Spectroscopic analysis of the interaction of human serum albumin with tricationic phosphorus porphyrins bearing axial pyridinio groups

Article abstract of DOI:10.1246/bcsj.20130191

Much attention has been paid to water-soluble porphyrins and metalloporphyrins as potential sensitizers for photodynamic therapy (PDT). Thus, water-soluble tricationic phosphorus porphyrin complexes were prepared via the introduction of an N-alkylpyridinio group on the axial ligands of phosphorus porphyrins. The resulting tricationic phosphorus porphyrins readily dissolve in aqueous solutions without the formation of aggregates, even at high concentration. Moreover, the affinity of the porphyrins to human serum albumin (HSA) is a key factor in their application to PDT since HSA plays an important role in the transport of drug molecules to a target cell. Interactions between the tricationic phosphorus porphyrins and HSA were examined on the basis of changes in the Soret bands of the absorption and fluorescence spectra of phosphorus porphyrin solutions in the presence of HSA and the fluorescence quenching of HSA by the tricationic phosphorus porphyrins. Tricationic phosphorus porphyrins with moderately long alkyl chains on the pyridinio group were strongly adsorbed on the HSA at a position near the tryptophan residue of HSA. This result suggests that tricationic phosphorus porphyrins containing alkyl groups are good candidates as PDT reagents because of their high water solubility and high affinity for HSA.

Full text of DOI:10.1246/bcsj.20130191

1240 Bull. Chem. Soc. Jpn. Vol. 86, No. 11, 1240-1247 (2013)
© 2013 The Chemical Society of Japan
BCSJ Award Article
Spectroscopic Analysis of the Interaction of Human Serum Albumin with
Tricationic Phosphorus Porphyrins Bearing Axial Pyridinio Groups
Jin Matsumoto,*1 Tomoya Kubo,1 Tomohiko Shinbara,1 Naomi Matsuda,1 Tsutomu Shiragami,1
Mamoru Fujitsuka,2 Tetsuro Majima,2 and Masahide Yasuda1
1Department of Applied Chemistry, Faculty of Engineering, University of Miyazaki,
Gakuen-Kibanadai, Miyazaki 889-2192
2The Institute of Scientific and Industrial Research (SANKEN), Osaka University,
8-1 Mihogaoka, Ibaraki, Osaka 567-0047
Received July 11, 2013; E-mail: jmatsu@cc.miyazaki-u.ac.jp
Much attention has been paid to water-soluble porphyrins and metalloporphyrins as potential sensitizers for
photodynamic therapy (PDT). Thus, water-soluble tricationic phosphorus porphyrin complexes were prepared via the
introduction of an N-alkylpyridinio group on the axial ligands of phosphorus porphyrins. The resulting tricationic phos-
phorus porphyrins readily dissolve in aqueous solutions without the formation of aggregates, even at high concentration.
Moreover, the affinity of the porphyrins to human serum albumin (HSA) is a key factor in their application to PDT since
HSA plays an important role in the transport of drug molecules to a target cell. Interactions between the tricationic
phosphorus porphyrins and HSA were examined on the basis of changes in the Soret bands of the absorption and
fluorescence spectra of phosphorus porphyrin solutions in the presence of HSA and the fluorescence quenching of HSA
by the tricationic phosphorus porphyrins. Tricationic phosphorus porphyrins with moderately long alkyl chains on the
pyridinio group were strongly adsorbed on the HSA at a position near the tryptophan residue of HSA. This result suggests
that tricationic phosphorus porphyrins containing alkyl groups are good candidates as PDT reagents because of their high
water solubility and high affinity for HSA.
Much attention has been paid to porphyrins and metal-
loporphyrins because of their potential for use as sensitizers for
photosterilization1,2 and photodynamic therapy (PDT).3-6 In
general, photosensitizers used in clinical applications should
meet the following requirements: i) sufficient solubility in
water for handling in aqueous media, ii) strong absorption
at longer wavelengths in the visible region, iii) the capability
to generate active oxygen species, such as singlet oxygen, via
energy transfer from the triplet state of the sensitizer and/or
to directly oxidize tumor cells via photoinduced electron
transfer,7,8 and iv) adsorption on the plasma proteins, such as
albumin, hemoglobin, and lipoproteins, which carry sensitizers
to tumor cells.9-11 Among these proteins, we focused on human
serum albumin (HSA), which is the most abundant plasma
protein, since HSA has the ability to bind many kinds of
drugs12,13 and is applied to drug delivery systems.14
buffer and saline solutions.17,22,23 To prevent this face-to-face
interaction, we have examined water solubilization via modi-
fication of the axial ligands of antimony- and phosphorus-
containing porphyrins.24-28 Recently, we prepared water-
soluble phosphorus porphyrins bearing axial ligands, such
as hydrophilic ethyleneoxy and/or hydrophobic alkoxy
groups.27,28 However, the presence of other components such
as salts in the aqueous solutions strongly lowered the water
solubility of these phosphorus porphyrins.27 Herein, we report
the preparation of six types of tricationic phosphorus porphy-
rins via the introduction of alkylpyridinio groups on the axial
ligands (1a-1f, Scheme 1), and the results of the examination
of their interactions with HSA in buffer solutions.
Results and Discussion
Preparation of Water-Soluble Porphyrins 1. The tri-
cationic phosphorus porphyrins 1a-1c were prepared via the
reaction of the appropriate 3-alkylpyridine with bis(5-bromo-
3-oxapentan-1-olato)tetraphenylporphyrinatophosphorus(V)
chloride (2c), which was synthesized from [(HO)2P(tpp)]Cl
(2b; tpp: tetraphenylporphyrino) and bis(2-bromoethyl) ether
(Scheme 2). The tricationic phosphorus porphyrins 1d-1f
In general, water solubilization of porphyrins has been
achieved through the introduction of ionic groups such as
pyridinium,7,15-18 sulfonate,19-21 and phosphonium18,22 in the
porphyrin rings. However, despite the presence of such ionic
groups, many types of water-soluble porphyrins tend to form
face-to-face aggregates, resulting in low water solubility in
Published on the web September 7, 2013; doi:10.1246/bcsj.20130191

J. Matsumoto et al.
Bull. Chem. Soc. Jpn. Vol. 86, No. 11 (2013) 1241
CnH2n+1
CnH2n+1
N
X
N
Br
O
Ph
N
O
P
O
Ph
N
+
+
N
N
N
N
P
Ph
Ph
Ph
Ph
N
N
Cl
Cl
Ph
Ph
O
O
O
Br
N
1a; n = 6
1b; n = 4
1c; n = 2
1d; n = 6, X = Br
1e; n = 4, X = Br
1f; n = 1, X = I
X
N
CnH2n+1
CnH2n+1
Scheme 1. Tricationic phosphorus porphyrins 1 bearing axial pyridinio groups.
O
Br
O
(CH2)n-H
HO
Ph
O
P
Ph
N
+
+
1a (40%)
1b (44%)
1c
N
Br
Br
N
N
N
N
N
N
P
Ph
Ph
Ph
Ph
N
80%
(58%)
Cl
Cl
Ph
Ph
OH
O
2b
2c
Br
O
H2O
MeCN
87%
N
N
Cl
P
Ph
Ph
N
O
+
+
1d (91%)
1e (93%)
H-(CH2)n-X
OH
N
N
N
N
N
N
Ph
Ph
P
Ph
Ph
N
1f
(95%)
Cl
Cl
Ph
Ph
Cl
O
2a
2d
N
Scheme 2. Synthesis of phosphorus porphyrins 1.
were prepared via N-alkylation of bis[3-(4-pyridyl)propan-
1-olato]tetraphenylporphyrinatophosphorus(V) chloride (2d),
which was synthesized from [Cl2P(tpp)]Cl (2a) and 3-(4-
pyridyl)-1-propanol.
The water solubilities (CW) of the pyridinio-substituted
phosphorus porphyrins are listed in Table 1. Porphyrins 1a-1c
containing both N-alkylpyridinio and ethylene glycol units
exhibited large CW values (>63 mM), whereas porphyrins 1d-
1f containing only the N-alkylpyridinio group had water solu-
bilities above 3 mM. These values are higher than those of
[P(OMe)2(tpp)]Cl without the pyridinio moiety (CW = 0.31
mM). Porphyrins tend to aggregate in aqueous solution, lead-
ing to a broadening of their absorption spectra.17 Therefore, the
absorption spectra of porphyrins 1a-1f in aqueous solutions
were evaluated to determine if aggregation occurred. We
previously reported that the half width (HW) of the Soret band
of phosphorus porphyrins is approximately 16 nm for the
monomer form and greater than 20 nm for aggregates.28 The
absorption spectra of 1a-1f showed the Soret bands with HWs
of 16.6-18.2 nm, indicating that no aggregation occurred, even

1242 Bull. Chem. Soc. Jpn. Vol. 86, No. 11 (2013) BCSJ AWARD ARTICLE
Table 1. The Axially Pyridinio-Coordinated Tricationic Phosphorus Porphyrins 1
¹1 b)
¾/104 M¹1 cm
log Ke)
c)
g)
CW
HWd)
/nm
KSV
¤
kQ
/1012
89i)
1
MWa)
n
f f)
¹1
¹1
Method Ah) Method B Method C
/mM
/104 M
M
¹1 s
Soret
Q-band
1a 1341.7
1b 1285.6
1c 1229.5
6
4
2
24.7
25.3
29.6
1.22
1.29
1.40
63.6
112
>120
17.7
16.8
16.6
6.1 (1.3)
4.4 (0.9)
3.9 (0.8)
6.3
4.3
4.1
5.8
4.5
4.1
43.7
2.57
1.11
0.6
1.0
1.0
5.2
2.3j)
1d 1281.6
1e 1225.5
6
4
1
31.3
23.1
26.9
1.45
1.18
1.38
5.84
6.10
3.35
17.2
17.7
18.2
®
®
®
4.6
4.7
4.5
4.6
4.7
5.1
3.52
4.30
12.5
0.6
0.6
0.2
7.2
8.8
26
1f
1235.4
a) Molecular weight. b) Molar absorption coefficient of the Soret band (429-431 nm) and the Q band (560-562 nm) in MeOH solution.
c) Water solubility. d) Peak width at half height for the Soret band in the absorption spectra of 1 in aqueous solution. e) Binding
constant (K) determined from the fluorescence quenching of HSA with 1 (Method A), from the shift of the Soret band in the
absorbance spectrum of 1 by addition of HSA (Method B), and from fluorescence quenching of 1 with HSA (Method C). f) The
f values were determined from the fluorescence quenching of 1 with HSA according to the modified S-V equation (eq 5). g) Rate
constants for the fluorescence quenching of 1 with HSA were calculated using 4.9 ns as the fluorescence lifetime. h) The value in the
parentheses is the number of binding sites (a) on HSA. i) The fluorescence lifetime was determined to be 4.91 ns. j) The fluorescence
lifetime was determined to be 4.82 ns.
3
2
1
0
400
300
200
100
0
(a)
1a
(b)
[1a]/μM
HSA
0
1.0
1.4
1.9
2.6
3.6
5.0
200
300
400
500
300
400
500
Wavelength/nm
Wavelength/nm
Figure 1. (a) Absorption spectra of HSA (10 ¯M) and 1a (5 ¯M) in a phosphate buffer solution. (b) Changes in the fluorescence
spectrum of HSA in the presence of 1a; excitation at 230 nm.
in a phosphate buffer solution (Table 1 and Figure S1 in SI).
Therefore, it was possible to unambiguously perform the
spectroscopic analysis of the interactions of 1a-1f with HSA
free of any effect of aggregation on the spectral changes.
Changes in the Fluorescence Spectrum of HSA by the
Addition of 1. The interaction of 1 with HSA was estimated
on the basis of the binding constant (K) for each 1 with HSA.
HSA has absorption bands at approximately 280 nm and below
250 nm that arise from the tryptophan and tyrosine residues
present at one and eighteen equivalents in HSA, respectively.
The quenching of HSA in the excited state with phosphorus
porphyrins occurs via photoinduced electron transfer (PET)
between 1 and the tryptophan and/or tyrosine residues, which
have lower oxidation potentials than those of other amino
acids. Although 1d-1f had considerable absorption in the range
from 220 to 260 nm, compounds 1a-1c exhibited only weak
absorption bands in this range (Figure 1a), and the absorbance
at 230 nm for HSA was much larger than that for 1a-1c by
a factor of 21. Therefore, the determination of the K values
based on the fluorescence quenching of HSA (Method A)
was applied for compounds 1a-1c. The fluorescence of HSA
appeared at an emission maximum of 330 nm when excited at
230 nm (Figure 1b). The K values were determined according
to eq 1:29
logðF0=F ꢀ 1Þ ¼ log K þ a log ½1ꢁ
ð1Þ
where F0 and F denote the fluorescence intensities of HSA in
the absence and presence of 1, respectively, and a denotes the
number of molecules of 1 bound to one molecule of HSA. F0
and F values were corrected for the inner filter effect according
to Gauthier’s method (See SI).30 As shown in Figure 2, the
values for log K and a were obtained from the intercepts and
slopes of the plots of log(F0/F ¹ 1) against log [1], respec-
tively, and the results are listed in Table 1. The a values were
determined to be 0.8-1.3, indicating that 1a-1c bonded to HSA
in approximately 1:1 stoichiometry.
Changes in the Absorption Spectrum of 1 by the
Addition of HSA.
The value of K for each phosphorus
porphyrin 1 was also measured on the basis of the shift in
each absorption spectrum by the addition of HSA (Method B).
The wavelength (-0) of the Soret band of 1 ([1] = 3.0 ¯M)
was approximately 430 nm. By the addition of HSA ([HSA] =

J. Matsumoto et al.
Bull. Chem. Soc. Jpn. Vol. 86, No. 11 (2013) 1243
0.25-60 ¯M), the Soret band shifted to a longer wavelength
(-): ¦- = - ¹ -0 (see SI, Figure S1). The value of K was then
determined using eq 2 (See SI):
sity (F) at different [HSA]. A typical example of the changes
observed in the fluorescence spectra can be observed in
Figure 4 for 1a. The value of K was obtained according to
eq 3, which was derived from the transformation of the change
in the absorption spectrum (¦-) in eq 2 to the change in the
fluorescence intensity ¦F (= F0 ¹ F) (See SI):
ꢀ
ꢁ
ꢀ-
ꢀ-
¼ K ½HSAꢁ ꢀ
½1ꢁ
-
1 ꢀ -
ꢀ- ¼ - ꢀ -0
-
1 ꢀ -0
ð2Þ
ꢀ
ꢁ
ꢀF
ꢀF
¼ K ½HSAꢁ ꢀ f
½1ꢁ
where -¨ denotes the wavelength at an infinite concentration
of HSA. Figure 3 shows the plots of ¦-/(-¨ ¹ -) against
[HSA] ¹ ¦-/(-¨ ¹ -0) [1]. The calculated K values using
this method are also listed in Table 1.
Changes in the Fluorescence Spectrum of 1 by the
Addition of HSA. Next, the value of K was determined on
the basis of the fluorescence quenching of 1 with HSA
(Method C). The fluorescence spectrum of each phosphorus
porphyrin 1 ([1] = 3 ¯M) was measured in a phosphate buffer
solution (3 mL) with excitation at 430 nm, and the intensity (F0)
at the maximum wavelength (614-616 nm) was recorded. The
fluorescence of 1 was quenched with various concentrations of
HSA ([HSA] = 0.25-30 ¯M) to obtain the fluorescence inten-
F ꢀ F
ꢀF ¼ F0 ꢀ F
F0 ꢀ F
1
1
ð3Þ
Here F¨ denotes the minimum fluorescence intensity at an
infinite concentration of HSA. The F¨ values were determined
by the curve fitting of the plot of F vs. [HSA] using an
equation: F = F¨ + A1 exp(¹[HSA]/B1) + A2 exp(¹[HSA]/
B2), where A1, A2, B1, and B2 were the constants. The f values
denote the ratio of the number of molecules of 1 adsorbed on
the quenching sites of HSA to the total number of molecules
of 1 adsorbed on the HSA, and were determined using eq 5
described below. Figure 5 shows the plots of ¦F/(F ¹ F¨)
against [HSA] ¹ f¦F/(F0 ¹ F¨) [1]. The K values are listed
in Table 1.
The fluorescence quenching of 1 provides useful information
about the binding sites because the excited singlet states of
phosphorus porphyrins are quenched only via PET31,32 with the
tryptophan residue, which is located at Site I and is known as a
-0.5
-1
600
[HSA]/μM
0
0.25
0.50
0.75
400
1.5
3.0
6.0
-1.5
15
200
-5.8
-5.6
-5.4
-5.2
0
log [1]
500
600
700
800
Figure 2. Determination of the binding constant (K)
according to eq 1 (Method A). The value of K was deter-
mined from the plot of log(F0/F ¹ 1) against log [1] for
the fluorescence quenching of HSA with 1a ( ), 1b ( ),
and 1c ( ); excitation at 230 nm.
Wavelength/nm
Figure 4. Changes in the fluorescence spectrum of 1a
(3 ¯M) in a phosphate buffer by the addition of HSA
(excitation at 430 nm).
(a)
(b)
(c)
2
1
0
1
0.5
0
1
λ
λ
λ
λ
λ
λ
0.5
0
0
0.5
1
0
20
40
60
0
10
20
30
[HSA] [1] λ/(λ∞ − λ0)/μM
[HSA] [1] λ/(λ∞ −λ0)/μM
[HSA] [1] λ/(λ∞ − λ0)/μM
−
Δ
−
Δ
−
Δ
Figure 3. Determination of the binding constant (K) according to eq 2 (Method B). The value of K was determined on the basis of the
change in the absorption spectrum of 1 in the presence of HSA: (a) 1a ( ); (b) 1b ( ) and 1c ( ); and (c) 1d ( ), 1e ( ), and 1f ( ).

1244 Bull. Chem. Soc. Jpn. Vol. 86, No. 11 (2013) BCSJ AWARD ARTICLE
(a)
(b)
(c)
8
6
4
2
0
4
3
2
1
0
1.5
1
0.5
0
0
5
10
0
20
40
60
0
10
20
30
[HSA] − f [1]ΔF/(F0 − F∞)/μM
[HSA] − f [1]ΔF/(F0 − F∞)/μM
[HSA] − f [1]ΔF/(F0 − F∞)/μM
Figure 5. Determination of the binding constant (K) according to eq 3 (Method C). The value of K was determined on the basis of the
changes in the fluorescence intensity of 1 in the presence of HSA: (a) 1a ( ); (b) 1b ( ) and 1c ( ); and (c) 1d ( ), 1e ( ), and 1f ( ).
The modified S-V plots show a linear relationship against
the reciprocal value of [HSA] (Figure 7). Using eq 5, the f
and KSV¤ (= kQ¸F) values were obtained from the inverse of
the intercept and ratio of the intercept to slope, respectively,
and are listed in Table 1. The fluorescence lifetime (¸F) of 1
was calculated to be 4.9 ns because the ¸F values of 1a and 1c
were found to be similar (4.9 and 4.8 ns) using single photon
counting. Using the KSV¤ and ¸F values, the rate constants (kQ)
for the fluorescence quenching of 1 with HSA were determined
(Table 1). These kQ values exceed the diffusion-controlled limit
in water (7.4 © 109 M¹1 s¹1) and suggest that 1 in the ground
state interacts with HSA to form a complex, and that the
quenching of 1 with HSA occurs through static quenching.
Moreover, it was found that the f values of 1a, 1d, and 1e were
approximately 0.6, whereas that of 1f was small (0.2) and those
of 1b and 1c were 1.0.
1.8
1.6
1.4
1.2
1
0
10
20
30
[HSA]/μM
Binding Properties of 1 with HSA. The affinity of 1 for
HSA was evaluated on the basis of three types of spectro-
scopic analyses (Methods A, B, and C). From these results, the
phosphorus porphyrins 1 were classified into three categories
according to their f values: 1, 0.6, and 0.2. The K values of 1b
and 1c, with f values of 1.0, were similar when determined
using Methods B and C, and these phosphorus porphyrins bind
to HSA site-specifically. However, 1a, 1d, and 1e, with f values
of 0.6, bind to HSA at two types of sites with similar affinities.
Finally, for 1f (n = 1) with an f value of 0.2, the K value
obtained using Method C was larger than that for Method B,
indicating that most molecules of 1f adsorbed on the HSA far
from the tryptophan residue leading to inefficient quenching.
Thus, the length of the alkyl chain, rather than the existence
of the pyridinio moiety, affects the site-specific binding of 1 to
HSA; the tricationic phosphorus porphyrins bind to HSA via
the interaction of the hydrophobic alkyl groups of 1 and the
hydrophobic pocket of HSA.
Figure 6. Stern-Volmer plots for the fluorescence quench-
ing of 1 with HSA according to eq 4: 1a ( ), 1d ( ), 1e
( ), and 1f ( ).
major drug-binding site.33 In fact, the fluorescence quenching
of 1c with L-tryptophan was confirmed to occur efficiently
¹1
in aqueous solution: KSV = 48 M
M
and kQ = 1.0 © 1010
¹1 s (see SI, Figure S2). Thus, it was demonstrated that
¹1
the quenching of phosphorus porphyrins 1 can take place via
PET with the tryptophan residue of HSA.
Next, the F0/F values were plotted against the [HSA]
according to the Stern-Volmer (S-V) equation (eq 4):
F0
¼ 1 þ KSV ½HSAꢁ
ð4Þ
F
The plots are convex upward for 1a and 1d-1f (Figure 6),
suggesting that these phosphorus porphyrins adsorbed on two
different types of binding sites of HSA. Therefore, the fluo-
rescence quenching of 1 with HSA was analyzed using the
modified S-V plots described by eq 5, where KSV¤ is the
modified S-V constant:34
Conclusion
Water solubilization of porphyrins was achieved via the
introduction of pyridinio groups on the axial ligands of phos-
phorus porphyrins, which were readily prepared using com-
mercially available tetraphenylporphyrins as the starting mate-
rials. The resulting tricationic phosphorus porphyrins 1 had
high CW values, formed no aggregates, even at high concen-
trations in buffer solutions, and interacted strongly with HSA.
F0
1
1
¼
þ
0
ꢀF
fKSV ½HSAꢁ
f
ꢀF ¼ F0 ꢀ F
ð5Þ

J. Matsumoto et al.
Bull. Chem. Soc. Jpn. Vol. 86, No. 11 (2013) 1245
(a)
(b)
15
15
10
5
10
5
0
0
0
1
2
3
4
0
0.05
0.1
0.15
[HSA]−1/106 M−1
[HSA]−1/106 M−1
Figure 7. Modified S-V plots for the fluorescence quenching of 1 with HSA according to eq 5: (a) 1a ( ); (b) 1b ( ), 1c ( ), 1d
( ), 1e ( ), and 1f ( ).
In addition, like other phosphorus porphyrins, the new com-
pounds are expected to serve as electron-transfer sensitizers
and energy-transfer sensitizers in photodynamic therapy32,35
because the reduction potentials of phosphorus porphyrins are
relatively shifted to positive values compared with those of
free-base porphyrins and metalloporphyrins based on divalent
metals, such as Zn(II) and Mg(II). Furthermore, it is possible
that the tricationic phosphorus porphyrins 1 can be delivered to
tumor cells via complexation with HSA in blood.
evaporated to provide dihydroxide(tetraphenylporphyrinato)-
phosphorus chloride (2b, [(HO)2P(tpp)]Cl) in 87% yield.37
Preparation of Bis(5-bromo-3-oxapentan-1-olato)tetra-
phenylporphyrinatophosphorus(V) Chloride (2c).
Com-
pound 2b (100 mg) was reacted with bis(2-bromoethyl) ether
(1.0 mL) in MeCN (10 mL) in the presence of K2CO3 (25 mg)
and 18-crown-6-ether (7.4 mg) at 50 °C for 5 h. Next, the
reaction mixture was concentrated on a rotary evaporator and
then poured into hexane to remove any unreacted bis(2-bromo-
ethyl) ether. The precipitated crude product was purified by
column chromatography on SiO2 with CHCl3-MeOH (100:
1-20:1 v/v) as the eluent to provide 2c.
Experimental
Instruments.
1H NMR (400 MHz) and 13C NMR (100
1
MHz) spectra were obtained with a Bruker AV 400M spec-
trometer using CDCl3 or CD3OD solutions with SiMe4 as the
internal standard. High-resolution mass spectra (HRMS) were
measured on a Thermo Scientific Q Exactive mass spectrometer
equipped with an electrospray ionization source at the Center
for Collaborative Research and Community Cooperation,
University of Miyazaki. The molar absorption coefficients (¾)
of 1 at the Soret and Q bands were determined from the visible
spectra measured in MeOH using a JASCO V-550 spectropho-
tometer. The fluorescence spectra of solutions were measured
on a Shimadzu RF-5300PC spectrometer.
Time-resolved fluorescence spectra were measured via single
photon counting using a streakscope (Hamamatsu Photonics,
C4334-01) equipped with a polychromator (Acton Research,
SpectraPro150).36 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 excita-
tion of the sample, the output of the Ti:sapphire laser was
converted to the second harmonic generation (SHG, 430 nm)
with a harmonic generator (Spectra-Physics, GWU-23FL).
Preparation of [(HO)2P(tpp)]Cl (2b). An MeCN-H2O
(3:1, 160 mL) solution of [Cl2P(tpp)]Cl (2a) was refluxed until
the Soret band shifted from 436 to 423 nm in the absorption
spectrum. After the evaporation of MeCN, the aqueous solu-
tion was extracted with CHCl3. The CHCl3 extract was then
2c. Yield 80%. H NMR (400 MHz, CDCl3): ¤ ¹2.21 (dt,
J
P-H = 10.7 Hz, J = 5.3 Hz, 4H), 0.66-0.71 (m, 4H), 2.33 (t,
J = 5.7 Hz, 4H), 2.43 (t, J = 5.7 Hz, 4H), 7.75-7.82 (m, 12H),
7.96-8.00 (m, 8H), 9.07 (d, JP-H = 2.9 Hz, 8H); 13C NMR
(100 MHz, CDCl3): ¤ 29.67, 60.91 (d, JP-C = 15.2 Hz), 66.91
(d, JP-C = 19.6 Hz), 69.83, 116.40, 128.51, 129.87, 133.23
(d, JP-C = 5.3 Hz), 133.51, 135.29, 139.23.
Preparation of the Tricationic Phosphorus Porphyrins
1a, 1b, and 1c. The product 2c (50 mg) was then reacted with
the appropriate 3-alkylpyridine (1.0 mL) in MeCN (10 mL) at
100 °C for 68, 50, and 20 h for the preparation of 1a, 1b, and
1c, respectively (Scheme 2). The reaction mixture was con-
centrated on a rotary evaporator and then poured into hexane to
remove any excess 3-alkylpyridine. Next, the crude precipitate
was dissolved into CHCl3 and extracted with water. Com-
pounds 1a-1c were isolated from the aqueous layers and dried
under reduced pressure.
Bis[5-(3-hexyl-1-pyridinio)-3-oxapentan-1-olato]tetra-
phenylporphyrinatophosphorus(V) Dibromide Chloride
1
(1a). Yield 44%. H NMR (400 MHz, CD3OD): ¤ ¹2.22-
2.17 (m, 4H), 0.66-0.70 (m, 4H), 0.86 (t, J = 6.6 Hz, 6H),
1.21-1.29 (m, 12H), 1.45 (quint, J = 7.4 Hz, 4H), 2.40 (t, J =
4.7 Hz, 4H), 2.53 (t, J = 7.4 Hz, 4H), 3.84 (t, J = 4.7 Hz, 4H),
7.47 (dd, J = 8.0 and 6.0 Hz, 2H), 7.74 (d, J = 6.0 Hz, 2H),
7.79-7.87 (m, 12H), 7.99-8.00 (m, 8H), 8.07 (s, 2H), 8.23
(d, J = 8.0 Hz, 2H), 9.17 (d, JP-H = 3.2 Hz, 8H); 13C NMR

1246 Bull. Chem. Soc. Jpn. Vol. 86, No. 11 (2013) BCSJ AWARD ARTICLE
(100 MHz, CD3OD): ¤ 14.35, 23.51, 29.68, 31.29, 32.50, Bis[3-(1-hexyl-4-pyridinio)propan-1-olato]tetraphenyl-
33.30, 60.92, 61.66 (d, JP-C = 14.8 Hz), 68.33 (d, JP-C
19.1 Hz), 68.81, 117.67, 129.64, 131.02, 134.59 (d, JP-C
=
=
porphyrinatophosphorus(V) Dibromide Chloride (1d).
Yield 91%. 1H NMR (400 MHz, CD3OD): ¤ ¹2.20 (dt,
5.2 Hz), 134.73, 136.70, 140.53, 142.86, 144.86, 144.92,
146.56; HRMS calcd for C74H78N6O4P3+ [M3+]: 1145.5822,
m/z 381.8602. Found: m/z 381.8603.
JP-H = 12.0 Hz, J = 6.0 Hz, 4H), ¹1.00-0.94 (m, 4H), 0.68
(t, J = 7.5 Hz, 4H), 0.85 (t, J = 6.7 Hz, 6H), 1.19-1.29 (m,
12H), 1.70-1.77 (m, 4H), 4.29 (t, J = 7.6 Hz, 4H), 6.53 (d,
J = 6.6 Hz, 4H), 7.82-7.89 (m, 12H), 8.06 (dd, J = 7.6, 2.2 Hz,
8H), 8.32 (d, J = 6.6 Hz, 4H), 9.21 (d, JP-H = 2.8 Hz, 8H);
13C NMR (100 MHz, CD3OD): ¤ 14.20, 23.39, 26.69, 27.90
(d, JP-C = 18.0 Hz), 31.16, 32.26, 61.53 (d, JP-C = 13.95 Hz),
Bis[5-(3-butyl-1-pyridinio)-3-oxapentan-1-olato]tetra-
phenylporphyrinatophosphorus(V) Dibromide Chloride
1
(1b). Yield 44%. H NMR (400 MHz, CD3OD): ¤ ¹2.22-
2.17 (m, 4H), 0.68-0.72 (m, 4H), 0.86 (t, J = 7.6 Hz, 6H), 1.21-
1.29 (m, 4H), 1.40-1.45 (m, 4H), 2.41 (t, J = 4.8 Hz, 4H), 2.53
(t, J = 7.6 Hz, 4H), 3.84 (t, J = 4.8 Hz, 4H), 7.46 (dd, J = 8.0
and 6.0 Hz, 2H), 7.73 (d, J = 6.0 Hz, 2H), 7.79-7.89 (m, 12H),
7.99-8.00 (m, 8H), 8.08 (s, 2H), 8.23 (d, J = 8.0 Hz, 2H), 9.18
(d, JP-H = 2.9 Hz, 8H); 13C NMR (100 MHz, CD3OD): ¤ 13.98,
22.98, 32.97, 33.31, 60.91, 61.66 (d, JP-C = 14.4 Hz), 68.31
(d, JP-C = 19.0 Hz), 68.81, 117.64, 127.95, 129.62, 130.99,
134.56 (d, JP-C = 5.2 Hz), 134.71, 136.67, 140.50, 142.84,
144.85, 144.87, 146.55; HRMS calcd for C70H70N6O4P3+
[M3+]: 1089.5196, m/z 363.1727. Found: m/z 363.1727.
Bis[5-(3-ethyl-1-pyridinio)-3-oxapentan-1-olato]tetra-
phenylporphyrinatophosphorus(V) Dibromide Chloride
62.01, 117.55, 128.00, 129.72, 131.05, 134.71 (d, JP-C
=
5.1 Hz), 134.90, 136.72, 140.58, 144.22, 162.52; HRMS calcd
for C72H74N6O2P3+ [M3+]: 1085.5611, m/z 361.8531. Found:
m/z 361.8532.
Bis[3-(1-butyl-4-pyridinio)propan-1-olato]tetraphenyl-
porphyrinatophosphorus(V) Dibromide Chloride (1e).
Yield 93%. 1H NMR (400 MHz, CD3OD): ¤ ¹2.20 (dt,
J
P-H = 12.1 Hz, J = 6.0 Hz, 4H), ¹1.00-0.94 (m, 4H), 0.68
(t, J = 7.5 Hz, 4H), 0.89 (t, J = 7.4 Hz, 6H), 1.25 (sext, J =
7.4 Hz, 4H), 1.68-1.75 (m, 4H), 4.30 (t, J = 7.6 Hz, 4H), 6.53
(d, J = 6.6 Hz, 4H), 7.81-7.87 (m, 12H), 8.06 (dd, J = 8.1,
2.1 Hz, 8H), 8.32 (d, J = 6.6 Hz, 4H), 9.21 (d, JP-H = 2.9 Hz,
8H); 13C NMR (100 MHz, CD3OD): ¤ 13.70, 20.28, 27.94
(d, JP-C = 16.7 Hz), 31.17, 34.23, 61.55 (d, JP-C = 14.7 Hz),
1
(1c). Yield 58%. H NMR (400 MHz, CD3OD): ¤ ¹2.22-
2.17 (m, 4H), 0.67-0.72 (m, 4H), 1.06 (t, J = 7.6 Hz, 6H), 2.45
(t, J = 4.5 Hz, 4H), 2.58 (q, J = 7.6 Hz, 4H), 3.88 (t, J =
4.5 Hz, 4H), 7.48 (dd, J = 7.9 and 6.0 Hz, 2H), 7.78 (d, J =
6.0 Hz, 2H), 7.79-7.87 (m, 12H), 7.99-8.02 (m, 8H), 8.09 (s,
2H), 8.25 (d, J = 7.9 Hz, 2H), 9.18 (d, JP-H = 2.9 Hz, 8H);
13C NMR (100 MHz, CD3OD): ¤ 14.56, 26.50, 60.86, 61.68
(d, JP-C = 14.6 Hz), 68.26 (d, JP-C = 19.0 Hz), 68.78, 117.61,
127.97, 129.61, 130.92, 134.56 (d, JP-C = 5.4 Hz), 134.72,
136.66, 140.46, 142.82, 144.58, 145.93, 146.17; HRMS calcd
for C66H62N6O4P3+ [M3+]: 1033.4570, m/z 344.4851. Found:
m/z 344.4851.
61.82, 117.59, 128.05, 129.75, 131.09, 134.74 (d, JP-C
=
5.1 Hz), 134.93, 136.76, 140.63, 144.29, 162.54; HRMS calcd
for C68H66N6O2P3+ [M3+]: 1029.4985, m/z 343.1656. Found:
m/z 343.1658.
Bis[3-(1-methyl-4-pyridinio)propan-1-olato]tetraphenyl-
porphyrinatophosphorus(V) Chloride Diiodide (1f). Yield
1
95%. H NMR (400 MHz, CD3OD): ¤ ¹2.20 (dt, JP-H = 12.0
Hz, J = 6.0 Hz, 4H), ¹0.90-0.92 (m, 4H), 0.69 (t, J = 7.4
Hz, 4H), 4.07 (s, 6H), 6.44 (d, J = 6.6 Hz, 4H), 7.84-7.89
(m, 12H), 8.06-8.08 (m, 8H), 8.19 (d, J = 6.6 Hz, 4H), 9.22
(d, JP-H = 2.9 Hz, 8H); 13C NMR (100 MHz, CD3OD): ¤ 27.89
(d, JP-C = 16.8 Hz), 31.20, 47.99, 61.57 (d, JP-C = 13.8 Hz),
117.52, 127.77, 129.74, 131.03, 134.73 (d, JP-C = 5.0 Hz),
134.94, 136.69, 140.55, 144.99, 162.21; HRMS calcd for
C62H54N6O2P3+ [M3+]: 945.4046, m/z 315.1343. Found: m/z
315.1343.
Measurement of Water Solubility. The solubility of 1 in
water, defined as the saturated concentration (CW), was deter-
mined using the following method.26 Compound 1 (10 mg) was
suspended in pure water (40 ¯L) and left to stand for 3 days.
A given volume of supernatant solution was then moved to
another vessel and diluted with MeOH to measure the absorp-
tion spectrum of the solution. The CW was calculated using the
absorbance and the ¾ value of the Soret band in MeOH.
Measurement of the Absorption Spectra of 1 in the
Preparation of Bis[3-(4-pyridyl)propan-1-olato]tetra-
phenylporphyrinatophosphorus(V) Chloride (2d).
An
MeCN (30 mL) solution of 2a (300 mg) was heated with
3-(4-pyridyl)-1-propanol (5 mL) at reflux for approximately
24 h until the Soret band shifted from 435 to 428 nm. After
evaporation of the reaction mixture, the residue was dissolved
into CHCl3 (20 mL), and the solution was washed three times
with water (20 mL). The washed CHCl3 solution was then
reprecipitated with hexane (200 mL) to remove the residual
alkanol and other impurities. Thereafter, the resulting precip-
itate of 2d was purified by column chromatography on SiO2
using CHCl3-MeOH (100:1-30:1 v/v). The purity of 2d was
1
confirmed by H NMR analysis.
1
2d. Yield 47%. H NMR (400 MHz, CDCl3): ¤ ¹2.44 (dt,
J
P-H = 13.0 Hz, J = 6.4 Hz, 4H), ¹1.18-1.11 (m, 4H), 0.55 (t,
J = 6.8 Hz, 4H), 5.51 (d, J = 5.7 Hz, 4H), 7.79-7.83 (m, 12H),
Presence of HSA.
HSA (199 mg, MW 66500, Wako
7.86 (d, J = 5.7 Hz, 4H), 7.91-7.93 (m, 8H), 9.04 (d, JP-H
2.8 Hz, 8H).
=
Chemicals, Japan) was dissolved in water (10 mL) to give a
0.3 M aqueous HSA solution. An aqueous solution of each
compound 1 (0.3 mM, 100 ¯L), the aqueous HSA solution
(200-1000 ¯L), and a phosphate buffer (0.1 M, 1 mL) were
added to separate measuring flasks (10 mL) and then diluted
with water to provide phosphate buffers (10 mM, 10 mL,
pH 7.6) containing the tricationic phosphorus porphyrins 1
(3 ¯M) and HSA (0.25-60 ¯M). After the solutions were
maintained at 25 °C for 1 h, their absorption spectra were
Preparation of the Tricationic Phosphorus Porphyrins
1d, 1e, and 1f. Compound 2d (50 mg) was reacted with the
corresponding alkyl halide (1 mL) in MeCN (25 mL) at reflux
for approximately 24 h (Scheme 2). After evaporation, the
residue was purified by column chromatography on SiO2 using
CHCl3-MeOH (100:1-30:1 v/v). The purities of 1d-1f were
1
confirmed by H NMR analysis.

J. Matsumoto et al.
Bull. Chem. Soc. Jpn. Vol. 86, No. 11 (2013) 1247
14 B. Elsadek, F. Kratz, J. Controlled Release 2012, 157, 4.
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observed at the Soret band of 1 (approximately 430 nm; see SI,
Figure S1). The absorption spectra were normalized at 1.0.
Fluorescence Quenching. Aqueous solutions of 1 (3.0 ¯M,
10 mL) in phosphate buffers (10 mM, pH 7.6) containing HSA
(0.25-60 ¯M) were prepared and maintained at 25 °C for 1 h.
Fluorescence spectra of the solutions of 1 were measured in the
absence and presence of HSA when excited at 430 nm to record
the intensity at maximum wavelength (614-616 nm). More-
over, fluorescence quenching of 1 (3 ¯M) was performed using
L-tryptophan (0-10 mM) as the quencher. In addition, the
fluorescence quenching of HSA was performed with excita-
tion at 230 nm in a phosphate buffer (10 mM, pH 7.6, 10 mL)
containing HSA (10 ¯M) and 1 (1-5 ¯M).
20 J. M. Dąbrowski, M. M. Pereira, L. G. Arnaut, C. J. P.
Monteiro, A. F. Peixoto, A. Karocki, K. Urbańska, G. Stochel,
Photochem. Photobiol. 2007, 83, 897.
This work was supported by a Grant-in-Aid for Young
Scientists (B) (No. 24750131) from the Japan Society for the
Promotion of Science (JSPS).
21 X. Zhang, K. Sasaki, Y. Kuroda, Bull. Chem. Soc. Jpn.
2007, 80, 536.
22 R.-H. Jin, S. Aoki, K. Shima, J. Chem. Soc., Faraday
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Supporting Information
23 S. M. Andrade, S. M. B. Costa, Chem.®Eur. J. 2006, 12,
1046.
Correction of the fluorescence intensity of HSA for inner
filter effect. Derivation of eq 2 and eq 3. The changes in the
absorption spectra of 1 by the addition of HSA and fluores-
cence quenching of 1c by the addition of L-tryptophan. This
material is available free of charge on the web at http://www.
csj.jp/journals/bcsj/.
24 J. Matsumoto, T. Shiragami, M. Yasuda, Chem. Lett. 2008,
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