Mechanism-based molecular design of highly selective fluorescence probes for nitrative stress

Article abstract of DOI:10.1021/ja061972v

Nitrative stress is implicated in various pathogenic processes, including neurodegenerative disorders, but there is no practical fluorescence probe which can monitor the generation of nitrative stress with high selectivity. To design a suitable fluorescence probe, we have first focused on the fluorescence quenching mechanism of the nitro group, which has been believed to be a unique quencher of fluorescent dyes. We found that nitro group-based fluorescence quenching could be explained in terms of an electron transfer process, from the excited fluorophore to the electron-deficient aromatic nitro moiety. By utilizing this result, we succeeded in developing novel fluorogenic probes, NiSPYs, which can selectively monitor the generation of nitrative stress based on aromatic nitration. NiSPYs showed strong fluorescence enhancement upon the reaction with nitrating agents, including peroxynitrite, but showed little or no fluorescence augmentation in the presence of other reactive oxygen species. NiSPYs should be potentially useful as tools to study the role of nitrative stress in various biological applications. Copyright

Full text of DOI:10.1021/ja061972v

Supporting Information
“Mechanism-Based Molecular Design of Highly Selective Fluorescence Probes for Nitrative Stress”
Tasuku Ueno,†,‡ Yasuteru Urano,†,§ Hirotatsu Kojima,†,‡ and Tetsuo Nagano†,‡,*
†Graduate School of Pharmaceutical Sciences, The University of Tokyo
Hongo, Bunkyo-ku, Tokyo,113-0033, Japan.
‡CREST, JST and §Presto, JST, 4-8-1 Honcho, Kawaguchi, Saitama, 332-0012, Japan
* To whom correspondence should be addressed. E-mail: tlong@mol.f.u-tokyo.ac.jp
Abbreviations
HOMO, highest occupied molecular orbital; LUMO, lowest unoccupied molecular orbital; PeT, photoinduced electron
transfer; d-PeT, donor excited photoinduced electron transfer: electron transfer from excited electron donor to electron
acceptor; a-PeT, acceptor excited photoinduced electron transfer: electron transfer from electron donor to excited
acceptor; ∆GeT, free energy change of electron transfer process; Φfl, fluorescence quantum efficiency; nitroBODIPY,
nitro-substituted BODIPY derivatives; DAMBO, 8-(3,4-diaminophenyl)-1,3,5,7-tetramethyl-4,4-difluoro-4-bora-3a,4a-
diaza-s-indacene and its derivatives; HPLC, high-performance liquid chromatography; NO2BF4, nitronium
tetrafluoroborate; CH3CN, acetonitrile; DMF, N,N-dimethylformamide; MeOH, methanol; ONOO-, peroxynitrite; H2O2,
-
hydrogen peroxide; OCl, hypochlorite; NO, nitric oxide; Eox, oxidation potential; Ered, reduction potential; ∆E0,0,
excitation
energy;
wp,
work
term;
TBAP,
tetrabutylammonium
perchlorate;
NOC-13:
1-hydroxy-2-oxo-3-(3-aminopropyl)-3-methyl-1- triazene; 1O2, singlet oxygen; SCE, saturated calomel electrode; PPTS,
pyridinium p-toluenesulfonate; TFA, trifluoroacetic acid; DDQ, 2,3-dichloro-5,6-dicyano-p-benzoquinone; MeMgBr,
methyl magnesium bromide; THF, tetrahydrofuran; AcOH, acetic acid: EtOH, ethanol; NaOEt, sodium ethoxide;
AcOEt, ethyl acetate: BF3•OEt2, boron trifluoride diethyl etherate; DIEA, diisopropylethylamine
S1

Figures and Tables
Table S1. Photophysical properties of nitroBODIPY derivatives.
e
reduction potential of oxidation potential of
absorption
maximum
emission
maximum
calcd ∆E 0,0
calculated relative
free energy change
Φfl
benzene moiety
BODIPY moiety
(V vs. SCE)a
-1.15
(V vs SCE)a
1.12
(nm)b
503
503
506
511
503
(nm)b
508
511
515
523
514
(eV)c
2.45
2.45
2.43
2.40
2.44
(eV)d
0
0.001
0.004
0.023
0.529
0.687
3
4
5
6
-1.15
1.12
0
-1.28
1.12
+0.15
+0.42
+0.79
-1.28
1.36
NiSPY-1 Nf
-1.42
1.63
a Measured in acetonitrile containing 0.1 M TBAP as a supporting electrolyte (see Figure S1). b Measured in methanol.
c
d
∆E0,0 values were determined from the average wave numbers of absorption maximum and emission maximum.
Relative ∆GeT values were calculated with respect to the value for 1 as a standard. e Calculated by using fluorescein as
a fluorescence standard (0.850). f All data were measured in acetonitrile.
(A)
(B)
O
NO2
NO2
NO
2
O
O
Ac
Ac
NC
CN
N
N
N
N
N
N
B
B
B
F
F
F
F
F
F
-1.15
-1.28
-1.42
1.12
1.36
1.63
Figure S1. (A) Reduction potentials of the benzene moiety and (B) Oxidation potentials of the BODIPY fluorophore of
nitroBODIPY derivatives in acetonitrile (V vs SCE).
(A)
(B)
[ONOO-]
1000
750
500
250
0
1000
750
500
250
0
5 µM
y = 178.67x - 7.2096
R2 = 0.9974
4
2
1
0
450
500
550
600
650
0
1
2
3
4
5
6
[ONOO-] (µM)
Wavelength (nm)
Figure S2. Detection of peroxynitrite using NiSPY-2. (A) Fluorescence spectra (excited at 505 nm) of NiSPY-2 (5
µM; 0.2% DMF as a cosolvent) in 0.1 M sodium phosphate buffer (pH 7.4) upon addition of peroxynitrite solution (final
0, 1, 2, 4 and 5 µM). (B) Relation between the final concentration of added peroxynitrite and the fluorescence intensity
determined at 518 nm (excited at 505 nm).
S2

(A)
(B)
NiSPY-3
NiSPY-2
200
150
100
50
60
50
40
30
20
10
0
0
0
5
10
15
0
5
10
15
Time (min)
Time (min)
NiSPY-2 + ONOO-
NiSPY-3+ ONOO-
200
150
100
50
60
50
40
30
20
10
0
0
0
5
10
15
0
5
10
15
Time (min)
Time (min)
200
150
100
50
NiSPY-2 N
NiSPY-3 N
250
200
150
100
50
0
0
0
5
10
15
0
5
10
15
Time (min)
Time (min)
Figure S3. HPLC chromatograms during the reaction of (A) NiSPY-2 and (B) NiSPY-3 with peroxynitrite. The
reaction of NiSPY-2 (5 µM, 0.1% DMF) with peroxynitrite (25 µM) was carried out in 0.1 M sodium phosphate buffer
(pH 7.4). The reaction of NiSPY-3 (10 µM, 0.1% DMF) with peroxynitrite (30 µM) was carried out in 0.1 M sodium
phosphate buffer (pH 7.4). The product was detected using HPLC with a linear gradient (eluent, 0 min, 32%
CH3CN/0.1% TFA aq. ~ 10 min, 80% CH3CN/0.1% TFA aq.; flow rate = 1.0 mL/min; detection wavelength, 500 nm
(absorbance, green solid line) and 500/510 nm (fluorescence, orange solid line)).
S3

(A)
(B)
13.2 min
m/z = 463
10000
8000
6000
4000
2000
0
250
200
150
100
50
15.1 min
0
-50
460
480
500
520
0
5
10
15
20
m/z
Time (min)
(C)
(D)
10000
8000
6000
4000
2000
0
OH
OH
NO2
HOOC
NC
O
HOOC
NC
O
CN
CN
N
N
N
N
B
B
m/z = 508
F
F
F
F
NiSPY-3
NiSPY-3 N
C23H19BF2N4O4
C23H18BF2N5O6
460
480
500
520
Exact Mass: 464.15
Exact Mass: 509.13
m/z
Figure S4. LC-MS analysis in the reaction of NiSPY-3 with peroxynitrite. The reaction of NiSPY-3 (10 µM, 0.1%
DMF) with peroxynitrite (10 µM) was carried out in 0.1 M sodium phosphate buffer (pH 7.4) at ambient temperature.
The product was detected using HPLC with a linear gradient (eluent, 0 min, 32% CH3CN/0.1% HCOOH aq. ~ 10 min,
80% CH3CN/0.1% HCOOH aq; flow rate, 0.2 mL/min; detection wavelength, 500 nm (absorbance)). (A) HPLC
chromatogram in the reaction of NiSPY-3 with peroxynitrite. Mass spectrum of the peak at (B) 13.2 minutes and (C)
15.1 minutes. The corresponding m/z values were analyzed by mass spectrometry in the negative ion mode. (D)
Calculated molecular weights of NiSPY-3 and NiSPY-3 N.
S4

a
b
c
d
e
f
750
500
250
0
g
h
450
500
550
600
650
Wavelength (nm)
Figure S5. Fluorescence response of NiSPY-3 in various reactive oxygen species generation systems. The dye (10
mM, 0.1% DMF as a cosolvent) was added to 0.1 M sodium phosphate buffer (pH 7.4). The fluorescence spectra of
•-
NiSPY-3 were measured with 505 nm excitation after stirring for 3 min (30 min for NO and O2 ) at room temperature
°
a
(37 C for NO).
•OH: Ferrous perchlorate (final, 100 µM) and H2O2 (final, 1 mM) were added at room temperature.
b 1O2: H2O2 (final, 1 mM) and NaClO (final, 100 µM) were added at room temperature.
H2O2: H2O2 (final, 1 mM)
c
d
e
was added at room temperature.
–OCl: NaClO (final, 10 µM) was added at room temperature.
NO:
°
1-hydroxy-2-oxo-3-(3-aminopropyl)-3-methyl-1-triazene (100 µM) was added and the mixture were stirred at 37 C for
f
g
•-
30 min.
oxidase system. h Blank.
ONOO–: Sodium peroxynitrite (final 10 µM) was added at room temperature.
O2 : xanthine/xanthine
NiSPY-3 (10 µM)
+ ONOO- (final 10 µM)
1
0.8
0.6
0.4
0.2
0
NiSPY-3 (10 µM)
+ 10 µM N-acetyltyrosine
+ ONOO- (final 10 µM)
450
500
550
600
650
wavelength (nm)
Figure S6. Fluorescence measurement for comparison of the relative reaction rate constants. To a mixture of
NiSPY-3 (10 µM) and N-acetyltyrosine (0 or 10 µM) was added sodium peroxynitrite (final 10 µM). The fluorescence
spectra of NiSPY-3 were measured with excitation at 505 nm after stirring for 1 min at room temperature. The
fluorescence response of NiSPY-3 was decreased to approximately 0.6 times by addition of the same concentration of
N-acetyltyrosine. This result showed that the reaction rate constants of these molecules are of the same order of
magnitude.
S5

140
120
A
B
C
D
Figure S7. Differential interference contrast (A), pseudo-color fluorescence (B, C) and merged images (D) of HeLa
cells loaded with NiSPY-3 in Hank’s balanced salts solution. Pseudo-color images were obtained before (B) and after
(C) addition of peroxynitrite. HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% (v/v) fetal bovine serum, penicillin (100 units/mL) and streptomycin (100 µg/mL) in a
humidified incubator containing 5% CO2 gas. For fluorescence microscopy, HeLa cells were plated in a 35-mm
glass-bottom dish (MatTek Corporation) and cultured overnight in DMEM. The medium was removed after 24 hr, and
the cells were washed with HBSS (2 x 1 mL). HBSS (2 mL) solution of 2 µM NiSPY-3 (0.01% DMF, 0.01%
Cremophor EL (SIGMA) as a cosolvent) was then added, and the cells were incubated for 20 min at room temperature.
The cells were then washed with 2 x 1 mL HBSS. Fluorescence images excited to 470-490 nm were obtained before
and after addition of peroxynitrite.
- 1 min
5 min
10 min
15 min
20 min
Figure S8. Differential interference contrast images of NiSPY-3 loaded HeLa cells in HBSS to evaluate the toxicity of
NiSYP-3. HeLa cells were exposed to the same experimental conditions as used for fluorescence imaging. No
apparent cytotoxicity was observed under these conditions.
S6

Experimental Section
Materials. General chemicals were of the best grade available, supplied by Tokyo Chemical Industries,
Wako Pure Chemical, Aldrich Chemical Co., Acros Organics and Lancaster Synthesis, and used without further
purification. Special chemicals were DMF (fluorometric grade, Dojindo) and TBAP, (electrochemical grade, Fluka).
All solvents were used after appropriate distillation or purification.
Instruments. NMR spectra were recorded on a JEOL JNM-LA300 instrument at 300 MHz for 1H NMR and
at 75 MHz for 13C NMR. Mass spectra (MS) were measured with a JEOL SX-102A for FAB and a JEOL JMS-T100LC
AccuToF for ESI. UV-visible spectra were obtained on a Shimadzu UV-1600. Fluorescence spectroscopic studies
were performed on a Hitachi F4500.
Fluorometric Analysis. The slit width was 2.5 nm for both excitation and emission. The photon multiplier
voltage was 700 V. Relative fluorescence quantum efficiency of BODIPY derivatives was obtained by comparing the
area under the emission spectrum of the test sample excited at 490 nm with that of a solution of fluorescein in 0.1 N
NaOH, which has a quantum efficiency of 0.850 according to the literature.1
In the experiment to measure the fluorescence response of NiSPYs, the slit width was 2.5 nm for excitation
and 2.5 nm for emission, and the photomultiplier voltage was 900 V. Compounds were dissolved in DMF to make a
stock solution, which was diluted to the required concentration for measurement.
Calculation of ∆GeT value. ∆GeT values were calculated from the Rehm-Weller equation: ∆GeT = Eox – Ered
–
∆E0,0 – wp, where Eox and Ered are oxidation and reduction potentials of electron donor and acceptor, respectively, ∆E0,0 is
the excited energy, and wp is the work term for the charge separation.2 Relative ∆GeT values were calculated with
respect to the value for 1 as a standard. wp values of each compounds were assumed to be the same, due to the
similarity of their structures.
Cyclic voltammetry. Cyclic voltammetry was performed on a ALS 600 A electrochemical analyzer.
A
three-electrode arrangement in a single cell was used for the measurements: a Pt wire as the auxiliary electrode, a Pt
electrode and a GC electrode as the working electrode, and a Ag/Ag+ electrode as the reference electrode. The sample
solutions contained 1.0 x 10-3 M sample and 0.1 M TBAP as a supporting electrolyte in acetonitrile, and argon was
bubbled for 2 min before each measurement. The scan rate was 0.1 V s-1. Obtained potentials (vs Ag/Ag+) were
converted to those vs a saturated calomel electrode (SCE) by adding 0.25 V.
HPLC analysis. HPLC analysis were performed on an Inertsil ODS-3 (4.6 x 250 mm) column using an
HPLC system composed of a pump (G1312A, Agilent) and a detector (G1315B or G1321A, Agilent).
In the LCMS experiment to analyze the reaction mixture of NiSPY-3 and peroxynitrite, LC analysis was
performed an Intertsil ODS-3 (2.1 x 150 mm) column using an HPLC system composed of a pump (G1312A, Agilent)
and a detector (G1314A, Agilent).
S7

Synthesis and characterization of compounds 3 – 6.
Scheme S1. Synthetic scheme of 3 – 6.
R1
R
1
R2
R
2
X
R1
NH
R3
R
3
R2
1) cat. TFA, CH2Cl2
2) DDQ
BF3•OEt2, DIEA
Toluene
X
X
X
X
R3
N
N
NH
N
B
CHO
F
F
7-10
3-6
R1
NO2
H
R2
R3
X
H
H
H
7 (crude)
8 (crude)
9 (crude)
10 (crude)
3. y. 24% (2 step)
4. y. 26% (2 step)
5. y. 41% (2 step)
6. y. 9% (2 step)
NO2
NO2
NO2
H
H
H
OMe
OMe
H
H
Ac
NitroBODIPY derivatives (3, 4,
5 and 6) were synthesized from commercially available compounds
(4-nitrobenzaldehyde, 3-nitrobenzaldehyde and 2,4-dimethylpyrrole, TCI; 3-acetyl-2,4-dimethylpyrrole and
2-methoxy-5-nitrobenzaldehyde, Aldrich) according to the following procedures.
Synthesis of 8-(4-nitrophenyl)-1,3,5,7-tetramethyl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (3). 4-Nitrobenzaldehyde
(302 mg, 2 mmol) and 2,4-dimethylpyrrole (380 mg, 4 mmol) were dissolved in 300 mL of CH2Cl2. A catalytic amount
of TFA was added, and the solution was stirred overnight at ambient temperature. When TLC monitoring showed
complete consumption of the aldehyde, a solution of DDQ (590 mg, 2.6 mmol) was added, and stirring was continued
for 10 min. The reaction mixture was washed with water (3 x 100 mL). The combined organic layers were washed
with brine (1 x 100 mL), dried over anhydrous Na2SO4, filtered, and evaporated. The crude product was purified by
alumina column chromatography (aluminium oxide 90, Merck), and dried under reduced pressure to constant weight to
afford 6 as a crude red amorphous solid. The crude dipyrromethene 6 and DIEA (4 mL, 23.0 mmol) were dissolved in
100 mL of toluene (100 mL) and stirred at ambient temperature for 5 min. BF3•OEt2 (3 mL, 23.7 mmol) was added,
and stirring was continued for 10 min. The reaction mixture was washed with water (3 x 80 mL), and brine (1 x 100
mL), dried over anhydrous Na2SO4, filtered, and evaporated. The crude compound was purified by silica gel column
chromatography (silica gel 60N, Kanto Chemical) to afford 3 as a red powder (177 mg, yield 24%). 1H NMR (300
MHz, CDCl3): δ 1.30 (s, 6H); 2.57 (s, 6H); 6.02 (s, 2H); 7.54 (d, J = 8.6 Hz, 2H); 8.41 (d, J = 8.6 Hz, 2H). 13C NMR
(75 MHz, CDCl3): δ 14.6, 121.8, 124.3, 129.6, 130.6, 138.3, 141.9, 142.5, 148.3, 156.6. HRMS (ESI-): calcd for
[M-H]-, 368.1382; found, 368.1384. Anal. Calcd for C19H18BF2N3O2: N, 11.38; C, 61.81; H, 4.91. Found: N, 11.26; C;
61.73; H, 5.04.
4 and 5 were similarly prepared from 3-nitrobenzaldehyde (4) and 2-methoxy-5-nitrobenzaldehyde (5) in 26% yield and
41% yield, respectively.
S8

4. 1H NMR (300 MHz, CDCl3): δ 1.36 (s, 6H); 2.57 (s, 6H); 6.02 (s, 2H); 7.69 (m, 1H); 7.73 (m, 1H); 8.23 (m, 1H);
8.37 (m, 1H). 13C NMR (75 MHz, CDCl3): δ 14.6, 14.8, 121.9, 123.5, 124.0, 130.4, 131.0, 134.5, 136.7, 137.8, 142.4,
148.6, 156.6. HRMS (ESI-): calcd for [M-H]-, 368.1382; found, 368.1405.
5. 1H NMR (300 MHz, CDCl3): 1.43 (s, 6H); 2.57 (s, 6H); 3.91 (s, 3H); 6.00 (s, 2H); 7.08 (d, J = 9.2 Hz, 1H); 8.14 (d,
J = 2.9 Hz, 1H); 8.40 (dd, J = 2.9, 9.2 Hz, 1H). 13C NMR (75 MHz, CDCl3): δ 14.1, 14.5, 56.6, 111.0, 121.4, 124.7,
125.8, 126.9, 131.0, 141.8, 141.9, 156.0, 161.7. HRMS (ESI+): calcd for [M+Na]+, 422.1464; found, 422.1436.
6 was similarly prepared from 2-methoxy-5-nitrobenzaldehyde and 3-acetyl-2,4-dimethylpyrrole in 9% yield.
1H NMR (300 MHz, CDCl3): δ 1.65 (s, 6H); 2.45 (s, 6H); 2.79 (s, 6H); 3.95 (s, 3H,); 7.14 (d, J = 9.2 Hz, 1H); 8.11 (d, J
= 2.9 Hz, 1H); 8.49 (dd, J = 2.9, 9.2 Hz, 1H). HRMS (FAB+): calcd for [M+H]+, 484.1856; found, 484.1844.
Synthesis and characterization of fluorescence probes for nitrative stress, NiSPYs.
Scheme S2. Synthetic scheme of 3-cyano-2,4-dimethyopyrrole
O
N
O
N
O
N
NC
NC
O
HBr
3-aminocrotonitrile
EtOH
MeMgBr
THF
NaOEt
EtOH
O
NC
O
O
O
N
H
N
H
O
N
H
N
H
BrH N
3
AcOH
11
y. 92%
12
13
14
y. 65%
y. 81% (crude)
y. 59% (2 step)
3-Cyano-2,4-dimethylpyrrole (14) was prepared from commercially available compounds, (N-tert-butoxycarbonyl-N-
methoxy-N-methyl-glycinamide, Aldrich; 3-aminocrotonitrile and MeMgBr, TCI), according to the following procedure.
Synthesis of 3-cyano-2,4-dimethylpyrrole (14). 13 was prepared according to the literature.3 13 (3.4 g, 18.6 mmol)
was dissolved in absolute THF (200 mL) under an argon atmosphere. To a vigorously magnetically stirred solution of
13 was added dropwise MeMgBr (3 M/diethyl ether, 18 mL, 54 mmol) at –10 °C. At the end of the reaction (monitored
by TLC), the mixture was concentrated in vacuo to approximately 20 mL, and the residue was poured into water (200
mL). The aqueous solution was extracted with AcOEt (3 x 150 mL). The combined organic layers were washed with
brine (1 x 100 mL), dried over anhydrous Na2SO4, filtered, and evaporated to afford crude 14. 14 was suspended in
absolute EtOH (100 mL). After addition of NaOEt (200 mg) under argon atmosphere at 0 °C, the reaction mixture was
stirred for 1 h, then pour into water (250 mL). The aqueous solution was extracted with AcOEt (3 x 150 mL) and brine
(1 x 100 mL), dried over anhydrous Na2SO4, filtrated, and evaporated. The crude compound was purified by silica gel
column chromatography (silica gel 60N, Kanto Chemical) to afford 14 as a white solid (1.3 g, yield 59% in 2step).
13. 1H NMR (300 MHz, DMSO-d6): δ 1.99 (s, 3H); 2.08 (s, 3H); 2.83 (s, 3H); 6.37 (m, 1H), 8.08 (br, 1H).
S9

14. 1H NMR (300 MHz, CDCl3): δ 2.18 (d, J = 5.9 Hz, 3H); 2.37 (s, 3H); 6.37 (m, 1H), 8.08 (br, 1H). 13C NMR (75
MHz, CDCl3): δ 10.7, 12.4, 92.9, 114.6, 116.9, 121.9, 136.6.
Scheme S3. Synthetic scheme of NiSPY-1
O
O
O
1) 14, cat. TFA, CH2Cl2
BF3•OEt2, DIEA
Toluene
O
O
O
NC
CN
NC
CN
2) DDQ
NH
N
N
N
CHO
B
F
F
15
NiSPY-1
y. 35% (2 step)
NiSPY-1 was synthesized from 14 and 2,4-dimethoxybenzaldehyde (TCI) according to the following procedure.
Synthesis of NiSPY-1. 2,4-Dimethoxybenzaldehyde (116 mg, 1.0 mmol) and 3-cyano-2,4-dimethylpyrrole (252 mg, 2.1
mmol) were dissolved in 200 mL of CH2Cl2. TFA (1 mL) was added dropwise into the vigorously stirred solution, and
the reaction mixture was stirred overnight at ambient temperature. When TLC monitoring showed complete
consumption of the aldehyde, DDQ (295 mg, 1.3 mmol) was added and stirring was continued for 20 min. The reaction
mixture was washed with water (3 x 100 mL). The combined organic layers were washed with brine (1 x 100 mL),
dried over anhydrous Na2SO4, filtered, and evaporated. The crude product was purified by alumina column
chromatography (alminium oxide 90, Merck), and dried under reduced pressure to constant weight to afford 15 as a
crude amorphous solid. The crude dipyrromethene and DIEA (4 mL, 17.3 mmol) were dissolved in 100 mL of toluene
(100 mL) and stirred at ambient temperature for 5 min. BF3•OEt2 (2 mL, 15.8 mmol) was added, and stirring was
continued for 10 min. The reaction mixture was washed with water (3 x 80 mL), and brine (1 x 100 mL), dried over
anhydrous Na2SO4, filtered, and evaporated. The crude compound was purified by silica gel column chromatography
(silica gel 60N, Kanto Chemical) to afford NiSPY-1 as an orange solid (152 mg, yield 35%). 1H NMR (300 MHz,
CDCl3): δ 1.70 (s, 6H); 2.71 (s, 6H); 3.76 (s, 3H); 3.90 (s, 3H); 6.60 (d, J = 2.1 Hz, 1H); 6.68 (dd, J = 2.1, 8.4 Hz, 1H);
6.95 (d, J = 8.4 Hz, 1H). 13C NMR (75 MHz, CDCl3): δ 13.5, 13.8, 55.6, 55.8, 99.5, 105.9, 106.1, 113.6, 113.8, 129.2,
132.2, 145.0, 149.0, 156.9, 159.0, 163.1. HRMS (ESI-): calcd for [M--1], 433.1647; found, 433.1634.
S10

Scheme S4. Synthetic scheme of NiSPY-2
OH
O
Benzyl bromide, Cs2CO3
1) 14, cat. TFA, CH2Cl2
O
O
DMF
2) DDQ
CHO
CHO
16
y. 80%
OH
O
O
BF3•OEt2, DIEA
H2/10% Pd-C
CHCl3/EtOH
O
O
O
Toluene
NC
CN
NC
CN
NC
CN
N
N
NH
N
N
N
B
B
F
F
F
F
17
crude
18
y. 62% (2 step)
NiSPY-2
y. 92%
NiSPY-2 was synthesized from 5-hydroxy-2-methoxybenzaldehyde (Acros Organics) according to the following
procedure.
Synthesis of 16. 4-Hydroxy-2-methoxybenzaldehyde (650 mg, 4.28 mmol) was dissolved in DMF (10 mL), and benzyl
bromide (878 mg, 5.14 mmol) and cesium carbonate (1.67 g, 5.14 mmol) were added. The reaction mixture was stirred
overnight at ambient temperature, water (150 mL) was added, and the reaction mixture was extracted with AcOEt (3 x
100 mL). The combined organic layers were washed with water (5 x 50 mL) and brine (1 x 100 mL), dried over
anhydrous Na2SO4, filtered, and evaporated to afford 17 as a crude amorphous solid. The crude product was purified
by silica gel column chromatography and recrystallized from n-hexane to afford 16 as a white powder (823 mg, yield
80%). 1H NMR (300 MHz, CDCl3): δ 3.86 (s, 3H): 5.11 (s, 2H); 6.52 (d, J = 2.2 Hz, 1H); 6.61 (dd, J = 2.2, 8.6 Hz,
1H); 7.39 (m, 5H); 7.80 (d, J = 8.6 Hz, 1H); 10.28 (s, 1H). 13C NMR (75 MHz, CDCl3): δ 55.5, 70.3, 98.8, 106.4,
119.1, 127.4, 128.3, 128.7, 130.6, 135.8, 163.5, 165.2, 188.2. Mp: 94.0 – 94.6 ºC. Anal. Calcd for C15H14O3: N, 0; C,
74.36; H, 5.82. Found: N, 0; C; 74.28; H, 5.92.
18 were prepared according to
a
similar synthetic procedure (see "Synthesis of NiSPY-1") from
4-benzyloxy-2-methoxybenzaldehyde (300 mg, 1.24 mmol), 3-cyano-2,4-dimethylpyrrole (354 mg, 2.6 mmol) and DDQ
(366 mg, 1.61 mg) in 62% yield (395 mg). 1H NMR (300 MHz, CDCl3): δ 1.69 (s, 6H); 2.71 (s, 6H); 3.74 (s, 3H); 5.14
(s, 2H); 6.69 (d, J = 2.2 Hz, 1H); 6.75 (dd, J = 2.2, 8.3 Hz, 1H); 6.95 (d, J = 8.3 Hz, 1H); 7.42 (m, 5H). 13C NMR (75
MHz, CDCl3): δ 13.4, 13.8, 55.8, 70.5, 100.4, 105.9, 107.1, 113.7, 113.9, 127.6, 128.4, 128.7, 129.2, 132.1, 135.9, 144.9,
148.9, 156.9, 159.0, 162.1. HRMS (ESI-): calcd for [M-H]-, 509.1960; found, 509.1915. Anal. Calcd for
C29H25BF2N4O5•0.25 H2O: N, 10.79; C, 67.07; H, 5.05. Found: N, 10.61; C; 67.38; H, 5.08.
Synthesis of NiSPY-2. 18 (53 mg, 0.10 mmol) was dissolved in CHCl3 (20 mL). The solution was diluted with EtOH
80 mL. After the addition of 10% Pd-C (10 mg), the mixture was vigorously stirred overnight under a H2 atmosphere.
The Pd-C was filtered off, and then filtrate was concentrated under reduced pressure to constant weight. The crude
S11

compound was purified by silica gel column chromatography (silica gel 60, Kanto chemical) to afford NiSPY-2 as an
orange powder (40 mg, 92%). 1H NMR (300 MHz, CDCl3): δ 1.71 (s, 6H); 2.72 (s, 6H); 3.75 (s, 3H); 6.02 (br, 1H);
6.59 (d, J = 2.2 Hz, 1H); 6.63 (dd, J = 2.2, 8.0 Hz, 1H); 6.88 (d, J = 8.0 Hz, 1H). 13C NMR (75 MHz, CDCl3): δ 13.4,
13.8, 55.8, 100.0, 105.7, 109.0, 113.2, 113.8, 129.2, 132.2, 145.1, 149.0, 157.1, 159.0, 159.6. HRMS (ESI-): calcd for
[M-H]-, 419.1491; found, 419.1464. Anal. Calcd for C22H19BF2N4O2•CH2Cl2: N, 11.09; C, 54.69; H, 4.19. Found: N,
11.00; C; 54.89; H, 4.38.
Scheme S5. Synthetic scheme of NiSPY-3
O
O
Benzyl bromoacetate, Cs2CO3
DMF
1) 14, cat. TFA, CH2Cl2
O
HO
O
2) DDQ
CHO
O
CHO
19
y. 93%
O
O
OH
O
O
O
BF3•OEt2, DIEA
Toluene
O
H2/10% Pd-C
CHCl3/EtOH
HOOC
NC
O
O
O
NC
NC
CN
CN
CN
NH
N
N
N
N
N
B
B
F
F
F
F
20
crude
21
y. 36% (2 step)
NiSPY-3
y. 59%
NiSPY-3 was synthesized from 4-benzyloxy-2-hydroxybenzaldehyde (Lancaster Synthesis) according to the following
procedure.
Synthesis of 19. 4-Benzyloxy-2-hydroxybenzaldehyde (1 g, 4.39 mmol) and benzyl bromoacetate (1.1 g, 4.82 mmol)
were dissolved in absolute DMF (8 mL), then cesium carbonate (1.57 g, 4.83 mmol) were added. The reaction mixture
was stirred overnight at ambient temperature, water (150 mL) was added, and the reaction mixture was extracted with
AcOEt (3 x 100 mL). The combined organic layers were washed with water (5 x 50 mL) and brine (1 x 100 mL), dried
over anhydrous Na2SO4, filtered, and evaporated. The crude product was purified by silica gel column chromatography
over neutral silica gel and recrystallized from n-hexane to afford 19 as a white powder (1.54 g, yield 93%). 1H NMR
(300 MHz, CDCl3): δ 4.73 (s, 2H); 5.03 (s, 2H); 5.23 (s, 2H); 6.35 (d, J = 2.0 Hz, 1H); 6.66 (dd, J = 2.0, 8.8 Hz, 1H);
7.33-7.40 (m, 10H); 7.94 (d, J = 8.8 Hz, 1H); 10.38 (s, 1H). 13C NMR (75 MHz, CDCl3): δ 65.4, 67.1, 70.3, 99.6,
107.6, 119.4, 127.5, 128.3, 128.4, 128.6, 128.7, 130.7, 134.8, 135.6, 161.6, 164.9, 167.8, 188.0. MS (ESI+): 399
[M+Na]+. Mp: 84.4 – 85.7 °C. Anal. Calcd for C23H20O5: N, 0; C, 73.39; H, 5.36. Found: N, 0; C; 73.18; H, 5.62.
21
was prepared according to
a
similar synthetic procedure (see "Synthesis of NiSPY-1") from
4-benzyloxy-2-methoxycarbonylmethoxybenzaldehyde (400 mg, 1.06 mmol), 3-cyano-2,4-dimethylpyrrole (262 mg,
2.18 mmol) and DDQ (314 mg, 1.38 mg) in 36% yield (245 mg, orange powder). 1H NMR (300 MHz, CDCl3): δ 1.54 (s,
6H); 2.70 (s, 6H); 4.61 (s, 2H); 5.05 (s, 2H); 5.17 (s, 2H); 6.42 (d, J = 2.0 Hz, 1H); 6.78 (dd, J = 2.0, 8.4 Hz, 1H); 7.00 (d,
S12

J = 8.4 Hz, 1H); 7.31-7.43 (m, 10H). 13C NMR (75 MHz, CDCl3): δ 13.4, 13.8, 64.7, 67.4, 70.4, 100.6, 106.0, 108.0,
113.8, 113.9, 127.7, 128.5, 128.6, 128.7, 128.8, 129.4, 132.1, 134.6, 135.7, 144.1, 149.4, 155.0, 159.1, 161.8, 167.7.
HRMS (ESI-): calcd for [M-H]-, 643.2328; found, 463.2334. Anal. Calcd for C37H31BF2N4O4: N, 8.69; C, 68.95; H,
4.85. Found: N, 8.39; C, 68.71; H, 5.02.
Synthesis of NiSPY-3. 21 (35 mg, 0.05 mmol) was dissolved in CHCl3 (10 mL). The solution was diluted with EtOH
40 mL. After the addition of 10% Pd-C (7 mg), the mixture was stirred vigorously under a H2 atmosphere overnight.
The Pd-C was filtered off, and the filtrate was concentrated under reduced pressure to constant weight. The crude
compound was purified by silica gel column chromatography (silica gel 60, Kanto Chemical) to afford NiSPY-3 as an
orange powder (15 mg, 59%). 1H NMR (300 MHz, CDCl3): δ 1.75 (s, 6H); 2.71 (s, 6H); 4.61 (s, 2H); 6.44 (d, J = 1.6
Hz, 1H); 6.68 (dd, J = 1.6, 8.2 Hz, 1H); 6.93 (d, J = 8.2 Hz, 1H). 13C NMR (75 MHz, CDCl3): δ 13.0, 13.5, 64.3, 100.3,
105.0, 109.4, 110.7, 113.6, 129.2, 131.6, 149.1, 155.0, 158.4, 160.7, 169.9. HRMS (ESI-): calcd for [M-H]-, 463.1389;
Found, 463.1342.
Scheme S6. Synthetic scheme of NiSPY-1 N
O
O
B
NO2
NO2BF4
CH3CN
O
O
NC
CN
NC
CN
N
N
N
N
B
F
F
F
F
NiSPY-1
NiSPY-1 N
NiSPY-1 N was synthesized from NiSPY-1 according to the following procedure.
Synthesis of NiSPY-1 N: NiSPY-1 was dissolved in absolute CH3CN (dried over molecular sieve). After addition of
nitronium tetrafluoroborate (30 mg), the reaction mixture was stirred at ambient temperature for 5 min, and then
saturated NaHCO3 (200 mL) was added. The aqueous mixture was extracted with CH2Cl2 (3 x 100 mL). The
combined organic layers were washed with water (2 x 100 mL) and brine (1 x 100 mL), dried over anhydrous Na2SO4,
filtered, and evaporated. The crude product was purified by chromatography on a preparative silica gel plate to afford
NiSPY-1 N as an orange powder. 1H NMR (300 MHz, CDCl3): δ 1.74 (s, 6H); 2.73 (s, 6H); 3.93 (s, 3H); 4.12 (s, 3H);
6.69 (s, 1H); 7.88 (s, 1H). 13C NMR (75 MHz, CDCl3): 13.9, 56.9, 60.0, 96.7, 106.7, 113.1, 113.4, 127.7, 131.8, 133.3,
140.7, 148.4, 157.4, 160.1, 160.8. HRMS (ESI-): calcd for [M-H]-, 478.1498; found, 478.1468.
S13

Scheme S7. Synthetic scheme of NiSPY-2 N
OH
OH
NO2
NO2
OH
OH
NO2
1) 14, cat. TFA, CH2Cl2
BF3•OEt2, DIEA
Toluene
c. HNO3
O
O
O
O
2) DDQ
NC
CN
NC
CN
CHO
CHO
NH
N
N
N
B
F
23
NiSPYF-2 N
22
y. 26%
crude
y. 15% (2 step)
NiSPY-2 N was synthesized from 4-hydroxy-2-methoxybenzaldehyde according to the following procedure.
Synthesis of 22. Conc. nitric acid (5 mL) was carefully added dropwise to 4-hydroxy-2-methoxybenzaldehyde (800 mg,
°
5.26 mmol) at 0 C. The reaction mixture was stirred at ambient temperature for 20 min, then diluted with water (150
mL). The aqueous mixture was extracted with CH2Cl2 (3 x 50 mL). The combined organic layers were washed with
water (1 x 100 mL) and brine (2 x 50 mL), dried over anhydrous Na2SO4, filtered, and evaporated. The crude product
was purified by silica gel column chromatography (silica gel 60, Kanto Chemical) to afford 22 as a pale yellow powder
(274 mg, yield 26%). 1H NMR (300 MHz, CDCl3): δ 4.03 (s, 3H); 6.63 (s, 1H); 8.67 (s, 1H); 10.27 (s, 1H); 11.29 (s,
º
1H). 13C NMR (75 MHz, CDCl3): δ 56.8, 100.9, 119.1, 127.7, 128.1, 161.0, 167.3, 186.3. Mp: 145.6 – 146.4 C.
Anal. Calcd for C8H7NO5: N, 7.10; C, 48.74; H, 3.58. Found: N, 7.01; C, 48.92; H, 3.75.
Synthesis of NiSPY-2 N. 22 (100 mg, 0.51 mmol) and 3-cyano-2,4-dimethylpyrrole (145 mg, 1.07 mmol) were
dissolved in 200 mL of CH2Cl2. TFA (1.5 mL) was added dropwise into the vigorously stirred solution, and the
reaction mixture was stirred overnight at ambient temperature. When TLC monitoring showed complete consumption
of the aldehyde, DDQ (189 mg, 0.58 mmol) was added and stirring was continued for 10 min. The reaction mixture
was washed with water (3 x 100 mL). The organic layers were washed with brine (1 x 100 mL), dried over anhydrous
Na2SO4, filtered, and evaporated. The crude product was purified by alumina column chromatography (aluminium
oxide 90 active neutral, Merck) to afford 23 as a crude red amorphous solid. The crude dipyrromethene 23 and DIEA
(4 mL, 17.3 mmol) were dissolved in 100 mL of absolute toluene (100 mL) and stirred at ambient temperature for 5 min.
BF3•OEt2 (2 mL, 15.8 mmol) was added, and stirring was continued for 10 min. The reaction mixture was washed with
water (3 x 80 mL), and brine (1 x 100 mL), dried over anhydrous Na2SO4, filtrated, and evaporated. The crude
compound was purified by silica gel column chromatography (silica gel 60, Kanto Chemical) to afford NiSPY-2 N as an
orange powder (36 mg, yield 15%). 1H NMR (300 MHz, CDCl3): δ 1.76 (s, 6H); 2.73 (s, 6H); 3.91 (s, 3H); 6.78 (s,
1H); 7.97 (s, 1H); 11.16 (s, 1H). 13C NMR (75 MHz, CDCl3): δ 13.9, 14.0, 57.3, 101.7, 106.8, 113.2, 115.0, 126.3,
128.1, 131.8, 140.2, 148.2, 159.1, 160.2, 163.1. HRMS (ESI-): calcd for [M-H]-, 464.1342; found, 464.1331.
S14

Scheme S8. Synthetic scheme of NiSPY-3 N
O
O
OH
2,3-dihydro-2H-pyran
PPTS
O
O
HCl
Benzyl bromoacetate, Cs2CO3
DMF
O
O
HO
HO
MeOH/H2O
CH2Cl2
O
CHO
CHO
CHO
24
y. 67%
25
y. 91%
OH
NO2
OH
OH
NO2
O
c. HNO3
1) 14, cat. TFA, CH2Cl2
BF3•OEt2, DIEA
CH2Cl2
O
O
O
O
NC
O
O
CN
2) DDQ
O
CHO
O
CHO
NH
N
26
27
28
quant
y. 19%
OH
OH
NO2
NO2
O
NaOH
O
HOOC
NC
O
O
NC
MeOH/H2O
CN
CN
N
N
N
N
B
B
F
F
F
F
29
y. 51% (2 step)
NiSPY-3 N
trace
NiSPY-3 N was synthesized from 2,4-dihydroxybenzaldehyde (TCI) according to the following procedure.
Synthesis of 24. 2,4-Dihydroxybenzaldehyde (2.0 g, 14.50 mmol) and 2,4-dihydro-2H-pyran (1.92 g, 22.78 mmol)
were dissolved in absolute CH2Cl2 (150 mL). After the addition of pyridinium para-toluenesulfonate (PPTS, 1.8 g,
14.50 mmol), the reaction mixture was stirred at ambient temperature for 4 h. The reaction mixture was washed with
sodium phosphate buffer pH 6.8 (3 x 100 mL) and brine (1 x 100 mL). The organic layers was dried over anhydrous
Na2SO4, filtered, and evaporated. The crude product was purified by silica gel column chromatography (silica gel 60N,
Kanto Chemical) to afford 24 as an oil (2.16 g, yield 67%). 1H NMR (300 MHz, CDCl3): δ 1.61-2.04 (m, 6H);
3.61-3.87 (m, 2H); 5.51 (m, 1H); 6.64 (m, 2H); 7.45 (d, J = 8.6 Hz, 1H); 9.73 (s, 1H); 11.36 (s, 1H). 13C NMR (75
MHz, CDCl3): δ 18.4, 24.9, 29.9, 62.1, 96.2, 103.6, 109.3, 115.7, 135.2, 164.1, 164.3, 194.5. HRMS (ESI-): calcd for
[M-H]-, 221.0814; found, 221.0801.
Synthesis of 25. 24. (2.16 g, 9.73 mmol) and methyl bromoacetate (1.64 g, 10.70 mmol) were dissolved in absolute
DMF (20 mL). Cesium carbonate (4.1, 12.65 mmol) was added, then the reaction mixture was stirred overnight at
ambient temperature, water (150 mL) was added, and the reaction mixture was extracted with AcOEt (3 x 100 mL).
The combined organic layers were washed with water (4 x 50 mL) and brine (1 x 100 mL), dried over anhydrous Na2SO4,
filtrated, and evaporated. The crude product was purified by silica gel column chromatography (silica gel 60N, Kanto
Chemical) and recrystallized from n-hexane to afford 25 as a white powder (2.60 g, yield 91%). 1H NMR (300 MHz,
CDCl3): δ 1.60-1.90 (m, 6H); 3.64 (m, 1H); 3.78 (m. 1H); 3.82 (s, 3H); 4.74 (s, 2H); 5.49 (t, J = 2.9 Hz, 1H); 6.50 (d, J =
S15

2.2 Hz, 1H); 6.77 (m, 1H); 7.83 (d, J = 8.8 Hz, 1H); 10.39 (s, 1H). 13C NMR (75 MHz, CDCl3): δ 18.2, 24.8, 29.9, 52.3,
61.9, 65.4, 96.2, 100.5, 109.5, 119.7, 130.2, 161.6, 163.4, 168.4, 188.1. HRMS (ESI+): calcd for [M+Na]+, 317.1001;
found, 317.1001.
Synthesis of 26. 25 (1.5 g, 5.10 mmol) was dissolved in MeOH (40 mL). After addition of 1 N HCl aq. (10 mL), the
reaction mixture was stirred at ambient temperature for 5 h. The reaction mixture was extracted with AcOEt (3 x 100
mL). The combined organic layers were washed with brine (1 x 100 mL), dried over anhydrous Na2SO4, filtrated, and
evaporated. The crude product was purified by silica gel column chromatography (silica gel 60, Kanto Chemical) to
afford 26 as a white powder (quant.). 1H NMR (300 MHz, CDCl3): δ 3.83 (s, 3H); 4.74 (s, 2H); 6.10 (br, 1H); 6.30 (d,
J = 2.2 Hz, 1H); 6.53 (m, 1H); 7.80 (d, J = 8.6 Hz, 1H); 10.36 (s, 1H). 13C NMR (75 MHz, DMSO-d6): δ 52.0, 65.0,
100.0, 109.4, 117.3, 129.9, 162.1, 165.0, 168.8, 186.9. HRMS (ESI+): calcd for [M+Na]+, 233.0426; found, 233.0383.
°
Synthesis of 27. Conc. nitric acid (10 mL) was carefully added dropwise to 26 (1.05 g, 5.02 mmol) at 0 C. The
reaction mixture was stirred at ambient temperature for 20 min, then diluted with water (150 mL). The aqueous
mixture was extracted with CH2Cl2 (3 x 50 mL). The combined organic layers were washed with water (1 x 100 mL)
and brine (2 x 50 mL), dried over anhydrous Na2SO4, filtered, and evaporated. The crude product was purified by silica
gel column chromatography (silica gel 60, Kanto Chemical) to afford 27 as a pale yellow powder (243 mg, yield 19%).
1H NMR (300 MHz, CDCl3): δ 3.86 (s, 3H); 4.84 (s, 2H); 6.49 (s, 1H); 8.70 (s, 1H); 10.35 (s, 1H); 11.24 (s, 1H). 13C
NMR (75 MHz, CDCl3): δ 52.8, 65.6, 101.7, 119.2, 127.7, 128.8, 160.6, 165.3, 167.1, 186.1. HRMS (ESI+): calcd for
[M+Na]+, 278.0277; found, 278.0271.
Synthesis of 28. 27 (135 mg, 0.53 mmol) and 3-cyano-2,4-dimethylpyrrole (134 mg, 1.11 mmol) were dissolved in 200
mL of CH2Cl2. TFA (1 mL) was added dropwise into vigorously stirred solution, and the reaction mixture was stirred at
ambient temperature overnight. When TLC monitoring showed complete consumption of the aldehyde, DDQ (189 mg,
0.58 mmol) was added and stirring was continued for 10 min. The reaction mixture was washed with water (3 x 100
mL). The organic layers were washed with brine (1 x 100 mL), dried over anhydrous Na2SO4, filtered, and evaporated.
The crude product was purified by alumina column chromatography over alminium oxide 90 active neutral (Merck) to
afford 28 as a red powder. 1H NMR (300 MHz, CDCl3): δ 1.57 (s, 6H); 2.46 (s, 6H); 3.79 (s, 3H); 4.71 (s, 2H); 6.51 (s,
1H); 7.91 (s, 1H). 13C NMR (75 MHz, CDCl3): δ 13.9, 15.2, 52.7, 65.2, 101.8, 104.9, 115.0, 118.1, 127.2, 128.4, 135.5,
136.8, 147.1, 155.1, 158.2, 162.0, 167.0. HRMS (ESI+): calcd for [M+H]+, 476.1570; found, 476.1535.
Synthesis of 29. 28 and DIEA (4 mL, 17.3 mmol) were dissolved in 100 mL of absolute CH2Cl2 (100 mL) and the
solution was stirred at ambient temperature for 5 min. BF3•OEt2 (2 mL, 15.8 mmol) was added, and stirring was
S16

continued for 10 min. The reaction mixture was washed with 1 N HCl aq. (3 x 80 mL) and brine (1 x 100 mL), dried
over anhydrous Na2SO4, filtered, and evaporated. The crude compound was purified by silica gel column
chromatography (silica gel 60, Kanto Chemical) to afford 29 as an orange powder (140 mg, yield 51%). 1H NMR (300
MHz, CDCl3): δ 1.84 (s, 6H); 2.73 (s, 6H); 3.82 (s, 3H); 4.75 (s, 2H); 6.55 (s, 1H); 8.01 (s, 1H); 11.10 (br, 1H). 13C
NMR (75 MHz, CDCl3): δ 13.9, 52.8, 65.1, 102.3, 106.7, 113.3, 115.0, 126.4, 128.6, 131.7, 139.6, 148.8, 158.5, 160.2,
161.1, 166.9. HRMS (ESI-): calcd for [M-H]-, 522.1397; found, 522.1342.
Synthesis of NiSPY-3 N. 29 was dissolved in 20 mL of THF, then 1 N NaOH was added, and the mixture was stirred at
º
0 C for 3 h. The reaction mixture was neutralized with 1 N HCl, and extracted with CH2Cl2 (3 x 100 mL). The
combined organic extracts were dried over Na2SO4, filtered, and evaporated. The crude compound was purified by
silica gel column chromatography (silica gel 60, Kanto Chemical) to afford NiSPY-3 N as an orange-red solid. 1H
NMR (300 MHz, CDCl3): δ 1.83 (s, 6H); 2.73 (s, 6H); 4.79 (s, 2H); 6.61 (s, 1H); 8.02 (s, 1H). HRMS (ESI-): calcd for
[M-H]-, 508.1240; found, 508.1205.
References
(1) Miura, T.; Urano, Y.; Tanaka, K.; Nagano, T.; Ohkubo, K.; Fukuzumi, S. J. Am. Chem. Soc. 2003, 125, 8666-8671.
(2) Rehm, D.; Weller,; Isr. J. Chem. 1970, 8, 259-271.
(3) Mendelson, W. L.; Homes, M; Dougherty, U. Synth. 1996, 26, 593-601
S17