Fundamental Reaction Mechanisms in Radiation Chemistry and Recent Examples

Abstract

By using radiation chemical methods, such as γ-ray radiolysis and pulse radiolysis, powerful oxidative and reductive reagents, which are not available with other methods, can be generated to promote various reactions. The reaction pathways caused by these oxidative and reductive reagents can be followed by various spectroscopic methods, such as transient absorption and EPR. Therefore, radiation chemical methods are applicable to various systems in a wide variety of fields. For example, our research group has studied the mechanisms of various phenomena by means of radiation chemical methods. In the present chapter, fundamental reaction mechanisms of radiation chemistry are introduced in the initial part. Additionally, our radiation chemical investigations on charge delocalization process, radical ions in the excited state, photocatalytic systems, emitting device, and biological systems are introduced as examples of wide applicability of radiation chemical methods.

1 Introduction

For many years, radiation chemical methods, such as γ-ray radiolysis and pulse radiolysis, have been employed by a variety of researchers as a well-established chemical method [1–5]. Generation of powerful oxidative and reductive reagents, which cannot be obtained by other methods, should be the most important characteristic of radiation chemical methods: these species can be used to initiate redox reactions of various molecules and materials. Therefore, radiation chemistry provides powerful techniques for investigating intermediate species such as radicals and radical ions. It is known that the radical ion species can be generated by other methods such as chemical and electrochemical reactions and photoinduced electron transfer. However, in the cases of chemical and electrochemical reactions, control of the oxidation or reduction states is a rather difficult task for the molecules with similar first and second oxidation or reduction potentials. In addition, heterogeneous generation of radical ion species by electrochemical reaction causes difficulty in the analysis of the reactions. Furthermore, radical ion generation by photoinduced electron transfer means formation of radical ion pair. Thus, contribution of the counter ion species cannot be neglected. On the other hand, radiation chemistry retains many advantages over these methods including high oxidation or reduction ability. Furthermore, the performance of radiation chemistry facilities has been significantly improved by continuous efforts by scientists in this field. These improvements made radiation chemical study more reliable and reproducible.

Our research group has continuously carried out mechanistic investigations of chemical reactions by means of radiation chemical methods [6, 7]. From these studies, we have reported reaction mechanisms of redox reactions of fundamental molecules, excited states, mechanisms of devices that are close to practical application, and biological processes caused by redox reactions. In the present chapter, fundamental reaction mechanisms of radiation chemistry are summarized in the initial part. In addition, our recent researches based on the radiation chemistry were summarized as examples of such studies to show the wide applicability of radiation chemical methods. In the later section, four topics are summarized. The first topic is charge delocalization over stacked or expanded chromophores. By employing structurally well-defined molecules, detailed insight into charge delocalization was obtained. Negative charge delocalization over organic chromophores was also revealed. Charge delocalization in two-dimensionally expanded oligomer is also introduced. The second topic involves the properties of radical ions in the excited state, which can be generated by a combination of pulse radiolysis and laser flash photolysis. The third topic is the investigation of reaction mechanisms of systems related to practical applications such as the photocatalysts and light emitting devices. The fourth topic concerns the effect of radiation to biological materials. In particular, studies on oxidation processes in DNA and structural change of proteins upon irradiation are summarized.

2 Fundamental Reaction Mechanisms in Radiation Chemistry

In this section, fundamental reaction mechanisms during γ-ray irradiation and pulse radiolysis are provided as a representative in radiation chemistry.

2.1 Generation of Radical Ion of Substrate by γ-Ray Irradiation

Generation of radical ions of solute molecule is normally carried out by γ-ray irradiation to low temperature (77 K or lower) glassy matrix of appropriate solvent, which has to be selected on the basis of preferred ion species, i.e., radical cation or radical anion. By employing rigid matrices, conventional steady-state spectroscopic measurements including absorption, fluorescence, and EPR become possible, because chemical reactions of generated intermediates are inhibited in the rigid matrices where translational and rotational motions of solute are highly restricted. As γ-ray source, ⁶⁰Co and ¹³⁷Cs are often employed (Fig. 1.1). ⁶⁰Co disintegrates by emitting two cascade γ-rays of 1.17 and 1.33 MeV, generating ⁶⁰Ni. The half-life of ⁶⁰Co is 5.27 years. ¹³⁷Cs emits γ-ray of 0.66 MeV to generate ¹³⁷Ba with half-life of 30 years. Because of the high energy, the γ-ray irradiation causes inner-shell ionization, the Compton effect, and electron-positron pair formation, generating electron ejection. Although these processes are possible with both solvent and solute, direct ionization of solute molecules is usually negligible due to quite small population compared to solvent molecule. This point is quite different from photochemistry using UV or visible excitation light (1–4 eV), which excites only solute. After ionization of solvent due to radiolysis, ionized molecule and ejected electrons are deactivated by various energy dissipation processes and thermalized within picoseconds. The thermalized species can generate radical ion species as indicated in what follows.

Fig. 1.1

γ-Ray source (60Co, 265.7 TBq) emitting Cerenkov light at Osaka University

For the formation of solute radical anion, 2-methyltetrahydrofuran (MTHF) is often used as a solvent. In MTHF, reduction of solute (S) by solvated electron (e^– _sol) occurs to form S radical anion (S^•−) according to the following scheme:

(1.1)

$$ \text{MTH}{{\text{F}}^{\bullet \text{+}}}\text{+MTHF}\to \text{MTHF(+H}{{)}^{\text{+}}}+\text{MTHF(-H}{{)}^{\bullet }}$$

(1.2)

$${{\text{e}}^{-}}_{\text{sol}}+\text{S}\to {{\text{S}}^{\bullet -}}$$

(1.3)

Reduction of S yielding S^•− by the similar reaction mechanism occurs in alkaline solutions such as ethers, amines, and alcohols.

Concentration of S^•− ([S^•−] (M)) generated by the γ-ray radiolysis can be estimated by the following equation:

$$ [{{\text{S}}^{\bullet -}}]=(GItd/N\text{)(}1000\text{ / }100) $$

(1.4)

where G is so-called G-value and defined as the total number of e^– _sol applicable to reduction of solute per 100 eV energy absorbed by the system, I is the radiation dose rate (eV g⁻¹ min⁻¹), t is the irradiation time (min), d is the density of solvent at the temperature or frozen matrix at 77 K (g cm⁻³), and N is the Avogadro number. In the case of the reduction of solute forming S^•− in MTHF, G = 2.55 can be applied.

For generation of solute radical cation (S^•+), saturated alkylhalides (RX) such as CCl₄ and buthylchloride (BuCl) are employed as a solvent. The oxidation reactions in BuCl are summarized as follows:

(1.5)

$${{\text{e}}^{-}}_{\text{sol}}+\text{BuCl}\to (\text{BuC}{{\text{l}}^{\bullet -}})\to \text{B}{{\text{u}}^{\bullet }}+\text{C}{{\text{l}}^{-}}$$

(1.6)

$$ \text{BuC}{{\text{l}}^{\bullet +}}+\text{S}\to \text{BuCl}+{{\text{S}}^{\bullet +}}. $$

(1.7)

The concentration of the resulted radical cation [S^•+] can be calculated on the basis of Eq. (1.4). G-value of s-BuCl is reported to be 3.25.

As indicated above, the employment of the glassy matrices realizes steady-state spectroscopic measurements of reactive intermediates due to restriction of molecular motion. Slight warming of the rigid glass causes softening of the matrices to start chemical reactions, and such processes can be also followed by various spectroscopic methods.

2.2 Generation of Radical Ions of Substrate by Pulse Radiolysis

In the pulse radiolysis, reactions are initiated by an electron pulse. The electron pulse can be generated by various types of radiation sources such as linear accelerator (linac) and Van de Graaf accelerator. The performance of accelerators depends on the facilities. For example, in the case of the L-band electron linac of Osaka University (Fig. 1.2), electrons injected from the gun (up to 91 nC) are bunched to 20–30 ps and accelerated up to 40 MeV through the acceleration tube. These values can be changed according to the condition of experiments. Furthermore, the laser-photocathode RF electron linear accelerator of Osaka University generates single bunch electron beam shorter than 100 fs. These electron pulses have been employed to follow the reactions in the time domain from sub-picosecond to millisecond.

Fig. 1.2

L-band linac of Osaka University

Fast electrons generated by an accelerator lose its kinetic energy during the passing through the sample by scattering due to atoms or molecules, which causes either electronic excitation or ionization initially. The excited states or ionized species generated initially produce secondary products such as ions and radicals. For example, pulse radiolysis of water caused following ionization (1.8) and electronic excitation (1.9) as the primary processes:

(1.8)

(1.9)

The ionized water undergoes rapid reaction with another water molecule to produce hydronium ion (H₃O⁺) and hydroxyl radical (OH) in 10⁻¹¹ s:

$$ {{\text{H}}_{\text{2}}}{{\text{O}}^{+}}+{{\text{H}}_{\text{2}}}\text{O}\to {{\text{H}}_{\text{3}}}{{\text{O}}^{+}}+\text{OH} $$

(1.10)

Ejected electrons are trapped by water after being thermalized to form thermalized electrons (e⁻ _thermal) and then hydrated electrons (e⁻ _aq).

$$ {{\text{e}}^{-}}\to {{\text{e}}^{-}}_{\text{thermal}}\to {{\text{e}}^{-}}_{\text{aq}}$$

(1.11)

The excited water (H₂O*) dissociates in 10⁻¹³ s to give a hydrogen atom and a hydroxyl radical.

$$ {{\text{H}}_{\text{2}}}\text{O*}\to \text{H}+\text{OH} $$

(1.12)

H₂O₂ is also a reactive intermediate generated from coupling of OH during the pulse radiolysis of water.

$$ \text{OH}+\text{OH}\to {{\text{H}}_{\text{2}}}{{\text{O}}_{\text{2}}}$$

(1.13)

These primary species are localized in the track, called as spur, in which the electron pulse passed through. The generated primary species can escape from the spur with time by diffusion to react with other primary species or solute in water. The G-values are G(e⁻ _aq) = 2.7, G(H) = 0.55, G(OH) = 2.75, G(H₂) = 0.45, G(H₂O₂) = 0.7, G(H⁺) = 2.9, and G(OH⁻) = 0.2 [5].

As indicated above, various species are generated by the initial processes. Thus, for the selective generation of reaction intermediate of the solute, so-called scavengers, which trap un-desirable intermediates, have to be included in the reaction system. For example, in order to achieve oxidation of DNA in water media, we employed a buffer solution including S₂O₈ ²⁻ and t-BuOH ((CH₃)₃COH), since t-BuOH traps hydroxyl radical and S₂O₈ ²⁻ generates oxidant SO₄ ^•− upon reaction with e_aq ⁻ as summarized in (1.14, 1.15 and 1.16) [8].

$$ {{\text{S}}_{\text{2}}}{{\text{O}}_{8}}^{2-}+{{\text{e}}^{-}}_{\text{aq}}\to \text{S}{{\text{O}}_{\text{4}}}^{\text{2}-}+\text{S}{{\text{O}}_{\text{4}}}^{\bullet -} $$

(1.14)

$$ \text{OH}+{{(\text{C}{{\text{H}}_{\text{3}}})}_{3}}\text{COH}\to {{\text{H}}_{\text{2}}}\text{O}{{+}^{\bullet }}\text{C}{{\text{H}}_{\text{2}}}{{(\text{C}{{\text{H}}_{\text{3}}})}_{2}}\text{COH} $$

(1.15)

$$ \text{S}{{\text{O}}_{\text{4}}}^{\bullet -}+\text{S}\to \text{S}{{\text{O}}_{\text{4}}}^{\text{2}-}+{{\text{S}}^{\bullet +}}$$

(1.16)

On the other hand, reduction of substrate can be achieved by e⁻ _aq as indicated by (1.17) in the presence of t-BuOH which traps hydroxyl radical according to (1.15).

$$ \text{e}_{_{\text{aq}}}^{-}+\text{S}\to {{\text{S}}^{\bullet -}}$$

(1.17)

Recently, we reported that guanidine HCl (H₂NC(=NH₂)⁺NH₂·Cl⁻), which is well known denaturant of protein, is effective to reduce solutes such as protein in solution according to the following scheme [9].

$$ {{\text{e}}^{^{-}}}_{\text{aq}}+{{\text{H}}_{\text{2}}}\text{NC(=N}{{\text{H}}_{\text{2}}}{{)}^{+}}\text{N}{{\text{H}}_{\text{2}}}\to {{\text{H}}_{\text{2}}}\text{NC(=N}{{\text{H}}_{\text{2}}}{{)}^{\bullet }}\text{N}{{\text{H}}_{\text{2}}}$$

(1.18)

$$ {{\text{H}}_{\text{2}}}\text{NC(=N}{{\text{H}}_{\text{2}}}{{)}^{\bullet }}\text{N}{{\text{H}}_{\text{2}}}\text{+ S}\to {{\text{H}}_{\text{2}}}\text{NC(=N}{{\text{H}}_{\text{2}}}{{)}^{\text{+}}}\text{N}{{\text{H}}_{\text{2}}}\text{+ }{{\text{S}}^{\bullet -}}$$

(1.19)

During the reduction process, Cl⁻ is used to trap hydroxyl radical according to the following scheme.

$$ \text{C}{{\text{l}}^{-}}\text{+ OH}\to \text{C}{{\text{l}}^{\bullet }}\text{+ O}{{\text{H}}^{\text{-}}}$$

(1.20)

$$ \text{C}{{\text{l}}^{\bullet }}{}\text{+ C}{{\text{l}}^{-}}\to \text{C}{{\text{l}}_{\text{2}}}^{\bullet -} $$

(1.21)

Although Cl₂ ^•− is a possible oxidizing regent, it was confirmed that its effect was negligible in the study of reduction of cytochrome c.

For the oxidation reaction by hydroxyl radical, e⁻ _aq should be trapped. For this purpose, N₂O is often employed due to the reaction indicated in (1.22). In the pulse radiolysis study on the TiO₂ reaction mechanism, we employed N₂O⁻ saturated water to generate hydroxyl radical according to following equation [10].

$$ {{\text{N}}_{\text{2}}}\text{O}\ \text{+}\ {{\text{e}}^{-}}_{\text{aq}}\text{+}\ {{\text{H}}_{\text{2}}}\text{O}\to \text{O}{{\text{H}}^{-}}\text{+}\ \text{OH+}\ {{\text{N}}_{\text{2}}}$$

(1.22)

For reduction and oxidation of organic substrate in organic solvent, reactions similar to those indicated in the section above are applicable. For oxidation, alkyl halides such as 1,2-dichloroethane (DCE) and dichloromethane and benzonitrile are employed as solvents. On the other hand, MTHF and dimethylformamide are used as solvents for the generation of radical anions.

When the excitation energy level of solvent in the singlet and triplet excited states (RH_S,_T*) is higher than that of solute (S), it can be transferred to solute to generate solute in the singlet or triplet excited states (S_S,_T*),

$$ \text{R}{{\text{H}}_{\text{S,T}}}\text{*+S}\to \text{RH+}{{\text{S}}_{\text{S,T}}}\text{*} $$

(1.23)

where RH is hydrocarbon solvent such as cyclohexane and benzene and subscripts S and T indicate singlet and triplet, respectively. In addition to the energy transfer (1.23), recombination of the solute ions provides the solute in the excited states (S_S,_T*) according to the following mechanisms (1.24–1.29):

(1.24)

$$ \text{R}{{\text{H}}^{\text{+}}}\text{+}\ \text{S}\to \text{RH}\ \text{+}\ {{\text{S}}^{\bullet \text{+}}}$$

(1.25)

$${{\text{e}}^{}}_{\text{sol}}\text{+ S}\to {{\text{S}}^{\bullet -}}$$

(1.26)

$$ {{\text{S}}^{\bullet +}}\text{+}\ {{\text{S}}^{\bullet \text{-}}}\to {{\text{S}}_{\text{S,T}}}\text{*}\ \text{+}\ \text{S} $$

(1.27)

$${{\text{S}}^{\bullet \text{+}}}\text{+ }{{\text{e}}^{}}_{\text{sol}}\to {{\text{S}}_{\text{S,T}}}\text{*}$$

(1.28)

$$ {{\text{S}}^{\bullet \text{-}}}\text{+}\ \text{R}{{\text{H}}^{\text{+}}}\to {{\text{S}}_{\text{S,T}}}\text{*}\ \text{+}\ \text{RH} $$

(1.29)

It should be noted that the formation of S_S,_T* by the recombination mechanisms (1.24–1.29) is proposed by the observation of radical ion species of the solute based on the highly time-resolved experiments [4].

As summarized in this section, various reactive intermediates can be generated during the pulse radiolysis. Since the reactive intermediates are generated instantaneously, deactivation or reaction pathways of intermediates can be followed by various experimental methods with better time resolution. Characteristics of the experimental methods are summarized briefly [4].

Electronic transition of intermediates can be measured with transient absorption spectroscopy. In order to follow the reactions of oxidized or reduced solute in solution, measurements in nanosecond to millisecond time scale are essential, because generation of the oxidized or reduced solute by initially generated solvent-related species takes a few tens nanoseconds due to limitation of diffusion in solution. Transient absorption measurements in nanosecond to millisecond time scale can be achieved by using instruments similar to nanosecond laser flash photolysis. That is, combination of steady state or pulsed Xe lamps and gated CCD or photodetectors such as photodiode and photomultiplier realizes the measurements. It is notable that employment of near-IR sensitive photodiode such as InGaAs detector is effective to expand the spectral region. Relatively better signal to noise ratio can be achieved with smaller number of accumulation when compared with other spectroscopic methods.

For the detection of kinetic processes faster than nanosecond such as reactions of the initial species generated by radiolysis like hydrated electron, techniques similar to femtosecond pump & probe, i.e. stroboscopic method, are often employed. In such systems, duration of probe light has to be short and delay time with respect to the electron pulse is controlled by an optical delay. Thus, white continuum or output of an optical parametric amplifier is often employed as a probe light. For determination of kinetics of the intermediate, a number of events have to be accumulated, which causes longer estimation time as well as damage of the sample, usually. In order to diminish these problems, a streak scope can be used as a detector for these measurements, although time resolution is not so high when compared to above pump & probe type measurement.

If intermediate generated during pulse radiolysis is emissive, its emission can be detected by CCD or photodetector. By employment of a streak scope, better time resolution can be expected.

Vibrational spectra of intermediate can be also measured. Raman spectrum of the intermediate generated during pulse radiolysis can be measured by synchronization of the accelerator and pulse laser, which is used as Raman probe. Raman signal can be detected by a gated CCD and the time resolution is determined by the laser pulse duration. For Raman spectrum measurement with better signal to noise ratio, accumulation of substantial number of events is required.

EPR measurement of the intermediate during the pulse radiolysis is also achieved by averaging a number of events. To take a spectrum at a certain delay with respect to an electron pulse, the EPR signals are sampled and averaged by using a box-car averager, under slow sweep of the magnetic field. Time profiles of the intermediate can be obtained by a transient digitizer. Thus, the time resolution is not better than other methods, while the information on the electronic population is quite informative as summarized in this book.

3 Recent Examples of Radical Chemical Works

In this section, some of our works are introduced as examples of the wide applicability of radiation chemical methods.

3.1 Charge Delocalization over Well-Defined Oligomer Systems

3.1.1 Positive and Negative Charge Delocalization on Stacked Benzene Rings

For years, charge transport in organic materials has been an attracting subject for a wide variety of researchers [11]. Recently, Miller et al. and Shanze et al. reported charge transport in conjugated polymers in the picosecond time scale using radiation chemical methods [12, 13]. In these studies, well-defined polymeric structures and high time resolution are indispensable. It should be noted that the charge carrier in organic molecular materials is always stabilized by surrounding molecules [11]. Furthermore, the charge stabilized in the molecular solid exhibits behaviors different from charged molecules isolated in solution or in the gas phase. For understanding the effect of the stabilization in molecular solids, dimer of molecules can be regarded as the simplest model. The charge resonance (CR) band, which manifests an interaction between the radical ion and neutral molecule in a dimeric molecule, is a characteristic property of the charge stabilized over chromophores. Dimer radical cations of aromatic molecules have been extensively studied since the initial observation of the CR band by γ-ray radiolysis of aromatic compounds by Badger and Brocklehurst in the 1960s [14]. It is well recognized from numerous studies that the stabilization energy of the dimer radical cation, which can be evaluated from the peak position of the CR band, is closely related to the conformations of chromophores that form the dimer radical cation [15]. That is, a larger molecular overlap and shorter distance between chromophores generate highly stabilized dimer radical cations, which show CR band at shorter wavelength side. In spite of this simple relation, quantitative analysis on the relation between the structure and amount of stabilization energy was difficult to obtain, because the structure of chromophores in solution was difficult to determine or be fixed. To clarify the relationship between conformation of chromophores and stabilization energy, a molecular system with fixed conformation has to be employed.

Cyclophanes, which possess two benzene rings connected by alkyl chains (Fig. 1.3), will be ideal molecules for studies on charge delocalization because of well-defined face-to-face structure. X-ray crystallographic studies revealed that the distance between the two benzene rings in cyclophanes depends on the number of bridging alkyl chains. In addition, the face-to-face structure is kept even in the oxidized state [16]. It was also confirmed that their molecular structure could be estimated adequately using theoretical calculations based on the density functional theory. In order to elucidate the relationship between structure and stabilization energy, we studied the dimer radical cation of cyclophanes using the radiation chemical methods [17–21].

Fig. 1.3

Molecular structures of cyclophanes (CP). (Reprinted with permission from [18]. Copyright (2006) American Chemical Society)

Among cyclophanes in the study, [3₆]CP is expected to have the shortest transannular distance. The absorption spectrum of the [3₆]CP radical cation, which was generated by pulse radiolysis using DCE as a solvent, showed a CR band at 667 nm, corresponding to 89.7 kJ mol⁻¹ of the stabilization energy [17]. The observed peak was located at a shorter wavelength than the commonly reported peak positions of the CR band (around 1–3 μm), indicating the formation of highly stabilized dimer radical cation. Similarly stable dimer radical cation was confirmed with [3₅]CP and [3₃](1,3,5)CP, although the peak position varied with the number of linker: CP with many linkers gave CR band at shorter wavelength. In order to explain this finding quantitatively, we measured the CR bands of 12 cyclophanes by means of pulse radiolysis [18]. A series of data indicated that the peak position of the CR band tends to shift to longer wavelength side with an increase in the distance between the two benzene rings. The exchange interaction is expected to be important in the interactions between the two benzene rings of [3 _n ]CPs. Therefore, the stabilization energy (E _CR) should be a function of the distance (r) between the two chromophores, i.e., E _CR ∝ exp(− βr), where β is a constant. The estimated stabilization energy was plotted against the transannular distance in Fig. 1.4. The resultant linear relation indicates the importance of the exchange interaction in charge delocalization. The β value was estimated to be 0.83 Å⁻¹. It was also confirmed that the electron-donating or electron-withdrawing nature of substituents does not significantly affect the stabilization energy.

Fig. 1.4

Distance (r) dependence on the stabilization energy (E _CR) for [3 _n ]CP and [4₂](1,4)CP (open circle), M₃CP (solid square), and F _n CPs (solid circle). Solid line is a fitted line for [3 _n ]CP and [4₂](1,4)CP. (Reprinted with permission from [18]. Copyright (2006) American Chemical Society)

For further understanding of charge delocalization over chromophores, the investigation of chromophore arrays with multi-layers is desirable. For cyclophanes, a series of multilayered cyclophanes were successfully synthesized. Therefore, we investigated the transient absorption spectra of three- and four-layered meta- and para-cyclophanes during the pulse radiolysis [19]. Local excitation (LE) and CR bands were successfully observed. It was revealed that the CR band shifted to longer wavelength side with the number of benzene rings. The stabilization energy estimated from the peak position of the CR band implied efficient charge delocalization over the multilayers of cyclophanes. Furthermore, the CR bands showed a slight peak position change attributable to change in the distribution of conformers. The CR band also exhibited a substantially long lifetime, which is due to the smaller charge distribution on the outer layers of multi-layered cyclophanes as expected from the theoretical calculation.

Although there are numerous studies on the delocalization of positive charge over chromophore arrays, the number of reports on delocalization of negative charge is quite small. In the case of the intermolecular dimer radical anions, Kochi et al. reported the CR band of some molecules with electron acceptor nature [22]. For cyclophane, in the 1960s, Ishitani and Nagakura reported an absorption spectrum of the radical anion of [2₂]paracyclophane generated by chemical reduction at low temperature; [2₂]paracyclophane showed a peak due to the dimer radical anion at 760 nm [23]. Despite these studies, a quantitative relation between structure and the stabilization energy of delocalized negative charge has not been estimated. Thus, we carried out γ-ray radiolysis of several cyclophanes and successfully observed the CR band due to the dimer radical anion (Fig. 1.5) [20]. It was confirmed that the peak position of the CR band shifts to longer wavelength side with an increase in the distance between the two benzene rings. This finding was also analyzed in terms of the exchange interaction indicated above. The β value of the dimer radical anion was estimated to be 0.62 Å⁻¹, which is slightly smaller than that of the dimer radical cation (0.83 Å⁻¹). This result indicates that negative charge delocalizes in stacked chromophores similarly to the positive charge; therefore, the negative charge is potentially an effective carrier in organic assemblies like positive charge.

Fig. 1.5

Absorption spectra of γ-ray irradiated cyclophanes in MTHF glassy matrix. 1: [3₂](1,3)CP, 2: [3₂](1,4)CP, 3: [3₃](1,3,5)CP, 4: [3₄](1,2,4,5)CP, 5: [3₄](1,2,3,5)CP, 6: [3₅] CP, and 8: [22](1,4)CP. (Reproduced from [20] with permission from The Royal Society of Chemistry)

The dimer radical anion was also observed with cyclophanes of benzothiadiazole, electron acceptor molecule [21]. In this case, two structural isomers, i.e., the syn- and anti-forms, were investigated. It was revealed that their stabilization energy depends largely on the overlap. These findings indicate that negative charge delocalization is possible in a variety of compounds when the two chromophores are held close to each other by alkyl chains.

3.1.2 Charge Delocalization on Two Dimensional Oligomers

As indicated in the above section, charge delocalized materials can be obtained by stacking of aromatic molecules. Connection of aromatic molecules by adequate covalent bond so as to expand the π-conjugation system is also effective to construct charge delocalized materials. To date various kinds of conjugated materials have been synthesized. Polyacetylene and polythiophene are well known polymeric materials with expanded π-conjugation systems. Among them, polyfluorenes have attracted wide attention due to their excellent optical and electric properties useful in various applications. To reveal their properties, an understanding of the charge states is important. From this viewpoint, radical ion species of oligofluorenes, which can be regarded as a model of polymers due to their well-defined structure, have been examined by means of radiation chemical methods [24].

These linear polymers and oligomers can be regarded as a conjugated material with a one-dimensional π-electron system. On the other hand, conjugated materials with a two-dimensional π-electron system have been realized by recent synthetic efforts. Among them, star-shaped oligofluorenes having a truxene (T) or isotruxene (IT) core are interesting (Fig. 1.6). Because T and IT can be regarded as three overlapping fluorene units, the conjugation pathway will be maintained through the core. To date various interesting optical and electronic properties have been reported for these star-shaped oligofluorenes. In order to understand these properties, we investigated radical ions species of these star-shaped oligofluorenes by means of γ-ray radiolysis to the glassy matrix [25].

Fig. 1.6

Molecular structures of star-shaped oligofluorenes having T or IT as a core. (Reprinted from [25] with permission from Elsevier)

The observed absorption spectra of radical ion species were in the range from the UV to near-IR regions. By using theoretical calculations, the observed peaks were assigned to electronic transitions. In the cases of radical cations, the near-IR bands were assigned to transitions from HOMO-n (n ≥ 1) to HOMO, while the major visible band was assigned to transition from HOMO to LUMO (Fig. 1.7). In the cases of radical anions, on the other hand, the near-IR bands were assigned to transitions from HOMO to LUMO + n (n ≥ 0), while the major visible band was assigned to transition from HOMO-1 to HOMO, which corresponds to HOMO to LUMO of the neutral molecules. Thus, it is indicated that the transition between HOMO and LUMO of the original neutral molecule is significant in the visible region both for radical cation and radical anion. Furthermore, structural changes generating more planar structure upon oxidation and reduction were indicated from the theoretical calculations. It is indicated that the charge is delocalized on the whole molecules. In addition, it is also indicated that the reduction induced larger structural change than oxidation.

Fig. 1.7

Absorption spectra of ITFn in BuCl (A: IT, B: ITF1, C: ITF2, D: ITF3, E: ITF4) at 77 K after γ-ray irradiation. Bar indicates oscillator strength evaluated using TDDFT at UB3LYP/6–31G(d) level. Oscillator strengths for syn- and anti-conformers were indicated by gray and black, respectively. (Reprinted from [25] with permission from Elsevier)

3.1.3 Dissociation Reactions of Charge Delocalized Phenyl-Substituted Ethanes

As indicated in the above section, application of structural constraint to a molecular assembly realizes intramolecular negative charge delocalization. To confirm this issue with more simple organic compounds, we have examined intramolecular negative charge delocalization on phenyl-substituted ethanes: The number of the phenyl rings was varied from three to six [26]. Upon γ-ray irradiation, these phenyl-substituted ethanes showed absorption bands attributable to two kinds of intramolecular charge delocalization, i. e., delocalization over phenyls on the same carbon or that on different carbon atoms of the ethane molecule (namely, 1,1-dimer or 1,2-dimer, respectively. Figure 1.8). It is notable that the peak position of the CR band of the 1,2-dimer is located at longer wavelength side than that of 1,1-dimer, because of weak interaction due to longer distance: In the case of radical anion of 1,1,2,2-tetraphenylethane at 77 K, CR bands due to 1,1- and 1,2-dimer appeared at ~1500 and >2000 nm, respectively. It was revealed from the transient absorption measurements during the pulse radiolysis and absorption measurement of warmed glassy matrix sample after γ-ray irradiation that radial anion of polyphenyl-substituted ethanes undergoes mesolysis generating phenyl-substituted methyl anion and phenyl-substituted methyl radical. Since no mesolysis occurred with disappearance of 1,2-dimer radical anion, the importance of an interaction between π* delocalized over 1,1-diphenyl and σ* of C-C of the ethane backbone was indicated.

Fig. 1.8

Intramolecular negative charge delocalization and mesolysis of 1,1,2,2-tetraphenylethane. I denotes 1,2 dimer. II and III are 1,1-dimer. (Reprinted with permission from [26]. Copyright (2013) American Chemical Society)

3.2 Dimer Radical Cation in the Excited State and Its Dissociation Reaction

During the pulse radiolysis, various intermediates are generated transiently. Since these transient species exhibit characteristic absorption bands different from the original molecule, selective excitation of intermediates to generate intermediates in the excited states is possible by using laser flash photolysis synchronized with pulse radiolysis. The excited intermediates are expected to possess various characteristic properties different from the ground state. The most important one should be electron donor- or acceptor-ability higher than those in the ground state. In addition, by combination of pulse radiolysis and laser flash photolysis, reactions can be caused site-selectively, because the excited intermediate can be generated at the intersection of the electron beam and laser pulse. Furthermore, the amount of the excited intermediate can be controlled by radiation timing of these pulses. We already reported numerous reports on hole and electron transfer from radical cations and anions in the excited states, respectively [6, 27, 28]. For example, hole transfer from stilbene radical cation in the excited state to anisole was reported [27]. By assuming a diffusion-controlled reaction, the lifetime of the radical cation in the excited state was determined. Additionally, we reported that the fluorescence intensity from an radical cation in the excited state depends on the irradiation timing of the laser pulse with respect to the electron pulse [29], indicating that the amount of excited intermediate can be controlled by the radiation timing.

Higher reactivities of the excited intermediate were also observed with other reactions [30]. For example, it was revealed that excitation of the radical ion species induced bond cleavage, even when such cleavage did not occur from the ground state radical ions.

Although the properties of dimer radical cations have been well investigated, those in the excited states have not been reported. This topic is informative, because charge injected to organic materials is expected to be a “hot” carrier corresponding to the excited state in many cases. From this viewpoint, we studied the properties of dimer radical cations in the excited state using the pulse radiolysis-laser flash photolysis combined method [31]. In order to study the excited dimer radical cation, the dimer radical cation of naphthalene (Np₂ ^•+) was selected, because Np₂ ^•+shows a CR band around 1000 nm, which can be selectively excited by the fundamental pulse of Nd:YAG laser (1064 nm).

Np₂ ^•+ was generated by the pulse radiolysis of N_p in DCE. When the CR band of Np₂ ^•+ was excited by the 1064 nm laser flash, the bleaching and recovery of ΔO.D. at 580 and 980 nm were observed with the concomitant growth and decay of ΔO.D. at 700 nm (Fig. 1.9). The immediate decay of the CR (980 nm) and LE (580 nm) bands of Np₂ ^•+ and the rise of the Np^•+ band (700 nm) indicate dissociation of Np₂ ^•+ from Np₂ ^•+* to generate Np^•+ and Np. On the other hand, recovery of these bands indicates quantitative regeneration of Np₂ ^•+ by the dimerization of Np^•+ and Np.

Fig. 1.9

Kinetic traces of ΔO.D. during the pulse radiolysis-laser flash photolysis of naphthalene in DCE at a 580, b 980, and c 700 nm. (Reprinted with permission from [31]. Copyright (2006) American Chemical Society)

It should be noted that the yield of the dissociation of Np₂ ^•+* is quite low (3.2 × 10⁻³), indicating that the main deactivation pathway of Np₂ ^•+* is internal conversion to generate the ground state. In addition, the recombination of the radical cation and neutral molecule in the solvent cage should be efficient and finished within laser duration. These findings indicate that the energy imparted to Np₂ ^•+ is only partly used for dissociation to Np and Np^•+; the majority of the excitation energy is dissipated by rapid internal conversion, which should be the important process when the fate of the “hot” carrier in the organic materials is considered.

Photodissociation of dimer radical cations was also examined by using pyrene (Py) [32]. In the case of Py₂ ^•+, the excitation pulse at 532 nm was absorbed by the LE band of the dimer radical cation, indicating the formation of dimer radical cation in the higher excited state. The quantum yield of photodissociation of Py₂ ^•+ was estimated to be 2.9 × 10⁻³ and the regeneration of Py₂ ^•+ occurred at the rate constant of 4.8 × 10⁹ M⁻¹ s⁻¹ in DCE which is almost equal to k _diff (8.5 × 10⁹ M⁻¹ s⁻¹). From the comparison with dissociation of Np₂ ^•+*, it was suggested that the photodissociation of Py₂ ^•+ occurred from the lowest-excited state Py₂ ^•+(D’₁), even though Py₂ ^•+ was initially excited to the higher-excited Py₂ ^•+(D’ _n ), due to the rapid internal conversion from Py₂ ^•+(D’ _n ) to Py₂ ^•+(D’₁) (Fig. 1.10). Consequently, the internal conversion rate is one of the major factors which govern the yield of the dissociation of Py₂ ^•+.

Fig. 1.10

Schematic diagram of dissociation process of Py dimer radical cation (Py₂ ^•+) via higher excited state. (Reprinted from [32], with permission from Springer)

3.3 Clarification of Photocatalytic Reaction by Radiation Chemistry

Titanium dioxide (TiO₂) has been widely used as a photocatalyst and charge transport material in dye-sensitized solar cells because of high photo-activity and preferable electric characteristics as well as high stability under various conditions. In the applications to photocatalysts, generation of the electron/hole pair upon absorption of a photon with a greater energy than the band gap (3.2 eV) is an initial important process. The electron/hole pair generates a trapped hole and surface-bound OH radicals, which are responsible for the initiation of a wide variety of photocatalytic one-electron redox reactions. However, information about the oxidizing ability of the surface-bound OH radicals is scarce. In order to clarify its oxidizing ability in physical chemical manner, pulse radiolysis was applied [10], since the generation of OH radicals is well established in the field of radiation chemistry as summarized in (1.8–1.13). The pulse radiolysis of N₂O-saturated colloidal TiO₂ aqueous solution generated OH radicals according to (1.22) which were strongly adsorbed on the TiO₂ particles with an apparent association constant of ~ 10⁶ M⁻¹. The OH radicals trapped on the TiO₂ surface exhibited an absorption with a maximum at 370 nm (Fig. 1.11). To clarify the oxidation ability of this species, the oxidation processes of 4-methylthiophenylmethanol (MTPM) and 2-phenylthioethanol (PTE) in an aqueous solution of the colloidal TiO₂ were studied on the basis of a kinetic analysis of the transient absorption. It was revealed that the oxidation ability of the surface-bound OH radical is high but still lower than that of free OH radicals in solution. On the basis of the oxidation potentials of MTPM and PET, it was proposed that the redox potential of surface-bound OH radicals is higher than 1.6 V vs. NHE. Therefore, using the pulse radiolysis method, the nature of surface-bound OH radicals was elucidated.

Fig. 1.11

a Transient absorption spectra at 1 μs after an electron pulse during the pulse radiolysis of colloidal TiO₂ aqueous solution (TiO₂ concentration: 0, 6.2 × 10⁻⁸ and 1.2 × 10⁻⁷ M). b Time profile of ΔO.D. at 370 nm (TiO₂ concentration: a 0, b 6.2 × 10⁻⁸, c 1.2 × 10⁻⁷, and 2.5 × 10⁻⁷ M). c The Benesi–Hildebrand plot for the [TiO₂]_p ⁻¹ vs k _obs ⁻¹, where [TiO₂]_p denotes concentration of TiO₂ particle. (Reprinted from [10], with permission from Elsevier)

3.4 Emission Process by Recombination of Radical Ion Species Generated during Pulse Radiolysis

Electrochemical luminescence (ECL) devices utilize the emission from excited molecules generated by the recombination process of radical cation and radical anion, which were formed by electrochemical oxidation and reduction, respectively. The emission is primarily resulted from the singlet excited state generated by recombination, while contribution of excimer and/or exciplex is also possible. In addition, the singlet excited state generated by triplet-triplet annihilation will be included in emission mechanisms. However, the mechanism for the formation of the singlet excited state and/or excimer as the emissive species was not fully understood. Especially, its kinetics is not known. As summarized in Eqs. (1.24–1.29), pulse radiolysis of nonpolar solvents such as benzene (Bz) generates the hole and electron pair, which undergoes fast geminate charge recombination to mainly produce solvent molecules in the excited states. In addition, a small fraction of the holes and electrons escape to generate radical cations and radical anions of the solute, respectively. Since the recombination of radical cation and radical anion of the solute generates the solute in the excited state, emission from the solute in the excited state is expected. These reactions are similar to the ECL mechanisms. Therefore, the kinetic aspects can be obtained from the study of the pulse radiolysis of the solute in nonpolar solvents [33–40].

We measured emission spectra of various aromatic hydrocarbons (AHs) in Bz during pulse radiolysis [33]. Generation of an emissive molecule can be attributed to charge recombination of radical cation and radical anion as indicated above. This emission mechanism is supported by the relationship between the annihilation enthalpy change (− H◦) for charge recombination and the excitation energies of ¹AH* (E _S1): It was confirmed that the ratio of fluorescence intensity (I) and fluorescence quantum yield (ϕ _fl), which relates to the rate constant of the charge recombination of AH^•+and AH^•−, increases with an increase of the energy difference between − H◦ and E _S1 (Fig. 1.12), which corresponds to the driving force for charge recombination. Therefore, the observed tendency accords with the electron transfer theory, supporting the emission mechanisms based on the charge recombination between radical cation and radical anion generated by the pulse radiolysis.

Fig. 1.12

Relation between ln(I/ϕ _fl) and – ΔH ⁰ -E _S1 , where I and ϕ _fl are emission intensity and fluorescence quantum yield, respectively. Characters close to marks indicate compounds name: An: anthracene, BF: 2,3-benzofluorene, bisPEB: 1,4-bis(phenylethynyl)benzene, B[b]Fl: benzo[b]fluoranthene, B[k]Fl: benzo[k]fluoranthene, Chry: chrysene, Cz: carbazole, DMeAn: 9,10-dimethylanthracene, DPhAn: 9,10-diphenylanthracene, Fl: fluoranthene, MePy: 1-methylpyrene, Pe: perylene, Py: pyrene, Te: tetracene, Th: thianthrene, TPh: triphenylene, Rub: rubrene. (Reprinted from [33], with permission from Elsevier)

Furthermore, we applied the pulse radiolysis technique to a variety of donor-acceptor compounds including compounds that were developed for ECL applications (Fig. 1.13) [34–37]. These molecules showed emission attributable to formation of the singlet excited state and excimer/exciplex. It was shown that both quantitative and qualitative explanations of the emission intensity can be given on the basis of the redox and structural properties. In the case of donor-acceptor quinolones with an ethynyl linkage (PnQ in Fig. 1.13), the emission due to the charge recombination was explained on the basis of positive and negative charge localization on donor and acceptor moieties, respectively, in the radical ion state, in accordance with the ECL mechanisms [34]. The localization of the charge in the radical ion state was also indicated for phenylethynyl substituted cyanoanthracenes (PEA etc. in Fig. 1.13) [35, 36]. In the case of PEA, which takes a planar structure, both monomer and excimer emission occurred upon charge recombination. On the other hand, DEA with twisted structure showed strong charge transfer emission. Excimer emission was also confirmed with arylethynyl substituted pyrenes [37].

Fig. 1.13

Structure of donor-acceptor molecules investigated using pulse radiolysis. (Reprinted with permission from [34–37]. Copyright (2005, 2006) American Chemical Society)

During the pulse radiolysis of the benzene solution of donor-acceptor substituted tetrakis(phenylethynyl)benzenes (TAEB, Fig. 1.14), emission with a peak, which largely depends on the CT character induced by substituents as well as its conjugation pathway, was observed [38, 39]. Because donor/acceptor substituted TAEBs possess three types of conjugation pathways (linear conjugated, cross-conjugated, and bent conjugated pathways between donor and acceptor substituents through ethynyl linkage), CT interaction largely depends on the substitution patterns. It was revealed that the emission peak varied from 405 to 640 nm depending on the substitution pattern [38, 39]. These results will be interesting examples showing that the substitution pattern allows one to control the emission peak precisely.

Fig. 1.14

Molecular structures of donor-acceptor substituted tetrakis(phenylethynyl)benzenes. (Reprinted with permission from [38, 39]. Copyright (2007, 2008) American Chemical Society)

It is interesting to note that a dimerization reaction of the radical anions of bis(phenylethynyl)benzenes was found during the study of their radical ion species [41] similar to that of the radical anion of biphenylacetylene [6].

3.5 Application to Biological Systems

3.5.1 Charge Transfer in DNA

For years, biological systems have been important subjects for radiation chemistry because of the significant interests on the effects of radiation to biological systems. One of the well investigated materials from the viewpoints of the radiation effects is DNA, because it is well known that the oxidation and reduction of DNA are closely related to DNA damage and repair, respectively [42–44]. To understand DNA damage or repair at a site apart from the initially oxidized or reduced sites, respectively, charge transfer along DNA has to be taken into account. Thus, charge transfer in DNA has been investigated by many researchers. Our research group has also investigated charge transfer in DNA using various methods including pulse radiolysis.

Nowadays, it is widely accepted that both tunneling and hopping mechanisms are included in hole transfer in DNA [45]. For the tunneling mechanism, the hole transfer rate (k _HT) can be expressed by a β factor of 0.6–0.7 Å⁻¹ in k _HT ∝ exp(− βr), where r is the distance between the donor and acceptor [46, 47], while the hopping mechanism provides a smaller β value [48]. In order to estimate the β value, our research group applied pulse radiolysis to pyrene (Py)-conjugated oligodeoxynucleotides (ODNs) in the presence of K₂S₂O₈, which generates oxidant SO₄ ^•− upon reaction with e_aq ⁻ to realize oxidation of DNA and/or Py according to (1.14, 1.15 and 1.16). Upon oxidation of ODN, hole transfer from G moiety in ODN to Py or from Py to 8-oxo-7,8-dihydroguanine (oxG) was expected [49]. From transient absorption measurements during the pulse radiolysis, both hole transfers were confirmed. From the distance dependence of the observed hole transfer rate from Py to oxG, the β value was estimated to be 0.6 Å⁻¹ in accordance with the reports by other research groups.

It is well known that multistep hopping mechanism makes long range hole transfer in DNA possible. In order to clarify the factors that govern the hopping rate, we employed Py-modified DNA in the pulse radiolysis study. By analyzing the kinetic trace of the transient absorption band of a Py radical cation (Py^•+) formed by hole transfer from DNA to Py, the rate constants for hole transfer in various sequences of DNA were determined [8]. It was revealed that the rate constants of the hole transfer from the nearest G to Py were weakly dependent on the distance between them, indicating the hole transfer in DNA by the multistep hopping mechanism. In contrast, in the hole transfer where the rate-determining step was the single-step hole transfer between guanines (Gs), the rate was strongly dependent on the distance. By comparing the intervening nucleobases between Gs, it was revealed that the rate constants of multistep hole transfer were in the order of G^•+AG > G^•+AC > G^•+TG. These results showed that A can mediate hole transfer between Gs effectively. The effect of multiple Gs (GGG) on multistep hole transfer was also examined. It was indicated that the hole transfer from DNA to Py became slow when the GGG site is located at a distant position from Py. The hole transfer rate in ODN with seven base pairs between Py and GGG decreased by one order when compared to that of ODN with one base pair between Py and GGG. The present finding indicates that the multiple G acts as an efficient hole trap site, which decreases the hole transfer rate in ODN.

In addition to the GGG consecutive site, we investigated the possibility of the planar G quartet in G quadruplex as a hole trapping site by means of the transient absorption measurement during the pulse radiolysis (Fig. 1.15) [50]. It was revealed that the spectrum of G + C observed from the G-quadruplex is red-shifted compared to the spectra with two or three consecutive G bases (Fig. 1.16), supporting that the hole trapping in the planar G quartet of the G-quadruplex is favored because of the delocalized positive charge along the more extended π orbitals. Meanwhile, the spectra with absorption maxima at 390 and 550 nm observed at longer delay times are due to the neutral radical of G [G + C(–H)], which is the deprotonated species of G + C. The rate constant for the formation of G + C(–H) was determined to be 4.0 × 10⁶ s⁻¹. Therefore hole trapping process in DNA was reasonably investigated by the radiation chemical method.

Fig. 1.15

Molecular structure of riboflavin labeled oligonucleotide and G-quadraplex. (Reprinted from [50], with permission from Wiley)

Fig. 1.16

a Transient absorption spectra during the pulse radiolysis of G-quadraplex (5′-TAGGG(TTAGGG)₃TT−3′) in potassium buffer. b Kinetic profiles of ΔO.D. at indicated wavelengths. Red curve indicates fitted curve obtained by global analysis. (Reprinted from [50], with permission from Wiley)

As indicated above, mechanisms of hole transfer are well established. On the other hand, the report on excess electron transfer is rather limited, probably because reduction of nucleobases is rather difficult due to their low reduction potentials. Thus, application of radiation chemical methods is effective in the study of the excess electron transfer in DNA. By applying EPR to the γ-ray-irradiated DNA at low temperature, Sevilla et al. reported the contribution of tunneling and hopping mechanisms in excess electron transfer in DNA [51]. Kobayashi et al. revealed the delocalization of the excess electron over nucleobases using pulse radiolysis [52]. Our research group investigated excess electron transfer in DNA conjugated with naphthalimide (NI) using the pulse radiolysis method [53]. Reduction of DNA was achieved according to (1.17). Although the formation of radical anion of NI (NI^•−) due to excess electron transfer from DNA to NI was expected, the component due to excess electron transfer was not confirmed in the time profile of NI^•−, indicating that excess electron transfer in DNA occurs very quickly. This assumption was confirmed by our recent laser flash photolysis studies on oligothiophene or N, N-dimethylaminopyrene conjugated DNA [54, 55].

3.5.2 DNA Motion

Conformational change of DNA is also an important subject in biological science because DNA is known to take a variety of structures, which relate to its biological functions. To reveal such DNA structures, various structural probes have been developed. For this purpose, the FRET system using fluorescence probes has been often employed. It should be noted that a probe which can monitor DNA structure in a long time domain is preferable. From this viewpoint, we examined to utilize the CR band which can be generated during the pulse radiolysis as a structure probe of DNA [56]. For this purpose, the formation of a Py dimer radical cation (Py₂ ^•+) in doubly Py-conjugated ODNs upon one-electron oxidation during the pulse radiolysis was investigated (Fig. 1.17). The formation of Py^•+ within 5 μs was confirmed by the observation of a 470 nm-band during the pulse radiolysis of doubly Py-conjugated ODN in D₂O in the presence of K₂S₂O₈. With the decay of Py^•+, the concomitant formation of Py₂ ^•+, which has an absorption peak at 1500 nm (CR band), was confirmed in the time range of ~ 100 μs. The rate of formation of Py₂ ^•+ in DNA reflects the dynamics of DNA, which allows the interaction between Py^•+ and Py, since the transient DNA structure is trapped by the attractive CR interaction to produce Py₂ ^•+. The formation rate of Py₂ ^•+, which has a characteristic CR absorption band in the near-IR region, is useful to obtain structural and dynamic information on transient DNA structures in the time range of 1 μs to 1 ms. Application of the CR band is also beneficial, because the CR bands usually appear at a wavelength region where other species do not exhibit absorption. These results indicate that pulse radiolysis is also useful for investigating the various dynamic aspects of DNA such as charge transfer and conformational motions.

Fig. 1.17

Formation of Py dimer radical cation (Py₂ ^•+) in Py-modified DNA (Py-Py-ODN) during the pulse radiolysis. (Reprinted with permission from [56]. Copyright (2003) American Chemical Society)

3.5.3 Protein Dynamics

Since the oxidation and reduction processes promote various biological process, pulse radiolysis will be one of the promising methods for these studies although examples of pulse radiolysis work on the biological systems are rather limited. We applied the pulse radiolysis to reveal the folding process of cytochrome c (Cyt c) [9]. In order to realize selective reduction of oxidized Cyt c, we applied high concentration of the denaturant, guanidine HCl (GdHCl, H₂NC(=NH₂)⁺NH₂·Cl⁻), which generates guanidine radical as a reducing reagent from the reaction between the solvated electron and guanidine as indicated in (1.18, 1.19, 1.20 and 1.21). From the kinetic profile of ΔO.D. at Q-band region during the pulse radiolysis of Cyt c, it was confirmed that ligation of Met80 and reduction of oxidized Cyt c occurred with time constants of 2.3 and 7.7 μs (Fig. 1.18). We also suggested that the dynamics which is characterized by 204 μs of the time constant is due to the intermediate-folding kinetics corresponding to the collapse of the unfolded Cyt c into a compact denatured structure. In addition, the slow dynamics (110 s⁻¹) attributable to the rearrangement to the native conformation was observed. These results indicate that application of pulse radiolysis is also effective to investigate the dynamics of proteins upon oxidation and reduction processes.

Fig. 1.18

Folding dynamics of oxidized Cyt c upon reduction during the pulse radiolysis. (Reprinted with permission from [9]. Copyright (2012) American Chemical Society)

By employing the reduction agent generated by the pulse radiolysis, we also investigated folding dynamics of myoglobin (Mb) upon reduction [57]. To obtain structural information, we combined pulse radiolysis and time-resolved resonance Raman spectroscopy. Upon reduction, the folded metmyoglobin (metMb), which has a six-coordinated heme geometry linked with a water molecule as a distal ligand, is structurally relaxed to the deoxymyoblobin (deoxyMb) form with a five-coordination heme geometry without water ligand. Meanwhile, the Raman spectrum of an unfolded metMb is almost identical to those of the unfolded deoxyMb formed by the reduction (Fig. 1.19), indicating that both unfolded metMb and deoxyMb have similar heme geometries. The results provided herein show that upon reduction, the folded metMb with a six-coordinated heme geometry is structurally relaxed to deoxyMb with a five-coordination heme geometry, while both unfolded metMb and deoxyMb have a six-coordinated heme geometry linked with a water molecule or histidine as a distal ligand (Fig. 1.20).

Fig. 1.19

Time-resolved resonance Raman spectra of metMb in the presence of GdHCl during the pulse radiolysis in buffer solution [57]

Fig. 1.20

Proposed structural change in the heme moiety of both folded (a ) and unfolded (b ) metMb followed by one-electron reduction [57]

4 Summary

In the initial part of the present chapter, fundamental reaction mechanisms in radiation chemistry are introduced. Because radiation chemical methods provide powerful oxidative and reductive species, radiation chemical methods can be applied to a variety of systems where oxidation and reduction processes play important roles. The time resolution of these systems is much improved nowadays. Furthermore, the application of various time-resolved spectroscopic techniques, such as IR, Raman, EPR, and so on, will enhance the availability of radiation chemical methods for researchers. Actually, we have been engaged to the studies on the variety of subjects as introduced in this section. Thus, the continuous contribution of the radiation chemical methods to a wide variety of research fields is expected.