Radiative relaxation quantum yields for synthetic eumelanin

We report absolute values for the radiative relaxation quantum yield of synthetic eumelanin as a function of excitation energy. These values were determined by correcting for pump beam attenuation and emission reabsorption in both eumelanin samples and fluorescein standards over a large range of concentrations. Our results confirm that eumelanins are capable of dissipating >99.9% of absorbed UV and visible radiation through nonradiative means. Furthermore, we have found that the radiative quantum yield of synthetic eumelanin is excitation energy dependent. This observation is supported by corrected emission spectra, which also show a clear dependence of both peak position and peak width on excitation energy. Our findings indicate that photoluminescence emission in eumelanins is derived from ensembles of small chemically distinct oligomeric units that can be selectively pumped. This hypothesis lends support to the theory that the basic structural unit of eumelanin is oligomeric rather than heteropolymeric.     

Quantitative fluorescence excitation spectra of synthetic eumelanin

Previously reported excitation spectra for eumelanin are sparse and inconsistent. Moreover, these studies have failed to account for probe beam attenuation and emission reabsorption within the samples, making them qualitative at best. We report for the first time quantitative excitation spectra for synthetic eumelanin, acquired for a range of solution concentrations and emission wavelengths. Our data indicate that probe beam attenuation and emission reabsorption significantly affect the spectra even in low-concentration eumelanin solutions and that previously published data do not reflect the true excitation profile. We apply a correction procedure (previously applied to emission spectra) to account for these effects. Application of this procedure reconstructs the expected relationship of signal intensity with concentration, and the normalized spectra show a similarity in form to the absorption profiles. These spectra reveal valuable information regarding the photophysics and photochemistry of eumelanin. Most notably, an excitation peak at 365 nm (3.40 eV), whose position is independent of emission wavelength, is possibly attributable to a 5,6-dihydroxyindole-2-carboxylic acid (DHICA) component singly linked to a polymeric structure.     

Radiative relaxation of Sepia eumelanin is affected by aggregation.

The steady‐state and time‐resolved emission properties of aqueous solutions containing different aggregation state distributions of eumelanin are reported. Excitation spectra of the size‐selected samples reveal, for the first time, differences in absorption bands due to varying levels of aggregation. These size‐dependent absorption properties result in size‐dependent emission band shapes and quantum yields. For all size fractions, absorption and emission overlap significantly. The emission yield for small eumelanin aggregates is 5.7 times greater than that for large eumelanin aggregates. Time‐resolved population decays reveal that small eumelanin aggregates are responsible for long‐lived emission dynamics (lifetimes greater than 1 ns), while large eumelanin aggregates are the source of short emission decay (lifetimes less than 1 ns). Polarized emission decays for the large and small aggregates reveal that energy transfer occurs both within the same and between the separate fundamental building blocks of eumelanin. The observed energy transfer dynamics can be accounted for using Förster theory.     

The action spectrum for generation of the primary intermediate revealed by ultrafast absorption spectroscopy studies of pheomelanin

The observation that fair-skinned individuals are more susceptible to skin cancers is commonly explained by invoking an enhanced photoreactivity of the red melanin, pheomelanin compared with the black melanin, eumelanin. For the wavelength range from 500 to 1000 nm, pump-probe spectroscopic measurements reveal the photoexcitation of pheomelanin by UVA light that generates an immediate (< 100 fs) transient absorption centered at 780 nm. Using a tunable femtosecond excitation source, the action spectrum between 300 and 390 nm for generation of the primary intermediate was measured. Similar action spectra are found for the sample with molecular weight (MW) between 1000 and 10 000 and the one with MW > 10 000 fractions of pheomelanin, indicating that the reactive chromophore has a low MW but is present and its photophysics is similar in the aggregated pigment. The shape of the action spectrum differs from the absorption spectrum of bulk melanin and mass-selected fractions but resembles reported absorption spectrum of benzothiazines, oxidation products of 5-S-cysteinyl-dopa, which are formed along the biosynthetic pathway of pheomelanin.     

The UV‐Absorption Spectrum of Human Iridal Melanosomes: A New Perspective on the Relative Absorption of Eumelanin and Pheomelanin and its Consequences†

Photoemission electron microscopy is used to measure the absorption coefficients, εc, of intact iridal stroma melanosomes isolated from dark brown and blue–green human irides for the spectral range λ = 244–310 nm. These iridal stroma melanosomes were chosen because different colored irides produce organelles of varying eumelanin:pheomelanin ratios with similar size and morphology. Similar absorption spectra are found for the two types of melanosomes. The experimental spectra measured within are compared with both the extinction coefficient spectra obtained on soluble synthetic model systems and the monomeric precursors to each pigment.     

Effect of dimerization on vibrational spectra of eumelanin precursors

We have synthesized a compound ideally suited to the study of structure-function relationships in eumelanin synthesis. N-methyl-5-hydroxy-6-methoxy-indole (MHMI) has key functional groups strategically placed on the indole framework to hinder binding in the 2, 5, 6 and 7 positions. Thus, the dimer bound exclusively in the 4-4' positions was isolated and characterized. In order to study the difference in vibrational structure between the MHMI monomer and dimer, Raman spectra were acquired of both compounds, as well as indole, indole-2-carboxylic acid and 5,6-dihydroxyindole-2-carboxylic acid (DHICA). Peaks were assigned to particular vibrational modes using B3LYP density functional theory calculations, and experimental and theoretical spectra displayed good agreement. Addition of functional groups to either benzene or pyrrole rings in the indole framework impacted vibrational spectra attributed to vibrations in either ring, and in some cases, peaks appearing unchanged between two compounds corresponded to different contributing vibrations. Dimerization resulted in an expected increase in the number of vibrational modes, but not a significant increase in the number of apparent peaks, as several modes frequently contributed to an individual observed peak. Comparison of spectral features of the monomer and dimer provides insight into eumelanin photochemistry, but final conclusions depend on the planarity of oligomeric structure in vivo.     

5,6-Dihydroxyindole Oxidation in Phosphate Buffer/Polyvinyl Alcohol: A New Model System for Studies of Visible Chromophore Development in Synthetic Eumelanin Polymers

The determinants of the broadband absorption spectrum of eumelanins are still largely unknown. Herein we report a novel approach to investigate eumelanin chromophore which is based on the biomimetic oxidation of the key monomer precursor, 5,6-dihydroxyindole (DHI, 1 ), with peroxidase/hydrogen peroxide in phosphate buffer, pH 7, containing 1–5% polyvinylalcohol (PVA, 27 000 Da). This approach relies on the discovery that as low as 1% PVA can prevent precipitation of the growing melanin polymer thus allowing investigation of the chromophoric phases accompanying oxidation of DHI without confounding scattering effects. Spectrophotometric monitoring showed the initial development of a band around 530 nm persisting for about 1 h before gradually changing into the typical broadband spectrum of eumelanin. Reductive treatment caused a significant absorbance decrease in the visible region without affecting an absorption band around 320 nm. Initial product analysis indicated an altered formation ratio of 2,4′-biindolyl ( 2 ) and 2,7′-biindolyl ( 3 ) relative to control experiments. Overall, these results demonstrate for the first time that the development in solution of visible chromophores since the early oligomer stages is independent of strong aggregation/precipitation phenomena.     

Characterization of the Fe(III)‐binding Site in Sepia Eumelanin by Resonance Raman Confocal Microspectroscopy¶

The resonance Raman spectrum of Sepia eumelanin is discussed by analogy to model compounds containing catechol (CAT)‐like structural units. These data are then compared with the analogous data on Fe(III)‐enriched Sepia eumelanin. In contrast to the natural eumelanin, the Fe(III)‐enriched samples exhibit absorption features in the visible and near‐IR spectral regions, which are attributed to ligand‐to‐metal charge‐transfer (LMCT) bands. Resonance Raman spectra collected by exciting these LMCT bands reveal bands at 580 and 1470 cm^?1; the intensity of these features increases wioth increasing Fe(III) content. The 580 and 1470 cm^?1 bands are assigned to Fe‐OR stretching and ring deformation modes, respectively. These data further substantiate that the Fe(III)‐ melanin‐binding site in melanin is composed of CAT‐like structural units.     

Chemical, pulse radiolysis and density functional studies of a new, labile 5,6-indolequinone and its semiquinone

The chemical and spectroscopic characterization of 5,6-indolequinones and their semiquinones, key transient intermediates in the oxidative conversion of 5,6-dihydroxyindoles to eumelanin biopolymers, is a most challenging task. In the present paper, we report the characterization of a novel, relatively long-lived 5,6-indolequinone along with its semiquinone using an integrated chemical, pulse radiolytic, and computational approach. The quinone was obtained by oxidation of 5,6-dihydroxy-3-iodoindole (1a) with o-chloranil in cold ethyl acetate or aqueous buffer: it displayed electronic absorption bands around 400 and 600 nm, was reduced to 1a with Na2S2O4, and reacted with o-phenylenediamine to give small amounts of 3-iodo-1H-pyrrolo[2,3-b]phenazine (2). The semiquinone exhibited absorption maxima at 380 nm (sh) and 520 nm and was detected as the initial species produced by pulse radiolytic oxidation of 1a at pH 7.0. DFT investigations indicated the 6-phenoxyl radical and the N-protonated radical anion as the most stable tautomers for the neutral and anion forms of the semiquinone, respectively. Calculated absorption spectra in water gave bands at 350 (sh) and 500 nm for the neutral form and at 310 and 360 (sh) nm for the anion. Disproportionation of the semiquinone with fast second-order kinetics (2k = 1.1 x 1010 M-1 s-1) gave a chromophore with absorption bands resembling those of chemically generated 1a quinone. Computational analysis predicted 1a quinone to exist in vacuo as the quinone-methide tautomer, displaying low energy transitions at 380 and 710 nm, and in water as the o-quinone, with calculated absorption bands around 400 and 820 nm. A strong participation of a p orbital on the iodine atom in the 360-380 nm electronic transitions of the o-quinone and quinone-methide was highlighted. The satisfactory agreement between computational and experimental electronic absorption data would suggest partitioning of 1a quinone between the o-quinone and quinone-methide tautomers depending on the medium.     

Synthesis and characterization of eumelanin‐inspired poly(indoylenearylenevinylene)s and poly(indoylenearyleneethynylene)s

Four novel conjugated polymers containing the eumelanin‐inspired indole core have been successfully synthesized using common cross coupling reactions. These polymers differed by the arylene and the carbon–carbon bond linkage. Optoelectronic experiments of these polymers suggest that the ethynylene linkage contributed to the red‐shifted absorption spectra and blue‐shifted emission spectra when compared to the vinylene linkage polymers. Furthermore, the optical bandgaps of the poly(indoylenearyleneethynylene)s (PIAEs) were smaller compared to the poly(indoylenearylenevinylene)s (PIAVs). Surprisingly, the HOMOs of these polymers were less affected by the nature of the carbon–carbon linkage. However, the LUMOs of the PIAEs were lower in comparison to the PIAVs. These eumelanin‐inspired PIAEs and PIAVs are good fluorophores with fluorescence quantum yields ranging from 0.12 to 0.67 and have good thermal stability for applications such as in organic light‐emitting diodes. © 2016 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2017 , 55, 457–463     

Radiative relaxation in synthetic pheomelanin

We report a detailed photoluminescence study of cysteinyldopa-melanin (CDM), the synthetic analogue of pheomelanin. Emission spectra are shown to be a far more sensitive probe of CDM's spectroscopic behavior than are absorption spectra. Although CDM and dopa-melanin (DM, the synthetic analogue of eumelanin) have very similar absorption spectra, we find that they have very different excitation and emission characteristics; CDM has two distinct photoluminescence peaks that do not shift with excitation wavelength. Additionally, our data suggest that the radiative quantum yield of CDM is excitation energy dependent, an unusual property among biomolecules that is indicative of a chemically disordered system. Finally, we find that the radiative quantum yield for CDM is approximately 0.2%, twice that of DM, although still extremely low. This means that 99.8% of the energy absorbed by CDM is dissipated via nonradiative pathways, consistent with its role as a pigmentary photoprotectant.     

Structure analysis of exfoliated unilamellar crystallites of manganese oxide nanosheets

Structure analysis of unilamellar manganese oxide nanosheets obtained via exfoliation of layered manganese oxides was carried out utilizing synchrotron radiation (SR) X-ray in-plane diffraction and polarization-dependent total reflection fluorescence X-ray absorption fine structure (PTRF-XAFS) analyses. A combination of SR excitation and the total reflection of incoming X-rays provides signals strong enough for both analyses even from a monolayer of the MnO(2) nanosheets having a concentration of 0.7 microg cm(-2). In addition, the mean oxidation state of constituent manganese ions in the MnO(2) sheets was estimated on the basis of XANES spectra, and bond valence sum calculations with the bond length obtained from the present EXAFS analyses. The obtained structural data revealed that the two-dimensional lattice of the MnO(2) sheets underwent a slight elongation upon delamination. These changes correspond to approximately 1% expansion of sheet area and 1-2% expansion of thickness, which can be understood by reduction of the mean oxidation number of manganese ions in the sheet through the exfoliation process.     

Structure-activity correlations in a nickel-borate oxygen evolution catalyst

An oxygen evolution catalyst that forms as a thin film from Ni(aq)(2+) solutions containing borate electrolyte (Ni-B(i)) has been studied by in situ X-ray absorption spectroscopy. A dramatic increase in catalytic rate, induced by anodic activation of the electrodeposited films, is accompanied by structure and oxidation state changes. Coulometric measurements correlated with X-ray absorption near-edge structure spectra of the active catalyst show that the nickel centers in activated films possess an average oxidation state of +3.6, indicating that a substantial proportion of nickel centers exist in a formal oxidation state of Ni(IV). In contrast, nickel centers in nonactivated films exist predominantly as Ni(III). Extended X-ray absorption fine structure reveals that activated catalyst films comprise bis-oxo/hydroxo-bridged nickel centers organized into sheets of edge-sharing NiO(6) octahedra. Diminished long-range ordering in catalyst films is due to their ostensibly amorphous nature. Nonactivated films display a similar oxidic nature but exhibit a distortion in the local coordination geometry about nickel centers, characteristic of Jahn-Teller distorted Ni(III) centers. Our findings indicate that the increase in catalytic activity of films is accompanied by changes in oxidation state and structure that are reminiscent of those observed for conversion of β-NiOOH to γ-NiOOH and consequently challenge the long-held notion that the β-NiOOH phase is a more efficient oxygen-evolving catalyst.     

Photoprotection by porcine eumelanin against singlet oxygen production

Melanin, a major pigment found in retinal pigment epithelium (RPE) cells, is considered to function in dual roles, one protective and one destructive. By quenching free radical species and reactive oxygen species (ROS) melanin counteracts harmful redox stress. However, melanin is also thought to be capable of creating ROS. In this destructive role, melanin increases redox strain in the cell. This study uses readily available eumelanin extracted from porcine RPE cells as a more authentic model than synthetic melanin to determine specific mechanisms of melanin activity with regard to singlet oxygen in the presence and absence of rose bengal, a singlet-oxygen photosensitizer. Optical detection of singlet-oxygen was determined by monitoring the bleaching of p-nitrosodimethylaniline in the presence of histidine. Production of singlet oxygen in aqueous oxygen-saturated solutions of rose bengal without eumelanin was readily accomplished. In contrast, detection of singlet oxygen in oxygen-saturated solutions of eumelanin without rose bengal failed, consistent with results of others. However, a significant decrease in singlet oxygen production by rose bengal was observed in the presence of eumelanin. After correction for light absorption and chemical bleaching of eumelanin, the results show that eumelanin also provides a photoprotective mode arising from chemistry, that is, not just the physical process of light absorption followed by energy dissipation as heat.     

Ultrafast spectral hole burning reveals the distinct chromophores in eumelanin and their common photoresponse

Eumelanin, the brown-black pigment found in organisms from bacteria to humans, dissipates solar energy and prevents photochemical damage. While the structure of eumelanin is unclear, it is thought to consist of an extremely heterogeneous collection of chromophores that absorb from the UV to the infrared, additively producing its remarkably broad absorption spectrum. However, the chromophores responsible for absorption by eumelanin and their excited state decay pathways remain highly uncertain. Using femtosecond broadband transient absorption spectroscopy, we address the excited state behavior of chromophore subsets that make up a synthetic eumelanin, DOPA melanin, and probe the heterogeneity of its chromophores. Tuning the excitation light over more than an octave from the UV to the visible and probing with the broadest spectral window used to study any form of melanin to date enable the detection of spectral holes with a linewidth of 0.6 eV that track the excitation wavelength. Transient spectral hole burning is a manifestation of extreme chemical heterogeneity, yet exciting these diverse chromophores unexpectedly produces a common photoinduced absorption spectrum and similar kinetics. This common photoresponse is assigned to the ultrafast formation of immobile charge transfer excitons that decay locally and that are formed among graphene-like chromophores in less than 200 fs. Raman spectroscopy reveals that chromophore heterogeneity in DOPA melanin arises from different sized domains of sp²-hybridized carbon and nitrogen atoms. Furthermore, we identify for the first time striking parallels between the excited state dynamics of eumelanin and disordered carbon nanomaterials, suggesting that they share common structural attributes.

Seeing the colors in black: ultrafast transient hole burning spectroscopy reveals the absorption properties of discrete chromophores and their interactions in the skin pigment eumelanin.     

Molecular packing effects on the optical spectra and triplet dynamics in oligofluorene films

We report on the triplet spectra and dynamics in two types of oligomeric films deposited by two different techniques: thermal evaporation and spin coating. The different molecular arrangement in both films is manifested in a red shift of the absorption, PL, and T1-Tn absorption spectra of the sublimated film relative to the spin-coated one. Triplet recombination dynamics studied with steady-state photoinduced absorption (PA) spectroscopy follow a dispersive bimolecular recombination model away from the trap filling regime. We obtained values for the triplet bimolecular recombination ratio (beta) of 3.4 x 10 (-14) and 1.1 x 10 (-15) cm3 s (-1) for evaporated and spin-coated film, respectively, the difference being attributed to diverse molecular arrangement in both films.     

Enhanced melanin fluorescence by stepwise three-photon excitation

The fluorescence of eumelanin (from Sepia officinalis and black human hair) was activated and enhanced by almost three orders of magnitude by exposure to near-infrared radiation. No activation or enhanced emission was observed when the samples were heated up to 100°C. The near-infrared irradiation caused obvious changes to the eumelanin and could be seen by fluorescence and bright field imaging. The area of enhanced emission appeared to originate from a region with changes in the morphology of the eumelanin's granule and increased with exposure time. At least two different components with enhanced fluorescence were activated and could be distinguished by their excitation properties. One component could be excited efficiently with wavelengths in the visible region and exhibited linear absorption dependence with respect to the laser power level. The second component could be excited efficiently using near-infrared wavelengths by a nonlinear process and exhibited a third-order dependence on the excitation. The third-order dependence is explained by a step-wise excited-state absorption process since the same third-order dependence was present when either continuous wave or femtosecond pulsed laser, with similar average-power levels, was used.     

Electron spin resonance (ESR/EPR) of free radicals observed in human red hair: a new,simple empirical method of determination of pheomelanin/eumelanin ratio in hair

The definition of the concentration of pheomelanin in the skin is an issue of great interest because in the case of being influenced by UV radiation, it manifests itself as a prooxidant, causing various skin disorders including melanoma that might help to explain the relatively high incidence of skin cancer among individuals with red hair. The ESR spectra of red hair samples were investigated. It was found that at low microwave power, they are characterized by two types of spectra. Red hair ESR signals result from a superposition of two spectral shapes, a singlet spectrum as a result of the existence of eumelanin and a triplet spectrum as a result of the existence of pheomelanin. At high microwave power, only triplet spectra shape was detected, caused by saturation of the eumelanin singlet. Using different concentration ratios of black to red hair, we measured ESR spectra and plotted the ratio values in each sample against a measured ‘g‐factor’ (experimental). We found that there is a linear relationship between these two parameters. So, it is evident that using these results, the concentration ratio of pheomelanin to eumelanin in a sample of hair can be easily determined by an almost noninvasive method. This can be considered a potential advantage for many practical activities compared with other invasive methods. The concentration dependence curve of pheomelanin (µg/mg) on g_exp‐factor in an ESR spectrum of hair has been designed, which allows the determination of the amount of pheomelanin in hair of any color. Copyright © 2014 John Wiley & Sons, Ltd.     

Ultrafast Radical Photogeneration Pathways in Eumelanin†

Eumelanin is a ubiquitous biological pigment that rapidly and efficiently deactivates excited states created by UV or visible radiation. Paradoxically, photoirradiation of eumelanin also generates radicals and harmful reactive oxygen species, but the relationship between these pathways and excited-state deactivation is uncertain. Here, greatly expanding the excitation tuning range (225–620 nm) and probing window (400–1500 nm) in femtosecond transient absorption spectroscopy of the synthetic eumelanin, DOPA melanin, enables the detection of photogenerated radials with ultrafast time resolution for the first time. Despite its heterogeneous nature, the transient absorption signals can be modeled by two spectral components assigned to solvated electrons and photogenerated radicals. Radical absorbance measured several nanoseconds after excitation increases exponentially with increasing photon energy, matching the trend in radical yields measured in electron paramagnetic resonance spectroscopy experiments. Spectral modeling of the transient signals reveals two radical generation mechanisms: (1) photoionization by UV light; and (2) photoinduced charge transfer among eumelanin chromophores by UVA and visible wavelengths capable of reaching the pigment in skin. Concurrent ultrafast relaxation and radical generation underlie the ability of eumelanin to be both photoprotective and photodamaging, and the branching between these pathways likely depends on the wavelength of the absorbed light.     

Towards Eumelanin@Zeolite Hybrids: Pore‐Size‐Controlled 5,6‐Dihydroxyindole Polymerization

5,6‐Dihydroxyindole ( 1 ) and its N‐methyl derivative ( 2 ), key eumelanin building blocks, were inserted into zeolite L by sublimation at 175 °C for 5 days. At a 10 mg/300 mg indole/zeolite ratio, the resulting hybrids displayed a stable deep red coloration. CP/MAS ¹³C NMR and UV/Vis spectroscopy of the red species suggested the generation and accommodation of quinonoid biindole derivative(s) within the void space of the acidic zeolite channels. Removal of the zeolite matrix by treatment with HF gave a stable species that could be separated by HPLC and characterized by mass spectrometry as an oxygenated biindole derivative (or a mixture of isomers), suggesting addition of water to the original dimer and subsequent re‐oxidation. The characterization was corroborated by optimized molecular geometries and simulated UV spectra with density functional calculations. Loading 1 or 2 into the larger pores of SBA‐15 type mesoporous silica resulted in black eumelanin‐type polymers, confirming channel size dependence over the polymerization process.