Identification of the radicals formed in the reactions of some endogenous photosensitizers with oleic acid under the UVA irradiation.

Electron spin resonance measurements were performed for the reactions of some endogenous photosensitizers (flavin mononucleotide or flavin adenine dinucleotide or folic acid or β-nicotinamide adenine dinucleotide or β-nicotinamide adenine dinucleotide phosphate or pyridoxal-5'-phosphate or urocanic acid) with oleic acid under the ultraviolet light A irradiation using α-(4-pyridyl-1-oxide)-N-tert-butylnitrone as a spin trap reagent. Of the endogenous photosensitizers, prominent electron spin resonance signals (α(N) = 1.58 mT and α(H)β = 0.26 mT) were observed for the reaction mixture of flavin mononucleotide (or flavin adenine dinucleotide or folic acid), suggesting that radical species form in the reaction mixtures. Singlet oxygen seems to participate in the formation of the radicals because the electron spin resonance peak heights increased for the reactions in D(2)O to a great extent. A high performance liquid chromatography-electron spin resonance-mass spectrometry was employed to identify the radicals formed in the reactions of the endogenous photosensitizers (flavin mononucleotide or flavin adenine dinucleotide or folic acid) with oleic acid under the ultraviolet light A irradiation. The high performance liquid chromatography-electron spin resonance-mass spectrometry analyses showed that 7-carboxyheptyl and 1-(3-carboxypropyl)-4-hydroxybutyl radicals form in the reaction mixture of flavin mononucleotide (or flavin adenine dinucleotide or folic acid).

Introduction

Of the various oxidative stresses, ultraviolet (UV) irradiation is one of the primary factors. The ultraviolet light A (UVA) component (320–400 nm) of solar radiation has been shown to produce deleterious biological effects in which singlet oxygen 1O2 (1Δg) plays a major role. Yasui and Sakurai( confirmed the generation of 1O2 (1Δg) in the skin of live rodents exposed to UVA using chemiluminescent detection and imaging. This must have an effect on all tissue that gets into contact with UVA, particularly the skin and the eye.

There seems to be a mechanism for the generation of 1O2 (1Δg) in biological systems. The type-II reaction of a photosensitizer transfers its energy to a ground state oxygen molecule to form 1O2 (1Δg).( Ragas et al.( showed that singlet oxygen is produced in gram-negative Escherichia coli bacteria in the presence of a cationic porphyrin by a combination of spectroscopic and time-resolved photophysical techniques. On the other hand, singlet oxygen was directly detected in the solution of endogenous photosensitizers such as riboflavin, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), β-nicotinamide adenine dinucleotide (NAD), β-nicotinamide adenine dinucleotide phosphate (NADP), pyridoxal-5'-phosphate (PLP), urocanic acid and cholesterol.(

Yamazaki et al.( found that the product of the 1O2 (1Δg)-specific oxygenation of cholesterol, 3β-hydroxy-5α-cholest-6-ene-5-hydroperoxide, accumulated in the skin of rats treated with an oral dose of a photosensitizer, pheophorbide and subsequently exposed to visible light. The UV-induced lipid peroxidation also occurs through a non-radical pathway by a direct reaction with singlet oxygen (1Δg)( in addition to a free radical chain reaction.(

Increasing electron spin resonance (ESR) evidences showed that free radicals may be involved in the acute sunburn reactions. Indeed, reactive oxygen speceis (ROS) such as hydroxyl radicals (HO•)( and superoxide anion radical (O2−•)( were detected under the UV irradiation using ESR spin trapping technique. Furthermore, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) or α-(4-pyridyl-1-oxide)-N-tert-butyl nitrone (4-POBN)/lipid-derived radical adducts were also observed in the epidermal homogenate of albino rat skins following UV exposure,( in UV light exposed mouse skin,( and in human skin exposed to solar-irradiation.(

In spite of the intensive ESR studies, chemical structures of the radical species formed in the epidermal homogenate of albino rat skins following UV exposure,( in UV light exposed mouse skin,( and in human skin exposed to solar-irradiation have not been determined.( In this study, identification of the lipid-derived radicals is performed for the reactions of some endogenous photosensitizers with oleic acid under the UVA irradiation (365 nm) using ESR, HPLC-ESR and HPLC-ESR-MS.( A spin trap reagent, 4-POBN was used to trap the lipid-derived radicals.

Materials and Methods

Materials

4-POBN, FMN, urocanic acid, NAD, NADP and oleic acid were purchased from Tokyo Kasei Kogyo, Ltd. (Tokyo, Japan). FAD, cholic acid, deuterium oxide (D2O) and thymidine-5'-monophospate (TMP) were purchased from Wako Pure Chem. Ind. Ltd. (Osaka, Japan). 2'-Deoxyguanosine-5'-monophospate (GMP), 2'-deoxycytidine-5'-monophospate (CMP), 4-oxo-2,2,6,6-tetramethylpiperidine (4-oxo-TEMP) and PLP were from Sigma-Aldrich Co. (St. Louis, MO). 2'-Deoxyadenosine-5'-monophospate (AMP) was from Oriental Yeast Co. Ltd. (Tokyo, Japan). Water used in these experiments was purified by passing through Milli-Q Jr. (Nihon Millipore Kogyo K.K., Yonezawa, Japan) after distillation. All other chemicals used were of analytical grade.

ESR, HPLC-ESR, and HPLC-ESR-MS analyses

The ESR, HPLC-ESR, and HPLC-ESR-MS analyses were performed as described in our previous paper.(

Standard reaction mixture

The standard reaction mixture contained 4.3 mM oleic acid, 25 µM FMN (or FAD or folic acid or urocanic acid or NAD or NADP or PLP), 40 mM phosphate buffer (pH 7.4), 10 mM cholic acid, 0.1 M 4-POBN and 1 mM FeSO4 in a quartz test tube (100 mm long × 8 mm i.d.). The standard reaction mixtures without 0.1 M 4-POBN and 1 mM FeSO4 were exposed to the 7.8 J/cm2 light of 365 nm using a REX 250 irradiation system with a LXO365 bandpass filter (ASAHI SPECTRA Co., Tokyo, Japan) unless otherwise noted. After the irradiation, 0.1 M 4-POBN was added. The reactions were started by adding 1 mM FeSO4. The reactions were performed at 25°C for 1 min. The reaction mixtures were aspirated into a Teflon tube centered in an ESR microwave cavity. And then, ESR spectra were measured.

Reaction in D2O

Reactions in D2O were performed as described in our previous paper.(

Ultraviolet and visible absorption spectra

Ultraviolet and visible absorption spectra were measured using a model UV-160A ultraviolet-visible spectrometer (Shimadzu Co., Kyoto, Japan). The spectrometer was operated from 300 to 550 nm. The measurements were performed at 25°C. The measurements were carried out in a cuvette with a 10 mm light path. In the reference cell, 200 mM phosphate buffer (pH 7.4) was contained. The ultraviolet and visible measurements were performed for the mixtures of 50 µM FMN in the 200 mM phosphate buffer.

Fluorescence spectroscopy

The fluorescence spectra of the samples were taken using a 650–10S fluorescence spectrophotometer (Hitachi, Ltd., Tokyo, Japan). The excitation wavelength used were 365 nm. The range of emission wavelength is 450 to 700 nm.

Results

ESR measurements of the standard reaction mixtures of some endogenous photosensitizers with oleic acid under the UVA irradiation

After 7.8 J/cm2 (or 23.4 J/cm2) irradiation of 365 nm, ESR spectra of the standard reaction mixtures of some endogenous photosensitizers with oleic acid were measured (Fig. 1). Prominent ESR signals (αN = 1.58 mT and αHβ = 0.26 mT) were observed for the standard reaction mixture of FMN (or FAD or folic acid) (Fig. 1 A–D), suggesting that free radicals form in the standard reaction mixture of FMN (or FAD or folic acid). The relative ESR peak height of FAD is 32 ± 9% of the standard reaction mixture of FMN (Table 1). The relative ESR peak heights of folic acid are 38 ± 7% (7.8 J/cm2) and 61 ± 13% (23.4 J/cm2) of the standard reaction mixture of FMN, respectively (Table 1). The ESR signals were hardly observed for the standard reaction mixture of FMN without the UVA (365 nm) irradiation (or Fe2+ or FMN or oleic acid) (Fig. 2 B–E). This is also the case for the reaction mixture of FAD (or folic acid) (data not shown). To know whether singlet oxygens are involved in the radical formation, the reactions were performed in D2O. The ESR peak heights of the reaction of FMN (7.8 J/cm2) [or FAD (7.8 J/cm2) or folic acid (23.4 J/cm2)] in D2O increased compared with the reactions in H2O (Table 1). Thus, singlet oxygen seems to be involved in the formation of the radicals because D2O increases the lifetime of singlet oxygen.(

Fig. 1

ESR spectra of the standard reaction mixtures of some endogenous photosensitizers under the UVA irradiation. The standard reaction mixtures of FMN under UVA irradiation (7.8 J/cm2) (A), FAD under UVA irradiation (7.8 J/cm2) (B), folic acid under UVA irradiation (7.8 J/cm2) (C), folic acid under UVA irradiation (23.4 J/cm2) (D), urocanic acid under UVA irradiation (23.4 J/cm2) (E), NAD under UVA irradiation (23.4 J/cm2) (F), NADP under UVA irradiation (23.4 J/cm2) (G), and PLP under UVA irradiation (23.4 J/cm2) (H).

Table 1

Relative ESR peak heights of various endogeneous photosensitizers

Photosensitizers(a)	Relative ESR peak height (%) ± SD (in H2O)(b)	Relative ESR peak height (%) ± SD (in D2O)(c)
FMN (7.8 J/cm2)	100	336 ± 78
FAD (7.8 J/cm2)	32 ± 9	89 ± 10
Folic acid (7.8 J/cm2)	38 ± 7	—
Folic acid (23.4 J/cm2)	61 ± 13	260 ± 33
Urocanic acid (23.4 J/cm2)	0	0
NAD (23.4 J/cm2)	0	0
NADP (23.4 J/cm2)	0	0
PLP (23.4 J/cm2)	0	0

The data presented are the percentages in comparison with the ESR peak height of the standard reaction mixture of FMN. The data are mean ± SD of three independent experiments. Irradiation energies are shown in the parentheses.(a) The reactions were performed in H2O(b) or D2O.(c)

Fig. 2

ESR spectra of the standard reaction mixtures of FMN under the UVA irradiation. The standard reaction mixtures of FMN (A), FMN without light (B), FMN without Fe2+ (C), FMN without FMN (D), and FMN without oleic acid (E).

The ESR peak heights under the UVA irradiations of the various wavelengths

After 7.8 J/cm2 irradiations of the various wavelengths (313, 334, 365, 405, and 436 nm), the ESR spectra were measured for the standard reaction mixtures of FMN (Fig. 3). The relative ESR peak heights were as follows, 46 ± 4% (313 nm), 74 ± 14% (334 nm), 88 ± 8% (405 nm) and 94 ± 17% (436 nm) of the ESR peak height (365 nm). Since absorbances of FMN at the respective wavelengths are 0.103 (313 nm), 0.246 (334 nm), 0.496 (365 nm), 0.361 (405 nm) and 0.588 (436 nm), the reaction is closely related to the ultraviolet-visible absorbance of FMN (Fig. 4). The FMN seems to work in the reactions as a photosensitizer.

Fig. 3

The ESR spectra under the UVA irradiation of various wavelengths. The standard reaction mixtures of FMN without both 0.1 M 4-POBN and 1 mM FeSO4 were exposed to the various wavelength lights (7.8 J/cm2) using a REX 250 irradiation system with a LXO 313 (313 nm) [or LXO334 (334 nm) or LXO365 (365 nm) or LHO405 (405 nm) or LHO436 (436 nm)] bandpass filter (ASAHI SPECTRA Co., Tokyo, Japan). (A) 313 nm. (B) 334 nm. (C) 365 nm. (D) 405 nm. (E) 436 nm.

Fig. 4

An ultraviolet and visible absorption spectrum of FMN.

Irradiation energy dependence of the ESR peak heights

Irradiation energy dependence of the ESR peak height was measured for the standard reaction mixtures of FMN (or FAD) (Fig. 5). The ESR peak heights increased with the increase of the irradiation energy for the standard reaction mixtures of FMN, and reached 2.8 cm at 15.6 J/cm2. On the other hand, the ESR peak height increased with increase of the irradiation energy for the standard reaction mixtures of FAD, and reached a plateau (1.0 cm) at 1.95 J/cm2, suggesting that FAD is a weak phtosensitizer compared with FMN.

Fig. 5

Irradiation energy dependence of the ESR peak heights. Standard reaction mixture of FMN (– I –). Standard reaction mixture of FAD (– ○ –).

Measurments of fluorescence spectra of FMN and FAD

To know whether or not adenosine moiety in the FAD quenches the singlet excited states of FAD, fluorescence spectra were measured for the standard reaction mixture of FMN (or FAD) without both ferrous ions and 4-POBN (Fig. 6). An intense fluorescence spectrum was observed for the solution of FMN. While, FAD showed a weak fluorescence spectrum. In order to clarify the effect of adenosine moiety in FAD on the fluorescence spectrum, fluorescence spectra were measured by adding various concentrations of AMP to the standard reaction mixture of FMN without both ferrous ions and 4-POBN (Fig. 7). The intensity of fluorescence spectra decreased with the increase of the concentration of AMP, suggesting that AMP quenches the fluorescence of FMN. At 26.7 mM of AMP, the intensity of the fluorescence decreased to 46.6 ± 1.6% of the standard reaction mixture without both ferrous ions and 4-POBN.

Fig. 6

Fluorescence spectra of FMN and FAD. FMN and FAD were dissolved in 0.2 M phosphate buffer (pH 7.4). 5 µM FMN ( — ). 5 µM FAD ( - - - ).

Fig. 7

The effect of AMP on the fluorescence spectra and the ESR peak height of the 4-POBN radical adduct and 4-oxo-TEMPO. The measurements of fluorescence spectra were done for the standard reaction mixtures of FMN without Fe2+ and 4-POBN with various concentration of AM P (– – – –). The data presented are the percentages in comparison with the one of the standard reaction mixtures of FMN without Fe2+, 4-POBN, and AMP. ESR measurements of 4-POBN radical adduct were performed for the standard reaction mixtures of FMN with various concentration of AMP ( - - - - - ). ESR spectra of 4-oxo-TEMPO were observed for the standard reaction mixture without Fe2+ and 4-POBN with 0.1 M 4-oxo-TEMP and various concentrations of AMP ( — ). The data presented for the 4-oxo-TEMPO and the 4-POBN radical adducts are the percentages in comparison with the one of the standard reaction mixtures of FMN without AMP.

The effect of AMP on the formation of 4-POBN radical adduct and 4-oxo-2,2,6,6-tetramethylpiperidine-N-oxyl (4-oxo-TEMPO)

Since AMP quenches the fluorescence of FMN, effects of AMP on the formation of the radicals and singlet oxygen 1O2 were examined. We used the trap probe 4-oxo-TEMP to detect 1O2. This probe, which has been shown to be specific for 1O2 detection,( reacts with 1O2 to yield a stable nitroxide radical 4-oxo-2,2,6,6-tetramethylpiperidine-N-oxyl (4-oxo-TEMPO), having a known three-line ESR spectrum. The stable nitroxide radical 4-oxo-TEMPO was detected in the standard reaction mixture of FMN without Fe2+ and 4-POBN with 0.1 M 4-oxo-TEMP. On addition of AMP to the standard reaction mixture without Fe2+ and 4-POBN with 0.1 M 4-oxo-TEMP, the ESR peak heights of 4-oxo-TEMPO decreased, AMP dose-dependently (Fig. 7). At 2 mM of AMP, the ESR peak heights of 4-oxo-TEMPO decreased to 27.3 ± 0.6% of the standard reaction mixture of FMN without Fe2+ and 4-POBN with 0.1 M 4-oxo-TEMP. On the other hand, the ESR peak height of the 4-POBN radical adduct also decreased with the increase of the concentration of AMP (Fig. 7). At 2 mM of AMP, the ESR peak heights of 4-POBN radical adduct decreased to 51 ± 6% of the standard reaction mixture of FMN.

The effects of the other nucleotides (GMP or CMP or TMP) on the ESR peak height of the 4-POBN radical adduct were investigated for the standard reaction mixtures of FMN with 2 mM GMP (or CMP or TMP) (Table 2). On addition of 2 mM GMP, the ESR peak heights of 4-POBN radical adduct decreased to 37 ± 11% of the standard reaction mixture of FMN. The CMP and TMP showed no inhibitory effect.

Table 2

Effects of nucleotids on the ESR peak heights of the 4-POBN radical adducts

Compounds added	Relative ESR peak height (%) ± SD
AMP	51 ± 6
GMP	37 ± 11
CMP	110 ± 2
TMP	94 ± 8

The reaction was performed for the standard reaction mixture of FMN with 2 mM AMP (or GMP or CMP or TMP). The data presented are mean ± SD of three independent experiments. The relative ESR peak heights (%) are percentages in comparison with the ESR peak height of the standard reaction mixture of FMN.

HPLC-ESR analyses of the standard reaction mixture of FMN under the UVA irradiation

In order to determine the structure of the radicals formed in the standard reaction mixtures of FMN under the UVA irradiation, the HPLC-ESR analyses were performed for the standard reaction mixtures of FMN. A prominent peak (peak 3) was observed on the HPLC-ESR elution profile of the standard reaction mixture of FMN under the UVA irradiation (Fig. 8A). The retention time of the peak 3 is 36.9 min. The peak 3 was hardly observed for the standard reaction mixture of FMN without the UVA irradiation (or Fe2+ or FMN or oleic acid) (Fig. 8 B–E).

Fig. 8

HPLC-ESR analyses of the standard reaction mixtures of FMN. The total volume of the reaction mixture was 3.0 ml. The standard reaction mixture of FMN (A), FMN without light (B), FMN without Fe2+ (C), FMN without FMN (D), FMN without oleic acid (E), and FMN in D2O (F).

The standard reactions of FMN were performed in D2O (Fig. 8F). The HPLC-ESR analyses of the reaction mixtures of FMN in D2O showed that peak height of the peak 3 increased to 297 ± 26% of the one in H2O (Fig. 8F). Thus, singlet oxygen seems to be involved in the formation of the radical of the peak 3 because D2O increases the lifetime of singlet oxygen.( Two new peaks (peak 1 and peak 2) appeared at the retention times of 26.8 and 27.3 min in the standard reaction mixture of FMN in D2O.

HPLC-ESR-MS analyses of the standard reaction mixture of FMN in D2O

In order to determine the structure of peak 1, peak 2 and peak 3 compounds observed on the HPLC-ESR elution profile of the standard reaction mixture of FMN in D2O, the HPLC-ESR-MS analyses were performed. The HPLC-ESR-MS analysis of the peak 1 compound gave ions at m/z 354 (Fig. 9A). We tentatively assigned the ions at m/z 354 to the protonated molecules of 4-POBN/•C8O3H15 adduct. The HPLC-ESR-MS analysis of the peak 2 compound gave also ions at m/z 354 (Fig. 9B). The HPLC-ESR-MS analysis of the peak 3 compound gave ions at m/z 251 and m/z 338 (Fig. 9C). The ions at m/z 338 correspond to the protonated molecules of the 4-POBN/7-carboxyheptyl radical adducts, [M+H]+. The fragment ions at m/z 251 correspond to the loss of (CH3)3C(O)N from the protonated molecules.

Fig. 9

HPLC-ESR-MS analyses of the peaks 1, 2, and 3 observed in the standard reaction mixtures of FMN in D2O. The total volume of the reaction mixture was 3.0 ml. (A) Peak 1. (B) Peak 2. (C) Peak 3. Chemical structures of 4-POBN/1-(3-carboxypropyl)-4-hydroxybutyl radical adducts and 4-POBN/7-carboxyheptyl radical adduct were inserted in (A), (B), and (C). Asymmetric carbon atoms of 4-POBN/1-(3-carboxypropyl)-4-hydroxybutyl radical adducts are indicated by * marks.

HPLC-ESR and HPLC-ESR-MS analyses of the standard reaction mixtures of FAD and folic acid in D2O under the UVA irradiation

To know which radicals formed in the standard reaction mixtures of FAD and folic acid under the UVA irradiation, the HPLC-ESR analyses were performed (Fig. 10) A peak (peak 3) appeared at the retention time of 37.0 min on the elution profile of the standard reaction mixtures of FAD in D2O (Fig. 10C). Based on the retention time, the peak 3 could be the 4-POBN/7-carboxyheptyl radical adducts. Three peaks (peak 1, peak 2, and peak 3) appeared at the retention times of 27.1 min, 27.7 min, and 36.9 min on the elution profile of the standard reaction mixtures of folic acid in D2O (Fig. 10D). The elution profile resembles the one of the HPLC-ESR analysis of FMN in D2O. The HPLC-ESR-MS analyses of the peak 1, peak 2, and peak 3 gave the ions at m/z 354, m/z 354, and m/z 338, respectively (data not shown), suggesting that the same radical species form in the standard reaction mixture of folic acid as FMN.

Fig. 10

HPLC-ESR analyses of the standard reaction mixtures of FMN, FAD, and folic acid under the UVA irradiation. Total volume of the reaction mixture was 3 ml. The reactions were performed in H2O (or D2O). HPLC-ESR analyses of the standard reaction mixtures of FMN in H2O (A), FMN in D2O (B), FAD in D2O (C), and folic acid in D2O (D).

Discussion

Our previous studies showed that 7-carboxyheptyl radicals form in the reaction mixture of riboflavin with oleic acid under UVA irradiation.( In this paper, we investigated the reactions of some endogenous photosensitizers (FMN or FAD or folic acid or NAD or NADP or PLP or urocanic acid) with oleic acid under UVA irradiation using ESR, HPLC-ESR, and HPLC-ESR-MS. Prominent ESR signals (αN = 1.58 mT and αHβ = 0.26 mT) were observed for the standard reaction mixture of FMN (or FAD or folic acid) (Fig. 1 A–D), suggesting that free radicals form in the standard reaction mixture of FMN (or FAD or folic acid). The HPLC-ESR and HPLC-ESR-MS analyses have shown that 7-carboxyheptyl and •C8O3H15 radicals form in the standard reaction mixtures of FMN (or FAD or folic acid) under the UVA irradiation. When the reactions were performed in D2O, the ESR and HPLC-ESR peak heights increased. Thus, singlet oxygen seems to be involved in the formation of the radicals because D2O increases the lifetime of singlet oxygen.( Of the photosensitizers, NAD (or NADP or PLP or urocanic acid) did not induce the formation of the radicals. These may be due to their low quantum yields of the singlet oxygen generation.( In spite of the low quantum yield of singlet oxygen generation,( folic acid enhanced the formation of the radicals. These might have occurred through the photodegradation products of folic acid, 6-formylpterin and 6-carboxypterin which show high quantum yields of the singlet oxygen generation.(

A possible reaction path for the formation of the 7-carboxyheptyl radical is as follows (Fig. 11). Since irradiation of UVA is essential for the formation of the radicals in the standard reaction mixture of FMN (Fig. 2 and 3), the excited singlet state of FMN 1(FMN)* seems to participate in the formation of the radicals. The excited singlet state sensitizer 1(FMN)*, which is produced under the UVA irradiation turns to the excited triplet state 3(FMN)* through intersystem crossing. The excited triplet state 3(FMN)* may reacts with triplet oxygen to form singlet oxygen (Eq. 1).(

Fig. 11

A possible reaction path for the formation of the 7-carboxyheptyl radical (Peak 3) in the standard reaction mixture of FMN.

The reaction of oleic acid with the singlet oxygen results in the formation of LOOH (9-hydroperoxy-10-octadecenoic acid or 10-hydroperoxy-8-octadecenoic acid) through the singlet oxygen ene reaction.( Since ESR signals were not observed in the standard reaction mixture of FMN without ferrous ions (Fig. 2), ferrous ions seem to participate in the formation of the radicals. The ferrous ions possibly catalyze decomposition of 9-hydroperoxy-10-octadecenoic acid. This reaction yields a radical intermediate LO• [1-(7-carboxyheptyl)-2-decenyloxyl radical]. The β-scession of the LO• could result in the generation of the 7-carboxyheptyl radical. The reaction of ferrous ion with 10-hydroperoxy-8-octadecenoic acid could result in the formation of octyl radicals. We could not detect the radicals. Since 4-POBN used in this experiment is a relatively polar molecule, the octyl radicals which are located at the non-polar area may not be trapped by 4-POBN.

Molecular structures of the peak 1 and peak 2 compounds, which were observed in the HPLC-ESR elution profiles of the standard reaction mixtures of FMN (or folic acid), may be assigned as the two diastereoisomers of 4-POBN/1-(3-carboxypropyl)-4-hydroxybutyl radical adducts (Fig. 9). A possible reaction path for the formation of the 1-(3-carboxypropyl)-4-hydroxybutyl radical is as follows (Fig. 12). The 8-hydroperoxyoctanoic acid could form through the reaction of 7-carboxyheptyl radicals, of which formation was described above (Fig. 11) with molecular oxygens. The ferrous ions possibly catalyze decomposition of the 8-hydroperoxyoctanoic acid. This reaction yields a radical intermediate, 7-carboxyheptyloxyl radical. Isomerization of the 7-carboxyheptyloxyl radicals by 1,5-hydrogen shift via a low-strain six-membered ring transition state possibly results in the formation of 1-(3-carboxypropyl)-4-hydroxybutyl radicals.(

Fig. 12

A possible reaction path for the formation of the 1-(3-carboxypropyl)-4-hydroxybutyl radical (peaks 1 and 2) in the standard reaction mixture of FMN.

To know why FMN works as a potent photosensitizer compared with FAD, effects of adenosine moiety on the formation of the radicals and singlet oxygen in addition to the fluorescence spectrum of FMN were examined. Formation of the radicals and singlet oxygen decreased with the increase of the AMP concentration in a similar manner. On addition of AMP to the reaction mixtures, peak height of the fluorescence spectra also decreased with the increase of the AMP concentration, but in a quite different manner as the ESR peak heights of the 4-POBN radical adducts and the 4-oxo-TEMPO, indicating that the quenching of the excited singlet 1(FMN)* does not seem to affect directly the formation of the oleic acid-derived radicals and singlet oxygen. Since singlet oxygen participates in the formation of the oleic acid-derived radicals (Table 1), the AMP may scavenge singlet oxygen. At higher concentrations of AMP, ESR peak heights of the 4-POBN radical adduct remained unchanged. The chemical species other than singlet oxygen may participate in the formation of the 4-POBN radical adducts at the higher concentration of AMP.

When irradiation energy dependence of the ESR peak height was measured for FMN and FAD, quite different dependences of the ESR peak height were observed for FMN and FAD (Fig. 5). FAD reached a plateau (1.0 cm) at 1.95 J/cm2, while FMN reached a plateau (2.8 cm) at 15.6 J/cm2. The difference seems to be due to the adenosine moiety in FAD. The reaction between the adenosine moiety and the excited isoalloxazine ring may result in the degradation of FAD and the irradiation energy dependence of the ESR peak height.