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Thieme
Supporting Information
for DOI: 10.1055/s-003
7
-
1
611662
© Georg Thieme Verlag KG Stuttgart · New
York 201
9
S
1
Supplementary Information
Engineered
C
ytochrome
c
-
C
atalyzed
L
actone
-
C
arbene B
‒
H
I
nsertion
Kai Chen,
a
Xiongyi Huang,
a
Shuo
-
Qing Zhang,
b
Andrew Z. Zhou,
a
S
.
B
.
Jennifer Kan,
a
Xin Hong,
*b
and
Frances H. Arnold
*a
a
Division of Chemistry and Chemical Engineering 210
-
41, California Institute of Technology,
Pasadena, CA 91125, USA.
b
Department of Chemistry, Zhejiang University, Hangzhou, Zhejiang 31007, P. R. China.
*
To whom correspondence
should be addressed. E
-
mail:
hxchem@zju.edu.cn
and
frances@cheme.caltech.edu
This PDF file includes:
I. General
Information
S
2
II.
General Protocol
S
3
-
S
4
III.
Synthesis of Lactone Diazos and NHC
-
Boranes
S
5
-
S
6
IV.
Synthesis of Organoborane Standard Products
S
7
-
S
9
V.
Enzymatic Synthesis of Lactone
-
Based Organoboranes in Analytical
and Preparative Scale
S
10
-
S
2
9
VI.
Computational Study of Carbene Intermediates
S
30
-
S
4
6
VII.
NMR Spectra
S
4
7
-
S
5
7
V
III. References
S
5
8
S
2
I.
General Information
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
,
13
C
and
11
B
NMR
spectra were recorded on a Bruker Prodigy 400 MHz instrument (400 MHz for
1
H and 100 MHz
for
13
C). Chemical shifts (δ) are reported in ppm downfield from tetramethylsilane, using the
solvent resonance as the internal standard. Sonication was
performed using 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 chromatography
(Merck 60 gel plates) using a UV
-
lamp
for visualization. Gas chromatography
-
mass spectrometry
(GC
-
MS) analyses were carried out using Shimadzu GCMS
-
QP2010SE system and J&W HP
-
5ms
column. Chiral normal
-
phase HPLC analyses were performed using an Agilent 1200 series
instrument with isopropanol a
nd hexanes as the mobile phase.
Plasmid
pET22
b(+)
was used as a cloning vector, and cloning was performed using
Gibson
assembly (
1
). The cytochrome
c
maturation plasmid pEC86 (
2
) was used as part of a two
-
plasmid system to express prokaryotic cytochr
ome
c
proteins
. Cells were grown using Luria
-
Bertani medium or HyperBroth (AthenaES) with 100
μg/
mL ampicillin and 20
μg/
mL
chloramphenicol (LB
amp/chlor
or HB
amp/chlor
). Cells without the pEC86 plasmid were grown with 100
μg/
mL ampicillin (LB
amp
or HB
amp
).
Primer sequences are available upon request.
Electrocompetent
Escherichia coli
cells were prepared following the protocol of Sambrook
et al.
(
3
). T5 exonuclease, Phusion polymerase, and
Taq
ligase were purchased from New England
Biolabs (NEB, Ipswich, M
A).
M9
-
N minimal medium (abbreviated as M9
-
N buffer; pH 7.4) was
used as a buffering system for whole c
ells
, 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
, a
nd 0.1 mM CaCl
2
.
S
3
II. General
Protocol
(A) Plasmid construction.
All variants described in this paper were clone
d and expressed
using the pET22b(
+
)
vector (Novagen).
The gene encoding
Rma
cyt
c
(UNIPROT ID B3FQS5)
was obtained as a single
gBlock (IDT), codon
-
optimized for
E. coli
, and cloned using Gibson
assembly (
1
) into
pET22b(
+
)
(Novagen) between restriction sites
Nde
I and
Xho
I
in frame with an
N
-
terminal pelB leader sequence (to ensure periplasmic
localization and proper maturation;
MKY
LLPTAAAGLLLLAAQPAMA) and a
C
-
terminal 6xHis
-
tag. This plasmid was co
-
transformed with the cytochrome
c
maturation plasmid pEC86 (
2
) into
E. cloni
®
EXPRESS
BL21(DE3) cells
(Lucigen).
(B)
Expression of c
ytochrome
c
variants.
25
mL
HB
amp/chlor
in a
125 mL
flask was
inoculated with an overnight culture (
0.
5
mL, LB
amp/chlor
) of recombinant
E.
cloni
®
EXPRESS
BL2
1(DE3) cells containing a pET22b(
+
)
plasmid encoding the cytochrome
c
variant, and the
pEC86 plasmid. The culture was shaken at 37 °C and 2
3
0 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 μM and 200 μM
,
respectively.
The incubator temperature was reduced
to 2
0
°C, and the culture was allowed to shake for 22 hours at
16
0 rpm. Cells were harvested by
centrifugation (4 °C, 5 min, 4,000
×
g)
.
The cell pellet was resuspended in
M9
-
N
buffer
.
(
C
) Hemochrome as
say.
A solution of
0.5 M
sodium dithionite
in 0.5 M NaOH
was
first
prepared
. Separately, a solution of 1 M NaOH (0.4 mL) was mixed with pyridine (1 mL), followed
by centrifugation (10,000
×
g, 30 seconds) to separate the excess aqueous layer
and
give
a p
yridine
-
NaOH solution. To a cuvette containing
4
00 μL protein solution (purified protein or heat
-
treated
lysate) in M9
-
N buffer,
40
0 μL pyridine
-
NaOH solution w
as
added
and mixed thoroughly.
2 μL
of sodium dithionite solution was added to the solution and
the
cuvette was sealed with Parafilm,
and the UV
-
Vis spectrum was recorded immediately. Cytochrome
c
concentration was determined
using ε
550
=
30
.
27
mM
‒
1
cm
‒
1
(
4
). Protein concentrations determined by the hemochrome assay
were in agreement with th
ose
determined by the bicinchoninic acid (BCA) assay (Thermo Fisher)
using bovine serum albumin (BSA) for standard curve preparation.
(
D
) Library construction.
Cytochrome
c
site
-
saturation mutagenesis libraries were
generated using a modified version of the
22
-
codon site
-
saturation method (
5
). For each site
-
saturation library, oligonucleotides were ordered such that the coding strand contained the
degenerate codon NDT, VHG or TGG. The reverse complements of these primers were also
ordered. The three forward p
rimers were mixed together in a 12:9:1 ratio, (NDT:VHG:TGG) and
the three reverse primers were mixed similarly. Two PCRs were performed, pairing the mixture
of
forward primers with a pET22b(
+
)
internal reverse primer, and the mixture of reverse primers wit
h
a pET22b
(+)
internal forward primer. The two PCR products were gel purified, ligated together
using Gibson assembly (
1
), and transformed into
E. cloni
®
EXPRESS BL21(DE3) cells. Primer
sequences are available upon request.
(
E
) Enzyme library screening.
Single colonies were picked with toothpicks off of
LB
amp/chlor
agar plates, and grown in deep
-
well (2 mL) 96
-
well plates containing LB
amp/chlor
(400
μ
L) at 37 °C, 250 rpm shaking, and 80% relative humidity overnight. After 16 hours, 30 μL
aliquots of these
overnight cultures were transferred to deep
-
well 96
-
well plates 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 glycer
ol (100
μ
L).
S
4
Glycerol stocks were stored at −78 °C in 96
-
well microplates. Growth plates were allowed to shake
for 3 hours at 37 °C, 250 rpm shaking, and 80% relative humidity. The plates were then placed on
ice for 30 min. Cultures were induced by adding
10 μL of a solution, prepared in sterile deionized
water, containing 2 mM
IPTG
and 20 mM ALA. The incubator temperature 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
,000
×
g, 5 min, 4 °C) and the plates containing the cell pellets were transferred
into an anaerobic chamber and then resuspended in 380 μL M9
-
N buffer. To deep
-
well plates of
cell suspensions were added the NHC
-
borane
substrate (10 μL per well, 400 mM in
MeCN) and
the diazo compound (10 μL per well, 400 mM in MeCN). The plates were sealed with aluminum
sealing tape
and shaken at 480 rpm for 12 h
in the anaerobic chamber
. After quenching with
hexanes/ethylacetate (4:6, 0.6 mL), internal standard was added (
20 μL of 20 mM
1,
3
,
5
-
trimethoxybenzene in toluene
). The plates were then sealed with sealing
mats and shaken
vigorously to thoroughly mix the organic and aqueous layers. The plates were centrifuged (4,000
×
g, 5 min) and the organic layer (300 μL) was tran
sferred to autosampler vials with inserts for
chiral HPLC analysis. Hits from library screening were confirmed by small
-
scale biocatalytic
reactions, which were analyzed by GC
-
MS
and
chiral HPLC
for accurate determination of
turnovers and enantioselectivit
ies.
S
5
III.
Synthesis of Lactone Diazos
and NHC
-
Boranes
3
-
Diazodihydrofuran
-
2
(
3
H
)
-
one
(
1
)
The preparation of the title compound
1
followed a modified procedure reported
by Sattely
et al
.
(
6
). Sodium azide (4.83 g, 74.3 mmol, 4 equiv.), sodium hydroxide (80
mL of 2 M in
water, 160 mmol), tetrabutylammonium bromide (60.0 mg, 0.190 mmol,
0.01 equiv.), and hexane (80 mL) were combined in a 500
-
mL flask with magnetic stir
bar open to the air and cooled to 0 °C. With vigorous stirring, triflic anhydride (6.20 mL, 37.1
mmol, 2
equiv.) was added dropwise. After 15 min, a solution of 2
-
acetyl
-
butyrolactone (2.00 mL,
18.6 mmol) in acetonitrile (70 mL) was poured into the vessel through a funnel, followed by an
additional 10 mL of acetonitrile to complete the transfer. The initially
colorless reaction mixture
immediately turned yellow. After stirring for 20 min at 0 °C, the mixture was diluted with ice
water (50 mL) and chilled EtOAc (50 mL) and transferred to a separatory funnel. After phase
separation and removal of the organic fra
ction, the aqueous layer was washed with chilled EtOAc
(50 mL × 5). The combined organic layer was dried over Na
2
SO
4
, filtered, and concentrated under
reduced pressure. The resulting crude product was purified through a silica column using hexane:
ethyl ac
etate (3:1 to 2:1) as eluents. The yellow
-
colored fractions were concentrated to afford the
product as a bright yellow crystalline solid (1.2 ‒ 1.6 g, 60 ‒ 75% yield).
3
-
Diazotetrahydro
-
2
H
-
pyran
-
2
-
one
(
4
) and
3
-
diazooxepan
-
2
-
one
(
6
)
The preparation of the title compounds
4
and
6
followed a
modified
procedure reported by
DeAngelis
et al
.
(
7
).
A flame
-
dried
round
-
bottomed flask was charged with diisopropylamine (1.88 mL,
13.41 mmol) under a nitrogen atmosphere. Anhydrous THF (40mL)
was added and the flask was cooled to 0 °C. A solution of
n
-
butyllithium (5.36 mL, 13.4 mmol)
(2.5M
solution in hexanes) wa
s added
dropwise and the mixture was stirred for 30 min and
subsequentl
y cooled to −78 °C by a bath of
dry ice/acetone. A solution of tetrahydro
-
2
H
-
pyran
-
2
-
one
or
oxepan
-
2
-
one
(11.2 mmol) in 27 mL of
anhydrous THF was added dropwise and the
reaction was st
irred at −78 °C for 15 minutes.
2,2,2
-
Trifluoroethyl trifluoroacetate (TFEA) (2.24
mL, 16.8 mm
ol) was then added dropwise and
the reaction was stirred at −78 °C for 30 minutes
and then allo
wed to warm to room temperature
over 1 hour.
The reaction was quenc
hed with
50
mL of 10% HCl and extract
ed with
Et
2
O
(50 mL
×
3)
.
The combined organic layer was
washed
with
brine
, dr
ied over anhydrous
MgSO
4
, and
concentrated under reduced pressure
. The crude
product
3
-
(2,2,2
-
trifluoroacetyl)tetrahydro
-
2
H
-
pyran
-
2
-
one
or
3
-
(2,2,2
-
trifluoroacetyl)oxepan
-
2
-
one
was used for the next step without further purification.
The
crude product
was dissolved in anhydrous CH
2
Cl
2
(110
mL), and
o
-
nitrobenzenesulfonyl
azide (
o
-
NBSA) (3.21 g, 1
4.1 mmol) was added followed by
dropwise addition
of
1,8
-
d
iazabicyclo(5.4.0)undec
-
7
-
ene
(
DBU,
2.
39
mL, 16
mmol) and the r
eaction was stirred at room
temperature for
3
hours. The
reaction
was
quenched by water (50 mL) and the product was
extracted by
ether
(40 mL
×
7).
The combined organic layer was
washed with
brine
, dr
ied over
anhydrous
MgSO
4
, and
concentrated under reduced pressure
.
The resulting crude product was
purified through a silica column using hexane: ethyl acetate (
2
:1 to
1
:
2
) as eluents. The yellow
-
colored fractions were concentrated to
afford product
4
as a bright yellow crystalline solid (1
.06
g, 75% yield)
and product
6
as an orange oil (1.28 g, 82% yield)
.
S
6
NHC
-
boranes were synthesized using the procedures
reported
in our previous paper (
8
).
S
7
IV.
Synthesis of Organoborane Standard Products
The preparation of racemic organoborane standard products follows the reported protocol
using rhodium acetate (
9
) or iodine (
10
) as catalyst for carbene
B‒H
insertion.
Iodine (10 mol%)
or Rh
2
(OAc)
4
(2 mol%) was added
to the solution
of
NHC
-
BH
3
(1.0 equiv) in dichl
oromethane.
The mixture was
allowed to stir for 5 min. A solution of diazo compound (1.2 equiv) in
dichloromethane was then added through a syringe pump. The reaction mixture was allowed to
stir for 10 h. Quick filtration and further purification through a silica column using hexane
: ethyl
acetate (2:
1 to 0:
1) or hexane
: (ethyl acetate
: acetone 5:
5) (2:
1 to 0:
1) elution system afforded
the organoborane standard products.
The
1
H NMR r
esonances of the B
‒
H protons are broad (due to geminal coupling with boron)
an
d generally in the range of 0.4
–
1.6 ppm. The
13
C NMR resonances of the boron
-
binding NHC
quaternary
carbons usually appear at around 170 ppm and are typically broad (due to germi
nal
coupling with boron) and weak; these signals are sometimes not visible in the
13
C NMR spectra.
(
1
,
3
-
D
imethyl
-
1
H
-
imidazol
-
3
-
ium
-
2
-
yl
)(
2
-
oxotetrahydrofuran
-
3
-
yl
)
dihydroborate
(
3
a
)
1
H NMR (400 MHz,
CDCl
3
) δ 6.83 (s, 2H), 4.45 (dddd,
J
= 10.7, 8.1, 6.8, 1.0
Hz, 1H), 4.27 (tq,
J
= 9.0, 1.1 Hz, 1H), 3.83
–
3.69 (m, 6H), 2.53
–
2.25 (m,
1H), 2.01
–
1.93 (m, 1H), 1.88
–
1.80 (m, 1H), 1.79
–
1.16 (m, 2H)
;
13
C NMR
(101 MHz, CDCl
3
) δ 187.91,
120.62, 67.86, 36.15, 30.86
;
11
B NMR (128
MHz, CDCl
3
) δ
-
27.08 (t,
J
= 89.6Hz).
HRMS (FAB+) m/z: 193.1144 ((M+H
+
)
–
H
2
); calc. for
C
9
H
14
O
2
N
2
B: 193.1148.
(
2
-
O
xotetrahydrofuran
-
3
-
yl
)(
1
,
3
,
5
-
trimethyl
-
1
H
-
imidazol
-
3
-
ium
-
2
-
yl
)
dihydroborate
(
3b
)
1
H NMR (400 MHz, CDCl
3
) δ 6.57 (q,
J
= 1.1 Hz, 1H), 4.47 (ddd,
J
= 10.8,
8.2, 6.8 Hz, 1H), 4.28 (ddd,
J
= 9.0, 8.2, 1.8 Hz, 1H), 3.70 (s, 3H), 3.64 (s,
3H), 2.45
–
2.33 (m, 1H), 2.18 (d,
J
= 1.2 Hz, 3H),
2.00
–
1.92 (m, 1H), 1.89
–
1.59 (m, 2H), 1.49
–
1.11 (m, 1H)
;
13
C NMR (101 MHz, CDCl
3
) δ 165.20,
128.29, 117.79, 67.89, 35.77, 32.59, 30.95, 9.67
;
11
B NMR (128 MHz, CDCl
3
)
δ
-
26.77 (t,
J
= 89.4 Hz)
.
HRMS (FAB+) m/z: 207.1317 ((M+H
+
)
–
H
2
); calc. for C
10
H
16
O
2
N
2
B:
207.1305.
(
1
,
3
-
D
imethyl
-
5
-
(
trifluoromethyl
)
-
1
H
-
imidazol
-
3
-
ium
-
2
-
yl
)(
2
-
oxotetrahydrofuran
-
3
-
yl
)
dihydroborate
(
3c
)
1
H NMR (400 MHz, CDCl
3
) δ 7.28 (q,
J
= 1.3 Hz, 1H), 4.45 (ddd,
J
= 10.4,
8.3, 7.0 Hz, 1H), 4.29 (ddd,
J
= 8.9, 8.3, 2.2 Hz, 1H), 3.87 (d,
J
= 0.8 Hz,
3H), 3.81 (s, 3H), 2.51
–
2.32 (m, 1H), 2.07
–
1.65 (m, 3H), 1.59
–
1.10 (m,
1H)
;
13
C NMR (101 MHz, CDCl
3
) δ 187.70, 122.82 (q,
J
= 4.2 Hz), 122.52
(q,
J
= 41.1 Hz), 119.39 (q,
J
= 268.7 Hz), 68.00, 36.81, 34.25, 34.23, 34.21,
34.19, 30.65
;
11
B NMR (128 MHz, CDCl
3
) δ
-
27.15 (t,
J
= 90.7 Hz)
.
HRMS
(FAB+) m/z: 261.1030
((M+H
+
)
–
H
2
); calc. for C
10
H
13
O
2
N
2
F
3
B: 261.1022.
S
8
(
1
-
E
thyl
-
3
-
methyl
-
1
H
-
imidazol
-
3
-
ium
-
2
-
yl
)(
2
-
oxotetrahydrofuran
-
3
-
yl
)
dihydroborate
(
3d
)
1
H NMR (400 MHz, CDCl
3
) δ 6.88 (d,
J
= 2.0 Hz, 1H), 6.84 (d,
J
= 2.0 Hz,
1H), 4.45 (ddd,
J
= 10.8, 8.2, 6.8 Hz, 1H), 4.27 (ddd,
J
= 8.9, 8.2, 1.8 Hz, 1H),
4.24
–
4.09 (m, 2H), 3.76 (s, 3H), 2.50
–
2.29 (m, 1H), 2.02
–
1.66 (m, 3H),
1.61
–
1.13 (m, 1H), 1.40 (t,
J
= 7.3 Hz, 3H)
;
13
C NMR (101
MHz, CDCl
3
) δ
187.77, 121.00, 118.45, 67.82, 43.72, 36.07, 30.93, 15.90
;
11
B NMR (128
MHz, CDCl
3
) δ
-
27.10 (t,
J
= 89.6 Hz)
.
HRMS (FAB+) m/z: 207.1296 ((M+H
+
)
–
H
2
); calc. for
C
10
H
16
O
2
N
2
B: 207.1305.
(
3
-
Hexyl
-
1
-
methyl
-
1
H
-
imidazol
-
3
-
ium
-
2
-
yl
)(
2
-
oxotetrahydrofuran
-
3
-
yl
)
dihydroborate
(
3e
)
1
H NMR (400 MHz, CDCl
3
) δ 6.86 (d,
J
= 1.9 Hz, 1H), 6.83 (d,
J
= 1.9 Hz,
1H), 4.45 (ddd,
J
= 10.8, 8.2, 6.8 Hz, 1H), 4.27 (ddd,
J
= 8.9, 8.2, 1.8 Hz, 1H),
4.17
–
4.01 (m, 2H), 3.76 (s, 3H), 2.48
–
2.31 (m, 1H), 2.13
–
1.64 (m, 5H),
1.58
–
1.14 (m, 7H), 0.88 (t,
J
= 7.0 Hz, 3H)
;
13
C NMR (101 MHz, CDCl
3
) δ
187.77, 120.82, 119.03, 67.82, 48.85, 36.12, 31.46, 30.95, 30.65, 26.36, 22.61,
14.12
;
11
B NMR (128 MHz, CDCl
3
) δ
-
27.09 (t,
J
= 90.3 Hz). HRMS (FAB+) m/z: 263.1927
((M+H
+
)
–
H
2
); calc. for C
14
H
24
O
2
N
2
B: 263.1931.
(
1
,
3
-
D
imethyl
-
1
H
-
imidazol
-
3
-
ium
-
2
-
yl
)(
2
-
oxotetrahydro
-
2
H
-
pyran
-
3
-
yl
)
dihydroborate
(
5a
)
1
H NMR (400 MHz,
CDCl
3
) δ 6.80 (s, 2H), 4.46 (dddd,
J
= 11.0, 7.4, 3.7, 1.0
Hz, 1H), 4.26
–
4.19 (m, 1H), 3.74 (s, 6H), 2.14
–
1.95 (m, 3H), 1.89
–
1.75
(m, 1H), 1.71
–
1.55 (m, 2H), 1.51
–
1.15 (m, 1H)
;
13
C
NMR (101 MHz,
CDCl
3
) δ 181.58, 120.48, 69.44, 36.10, 27.17, 22.39
;
11
B NMR (128 MHz,
CDCl
3
) δ
-
24.71 (t,
J
= 90.4 Hz)
.
HRMS (FAB+) m/z: 207.1330 ((M+H
+
)
–
H
2
); calc. for
C
10
H
16
O
2
N
2
B: 207.1305.
(
1
-
E
thyl
-
3
-
methyl
-
1
H
-
imidazol
-
3
-
ium
-
2
-
yl
)(
2
-
oxotetrahydro
-
2
H
-
pyran
-
3
-
yl
)
dihydroborate
(
5b
)
1
H NMR (400 MHz,
CDCl
3
) δ 6.86 (d,
J
= 2.0 Hz, 1H), 6.82 (d,
J
= 1.9 Hz,
1H), 4.46 (dddd,
J
= 10.8, 7.4, 3.7, 1.0 Hz, 1H), 4.26
–
4.10 (m, 3H), 3.75 (s,
3H), 2.14
–
1.94 (m, 3H), 1.86
–
1.74 (m, 1H), 1.70
–
1.10 (m, 3H), 1.43
–
1.38 (m, 3H)
;
13
C NMR (101 MHz, CDCl
3
) δ 181.45, 120.88, 118.29, 69.42,
43.64, 36.02, 27.22, 22.40, 15.88
;
11
B NMR
(128 MHz, CDCl
3
) δ
-
24.73 (t,
J
= 90.4 Hz)
.
HRMS
(FAB+) m/z: 221.1452 ((M+H
+
)
–
H
2
); calc. for C
11
H
18
O
2
N
2
B: 221.1461.
(
1
,
3
-
D
imethyl
-
5
-
(
trifluoromethyl
)
-
1
H
-
imidazol
-
3
-
ium
-
2
-
yl
)(
2
-
oxotetrahydro
-
2
H
-
pyran
-
3
-
yl
)
dihydroborate
(
5c
)
1
H NMR (400 MHz, CDCl
3
) δ 7.26 (s, 1H), 4.43 (dddd,
J
= 10.7, 7.1, 3.7,
1.1 Hz, 1H), 4.30
–
4.19 (m, 1H), 3.90
–
3.82 (m, 3H), 3.79 (s, 3H), 2.13
–
1.96 (m, 3H), 1.90
–
1.59 (m, 3H), 1.54
–
1.11 (m, 1H)
;
13
C N
MR (101
MHz, CDCl
3
) δ 181.22, 122.70 (q,
J
= 4.1 Hz), 122.32 (q,
J
= 40.8 Hz),
S
9
119.44 (q,
J
= 267.6 Hz), 69.76, 36.71, 34.14, 34.12, 27.29, 22.32
;
11
B NMR (128 MHz, CDCl
3
)
δ
-
24.93 (t,
J
= 91.3 Hz)
.
HRMS (FAB+) m/z: 275.1170 ((M+H
+
)
–
H
2
); calc. for C
11
H
15
O
2
N
2
F
3
B:
275.1179.
S
10
V.
Enzymatic Synthesis of
Lactone
-
Based Organoboranes
in A
nalytical and Preparative
Scale
All enzymatic reactions for lactone
-
based organoborane
formation in analytical scale were
conducted following the general procedure described below
and analyzed with gas
chromatography
-
mass spectrometry
(GC
-
MS
). All TTNs for the different products were
determined using the GC standard curve of the corresponding racemic standard product made with
Rh
2
(OAc)
4
or I
2
.
General procedure for analytical
-
scale
reactions:
To a 2 mL vial were added degassed suspension of
E. coli
expressing
R
m
a
cytochrome
c
variant in M9
-
N buffer (OD
600
= 15,
40
0 μL),
borane
(10 μL of 400 mM stock solution in
MeCN
,
10 mM),
and
lactone
-
diazo
(10 μL of 400 mM stock solution in
MeCN
, 10
mM
)
under anaerobic
conditions. The vial was capped and shaken at
52
0 rpm at room temperature for 1
8
h. After the
reaction was completed, internal standard 1,3,5
-
trimethoxybenzene (20 μL of 20 mM stock
solution in toluene) was added to the reaction vi
al followed by mixed solvent (hexane / ethyl
acetate =
3 : 7
, 1 mL). The mixture was transferred to a 1.5 mL microcentrifuge
,
vortexed (15
seconds × 3) and centrifuged (14,000 rpm, 5 min) to completely separate the organic and aqueous
layers. 0.8 mL of org
anic layer was taken for GC
-
MS
analysis. TTN was calculated based on
measured protein concentration. Enantiomeric excess was measured by chiral HPLC. Reactions
for every substrat
e were set up in quadruplicate.
GC standard curve:
All data points represent t
he average of duplicate runs. The calibration curves depict product
concentration in mM (y
-
axis) against the ratio of product area to internal standard area on the GC
(x
-
axis).
All enzymatic reactions for
lactone
-
based organoborane
formation in
preparative scale were
conducted following the general procedure described below
,
and the corresponding
lactone
-
based
organoborane
products were isolated. Detailed conditions for preparative
-
scale reactions of
different subst
rates are indicated separately.
General procedure for preparative
-
scale reactions:
To a
20
mL vial were added degassed suspension of
E. coli
expressing
R
m
a
cytochrome
c
(
5
mL,
OD
600
=
15
),
borane
(
150 μL of 400 mM
stock solution in MeCN
,
0.
06
mmol),
lactone diazo
(
150 μL of 400 mM
stock solution in MeCN
, 0.
06
mmol
),
D
-
glucose (
50 mM
),
in
M9
-
N buffer
(pH 7.4) under anaerobic conditions. The vial was capped and shaken (
48
0 rpm) at room
temperature for 1
8
h.
Reactions for each substrate were set up in
quadruplicate
.
After the reaction
was completed,
reactions in replicate
were
combined and
transferred to 50
mL centrifuge tubes. The reaction
vials
w
ere
washed with water (2 mL × 2) followed by mixed
organic solvent (hexane / ethyl acetate = 1 : 1, 2 mL × 3). The washing solution was comb
ined
with the reaction mixture in the centrifuge tubes. An additional 1
5
mL of hexane / ethyl acetate
solvent was added
to every tube.
T
he tube
was
then
vortexed (1 min × 3) and shake
n vigorously,
and centrifuged (5
,000 × g, 5 min). The organic layer was s
eparated and the aqueous layer was
subjected to three more rounds of extraction. The organic layers were combined, dried over
Na
2
SO
4
and concentrated under reduced pressure. Purification by silica column chromatography
with hexane /
(
ethyl acetate
: acetone
7:3)
as eluent afforded the desired
lactone
-
based
S
11
organoboranes
.
E
nantiomeric excess (
ee
) was measured by chiral HPLC. TTNs were calculated
based on measured protein concentration and isolated product yield.
(
1
,
3
-
Dimethyl
-
1
H
-
imidazol
-
3
-
ium
-
2
-
yl
)(
2
-
oxotet
rahydrofuran
-
3
-
yl
)
dihydroborate
(
3a
)
GC calibration curve:
Analysis Data:
Pdt
-
Entries
Pdt
Std
Pdt/Std
[Pdt]/mM
[PC]/μM
TTN
Avg.
TTN
Avg.
yield
3a
-
(1)
38262
5643
6.780
7.45
8.30
898
3a
-
(2)
40969
5630
7.277
7.94
8.30
957
3a
-
(3)
43223
5984
7.223
7.89
8.30
950
3a
-
(4)
49313
5559
8.871
9.46
8.30
1140
986
81.8%
Chiral HPLC trace:
Chiralpak IC, 40%
i
-
PrOH in hexane, 1.5 mL/min, 32 °C, 235 nm
y =
-
0,0156x
2
+ 1,2046x
R² = 0,9996
0
3
6
9
12
0
3
6
9
12
Concentration / mM
Product / Standard
Lactone
-
BH
-
NHC Product