Q. Lam et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
3
recrystallized in DMSO/water mixture (C6 (406 mg, 77%), C11 (340 mg,
7.34 ppm (s, 1H, benzyl), 6.94 ppm (d, 2H, phenyl-O-), 5.13 ppm (q,
2H); C NMR: δ = 163.8 ppm (C–NO ), 141.8 ppm (Cphenyl–O),
2
135.6 ppm (Cbenzyl), 129.0 ppm (Cbenzyl), 128.7 ppm (Cbenzyl),
127.7 ppm (Cbenzyl), 126.1 ppm (Cphenyl), 115.0 ppm (Cphenyl),
2
70.9 ppm (–O–CH –).
1
13
13
6
2%), C16 (416 mg, 74%)). The H and C NMR chemical shifts for 6-
and 11-pNCA match the previously reported values (see Figs. S7–S9)
12].
p-Nitrophenoxyhexadecanoic acid (16-pNCA, 3): mp = 105–107 °C;
H NMR (CDCl ): δ = 8.15 ppm (d, 2H, NO -phenyl-), 6.91 ppm (d, 2H,
phenyl-O-), 4.03 ppm (t, 2H, phenyl-O–CH
COO), 1.78 ppm (m, 2H, –O–CH –CH –), 1.58 ppm (m, 2H, –CH
[
1
3
2
2
–), 2.32 ppm (t, 2H, –CH
2
–
–
2.3. Photocatalytic reactions and optimization of the conditions
2
2
2
–CH
2
13
COO), 1.41 ppm (m, 2H), 1.25 ppm (m, 20H); C NMR: δ =
Photocatalytic reactions are typically run as follows: 1 μM of enzyme
with 100 mM diethyldithiocarbamate and 400 μM of substrates in a
500 μL total reaction volume. The solution, maintained at 30 °C in a
water bath, is then irradiated for 30 min before being quenched by the
addition of 10 μL of 6 M NaOH. For the optimization of the reaction con-
ditions, individual parameters in the light-driven process were varied
one at a time including the nature and concentration (0 mM to
150 mM) of reductive quencher, reaction pH (7.0 to 9.0), temperature
(10 °C to 35 °C), buffer concentration (10 mM to 100 mM), and light
intensity.
1
1
4
2
80.0 ppm (–COOH), 164.2 ppm (C–NO
26.1 ppm (Cphenyl), 114.6 ppm (Cphenyl), 69.1 ppm (–O–CH
0.6 ppm (−CH –), 34.0 ppm (–CH –COOH), 29.1–29.8 ppm (–CH
6.1 ppm (–CH –), 24.9 (–CH –).
2
), 141.3 ppm (Cphenyl–O),
–),
–),
2
2
2
2
2
2
2
.2.2. Synthesis of the 1-alkoxy-4-nitrobenzene (5–9)
A DMSO solution (20 mL) of the corresponding bromoalkyl com-
pounds (1.1 eq.) was added to a solution (20 mL) containing p-
nitrophenolate (1 eq.) and potassium carbonate (1 eq.). The reaction
mixture was stirred at 70 °C for 4 to 6 h. Upon cooling and addition of
water (50 mL), the aqueous solution was extracted three times with di-
chloromethane. The desired products were recovered after evaporation
of the organic layer. For 5, white crystalline material was obtained after
recrystallization in DMSO/water (65%). For 6–9, the desired compound
was recovered as a clear oil (60–85%) after purification by flash column
2.4. Control reactions
Two sets of control reactions were performed to ensure that the ob-
served color in the assay is not generated from background reactions or
through the hydrogen peroxide shunt. The first set involved the system-
atic removal of individual components necessary for the light-driven
process (i.e. reductive quencher, photosensitizer covalent attachment,
hybrid enzyme). The second set uses peroxide shunt reactions with
10 mM hydrogen peroxide.
chromatography using silica and petroleum/ether (8:2) as eluent.
1
1
-ethyl-4-nitrobenzene (5): mp = 58–61 °C. H NMR (CDCl
3
): δ =
8
4
NO
6
.14 ppm (d, 2H, NO
.08 ppm (q, 2H), 1.42 ppm (t, 3H); C NMR: δ = 164.2 ppm (C–
), 141.1 ppm (Cphenyl–O), 126.1 ppm (Cphenyl), 114.6 ppm (Cphenyl),
4.6 ppm (–O–CH –), 14.7 ppm (–CH ).
2
-phenyl-), 6.88 ppm (d, 2H, phenyl-O-),
1
3
2
2
3
2.5. Substrate binding determination
1
3
1-isopropyl-4-nitrobenzene (6): H NMR (CDCl ): δ = 8.04 ppm (d,
2
H, NO
2
-phenyl-), 6.83 ppm (d, 2H, phenyl-O-), 4.56 ppm (m, 1H),
d
The dissociation constants, K , were determined by optical titration
1
3
1
.29 ppm (d, 6 H); C NMR: δ = 163.4 ppm (C–NO
phenyl–O), 126.0 ppm (Cphenyl), 115.3 ppm (Cphenyl), 71.1 ppm (–O–
CH–), 21.9 ppm (–CH ).
-propyl-4-nitrobenzene (7): H NMR (CDCl
2
), 141.0 ppm
with DMSO solutions of the respective substrates, keeping the maxi-
mum concentration of DMSO below 2.5% (v/v). All hybrid enzyme solu-
tions were at 2 μM in 50 mM Tris at pH 8.5 and room temperature. The
difference spectra between 350 and 650 nm were recorded 30 s after
each addition, using the substrate-free hybrid enzyme solution as the
reference spectrum. For the substrates displaying binding, the peak-
to-trough difference between 390 and 420 nm was plotted against sub-
strate concentrations (see Fig. S1). Substrate dissociation constants
were extracted from fitting the data of triplicate experiments to a rect-
angular hyperbolic function of the type Y = Bmax ∗ X / (Kd + X) for spe-
cific substrate binding using the GraphPad Prism® software.
(
C
3
1
1
3
): δ = 8.14 ppm (d,
-phenyl-), 6.91 ppm (d, 2H, phenyl-O-), 3.99 ppm (q, 2H),
2
1
1
1
1
H, NO
2
13
13
.81 ppm (m, 2H), 1.02 ppm (t, 3H); C NMR: C NMR: δ =
64.4 ppm (C–NO ), 141.3 ppm (Cphenyl–O), 125.9 ppm (Cphenyl),
14.5 ppm (Cphenyl), 70.4 ppm (–O–CH –), 22.4 ppm (–CH –),
0.5 ppm (–CH ).
-hexyl-4-nitrobenzene (8): H NMR (CDCl
-phenyl-), 6.88 ppm (d, 2H, phenyl-O-), 3.98 ppm (t, 2H), 1.75 ppm
2
2
2
3
1
1
3
): δ = 8.11 ppm (d, 2H,
NO
2
13
(
t, 2H), 1.39 ppm (t, 2H), 1.27 ppm (t, 4H), 0.84 ppm (t, 3H); C NMR:
δ = 164.4 ppm (C–NO ), 141.4 ppm (Cphenyl–O), 126.0 ppm (Cphenyl),
14.5 ppm (Cphenyl), 69.1 ppm (–O–CH –), 31.6 ppm (–CH –),
–), 22.7 ppm (–CH –), 14.1 ppm
2
2.6. Enzyme kinetic parameters
1
2
2
2
9.1 ppm (–CH
).
2
–), 25.7 ppm (–CH
2
2
Kinetics of the hybrid mutants were measured by monitoring
(–CH
3
the product formation at 410 nm (extinction coefficient =
1
−1
−1
1-octyl-4-nitrobenzene (9): H NMR (CDCl
3
): δ = 8.11 ppm (d, 2H,
13,200 M
cm ) [3]. The substrate concentrations are set from
NO
2
-phenyl-), 6.88 ppm (d, 2H, phenyl-O-), 3.98 ppm (t, 2H), 1.75 ppm
5 μM to 700 μM with either 2 μM of tK97C-1 or 1 μM of the sL407C hy-
brid enzymes. Each reaction, with a total reaction volume of 500 μL
containing substrate, hybrid enzyme, and 100 mM DTC in 50 mM
tris pH 8.2, is irradiated with visible light (IR and UV-cutoff filters)
from an Orion 1000 W Xenon arc lamp and stopped using 5 μL of
6 M NaOH after 1 min to eliminate overlapping absorbance from
the hybrid enzymes.
13
(
t, 2H), 1.39 ppm (t, 2H), 1.22 ppm (t, 8H), 0.82 ppm (t, 3H); C NMR:
δ = 164.4 ppm (C–NO ), 141.4 ppm (Cphenyl–O), 126.0 ppm (Cphenyl),
14.5 ppm (Cphenyl), 69.0 ppm (–O–CH –), 31.9 ppm (–CH –),
9.1 ppm (–CH –), 26.1 ppm (–CH –), 22.8 ppm (–CH –), 14.2 ppm
).
2
1
2
2
2
2
2
2
(–CH
3
2
.2.3. Synthesis of 1-benzoxy-4-nitrobenzene (10)
A DMSO solution (20 mL) of benzyl bromide (1.2 eq) was added to a
2.7. 96-well plate assay
DMSO solution (20 mL) containing p-nitrophenolate (1 eq.) and potas-
sium carbonate (1 eq.). The reaction mixture was then stirred at 120 °C
for 4 h. Upon cooling, the desired product was precipitated from the so-
lution by addition of water and collected by vacuum filtration. White
crystalline material was obtained after recrystallization from DMSO/
water (75% yield).
Each well, with a total reaction volume of 200 μL, contains 1 μM en-
zyme, 400 μM substrate and 100 mM DTC in 50 mM tris pH 8.2. The
plate is then irradiated using LED lamps (Apollo horticulture 100 ×
3 W). After 30 min of reaction, 10 μL of 6 M NaOH is added to each
well to stop the reaction. After spinning and transferring the reaction
mixture to a new plate, the absorbance at 405 nm is read using a
Vmax microplate reader (Molecular Devices).
1
-benzoxy-4-nitrobenzene (10): mp = 112–113 °C. 1H NMR
CDCl ): δ = 8.16 ppm (d, 2H, NO -phenyl-), 7.40 ppm (d, 4H, benzyl),
(
3
2