Photochemistry and photophysics of mycosporine-like amino acids and gadusols, nature’s ultraviolet screens

UV-Vis absorption

One of the most relevant features of the MAAs is their high UV-absorbing capability, due to a single band centered in the 310–360 nm range, partially covering the harmful UVA and UVB radiations zones. This strong absorption reaches molar absorption coefficients between 20 000 and 40 000 L cm⁻¹ mol⁻¹ which may rise over 50 000 in cases such as that of palythene [7]. Depending on the origin, relevant differences between the compounds found in algae and those found in fungi have been reported. In fungi metabolites, the chromophore is less conjugated and in all cases it shows a maximum near 310 nm, practically without dependence on the amino-substitution [9].

In all marine MAAs a similar behavior is observed with an absorption maximum ca. 320 nm for the non-substituted imino-MAAs and ca. 360 nm for the substituted ones. In this case, the substituents may be important as they can modify the chromophore’s conjugation. In turn, this allows for the modulation of their UV-Vis spectral properties, yielding a bathochromic shift when conjugation increases. When conjugated C=C bonds are directly linked to the chromophore, the Z-isomer shows an hypsochromic shift relative to the E-isomer of 2–3 nm, as in usujirene and palythene (Fig. 1) [23]. A non-common behavior is found for the E–Z-palythenic acid pair, in which the E-isomer is 2 nm blue-shifted [24].

Due to the amino acidic structure, a zwitterionic character is expected. It is unclear if the relevant form in vivo is the anionic, the “neutral” or zwitterionic or the protonated one. To try to clarify this aspect porphyra-334 was studied at different pH values [25]. It was found that in high acidic aqueous solution (pH 1–3) the absorption maximum of porphyra-334 shows a hypsochromic shift from 332 nm at pH 3 and to 330 nm at pH 1 and pH 2. Under these conditions, the molar absorption coefficient also decreases with higher acidity. As other MAAs, porphyra-334 is a zwitterion that presents two acidic groups and one iminic nitrogen as shown in Fig. 2. When the pH is below 3, the π delocalization is not possible due to the protonation of the non-bonding pair in the nitrogen atom. Subsequent protonation of the acidic groups may take place at lower pH values.

Fig. 2:

Molecular structures of porphyra-334 at different pH values.

On the other hand, the absorption maximum and molar absorption coefficient of porphyra-334 do not change in alkaline solutions. This is reasonable since both acidic groups do not alter the electronic properties of the chromophore as they are not directly bonded to it.

The behavior of porphyra-334 under acidic conditions was further explored by computing the gas phase proton affinity [26]. For this compound, a value of 266 kcal mol⁻¹ was found which is in the range of the gas phase proton affinity of certain artificial super-bases. This turns porphyra-334 into a so-called “proton sponge.” The term refers to species with exceptionally high proton affinities which seem to be related to the high stability of this compound in very acidic water solutions. In contrast, under relatively low basic conditions of pH 12, porphyra-334 decomposes rapidly to give unknown products [25]. The reasons behind this feature were explained in terms of positive charge delocalization between the two nitrogen atoms and the alkene moiety that connects them. This type of push–pull charge stabilization has been previously proposed for other types of compounds showing also high proton affinity. Significantly, this proton-capturing ability is comparable to that one of other artificial systems specifically designed to behave like this [27]. Other MAAs show also small solvent dependence in the UV absorption with changes within 2–4 nm in many of them [28, 29].

Fluorescence emission

In contrast to the UV absorption properties, results from fluorescence measurements on MAAs are only available for the most studied compounds, namely porphyra-334, shinorine and palythine. These three molecules were characterized by steady-state spectrofluorometry in aqueous solutions with monochromatic irradiation at the absorption maximum and by time-correlated single photon counting (TCSPC) for the determination of the singlet state lifetimes.

One of the better known MAAs, porphyra-334, was first isolated from the red alga Porphyra tenera [30]. However, it took more than 20 years to thoroughly explore its excited-state properties [28]. The results from TCSPC for porphyra-334 in aqueous solution evidence an excited-state with a short lifetime of 0.4 ns. Besides, no longer lived transient species were detected after irradiation at the absorption maximum. The emission spectrum consists of a weak band centered at 395 nm which amounted a very low fluorescence quantum yield (Φ_F = 2.0 × 10⁻⁴) [28, 31].

Structurally very similar to porphyra-334, shinorine is usually found in the same organisms but sometimes in larger amounts (Fig. 1). It has been obtained from adult sea urchins eggs as methanolic extracts [32]. Detailed photophysical studies on shinorine indicated that, as porphyra-334, this metabolite efficiently dissipates light energy into heat [31]. Shinorine also shows a very low fluorescence quantum yield (Φ_F = 1.6 × 10⁻⁴) and a short fluorescence lifetime (0.35 ns). The emission spectrum exhibits a very weak maximum around 386 nm.

Palythine is the MAA with the simplest structure. It was first isolated from the zoanthid Palythoa tuberculosa [33]. The structures of palythine and other related compounds (mycosporine-glycine, palythinol and palythine) obtained from the same source were later proposed (Fig. 1) [34]. A very detailed photophysical analysis of palythine in water has been performed [35]. The lack of measurable luminescence implies a very efficient deactivation pathway. The weak emission band experimentally observed was attributed to an impurity and not to palythine.

According to these results, the suggested role of MAAs as transducers of the UV light to more useful wavelengths in the photosynthesis seems quite improbable due to the very low observed fluorescence quantum yields.

Triplet excited state

As with fluorescence, the absorption features, formation yield and the energy of the triplet state have been only determined for a few MAAs.

Laser-flash photolysis experiments have allowed for the characterization of the excited triplet state of shinorine [31], porphyra-334 [28] and palythine [35]. Data for the triplet-triplet energy transfer from 1-naphthalene-methanol to porphyra-334 were used to assess the quantum yield of the triplet production in aqueous solution, affording an upper limit of 3 % [31]. This result accounts for the low probability of triplets to generate reactive intermediates which might start further chemical reactions, in agreement with the lack of radical detection previously reported [36].

The triplet–triplet absorption spectrum of porphyra-334 was determined by sensitization with benzophenone and consisted of a broad band centered at around 420 nm. Similar results were obtained for shinorine and palythine, both sensitized by acetone [31, 35].

The energy of the triplet state was estimated from the evaluation of the rate constants for the energy transfer processes with different donors with variable triplet energies. Thus, triplet energy for porphyra-334 in water solution is delimited to be <250 kJ mol⁻¹ whereas the triplet energy for palythine is approximately 330 kJ mol⁻¹ [28, 35]. In the cases of shinorine and palythine, small quantum yields for triplet formation were also obtained (Φ_T < 5 × 10⁻²) by using acetone as sensitizer. The triplet life times were determined under the same conditions. Thus, for porphyra-334 a value of 14 μs was obtained, while shinorine and palythine yielded 11 μs and 9 μs, respectively.

Non radiative deactivation

Photoacoustic calorimetry studies allowed the direct quantification of non-radiative deactivation pathways of the excited species [31]. Laser induced experiments at two different temperatures on porphyra-334 and shinorine aqueous solutions led to conclude that around 98 % of the absorbed energy was promptly dissipated as heat to the environment. For palythine, analogous determinations showed that ca. 90 % of the light energy is transferred to the medium as heat [35]. Thus, these compounds proved to have all the features that should be desirable for an ideal sunscreen from the photophysical point of view.

Photostability

As previously stated, MAAs have been suggested to play a number of different roles. While some of them are probably speculative, the photoprotective capabilities of these species have been analyzed in detail. The role in UV-protoprotection is supported by several characteristics that turn MAAs into compounds capable of dealing very efficiently with the potentially damaging effects of radiation. First, MAAs evidence a strong UV absorption in the UVA-UVB region of the spectrum as described in the previous section. Second, these compounds also show a very low fluorescence emission yield. Nevertheless, a number of natural compounds can also share these two properties. In the case of MAAs, these features are also accompanied by a high photostability that greatly increases their relevance.

Preliminary results on the photostability of MAAs were obtained through UV irradiation of eggs of the green sea urchin [32]. The concentration of shinorine (the predominant MAA in the samples) remained unaltered for short-term UV irradiation in vivo and long-term exposure in vitro as determined by HPLC. Also, extracts from the red alga Gracilaria cornea (mainly containing porphyra-334 and shinorine) showed no change in the in vitro absorption properties when exposed to UVB as demonstrated by UV absorption spectroscopy [37]. More recently, a number of reports by Rastogi and Incharoensakdi have addressed the stability of MAAs occurring in different cyanobacteria species with the purpose of evaluating the photoprotective ability of these ancient organisms against damaging effects of UV radiation. They showed that palythine, asterina and a compound designated as M-312 which could not be identified, all present in a partially purified aqueous extract from Lyngbya sp., were highly resistant to UVB [38]. Similarly, the absorbance of mycosporine-glycine obtained from the cyanobacterium Arthrospira (Spirulina) was found to slightly decrease upon exposure to different regions of UV radiation [39]. Shinorine and a non identified compound with maximal absorbance at 307 nm were detected in Gloeocapsa sp. isolated from the autotrophic biofilm covering stone monuments. The extent of degradation of the compounds in a partially purified extract under spectrally differentiated UV sources was also qualitatively assessed by HPLC analysis [40].

Further detailed studies on diverse MAAs provided quantitative evidences on the high degree of photostability of these compounds in vitro. The photodegradation of aqueous solutions of porphyra-334 was explored by following the spectral changes as a function of time when irradiating with a medium-pressure Hg lamp [28]. The photodecomposition quantum yield was determined to be Φ_R = 1.9 × 10⁻⁴ under N₂ atmosphere and Φ_R = 3.4 × 10⁻⁴ in 1 atm O₂-equilibrated aqueous solutions, by using valerophenone as actinometer. On one hand, this MAA was found also to be very photostable. On the other hand, the small effect of oxygen in the photodecomposition quantum yield seems to suggest a scarce contribution of the triplet state in the photochemistry of these compounds. In agreement with the results from photoacoustic calorimetry described above, shinorine and porphyra-334 dissipate most of the absorbed energy as heat into the environment consistently with the very low photodecomposition quantum yield [28, 31]. Palythine was also studied in order to confirm its photostability [35]. Continuous irradiation at 320 nm (absorption maximum) of aqueous solutions showed no significant changes in the pH nor the absorbance. The photodecomposition quantum yield for this MAA was found to be Φ_R = 1.2 × 10⁻⁵.

These results suggest that the photostability of MAAs is related to their core structure and the variability of substituents among different MAAs only have a minor effect on the photochemical properties. Interestingly, the photostability of these compounds is also maintained when incorporated in more complex structures. This is illustrated by mycosporine-glutaminolglucoside (Fig. 3) [41], a mycosporine metabolite present in several yeast species, which was also found to be very photostable (Φ_R = 1.2 × 10⁻⁵) most probably due to the mycosporine moiety [42].

Fig. 3:

Molecular structure of mycosporine-glutaminolglucoside.

Photoisomerization

The high photostability of MAAs seems to be caused by the general structure of aminocyclohexenimine or aminocyclohexenone. Beyond this common moiety, the different substituents in the basic core of MAAs appear to be related with the metabolic routes but most of them have a minor effect in the photophysical and photochemical properties of these compounds. The amino and alcohol groups directly bonded to the chromophore only affect the wavelength of absorption while keeping the photostability almost unchanged. The rest of substituents do not have any relevant influence in the chromophore. Acid, amino, and alcohol groups present in the lateral chains have probably their origin in the biosynthetic routes and seem to have minor effects on the solubility. However, some differences may arise when the substituents directly affect the chromophore. Among the different isolated MAAs, this may happen in the pair palythene–usujirene (Fig. 1) [33]. In these two compounds, an additional double C=C bond is conjugated with the common chromophore. Thus, the absorption occurs at higher wavelengths: 360 nm for palythene and 357 nm for usujirene, in contrast with the absorption at 320 nm for palythine, the equivalent MAA without the C=C bond. Besides this evident consequence of the extra double bond, a higher photoreactivity was also expected.

In a careful study of the reactivity of this couple of compounds, continuous irradiation of usujirene at 366 nm and HPLC quantitative analysis allowed for the evaluation of its photodecomposition quantum yield, amounting Φ_R = 2.9 × 10⁻⁵ [43]. However, most of the disappearance of usujirene was due to the cis-trans isomerization of the exocyclic C=C double bond to yield palythene with an only slightly lower quantum yield (Φ_iso = 1.7 × 10⁻⁵). Thus, most of the reacting usujirene leads to the formation of palythene, its photostable isomer. Under these conditions, a photostationary state composed by palythene and usujirene (11:1) was found, probably explained by the intrinsic higher stability of the trans C=C double bond in palythene. Thus, usujirene and palythene undergo photoisomerization as an additional reactivity in the excited state. This reactive pathway converts one isomer into the other affecting the mixture composition while keeping the global photoprotective capacities.

Photosensitized processes

As described in the previous sections, the high photostability of MAAs is a key aspect for their photoprotective capability together with the strong absorption in the relevant UV regions. From a purely photochemical point of view this implies that direct absorption of a photon populates excited electronic states of singlet multiplicity that eventually decay to the ground state mainly through non-reactive and non-radiative pathways. However, it should be noted that other reactive possibilities may also influence the protection achieved in vivo. Among them, the photosensitized processes are viable photodegradation routes provided that different sensitizers may be available in biological media. For instance, flavin-mediated photodegradation of mycosporine glutamine leads to the formation of an aminocyclohexenone and 2-hydroxyglutaric acid [44]. This reactivity is independent of the pH but temperature and light wavelength have a clear effect. Thus, a mixture of photochemical and thermal degradation paths seems to be operating at the same time. Different sensitizers such as erythrosine, methylene blue or Rose Bengal (RB) are also effective.

Further comparative studies with up to seven different MAAs evidenced photosensitized decomposition in the presence of riboflavin, RB or simply natural seawater [45]. However very different decomposition rates were observed when RB (an efficient singlet oxygen source) and riboflavin (a photosensitizer) were used. A higher rate of photodecomposition was observed when riboflavin was present, this indicating that a type I photooxidation mechanism may be responsible for the degradation. Also, the requirement of a strong photosensitizer such as riboflavin and the low decomposition rates when other sensitizers are present further confirms the high photostability of MAAs even under indirect irradiation.

It was already mentioned that several MAAs have been the subject of detailed analysis in photosensitization experiments that enabled the characterization of their triplet states. In fact, neither porphyra-334 nor shinorine showed any transient absorption in laser flash photolysis measurements within the microsecond time range. In contrast, the triplet states were detected upon sensitization with benzophenone or 1-naphtalene-methanol for porphyra-334 and with acetone for shinorine [28, 31]. Palythine also forms triplets under sensitization with acetone [35].

The energy of these triplets states were respectively estimated by considering the efficiency of the energy transfer in relation with the triplet energy of the donor. These triplet energy values are relevant for the discussion of the complementary role of MAAs that consists of avoiding the formation of pyrimidine dimmers [46]. These species are associated with molecular damages caused in DNA through photochemical reactions. When thymine or cytosine bases absorb UV light, the formation of covalent linkages between two of these moieties by reactions localized on the C=C double bonds may take place [47]. Thus, MAAs were proposed not only to protect against the UV damage by direct absorption and filtering of the light, but also through quenching the excited state of thymine thus avoiding the formation of the dimers. However, as the value for the triplet energy of thymine in DNA was estimated to be 270 kJ mol⁻¹ [48], it seems that this possibility could be ruled out in the case of palythine (triplet energy ca. 330 kJ mol⁻¹) as no energy transfer seems possible from the thymine excited state. In contrast, porphyra-334, with triplet energy of 250 kJ mol⁻¹, could quench the excited thymine and contribute to the photoprotection through this additional mechanism.

UV-Vis absorption

The electronic absorption characteristics of gadusol extracted from marine fish roes were examined in different solvents. In water solution, the maximum shifts reversibly from 268 nm to 296 nm and the intensity increases on going from acidic to neutral pH [57]. Previous publications by Grant et al. [50] and by Bandaranayake et al. [51] report respectively the values 269 (12 400 M⁻¹ cm⁻¹) and 264 nm (12 900 M⁻¹ cm⁻¹) for the peak wavelength and maximal molar absorption coefficient, ε_max, of the band in the acidic medium. For the absorption band at higher pH the informed values for ε_max range from 21 800 to 22 750 M⁻¹ cm⁻¹ [11, 50, 51].

The effect of the pH on the absorption spectrum of the metabolite has been ascribed to the deprotonation of the enol form of the molecule (gadusol) to give the resonance-stabilized enolate (gadusolate) as depicted in Fig. 4 [11, 50, 57].

Fig. 4:

Gadusol–gadusolate equilibrium.

The solvent effect was evaluated for the enolic species in acetonitrile−water mixtures, various alcohols, 1,4-dioxane, and organic acids. A tendency to bathochromic shifts was observed for the absorption maxima with increasing polarity of the solvent, pointing to a relative stabilization of the excited state which suggests its π−π* nature. The results are consistent with specific interactions involving hydrogen bonds between gadusol and the solvent molecules. Correlation of the data according to the Kamlet−Taft empirical approach indicates comparable contributions of the hydrogen donor and acceptor characters of such interactions, probably implying the hydroxylic and ketone groups of gadusol [57].

Radiative and non-radiative deactivations

Steady state fluorescence measurements evidenced no net emission from gadusol in acid water by excitation at 268 nm or from gadusolate under neutral pH by excitation at 296 nm [57].

Non-radiative relaxation pathways were quantified for gadusolate, the physiologically relevant species, in aqueous phosphate solution by photoacoustic calorimetry. The photoacoustic signals for gadusolate with laser excitation at 266 nm matched exactly those from the calorimetric reference; potassium dichromate dissolved in the same medium, no matter the temperature of the experiment (298 K or 283 K). This indicated that excited gadusolate promptly releases the complete amount of absorbed UV energy as heat to the surroundings.

No transient absorption was detected by direct laser flash photolysis of gadusolate at 266 nm that could be attributed to T–T transitions [57]. The result contrasts the production of stabilized triplets reported for β-carbon substituted α,β-unsaturated ketones [59]. However, in the case of gadusolate, the formation of long lived triplets is probably avoided under the competition with the extremely rapid internal conversion involving allowed π → π* transitions.

Photostability

The photostability of both forms of gadusol in aqueous solution was assessed by monochromatic steady irradiation and chemical actinometry with phenylglyoxylic acid. The photodecomposition quantum yields amounted Φ_R = 3.6 × 10⁻² for gadusol at 254 nm and Φ_R = 1.4 × 10⁻⁴ for gadusolate at 303 nm. No significant effect was found for the presence of oxygen on these yields [57]. Both values denote a high photostability, although the photodecomposition yield for the neutral form of gadusol is two orders of magnitude larger. Thus, the most photostable species from both is the anionic form, gadusolate, which dominates the acid base equilibrium under physiological pH. Moreover, gadusolate photodecomposition quantum yield compares better than gadusol’s with the values for MAAs in solution [28, 31, 35]. This result accounts for the favorable conditions supporting a UV-screening role of the metabolite, particularly relevant in prebiotic environments.

Photosensitized processes

A series of laser flash photolysis studies on gadusolate in the presence of efficient triplet sensitizers were carried out in polar media and anoxic conditions by Arbeloa et al. [57]. The studies confirmed the ability of the natural molecule to react as a reductive quencher. The transient spectra obtained for experiments in methanol with excitation at 355 nm and benzophenone or acridine as sensitizers are consistent with the occurrence of electron transfer processes. In fact, the spectral features of benzophenone ion radical, and a neutral “C” radical from acridine were respectively evidenced in these studies.

On the other hand, RB was used as triplet donor in water solution with laser excitation at 532 nm. The kinetic analysis of the transient bands clearly showed the decrease of the absorbance of RB triplet state at 590 nm and the concomitant enhancement of the signal at 410 nm due to the semireduced form of the sensitizer. The time constant of the decay of triplet RB as a function of gadusolate concentration followed the Stern-Volmer dependence with a quenching rate constant k_q = 2 × 10⁸ M⁻¹ s⁻¹, thus below the diffusion limit in water. However, the presence of gadusolate at concentrations as large as 2.3 × 10⁻³ M did not affect the transient absorption spectrum of anthracene in methanol obtained by excitation at 355 nm. These findings were rationalized in terms of the following one electron transfer from gadusolate (Gad⁻) to the triplet state sensitizer (³Sens) yielding the sensitizer radical anion (Sens^•−), and the neutral gadusol radical (Gad^•):

An energetic analysis of reaction (1) requires taking into account the reduction potentials of the sensitizer and of gadusol, and the triplet energy of the sensitizer. On this basis, a less favourable thermodynamic balance explains the lack of reaction with anthracene triplets and predicts the feasibility of gadusol to quench natural sensitizers such as riboflavin. Thus, the reductive quenching reactivity of gadusol may be considered as one of the potential contributions to the antioxidant mechanisms involving the metabolite in biological environments.