Free radical formation in novel carotenoid metal ion complexes of astaxanthin

Abstract

The carotenoid astaxanthin forms novel metal ion complexes with Ca(2+), Zn(2+), and Fe(2+). MS and NMR measurements indicate that the two oxygen atoms on the terminal cyclohexene ring of astaxanthin chelate the metal to form 1:1 complexes with Ca(2+) and Zn(2+) at low salt concentrations <0.2 mM. The stability constants of these complexes increased by a factor of 85 upon changing the solvent from acetonitrile to ethanol for Ca(2+) and by a factor of 7 for Zn(2+) as a consequence of acetonitrile being a part of the complex. Optical studies showed that at high concentrations (>0.2 mM) of salt, 2:1 metal/astaxanthin complexes were formed in ethanol. In the presence of Ca(2+) and Zn(2+), salts the lifetime of the radical cation and dication formed electrochemically decreased relative to those formed from the uncomplexed carotenoid. DFT calculations showed that the deprotonation of the radical cation at the carbon C3 position resulted in the lowest energy neutral radical, while proton loss at the C5, C9, or C13 methyl groups was less favorable. Pulsed EPR measurements were carried out on UV-produced radicals of astaxanthin supported on silica-alumina, MCM-41, or Ti-MCM-41. The pulsed EPR measurements detected the radical cation and neutral radicals formed by proton loss at 77 K from the C3, C5, C9, and C13 methyl groups and a radical anion formed by deprotonation of the neutral radical at C3. There was more than an order of magnitude increase in the concentration of radicals on Ti-MCM-41 relative to MCM-41, and the radical cation concentration exceeded that of the neutral radicals.

Figure 1

¹H NMR (360 MHz) spectra of 1 mM astaxanthin in ethanol in the absence and in the presence of 30 mM Ca(ClO₄)₂ or Zn(ClO₄)₂ salts at room temperature.

Figure 2

ESI MS spectrum of astaxanthin in the presence of Ca(ClO₄)₂ in acetonitrile. The main peaks correspond to m/z = 318.2 [Ast+Ca]²⁺ and m/z = 597.4 [Ast+H]⁺. The complex was first prepared with carotenoid concentration of 0.1 mM with excess of the salt, and then was dissolved by a factor of 10 to obtain a good signal of the spectrum.

Figure 3

Optical absorption spectra of 0.01 mM astaxanthin in ethanol in the presence of different concentrations of Fe(ClO₄)₂.

Figure 4

Optical absorption spectra of 0.01 mM astaxanthin in CH₂Cl₂ in the presence of 0.01 mM CuCl₂.

Figure 5

Benesi-Hildebrant plots of astaxanthin optical density in ethanol and acetonitrile at 550 nm on Ca(ClO₄)₂ salt concentration.

Figure 6

CV plots of astaxanthin in methylene chloride at different scan rates.

Figure 7

Relative scavenging rates of carotenoids toward peroxyl radicals.

Figure 8

Pulsed Mims ENDOR spectra of astaxanthin radicals as a function of τ (220 nm and 200 nm). (a) The red trace is the experimental spectrum produced in activated silica-alumina after UV irradiation; ENDOR parameters: T = 20 K, B = 3475 G, ν = 9.76 GHz, τ = 220 ns. (b) The black trace is the simulated spectrum using isotropic and anisotropic DFT-calculated couplings of Ast^•+ only. (c) The blue trace is the simulated spectrum using isotropic and anisotropic DFT-calculated hyperfine couplings for Ast^•+, Ast^•−, #Ast^•(3)b, #Ast^•(5), #Ast^•(9) and #Ast^•(13) in a 1:1:1:1:1:1 ratio. Note: ENDOR lines occur at ν_n = ± A/2, where ν_n is the proton frequency situated at the center of the ENDOR spectrum (proton frequency ν_n = 14.793571 MHz). Note: the outer peaks indicated by * are due to the neutral radicals. Below 6 MHz the baseline includes an artifact from the non linearity of the ENDOR amplifier and thus interferes with low-frequency ENDOR lines due to neutral radicals. The simulation for τ = 200 ns is missing Ast^•− and #Ast^•(3)b and which contribute to the center of the spectrum.

Figure 9

Simulated Mims ENDOR spectra (τ = 220 ns) of the individual radicals using DFT proton hyperfine couplings listed in Tables S8–S14 in supporting information for Ast^•− orange, Ast^•+ black, #Ast^•(3)b blue, #Ast^•(5) red, #Ast^•(9) green and #Ast^•(13) violet. Note: the outer peaks indicated by * are due to the neutral radicals.

Figure 10

Powder X-band cw ENDOR spectrum at 130 K of astaxanthin on silica-alumina in the absence of light irradiation. Assigments of the DFT generated couplings for the radical cation for protons located at C1(1′)-CH₃, C2(2′)-H_α, C3(3′)-H_α, C5(5′)-CH₃, C9(9′)-CH₃ and C13(13′)-CH₃ positions account for the observed lines. ENDOR lines above 20 MHz and below 9 MHz for the neutral radicals are missing.

Figure 11

Mims ENDOR spectra of astaxanthin on Ti-MCM-41 at τ = 200 ns (A) and τ = 220 ns (B). The concentration of the radical cations (grey area) exceeds the concentration of the neutral radicals in the absence of Ti(IV) (C).

Scheme 1

Stability constants K₁ and K₂ for the complexation reactions of astaxanthin with metal ions

Scheme 2

Possible radical intermediates of astaxanthin formed upon the proton loss (indicated by blue circle) during the photolysis in the presence of metal ions. On the right: unpaired spin distribution for astaxanthin radicals Ast^•+, #Ast^•(5), #Ast^•(9), #Ast^•(13), #Ast^•(3)a (not detected by EPR), #Ast^•(3)b (couplings similar to #Ast^•(5)) and Ast^•− (small couplings, ENDOR 13 to 17 MHz region) from DFT.