A Photochemical Study of Photo-Induced Electron Transfer from DNAs to a Cationic Phthalocyanine Derivative

Water-soluble cationic gallium(III)-Pc complex (GaPc) is capable of photogenerating ROSs but does not exhibit photocytotoxicity in vivo. GaPc binds selectively, through a π-π stacking interaction, to the 5’-terminal G-quartet of a G-quadruplex DNA. The photo-excited state of GaPc of the complex is effectively quenched through electron transfer (ET) from the ground state of DNA guanine (G) bases to the photo-excited state of GaPc (ET(G-GaPc)). Hence the loss of the photocytotoxicity of GaPc in vivo is most likely to be due to the effective quenching of its photo-excited state through ET(G-GaPc). In this study, we investigated the photochemical properties of GaPc in the presence of duplex DNAs formed from a series of sequences to elucidate the nature of ET(G-GaPc). We found that ET(G-GaPc) is allowed in electrostatic complexes between GaPc and G-containing duplex DNAs and that the rate of ET(G-GaPc) (kET(G-GaPc)) can be reasonably interpreted in terms of the distance between Pc moiety of GaPc and DNA G base in the complex. We also found that the quantum yields of singlet oxygen (1O2) generation (ΦΔs) determined for the GaPc-duplex DNA complexes were similar to the value reported for free GaPc (Fujishiro R, Sonoyama H, Ide Y, et al (2019) J Inorg Biochem 192:7–16), indicating that ET(G-GaPc) in the complex is rather limited. These results clearly demonstrated that photocytotoxicity of GaPc is crucially affected by ET(G-GaPc). Thus elucidation of interaction of a photosensitizer with biomolecules, i.e., an initial process in PDT, would be helpful to understand its subsequent photochemical processes.


Introduction
Phthalocyanine (Pc) and its derivatives have been used extensively in materials and biomedical engineering because of their unique physicochemical properties, in addition to ease of chemical transformation and high thermal and chemical stability [1].In biomedical applications, watersoluble Pc derivatives [2][3][4][5] are recognized as promising photosensitizers for photodynamic therapy (PDT) [6][7][8][9].The advantages of Pc as a PDT photosensitizer are primarily its strong absorption band in the long-wavelength region, i.e., ~680 nm, reflecting high tissue permeability, and its highly efficient photogeneration of reactive oxygen species (ROSs), due to its macrocyclic 18 electron π-conjugated system and high intersystem crossing efficiency.Furthermore, coordination of Pc to diamagnetic metal ions possessing closed-shell valence electron configurations such as Zn(II) and Ga(III) yields metal-Pc complexes exhibiting high triplet quantum yields and long triplet state lifetimes [10,11], which are essential for efficient photosensitization in PDT.Especially compared with a Zn(II)-Pc complex, a Ga(III)-Pc one has relatively high water-solubility desirable for a PDT photosensitizer due to the presence of an axially coordinated chloride ion, which prevents self-aggregation [12,13].
The photochemistry and photophysics of Ga(III)-Pc complexes have been well characterized, and studies have demonstrated that the complexes are promising PDT photosensitizers [14][15][16][17].Incidentally, since oxidation reactions 1 3 with ROSs have been shown to occur exclusively at the site where they are generated, i.e., within the vicinity of the photosensitizer, knowledge about intermolecular interaction of the photosensitizers with other molecules in cells is expected to be extremely useful for molecular design of PDT photosensitizers to enhance their PDT efficacy [18,19].
Water-soluble Ga(III)-Pc complex possessing N-methylpyridinium groups at eight peripheral β-positions(GaPc, Fig. 1) is capable of photogenerating singlet oxygen ( 1 O 2 ) and is well taken up by cells, but exhibited no photocytotoxicity in vivo [20].Since the photo-induced electron transfer (ET) from biomolecules such as DNA to photosensitizers is thought to be associated with the early stages of so-called Type I reaction, one of the major pathways for the generation of ROSs in PDT processes [21][22][23], it is of particular importance to elucidate the interaction of photosensitizers with biomolecules and the nature of the photo-induced ET between them, if any.We found that GaPc binds selectively, through a π-π stacking interaction, to the 5'-terminal G-quartet of a G-quadruplex DNA [24] to form a complex and that the photo-excited state of GaPc of the complex is effectively quenched through ET from the ground-state of DNA guanine (G) bases to the photo-excited state of GaPc (ET (G-GaPc) , Fig. 1) [25].
In this study, we characterized the photochemical properties of GaPc in the presence of duplex DNAs formed from various G-containing sequences as well as a G-free one to evaluate the nature of ET (G-GaPc) .We found that ET (G-GaPc) is allowed in electrostatic complexes between GaPc and G-containing DNAs and that the rate of ET (G-GaPc) (k ET(G-GaPc) ) can be reasonably interpreted in terms of the distance between Pc moiety of GaPc and DNA G base in the complex (GaPc-G distance).Moreover, the quantum yields of singlet oxygen ( 1 O 2 ) generation (Φ Δ s) determined for the GaPc-duplex DNA complexes were comparable to the value reported for free GaPc [20], indicating that the ET (G-GaPc) process is restricted in the complex.This is in sharp contrast to the GaPc-quadruplex DNA complex where 1 O 2 generation is significantly inhibited by the ET (G-GaPc) process.These findings provided a novel insight into the molecular design for Pc-based PDT photosensitizing agents.

UV-Vis Absorption and Fluorescence Spectra, and Fluorescence Lifetime Measurements
Absorption spectra were measured with a JASCO V750 UV-Vis spectrometer (JASCO, Tokyo, Japan) at 25 °C using a quartz cell of 1.0 cm path length.Fluorescence spectra were measured with an FP-6500 spectrofluorometer (JASCO, Tokyo, Japan) using a quartz cell of 1.0 cm path length.Fluorescence lifetimes were measured with a Fluorolog-3 fluorescence spectrometer (Horiba, Kyoto, Japan) using a quartz cell of 1.0 cm path length.An excitation wavelength of 634 nm was used for selective excitation of GaPc, followed by monitoring of the fluorescence of GaPc at 688 nm.

Determination of the Rate Constant of the Electron Transfer (k ET(G-GaPc) ) and Quantum Yield of the Electron Transfer (Φ ET(G-GaPc) ) From the Ground State of DNA Guanine (G) Base(s) to the Photo-excited State of GaPc
The fluorescence emission rate constant of GaPc (k f ) can be obtained from the fluorescence lifetime (τ 0 ) and fluorescence quantum yield (Φ f ) of free GaPc through Eq. ( 1), and has been determined to be 3.68 × 10 7 s -1 [25].τ 0 is expressed in terms of k f and the nonradiative deactivation rate constant of free GaPc (k nr0 ) using Eq. ( 2).
No photo-induced electron transfer is expected to occur even if GaPc binds to G-free duplex DNA (G 0 ) (see below), and hence the fluorescence lifetime of GaPc bound to G 0 (τ 1 ) is expressed by k f and the nonradiative deactivation rate constant of GaPc bound to G 0 (k nr1 ) using Eq.(2').
When GaPc is bound to a G-containing duplex DNA such as G 2 -G 12 , the photo-induced ET from the ground-state of DNA G base(s) to the excited state of GaPc (ET (G-GaPc) ) occurs (see below).Hence, the fluorescence lifetime of GaPc bound to G-containing DNA (τ 2 ) is expressed as follows.
where k ET(G-GaPc) is the rate constant of ET (G-GaPc) in a GaPc-DNA complex.

O 2 Detection
1 O 2 generation by GaPc via photo-irradiation in the absence and presence of duplex (G 0 , G 6a ) or G-quadruplex (GQ) DNAs in potassium phosphate buffer was examined.Sample preparation was carried out in the dark.9,10-Antracenediyl-bis(methylene) dimalonoic acid (ADMA) (Funakoshi Co. Ltd., Tokyo, Japan) was used as a scavenger of ROS.Samples containing 2 μM GaPc and 20 μM ADMA, in the absence and presence of 10 μM DNA, in quartz cuvettes were irradiated with monochromatic (1) light (690 nm) using a Xe lamp of a JASCO FP-6500 spectrofluorometer (JASCO, Tokyo, Japan).The absorption spectra of an irradiated sample were measured at 0, 180, 360, 600, 1200, 1800, 2400, 3000, 3600, 5400 s of irradiation.The photochemical reactions were followed spectrophotometrically by observing the decrease in the absorbance at 380 nm of ADMA.The quantum yield for 1 O 2 generation (Φ Δ ) from GaPc-DNA complexes was determined by a relative method using free GaPc as a reference, of which Φ Δ has been reported to be 0.029 [20].

Photochemical Properties of GaPc in the Presence of Duplex DNAs
The impact of DNA quenching on GaPc fluorescence was assessed using the Gibbs energy (ΔG ET(G-GaPc) ) for electron transfer from the ground state of the G base to the photoexcited state of GaPc, which was calculated by applying the Rehm-Weller equation (Eq.( 6) [26], where E ox and E red are the first oxidation potential of the G base (electron donor) and the first reduction potential of GaPc (electron acceptor), respectively, E 0,0 the excited singlet energy of GaPc, and w the work term for the charge separation state, which can be assumed to be −0.1 V for a quenching reaction in aqueous solution [26].E ox of G base was reported to be 1.47 V vs standard hydrogen electrode (NHE) [27].E red and E 0,0 values for GaPc was reported to be −0.14V vs NHE and 1.82 V, respectively [25].The substitution of these values into Eq.( 6) yielded the value of −0.31 V for the ΔG ET(G-GaPc) , indicating that the fluorescence quenching occurs exergonically through ET (G-GaPc) [25].
We first examined the interaction of GaPc with duplex DNAs using fluorescence spectroscopy (Fig. 2).The GaPc fluorescence was not largely affected by the addition of G-free G 0 (Fig. 2A).In contrast, the GaPc fluorescence was quenched by the addition of G-containing G i (i ≠ 0), although the extent of quenching varied with the DNA sequence (Figs.2B and S1).Quenching of the photo-excited state of GaPc by G-containing DNA is most likely to be due to ET (G-GaPc) .
The results obtained from the characterization of the effects of duplex DNAs, i.e., G i , on the GaPc fluorescence (Fig. 3) showed a unique relationship between the efficiency of quenching of the excited state of GaPc by the DNAs and percentage composition of G base(s) in the DNAs (G(%)) such that the efficiency of quenching increases with increasing G(%), up to ~30%, as observed for G 0 -G 6n , where subscript n represents a, b, c, or d, and then reaches a steady is >33%, as observed for G 6n -G 12 (Fig. 3).The G(%)-dependent quenching of the excited state of GaPc by the DNAs could be reasonably interpreted in terms of the GaPc-G distance in the GaPc-DNA complex because the interchromophore distance practically shortens with increasing G(%) up to ~30% and is almost independent of the G(%) which is >33% (Table 1).

Fluorescence Lifetime and Photo-induced Electron Transfer Rate of GaPc Bound to Duplex DNAs
We then performed fluorescence lifetime analysis of GaPc in the presence of the DNAs (Table 2) to estimate k ET(G-GaPc) of the GaPc-DNA complexes.In the presence of an excess amount of G-free G 0 , the fluorescence decay of GaPc was fitted well with a single exponential function with lifetime of τ 1 = 4.9 ns, which was longer by ~1 ns than that of free GaPc, i.e., τ 0 = 3.8 ns, probably due to suppression of nonradiative relaxation as a result of the immobilization of GaPc Fig. 2 Fluorescence spectra of 2 μM GaPc in the presence of 0-3 equivalent of guanine(G)-free G 0 (A) and G-containing G 6a (B) in 50 mM potassium phosphate buffer, pH 6.80, and 300 mM KCl at 25°C.The spectra of GaPc in the absence and presence of 3.0 equivalent DNAs are shown in blue and red, respectively.Excitation wavelengths were selected on the basis of the isosbestic points of the UV spectral change of GaPc observed upon the addition of duplex DNAs and were 336 and 308 nm for (B) and (C), respectively in the complex. 1 H NMR signals due to imino protons of G 6d were not affected by the addition of GaPc (Fig. S2), indicating that GaPc does not intercalate between base pairs of the DNA.Consequently, cationic GaPa is thought to bind electrostatically to duplex DNAs, as depicted in Fig. 4. On the other hand, the fluorescence decay curves of GaPc in the presence of G i (i ≠ 0) were all fitted reasonably well by a double exponential function with lifetimes of τ 1 = 4.5-4.7 ns and τ 2 = 2.1-3.0 ns (Table 2).It is noteworthy that the slowdecaying component was always observed for the GaPc-G i complexes regardless of the G(%) and the τ 1 was essentially independent of the G(%), indicating that the slow-decaying component is due the complex where ET (G-GaPc) is not allowed.On the other hand, the τ 2 of the GaPc-G i (i ≠ 0) complex shortened from 3.0 ns to 2.1 ns with increasing G(%) which is ≤ 33% and then reached a steady value of 2.1 ns for the DNAs of which the G(%)s are >33%.Therefore the fast-decaying component is most likely to be due to the GaPc-G i (i ≠ 0) complex where ET (G-GaPc) is allowed.
In addition, by substituting τ 2 = 2.1 ns into Eq.( 3), value of ~2.7×10 8 s -1 was obtained for k ET(G-GaPc) of the GaPc bound to the DNAs with G(%) >33%.Thus k ET(G-GaPc) of the GaPc-G i (i ≠ 0) complex was found to be independent of both the positions of G bases in the sequence and the location of the GaPc binding site in the complex, provided that the G(%) is sufficiently large.These results indicated that the GaPc-DNA complex exhibiting τ 1 = 4.5-4.7 ns could have a promising PDT effect due to the absence of ET (G-GaPc) .
We investigated the effect of complex formation with duplex and quadruplex DNA on the quantum yield of 1 O 2 generation (Φ Δ ), a measure of the PDT effect of GaPc (Table 3 and Figure S3).Interestingly, the GaPc-G 0 and GaPc-G 6a complexes exhibited almost identical Φ Δ values, i.e., 0.032 and 0.031, respectively (Table 3), which were also similar to the value reported for GaPc, i.e., 0.029 [20], indicating that ET (G-GaPc) in the GaPc-G i complex is rather limited.Therefore, it is concluded that the binding of GaPc to G-containing duplex DNA does not significantly interfere with its PDT effect.In contrast, the Φ Δ value of 0.011 was obtained for the GaPc-GQ complex, indicating that the 1 O 2 generation in the complex is considerably inhibited by the ET (G-GaPc) process (vide infra).

Fluorescence Lifetime and Photo-induced Electron Transfer Rate of GaPc Bound to Various DNAs
Various cancer-related genes with G-quadruplex structures have been extensively studied as therapeutic targets [28][29][30].Since oxidation reactions with ROSs occur selectively in Fig. 3 Effect of complexation with various DNAs on the fluorescence of GaPc.The value (f/f 0 ) was obtained as the ratio between the GaPc fluorescence intensities of GaPc in the absence (f 0 ) and with the addition of 3.0 eq. of DNAs (f).The remarkably small f/f 0 value of GQ is due to preferential π-π stacking of GaPc on the 5'-terminal G quartet (see "type-a" of the GaPc-GQ complex in Fig. 5A)

Table 1 G-quadruplex and duplex DNAs used in this study
Name DNA the vicinity of photosensitizers, it is crucial to understand the photochemical and photophysical processes of photosensitizers bound to G-quadruplex DNAs.We previously reported that GaPc binds selectively to GQ at the A3G4 step, forming a π-π stacking complex with a binding constant of (21 ± 2) × 10 6 M -1 [24].The GaPc fluorescence was quenched by 96% in the presence of GQ (Figs. 3 and S4) and the fluorescence decay curve of the GaPc-GQ complex could be fitted well with a double exponential function, with slow-and fast-decaying components with lifetimes of τ 2 = 2.8 ns and τ 3 = 0.17 ns, respectively (Table 2), both of which were shorter than that of free GaPc, i.e., τ 0 = 3.8 ns [25].These results indicated the existence of two different types of the GaPc-GQ complexes where ET (G-GaPc) is allowed.As Table 2 Fluorescence lifetimes (τ 0 -τ 3 ) of GaPc and its complexes with various DNAs, electron transfer rate constants from guanine bases to the excited state GaPc (k ET(G-GaPc) ), and the quantum yields for electron transfer (Φ ET ) a The fluorescence lifetime values of free GaPc and GaPc-GQ complexes were taken from reference [25] Fluorescence lifetime/ns (fraction %) 0.17 (24) a 5.7×10 9 0.97 Fig. 4 Schematic representation of binding mode between GaPc and duplex DNA depicted in Fig. 5A, it would be reasonable to assume that, in addition to the "type-a" complex revealed by NMR, the "type-b" complex in which GaPc binds electrostatically to the surface of GQ is thought to be formed as in the case of the GaPc-G i complex.The value of 5.6 × 10 9 s -1 has been determined for k ET(G-GaPc) of the type-a complex.On the other hand, k ET(G-GaPc) of the type-b complex could be calculated as follows.Assuming that k f is not largely affected by the experimental conditions used in the study, substitution of k f =3.68 × 10 7 s -1 and τ 1 = 4.9 ns, obtained for the GaPc-G 0 complex where ET (G-GaPc) is not allowed, into Eq.(2') yielded k nr1 = 1.7 × 10 8 s -1 .Then the value of 1.5 × 10 8 s -1 was obtained for k ET(G-GaPc) of the type-b complex from τ 2 = 2.8 ns, k f =3.68 × 10 7 s -1 , and k nr1 = 1.7 × 10 8 s -1 using Eq. ( 3).Thus, k ET(G-GaPc) in the type-a complex was found to be greater by a factor of ~37 than that in the type-b complex and approximately 20-40 greater than those in GaPc-G i (i ≠ 0) complex.Based on a model study, the distance between the π-systems of GaPc and the G-quartet in the type-b complex is estimated to be at least ~0.7 nm [31].Therefore, the finding that k ET(G-GaPc) of the type-a complex is considerably greater than that of the type-b one could be reasonably interpreted in terms of the difference in the GaPc-G distance between the type-a and type-b complexes, i.e., ~0.4 and at least ~0.7 nm for the former and latter, respectively.Thus, k ET(G-GaPc) was found to be affected by the GaPc-G distance.
We next attempted to quantitatively determine the ratio of the type-a complex to the type-b one in the GaPc-GQ complex using a photochemical approach.First, substituting k ET(G-GaPc) s, together with k f =3.68 × 10 7 s -1 and k nr1 = 1.7 × 10 8 s -1 , into Eq.( 5), the Φ f s were calculated to be 6.3 × 10 -3 and 7.8 × 10 -2 , for the type-a and type-b complexes, respectively.Then, we performed convolution of the fluorescence spectrum of the GaPc-GQ complex (Fig. 5B and B').A comparative study between the normalized fluorescence spectra of free GaPc and the GaPc-GQ complex indicated that the binding of GaPc to the DNA results in the observation of a new band at a longer wavelength in the spectrum (Fig. 5B).The bands of the GaPc-GQ complex could be reasonably fitted with two components centered at 687 and 703 nm, with an intensity ratio of 1.00:1.16,respectively (Fig. 5B' and  B").Based on the similarity in shape to the bands observed for the GaPc-G i complexes (Fig. 5B"), the band at 687 nm could be attributed to the type-b complex and hence that at 703 nm is attributable to the type-a one.From the intensity ratio between the two bands and their fluorescence quantum yields, the ratio of incident light absorbed by the type-a and type-b complexes can be calculated to be 95:5, in favor of the type-a complex.
Red light (699 nm) irradiation on the GaPc-GQ complex did not result in photo-degradation of GQ (Fig. S5), indicating that the ET (G-GaPc) process in the complex suppresses subsequent photochemical reaction.In general, the primary photocytotoxic pathway in PDT, i.e., Type I reaction, is initiated by ET between a photosensitizer in the photo-excited triplet state and biomolecules in cell, while the ET (G-GaPc) process Table 3 The quantum yields of singlet oxygen generation (Φ Δ s) of GaPc and its complexes with various DNAs a The value was taken from reference [20] Φ Δ GaPc 0.029 a GaPc-G 0 0.032 GaPc-G 6a 0.031 GaPc-GQ 0.011 Fig. 5 Schematic representation of the formation of GaPc-GQ complex through π-π stacking interaction at A3G4 step "type-a" and through electrostatic interaction "type-b" (A).The normalized fluorescence spectra of 2 μM free GaPc (black) and GaPc-GQ complex (red, magnified 57 times on the y-axis), including 2 μM GaPc and 2 μM GQ in 50 mM potassium phosphate buffer, pH 6.80, and 300 mM KCl at 25 °C (B).In (B'), fluorescence spectrum of GaPc-GQ (red) was well fitted by two components (blue), that resolved into two bands (B").Green line in B' indicates the base line.The simulated band at shorter wavelength (red-broken line, λ max 687 nm) was well overlapped with the observed fluorescence band of GaPc-G 6a complex (orange), and can be assigned for "type-b".Another simulated bands (blue broken line λ max 703 nm) can be assigned to the "type-a" in the GaPc-GQ complex proceeds as an excited singlet state reaction, i.e., a simple back ET reaction (Fig. 6).Thus the photocytotoxicity of GaPc in vivo could be suppressed by not only G-quadruplex DNAs, but also G-quadruplex RNAs.These results again highlighted the significant effects of the interaction between photosensitizers and other molecules in cell on their PDT efficacy.

Conclusion
We characterized photochemical properties of GaPc in the presence of various DNAs to elucidate the nature of ET (G-GaPc) .We found that GaPc forms an electrostatic complex with a duplex DNA where ET (G-GaPc) is allowed as long as guanine is present in the sequence and that the rate of ET (G-GaPc) (k ET(G-GaPc) ) can be reasonably interpreted in terms of the distance between Pc moiety of GaPc and DNA G base in the complex.The determination of the quantum yields of singlet oxygen ( 1 O 2 ) generation (Φ Δ s) from the GaPc-DNA complexes clearly demonstrated that the photocytotoxicity of GaPc is crucially affected by ET (G-GaPc) .In addition, the red-light irradiation on the GaPc-GQ complex did not lead to decomposition of the G-quadruplex DNA, highlighting the importance of unraveling photochemical properties of photosensitizers in the presence of biomolecules for the molecular design to enhance their PDT efficacy.

Fig. 6
Fig.6 Reaction scheme for the photo-induced electron transfer from the guanine bases to GaPc in the excited state (ET (G-GaPc) ) to generate radical ion pair (D •+ A •− ) in the GaPc-GQ complex.Decay of the generated radical ion pair proceeds through back electron transfer yielding the ground state.The parameters are as follows: k nr : the rate constant of nonradiative deactivation of GaPc.k f : the fluorescence emission rate constant of GaPc.The k f contribution was calculated from τ f0 = 3.8 ns and Φ f = 0.14.k ET(G-GaPc) : the rate constant of ET (G-GaPc).k BET : the rate constant of the back electron transfer.k isc : the rate constant of intersystem crossing of GaPc