Synthesis and spectroscopic analysis of benzylidene imidazolone linked to P-porphyrins through axial ligand

Article abstract of DOI:10.1007/s00044-018-2255-0

Tetraphenylporphyrinatophosphorus(V) complexes (1) comprising two axially linked benzylidene imidazolone (Biz) moieties, which are chromophores of the green fluorescent protein, were prepared. In medical applications such as photodynamic therapy, the P-po

Full text of DOI:10.1007/s00044-018-2255-0

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-018-2255-0
ORIGINAL RESEARCH
Synthesis and spectroscopic analysis of benzylidene imidazolone
linked to P-porphyrins through axial ligand
1
Kyosuke Takemori1 Jun Ishikawa1 Yu Nabetani1 Mamoru Fujitsuka2 Tetsuro Majima2
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Jin Matsumoto
Masahide Yasuda1
Received: 24 May 2018 / Accepted: 19 October 2018
© Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
Tetraphenylporphyrinatophosphorus(V) complexes (1) comprising two axially linked benzylidene imidazolone (Biz)
moieties, which are chromophores of the green fluorescent protein, were prepared. In medical applications such as
photodynamic therapy, the P-porphyrin part (Ptp) is expected to sensitize to generate singlet oxygen, whereas the Biz units
act as fluorescent probes. The fluorescence spectra of 1 were analyzed under the excitations of Biz at 370 nm. Fluorescence
stemming from the excited states of Biz and Ptp was observed at 460 and 610 nm, respectively. The intramolecular
quenching of Biz in the excited singlet state by Ptp occurred, resulting in weak fluorescence from Biz. The introduction of a
cyano group in the Biz units of 1 enhanced their fluorescence quantum yield up to 7.7 × 10−4. The fluorescence spectra of 1
under the excitations of Ptp at 550 nm were extremely similar to that of a reference compound of P-porphyrin without the
Biz chromophore, dimethoxy(tetraphenylporphyrinato)phosphorus chloride. The physicochemical parameters of Ptp
remained unaltered following the introduction of Biz on the axial positions of P-porphyrin.
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Keywords Benzylidene imidazolone P-porphyrin Intermolecular electron transfer Fluorescent probe
Introduction
acted as a fluorescent probe. Previously, we have exten-
sively studied the photodynamic activity of phosphorus
porphyrins (P-porphyrins) using Saccharomyces cerevisiae
(Matsumoto et al. 2016; Matsumoto et al. 2011), E. coli
(Matsumoto et al. 2017a; Matsumoto et al. 2017b), and
cancer cells (Matsumoto et al. 2017c). On the other hand,
the green fluorescent protein (GFP) is a biologically
applicable fluorescent protein that contains a benzylidene
imidazolone (Biz) chromophore, which is constructed by
the condensation of glycine, tyrosine, and serine (Craggs
2009; Phillips Jr 1997) and whose fluorescence properties
can be tuned via chemical modifications (Follenius-Wund
et al. 2003; Walker et al. 2015). Bearing this in mind, we
selected the Biz chromophore as the second fluorescent
probe, which was connected to a P-porphyrin complex
through linkers (L) in the axial positions, affording P-
Photodynamic therapy (PDT) has become a well-recognized
cancer therapy that uses singlet-oxygen photosensitizers
such as porphyrin derivatives (Dąbrowski et al. 2016;
Gomer and Ferrario 1990; Mehraban and Freeman 2015). In
PDT, the porphyrin derivatives function as both sensitizers
and fluorescent probes (Ethirajan et al. 2011; Josefsen and
Boyle 2012). These sensitizers are required to absorb in the
optical window between 650 and 850 nm to avoid inter-
ference with absorption of light by tissue chromophores
(Plaetzer et al. 2009). This causes difficulties in detecting
the presence of these sensitizers in tumor by fluorescence,
which shifts from visible region to near infrared region.
With the aim of overcoming this problem, we intended to
develop a porphyrin bearing a second chromophore that
* Jin Matsumoto
2
The Institute of Scientific and Industrial Research (SANKEN),
Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan
jmatsu@cc.miyazaki-u.ac.jp
1
Department of Applied Chemistry, Faculty of Engineering,
University of Miyazaki, Gakuen-Kibanadai, Miyazaki 889-2192,
Japan

Medicinal Chemistry Research
Scheme 1 P-porphyrins 1a–1c
having benzylidene imidazolone
(Biz) units linked via axial
ligands
porphyrins 1a–1c (Scheme 1). Herein, we examined the
effects of the substituents (R and Ar) of Biz and L on the
fluorescence properties of 1.
p-Trifluoromethylbenziyidene-5-phenyl-1-oxazolone (3a)
Pale yellowish needles; Yield 80%; mp 171–172 °C
(CHCl3–Hexane); IR (KBr) νmax 3044, 1802, 1657, 1558,
1324, 1168 cm−1 1H NMR δ = 7.25 (s, 1H, -CH=C<),
;
Instruments
7.55–7.58 (m, 2H, H-3, and H-5 of C6H5), 7.64–7.68 (m,
1H, H-4 of C6H5), 7.73 (d, J = 8.2 Hz, 2H, H-3, and H-5 of
C6H4CF3), 8.20–8.22 (m, 2H, H-2, and H-6 of C6H5), 8.32
(d, 2H, H-2, and H-6 of C6H4CF3); HRMS Calcd for
1H NMR (400 MHz) and 13C NMR (100 MHz) spectra
were recorded with a Bruker AV 400 M spectrometer,
using CDCl3 or CD3OD as solvents and SiMe4 as the
internal standard. In 13C NMR, substitution level of each
carbon was determined by DEPT method and described as
CH3 (primary), CH2 (secondary), CH (tertiary and vinyl),
and C (quaternary and ipso) in spectral data. High-
resolution mass spectra (HRMS) were measured on a
Thermo Scientific Q Exactive mass spectrometer equipped
with an electrospray ionization source. Absorption spectra
were measured in MeOH using a JASCO V-550 spectro-
photometer. Redox potentials were determined via cyclic
voltammetry in MeCN (10 mM) in the presence of a sup-
porting electrolyte (Et4NBF4; 0.1 M); these measurements
were performed on a BAS CV-50W cyclic voltammeter at
a scan rate of 300 mV s−1 at 25 °C using a Pt working
electrode, a Pt counter electrode, and an Ag/AgNO3
reference electrode.
+
C17H11F3NO2 [M+H]+: 318.0736, m/z 318.0736. Found:
318.0726.
p-Cyanobenziyidene-5-phenyl-1-oxazolone (3b)
White powder; Yield 40%; mp > 157 °C (dec); IR (KBr)
νmax 3066, 2222, 1796, 1661, 1561, 1165 cm−1; 1H NMR δ
= 7.20 (s, 1H, -CH=C<), 7.55–7.59 (m, 2H, H-3, and H-5
of C6H5), 7.65–7.70 (m, 1H, H-4 of C6H5), 7.76 (d, J = 8.4
Hz, 2H, H-3, and H-5 of C6H4CN), 8.19–8.23 (m, 2H, H-2,
and H-6 of C6H5), 8.31 (d, J = 8.4 Hz, 2H, H-2, and H-6 of
+
C6H4CN); HRMS Calcd for C17H11N2O2 [M+H]+:
275.0815, m/z 275.0815. Found: 275.0809.
p-Cyanobenziyidene-5-(4-cyanophenyl)-1-oxazolone (3c)
1
Pale yellow solid; Yield 52%. H NMR δ = 7.30 (s, 1H,
Preparation of 4-substituted benzylidene-2-
phenyloxazolone (3)
-CH=C<), 7.77–7.89 (m, 2H, H-3, and H-5 of C6H4CN on
olefin), 7.85–7.88 (m, 2H, H-3, and H-5 of C6H4CN on
oxazolone), 8.29–8.31 (m, 2H, H-2, and H-6 of C6H4CN on
olefin), 8.31 (d, J = 8.4 Hz, 2H, H-2, and H-6 of C6H4CN
on oxazolone).
To a solution of p-trifluoromethylbenzaldehyde (1.0 mL) in
tetrahydrofuran (20 mL), N-benzoylglycine(1.0 g), and cat-
alytic Zn(OAc)2 (1.1 g) were added. The reaction mixture
was refluxed for 4 h at 80 °C (Scheme 2). After the solvent
of the reaction mixture was evaporated under vacuum,
water (120 mL) was added and the solution was allowed to
stand overnight at room temperature. Compound 3a was
collected from the solution as a precipitate. Following
recrystallization from CH2Cl2 (100 mL), 3a was isolated in
80% yield (1.41 g). A similar procedure was followed for
the preparation of the other oxazolone derivatives (3b–3d).
The spectral data corresponding to compounds 3a–3d are
listed below.
p-Hydroxybenziyidene-5-phenyl-1-oxazolone (3d)
Yellow powder; Yield 50%; mp > 180 °C (dec); IR (KBr)
1
νmax 3443, 3069, 1752, 1656, 1557, 1362, 1165 cm−1; H
NMR δ = 6.93–6.95 (m, 2H, H-3, and H-5 of C6H4-OH),
7.22 (s, 1H, -CH=C<), 7.51–7.55 (m, 2H, H-3, and H-5 of
C6H5), 7.58–7.63 (m, 1H, H-4 of C6H5), 8.15–8.18 (m, 2H,
H-2, and H-6 of C6H4OH), 8.16–8.19 (m, 2H, H-2, and H-6
+
of C6H5); HRMS Calcd for C16H12NO3 [M+H]+:
266.0812, m/z 266.0812. Found: 266.0807.

Medicinal Chemistry Research
Scheme 2 Synthetic routes of
2a–2d and 1a–1c
General procedure for the preparation of
imidazolone derivatives 2
NMR δ = 3.45–3.48 (m, 2H, -OCH2CH2OH), 3.59–3.62
(m, 2H, -OCH2CH2OH), 3.66 (t, J = 5.4 Hz, 2H,
>NCH2CH2O-), 4.04 (t, J = 5.4 Hz, 2H, >NCH2CH2O-),
7.18 (s, 1H, -CH=C<), 7.54–7.64 (m, 3H, H-3, H-4, and H-
5 of C6H5 on Biz), 7.68–7.70 (m, 2H, H-3, and H-5 of
C6H4CN), 7.87–7.90 (m, 2H, H-2, and H-6 of C6H5 on Biz),
8.31–8.32 (m, 2H, H-2, and H-6 of C6H4CN); HRMS Calcd
The linker was introduced via aminolysis of 3a with 3-amino-
1-propanol (70 μL) in the presence of 4-(dimethylamino)pyr-
idine (DMAP, 10.8 mg) in CH2Cl2 (6.0 mL) at room tem-
perature for 24 h to afford 4a (R=CF3, Ar=Ph, L=OCH2CH2)
in 98% yield (340 mg). Crude 4a (200 mg) was heated in
DMF (10 mL) at 180 °C for 6 h in a pressure bottle, affording
a crude residue, which was purified via column chromato-
graphy on silica gel to yield 2a (120 mg, 51%). Other imi-
dazolone derivatives 2b–2d were prepared following a similar
procedure. For 2b and 2c, 2-(2-aminoethoxy)ethanol (70 μL)
was used for aminolysis instead of 3-amino-1-propanol. The
spectral data of 2a–2d are listed below.
+
for C21H20N3O3 [M+H]+: 362.1499, m/z 362.1499.
Found: 362.1496.
5-[3-(p-Cyanobenzylidene)-5-(4-cyanophenyl)-1-
imidazolonyl)-3-oxypentanol (2c)
Yellow powder. Yield 12% (10% from p-cyanobenzalde-
hyde), mp > 176 °C (dec); IR (KBr) νmax 3425, 2959, 2227,
1720, 1644, 1165, 1053 cm−1; 1H NMR δ = 3.49 (t, J = 4.9
Hz, 2H, -OCH2CH2OH), 3.63 (t, J = 4.9 Hz, 2H, -OCH2-
CH2OH), 3.78 (t, J = 5.1 Hz, 2H, >NCH2CH2O-), 3.93 (t, J
= 5.1 Hz, 2H, >NCH2CH2O-), 7.22 (s, 1H, -CH=C<),
7.69–7.71 (m, 2H, H-3, and H-5 of C6H4CN on olefin),
7.82–7.84 (m, 2H, H-3, and H-5 of C6H4CN on Biz), 8.18–
8.20 (m, 2H, H-2, and H-6 of C6H4CN on Biz), 8.29–8.30
(m, 2H, H-2, and H-6 of C6H4CN on olefin); HRMS Calcd
3-[3-(p-Trifluoromethylbenzylidene)-5-phenyl-1-
imidazolonyl]propanol (2a)
Orange solid. Yield 17%; mp = 99–101 °C; IR (KBr) νmax
3400, 2931, 1715, 1645, 1325, 1165, 1070 cm−1; H NMR
1
δ = 1.80–1.86 (m, 2H, -CH2CH2CH2OH), 3.62 (t, J = 5.7
Hz, 2H, -CH2CH2CH2OH), 3.95 (t, J = 6.5 Hz, 2H,
-CH2CH2CH2OH), 7.26 (s, 1H, -CH=C<), 7.54–7.64 (m,
3H, H-3, H-4, and H-5 of C6H5 on Biz), 7.67 (d, J = 8.4 Hz,
2H, H-3, and H-5 of C6H4CF3), 7.82–7.84 (m, 2H, H-2, and
H-6 of C6H5 on Biz), 8.33 (d, J = 8.4 Hz, 2H, H-2, and H-6
+
for C22H19N4O3 [M+H]+: 387.1452, m/z 387.1452.
Found: 387.1453.
3-[3-(p-Hydroxybenzylidene)-5-phenyl-1-imidazolonyl]
propanol (2d)
+
of C6H4CF3); HRMS Calcd for C20H18F3N2O2 [M+H]+:
375.1315, m/z 375.1315. Found: 375.1315.
Yellow powder. Yield 22%; mp > 153 °C (dec); IR (KBr) νmax
5-[3-(p-Cyanobenzylidene)-5-phenyl-1-imidazolonyl]-3-
oxypentanol (2b)
3349, 3150, 2946, 1682, 1640, 1377, 1293, 1172, 1161, 1057
1
cm−1; H NMR δ = 1.74–1.82 (m, 2H, -CH2CH2CH2OH),
3.51 (t, J = 6.0 Hz, 2H, -CH2CH2CH2OH), 3.90 (t, J = 7.6 Hz,
2H, -CH2CH2CH2OH), 6.85 (d, J = 8.8 Hz, 2H, H-3, and H-5
of C6H4OH), 7.19 (s, 1H, -CH=C < ), 7.56-7.63 (m, 3H, H-3,
Yellow powder. Yield 20%; mp = 120–121 °C; IR (KBr)
1
νmax 3425, 2939, 2226, 1715, 1646, 1165, 1051 cm−1; H

Medicinal Chemistry Research
H-4, and H-5 of C6H5), 7.83–7.85 (m, 2H, H-2, and H-6 of
C6H5 on Biz), 8.12 (d, J = 8.8 Hz, 2H, H-2, and H-6 of
Bis[5-{3-(p-cyanobenzylidene)-5-phenyl-1-imidazolonyl}-3-
oxypentyloxo]tetraphenylporphyrinatophosophorus
chloride (1b)
−
C6H4OH); HRMS Calcd for C19H17N2O3 [M−H]−:
321.1245, m/z 321.1245. Found: 321.1245.
Purple solid. Yield 14%. mp > 300 °C; IR (KBr) νmax 3057,
1
Preparation of P-porphyrins (1a–1c) with
benzylidene imidazolone (Biz) units linked through
the axial ligands
2939, 2226, 1714, 1644, 1022, 803, 759, 704 cm−1; H
NMR δ = −2.30 (dt, JP–H = 12.4 Hz, J = 5.2 Hz, 4H, P-
OCH2-), 0.56–0.59 (m, 4H, P-OCH2CH2-), 2.33 (t, J = 5.4
Hz, 4H, -O CH2CH2N-), 2.91 (t, J = 5.4 Hz, 4H,
-OCH2CH2N-), 6.80 (s, 2H, -CH=C<), 6.84–6.88 (m, 4H,
H-3, and H-5 of C6H5 on Biz), 6.92–6.94 (m, 4H, H-2, and
H-6 of C6H5 on Biz), 7.37–7.41 (m, 2H, H-2 of C6H5 on
Biz), 7.69–7.78 (m, 8H, H-3, H-4, and H-5 of C6H5 at meso
position), 7.71 (d, J = 8.3 Hz, 2H, H-3, and H-5 of
C6H4CN), 7.85–7.87 (m, 8H, H-2, and H-6 of C6H5 at meso
position), 8.24 (d, J = 8.3 Hz, 2H, H-2, and H-6 of
C6H4CN), 8.94 (d, JP–H = 2.9 Hz, 8H, pyrrole β); 13C NMR:
1a was prepared by heating tetraphenylporphyrinato
(dichloro)phosphorus(V) chloride ([Cl2P(tpp)]Cl, 30 mg)
with 2a (200 mg) in a mixed solvent of MeCN (10 mL) and
pyridine (1.0 mL) at 110 °C for 5 days in a pressure bottle.
The crude product was purified via column chromatography
on silica gel to afford 1a (8.0 mg) in 14% yield. Similarly,
1b and 1c were prepared from 2b and 2c, respectively.
Bis[3-{3-(p-trifluoromethylbenzylidene)-5-phenyl-1-
imidazolonyl}propyloxo]
tetraphenylporphyrinatophosophorus chloride (1a)
δ = 40.95 (CH2, -O-CH2-CH2-N), 60.31 (CH2, d, JP–C =
15.2 Hz, P-O-CH2-CH2-), 66.60 (CH2, O-CH2-CH2-N),
66.85 (CH2, d, JP–C = 18.3 Hz, P-O-CH2CH2-), 112.88 (C,
C-4 of C6H4CN), 116.28 (C, meso of porphyrin ring),
118.63 (C, -CN), 124.92 (CH, -CH=C<), 128.10 (C, C-1 of
C6H5 on Biz), 128.32 (CH, C-2, and C-6 of C6H5 on Biz),
128.32 (CH, C-3, and C-5 of C6H5 on Biz), 128.57 (CH, C-
3, and C-5 of C6H5 at meso position), 129.84 (CH, C-4 of
C6H5 at meso position), 131.50 (CH, C-4 of C6H5 on Biz),
132.30 (CH, C-3, and C-5 of C6H4CN), 132.38 (CH, C-2,
and C-6 of C6H4CN), 133.05 (CH, d, JP–C = 5.3 Hz, pyrrole
β), 133.46 (CH, C-2, and C-6 of C6H5 at meso position),
135.14 (C, C-1 of C6H5 at meso position), 138.41 (C, C-1 of
C6H4CN), 139.08 (C, pyrrole α), 140.46 (C, C-3 of imi-
dazolone), 164.13 (C, C-5 of imidazolone), 170.78 (C, C-2
of imidazolone); HRMS Calcd for C86H64N10O6P+ [M+]:
1363.4742, m/z 1363.4742. Found: 1363.4763.
Purple solid. Yield 14%. mp > 300 °C; IR (KBr) νmax 3058,
2931, 1714, 1644, 1323, 1168, 1124, 1068, 1022, 803, 758,
702 cm−1; 1H NMR δ = −2.47 (dt, JP–H = 14.5 Hz, J = 6.5
Hz, 4H, P-OCH2-), −1.27 (t, J = 6.5 Hz, 4H, P-OCH2CH2-
), 1.84 (t, J = 6.5 Hz, 4H, P-OCH2CH2CH2-), 6.66 (s, 2H,
-CH=C<), 6.83–6.85 (m, 4H, H-2, and H-6 of C6H5 on
Biz), 7.07–7.10 (m, 4H, H-3, and H-5 of C6H5 on Biz),
7.35–7.39 (m, 2H, H-2 of C6H5 on Biz), 7.65–7.71 (m, 8H,
H-3, and H-5 of C6H5 at meso position), 7.71 (d, J = 8.3 Hz,
2H, H-3, and H-5 of C6H4CF3), 7.74–7.78 (m, 4H, H-4 of
C6H5 at meso position), 7.82–7.84 (m, 8H, H-2, and H-6 of
C6H5 at meso position), 8.19 (d, J = 8.3 Hz, 2H, H-2 and H-
6 of C6H4CF3), 8.99 (d, JP–H = 2.8 Hz, 8H, pyrrole β); 13C
NMR: δ = 26.66 (CH2, d, JP–C = 15.2 Hz, P-O-CH2-CH2-),
37.16 (CH2, P-O-CH2-CH2-CH2-), 59.03 (CH2, d, JP–C
=
Bis[5-{3-(p-cyanobenzylidene)-5-(p-cyanophenyl)-1-
imidazolonyl}-3-oxypentyloxo]
tetraphenylporphyrinatophosophorus chloride (1c)
14.1 Hz, P-O-CH2-), 116.19 (C, meso of porphyrin ring),
123.89 (C, q, JF–C = 271.8 Hz, -CF3), 125.48 (CH,
-CH=C<), 125.67 (CH, q, JF–C = 3.7 Hz, C-3, and C-5 of
C6H4CF3), 127.57 (CH, C-2, and C-6 of C6H5 on Biz),
127.71 (C, C-1 of C6H5 on Biz), 128.45 (CH, C-3, and C-5
of C6H5 at meso position), 128.59 (CH, C-3 and C-5 of
C6H5 on Biz), 129.76 (CH, C-4 of C6H5 at meso position),
131.46 (C, q, JF–C = 32.5 Hz, C-4 of C6H4CF3), 131.76
(CH, C-4 of C6H5 on Biz), 132.37 (CH, C-2, and C-6 of
C6H4CF3), 133.11 (CH, d, JP–C = 5.1 Hz, pyrrole β), 133.50
(CH, C-2, and C-6 of C6H5 at meso position), 135.09 (C, C-
1 of C6H5 at meso position), 137.32 (C, C-1 of C6H4CF3),
139.15 (C, C-3 of imidazolone), 139.23 (C, pyrrole α),
162.08 (C, C-5 of imidazolone), 169.72 (C, C-2 of imida-
zolone); 19F NMR (376 MHz): δ = −62.82; HRMS Calcd
for C84H60F6N8O4P+ [M+]: 1389.4374, m/z 1389.4374.
Found: 1389.4401.
Purple solid. Yield 3.4%. mp > 300 °C; IR (KBr) νmax 3058,
2959, 2226, 1716, 1661, 1443, 1384, 1022, 804, 759, 703
1
cm−1; H NMR δ = −2.23 (dt, JP–H = 13.3 Hz, J = 4.8 Hz,
4H, P-OCH2-), 0.63–0.66 (m, 4H, P-OCH2CH2-), 2.43 (t, J
= 4.9 Hz, 4H, -OCH2CH2N-), 2.90 (t, J = 4.9 Hz, 4H,
-OCH2CH2N-), 6.86 (s, 2H, -CH=C<), 6.89 (d, J = 8.5 Hz,
4H, H-3, and H-5 of C6H4CN on Biz), 6.98 (d, J = 8.5 Hz,
4H, H-2, and H-6 of C6H4CN on Biz), 7.70–7.79 (m, 8H,
H-3, H-4, and H-5 of C6H5 at meso position), 7.75 (d, J =
8.3 Hz, 2H, H-3, and H-5 of C6H4CN on olefin), 7.85–7.87
(m, 8H, H-2, and H-6 of C6H5 at meso position), 8.24 (d, J
= 8.3 Hz, 2H, H-2, and H-6 of C6H4CN on olefin), 8.97 (d,
JP–H = 2.9 Hz, 8H, pyrrole β); HRMS Calcd for

Medicinal Chemistry Research
Table 1 Characterization of the
Biz chromophore (2)
2
R
Ar
L
λmax/nma λEM/nm (E0–0/eV)b Φ2/10–2c τ2/nsd E1/2ox/Ve
2a CF3 Ph
2b CN Ph
CH2
374
463 (2.68)
472 (2.63)
451 (2.75)
453 (2.74)
1.03
7.45
7.63
0.014
0.23
0.50
0.78
—
1.43
1.26
1.35
0.95
OCH2CH2 384
2c CN 4-NCC6H4 OCH2CH2 382
2d OH Ph
CH2
394
aAbsorption maxima
bEmission maxima. The values in parentheses correspond to the excitation energy calculated from the
emission spectra
cFluorescence quantum yields from the Biz chromophore
dFluorescence lifetime
eOxidation potential vs. Ag/AgNO3
C88H62N12O6P+ [M+]: 1413.4647, m/z 1413.4647. Found:
Table 2 Quantum yield and lifetime of the fluorescence of 1a–1c
Excitation of Biz at 370 nm
Biz/10−3a Φ2Ptp/10−3 (τ2Ptp/ns)b Φent
Excitation of Ptp at 550 nm
1413.4646.
c
Φ
Φ1Ptp/10−2 (τ1Ptp/ns)d
Measurement of fluorescence quantum yields
1a 0.062
1b 0.47
1c 0.77
2.05 (5.08)
1.87 (5.08)
1.98 (5.08)
0.044 4.62 (5.13)
0.046 4.07 (5.08)
0.051 4.29 (4.50)
The fluorescence (FL) spectra of solutions were measured
using a Shimadzu RF-5300PC spectrometer. The FL spectra
of 2a–2d were measured at room temperature under exci-
tation at 370 nm. The emission maxima (λEM) appeared near
460 nm. The concentrations of the solutions of 2 were
adjusted so that the absorbance was <0.10 at the excitation
wavelength. According to the reported method (Birks
1970), the quantum yield (Φ2) of 2 was determined in
MeOH using a solution of quinine bisulfate in 0.5 M H2SO4
with a quantum yield of 0.546 under excitation at 370 nm
(Brouwer 2011). Table 1 shows the Φ2 values.
aFL quantum yield (ΦBiz) from Biz (460 nm) under excitation of Biz at
370 nm. Lifetime of FL from Biz was not measured due to weak FL
intensity
bFL appeared at 610 nm. Quantum yield (Φ2Ptp) and lifetime (τ2Ptp) of
the FL from Ptp under excitation of Biz at 370 nm
cEnergy transfer efficiency (Φent) from Biz* to Ptp
dFL appeared at 610 nm. Quantum yield (Φ1Ptp) of FL from Ptp under
excitation at 550 nm. The lifetime (τ1Ptp) was measured under
excitation at 430 nm
In addition, the FL spectra of 1a–1c were measured in
MeOH under excitation of the P-porphyrin (Ptp) and Biz
units at 550 and 370 nm, respectively. An MeCN solution
of Zn (II) tetraphenylporphyrin (Zn(tpp)) with a fluores-
cence quantum yield of 0.029 (Sirish and Maiya 1994) was
used as an actinometer for excitation at 550 nm. Table 2
laser was converted to second harmonic generation (430 or
370 nm) using a harmonic generator (Spectra-Physics,
GWU-23FL) or a type I BBO (β-BaB2O4) crystal, respec-
tively. The fluorescence lifetime of 2 (τ2) was measured
under excitation at 370 nm. The lifetimes (τ1Ptp and τ2Ptp) of
the fluorescence from the Ptp moiety of 1 were measured
under excitation of Ptp and Biz at 430 and 370 nm,
respectively. Tables 1 and 2 summarize the obtained results.
Ptp
lists the quantum yields (Φ1 and Φ2Ptp) of FL data from
Ptp at 610 nm under excitation of Ptp of 1 at 550 nm and
370 nm, respectively. The quantum yield of FL stemming
from Biz at 460 nm under excitation of 1 at 370 nm is
Biz
denoted as Φ2
.
Results and discussion
Measurement of fluorescence lifetimes
Design of P-porphyrins (1) with Biz on the axial
positions
Time-resolved fluorescence spectra were measured via the
single photon counting method, using a streakscope
(Hamamatsu Photonics, C4334-01) equipped with a poly-
chromator (Acton Research, SpectraPro150) (Fujitsuka
et al. 2004). An ultrashort laser pulse was generated using a
Ti:sapphire laser (Spectra-Physics, Tsunami 3941-M1BB,
full width at half maximum = 100 fs) pumped with a diode-
pumped solid-state laser (Spectra-Physics, Millennia VIIIs).
For excitation of the sample, the output of the Ti:sapphire
GFP is a biologically applicable fluorescent protein that
contains the p-hydroxybenzylidene imidazolonyl chromo-
phore depicted in Scheme 3, which is constructed via the
condensation of glycine, tyrosine, and serine (Craggs 2009;
Phillips Jr 1997). However, in the case of 2d with R=OH,
the fluorescence quantum yield (Φ2d) was very weak (1.4 ×
10−4), as shown in Table 1. The P-porphyrin is a strong
electron-accepting porphyrin due to the presence of

Medicinal Chemistry Research
25
20
15
10
5
1a
1b
1c
Scheme 3 Chromophore in GFP
0
400
450
500
550
600
650
700
Wavelength/nm
Fig. 1 Fluorescence spectra of 1a–1c under excitation of Biz at 370
nm. The emissions at 450 and 610 nm can be attributed to the fluor-
escence from the Biz and Ptp chromophores, respectively
were dependent on R, with the largest value corresponding
to 1c (7.7 × 10−4). Under excitation of Biz at 370 nm, the
FL from Ptp* appeared at 610 nm in Φ2Ptp. Ptp* is most
likely formed by energy transfer from Biz* to Ptp; the
energy transfer efficiency (Φent) was calculated to be 4.4–
Scheme 4 Fluorescence parameters of 2c and 5 in MeOH
pentavalent phosphorus. Indeed, the reduction potential of
P-porphyrin was positively shifted compared with that of
Ptp
5.1% for 1a–1c according to Eq. (1), using Φ1 for FL
from Ptp under excitation of the Ptp unit of 1.
red
other metalloporphyrins; e.g., the E1/2 values of dime-
thoxy(tetraphenylporphyrinato)phosphorus chloride (5,
Scheme 4) and Zn(tpp) are −0.82 V (Shiragami et al. 2005)
and −1.31 V (Felton and Linschitz 1966), respectively. This
suggests that electron transfer between Biz and Ptp moieties
may readily occur. Therefore, we introduced the electron-
accepting CF3 group in Biz (Follenius-Wund et al. 2003),
obtaining 2a. However, the Φ2a of 2a was still low (1.03 ×
10−2, Table 1). Therefore, R of Biz was changed from CF3
to CN, thereby producing 2b. In the case of 2c, the CN
group was introduced on both benzylidene and 5-phenyl
moieties, and the Φ2 values for both 2b and 2c were
enhanced to 7.45–7.63 × 10−2. Then next, the prepared 2a–
2c compounds were connected with Ptp through the linker
(L) to produce 1a–1c, respectively. In the cases of 1b and
1c, di(ethylenedioxy) group was used as L to weaken the
intramolecular interaction between Biz and Ptp effectively.
ΦP2tp
Φent
¼
ð1Þ
ΦP1tp
Analysis of the fluorescence spectra of 1c
Kinetic analysis was performed on 1c due to its highest ΦBiz
among the P-porphyrins 1. Compound 2c was used as a
reference of Biz without the Ptp chromophore. The fluor-
escence rate constants (kf2c) from the excited singlet states
of 2c were calculated to be 1.23 × 108 s−1 using Eq. (2). The
rate constant (kq) for the intramolecular quenching of Biz*
by the Ptp unit in 1c was calculated to be 1.26 × 1011 s−1 (=
(0.0763/0.00077−1)/0.78 × 109) according to Eq. (3). The
kq value was extremely large compared with kf2c. In the
quenching process, the participation of the energy transfer
from Biz* to Ptp was small, considering the small Φent
value. Therefore, it seems reasonable to assume that the
intramolecular quenching of Biz* by Ptp occurred mainly
through electron transfer, which was an exothermic process
with −0.58 eV according to the Rehm–Weller equation Eq.
(4) (Rehm and Weller 1970), in which the oxidation
potential of 2c (E1/2ox = 1.35 V, Table 1), the reduction
potential (E1/2red = −0.82 V) of [(MeO)2P(tpp)]Cl (5)
(Shiragami et al. 2005), and the excitation energy of 2c (E0–
Fluorescence of 1 under excitation of Biz
Figure 1 shows the FL spectra of 1a–1c under selective
excitation of the Biz moiety with 370 nm light. Two peaks
attributable to the excited singlet state of Biz and Ptp (Biz*
and Ptp*) at 460 and 610 nm, respectively, were observed.
As can be seen in Table 2, the FL quantum yields (ΦBiz) for
the FL from the Biz* unit of 1a–1c are about 1/100 of the
Φ2 values of 2a–2c, suggesting the occurrence of the
intramolecular quenching of Biz* by Ptp. The ΦBiz values

Medicinal Chemistry Research
efficiently quenched by the Ptp moiety, resulting in weak
fluorescence from Biz. The fluorescence quantum yield
from Biz in 1 was enhanced up to 7.7 × 10−4 by introducing
the CN group in Biz.
Acknowledgements This work was supported by a Grant-in-Aid for
Scientific Research (C) (16K05847) from the Japan Society for the
Promotion of Science (JSPS).
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.
Scheme 5 Energy diagram for the decay processes of 1c under exci-
tation of Biz at 370 nm
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