Genetically programmed chiral organoborane synthesis
S. B. Jennifer Kan,
†
Xiongyi Huang,
†
Yosephine Gumulya, Kai 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
†
= These authors contributed equally to this work.
This PDF includes:
I.
Materials and Methods
2
II.
Kinetics Studies
3
-
4
III.
Inhibition Studies
4
IV.
Substrate Synthesis and Characterization
4
-
6
V.
Synthesis and Characterization of Authentic Organoborane Products
6
-
9
VI.
GC-MS Standard Curves for Organoborane Products
10
-
15
VII.
Determination of Enantioselectivity
16
-
25
VIII.
Preparative Scale Enzymatic Reactions
26
-
27
IX.
Derivatization of Enzymatic Borylation Product
18
28
-
30
X.
NMR Spectra
31
-
56
XI.
X-ray Crystallography and the Assignments of Absolute Configuration
57
-
63
XII.
Supplemental References
64
-
65
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PPLEMENTAR
Y INFORMATION
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I. Materials and Methods
Unless otherwise noted, all chemicals and reagents were obtained from commercial
suppliers (Sigma-Aldrich, VWR, Alfa Aesar, Acros) and used without further purification. Bovine
serum albumin (BSA) was purchased from Sigma-Aldrich.
Silic
a gel chromatography
was 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 (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 (
1
H NMR:
δ
= 7.26,
13
C NMR:
δ
= 77.36 for CDCl
3
).
19
F NMR and
11
B NMR data were
collected on a VARIAN 300 MHz spectrometer (101 MHz for
19
F NMR) and a Bruker Prodigy
400 MHz instrument (128 MHz for
11
B NMR), respectively. Sonication was performed using a
Qsonica Q500 sonicator. High-resolution mass spectra were obtained at the California Institute of
Technology Mass Spectral Facility. Chemical reactions were monitored using thin layer
chromatography (Merck 60 gel plates) using 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 column (30 m x 0.32 mm, 0.25 μm film). Gas chromatography-mass spectrometry
(GC-MS) analyses were carried out using Shimadzu GCMS-QP2010SE system and J&W HP-5ms
column. 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. Chiral normal-
phase HPLC analyses were performed using an Agilent 1200 series instrument with
i
-PrOH and
hexanes as the mobile phase. Chiral GC was performed on an Agilent 6850 GC with FID detector
using a Chiraldex GTA column (30.0 m
×
0.25 mm) at 1.0 mL/min He carrier gas flow.
Biological materials and methods are described in the Methods section of the manuscript.
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II. Kinetic Studies
Comparison of carbon–boron bond forming rates of BOR
WT
and BOR
R1
as whole-cell
catalysts, cell lysates, or purified proteins.
Biocatalysts
turnover frequency (TOF) / h
–1
BOR
WT
purified protein
3 ± 2
BOR
WT
cell lysate
4 ± 1
BOR
WT
whole cell
410 ± 250
BOR
R1
purified protein
30 ±
2
BOR
R1
cell lysate
160 ± 100
BOR
R1
whole cell
6100 ± 700
TOFs reported represent mean values averaged over four experiments. Errors quoted indicate
one standard deviation.
Whole cell-catalysed reaction
: Experiments were performed using whole
E. coli
cells
harbouring BOR
WT
or BOR
R1
(with the BOR protein concentration normalised to 10 μM), 10 mM
borane, 10 mM diazo ester, 5 vol% MeCN, M9-N buffer at room temperature under anaerobic
conditions for various time intervals.
Cell lysate-catalysed reaction
: Experiments were performed using cell lysate of
E. coli
harbouring BOR
WT
or BOR
R1
(with the BOR protein concentration normalised to 10 μM), 10 mM
borane, 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 various time intervals. See Methods section of the manuscript for
cell lysate preparation procedure.
Purified protein-catalysed reaction
: Experiments were performed using purified BOR
WT
or BOR
R1
(10 μM), 10 mM borane, 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 various time intervals. See Methods
section of the manuscript for purified protein preparation procedure
General procedure for carrying out timed experiments:
In an anaerobic chamber, 3.8
mL of whole
E. coli
cells
harboring BOR variant, or a solution of 3.4 mL of BOR variant cell
lysate / purified protein and 0.4 mL Na
2
S
2
O
4
(100 mM in M9-N buffer), was added to a 10 mL
glass vial. After charging NHC-borane
1
(100 μL, 400 mM in MeCN) and Me-EDA
2
(100 μL,
400 mM in MeCN), the vial was capped and the reaction was shaken at 600 rpm on an orbital
shaker. At regular time intervals (see table below), 400 μL of the reaction mixture was removed
from the vial and added to a 2 mL microcentrifuge tube containing 600 μL cyclohexane / EtOAc
Me
N
2
O
OEt
+
E. coli
harbouring
Rma
cyt
c
variant
Me
BH
2
O
OEt
N
N
BH
3
N
N
M9-N buffer (pH 7.4)
room temperature
Me-EDA
(
2
)
NHC-borane (
1
)
3
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(1:1 v/v) and internal standard (20 μL, 20 mM 1,2,3-trimethoxybenzene in toluene). After
vortexing for 20 seconds, 200 μL of the organic layer was immediately removed for GC analysis.
Biocatalys
ts
Sampling time
BOR
WT
purified protein
Every hour from t = 1 to 4 h
BOR
WT
cell lysate
Every hour from t = 1 to 4 h
BOR
WT
whole cell
Every minute from t = 1 to 4 min
BOR
R1
purified protein
Every minute from t = 1 to 4 min
BOR
R1
cell lysate
Every minut
e from t = 0.5 to 3.5 min
BOR
R1
whole cell
Every minute from t = 0.5 to 3.5 min
Table above shows time points at which the biocatalytic reaction was sampled to determine the
reaction initial rate.
III.
Inactivation Studies
Inactivation studies of BOR
R1
were carried out using purified protein or whole cell
E. coli
harbouring BOR
R1
. Effects of NHC-borane
1
, Me-EDA
2
, or organoborane
3
were determined
by preincubating the biocatalyst with either one of these reagents (10 mM) for 15 min before the
catalyst was used for borylation, and by comparing the TTN of the resulting catalyst (TTN
incub
)
with that of an untreated biocatalyst (TTN
control
), as described in Figure 2f.
Purified protein-catalysed reactions were performed using purified BOR
R1
(10 μM), 10
mM borane, 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 30 min. See
Methods section of the manuscript for
purified protein preparation procedure
Whole cell-catalysed reaction
s
were performed using whole
E. coli
cells harboring BOR
R1
(with the BOR protein concentration normalised to 10 μM), 10 mM borane, 10 mM diazo ester, 5
vol% MeCN, M9-N buffer at room temperature under anaerobic conditions for 30 min.
IV. Substrate Synthesis and Characterization
Picoline borane substrate was obtained from Sigma-Aldrich. Ethyl 2-diazopropanoate (Me-
EDA) was obtained from Arch Bioscience. All commercially available reagents were used as
received. The following diazo compounds are known and prepared according to literature
procedures: methyl 2-diazopropanoate
1
, isopropyl 2-diazopropanoate
2
, benzyl 2-diazopro-
panoate
3
, ethyl 2-phenyldiazoacetate (Ph-EDA)
4
, ethyl 2-diazo-3,3,3-trifluoropropanoate (CF
3
-
EDA)
5
, and (1-diazo-2,2,2-trifluoroethyl)benzene (CF
3
-DMB)
6
.
Other
NHC-BH
3
substrates were synthesized from corresponding imidazolium iodide salts
as reported
7
. Namely, imidazolium iodide
salts
(5 mmol) were resuspended in 5 mL THF.
A
solution of NaHMDS (1M in THF, 1.05 equiv.) was then added at
‒
78
o
C under Ar and shaken for
1 h at
‒
78
o
C. Afterwards, a solution of BH
3
-THF (1M in THF, 1 equiv.) was added to the reaction
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and the reaction mixture was allowed to warm from
‒
78
o
C to rt and stirred overnight. The solvent
was removed under reduced pressure and the residue was purified by flash column
chromatography to give the
NHC-BH
3
complexes. The
1
H NMR resonances of the B
‒
H
protons
are broad (due to geminal coupling with boron) and generally in the range of 0.4 – 1.6 ppm. T
he
13
C NMR resonances of
the boron-binding NHC quarternary carbons usually appear at around 170
ppm and are typically broad (due to germinal coupling with boron) and weak; these signals are
sometimes not visible in the
13
C NMR spectra.
This compound is known
8
.
1
H NMR (400 MHz, Chloroform-
d
)
δ
6.91 –
6.66 (m, 2H), 3.71 (s, 6H), 0.99 (dd,
J
= 172.7, 86.3 Hz, 3H).
This compound is known
9
.
1
H NMR (400 MHz, Chloroform-
d
)
δ
6.87 –
6.65 (m, 2H), 4.00 (q,
J
= 7.3 Hz, 2H), 3.57 (s, 3H), 1.22 (t,
J
= 7.3 Hz,
3H), 1.44 – 0.30 (m, 3H).
This compound is known
8
.
1
H NMR (400 MHz, Chloroform-
d
)
δ
6.84 –
6.79 (m, 2H), 5.91 (ddt,
J
= 17.1, 10.2, 6.1 Hz, 1H), 5.30 – 5.06 (m, 2H),
4.71 (dt,
J
= 6.1, 1.5 Hz, 2H), 3.71 (s, 3H), 1.43 – 0.35 (m, 3H).
1
H NMR (400 MHz, Chloroform-
d
)
δ
6.82 – 6.76 (m, 2H), 4.13 – 3.97 (m,
2H), 3.69 (s, 3H), 1.83 – 1.63 (m, 2H), 1.42 – 1.19 (m, 6H), 0.97 – 0.75 (m,
3H), 1.46 – 0.41 (m, 3H);
13
C NMR (101 MHz, CDCl
3
)
δ
171.0, 119.9, 118.7,
48.8, 35.8, 31.3, 30.1, 26.1, 22.5, 14.0;
11
B NMR (128 MHz, Chloroform-
d
)
δ
‒
37.4 (q,
J
= 86
Hz)
;
MS (FAB)
m/z
[(M + H)
+
‒
H
2
]
calcd for
C
10
H
20
N
2
B:
179.1720
, found:
179.1707
.
This compound is known
8
.
1
H NMR (400 MHz, Chloroform-
d
)
δ
7.85 (s,
1H), 3.94 (s, 3H), 3.74 (s, 3H), 1.45 – 0.42 (m, 3H).
This compound is known
10
.
1
H NMR (400 MHz, Chloroform-
d
)
δ
6.49 (q,
J
= 1.2 Hz, 1H), 3.56 (s, 3H), 3.50 (s, 3H), 2.07 (d,
J
= 1.3 Hz, 3H), 1.31 –
0.43 (m, 3H).
This compound is known
11
.
1
H NMR (400 MHz, Chloroform-
d
)
δ
3.72 (s,
6H), 1.44 – 0.41 (m, 3H).
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1
H NMR (400 MHz, Chloroform-
d
)
δ
7.23 (q,
J
= 1.5 Hz, 1H), 3.82 (s, 3H),
3.77 (s, 3H), 1.49 – 0.54 (m, 3H).
13
C NMR (101 MHz, CDCl
3
)
δ
177.1,
122.2, 121.8, 119.40 (q,
J
= 267.3 Hz), 36.5, 34.0.
11
B NMR (128 MHz,
Chloroform-
d
)
δ
‒
37.4 (q,
J
= 88 Hz).
19
F NMR (282 MHz, Chloroform-
d
)
δ
‒
61.2 (d,
J
= 3
Hz)
;
MS (FAB)
m/z
[(M+H)
+
‒
H
2
]
calcd for
C
6
H
9
F
3
N
2
B:
177.0811
, found:
177.0815
.
1
H NMR (400 MHz, Chloroform-
d
)
δ
6.78 (s, 1H), 3.71 (s, 3H), 3.67 (s, 3H),
1.45 – 0.51 (m, 3H).
13
C NMR (101 MHz, CDCl
3
)
δ
13
C NMR (101 MHz,
CDCl
3
)
δ
173.5, 119.3, 116.9, 36.4, 33.0.
11
B NMR (128 MHz, Chloroform-
d
)
δ
‒
36.9 (q,
J
= 87
Hz)
;
MS (FAB)
m/z
[(M+H)
+
‒
H
2
]
calcd for
C
5
H
9
N
2
BCl:
143.0547
, found:
143.0547
.
V. Synthesis and Characterization of Authentic Organoborane Products
Racemic standard references of organoborane products were prepared
via
Rh-catalyzed B
‒
H insertion reactions with procedures slightly modified from a previously reported method
8
.
Namely,
a 4 mL vial with screw cap and PTFE septum was charged with a borane substrate (1.0
mmol, 1 equiv.) and Rh
2
(OAc)
4
(11 mg, 2.5 mol%). The vial was evacuated and backfilled with
Ar three times and 2 mL of anhydrous CH
2
Cl
2
was added. The vial was placed in a 38
o
C water
bath. A CH
2
Cl
2
solution (1 mL) of diazo compound (1.0 mmol) was slowly added to the reaction
mixture over 4 hours. Afterwards, the reaction mixture was allowed to further react overnight. The
crude reaction mixture was purified by flash chromatography (dry loading) using EtOAc and
hexanes as eluents and afforded organoborane products in 30 - 75% yield. The
1
H NMR resonances
of the B
‒
H protons are broad (due to geminal coupling with boron) and generally in the range of
0.4 – 1.6 ppm. T
he
13
C NMR resonances of the boron-binding NHC quarternary carbons usually
appear at around 170 ppm and are typically broad (due to germinal coupling with boron) and weak;
these signals are sometimes not visible in the
13
C NMR spectra.
(
1
,
3
-
Dimethyl
-
1
H
-
imidazol
-
3
-
ium
-
2
-
yl
)(
1
-
ethoxy
-
1
-
oxopropan
-
2
-
yl
)
dihydroborate
(
3
)
1
H NMR (400 MHz, Chloroform-
d
)
δ
6.82 (s, 2H), 3.98 – 3.78 (m, 2H), 3.75 (s,
6H), 1.95 – 1.10 (m, 2H), 1.88 (br s, 1H), 1.10 (d,
J
= 6.2 Hz, 3H), 1.06 (t,
J
=
7.1 Hz, 3H);
13
C NMR (101 MHz, Chloroform-
d
)
δ
183.5, 120.4, 58.7, 36.2, 30.5,
17.8, 14.6. The boron-bound NHC quarternery carbon
was
not resolved;
11
B
NMR (128 MHz, Chloroform-
d
)
δ
‒
24.6 (t,
J
= 90
Hz)
;
MS (FAB)
m/z
[(M +
H)
+
‒
H
2
]
calcd for
C
10
H
18
O
2
N
2
B: 209.1461
, found:
209.1456
.
(
1
-
Ethoxy
-
1
-
oxopropan
-
2
-
yl
)(
3
-
ethyl
-
1
-
methyl
-
1
H
-
imidazol
-
3
-
ium
-
2
-
yl
)
dihydroborate
(
4
)
1
H NMR (400 MHz, Chloroform-
d
)
δ
6.91 – 6.82 (m, 2H), 4.28 – 3.97 (m, 2H),
3.93 – 3.73 (m, 2H), 3.70 (s, 3H), 1.84 (br s, 1H), 1.95 – 1.10 (br m, 2H), 1.34 (t,
J
= 7.3 Hz, 3H), 1.05 (d,
J
= 6.7 Hz, 3H), 0.98 (t,
J
= 7.2 Hz, 3H);
13
C NMR (101
MHz, Chloroform-
d
)
δ
183.3, 170.0, 120.7, 118.2, 58.4, 43.5, 35.9, 30.4, 17.6,
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15.8, 14.4;
11
B NMR (128 MHz, Chloroform-
d
)
δ
–24.5 (t,
J
= 89 Hz).
MS (FAB)
m/z
[M
+
·
]
calcd
for
C
11
H
21
O
2
N
2
B: 224.1696
, found:
224.1693
.
(
3
-
Allyl
-
1
-
methyl
-
1
H
-
imidazol
-
3
-
ium
-
2
-
yl
)(
1
-
ethoxy
-
1
-
oxopropan
-
2
-
yl
)
dihydroborate
(
5
)
1
H NMR (400 MHz, Chloroform-
d
)
δ
6.85 (AB q,
J
= 2.0 Hz, 2H), 5.94 (ddt,
J
=
17.1, 10.2, 6.1 Hz, 1H), 5.39 – 5.17 (m, 2H), 4.82 (ddt,
J
= 15.3, 6.0, 1.5 Hz, 1H),
4.68 (ddt,
J
= 15.3, 6.2, 1.4 Hz, 1H), 3.99 – 3.78 (m, 2H), 3.76 (s, 3H), 1.92 – 1.05
(m, 2H), 1.87 (br s, 1H), 1.09 (d,
J
= 6.6 Hz, 3H), 1.05 (t,
J
= 7.1 Hz, 3H);
13
C
NMR (101 MHz, Chloroform-
d
)
δ
183.6, 132.9, 120.9, 119.7, 119.0, 58.8, 51.4,
36.4, 32.0, 17.9, 14.8. The boron-bound NHC quarternery carbon
was
not resolved;
11
B NMR (128 MHz, Chloroform-
d
)
δ
–24.6 (t,
J
= 90 Hz).
MS (FAB)
m/z
[M
+
H
+
]
calcd for
C
12
H
22
O
2
N
2
B: 237.1774
, found: 237.1783
.
(
1
-
Ethoxy
-
1
-
oxopropan
-
2
-
yl
)(
3
-
hexyl
-
1
-
methyl
-
1
H
-
imidazol
-
3
-
ium
-
2
-
yl
)
dihydroborate
(
6
)
1
H NMR (400 MHz, Chloroform-
d
)
δ
6.88 – 6.79 (m, 2H), 4.20 – 4.06 (m,
1H), 3.99 (m, 1H), 3.93 – 3.74 (m, 2H), 3.72 (s, 3H), 1.93 – 1.79 (m, 1H),
1.72 (dt,
J
= 13.8, 6.9 Hz, 2H), 1.71 – 1.20 (m, 8H), 1.12 – 1.05 (m, 3H), 1.01
(td,
J
= 7.2, 2.4 Hz, 3H), 0.90 – 0.80 (m, 3H);
13
C NMR (101 MHz,
Chloroform-
d
)
δ
183.6, 120.7, 119.0, 58.7, 48.9, 36.2, 31.6, 30.8, 30.5, 26.5,
22.7, 17.9, 14.7, 14.2. The boron-bound NHC quarternery carbon
was
not
resolved;
11
B NMR (128 MHz, Chloroform-
d
)
δ
–24.5 (t,
J
= 90 Hz);
MS (FAB)
m/z
[M
+
·
]
calcd for
C
15
H
29
O
2
N
2
B: 280.2322
, found: 280.2330
.
(
1
-
Ethoxy
-
1
-
oxopropan
-
2
-
yl
)(
1
,
3
,
4
-
trimethyl
-
1
H
-
imidazol
-
3
-
ium
-
2
-
yl
)
dihydroborate
(
7
)
1
H NMR (400 MHz, Chloroform-
d
)
δ
6.55 (q,
J
= 1.2 Hz, 1H), 3.95 – 3.77 (m,
2H), 3.66 (s, 3H), 3.61 (s, 3H), 2.16 (d,
J
= 1.1 Hz, 3H), 1.84 (br
s, 1H), 1.93 –
1.10 (m, 2H), 1.07 (s, 3H), 1.12 – 1.02 (m, 3H);
13
C NMR (101 MHz, Chloroform-
d
)
δ
183.7, 170.0, 128.3, 117.6, 58.7, 35.9, 32.7, 32.0 – 29.5 (m), 17.9, 14.7, 9.7;
11
B NMR (128 MHz, Chloroform-
d
)
δ
–24.2 (t,
J
= 89 Hz);
MS (FAB)
m/z
[M
+
·
]
calcd
for
C
11
H
21
O
2
N
2
B: 224.1696
, found: 224.1695
.
(
1
,
3
-
Dimethyl
-
4
-(
trifluoromethyl
)-
1
H
-
imidazol
-
3
-
ium
-
2
-
yl
)(
1
-
ethoxy
-
1
-
oxopropan
-
2
-
yl
)
dihydroborate
(
8
)
1
H NMR (400 MHz, Chloroform-
d
)
δ
7.29 (q,
J
= 1.3 Hz, 1H), 4.00 – 3.70 (m,
2H), 3.85 (s, 3H), 3.81 (s, 3H), 1.88 (br s, 1H), 1.85 – 1.05 (m, 2H), 1.12 (d,
J
=
6.6 Hz, 3H), 1.05 (t,
J
= 7.1 Hz, 3H);
13
C NMR (101 MHz, Chloroform-
d
)
δ
183.2, 123.6, 122.8 – 122.5 (m), 119.6 (q,
J
= 267.6 Hz), 59.0, 36.9, 34.4, 30.0,
17.9, 14.7. The boron-bound NHC quarternery carbon
was
not resolved;
11
B
NMR (128 MHz, Chloroform-
d
)
δ
–24.7 (t,
J
= 91 Hz).
19
F NMR (282 MHz,
Chloroform-
d
)
δ
–61.1;
MS (FAB)
m/z
[M
+·
] calcd for C
11
H
18
O
2
N
2
BF
3
:
278.1414, found: 278.1405.
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24996
(
4
-
Chloro
-
1
,
3
-
dimethyl
-
1
H
-
imidazol
-
3
-
ium
-
2
-
yl
)(
1
-
ethoxy
-
1
-
oxopropan
-
2
-
yl
)
dihydroborate
(
9
)
1
H NMR (400 MHz, Chloroform-
d
)
δ
6.83 (s, 1H), 3.97 – 3.79 (m, 2H), 3.73
(s, 3H), 3.70 (s, 3H), 2.00 – 1.10 (m, 2H), 1.94 – 1.76 (m, 1H), 1.13 – 1.02 (m,
6H);
13
C NMR (101 MHz, Chloroform-
d
)
δ
183.4, 173.0, 119.9, 117.3, 58.9,
36.6, 33.3, 30.3, 17.9, 14.7;
11
B NMR (128 MHz, Chloroform-
d
)
δ
–24.2 (t,
J
= 90
Hz)
;
MS (FAB)
m/z
[M
+
·
]
calcd for
C
10
H
18
O
2
N
2
BCl: 244.1150
, found:
244.1154
.
(
4
,
5
-
Dichloro
-
1
,
3
-
dimethyl
-
1
H
-
imidazol
-
3
-
ium
-
2
-
yl
)(
1
-
ethoxy
-
1
-
oxopropan
-
2
-
yl
)
dihydroborate
(
10
)
1
H NMR (400 MHz, Chloroform-
d
)
δ
3.95 – 3.78 (m, 2H), 3.72 (s, 6H), 1.83
(br s, 1H), 1.99 – 1.05 (m, 2H), 1.10 (d,
J
= 11.9 Hz, 3H), 1.05 (t,
J
= 7.1 Hz,
3H);
13
C NMR (101 MHz, Chloroform-
d
)
δ
183.1, 172.0, 116.6, 59.0, 34.1,
30.0, 17.8, 14.7;
11
B NMR (128 MHz, Chloroform-
d
)
δ
–23.9 (t,
J
= 91 Hz);
MS (FAB)
m/z
[M
+ H
+
]
calcd for
C
10
H
18
O
2
N
2
BCl
2
: 279.0838
, found: 279.0846.
(
1
,
4
-
Dimethyl
-
4
H
-
1
,
2
,
4
-
triazol
-
1
-
ium
-
5
-
yl
)(
1
-
ethoxy
-
1
-
oxopropan
-
2
-
yl
)
dihydroborate
(
11
)
1
H NMR (400 MHz, Chloroform-
d
)
δ
7.92 (s, 1H), 3.95 (s, 3H), 3.94 – 3.79 (m,
2H), 3.78 (s, 3H), 1.89 (br s, 1H), 2.00 – 1.05 (m, 2H), 1.13 – 1.09 (m, 3H), 1.05
(t,
J
= 7.1 Hz, 3H).
13
C NMR (101 MHz, Chloroform-
d
)
δ
183.2, 141.7, 59.0 (d,
J
= 8.0 Hz), 38.6, 34.1, 30.0, 17.9, 14.7. The boron-bound NHC quarternery carbon
was
not resolved;
11
B NMR (128 MHz, Chloroform-
d
)
δ
–25.0 (t,
J
= 91 Hz).
MS
(FAB)
m/z
[M
+ H
+
]
calcd for
C
9
H
19
O
2
N
3
B: 212.1570
, found: 212.1570
.
Ethyl
2
-((
2
-
methyl
-
pyridin
-
1
-
yl
)
boraneyl
)
propanoate
(
12
)
1
H NMR (400 MHz, Chloroform-
d
)
δ
8.53 (dd,
J
= 6.0, 1.6 Hz, 1H), 7.84 (td,
J
= 7.7, 1.7 Hz, 1H), 7.42 – 7.36 (m, 1H), 7.33 – 7.28 (m, 1H), 3.79 (AB qq,
J
=
10.8, 7.1 Hz, 2H), 3.30 – 2.15 (m, 2H), 2.77 (s, 3H), 2.05 – 1.92 (m, 1H), 1.05
(d,
J
= 6.8 Hz, 3H), 0.94 (t,
J
= 7.1 Hz, 3H);
13
C NMR (101 MHz, Chloroform-
d
)
δ
182.1, 157.9, 149.4, 140.2, 127.7, 122.6, 58.8, 32.8, 22.8, 15.2, 14.6;
11
B
NMR (128 MHz, Chloroform-
d
)
δ
–5.1 (t,
J
= 103 Hz);
MS (FAB)
m/z
[M
+
·
]
calcd
for
C
11
H
18
O
2
NB: 207.1431
, found: 207.1431
.
(
1
,
3
-
Dimethyl
-
1
H
-
imidazol
-
3
-
ium
-
2
-
yl
)(
1
-
methoxy
-
1
-
oxopropan
-
2
-
yl
)
dihydroborate
(
13
)
1
H NMR (400 MHz, Chloroform-
d
)
δ
6.82 (s, 1H), 3.72 (s, 6H), 3.43 (s, 2H),
1.99 – 1.08 (m, 3H), 1.06 (d,
J
= 6.8 Hz, 2H);
13
C NMR (101 MHz, Chloroform-
d
)
δ
183.9, 170.0, 120.6, 50.7, 36.2, 30.5, 17.8;
11
B NMR (128 MHz,
Chloroform-
d
)
δ
–24.6 (t,
J
= 90 Hz);
MS (FAB)
m/z
[M
+
·
]
calcd for
C
9
H
17
O
2
N
2
B:
196.1383
, found: 196.1388
.
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(
1
,
3
-
Dimethyl
-
1
H
-
imidazol
-
3
-
ium
-
2
-
yl
)(
1
-
isopropoxy
-
1
-
oxopropan
-
2
-
yl
)
dihydroborate
(
14
)
1
H NMR (400 MHz, Chloroform-
d
)
δ
6.81 (s, 2H), 4.76 (hept,
J
= 6.2 Hz, 1H),
3.75 (s, 6H), 1.86 (br s, 1H), 2.00 – 1.10 (m, 2H), 1.09 (d,
J
= 6.2 Hz, 6H), 0.94
(d,
J
= 6.3 Hz, 3H);
13
C NMR (101 MHz, Chloroform-
d
)
δ
183.3, 170.0, 120.6,
65.1, 36.4, 30.7, 22.5, 22.2, 18.1;
11
B NMR (128 MHz, Chloroform-
d
)
δ
–24.5
(t,
J
= 90 Hz);
MS (FAB)
m/z
[M
+
·
]
calcd for
C
11
H
21
O
2
N
2
B: 224.1696
, found:
224.1703
.
(
1
-(
Benzyloxy
)-
1
-
oxopropan
-
2
-
yl
)(
1
,
3
-
dimethyl
-
1
H
-
imidazol
-
3
-
ium
-
2
-
yl
)
dihydroborate
(
15
)
1
H NMR (400 MHz, Chloroform-
d
)
δ
7.36 – 7.17 (m, 5H), 6.71 (s, 2H), 4.92 (s,
2H), 3.62 (s, 6H), 2.10 – 1.15 (m, 2H), 1.97 (br s, 1H), 1.16 (d,
J
= 6.5 Hz, 3H);
13
C NMR (101 MHz, Chloroform-
d
)
δ
183.2, 170.0, 137.6, 128.4, 128.1, 127.7,
120.5, 64.7, 36.1, 30.8, 17.9;
11
B NMR (128 MHz, Chloroform-
d
)
δ
–24.5 (t,
J
= 88 Hz);
MS (FAB)
m/z
[(M
+
H)
+
‒
H
2
]
calcd for
C
15
H
20
O
2
N
2
B: 271.1618
,
found: 271.1616
.
(
1
,
3
-
Dimethyl
-
1
H
-
imidazol
-
3
-
ium
-
2
-
yl
)(
3
-
ethoxy
-
1
,
1
,
1
-
trifluoro
-
3
-
oxopropan
-
2
-
yl
)
dihydroborate
(
16
)
1
H NMR (400 MHz, Chloroform-
d
)
δ
6.88 (s, 2H), 4.15 – 3.97 (m, 2H), 3.76 (s,
6H), 2.65 (s, 1H), 2.10 – 1.25 (m, 2H), 1.18 (t,
J
= 7.1 Hz, 3H);
13
C NMR (101
MHz, Chloroform-
d
)
δ
174.2 (d,
J
= 5.2 Hz), 168.0, 128.7 (q,
J
= 276.2 Hz), 121.2,
60.0, 42.6, 36.3, 14.6;
11
B NMR (128 MHz, Chloroform-
d
)
δ
–
28.6 (t,
J
= 92 Hz).
19
F NMR (282 MHz, Chloroform-
d
)
δ
–
62.5 (d,
J
= 10 Hz);
MS (FAB)
m/z
[(M
+
H)
+
‒
H
2
]
calcd for
C
10
H
15
O
2
N
2
BF
3
: 263.1179
, found: 263.1167
.
(
1
,
3
-
Dimethyl
-
1
H
-
imidazol
-
3
-
ium
-
2
-
yl
)(
2
-
ethoxy
-
2
-
oxo
-
1
-
phenylethyl
)
dihydroborate
(
17
)
1
H NMR (400 MHz, Chloroform-
d
)
δ
7.35 – 7.24 (m, 2H), 7.19 – 7.11 (m, 2H),
7.07 – 6.99 (m, 1H), 6.77 (s, 2H), 4.24 – 3.93 (m, 2H), 3.46 (s, 6H), 3.35 – 3.22 (m,
1H), 2.34 – 1.41 (m, 2H), 1.21 (t,
J
= 7.1 Hz, 3H);
13
C NMR (101 MHz,
Chloroform-
d
)
δ
179.7, 145.8, 127.9, 127.8, 124.1, 120.7, 120.2, 59.3, 45.6 (d,
J
=
44.3 Hz), 36.0, 14.8, (the NHC quarternary carbon was too broad to be visible due
to coupling with B);
11
B NMR (128 MHz, Chloroform-
d
)
δ
–23.2 (t,
J
= 93 Hz);
MS (FAB)
m/z
[M
+
·
]
calcd for
C
15
H
21
O
2
N
2
B: 272.1696
, found: 272.1687
.
(
1
,
3
-
Dimethyl
-
1
H
-
imidazol
-
3
-
ium
-
2
-
yl
)(
2
,
2
,
2
-
trifluoro
-
1
-
phenylethyl
)
dihydroborate
(
18
)
1
H NMR (400 MHz, Chloroform-
d
)
δ
7.23 – 7.05 (m, 5H), 6.76 (s, 2H), 3.52 (s, 6H),
2.90 – 2.60 (m, 1H), 2.25 – 1.40 (m, 2H);
13
C NMR (101 MHz, Chloroform-
d
)
δ
169.1, 143.7 (d,
J
= 3.5 Hz), 131.4 (q,
J
= 278.0 Hz), 128.4, 128.3, 125.2, 120.8, 43.5,
36.0;
11
B NMR (128 MHz, Chloroform-
d
)
δ
‒
26.7 (t,
J
= 90 Hz);
19
F NMR (282 MHz,
Chloroform-
d
)
δ
–61.8 (d,
J
= 13 Hz);
MS (ESI)
m/z
[M
+ H
+
]
calcd for
C
13
H
17
N
2
BF
3
:
269.1437
, found: 269.1440
.
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VI.
GC-MS Standard Curves for Organoborane Products
Product formation in enzymatic reactions was quantified by GC-MS based on standard
curves. To determine the standard calibration curves, stock solutions of chemically synthesized
organoborane products were prepared at various concentrations (1 - 7 mM in 4:6 hexanes/EtOAc)
with added internal standard 1,2,3-trimethoxybenzene with a final concentration of 6.45 mM in
the stock solutions of organoborane products. Individual data point for each duplicate run is
marked as triangle, the average of duplicate runs is marked as red dot. The standard curves plot
product concentration in mM (y-axis) against the average ratio of product area to internal standard
area on GC-MS (x-axis). The quantification of organoborane
12
was determined by preparative
scale reactions as this compound cannot be identified by GC-MS.
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