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ArticlePhotooxidation Activity Control of Dimethylaminophenyltris‑(N‑methyl-4-pridinio)porphyrin by pH
Kazutaka Hirakawa,* Syunsuke Takai, Hiroaki Horiuchi, and Shigetoshi Okazaki
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sı Supporting Information*

ABSTRACT: To control the activity of photodynamic agents by pH, an electron donor-connecting cationic porphyrin, meso(N′,N′-dimethyl-4-aminophenyl)-tris(N-methyl-p-pyridinio)porphyrin (DMATMPyP), was designed and synthesized. The
photoexcited state (singlet excited state) of DMATMPyP was deactivated through intramolecular electron transfer under a neutral
condition. The pKa of the protonated DMATMPyP was 4.5, and the ﬂuorescence intensity and singlet oxygen-generating activity
increased under an acidic condition. Furthermore, the protonation of DMATMPyP enhanced the biomolecule photooxidative
activity through electron extraction. Photodamage of human serum albumin (HSA) was observed under a neutral condition because
a hydrophobic HSA environment can reverse the deactivation of photoexcited DMATMPyP. However, an HSA-damaging
mechanism of DMATMPyP under a neutral condition was explained by singlet oxygen production. Therefore, it is indicated that theprotein photodamaging activity of DMATMPyP goes into an OFF state under a neutral hypoxic condition. Under an acidic
condition, the HSA photodamaging quantum yield by DMATMPyP through electron extraction could be preserved in the presenceof a singlet oxygen quencher. Photooxidation of nicotinamide adenine dinucleotide by DMATMPyP was also enhanced under anacidic condition. This study demonstrated the concept of using pH to control photosensitizer activity via inhibition of theintramolecular electron transfer deactivation and enhancement of the oxidative activity through the electron extraction mechanism.
Speciﬁcally, biomolecule oxidation through electron extraction may play an important role in photodynamic therapy to treat tumors
under a hypoxic condition.

■

conditions.5,23−27 Administered photosensitizers are illuminated by nonthermal visible light, resulting in biomoleculedamage through photochemical reactions. In general, 1O2
production through energy transfer from the excited photosensitizer to oxygen molecules is considered to be an importantmechanism in biomolecule damage.28,29 Electron transfer frombiomolecules to the photoexcited photosensitizers can be analternate mechanism of PDT.30−32 It has been reported that

INTRODUCTION
Electron transfer is the key factor in controlling thephotochemical process of photoexcited molecules.1−6 Fluorescence sensing,7−9 bioimaging,10−14 singlet oxygen (1O2)
production,15−21 and photooxidative activities22 of photoexcited molecules can be controlled through intramolecularelectron transfer. Changes in the redox potential of a moleculeby its surroundings, for example, pH,9,18−22 metal ion
concentration,7,8 and interaction with macromolecules, such
as DNA,15−17 can inhibit the electron-transfer quenching,leading to an active (ON) state. One important application of
the OFF → ON control is in the photomedicinal property ofmolecules. For example, cancer-selective photodynamic
therapy (PDT) can be realized by controlling the activity ofphotosensitizing molecules (photosensitizers). PDT is a lessinvasive treatment for cancer and other nonmalignant© 2020 American Chemical Society

Received: September 3, 2020
Accepted: September 29, 2020
Published: October 12, 2020

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Articlecalculated. These calculations showed that the highest
occupied molecular orbital (HOMO) of DMATMPyP is
located on the DMA moiety (Supporting Information).
Consequently, it is speculated that the photoexcited state of
DMATMPyP can be deactivated via intramolecular electrontransfer from the DMA moiety to the porphyrin ring, forming acharge-transfer (CT) state (Figure 1). In the case of H+DMATMPyP, the HOMO is located on the porphyrin ring,suggesting that the electron transfer-mediated quenching canbe suppressed under an acidic condition. In addition, thehighest molecular orbital energy of the porphyrin ring of H+DMATMPyP (−17.4 eV, in vacuum, calculated using the DFT
method) became lower than that of DMATMPyP (−12.9 eV).
Gibbs Energy of the Intramolecular Electron Transfer. The Gibbs energy (ΔGiet) of the intramolecular electrontransfer from the DMA moiety to the porphyrin ring wasroughly calculated using the redox potentials and excitationenergy of DMATMPyP (Supporting Information). The
obtained value (ΔGiet = −0.73 eV) supports the possibilityof quenching through intramolecular electron transfer.
pKa of the Protonated DMATMPyP. The UV−Vis
absorption spectra of DMATMPyP shifted depending on pH(Figure 2A). This absorption spectral change of DMATMPyP
by pH could be explained by the protonation of the DMA
moiety and the central pyrrole nitrogen atoms.37 Analysis of
the relationship between absorbance and pH (Figure 3) was

the pH of cancer cells is slightly acidic because of theenhancement of glycolysis.33−35 As mentioned above, electrontransfer can be controlled by pH. Furthermore, in general, aprotonated cationic compound becomes a stronger electron
acceptor than its nonprotonated form.36 Therefore, the pHinduced change in the intramolecular electron transfer can beapplied to cancer-selective PDT.18−22 The aim of this study is
to examine the concept of using pH to control the activity of acationic water-soluble porphyrin photosensitizer. Tetrakis(Nmethyl-p-pyridinio)porphyrin (TMPyP) has been studied as
the model compound for PDT.17,29 Because the watersolubility of previously reported porphyrins was relatively
low,18−20,22 the use of TMPyP-based porphyrin may be
advantageous for the application. To control the photosensitizing activity of TMPyP, meso-(N′,N′-dimethyl-4-aminophenyl)tris(N-methyl-p-pyridinio)porphyrin (DMATMPyP, Figure 1)

Figure 1. Molecular structure and the proposed relaxation schemes of
DMATMPyP and the protonated form (H+-DMATMPyP).

was designed and synthesized. The dimethylaniline (DMA)
moiety was used as a pH-dependent electron donor. The
photochemical property and biomolecule-damaging activity of
DMATMPyP were examined.

Figure 3. Relationship between the photochemical parameters of
DMATMPyP (absorbance at 440 nm and ﬂuorescence quantum
yield) and the pH of the solvent. The sample solution contained 5 μM
DMATMPyP in a 10 mM sodium phosphate buﬀer (indicated pH).
The excitation wavelength was 550 nm. The method of analysis is
described in the Supporting Information.

■

RESULTS
Energy Level of the Intramolecular Charge Transfer
State. The structures of DMATMPyP and its protonated form
(H+-DMATMPyP) were optimized using density functional
theory (DFT) calculation at the ωB97X-D/6-31G* level(Supporting Information). Their orbital energies were also

Figure 2. (A) Absorption and (B) ﬂuorescence spectra of DMATMPyP. The sample solution contained 5 μM DMATMPyP in a 10 mM sodium
phosphate buﬀer (indicated pH). The excitation wavelength was 550 nm.
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ArticleTable 1. Quantum Yields of Biomolecule Damage and 1O2 Production Photosensitized by DMATMPyPapH

ΦHSA(T)ΦHSA(ET)ΦHSA(Δ)

ΦNADH(T)ΦNADH (ET)ΦNADH(Δ)

ΦΔ

7.6
3.2

4.4 × 10−4
1.6 × 10−4∼0
1.4 × 10−4

4.4 × 10−4
0.2 × 10−4

0.03
0.18

0.023
0.14

0.007
0.044

0.18
0.36

a
ΦHSA(T): total quantum yields of HSA damage; ΦHSA(ET): quantum yield of HSA damage through electron transfer; ΦHSA(Δ): quantum yield of
HSA damage through 1O2 production; ΦNADH(T): total quantum yields of NADH decomposition; ΦNADH (ET): quantum yield of NADH
decomposition through electron transfer; ΦNADH(Δ): quantum yield of NADH decomposition through 1O2 production.

M−1, pH 3.2). The distance between the binding DMATMPyP
molecule and the tryptophan residue of HSA, which is locatedalmost at the center of HSA, was estimated based on an
analysis using the Förster resonance energy transfer (FRET)
method based on the energy transfer theory42 (Supporting
Information). The obtained values were 57 and 55 Å under pH
7.6 and 3.2, respectively.
Photosensitized Protein Oxidation by DMATMPyP.
The photosensitized protein oxidation activity of DMATMPyP
was examined by a ﬂuorescence intensity of HSA, as previously
reported.30−32 The abovementioned Kbc values showed thatthe binding ratios of DMATMPyP to HSA were 95% (pH 7.6)
and 60% (pH 3.2) in this experimental condition (10 μM HSA
and 5 μM DMATMPyP). The tryptophan residue of HSA canbe an important indicator of protein oxidation. HSA has one
tryptophan,43 which is easily oxidized through electron transfer
and 1O2 generation, leading to the diminishment of its intrinsic
ﬂuorescence.30−32 We observed that HSA photooxidation wasirradiation-dose dependent (Figure 4). The extent of photo-performed using a method similar to that in previous reports(Supporting Information).22 The obtained pKa values were 4.5
and 2.1. These values could be explained by the fact that thepKa of H+-DMATMPyP is 4.5 and that of the central nitrogenatoms is 2.1. Indeed, a similar pKa value of the central nitrogenatoms has been reported in the case of a similar porphyrin.37
pH-Dependent Fluorescence Change. The ﬂuorescenceintensity was also changed by pH, and a relationship similar tothat with the absorbance change was observed (Figure 3). A
relatively small ﬂuorescence quantum yield (Φf) value (0.037
at pH 7.6 and pH 6.4) under neutral conditions suggests thequenching of the singlet excited (S1) state of the porphyrin
ring through intramolecular electron transfer. The observed Φfvalue at pH 3.2 (0.12) was signiﬁcantly larger than that under
the neutral condition and suggests the inhibition of electrontransfer-mediated quenching.
Triplet Excited-State Formation and 1O2 Production.
Transient absorption measurements showed the typical peak ataround 470 nm,38 which was assigned to the absorption
spectrum of the triplet excited (T1) state (Supporting
Information). Enhancement of the T1-state formation underan acidic condition was qualitatively demonstrated. 1O2
production photosensitized by DMATMPyP was conﬁrmed
by near-infrared emission measurement, as previously reported(Supporting Information).39 The calculated values of 1O2
production quantum yield (ΦΔ) are listed in Table 1. The
analyzed time constant of 1O2 emission showed that the
lifetimes of 1O2 were 3.8 μs (pH 7.6) and 3.9 μs (pH 3.2).
These values were similar to the reported values for the 1O2
lifetime in an aqueous solution.17 Furthermore, these results
showed that the lifetime of 1O2 in an aqueous solution is barely
aﬀected by pH in this experimental region (pH 7.6−3.2).
Indeed, the previous reports demonstrated that 1O2 lifetime indeuterium oxide (72−78 μs)40 is independent of acidconcentration (72−77 μs in the presence of 5 mM triﬂuoroacetic acid).36 The T1 lifetime was also estimated from theseemissions using the equation in the literature41 (Supporting
Information). The obtained values of T1-state lifetimes were
1.6 μs (pH 7.6) and 1.3 μs (pH 3.2). These values were almost
the same as the decay time of the T1 transient absorption(about 1 μs, Supporting Information). The T1-state lifetime ismostly determined by the quenching by oxygen molecules andbecomes about 1−2 μs in an aqueous solution.17,30
Interaction between Human Serum Albumin and
DMATMPyP. To investigate the protein photodamage by
DMATMPyP using human serum albumin (HSA), theirinteraction was studied. The absorption spectra of DMATMPyP in the visible region were changed by the addition of HSA(Supporting Information). Since HSA has no absorption in thevisible region, the absorption change can be explained by thebinding interaction.30−32 Analysis of the absorbance change by
the previously reported method30−32 showed that the binding
constant (Kbc) under a neutral condition (3.3 × 106 M−1, pH
7.6) was larger than that under an acidic condition (2.1 × 105

Figure 4. (A) Time proﬁle of HSA damage photosensitized by
DMATMPyP and (B) the eﬀect of a 1O2 quencher. The sample
solution containing 10 μM HSA and 5 μM DMATMPyP with orwithout 10 mM NaN3 in a 10 mM sodium phosphate buﬀer(indicated pH) was irradiated with an LED (585 nm, 2.0 mW cm−2).
The eﬀect of NaN3 was examined for 30 min photoirradiation. Data
are presented as mean ± standard error (SE) (n = 3).

sensitized HSA oxidation caused by DMATMPyP increased
under a neutral condition. The quantum yields of HSA
photodamage through 1O2 generation and the electrontransfer-mediated mechanism were examined in light of the
scavenger eﬀect of NaN3, a strong physical quencher of 1O2,44
as in previous reports.30−32 The photosensitized HSA damage
increased linearly within 30 min. Therefore, the amount of
HSA damage by DMATMPyP with NaN3 for 30 min ofphotoirradiation was compared with that without NaN3. The
estimated quantum yields are listed in Table 1.
Photosensitized NADH Oxidation by DMATMPyP.
Nicotinamide adenine dinucleotide (NADH) was used as thetarget biomolecule to examine the photosensitizing activity of
DMATMPyP. NADH can be oxidized by 1O2 production andelectron transfer reactions.22 Because NADH is a relatively
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Articleprotonated DMA moiety of H+-DMATMPyP was 4.5, and theﬂuorescence intensity was recovered in an acidic solution. DFT
calculation predicted that the intramolecular electron transfer
in H+-DMATMPyP is suppressed because the highest
occupied orbital energy of the DMA moiety becomes lower
than that of the porphyrin ring. These results indicated that therelaxation process of the DMATMPyP S1 state could becontrolled by pH through protonation of the electron-donating
moiety, as proposed in Figure 1. The photosensitized 1O2generating activity of DMATMPyP was increased under anacidic condition. Transient absorption spectrum measurements
veriﬁed that T1-state formation is enhanced under an acidic
condition (Supporting Information). Inhibition of the S1-state
quenching leads to the increase in intersystem crossing
eﬃciency to the T1 state and 1O2 generation.
The absorption spectral change showed that DMATMPyP
bound to HSA. The tryptophan residue is located near thecenter of HSA,43 and HSA has pockets as drug-binding sites(Sudlow’s site I and site II).45 The FRET method suggests thatthe distance between DMATMPyP and the tryptophan residue
is almost 55 Å. Since the size of HSA is almost 100 Å,43 thisresult suggests that DMATMPyP binds mostly to the surfaceof HSA pockets. The estimated binding constants between
DMATMPyP and HSA under neutral (pH 7.6) and acidic (pH3.2) conditions are 3.3 × 106 M−1 and 2.1 × 105 M−1,
respectively. HSA that is positively charged by the amino acidprotonation under an acidic condition should inhibit theinteraction with H+-DMATMPyP.
Photoirradiation of DMATMPyP induced tryptophan
residue damage. The estimated quantum yield of HSA damage
at pH 7.6 was larger than that at pH 3.2. As mentioned above,a suppression of the interaction under an acidic condition may
decrease the HSA damage by H+-DMATMPyP at pH 3.2.
Furthermore, a hydrophobic environment of protein is not
appropriate for the intramolecular electron transfer because theCT-state energy level depends on the surrounding dielectric
constant and increases in a less-polar environment.46 The
environment of HSA may lead to recovery of the photosensitizing activity of porphyrin. Therefore, the total HSA
damage was decreased under an acidic condition. However,small molecule and the binding interaction with DMATMPyP
is considered to be negligibly small, an environmental eﬀect
such as the case of HSA does not occur. Therefore, relatively
high concentration of NADH (100 μM) against DMATMPyP(5 μM) was used. The typical absorption spectrum of NADH,
around 340 nm, was decreased by photoirradiation withDMATMPyP. The NADH photooxidation was increased
under an acidic condition (Figure 5). Similar to the analysis

Figure 5. (A) Time proﬁle of NADH oxidation photosensitized by
DMATMPyP and (B) the eﬀect of a 1O2 quencher. The sample
solution containing 100 μM NADH and 5 μM DMATMPyP with orwithout 10 mM NaN3 in a 10 mM sodium phosphate buﬀer(indicated pH) was irradiated with an LED (585 nm, 2.0 mW cm−2).
The eﬀect of NaN3 was examined for 5 min photoirradiation. Data arepresented as mean ± SE (n = 3).

of HSA damage, the NADH photooxidation by DMATMPyP
with NaN3 for 5 min of photoirradiation was compared withthat without NaN3. The estimated quantum yields of NADH
oxidation photosensitized by DMATMPyP are listed in Table
1.

■

DISCUSSION
The photoexcited state (S1 state) of DMATMPyP wasdeactivated through intramolecular electron transfer from the
DMA moiety, the electron-donating moiety, in an aqueous
solution. DFT calculation and the observed redox potentials
supported this intramolecular electron transfer. The pKa of the

Figure 6. Diagram of the pH-controlled activity of the DMATMPyP photosensitizer and the relative energy levels of the highest occupied orbitals
for the porphyrin moiety, DMA moiety, and targeting biomolecule.
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Articlewere from Kanto Chemical Co., Inc. (Tokyo, Japan). HSA waspurchased from Sigma-Aldrich Co. LLC. (St. Louis, MO,
USA). These reagents were used as received. A sodium
phosphate buﬀer (pH 6.4) was prepared from disodiumhydrogen phosphate dodecahydrate and sodium dihydrogen
phosphate dihydrate (Nacalai Tesque, Inc., Kyoto, Japan).
Sodium phosphate buﬀers (pH 4.4 and 3.2) were preparedfrom sodium dihydrogen phosphate dihydrate (Nacalai
Tesque, Inc.) and 0.05 M phosphoric acid (Kanto Chemical,
Co. Inc., Tokyo. Japan). An acidic solution (pH 2.0) was
prepared from 0.1 M hydrochloric acid solution (FUJIFILM
Wako Pure Chemical Co.) and distilled water (FUJIFILM
Wako Pure Chemical Co.).
Measurements. The absorption spectra of DMATMPyP
were measured with a UV−Vis spectrophotometer UV1650PC (Shimadzu, Kyoto, Japan). The ﬂuorescence spectraof samples were measured with an F-4500 ﬂuorescence
spectrophotometer (Hitachi, Tokyo, Japan). The Φf wasmeasured with an absolute photo-luminescence quantum yieldmeasurement system (C9920-02, Hamamatsu Photonics KK,
Hamamatsu, Japan). The 1O2 formation photosensitized by
DMATMPyP was directly measured by near-infrared luminescence around at 1270 nm from deactivated 1O2, whichcorresponds to the 1O2 (1Δg)-3O2(3Σg−) transition aspreviously reported.39 The ΦΔ was determined from thecomparison of the 1O2 emission intensities by DMATMPyP
solution and methylene blue (ΦΔ = 0.52 in water).50A cyclic
voltammogram was measured with a potentiostat/galvanostat(HA-301, Hokuto Denko Co., Tokyo, Japan), a function
generator (DF1906, NF Co., Yokohama, Japan), and a datalogger (midi LOGGER, GL900-4, Graphtec Co., Yokohama,Japan) using a platinum working electrode (ALS Co., Ltd.,Tokyo, Japan), a platinum counterelectrode (ALS Co., Ltd),and saturated calomel electrode (SCE, ALS Co., Ltd).
Calculations. The optimized structure and energy of
DMATMPyP and H+-DMATMPyP were calculated by the
DFT method at the ωB97X-D/6-31G* level utilizing theSpartan 18′ (Wavefunction Inc., CA, USA).

electron transfer-mediated HSA damage could be enhanced
under an acidic condition. Analysis of the HSA-damaging
quantum yield showed that the mechanism of HSA damage
photosensitized by DMATMPyP at pH 7.6 is mainly explainedby 1 O 2 generation. The protein damage through 1 O 2
production is suppressed under hypoxia in both cases of acidic
and neutral conditions. However, the oxidative activity of
DMATMPyP through the electron-transfer mechanism wasenhanced under an acidic condition. DFT calculation showedthat the molecular orbital energy level of the porphyrin ring
becomes lower in the protonated state, H+-DMATMPyP(Supporting Information). Consequently, photosensitized
HSA damage by DMATMPyP through electron transfer
could be preserved at pH 3.2 in the presence of a 1O2
quencher. It is speculated that DMATMPyP can selectively
oxidize proteins under an acidic hypoxic condition (Figure 6).
NADH photooxidation was also induced by DMATMPyP.
NADH is a relatively small biomolecule and does not form abinding complex with DMATMPyP. The results of NADH
oxidation showed that the photooxidative activity of
DMATMPyP can be increased under an acidic condition ina simple aqueous solution. Because NADH is easily oxidized,
the observed quantum yield through the electron-transfer
mechanism was not signiﬁcantly increased under an acidic
condition.
In summary, the relaxation process of DMATMPyP can becontrolled by pH through the protonation of the DMA moiety.
Fluorescence intensity, T1-state formation, and the resultingΦΔ are increased in the H+-DMATMPyP state. The total
quantum yield of HSA photodamage was decreased under anacidic condition because the interaction between protonated
HSA and H+-DMATMPyP is weaker than that under a neutral
condition and a hydrophobic environment reverses thephotodamaging activity of DMATMPyP. However, under ahypoxic condition, DMATMPyP can selectively photosensitize
protein damage through the electron transfer-mediatedmechanism under an acidic condition since 1O2-mediatedbiomolecule damage is not eﬀective, whereas the electrontransfer-mediated mechanism is enhanced by the protonation.
Although the pKa 4.5 of H+-DMATMPyP is smaller than thephysiological pH (around 7)35 and not an ideal value forclinical use, this study demonstrated the concept of activity
control based on inhibition of the intramolecular electrontransfer deactivation and enhancement of the oxidative activity
through protonation of the photosensitizer. Speciﬁcally,
electron transfer-mediated biomolecule oxidation can become
an important PDT mechanism to treat tumors under a hypoxic
condition.

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ASSOCIATED CONTENT

sı Supporting Information*

The Supporting Information is available free of charge athttps://pubs.acs.org/doi/10.1021/acsomega.0c04303.

■

EXPERIMENTAL SECTION
Materials. DMATMPyP was obtained by the methylationof meso-(N,N-dimethyl-4-aminophenyl)-tris(p-pyridyl)porphyrin (DMATPyP). Synthesis of DMATPyP was
according to the previously reported method.47 To obtain
DMATMPyP, the methylation of DMATPyP was carried out
according to the literature.48,49 Details about the synthesis andcharacterization of DMATMPyP are described in the
Supporting Information. The spectroscopic-grade distilledwater was purchased from Dojin Chemicals Co. (Kumamoto,Japan). Sodium phosphate buﬀer (pH 7.6) and NADH werefrom Nakalai Tesque Inc. (Kyoto, Japan). Dimethyl sulfoxided6 and sodium azide were from FUJIFILM Wako Pure
Chemical Co. (Osaka, Japan). Methyl iodide and diethyl ether■

Synthesis of DMATPyP and DMATMPyP, DFT
calculation, calculation of the driving force of electrontransfer, analysis of the pKa values of DMATMPyP,
transient absorption spectra of DMATMPyP, photosensitized singlet oxygen production by DMATMPyP,
analysis of the interaction between DMATMPyP and
HSA, and analysis of the binding position by the FRET
method (PDF)

AUTHOR INFORMATION

Corresponding Author

Kazutaka Hirakawa − Applied Chemistry and Biochemical
Engineering Course, Department of Engineering, Graduate
School of Integrated Science and Technology and Department ofOptoelectronics and Nanostructure Science, Graduate School of
Science and Technology, Shizuoka University, Hamamatsu 4328561, Japan; orcid.org/0000-0002-3694-8165;
Phone: +81-53-478-1287; Email: hirakawa.kazutaka@shizuoka.ac.jp; Fax: +81-53-478-1287

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Authors

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Syunsuke Takai − Applied Chemistry and Biochemical
Engineering Course, Department of Engineering, Graduate
School of Integrated Science and Technology, Shizuoka
University, Hamamatsu 432-8561, Japan
Hiroaki Horiuchi − Division of Molecular Science, Graduate
School of Science and Technology, Gunma University, Kiryu
376-8515, Japan
Shigetoshi Okazaki − Preeminent Medical Photonics Education& Research Center, Hamamatsu University School of Medicine,
Hamamatsu 431-3192, Japan
Complete contact information is available at:https://pubs.acs.org/10.1021/acsomega.0c04303
Notes

The authors declare no competing ﬁnancial interest.

■

ACKNOWLEDGMENTS
The authors wish to thank Dr. Hirohiko Watanabe and Dr.
Kiyotada Hosokawa (Hamamatsu Photonics K.K.) for
transient absorption measurement. The presented work waspartially supported by Grants-in-Aid for Scientiﬁc Research(B) from Japan Society for the Promotion of Science (JSPS
KAKENHI 17H03086) and Futaba Electronics Memorial
Foundation (10407).

■

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