A new Phenol Red-modified porphyrin as efficient protein photocleaving agent

Article abstract of DOI:10.1039/c0cp00012d

Protein affinity is of importance for porphyrins in their application in photodynamic therapy (PDT). A new Phenol Red-modified porphyrin (R-TPP) was designed and synthesized to fully take advantage of the binding character of Phenol Red towards protein. Detailed comparisons of absorption spectra, fluorescence spectra, n-octanol/water partition coefficients, ¹O ₂ quantum yields, as well as protein photocleaving abilities between R-TPP and its parent porphyrin Br-TPP clearly demonstrate the benefits stemming from the modification of Phenol Red. On one hand, the presence of Phenol Red moiety greatly enhances the binding affinity of R-TPP towards model proteins (bovine serum albumin and hen egg lysozyme), and therefore improves the availability of ¹O₂. On the other hand, the presence of Phenol Red moiety provides R-TPP with amphiphilic character, and therefore restricts aggregation and favors the generation of ¹O₂. As a result, R-TPP photocleaves proteins efficiently, showing promising application potential in PDT.

Full text of DOI:10.1039/c0cp00012d

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www.rsc.org/pccp | Physical Chemistry Chemical Physics
A new Phenol Red-modified porphyrin as efficient protein photocleaving
agentw
Guo-Yu Jiang,ab Wan-Hua Lei,a Qian-Xiong Zhou,ab Yuan-Jun Hou,a
Xue-Song Wang*a and Bao-Wen Zhang*a
Received 24th March 2010, Accepted 24th June 2010
DOI: 10.1039/c0cp00012d
Protein affinity is of importance for porphyrins in their application in photodynamic therapy
(PDT). A new Phenol Red-modified porphyrin (R-TPP) was designed and synthesized to fully
take advantage of the binding character of Phenol Red towards protein. Detailed comparisons of
1
absorption spectra, fluorescence spectra, n-octanol/water partition coefficients, O2 quantum
yields, as well as protein photocleaving abilities between R-TPP and its parent porphyrin
Br-TPP clearly demonstrate the benefits stemming from the modification of Phenol Red. On one
hand, the presence of Phenol Red moiety greatly enhances the binding affinity of R-TPP towards
model proteins (bovine serum albumin and hen egg lysozyme), and therefore improves the
1
availability of O2. On the other hand, the presence of Phenol Red moiety provides R-TPP
1
with amphiphilic character, and therefore restricts aggregation and favors the generation of O2.
As a result, R-TPP photocleaves proteins efficiently, showing promising application potential
in PDT.
protein binding capability may be useful for structure–activity
Introduction
studies of proteins by spatio-temporal control of protein
cleavage.7
Photodynamic therapy (PDT) is a dual-selective medicinal
treatment, which combines light and a photosensitizer to bring
about cytotoxic effects to cancer cells.1 Among the different
PDT agents inspired extensive studies on the interactions
The important role of protein-binding in porphyrin-based
between porphyrins and proteins,8 mainly bovine serum
types of photosensitizers being used in PDT, porphyrins are
the most extensively studied, and several of them have gained
albumin (BSA), a serum protein that not only is the most
approval for clinical use.1 The porphyrin-based PDT mainly
common model protein but also has been utilized in targeting
relies on the singlet oxygen (1O2) production. O2 is a highly
1
delivery of phototherapeutic sensitizers.9 However, just
reactive species, however, has a very short lifetime in biological
recently, efforts are emerging to pursue novel porphyrins with
high protein affinity by intended structure design.10 For
example, Anzenbacher10a introduced phosphonate groups to
modulate the binding of the corresponding porphyrins to
BSA. Yano10b found that the binding numbers of the glyco-
conjugated porphyrins to BSA depended on the sugar moieties.
Only Toshima10c clearly disclose, for the first time, that
5,10,15,20-tetra(4-hydroxyphenyl) porphyrin effectively and
selectively degraded the target transcription factor, human
estrogen receptor-a (hER-a), upon visible light irradiation,
due to the similarity of its structure to an estradiol, which has
high affinity for hER.
systems (o0.04 ms) and therefore, a very limited diffusion
range in tumor cells (o0.02 mm),2 implying the importance of
the binding ability of porphyrins towards 1O2 biotarget.
Intracellular proteins are expected to be the major targets of
1O2, due to their abundance within cells (ca. 70% of the dry
mass of most cells) and their high reaction rate constants with
1O2.3 The oxidative damage to proteins can regulate cellular
signaling events, including apoptosis.4 In this regard, protein-
binding ability is of importance in development of new
porphyrins for PDT application. On the other hand,
serum albumin is known to be the most abundant circulatory
protein and can accumulate in malignant and inflamed tissue
due to a leaky capillary combined with an absence or defective
lymphatic drainage system and thus is emerging as a versatile
targeting carrier for drugs, including PDT agents.5,6 Accordingly,
protein affinity may also benefit tumor targeting of porphyrins
by aiding their coupling to either endogenous or exogenous
serum albumin. Additionally, porphyrins possessing
Phenol Red is a commonly used pH indicator which belongs
to the well known triarylmethane dyes. It is used in biology to
estimate the physiological function of the kidney and for
colorimetric or spectrophotometric determination of blood
or plasma pHs.11 It is also used as a pH indicator in tissue
culture media. Phenol Red was known to be able to bind to
BSA, with a binding constant of 1.1 ꢀ 105 Mꢁ1 12
.
Taking
advantage of the protein-binding character of Phenol Red,
herein, we designed and synthesized a porphyrin-Phenol Red
conjugate (R-TPP, Scheme 1), expecting that the Phenol
Red moiety may enhance the binding of the porphyrin with
proteins. UV-visible absorption, fluorescence emission,
time-resolved absorption spectra, and protein photocleavage
measurements indeed show an improved protein affinity of
a Key Laboratory of Photochemical Conversion and Optoelectronic
Materials, Technical Institute of Physics and Chemistry,
Chinese Academy of Sciences, Beijing 100190, P. R. China.
E-mail: xswang@mail.ipc.ac.cn, g203@mail.ipc.ac.cn
b Graduate School of Chinese Academy of Sciences, Beijing 100049
w Electronic supplementary information (ESI) available: NMR and
HRMS spectra. See DOI: 10.1039/c0cp00012d
c
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Results and disscussion
Synthesis of a Phenol Red modified porphyrin (R-TPP)
HO-TPP was prepared by condensation of pyrrole, benzalde-
hyde and 4-hydroxybenzaldehyde in refluxing propanoic acid.
Br-TPP was prepared from the reaction of HO-TPP and 1,4-
dibromobutane in the presence of potassium carbonate.
R-TPP was prepared from Br-TPP and Phenol Red in a
similar procedure, using potassium hydroxide instead of
potassium carbonate (Scheme 1).
Photophysical and photochemical properties of the examined
porphyrins
Fig. 1a shows the absorption and emission spectra (lex = 415 nm)
of the two porphyrins, Br-TPP and R-TPP, in acetonitrile and
Table 1 lists the corresponding spectra data. The spectra data
of the two porphyrins in CH2Cl2 are also included in Table 1.
Both Br-TPP and R-TPP show typical UV-Vis spectra of free
base porphyrins, with one intense peak (Soret band) at ca. 415 nm
and four vibronic peaks (Q bands) at ca. 513, 547, 590
and 645 nm, respectively. The emission spectra of the two
porphyrins in acetonitrile are quite similar too, showing the
fine structures peaked at about 660 nm and 720 nm. The
fluorescence quantum yields of Br-TPP and R-TPP in the three
examined solvents were measured using TMPyP as reference,
whose fluorescence quantum yield was reported to be 0.017 in
PBS.13 The high extent of similarity in absorption spectra,
emission spectra, as well as fluorescence quantum yields of
Br-TPP and R-TPP indicates the lack of intramolecular inter-
actions between porphyrin and Phenol Red moieties in both
ground and singlet excited state of R-TPP.
Scheme 1 Synthetic route of R-TPP.
R-TPP towards two model proteins, both BSA and hen egg
lysozyme (Lyso). As a result, R-TPP can effectively photo-
cleave BSA and Lyso.
Since the application of the photodynamic properties of
porphyrins is in aqueous solutions, the photophysical and
photochemical properties of Br-TPP and R-TPP were also
compared in PBS. Due to the poor solubility of Br-TPP in
aqueous solution, a PBS/DMSO (3 : 1 in volume ratio) mixed
solution was used in our studies. The absorption and emission
spectra (lex = 420 nm) of Br-TPP and R-TPP in aqueous
solutions are shown in Fig. 1b. Obviously, the Soret bands of
both porphyrins in PBS became red-shifted and much broader
compared to those in acetonitrile (Table 1), which implies the
aggregation of the two porphyrins in aqueous solutions. For
Br-TPP this broadening is more remarkable, in line with its
highly hydrophobic character. The n-octanol/water partition
coefficients of Br-TPP and R-TPP were measured to be 2.17
and 0.12, respectively (Table 2), fully demonstrating the
capability of the Phenol Red moiety in making a hydrophobic
photosensitizer become an amphiphilic. It is well accepted
that amphiphilicity is of importance for an ideal PDT photo-
sensitizer.14 The aggregation behavior of Br-TPP and R-TPP
may also partly account for their much lower fluorescence
quantum yields in PBS than in acetonitrile (Table 1).
The 1O2 production abilities of the two porphyrins in
acetonitrile were evaluated by measuring the degradation rate
of 1,3-diphenylisobenzofuran (DPBF) (Scheme 2) by 1O2
generated upon photoirradiation and using 5,10,15,20-tetra-
phenyl porphyrin (TPP) as reference (Fig. 2a, Table 2). The
1O2 quantum yield of R-TPP is as high as that of Br-TPP,
Fig. 1 The absorption and emission spectra (lex = 415 nm in
CH3CN, and lex = 420 nm in PBS/DMSO) of Br-TPP (solid lines)
and R-TPP (dotted lines) in CH3CN (a) and in PBS/DMSO (3 : 1 in
volume ratio) (b). Insets show the expanded spectra of the Q-bands.
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Table 1 Photophysical properties of Br-TPP and R-TPP in CH2Cl2, CH3CN (5 mM) and in aqueous solutions (10 mM)
l
max/nm (e ꢀ 10ꢁ3/Mꢁ1 cmꢁ1
)
Solvent
CH2Cl2
Soret band
Q bands
lem/nm
Fluorescence quantum yield
0.023
Br-TPP
418.0
(265.0)
414.0
(343.4)
429.0
(80.0)
418.0
(288.2)
414.5
515.5
(10.6)
513.0
(23.0)
520.0
(15.4)
515.5
(11.4)
512.5
(21.8)
519.5
(10.7)
550.5
(5.2)
547.5
(16.2)
555.0
(8.4)
550.5
(5.8)
547.5
(14.8)
554.5
(2.5)
590.5
(3.4)
589.5
(13.6)
593.5
(5.0)
592.0
(3.4)
590.0
(12.0)
592.0
(1.5)
645.5
(2.8)
644.5
(13.6)
650.0
(3.4)
646.0
(2.6)
645.0
(11.4)
648.0
(1.2)
659.6
658.0
663.2
658.6
657.2
662.4
720.4
719.6
724.2
721.0
721.2
724.0
CH3CN
PBSa
0.033
0.0085
0.021
0.034
0.0095
CH2Cl2
CH3CN
PBSa
R-TPP
(365.4)
417.5
(60.0)
a
Due to the poor solubility of Br-TPP in aqueous solution, a PBS/DMSO (3 : 1 in volume ratio) was used.
Table 2 Singlet oxygen quantum yields (FD) in CH3CN, n-octanol/
water partition coefficients (log P) and triplet excited state lifetimes of
Br-TPP and R-TPP
Br-TPP
R-TPP
a
FD
0.59
2.17
4.2
0.59
0.12
3.8
log Pb
tTc/ms
a
b
TPP in acetonitrile was used as reference (0.60). SD o 5%.
c
In CH3CN.
suggesting that no intramolecular interactions between
porphyrin and Phenol Red moieties in the triplet excited state
of R-TPP. The similar triplet excited state lifetimes of Br-TPP
and R-TPP also support this conclusion (Table 2).
The singlet oxygen production abilities of Br-TPP and
R-TPP were also compared in PBS by measuring the degradation
rate of 9,10-anthracenediylbis(methylene)dimalonic acid
(AMDA) (Scheme 3, Fig. 2b). Though both porphyrins
exhibit the same 1O2 quantum yield in CH3CN, in aqueous
1
solutions, Br-TPP showed much lower O2 generation ability
than R-TPP, most likely originating from its lower water
solubility and thus higher aggregation extent.
The spin trapping technique was also used to confirm the
1
production of O2 and other reactive oxygen species, such as
hydroxyl radical (Fig. 3). Upon irradiation of oxygen-saturated
PBS (5 mM, pH = 7.0)/DMSO (3 : 1 in volume ratio) of
R-TPP (1mM) and 2,2,6,6-tetramethyl-4-piperidone (TEMP)
(50 mM) with 532 nm laser, a three-line EPR spectrum with
equal intensity and hyperfine coupling constant of aN = 16.0 G
was observed which could be assigned to the TEMPO
(TEMP-1O2 adduct) signal. However, whenꢂ 5,5-dimethyl-1-
pyrroline-N-oxide (DMPO) was used as the OH trap in the
same experimental conditions, no ESR signal was observed
Fig. 2 Degradation of DPBF (a) and AMDA (b) by 1O2 generated by
Br-TPP and R-TPP upon irradiation.
after irradiation, indicating that R-TPP did not have the
ꢂ
ability to generate OH.
Protein photocleavage
Protein electrophoresis experiments were carried out to
investigate the protein photocleaving abilities of Br-TPP and
R-TPP, using both BSA and Lyso as model proteins (Fig. 4).
Br-TPP exhibited very weak photocleavage ability towards
both BSA and Lyso (lane 2). In stark contrast, R-TPP led to
significant photocleavage of BSA and Lyso at the concentration
of 25 mM (lane 4). The photocleavage was either not observed
or greatly diminished in the dark or in the presence of NaN3,
Scheme 2 Reaction of DPBF with singlet oxygen (1O2).
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targeting property of Phenol Red may also play an important
role in the protein photocleavage by R-TPP. To examine this
possibility, control experiment as shown in lane 5 was carried
out. The addition of excess amount of Phenol Red can greatly
inhibit the photocleavage of both proteins. This finding may
be reasonably explained by the competitive binding of R-TPP
and Phenol Red towards BSA or Lyso, as a result, the
displacement of the bound R-TPP by Phenol Red decreases
1
the availability of O2 in light of the short lifetime character
1
of O2.
Scheme
acetonitrile.
3
Reaction of AMDA with singlet oxygen (1O2) in
Binding of porphyrins towards BSA or Lyso
In order to disclose the role of Phenol Red in the much
improved protein photocleavage activity of R-TPP, we
investigated the binding properties of Br-TPP and R-TPP
towards both BSA and Lyso.
The addition of increasing aliquots of BSA or Lyso to the
PBS solutions of the two porphyrins resulted in obvious
changes in decrease in the emission intensity (Fig. 5a). The
addition of Lyso into Br-TPP gave similar results, i.e. a 4.5 nm
red shift and a 16% hypochromic effect in the Soret band and
a 35% reduction in the emission intensity (Fig. 5c). In the case
of R-TPP, no significant red shift or hypochromicity in the
Soret band was observed upon the addition of either BSA or
Lyso, while the fluorescence intensity underwent a remarkable
increase rather than decrease, 95% increase for BSA and 30%
increase for Lyso (Fig. 5b and d). The remarkably varied water
solubilities of Br-TPP and R-TPP may be responsible for the
markedly different effects of the proteins. Due to the highly
hydrophobic character of Br-TPP, the presence of protein may
further promote its aggregation inside the hydrophobic pockets,
i.e., the hydrophobic pocket of the protein may trap two or
more Br-TPP molecules simultaneously, thus leading to the
red shift and hypochromic effect of the Soret band and
fluorescence quenching. This will not occur for R-TPP due
to its amphiphilic feature. Contrarily, the binding of R-TPP
towards proteins may favor the monomerization of R-TPP,
and enhance the fluorescence intensity. The enhancement of
the fluorescence intensity of water soluble porphyrins
upon addition of BSA were also found in the literature.15
Consequently, the binding of Br-TPP towards proteins promotes
Fig. 3 EPR signal of R-TPP (1 mM) in PBS (5 mM, pH = 7.0)/
DMSO (3 : 1 in volume ratio) upon irradiation with 532 nm laser: (a) in
the presence of 50 mM TEMP; and (b) in the presence of 50 mM
DMPO.
Fig. 4 Photo-cleavage of BSA (a, 2 mM) and Lyso (b, 5 mM) in PBS
(5 mM, pH = 7.0)/DMSO (3 : 1 in volume ratio) upon 3 h of irradiation
of a broadband light (>550 nm) and analyzed by 8% SDS-PAGE:
lane 1, protein alone; lane 2, protein + Br-TPP; lane 3, protein +
Br-TPP + Phenol Red (25 mM); lane 4, protein + R-TPP; lane 5,
protein + R-TPP + excess Phenol Red; lane 6, protein + R-TPP,
but without light; lane 7, protein + R-TPP + NaN3 (0.1 M). The
concentrations of Br-TPP and R-TPP were fixed at 25 mM.
1
aggregation and has a negative effect for O2 generation and
protein cleavage, while the binding of R-TPP towards proteins
restricts aggregation and exhibits a positive effect for 1O2
generation and protein cleavage.
The binding of porphyrins towards proteins not only
influences the photophysical properties of porphyrins as
mentioned above, but also varies the photophysical characters
of proteins, e.g. quenches the fluorescence emission of
proteins, from which both the binding mode and binding
strength can be deduced. As shown in Fig. 6, the presence of
R-TPP quenched the intrinsic fluorescence of both BSA and
Lyso significantly, while Br-TPP quenched the protein
fluorescence with a lower efficiency.
indicative of the involvement of 1O2 (lane 6 and 7). The
presence of 25 mM of Phenol Red did not improve the
photocleavage ability of Br-TPP (lane 3), indicating that
Phenol Red itself cannot result in protein cleavage under
experimental conditions and the attachment of Phenol Red
onto porphyrin is critical for the observation of improved
photocleavage. R-TPP possesses higher 1O2 generation
efficiency in aqueous solutions than Br-TPP, undoubtedly
facilitating the photocleavage of proteins. Additionally, the
The fluorescence quenching rate constants can be calculated
based on the Stern–Volmer equation (eqn (1)),16 where kq, t0,
and [Q] are the fluorescence quenching rate constant, the
fluorescence lifetime of protein in the absence of the quencher,
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Fig. 5 Absorption and emission spectra of Br-TPP and R-TPP (10 mM) in PBS/DMSO (3 : 1 in v/v) upon addition of increasing amounts of
protein. a: Br-TPP + BSA; b: R-TPP + BSA; c: Br-TPP + Lyso; d: R-TPP + Lyso.
Fig. 6 Fluorescence quenching (lex = 280 nm) of BSA (2 mM) or Lyso (2 mM) by porphyrins (0–5 mM). (a) BSA + Br-TPP; (b) BSA + R-TPP;
(c) Lyso + Br-TPP; (d) Lyso + R-TPP.
and the concentration of the quencher, respectively. F0 and F
are the fluorescence intensities in the absence and presence of
the quencher, respectively.
i.e. the binding of Br-TPP and R-TPP towards BSA, can be
concluded.
ꢀ
ꢁ
ꢀ
ꢁ
n
F0 ꢁ F
F ꢁ F
½Porphyrinꢃ
¼
ð2Þ
Kd
F0/F = 1 + kqt0[Q]
(1)
1
Using the corrected fluorescence intensity data and the
lifetime of 10ꢁ8 s for BSA,17 the quenching rate constants of
Br-TPP and R-TPP are calculated to be 3.36 ꢀ 1012 and 1.43 ꢀ
The binding constants (Kb) can be obtained according to
eqn (2),8g in which Kd is the dissociation constant (1/Kb), n is
the number of porphyrin molecules bound to each protein
molecule, F0 and F are the corrected fluorescence of protein in
the absence or presence of porphyrin, FN is the fluorescence at
infinite concentration of porphyrin (calculated as the intercept of
1/(F0 ꢁ F) vs. 1/[Porphyrin]). The plot of log [(F0 ꢁ F)/(F ꢁ FN)]
versus log[porphyrin] yields the number of binding sites and the
1013
M
ꢁ1 sꢁ1, respectively, from the slops of the corresponding
Stern–Volmer plots (Fig. 7). Since the quenching rate
constants are 2–3 orders of magnitude higher than the collision
17
quenching constant (2.0 ꢀ 1010 Mꢁ1 sꢁ1
)
for various
quenchers with biomolecules, a static quenching mechanism,
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1
improves the availability of O2 towards proteins, the major
biotarget of 1O2 in PDT. Secondly, the presence of Phenol Red
moiety renders R-TPP amphiphilic character, and therefore
1
restricts aggregation and favors O2 generation. As a result,
R-TPP can photocleave proteins efficiently, showing
promising application potential in PDT.
Experimental
General remarks
1H/13C NMR spectra were obtained on a Bruker DMX-400 MHz
spectrophotometer. High resolution mass spectra were
obtained on a Bruker APEX IV FT_MS. UV-Vis absorption
spectra and fluorescence emission spectra were recorded on a
Shimadzu UV-1601PC spectrophotometer and a Hitachi
F-4500 fluorescence spectrophotometer, respectively.
Fig. 7 Stern–Volmer plot of the BSA fluorescence quenching by
Br-TPP (square) and R-TPP (circle).
value of Kb (Fig. 8 and Table 3). Clearly, the attachment of
Phenol Red moiety greatly enhances the binding affinity of
the corresponding porphyrin, R-TPP, than its parent porphyrin,
Br-TPP.
Materials
Pyrrole, benzaldehyde, 4-hydroxybenzaldehyde, 1,4-dibromo-
butane, Phenol Red, 1,3-diphenylisobenzofuran (DPBF),
9,10-anthracenediyl-bis(methylene)dimalonic acid (AMDA),
sodium azide (NaN3), 5,5-dimethyl-1-pyrroline-N-oxide
(DMPO), 2,2,6,6-tetramethyl-4-piperidone (TEMP) and
trishydroxymethylaminomethane (Tris base) were purchased
from Sigma-Aldrich. All other chemicals of analytical grade
were from Beijing Chemical Plant. Water is freshly distilled
twice before use. Stock solutions of photosensitizers were
dissolved in DMSO and kept in freezer (4 1C) until use. BSA
and Lyso were purchased from Sigma Aldrich.
Conclusions
In summary, we synthesized a Phenol Red modified porphyrin,
R-TPP. Compared to its parent porphyrin, Br-TPP, R-TPP
exhibits two distinct advantages. At first, the presence of
Phenol Red moiety greatly enhances the binding affinity of
R-TPP towards proteins (BSA and Lyso), and therefore
Synthesis of porphyrins
Synthetic route of R-TPP is shown in Scheme 1.
5-(40-Hydroxyphenyl)-10,15,20-triphenylporphyrin (HO-TPP).
HO-TPP was synthesized according to a reported method,18
by reacting 4-hydroxybenzaldehyde (1.83 g, 15 mM), benzal-
dehyde (4.57 mL, 45 mM), and pyrrole (4.15 mL, 60 mM), in
refluxing propanoic acid. The crude product was purified on a
silica gel column with chloroform as eluent. Yield 10%.
1H-NMR (400 MHz in CDCl3), d ppm: 8.88 (m, 8 H), 8.23
(m, 6 H), 7.78 (m, 9 H), 8.07 (d, 2 H, J = 8.28 Hz), 7.17
(d, 2 H, J = 8.08 Hz), ꢁ2.75 (s, 2 H).
5-(40-Bromobutoxyphenyl)-10,15,20-triphenylporphyrin (Br- TPP).
Br-TPP was synthesized by reacting HO-TPP with an excess of
1,4-dibromobutane in the presence of potassium carbonate at
150 1C for 24 h. Crude Br-TPP was purified on a silica gel
column with chloroform–ethanol (100 : 2 in v/v) as eluent.
Yield 85%. 1H NMR (400 MHz in CDCl3), d ppm: 8.87
(m, 8 H), 8.22 (m, 6 H), 7.78 (m, 9 H), 8.13 (d, 2 H, J =
8.40 Hz), 7.28 (d, 2 H, J = 8.88 Hz), 4.31 (t, 2 H, J = 11.80 Hz),
3.65 (t, 2 H, J = 13.00 Hz), 2.26 (d, 2 H, J = 7.68 Hz), 2.17
(d, 2 H, J = 8.04 Hz), ꢁ2.75 (s, 2 H).
Synthesis of the porphyrin-Phenol Red conjugate (R-TPP).
R-TPP was synthesized by reacting Br-TPP and an excess of
Phenol Red in DMF in the presence of potassium hydroxide at
150 1C for 24 h. Crude R-TPP was purified on a silica gel
column with chloroform–ethanol (100 : 10, v/v) as eluent.
Fig. 8 Double logarithmic plots of BSA (a) and Lyso (b) fluorescence
quenching by Br-TPP (square) and R-TPP (circle).
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Table 3 Binding constants of Br-TPP and R-TPP towards BSA and Lyso
Br-TPP + BSA
R-TPP + BSA
Br-TPP + Lyso
R-TPP + Lyso
n
0.99
3.32 ꢀ 104
0.99
8.49 ꢀ 104
1.40
2.11 ꢀ 104
1.59
1.40 ꢀ 105
Kb/Mꢁ1
Yield 15%. 1H NMR (400 MHz in CDCl3), d ppm: 8.70
(m, 8H), 8.03 (m, 6H), 7.55–7.42 (m, 9 H), 7.79 (d, 2 H,
J = 6.56 Hz), 6.99 (d, 2 H, J = 6.96 Hz), 7.08–6.52 (m, 12 H),
3.65 (s, br, 4 H), 1.26 (s, br, 4 H), ꢁ2.75 (s, 2 H). 13C NMR
(400 MHz in CDCl3), d ppm: 188.2, 161.1, 158.7, 142.2, 135.6,
134.6, 131.5, 131.1, 129.9, 129.4, 127.7, 126.7, 120.2, 114.4,
112.7, 67.7, 67.5, 29.9, 26.1. HRMS, m/z: calculated for
(M+H) = 1039.3524, found: 1039.3486.
Triplet excited state lifetime
The lifetime of the triplet excited-state was measured using an
Edinburgh LP920 laser flash photolysis spectrometer. The
third harmonic output (l = 355 nm, 5 ns of fwhm) of a
Nd:YAG laser was used as the excitation source, and a pulsed
flashlamp (Xe 900) was used as the analyzing light. All samples
were degassed with argon for 20 min before each
measurement.
Fluorescence quantum yield
n-Octanol/water partition coefficients (log P)
The fluorescence quantum yields of the synthesized porphyrins,
Br-TPP and R-TPP, were evaluated using 5,10,15,20-tetrakis-
(4-N-methylpyridyl)porphyrin (TMPyP) as reference, whose
fluorescence quantum yield was reported to be 0.017 in PBS.13
In all cases, the absorbance of the sample and the reference
solutions were kept below 0.1 at the excitation wavelength to
minimize inner filter effects. Eqn (3) was applied to calculate
the fluorescence quantum yields of the porphyrins, where Q, I,
OD, n and QR, IR, ODR, nR, are the fluorescence quantum
yields, the area of the emission spectra, the optical densities
and the refractive indexes of the solutions for the examined
porphyrin and the reference, respectively.
The n-octanol/water partition coefficients were measured at
room temperature following a reported method.21 Typically,
solutions of each porphyrin (100 mM) in equal volumes of
5 mM PBS, pH 7.0 (1 mL) and n-octanol (1 mL) were mixed
and sonicated for 30 min. After separation by centrifugation,
the amounts of porphyrin in each phase were determined
after dilution with DMSO by fluorescence intensity at 658 nm
(420 nm excitation) on a F-4500 Fluorescence Spectro-
photometer (Hitachi, Japan) and the results were the average
of three independent measurements.
Protein photo-degradation
I ODR n2
An Oriel 91192 Solar simulator was used as the light source for
the irradiation of the proteins and a 550 nm cut-off filter was
used to remove the short wavelength light. Each protein
sample (2 mM for BSA and 5 mM for Lyso) in a total volume
of 200 mL of 5 mM PBS (pH = 7.0) containing 25% DMSO
was irradiated for 3 h. Porphyrin concentrations were fixed at
25 mM.
Q ¼ QR
ð3Þ
nR2
IR OD
Singlet oxygen (1O2) quantum yield
The reaction of 1O2 with DPBF19a (Scheme 2) was adopted to
measure the 1O2 quantum yields of the synthesized porphyrins,
using tetraphenylporphyrin (TPP) as the reference, whose 1O2
quantum yield was measured19b to be 0.60 in air- saturated
CH3CN. A series of 2 mL of air-saturated acetonitrile
solutions containing DPBF and porphyrins, of which the
absorbance at 512 nm originating from the absorption of the
examined porphyrin was adjusted to the same (OD512nm = 0.15),
were separately charged into an opened 1 cm path fluorescence
cuvette and illuminated with the light of 512 nm (obtained
from a Hitachi F-4500 fluorescence spectrophotometer).
The consumption of DPBF was followed by monitoring its
fluorescence intensity decrease at the emission maximum
(lex = 440 nm, lem = 479 nm).
Protein electrophoresis
SDS/polyacrylamide gel electrophoresis (SDS-PAGE) experiments
were performed as reported.22 After addition of 5 mL loading
buffer, 20 mL of irradiated protein samples was steamed in
boiling water for 2 min. Gel electrophoresis was run by
applying 100 V for about 1.5 h. The gels were then stained
with Coomassie Brilliant Blue R-250 solution for 2 h, distained
in a mixture of acetic acid, ethanol and water (10 : 5 : 85 in
volume ratio) overnight, washed with water, and then scanned
on a CanoScan LiDE 700F scanner and processed using
Adobe Photoshop software.
The reaction of 1O2 with 9,10-anthracenediylbis(methyl-
ene)dimalonic acid (AMDA)20 (Scheme 3) was adopted to
measure the relative 1O2 quantum yields of the synthesized
porphyrins in PBS (5 mM, pH = 7.0) containing 25% DMSO.
The process was similar to DPBF method, except that a
medium pressure sodium lamp (500 W, with cut off glass filter
l > 550 nm) in a ‘‘merry-go-round’’ apparatus was used as
irradiation light source and the consumption of AMDA was
followed by monitoring its absorbance decrease at 380 nm.
EPR measurement
The EPR spectra were recorded at room temperature on a
Bruker-ESP-300E spectrometer at 9.8 GHz, X-band with
100 Hz field modulation. Samples were injected quantitatively
into quartz capillaries and purged with oxygen for 15 min in
dark, respectively, and illuminated in the cavity of the EPR
spectrometer with a Nd : YAG laser at 532 nm (5–6 ns of pulse
width, 10 Hz of repetition frequency, 30 mJ/pulse energy).
c
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Absorption and emission measurements of the bound porphyrins
7 (a) C. V. Kumar and A. Buranaprapuk, J. Am. Chem. Soc., 1999,
121, 4262–4270; (b) C. V. Kumar, A. Buranaprapuk, H. C. Sze,
S. Jockusch and N. J. Turro, Proc. Natl. Acad. Sci. U. S. A., 2002,
99, 5810–5815; (c) A. Suzuki, M. Hasegawa, M. Ishii,
S. Matdumura and K. Toshima, Bioorg. Med. Chem. Lett., 2005,
15, 4624–4627; (d) A. Suzuki, K. Tsumura, T. Tsuzuki,
S. Matsumura and K. Toshima, Chem. Commun., 2007,
4260–4262.
A 2 mL of porphyrin solution (10 mM) in PBS/DMSO (3 : 1 in
volume ratio) was titrated with a protein solution (2 mM in
5 mM PBS, pH = 7.0) and the absorption and fluorescence
spectra were recorded after each addition of the protein.
Protein fluorescence quenching
8 (a) M. Beltramini, P. A. Firey, M. A. J. Rodgers and G. Jori,
Biochemistry, 1987, 26, 6852–6858; (b) B. M. Aveline, T. Hasan
and R. W. Redmond, J. Photochem. Photobiol., B, 1995, 30,
161–169; (c) I. M. Borissevitch, T. T. Tominaga and
C. C. Schmitt, J. Photochem. Photobiol., A, 1998, 114, 201–207;
(d) S. M. Andrade and S. M. B. Costa, Biophys. J., 2002, 82,
1607–1619; (e) N. H. Karapetyan and V. N. Masakyan, Russ. J.
Bioorg. Chem., 2004, 30, 172–177; (f) Y.-B. Yin, Y.-N. Wang and
J.-B. Ma, Spectrochim. Acta, Part A, 2006, 64, 1032–1038;
(g) F. Tian, E. M. Johnson, M. Zammarripa, S. Samsone and
L. Brancaleon, Biomacromolecules, 2007, 8, 3767–3778;
(h) X.-L. Lu, J.-J. Fan, Y. Liu and A.-X. Hou, J. Mol. Struct.,
A 2 mL of BSA or Lyso solution (2 mM in PBS) was titrated by
a porphyrin solution (10ꢁ4 M in DMSO) and the protein
fluorescence was recorded by excitation at 280 nm. The raw
fluorescence intensities, the integral of the emission spectrum
in the range of 290–420 nm, were corrected with eqn (4) by
considering the inner filter effect of porphyrin,8g where Aex and
Aem are the optical density of porphyrin at the excitation
(280 nm) and emission wavelength maximum (345 nm) of
protein and Fraw is the raw emission intensity of the protein.
Binding constants were calculated according to the corrected
fluorescence quenching data.
2009, 934, 1–8; (i) E. Alarcon, A. M. Edwards, A. Aspee,
C. D. Borsarelli and E. A. Lissi, Photochem. Photobiol. Sci.,
2009, 8, 933–943.
´
´
9 (a) N. Brasseur, R. Langlois, C. L. Madeleine, R. Ouellet and
J. E. van Lie, Photochem. Photobiol., 1999, 69, 345–352;
(b) W. M. Sharman, J. E. van Lie and C. M. Allen, Adv. Drug
Delivery Rev., 2004, 56, 53–76; (c) B. Biplab Bose and A. Dube,
J. Photochem. Photobiol., B, 2006, 85, 49–55.
ex+Aem)/2
Fcorr = 10(A
F
(4)
raw
10 (a) P. Kubat, K. Lang, Jr and P. Anzenbacher, Biochim. Biophys.
´
Acta, Gen. Subj., 2004, 1670, 40–48; (b) M. Obata, S. Hirohara,
K. Sharyo, H. Alitomo, K. Kajiwara, S. Ogata, M. Tanihara,
C. Ohtsuki and S. Yano, Biochim. Biophys. Acta, Gen. Subj., 2007,
1770, 1204–1211; (c) S. Tanimoto, S. Matsumura and K. Toshima,
Chem. Commun., 2008, 3678–3680.
Acknowledgements
We greatly appreciate the financial support from NNSFC
(20772133, 20873170) and CAS (KJCX2.YW.H08).
11 F. L. Rodkey, J. Biol. Chem., 1961, 236, 1589–1594.
12 F. L. Rodkey, Arch. Biochem. Biophys., 1961, 94, 38–47.
13 (a) S. Fery-Forgues and D. Lavabre, J. Chem. Educ., 1999, 76,
1260–1264; (b) E. Reddi, M. Ceccon, G. Valduga, G. Jori,
J. C. Bommer, F. Elisei, L. Latterini and U. Mazzucato, Photo-
chem. Photobiol., 2002, 75, 462–470.
14 (a) N. Cauchon, H.-J. Tian, R. Langlois, C. L. Madeleine,
S. Martin, H. Ali, D. Hunting and J. E. van Lie, Bioconjugate
Chem., 2005, 16, 80–89; (b) A. Sholto, S.-W. Lee, B. M. Hoffman,
A. G. M. Barrett and B. Ehrenberg, Photochem. Photobiol., 2008,
84, 764–773.
15 (a) I. E. Borissevitch, T. T. Tominaga, H. Imasato and M. Tabak,
J. Lumin., 1996, 69, 65–76; (b) F. Ricchelli, D. Stevanin and
G. Jori, Photochem. Photobiol., 1988, 48, 13–18.
16 B. Valeur, Molecular Fluorescence. Principles and Applications,
Wiley Press, NY, 2001, p. 84.
17 J. R. Lakowicz and G. Weber, Biochemistry, 1973, 12, 4161–4170.
18 F. D’Souza, G. R. Deviprasad, M. E. El-Khouly, M. Fujitsuka
and O. Ito, J. Am. Chem. Soc., 2001, 123, 5277–5284.
19 (a) R. H. Young, K. Wehrly and R. L. Martin, J. Am. Chem. Soc.,
1971, 93, 5774–5779; (b) F. Wilkinson, W. P. Helman and
A. B. Ross, J. Phys. Chem. Ref. Data, 1993, 22, 113–262.
20 N. A. Kuznetsova, N. S. Gretsova, O. A. Yuzhakova,
V. M. Negrimovskii, O. L. Kaliya and E. A. Luk’yanets, Russ. J.
Gen. Chem., 2001, 71, 36–41.
Notes and references
1 (a) E. D. Stemberg and D. Dolphin, Tetrahedron, 1998, 54,
4151–4202; (b) T. J. Dougherty, C. J. Gomer, B. W. Henderson,
G. Jori, D. Kessel, M. Korbelik, J. Moan and Q. Peng, J. Natl.
Cancer Inst., 1998, 90, 889–905; (c) M. R. Detty, S. L. Gibson and
S. Wagner, J. Med. Chem., 2004, 47, 3897–3915; (d) E. S. Nyman
and P. H. Hynninen, J. Photochem. Photobiol., B, 2004, 73, 1–28;
(e) A. E. O’Connor, W. M. Gallagher and A. T. Byme, Photochem.
Photobiol., 2009, 85, 1053–1074.
2 J. Moan and K. Berg, Photochem. Photobiol., 1991, 53, 549–553.
3 (a) M. J. Davies, Biochem. Biophys. Res. Commun., 2003, 305,
761–770; (b) M. J. Davies, Photochem. Photobiol. Sci., 2004, 3,
17–25; (c) M. Gracanin, C. L. Hawkins, D. I. Pattison and
M. J. Davies, Free Radical Biol. Med., 2009, 47, 92–102.
4 (a) B. Magi, A. Ettorre, S. Liberatori, L. Bini, M. Andreassi,
S. Frosali, P. Neri, V. Pallini and A. D. Stefano, Cell Death Differ.,
2004, 11, 842–852; (b) P. A. Tsaytler, M. C. O’Flaherty,
D. V. Sakharov, J. Krijgsveld and M. R. Egmond, J. Proteome
Res., 2008, 7, 3868–3878.
5 (a) F. Kratz, J. Controlled Release, 2008, 132, 171–183;
(b) M. J. Hawkins, P. Soon-Shiong and N. Desai, Adv. Drug
Delivery Rev., 2008, 60, 876–885.
6 R. K. Pandey, S. Constantine, T. Tsuchida, G. Zheng,
C. J. Medforth, M. Aoudia, A. N. Kozyrev, M. A. J. Rodgers,
H. Kato, K. M. Smith and T. J. Dougherty, J. Med. Chem., 1997,
40, 2770–2779.
21 M. Kepczyn
Photochem. Photobiol., 2002, 76, 127–134.
´
ski, R. P. Pandian, K. M. Smith and B. Ehrenberg,
22 H. Schagger and G. Von Jagow, Anal. Biochem., 1987, 166,
¨
368–379.
c
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