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Spectroscopic Characterizations of Semiquinone Anion Radical Formation and Autosensitized Photooxidation for Elsinochrome A.

Byline: Wenjian Lao, Cong Li, Hong Tai andJinmao You

Summary: Elsinochrome A (EA) is a natural perylenequinone known for high photosensitization activity. There are a limited number of studies on its photophysical and photosensitive properties. In this study, formation of semiquinone anion radical and EA autosensitized photooxidation reactions were examined. Rate constant of semiquinone anion radical formation under anaerobic condition, and rate constant of autosensitized photooxidation reaction under aerobic condition were measured for Elsinochrome A (EA) by electron paramagnetic resonance and UV-visible spectra, respectively. The rate constant of formation was estimated at 0.767 min-1 (R2 = 0.963) using the zero-order reaction equation. The autosensitized photooxidation reaction occurred only when irradiation and oxygen co-existed in the EA solution. The average reaction rate constant was 3.22 0.36 (A-10-9, mol.s-1), and EA half-life was 4.48 0.52 (h) in the tetrachloromethane solution.

Keywords: Elsinochrome A; EPR spectra; Semiquinone anion radical; Autosensitized photooxidation

Introduction Natural perylenequinone chemicals such as hypocrellin, cercosporin, erythroaphin, calphostin, hypomycin, phleichrome, and elsinochrome are highly effective photosensitizers and share common photosensitization mechanisms in photodynamic process [1]_ENREF_1. The photosensitizer can be activated to singlet excited state by visible light absorption and then partial excited-state molecules convert into longer-lived triplet state through intersystem crossing. The excited-state molecule can conduct two favorable reactions, i.e., Type-I and Type-II, in photodynamic interaction [2]. In Type-I reaction, the excited-state molecule reacts directly with another reductive substrate to yield semiquinone anion radical that can react with other compounds causing oxidative damage or free radical. Photoinduced electron-transfer is a predominant pathway in the radical formation.

When oxygen exists, the semiquinone anion radical can transfer energy to ground state oxygen (3O2) to form superoxide (O2 ), whereby other active oxygen species such as hydroxyl radical (OH) and hydrogen peroxide (H2O2) can be generated. These highly active oxygen species can oxide other compounds, leading to toxicity. In Type-II reaction, the excited- state molecule reacts directly with 3O2 to generate highly toxic triplet state oxygen (1O2) [1, 3]. The photosensitizer, light, and oxygen are the three necessary components in the photosensitization process [4]. The photoinduced electron-transfer interactions have been studied for hypocrellin A (HA) and hypocrellin B (HB) with a variety of electron-donor substrates such as N,N-diethylaniline (DEA) [5], 1-Benzyl-1,4-dihydronicotinamide (BNAH) [6], and aliphatic amines [7].

Besides Type I and II reactions, a side- reaction known as autosensitized photooxidation can also occur in the photodynamic process [1]. The autosensitized photooxidation reaction is harmful to photodynamic interaction because it destroys the large-ring molecule structure of the photosensitizer and causes quick consumption of the oxygen species. For example, it has been elucidated that HA and HB underwent autosensitized photooxidation, yielding unstable peroxide intermediate that further converted partially into a stable compound with a binaphthyl quinine structure that had no photosensitization activity under long-wavelength visible light irradiation [7].

Elsinochrome A (EA) is one of the four derivatives of elsinochrome pigment that was originally isolated from natural fungus (Fig.1) [8]. Because of it possesses high quantum yield of 1O2 [9], EA has been not only investigated to be a promising pharmaceutical in photodynamic therapy against tumor [10], but also has toxicity to plant cells [11]. Even though EA was found as early as in 1957 [8], relatively little information was available regarding its properties [12]; a partial reason was its limited source of availability [13]_ENREF_11. However, the situation has been improved since Li et al. successfully developed an EA biosynthetic approach in 1998 [14, 15]. After that, Li and his collaborators have conducted a series of investigations including EA structural characterization [16], photophysical and photosensitive properties examination [17, 18], photodynamic activity assessment on inhibition of tumor cell and bacterial growth [10, 19, 20].

On the other hand, Wang and Zhang, and Shen performed theoretical studies on EA photochemical properties in ground state and excited state, respectively [21, 22]_ENREF_17. Recently, Ma et al., studied the interaction between EA and myoglobin [23]. Zhou et al. prepared and characterized an EA embedded silica nano-colloid to improve photostability and aqueous solubility [16, 24]. However, there are still a limited number of studies on EA photophysical and photosensitive properties.

In the present study, the first objective was to estimate rate constant of semiquinone anion radical formation under anaerobic condition by electron paramagnetic resonance (EPR). Dibenzylamine DBA) was utilized as an electron-donor (Fig. 1). The second objective was to examine EA autosensitized photooxidation reaction under UV-visible light irradiation and to estimate rate constant of the reaction.

Experimental

EA was produced by a biosynthetic method that was previously reported [14]. All reagents were optima grade or better. All stock solutions were purged with argon prior to storage in the dark at 4 C until use.

Formation of Semiquinone Anion Radical Under Anaerobic Condition

EPR spectra were recorded at X-band (9.8 GHz) on a Varian E-115 spectrometer at 20 C. The instrumental settings were: microwave power, 5mW; modulation frequency, 100 kHz; sweep width, 200G; modulation amplitude, 1.0G; receiver gain, 2.5 A- 104; time constant, 0.25 second; operation temperature: 20 C. The EA and DBA stock solutions were separately prepared in argon-gassed chloroform at 1 A- 10-3 mol l-1. The working solution was an EA-DBA mixture (1:1, v/v). A high-pressure mercury lamp (500 W) was used as the light source irradiating at 70 cm away from the EA-DBA solution. EPR spectra were recorded for the solution that was bubbled with air or argon after a 30 min light irradiation. In the kinetic experiment, EPR spectra were recorded every 2 min to 30 min for the argon-gassed solution under the light irradiation. Triplicate experiments were conducted.

Autosensitized Photooxidation Reaction Under Aerobic Condition

UV-Vis absorption spectra were recorded in a cuvette (1A- 1A- 4 cm) on a HP 8453 UV-Vis Spectrophotometer (Agilent, USA) at room temperature (E22C). The EA stock solution was prepared at 1 A- 10-4 mol.l-1 in tetrachloromethane (CCl4). After adding 3 mL of the stock solution into a cuvette, keeping the cuvette open and purging oxygen gas into the solution in the dark, the UV-Vis spectra was recorded every 6 minutes. At the 48th minute, after capping the cuvette to prevent solvent from evaporation, the solution was irradiated by a high pressure mercury lamp (250 W) from a distance of 30 cm. The UV-Vis spectrum scan was not stopped until the 114th minute. Triplicate experiments were performed.

Results and Discussion

Formation of Semiquinone Anion Radical Under Anaerobic Condition

No EPR signal was detected in blank chloroform solution, or in the stock solution of DBA or EA without UV-Vis light irradiation (Fig. 2). In the oxygen-saturated EA-DBA solution, the UV-Vis light irradiation excited EA molecules that reacted directly with 3O2 to generate triplet state oxygen (1O2). The 1O2 specie was captured by DBA to yield DBA nitroxide radical (DBA-O -) that created EPR signal [25]. This was a typical Type-II reaction.

In the deoxygenated EA-DBA solution, the UV-Vis light irradiation alternatively produced EPR signal of semiquinone anion radical (EA -) shown in Fig. 2. The EA - was yielded by electron transfer from DBA to the excited-state EA molecule. Because of the absence of 3O2, in the solution, the EA - could remain in the system to create the EPR signal. This photochemical interaction under the anaerobic condition was assigned as a Type-I reaction.

The EPR signal intensity of EA - increased over the irradiation time in the 30-minute kinetic experiment (Fig. 3). These EPR signals were fitted well against irradiation time in the zero-order reaction equation: Equation

where I represents the EPR signal intensity; I0 is the initial EPR signal intensity (here the EPR signal intensity at the 2 minute was used as I0), k is rate constant; t is irradiation time (min). The k value was then solved as 0.767 min-1 (R2 = 0.963). Previously, coauthors of the present work had presented EPR signals of HA semiquinone anion radical (HA - ) recorded under exactly the same experimental conditions, e.g., solvent, concentration, light irradiation, and instrumental setup, as in the present study [19]. For comparison purposes, the HA - time- series signals were also depicted in Fig. 3. Similarly, the k value was calculated as 0.163 min-1 (R2 = 0.986) using Eq.1.

The EPR kinetic results validated that EA could form semiquinone anion radical quickly in the photodynamic interaction. Comparison of k values for EA and HA indicated that the EA - was generated 5 times faster than the HA -. In a recent report, an EA derivative, 5-(3-mercapto-1- propanesulfonic acid)-substituted EA (MPEA), was prepared to improve aqueous solubilityand to be evaluated for photodynamic activity. Its semiquinone anion radical productivity was about 70% of EA in argon-saturated DMSO solutions [19].

Autosensitized Photooxidation Reaction Under Aerobic Condition

EA inherently owns good planarity that facilitates to form a large delocalized p bond and yield the long-wavelength absorption bands. Three major UV-Vis absorption of EA peaks and their logarithm molar extinction coefficients (Log e, in parentheses) in CCl4 were 460 (4.08), 530 (3.88), and 571 nm (3.79). In a preliminary test of the present study, the absorption peaks in some of the other solvents were recorded as 457, 527 and 567 nm in methanol; 459, 528 and 568 nm in ethanol; and 458, 528 and 571 nm in chloroform. The peak around 460 nm was generated by p-p electronic transition of perylenequinone ring, while the peaks at ~528 and ~570 nm were caused by intramolecular proton transfers between phenolic hydroxyl and quinone carbonyl groups [26]. The UV-Vis absorption peaks and Log e has been previously reported for EA as 457(4.11), 527(3.87), and 567nm (3.94) in methanol; 457 (4.31), 528 (4.00) and 571 nm (4.07) in chloroform; and 468.5 nm (4.30) in DMSO[17].

Under aerobic condition, the visible-light absorbance from 460 to 570 nm gradually increased during the oxygen-purging period since the beginning of the EA photooxidation experiment (Fig. 4). Once the light irradiation to the oxygen-saturated solution in the sealed cuvette started, the absorbance declined apparently at the three long-wavelength peaks (460, 530 and 571 nm) but increased at a short- wavelength region around 340 nm (Fig. 5). The absorbance increase at the oxygen-purging period was due to solvent (CCl4) evaporation to raise the solution concentration. The absorbance decrease with the light irradiation indicated EA amount was reduced by a photoinduced reaction, i.e., autosensitized photooxidation, because of only EA and oxygen in the solvent. The absorbance increase at 340 nm was an effect of forming binaphthyl quinone structure from the photooxidation reaction [7].

The absorbance values for each of the three peaks at 460, 530 and 571 nm fitted well in the zero-order kinetic reaction equation:Equation

where C0 and A0 are concentration and absorbance at the beginning of irradiation, respectively; At is absorbance at time t; K is the rate constant of the autosensitized photooxidation reaction. The C0 value was calculated with the BeerLambert law from the initial concentration (1 A- 10-4 mol.l-1). EA absorption half-life in the autosensitized photooxidation was estimated (Half-life = C0 / 2K) for the three peaks (Table-1). The correlation coefficients (Rs) were greater than 0.99 concurring with the zero-order reaction mechanism. The larger K values and shorter absorption half-live at 530 and 571 nm than at 460 nm indicated the intramolecular proton transfer between phenolic hydroxyl and quinone carbonyl groups was more vulnerable to decrease of photosensitivity than the perylenequinone ring.

The average K value and half-life of EA were estimated at 3.22 0.36 (A-10-9, mol.s-1) and 4.48 0.52 (h) from the three absorbance peaks in the autosensitized photooxidation reaction.

Table-1: Characteristics of EA autosensitized photooxi dation reaction in carbon tetrachl oride.

Wavelength (nm)###460###530###571

K (A- 10-9, mol.s-1)###2.83###3.55###3.29

R###0.9996###0.9990###0.9997

Half-life (h)###5.06 0.35###4.03 0.28###4.35 0.33

Under aerobic and illumination condition, EA was prone to fast autosensitized photooxidation, resulting in a loss of EA amount. The K value of EA autosensitized photooxidation was respectively 4.0 and 169 times higher than that of HA (K = 8.1A-10-10, mol.s-1) and HB (K = 1.9A-10-11, mol.s-1) measured in chloroform in an earlier study [27]. This was consistent with an order (EA greater than HA greater than HB) of their quantum yield of 1O2 , despite there was possible uncertainty arisen from solvent difference [17]. Although the large K value demonstrated high photosensitization activity, the EA autosensitized photooxidation reaction could compete against the preferred Type-I or type-II photosensitization process to cause adverse effect to photodynamic action. It has been reported that the K value of HA decreased when HA irradiated under long- wavelength (greater than 530 nm) light [17].

Therefore, EA autosensitized photooxidation reaction is expected to be further examined under the long- wavelength light irradiation with isolation and characterization of photooxidation product.

The autosensitized photooxidation mechanism has been elucidated for hypercrelin and its derivatives [22, 27]. Briefly, under light irradiation, hypercrelin generated 1O2 and O2 - which in turn reacted with hypercrelin to yield light-unstable peroxide intermediate. A fraction of the intermediate decomposed into stable binaphthyl quinone product, while the rest returned back to ground state by releasing 1O2. The peroxide intermediate could dominantly transfer to the stable photooxidation product under short-wavelength light irradiation. The absorption increase at around 340 nm was caused by formation of binaphthyl quinone structure from the photooxidation reaction. Therefore, EA likely followed the similar pathway in the autosensitized photooxidation reaction.

Conclusion

The formation kinetics of semiquinone anion radical and the reaction of autosensitized photooxidation demonstrated EA possessed a very high photosensitization activity. The EPR kinetic experiment validated the capability in fast formation of EA semiquinone anion radical under anaerobic scenario. Under the condition of aerobic illumination, EA was liable to conduct the fast autosensitized photooxidation reaction, which would be unfavorable to the photodynamic action.

Acknowledgments

This work was supported by the Director Foundation of Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences.

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