(
1
7
)
:
1
H
NMR
(
3
00 MHz, CDC
l
3
)
δ
7.
75
(d,
J
= 8.3 Hz, 2H),
7.29
–
7.23 (m, 2H), 5.77 (dd,
J
= 17.3
, 10.6 Hz, 1H),
5.08 (dd,
J
= 17.3, 0.7 Hz, 1H),
4.97
–
4.89 (m, 2H),
2.41 (s, 3H), 1.29 (s, 6H).
S
-
(
n
-
Propyl)
-
S
-
ph
enyl
-
N
-
(
4
-
methylbenzene
sulfonyl)sulfimide
(
1
8
)
:
1
H
NMR (300 MHz, CDC
l
3
)
δ
7.
74
(d,
J
= 8.2 Hz, 2H), 7.70
–
7.65 (m, 2H),
7.56
–
7.45 (m, 3H), 7.16
–
7.12 (m, 2H), 3.08 (ddd,
J
= 12.7, 8.3, 5.6 Hz, 1H),
HN
Me
Ts
HN
Ts
Me
Me
HN
Ts
Me
Me
NHTs
Ph
S
N
Ts
Me
10
2.85 (ddd,
J
= 12.7, 8.6, 7.1 Hz, 1H),
2.34 (s, 3H), 1.75
–
1.55 (m, 2H), 0.95
(t,
J
= 7.4 Hz, 3H).
S
-
(
n
-
Butyl)
-
S
-
phenyl
-
N
-
(
4
-
methylbenzene
sulfonyl
)sulfimide
(
1
9
)
:
1
H NMR
(
3
00 MHz
, CDCl
3
)
δ
7.77
–
7.64
(m
, 4
H),
7.55
–
7.44 (m, 3H), 7.18
–
7.12 (m,
2H),
3.07 (ddd,
J
= 12.8, 8.5, 5.6 Hz, 1H), 2.87 (ddd,
J
= 12.7, 8.8, 7.0 Hz,
1H), 2.34 (s, 3H),
1.70
–
1.43 (m, 2H), 1.42
–
1.20 (m, 2H), 0.82 (t,
J
= 7.3 Hz,
3H).
Ph
S
Me
N
Ts
11
Table
S1
.
Mutations
present in P450
BM3
variants used in this work.
Variant
Mutations relative to wild
-
type P450
BM3
P411
BM3
-
CIS T438S
(P)
V78A, F87V, P142S, T175I, A184V, S226R, H236Q, E252G, T268A,
A290V, L353V, I366V, C400S,
T438S,
E442K
P
-
I263F
V78A,
F87V, P142S, T175I, A184V, S226R, H236Q, E252G, I263F,
T268A, A290V, L353V, I366V, C400S, T438S, E442K
P
-
I263F A328V
V78A, F87V, P142S, T175I, A184V, S226R, H236Q, E252G, I263F,
T268A, A290V, A328V, L353V, I366V, C400S, T438S, E442K
P
-
I263F V87A
V78A, F8
7A, P142S, T175I, A184V, S226R, H236Q, E252G, I263F,
T268A, A290V, L353V, I366V, C400S, T438S, E442K
P
-
I263F V87A
A328V
V78A, F87A, P142S, T175I, A184V, S226R, H236Q, E252G, I263F,
T268A, A290V, A328V, L353V, I366V, C400S, T438S, E442K
P
-
I263F V87A
A328V
A268G
V78A, F87A, P142S, T175I, A184V, S226R, H236Q, E252G, I263F,
T268G, A290V, A328V, L353V, I366V, C400S, T438S, E442K
P
-
I263F V87A
A328V A268G A82L
V78A, A82L, F87A, P142S, T175I, A184V, S226R, H236Q, E252G,
I263F, T268G, A290V, A328V, L353V, I366V,
C400S, T438S, E442K
P
-
I263F V87A
A328V A268G A82I
V78A, A82I, F87A, P142S, T175I, A184V, S226R, H236Q, E252G,
I263F, T268G, A290V, A328V, L353V, I366V, C400S, T438S, E442K
12
Table S2.
Yields
and enantioselectivities of
whole cell bioconversions
re
presented
in Figure
s 1
and 2 (and some not shown elsewhere)
.
a
Thioanisole
Phenyl ethyl
sulfide
Phenyl allyl
sulfide
Phenyl crotyl
sulfide
(3:1
E:Z
)
Phenyl crotyl
sulfide
(>15
:1
Z:E
)
P
19
% yield
,
660
TTN
1.1
% yield
,
38
TTN
1.7
% yield
,
59
TTN
0.2
%
yield
,
7
TTN
0.
3
% yield
,
9
TTN
P
-
I263F
51
% yield
,
280
0
TTN
12
% yield
,
650
TTN
3.1
% yield
,
170
TTN
0.8
% yi
eld
,
45
TTN
1.4
%
yield,
77
TTN,
57%
ee
P
-
I263F
A328V
63
% yield
,
160
0
TTN
27
% yield
,
680
TTN
9.0
% yield
,
230
TTN
4.6
% yield
,
120
TTN
10
% yield,
260
TTN,
66% ee
P
-
I263F
V87A
51
% yield
,
430
0
TTN
43
% yield
,
3600
TTN
,
48
% ee
24
% yield
,
200
0
TTN
4.5
% yield
,
380
TTN
5.5
% yield,
460
TTN,
47% ee
P
-
I263F
V87A
A328V
46
% yield
,
160
0
TTN
54
% yield
,
180
0
TTN,
82%
ee
27
% yield
,
930
TTN
6.6
% yield
,
220
TTN
14
% yield,
490
TTN,
66% ee
P
-
I263F
V87A
A328V
A268G
N.D.
75% y
ield,
2500
TTN,
39%
ee
N.D.
17% yield,
580 TTN
44
% yield,
180
0
TTN,
66% ee
P
-
I263F
V87A
A328V
A268G
A82L
N.D.
N.D.
N.D.
N.D.
70
%
yield,
1500
TTN,
69%
ee
P
-
I263F
V87A
A328V
A268G
A82I
N.D.
86
%
yield,
2
20
0
TTN,
88%
ee
N.D.
N.D.
77
%
yield,
2
20
0
TTN,
68% ee
13
a
Experiments were performed using whole cells overexpressing the P411 variant, resuspended to
OD
600
= 30, with 5 mM sulfide and 5 mM tosyl azide. Results are the average of experiments
performed with duplicate cell cultures, each used to perform duplicate chemical reactions (total
of four reactions).
N.D. = Not determined; ee = enantiomeric excess.
Tab
le
S3
.
Reactions
of other sulfide substrates with intermediate P411 variants.
Conditions as in
Table S2.
The mutations introduced in the course of evolving a catalyst for amination of sulfide
1
are also strongly activating
toward amination of related
substrates
.
P
-
I263F V87A
A328V
3.8% yield,
130
TTN
0.4
% yield,
14
TTN
P
-
I263F V87A
A328V A268G
18% yield, 600 TTN,
71% ee
7.9% yield, 260 TTN
S
Ph
Me
S
Ph
Me
Me