Electron transfer mediated decomposition of folic acid by photoexcited dimethoxophosphorus(V)porphyrin

Article abstract of DOI:10.1016/j.jphotochem.2015.11.028

A water soluble porphyrin, dimethoxophosphorus(V)tetraphenylporphyrin (MP(V)TPP) photosensitized folic acid decomposition in aqueous solution, resulting in the formation of a strongly fluorescent pteridine compound. The quantum yield of folic acid decomposition by photoexcited MP(V)TPP could be determined by a flurometry. A possible mechanism of the initial process of this photodecomposition is direct oxidation through an electron transfer from folic acid to the excited singlet state of MP(V)TPP. It is considered that this electron transfer proceeds as a diffusion-controlled reaction. The quantum yields of the electron transfer and the following process of folic acid decomposition could be determined. In deuterium oxide, folic acid decomposition through the photosensitized singlet oxygen generation by MP(V)TPP was also observed. A fluorometry based on the formation of a strong fluorescent compound through the oxidized decomposition of folic acid may be applied to evaluate the biomolecule damaging-activity of photosensitizers. In addition, a protocol of this assay is proposed. This simple photosensitized reaction in aqueous solution may be used for a first screening of a photosensitizer for phototherapy.

Full text of DOI:10.1016/j.jphotochem.2015.11.028

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Journal of Photochemistry and Photobiology A: Chemistry 318 (2016) 1–6
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology A:
Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
Electron transfer mediated decomposition of folic acid by photoexcited
dimethoxophosphorus(V)porphyrin
a
Kazutaka Hirakawaa,b, , Shu Morimoto
*
a
Applied Chemistry and Biochemical Engineering Course, Department of Engineering, Graduate School of Integrated Science and Technology, Shizuoka
University, Johoku 3-5-1, Naka-ku, Hamamatsu, Shizuoka 432-8561, Japan
b
Department of Optoelectronics and Nanostructure Science, Graduate School of Science and Technology, Shizuoka University, Johoku 3-5-1, Naka-ku,
Hamamatsu, Shizuoka 432-8561, Japan
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 22 October 2015
Received in revised form 27 November 2015
Accepted 30 November 2015
Available online 2 December 2015
A water soluble porphyrin, dimethoxophosphorus(V)tetraphenylporphyrin (MP(V)TPP) photosensitized
folic acid decomposition in aqueous solution, resulting in the formation of a strongly fluorescent
pteridine compound. The quantum yield of folic acid decomposition by photoexcited MP(V)TPP could be
determined by a flurometry. A possible mechanism of the initial process of this photodecomposition is
direct oxidation through an electron transfer from folic acid to the excited singlet state of MP(V)TPP. It is
considered that this electron transfer proceeds as a diffusion-controlled reaction. The quantum yields of
the electron transfer and the following process of folic acid decomposition could be determined. In
deuterium oxide, folic acid decomposition through the photosensitized singlet oxygen generation by MP
(V)TPP was also observed. A fluorometry based on the formation of a strong fluorescent compound
through the oxidized decomposition of folic acid may be applied to evaluate the biomolecule damaging-
activity of photosensitizers. In addition, a protocol of this assay is proposed. This simple photosensitized
reaction in aqueous solution may be used for a first screening of a photosensitizer for phototherapy.
ã 2015 Elsevier B.V. All rights reserved.
Keywords:
Folic acid
Phosphorus(V)porphyrin
Photosensitized reaction
Electron transfer
Fluorescence enhancement
1. Introduction
photosensitizer absorbing visible-light is relatively small and the
electron transfer-mediated oxidation of biomolecules is difficult.
Photochemical processes such as photosensitized singlet
oxygen (1O2) generation and photo-induced electron transfer are
important mechanisms of photodynamic therapy (PDT) of cancer
[1–3]. In general, PDT process can be explained by the 1O2
generation. Administered photosensitizers in tumor photosensi-
tize 1O2 generation, resulting in oxidative damage to biomacro-
molecules of cancer cells. However, the PDT effect by 1O2
generation is insufficient, because the oxygen concentration in
tumor is lower than that in normal cells [4–6]. Thus, we have
studied the development of photosensitizer, which can induce
biomolecule damage through photo-induced electron transfer
mechanism [7]. Many photosensitizers absorbing ultraviolet ray
can oxidize biomacromolecules, such as DNA, through the electron
transfer from the targeting molecule to photoexcited state of
photosensitizer [8]. However, the oxidative activity of
For PDT, porphyrins have been extensively studied as photo-
sensitizers [9–12]. Since porphyrins are visible light photosensi-
tizers, the electron transfer-mediated mechanism is not
appropriate. However, phosphorus(V) complex of porphyrins
demonstrate
a relatively strong oxidative activity in their
photoexcited state [7,8,12]. To evaluate the biomacromolecule
damaging activity of photosensitizers through electron transfer
mechanism, development of a simple analysis method is expected.
Folic acid (Fig. 1), a B vitamin, can be decomposed by oxidation
through photo-induced electron transfer [13–18]. Since the highest
occupied molecular orbital (HOMO) of folic acid is located on its
aminobenzoyl moiety, the electron transfer from this moiety to the
photoexcited pteridine moiety can occur [19]. The electron
transfer-mediated oxidation of aminobenzoyl moiety leads to
the decomposition of folic acid and the formation of strongly
fluorescent pteridine compounds [13–18,20,21]. Recently, we
reported the decomposition of folic acid through photo-induced
electron transfer by rhodamine-6G [22], a fluorescent dye, and the
resulting fluorescence enhancement. Although a reaction of PDT
occurs in the complex biological system, similar method may be
applied to evaluate the activity of PDT photosensitizers simply, for
* Corresponding author at: Applied Chemistry and Biochemical Engineering
Course, Department of Engineering, Graduate School of Integrated Science and
Technology, Shizuoka University, Johoku 3-5-1, Naka-ku, Hamamatsu, Shizuoka
432-8561, Japan. Fax: +81 53 478 1287.
E-mail address: hirakawa.kazutaka@shizuoka.ac.jp (K. Hirakawa).
http://dx.doi.org/10.1016/j.jphotochem.2015.11.028
1010-6030/ã 2015 Elsevier B.V. All rights reserved.

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K. Hirakawa, S. Morimoto / Journal of Photochemistry and Photobiology A: Chemistry 318 (2016) 1–6
1.2
1
(A)
Folic acid
PCA
MP(V)TPP
0.8
0.6
0.4
0.2
0
Fig. 1. Structures of MP(V)TPP, folic acid, and PCA.
example, a first screening of PDT drug. In the present study, to
develop the simple method to evaluate the activity of electron
transfer photosensitizer, the photosensitized decomposition of
folic acid by a water soluble porphyrin, dimethoxophosphorus(V)
tetraphenylporphyrin (MP(V)TPP, Fig. 1) was examined. MP(V)TPP
can photosensitize protein damage through electron transfer and
1O2 generation [23]. The synthesis of this porphyrin is relatively
easy and its water-solubility is relatively large. Therefore, MP(V)
TPP was used as a model of electron transfer photosensitizer.
250
350
450
550
650
Wavelength / nm
0.3
0.2
0.1
0
(B)
Folic acid
PCA
MP(V)TPP
2. Experimental
MP(V)TPP was synthesized according to the previous reports
[24,25]. Folic acid, sodium azide (NaN3), and potassium iodide (KI)
were purchased form Wako Pure Chemical Industries, Ltd., (Osaka,
Japan). 2-Amino-4-oxo-3H-pterine-6-carboxylic acid (PCA) was
from Sigma–Aldrich Co., LLC. (St. Louis, MO, USA). Sodium
phosphate buffer (pH 7.6) was from Nakalai Tesque Inc. (Kyoto,
Japan). Distilled water (H2O) was from Kanto Chemical Co., Inc.,
(Tokyo Japan). Deuterium oxide (D2O) was from Acros Organics
(Geel, Belgium).
250
350
450
550
650
Wavelength / nm
Fig. 2. Absorption spectra of folic acid, PCA, and MP(V)TPP. The sample solution
contained 10 M folic acid, 10 M PCA, or 5 M MP(V)TPP in a 10 mM sodium
phosphate buffer (pH 7.6). (B) is the magnified figure of (A).
m
m
m
The sample solution containing 5
folic acid in a 10 mM sodium phosphate buffer (pH 7.6) was
irradiated with light-emitting diode (LED) lmax = 585 nm,
mM MP(V)TPP and 10 mM
a
(
1 mW cmꢀ2, CCS Inc., Kyoto, Japan). The intensity of the LED light
source was measured with a photo-power meter (8230E, ADCMT,
Tokyo, Japan). After photo-irradiation, the fluorescence spectrum
of folic acid was measured with an F-4500 fluorescence spectro-
photometer (Hitachi, Tokyo, Japan). To evaluate the decomposed
folic acid by a fluorometry, the fluorescence intensity at 450 nm
was measured with a fluorescence photometer 650-60 (Hitachi).
The excitation wavelength for these measurements is 350 nm.
Because oxygen molecule is required in the decomposition process
of folic acid, the fluorescence enhancement of folic acid is not
observed under anaerobic condition. Therefore, this assay was
performed under air saturated condition. To evaluate the oxygen-
independent electron transfer reaction, an effect of 1O2 scavenger,
NaN3, was investigated. The above mentioned sample solution
with or without NaN3 was used for the photosensitized reaction.
Absorption spectra of folic acid, PCA, and MP(V)TPP in a 10 mM
sodium phosphate buffer (pH 7.6) were measured with a UV–vis
spectrophotometer UV-1650PC (Shimadzu, Kyoto, Japan). Fluores-
cence decay was measured using a time-correlated single-photon
counting method with a Fluorescence Lifetime System TemPro
(HORIBA, Kyoto, Japan). Laser excitation at 394 nm (or 571 nm) was
achieved by using a diode laser (NanoLED-390 (or NanoLED-570),
HORIBA) in a repetition rate of 1.0 MHz. The detection wavelength
is >640 nm by using a long-pass filter SCF-50S-64R (SIGMAKOKI
Co., Ltd., Tokyo, Japan). The experimental error of this measure-
3. Results and discussion
3.1. Photosensitized decomposition of folic acid by MP(V)TPP
The absorption spectra of folic acid, PCA, and MP(V)TPP in a
10 mM sodium phosphate buffer (pH 7.6) were shown in Fig. 2. In
general, PDT photosensitizers including porphyrins can absorb
light of longer wavelengths than that by folic acid, up to around
600–700 nm. Therefore, selective photoexcitation of porphyrins is
possible. The absorption spectrum of MP(V)TPP was not affected by
folic acid (data not shown), suggesting that the static interaction
between MP(V)TPP and folic acid is negligibly small. Therefore, a
diffusion process is necessary for the photosensitized decomposi-
tion of folic acid by MP(V)TPP.
The fluorescence intensity assigned to the pteridine moiety of
folic acid was increased by the photosensitized reaction of MP(V)
TPP (Fig. 3). It was confirmed that MP(V)TPP without photo-
irradiation did not enhance the fluorescence of folic acid.
Consequently, this fluorescence enhancement indicates the
decomposition of folic acid through the photosensitized reaction
by MP(V)TPP. The decomposed folic acid was calculated using the
calibration curve method on the fluorescence intensity of PCA
which is the fluorescent decomposed product of folic acid (Fig. 1
and Supplementary information). The quantum yield of the
photosensitized folic acid decomposition in sodium phosphate
buffer (pH 7.6, H2O) (FD) was estimated as follows:
ment is within 0.01 ns. The sample solution contained 5 mM MP(V)
PP and indicated concentration of folic acid in a 10 mM sodium
phosphate buffer (pH 7.6).
½PCAꢁ ꢂ V
FD
¼
ð1Þ
R
ð1 ꢀ 10ꢀAbsðvÞÞIðvÞSdv ꢂ T

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K. Hirakawa, S. Morimoto / Journal of Photochemistry and Photobiology A: Chemistry 318 (2016) 1–6
3
140
120
100
80
Irradiation time
120 min
90 min
60 min
30 min
without
60
40
20
0
350
400
450
500
550
600
Wavelength / nm
Fig. 5. Effects of sodium azide and potassium iodide on the photosensitized
decomposition of folic acid by MP(V)TPP. The sample solution containing 5
mM MP
Fig. 3. Fluorescence spectra of folic acid photosensitized by MP(V)TPP. The sample
(V)TPP and 10 M folic acid with or without 1 mM NaN3 or 1 mM KI in a 10 mM
m
solution containing 5 mM MP(V)TPP and 10 mM folic acid in a 10 mM sodium
sodium phosphate buffer (pH 7.6) was irradiated with an LED (lmax = 585 nm,
1 mW cmꢀ2). The concentration of decomposed folic acid was determined by a
fluorometry. The excitation wavelength is 350 nm.
phosphate buffer (pH 7.6) was irradiated with an LED (lmax = 585 nm, 1 mW cmꢀ2).
The excitation wavelength is 350 nm.
the quantum yield of 1O2 generation by MP(V)TPP is relatively large
(0.64) [23]. These findings suggest that 1O2 generation can be an
important mechanism of biomolecules damage by MP(V)TPP in a
certain condition. However, NaN3, a typical quencher of 1O2 [28],
did not inhibit the folic acid decomposition photosensitized by MP
(V)TPP and rather enhanced this photo-decomposition (Fig. 5).
Potassium iodide, a typical quencher of excited triplet (T1) state,
also enhanced this photo-decomposition (Fig. 5). These strange
results could be reasonably explained as follows: 1O2-mediated
damage of folic acid in aqueous solution is negligible and the
electron transfer mechanism is mainly responsible for the photo-
sensitized folic acid decomposition. Indeed, a previous report
demonstrated that folic acid cannot be decomposed by 1O2 in
aqueous solution of H2O due to its short lifetime [29]. Because MP
(V)TPP does not statically interact with folic acid, a diffusion
process is necessary to decompose folic acid. The lifetime of 1O2 is
short in H2O and most 1O2 generated by MP(V)TPP deactivates in
solution. Furthermore, some part of the generated 1O2 around MP
(V)TPP molecule induces oxidative self-degradation of MP(V)TPP,
resulting in the deactivation. Therefore, the addition of NaN3
slightly recovered the photosensitized folic acid decomposition by
MP(V)TPP. Because, in general, 1O2 is formed through energy
transfer from the T1 state of photosensitizer, the similar effect of KI
could be explained by the similar mechanism to that of NaN3. In
addition, the effect of KI suggests that the contribution of the T1
state of MP(V)TPP to folic acid photo-decomposition is negligible.
Since the oxidative activity of T1 state is smaller than that of excited
singlet (S1) state, the electron transfer-mediated oxidation of folic
acid by the T1 state may be thermodynamically difficult (described
in later).
where, [PCA] represents the concentration of PCA formed, V, the
volume of the sample solution (1.2 mL), Abs(
MP(V)TPP, I( ), the photon fluence of the light source at the
indicated light frequency the light
mol cmꢀ2 minꢀ1 s),
n), the absorbance of
n
(
m
n,
frequency (sꢀ1), S, the irradiated area (1 cm2), and T, the irradiation
time (min). The obtained value of FD was 1.1 ꢂ10ꢀ4
.
3.2. Mechanism of the photosensitized decomposition of folic acid by
MP(V)TPP
The sample solutions containing 5 mM MP(V)TPP with 10 mM
folic acid in H2O or D2O were prepared. These samples were
irradiated with an LED (lmax = 585 nm, 1 mW cmꢀ2) and the folic
acid decomposition was examined by a fluorometry. The decom-
posed folic acid (formed PCA) increased in a dose-dependent
manner in H2O or D2O (Fig. 4). This result demonstrated that the
photodecomposition rate of folic acid in D2O is significantly larger
than that in H2O. Because the lifetime of 1O2 is markedly elongated
in D2O (about 3.5 ms in H2O [26] to more than 70 ms in D2O [27]),
the rate of the 1O2-mediated reaction is accelerated in D2O. Indeed,
The initial process of photosensitized decomposition of folic
acid by MP(V)TPP can be explained by the photo-induced electron
transfer from folic acid to the S1 state of MP(V)TPP. The HOMO of
folic acid is located on its aminobenzoyl moiety (Fig. 1) [19], and
the electron abstraction from this moiety should trigger the
oxidative decomposition of folic acid (Supplementary information)
[13–18]. The Gibbs free energy (DG) of this electron transfer can be
calculated by the following equation:
DG ¼ eðEox ꢀ EredÞ ꢀ ES
ð2Þ
1
Fig. 4. Solvent deuterium effect on the photosensitized decomposition of folic acid
by MP(V)TPP. The sample solution containing 5 mM MP(V)TPP and 10 mM folic acid
where, e is the charge of the electron, Eox and Ered are the redox
potentials of one-electron oxidation of the aminobenzoyl moiety of
folic acid (0.84 V vs. saturated calomel electrode (SCE)) [30] and
in H2O or D2O was irradiated with an LED (lmax = 585 nm, 1 mW cmꢀ2). The
concentration of decomposed folic acid was determined by a fluorometry. The
excitation wavelength is 350 nm.

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K. Hirakawa, S. Morimoto / Journal of Photochemistry and Photobiology A: Chemistry 318 (2016) 1–6
one-electron reduction of MP(V)TPP (ꢀ0.50 V vs. SCE) [23],
equation:
respectively, and ES is theS1energy of MP(V)TPP (2.05 eV, estimat-
1
1
tf
1
tf0
¼
þ kET½FAꢁ
ð3Þ
ed from the fluorescence maximum: 606 nm). The estimated value
(ꢀ
theS1of MP(V)TPP is possible in the terms of thermodynamics.
G value of the electron transfer from folic acid to
DG = 0.71 eV) supported that electron transfer from folic acid to
where, t0f is the tf without folic acid and [FA] is the concentration of
The ꢀD
folic acid. The resulting value of kET (4.2 ꢂ109 Mꢀ1 sꢀ1) was
comparable with the value of diffusion control limit (kdif = 7.4
ꢂ 109 Mꢀ1 sꢀ1; Supplementary information). Consequently, the
quantum yield of the electron transfer process (FET) can be
expressed as follows:
theT1state of MP(V)TPP can be estimated from the similar equation
of Eq. (2), of which theES is replaced with theT1 energy (ET ). Since
1
1
theET of similar porphyrin is around 1.5 eV [31,32], the roughly
1
estimated value of ꢀ
DG is 0.2 eV, suggesting that the electron
transfer-mediated oxidation of folic acid by the T1state of MP(V)
TPP is exothermic reaction. However, in this experimental
condition, the photosensitized folic acid decomposition by the
T1state of MP(V)TPP could not be demonstrated. The driving force
of around 0.2 eV might be insufficient (described in Section 3.3).
The MP(V)TPP radical, formed through the electron transfer
from folic acid, should be returned to the original cationic form of
MP(V)TPP possibly by molecular oxygen under aerobic condition.
In the biological system, other oxidative materials such as metal
ions may assist the re-oxidation of this MP(V)TPP radical. The
photoreaction of MP(V)TPP should proceed catalytically.
kET½FAꢁ
FET
¼
ð4Þ
1
þ kET½FAꢁ
tf0
In this experimental condition ([FA] = 10
m
M), the value of FET
is 2.2 ꢂ10ꢀ4. The formed charge-transfer (CT) state undergoes
further reaction, leading to the decomposition of folic acid [13–18].
Alternatively, some part of the CT state can return to the ground
state through reverse-electron transfer. The reaction quantum
yield of the CT state to the decomposed product (Fr) can be
calculated using the following equation:
The sample solution containing 5 mM MP(V)TPP with or
FD
FET
Fr
¼
ð5Þ
without folic acid in a 10 mM sodium phosphate buffer (pH 7.6)
were prepared to measure the time profile of fluorescence
intensity. The time profile of MP(V)TPP fluorescence intensity
could be analyzed by a single exponential function (Supplementary
information). The obtained fluorescence lifetime (tf) was 5.2 ns in a
10 mM sodium phosphate buffer (pH 7.6). The electron transfer
should occur within this lifetime. To evaluate the electron transfer
rate constant, the tf with folic acid was measured. A significant
change of tf was not observed in the presence of relatively small
The calculated value of Fr (0.50) suggests that the efficiency of
the reverse-electron transfer (FRET = 0.50) is relatively large.
3.3. Protocol to evaluate the activity of photosensitizer using folic acid
This photosensitized reaction of folic acid decomposition and
resulting fluorescence enhancement can be applied to the
evaluation of the biomolecule damaging-activity of photosensi-
tizers. Because the PDT reaction occurs in the complex biological
system, this method may be used for a first screening process of
PDT drug. In general, the mechanisms of photosensitized
biomolecule damage can be explained by two processes, electron
transfer (Type I) and 1O2 generation (Type II) [33]. It has been
reported that 1O2 cannot decompose folic acid in aqueous solution,
whereas it is possible in D2O [29]. This study also demonstrated
that a 1O2 quencher does not inhibit the photosensitized folic acid
decomposition by MP(V)TPP. Therefore, the electron transfer-
mediated biomolecule damaging-activity of photosensitizers can
be simply evaluated by a fluorometry using folic acid in aqueous
solution.
concentration (around 10 mM) of folic acid, which is a similar
condition of the photosensitized reaction (Supplementary infor-
mation). Thus, the values of tf with relatively higher concentration
of folic acid were measured (Supplementary information). The
time profiles of fluorescence decay of MP(V)TPP with high
concentration of folic acid could be also fitted by single exponential
function. The analyzed values of tf were slightly and gradually
decreased depending on the concentration of folic acid. To obtain
the electron transfer rate coefficient (kET), these values were
analyzed by the Stern–Volmer plot (Fig. 6) using the following
For this fluorometry, a sample solution containing 10–20 mM
folic acid and photosensitizers are mixed in aqueous solution.
Because folic acid solution exhibits optimum stability at pH 7.6, the
pH range 6.0–8.0 is recommend. For example, sodium phosphate
buffer (pH 7.6) is appropriate. The concentration of the photo-
sensitizers should be adjusted to the appropriate absorbance of the
fluorescence apparatus used for this assay. A visible-light-emitting
diode (LED) is recommended as the light source. The sensitivity of
this analysis is proportional to the irradiation time, and several
minutes (10–30 min) may be sufficient for a general photosensi-
tizer. The biomolecules damaging-activity of photosensitizers can
be evaluated by the determination of the FD, which can be
estimated by Eq. (1).
Relevantly, photosensitized folic acid decomposition by other
compounds, riboflavin [15,17,34] and rhodamine-6G [22] were
reported. The folic acid decomposition by these molecules can be
explained by electron transfer mechanism. The ꢀ
riboflavin and rhodamine-6G are 0.81 eV and 0.34 eV [22],
G value of the case of riboflavin was estimated
from the reported redox potential [35] and excitation energy [36]
using the Eq. (2). In the case of rhodamine-6G, the reported value of
DG values of
respectively. The ꢀ
D
Fig. 6. Stern–Volmer plot of the fluorescence quenching of MP(V)TPP by folic acid.
The sample solution contained 5 mM MP(V)TPP and the indicated concentration of
folic acid in a 10 mM sodium phosphate buffer (pH 7.6). The excitation wavelength is
571 nm. The detection wavelength is >640 nm.