Journal of the American Chemical Society
Article
mixture was stirred at room temperature fore 20 min. Pyrrole d/e/f
2.88−2.79 (m, 2H), 2.72 (dd, J = 8.5, 6.4 Hz, 2H), 1.22 (t, J = 7.1 Hz,
1
3
(
1.0 equiv) and acetic anhydride (4 mL per mmol of pyrrole) were
6H). C NMR (126 MHz, CDCl ): δ 160.45, 158.77, 151.00, 149.79,
3
added. The reaction mixture was stirred for 20 min at room
temperature and then 30 min at 80 °C. The reaction mixture was
cooled, diluted with a cold saturated solution of sodium bicarbonate,
and filtered to isolate the heterodimer as a dark solid. The solid was
suspended in dichloroethane (20 mL per mmol of dimer), and
triethylamine (17 equiv) and BF OEt (19 equiv) were added. The
145.45, 142.71, 138.18, 137.64, 137.51, 134.93, 132.71, 131.95,
131.67, 131.06, 130.36, 128.99, 128.48, 128.26, 128.12, 127.42,
127.12, 127.02, 124.29, 117.23, 113.72, 110.87, 55.40, 44.66, 30.03,
1
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29.71, 21.92, 20.96, 12.85. F NMR (471 MHz, CDCl ): δ −131.44
3
1
1
(dd, J = 63.5, 31.5 Hz). B NMR (161 MHz, CDCl ): δ 0.89 (t, J =
3
31.8 Hz).
3
2
reaction mixture was stirred for 20 min at room temperature and 30
min at 80 °C. The reaction mixture was then cooled, diluted with
water, and extracted with dichloromethane. The organic layer was
washed with saturated sodium bicarbonate, dried over sodium sulfate,
and concentrated. The resulting solid was purified as described below.
Compound 1. Compound 1 was synthesized from pyrroles b and e
Compound 5. Compound 5 was synthesized from pyrroles c and e
(0.090 mmol each) using the general procedure. The product was
purified by alumina column chromatography (2:1 chloroform/
hexanes) to yield a deep purple solid (44.8 mg, 0.069 mmol, 76%
1
yield over two steps). H NMR (500 MHz, CDCl
): δ 8.61 (d, J = 9.3
3
Hz, 1H), 8.42−8.37 (m, 1H), 7.81−7.77 (m, 2H), 7.75 (d, J = 8.6 Hz,
2H), 7.52 (dd, J = 8.4, 6.9 Hz, 2H), 7.48−7.42 (m, 1H), 7.24−7.13
(m, 3H), 7.04−6.99 (m, 2H), 6.73−6.66 (m, 1H), 6.52 (s, 1H), 3.89
(s, 3H), 3.47 (q, J = 7.1 Hz, 4H), 2.98 (t, J = 7.0 Hz, 2H), 2.91 (t, J =
7.4 Hz, 4H), 2.71 (dd, J = 8.5, 6.3 Hz, 2H), 1.24 (t, J = 7.1 Hz, 6H).
(
0.20 mmol each) using the general procedure. The product was
purified by recrystallization in dichloromethane/hexanes (85.0 mg,
0
1
.13 mmol, 65% yield over two steps). H NMR (500 MHz, DMSO-
d ): δ 8.58 (d, J = 9.4 Hz, 1H), 8.45 (d, J = 8.9 Hz, 1H), 7.78−7.73
6
1
3
(
m, 2H), 7.73−7.66 (m, 2H), 7.51 (dd, J = 8.4, 7.0 Hz, 2H), 7.47 (t, J
C NMR (126 MHz, CDCl ): δ 159.84, 156.68, 151.21, 148.03,
3
=
7.7 Hz, 2H), 7.44−7.40 (m, 1H), 7.37−7.32 (m, 1H), 7.03−6.96
146.03, 141.44, 137.79, 137.30, 133.50, 132.28, 131.60, 131.54,
130.57, 128.46, 128.23, 128.06, 127.38, 126.81, 126.79, 126.51,
(
m, 2H), 6.93 (dd, J = 9.4, 2.7 Hz, 1H), 6.81 (d, J = 2.7 Hz, 1H), 3.86
(
s, 3H), 3.57 (q, J = 7.1 Hz, 4H), 2.98−2.77 (m, 8H), 1.21 (q, J = 6.8
125.16, 113.60, 111.80, 110.75, 55.37, 44.97, 31.07, 30.27, 22.29,
13
19
Hz, 7H). C NMR (126 MHz, DMSO-d ): δ 159.91, 154.15, 151.18,
20.94, 12.96. F NMR (471 MHz, CDCl ): δ −134.39 (dd, J = 64.9,
6
3
1
1
1
1
1
2
−
46.61, 146.31, 143.48, 142.15, 142.04, 136.00, 133.57, 132.64,
31.49, 131.33, 130.03, 129.91, 128.37, 128.14, 128.03, 127.42,
20.97, 114.25, 113.09, 112.53, 111.71, 111.23, 55.32, 44.33, 29.99,
31.1 Hz). B NMR (161 MHz, CDCl ): δ 1.23 (t, J = 32.9 Hz).
3
Compound 6. Compound 6 was synthesized from pyrroles b and f
(0.10 mmol each) using the general procedure. The product was
purified by slow-drip alumina column chromatography (first
purification, 1:1 dichloromethane/hexanes; second purification, 8:1
hexanes/acetone, flushed off with dichloromethane) to yield a deep
1
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9.87, 21.51, 20.98, 12.81. F NMR (471 MHz, DMSO-d ): δ
6
1
1
134.40 (dd, J = 68.0, 29.5 Hz). B NMR (161 MHz, DMSO-d ): δ
6
1
.42 (t, J = 34.1 Hz).
1
Compound 2. Compound 2 was synthesized from pyrroles a and e
purple solid (52.9 mg, 0.088 mmol, 82% yield over two steps). H
(
0.4 mmol each) using the general procedure. The product was
NMR (500 MHz, CDCl ): δ 8.63 (d, J = 8.9 Hz, 1H), 8.45 (ddt, J =
3
purified by recrystallization in dichloromethane/hexanes (90 mg, 0.14
mmol, 36% yield over two steps). H NMR (500 MHz, CDCl ): δ
6.4, 4.6, 2.3 Hz, 1H), 7.86−7.80 (m, 2H), 7.79−7.73 (m, 2H), 7.52
(t, J = 7.7 Hz, 2H), 7.46−7.39 (m, 1H), 7.25−7.20 (m, 3H), 6.91 (dd,
J = 8.9, 2.7 Hz, 1H), 6.81 (d, J = 2.7 Hz, 1H), 6.78 (d, J = 8.7 Hz,
2H), 3.87 (s, 3H), 3.47 (q, J = 7.1 Hz, 4H), 2.92 (d, J = 8.2 Hz, 6H),
1
3
8
2
1
6
3
2
1
1
1
1
.69 (d, J = 9.3 Hz, 1H), 8.11−8.04 (m, 2H), 8.01 (d, J = 8.3 Hz,
H), 7.80−7.73 (m, 2H), 7.49 (t, J = 7.6 Hz, 2H), 7.45−7.40 (m,
H), 7.36 (dd, J = 8.4, 6.9 Hz, 2H), 7.28 (d, J = 7.4 Hz, 1H), 7.04−
.98 (m, 2H), 6.83 (s, 1H), 6.74 (d, J = 9.0 Hz, 1H), 6.54 (s, 1H),
.89 (s, 3H), 3.49 (s, 4H), 2.99 (s, 2H), 2.92 (dd, J = 7.9, 5.6 Hz,
1
3
2.82 (t, J = 7.5 Hz, 2H), 1.25 (t, J = 7.0 Hz, 6H). C NMR (126
MHz, CDCl ): δ 161.63, 155.31, 151.36, 149.23, 145.52, 144.52,
3
143.86, 137.41, 136.45, 132.87, 132.33, 131.10, 130.59, 130.29,
128.52, 128.22, 128.10, 128.00, 127.26, 126.97, 121.01, 117.84,
114.44, 112.97, 110.86, 55.53, 44.61, 30.84, 29.88, 21.88, 21.73, 12.93.
1
3
H), 1.26 (t, J = 7.1 Hz, 6H). C NMR (151 MHz, CDCl ): δ
3
60.27, 157.01, 151.42, 149.44, 148.02, 146.29, 142.72, 137.93,
35.75, 134.13, 133.91, 133.59, 133.51, 132.11, 130.76, 130.55,
28.68, 128.52, 128.41, 128.22, 127.59, 126.37, 115.16, 114.27,
1
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11
F NMR (376 MHz, CDCl ): δ −133.82 (dd, J = 65.3, 32.2 Hz).
B
3
NMR (128 MHz, CDCl ): δ 1.33 (t, J = 32.7 Hz).
3
1
9
13.94, 111.86, 110.80, 55.43, 45.00, 31.07, 22.36, 13.02, 12.95.
F
B
Maximum Ratiometric Fold Turn-on in Vitro. HyP-1, red-
HyP-1, CRaB-HyP, red-CRaB-HyP, OMe-APC, t-OMe-APC, CRaB-
OMe-APC, and t-CRaB-OMe-APC were dissolved in aqueous
solution (5 μM in 1:1 mixture of acetonitrile and PBS without
calcium or magnesium, pH adjusted to 7.4). These solutions were
pipetted into FEP tubes and imaged through a custom tissue-
mimicking phantom, and their PA signals were measured upon
irradiation at their respective PA maxima. PhotoNOD-1, rNOD-1,
CRaB-photoNOD, and r-CRaB-NOD were dissolved in chloroform
(5 μM) and imaged as above. Each experiment was repeated in
triplicate.
Tissue Phantom Preparation. Tissue phantoms were prepared
by suspending agarose LE (4 g) in a solution of 2% milk (2 mL) and
deionized water (78 mL). The suspension was heated in a microwave
until a viscous, translucent gel was produced (30−45 s). The hot gel
was poured into a custom Teflon mold containing two pieces of
hypodermic steel tubing and cooled at 4 °C for at least 2 h. After
cooling, the steel tubes were removed and the gel was removed from
the mold, yielding a tissue phantom with two parallel channels for the
placement of FEP tubes containing sample solutions.
1
1
NMR (471 MHz, CDCl ): δ −134.18 (dd, J = 65.7, 31.9 Hz).
NMR (161 MHz, CDCl ): δ 1.37 (t, J = 33.1 Hz).
3
3
Compound 3 (red-CRaB-HyP). Compound 3 was synthesized from
pyrroles b and d (0.30 mmol each) using the general procedure. The
product was purified by silica column chromatography (3:1
dichloromethane/hexanes with 0.1% Et N) to yield the product
3
1
(
112 mg, 0.18 mmol, 60% yield over two steps). H NMR (500 MHz,
CDCl ): δ 8.72 (d, J = 8.9 Hz, 1H), 8.23−8.16 (m, 2H), 8.13−8.06
(
3
m, 2H), 7.77−7.70 (m, 2H), 7.48 (t, J = 7.6 Hz, 2H), 7.43−7.37 (m,
3
H), 7.37−7.33 (m, 1H), 7.15 (s, 1H), 6.97 (dd, J = 8.9, 2.7 Hz, 1H),
.83 (d, J = 2.7 Hz, 1H), 6.78 (d, J = 8.7 Hz, 2H), 3.89 (s, 3H), 3.48
6
1
3
(
q, J = 7.1 Hz, 4H), 2.93 (s, 4H), 1.25 (t, J = 7.1 Hz, 6H). C NMR
(
126 MHz, CDCl ): δ 161.43, 157.24, 150.28, 149.98, 146.09, 144.99,
3
1
1
1
43.70, 141.35, 135.82, 132.91, 132.88, 132.33, 130.71, 130.52,
30.31, 129.11, 128.77, 128.49, 128.15, 127.96, 121.17, 118.26,
1
9
18.13, 114.41, 112.97, 111.58, 55.52, 44.75, 30.88, 21.90, 12.92.
F
B
1
1
NMR (471 MHz, CDCl ): δ −134.18 (dd, J = 66.5, 33.2 Hz).
NMR (161 MHz, CDCl ): δ 1.51 (t, J = 33.4 Hz).
3
3
Compound 4. Compound 4 was synthesized from pyrroles c and f
(
0.96 mmol each) using the general procedure. The product was
In Vitro Hypoxia Turnover Experiments. HyP-1 and CRaB-
HyP were each dissolved into 100 mM potassium phosphate buffer to
a final concentration of 10 μM in the presence of 15 μL of RLM/mL
and 100 μM NADPH and stirred at 37 °C for 2 h. Normoxic controls
were stirred under atmosphere. Hypoxic samples were stirred under
nitrogen. Upon the conclusion of the incubations, the samples were
diluted 1:1 with acetonitrile, pipetted into FEP tubes, and imaged in a
custom tissue-mimicking phantom.
purified by alumina column chromatography (1:1 dichloromethane/
hexanes) to yield a deep purple solid (33.2 mg, 0.51 mmol, 53% yield
over two steps). H NMR (500 MHz, CDCl ): δ 8.61 (dd, J = 7.7, 1.3
1
3
Hz, 1H), 8.55 (d, J = 7.6 Hz, 1H), 7.83−7.77 (m, 2H), 7.75−7.70 (m,
2
1
2
H), 7.37 (dtd, J = 8.8, 7.4, 2.7 Hz, 2H), 7.31 (td, J = 7.4, 1.4 Hz,
H), 7.29−7.26 (m, 3H), 7.01−6.96 (m, 2H), 6.71 (d, J = 8.7 Hz,
H), 3.86 (s, 3H), 3.43 (q, J = 7.1 Hz, 4H), 2.94 (q, J = 7.3 Hz, 4H),
G
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX