Melatonin: Free Radicals and Metabolites Resulting by Emission and Consumption of Solvated Electrons (e_aq⁻): Reaction Mechanisms

Results

Emission of e_aq⁻ from melatonin. The emission of solvated electrons, e_aq⁻, from melatonin dissolved in air-free 40/60=water/ethanol mixture and photo-excited in the singlet state (UV: λ=254 nm) was studied for three melatonin concentrations (1×10⁻⁵, 5×10⁻⁵ and 1×10⁻⁴ mol/l). Firstly, the formation of unstable melatonin complexes similar to other hormones (15, 16) was examined. Hence, the molar extinction coefficient, ε254 nm (l/mol/cm) depends on the melatonin concentration (Figure 1). This indicates that some of the emitted e_aq⁻ are scavenged by melatonin molecules in the ground state, contained in the complexes, where k (melatonin + e_aq⁻)=4.2×10⁸ l/mol/s (10). Therefore, the proportion of e_aq⁻ scavenged by melatonin in complexes rises with increasing melatonin concentration, leading to a decrease of the corresponding e_aq⁻ quantum yield, Q (e_aq⁻), and the formation of free radicals. Photodegradation of substrate progresses simultaneously to the formation of free radicals (Figure 1).

Studying the emission of e_aq⁻ from 1×10⁻⁵ mol/L melatonin as a function of absorbed UV dose, two peaks were observed (Figure 2), similar to the findings for other hormones (15, 16). This effect can be attributed to the fact that the primary metabolites are excited in the solution with higher concentration and are able to eject e_aq⁻, but with much lower Q (e_aq⁻) (Figure 2). Simultaneously, the pH of the irradiated media decreased significantly, which indicates that the e_aq⁻ emission is related to H⁺ ion formation. Thereby some of the ejected e_aq⁻ are scavenged by the H⁺ ions leading to Q (e_aq⁻) reduction: Eq.3 A similar course of the e_aq⁻ emission from 5×10⁻⁵ mol/l melatonin was observed depending on the absorbed UV dose, depicted in Figure 3. Here, only a slight pH-decrease with the increase of the absorbed UV dose was registered, indicating the role of the process expressed by Eq.3. Moreover the determined Q (e_aq⁻) values for peaks A and B are one order of magnitude lower compared to those of 1×10⁻⁵ mol/l melatonin (Figure 2). This observation underlines once more the scavenging effect of complexes and H⁺ ions on the e_aq⁻.

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Figure 1.

Photo-induced (UV: λ=254 nm) degradation of 5×10⁻⁵ mol/l melatonin as a function of absorbed UV-dose (hν/l). Inset: Molar extinction coefficient, ε_254nm (l/mol/cm) for three melatonin concentrations. Structure formula of melatonin.

Using 1×10⁻⁴ mol/l melatonin, a further decrease of Q(e_aq⁻) for peaks A and B was ascertained (Figure 4) but the pH of the irradiated solution hardly decreased. This indicates that associates were formed and that Eq.1 is valid.

Sites of the melatonin molecule ejecting and consuming e_aq⁻. The structure of melatonin molecule is complex (Figure 1). There are spots, which emit e_aq⁻ and there are other sites which act as scavengers for e_aq⁻. Considering the structural formula of melatonin, it is clear that the first ring corresponds to anisole. On the other hand, it has been previously reported (18) that UV excitation in of anisole in the S1 state in aqueous solution gave Q (e_aq⁻)=0.025 under formation of a radical cation.

Eq.4

Eq.5

Eq.6

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Figure 2.

Emission of e_aq⁻ (mol/l) from 1×10⁻⁵ mol/l melatonin in 40/60=water/ethanol at 37°C as a function of absorbed UV-dose (hν/l) of monochromatic UV-light (λ=254 nm). Q(e_aq⁻)-yields at peak A and B: 0.1543 and 0.0210 respectively. Inset: pH-change in dependence of absorbed UV-dose.

Further, it has been previously found (18) that the iminogroups of the pyrol ring as well as of the side chain can act as e_aq⁻ emission spots also, where on the other hand carbonyl group scavenges e_aq⁻ [k=1.7×10⁹ l/mol/s] (17)]. Based on these facts a simplified scheme of the competing processes is presented in Figure 5.

Eq.7

Eq.8

Eq.9

The resulting free radicals lead to the formation of various metabolites dependent on the given medium conditions.

Metabolite analysis. The degradation of 5×10⁻⁵ mol/L melatonin as a function of absorbed UV-dose is illustrated in Figure 1. As expected the number of metabolites and degradation products increases with increasing absorbed UV dose. This also indicates that, by the emission of e_aq⁻, the melatonin free radicals expected to be formed (Figure 5) undergo reactions with each other, resulting in various metabolites. Simultaneously with these reactions, some of the primary-formed melatonin metabolites are decomposed by emitting e_aq⁻ (Figures 2, 3 and 4), leading to the formation of secondary metabolites and to numerous low molecular weight degradation products. All these processes are superimposed and depend on the specific concentrations of the reaction partners and the corresponding reaction rate constants. In other words, the increase of the absorbed UV dose results in propagation of the number of final products with different molecular weight. The structure formulas of main metabolites formed by using 5×10⁻⁵ mol/l melatonin with various UV doses was determined (Figure 6). In addition to these, dimers with molecular weight of 437 and 473, as well as degradation products with lower molecular weights 32.02, 58.03, 175.0 etc. were also observed. This again demonstrates the complexity of the involved reaction mechanisms.

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Figure 3.

Emission of e_aq⁻ (mol/l) from 5×10⁻⁵ mol/l melatonin in 40/60=water/ethanol at 37°C as a function of absorbed UV-dose (hν/l) of monochromatic UV-light (λ=254 nm). Q(e_aq⁻)-yields at peak A and B: 0.0241 and 0.0047 respectively. Inset: pH-change in dependence of absorbed UV-dose.

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Figure 4.

Emission of e_aq⁻ (mol/l) from 5×10⁻⁴ mol/l melatonin in 40/60=water/ethanol at 37°C as a function of absorbed UV-dose (hν/l) of monochromatic UV-light (λ=254 nm). Q(e_aq⁻)-yields at peak A and B: 0.0113 and 0.0021 respectively. Inset: pH-change in dependence of absorbed UV-dose.

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Figure 5.

Simplified scheme of the e_aq⁻-emitting positions of melatonin and resulting transients R1 – R3 as well as R4 formed as e-aq-scavenging position.