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

TLDR: In this article, experimental and theoretical studies on model mycosporine-like amino acids and gadusol offer a helpful description of the photoprotective mechanism at the molecular level.

Abstract: Mycosporine-like amino acids (MAAs) and related gadusols are among the most prominent examples of metabolites suggested to act as UV-sunscreens. This review illustrates how experimental and theoretical studies on model MAAs and gadusol offer a helpful description of the photoprotective mechanism at the molecular level. This knowledge may contribute to the rational design of chemical systems with predictable and tuneable response to light stimulus. Synthetic efforts to obtain MAAs and simplified related structures are also discussed. © IUPAC & De Gruyter 2015.

Figures

Fig. 3: Molecular structure of mycosporine-glutaminolglucoside.

Fig. 6: Critical points along the potential energy surface and MEP for the palythine protonated form. Energies in kcal mol−1 relative to ground state minimum. Sampedro, D. Phys. Chem. Chem. Phys. 2011, 13, 5584. Reproduced by permission of the PCCP Owner Societies.

Fig. 7: S1/S0 conical intersection geometry and conformers obtained after ground state relaxation for the palythine neutral form. Sampedro, D. Phys. Chem. Chem. Phys. 2011, 13, 5584. Reproduced by permission of the PCCP Owner Societies.

Fig. 14: Retrosynthesis proposed in the WO0239974A1 patent.

Fig. 15: Synthesis of rigid core vinamidinium salts.

Fig. 8: Computed anionic species for gadusol.

Full text

Pure Appl. Chem. 2015; 87(9-10): 979–996
Conference paper
Raul Losantos, Diego Sampedro and María Sandra Churio*
Photochemistry and photophysics
ofmycosporine-like amino acids and
gadusols, nature’s ultraviolet screens
DOI 10.1515/pac-2015-0304
Abstract: Mycosporine-like amino acids (MAAs) and related gadusols are among the most prominent exam-
ples of metabolites suggested to act as UV-sunscreens. This review illustrates how experimental and theoreti-
cal studies on model MAAs and gadusol offer a helpful description of the photoprotective mechanism at the
molecular level. This knowledge may contribute to the rational design of chemical systems with predictable
and tuneable response to light stimulus. Synthetic efforts to obtain MAAs and simplified related structures
are also discussed.
Keywords: computational chemistry; laser spectroscopy; natural products; organic chemistry; Photo-
biology-16; photoprotection; sunscreens; synthesis.
Introduction
Since the late 1970s, hundreds of publications have addressed the isolation, structural elucidation, occur-
rence and roles of mycosporine and mycosporine-like amino acids (MAAs). These are two groups of secondary
metabolites synthesized by cyanobacteria, fungi, algae and ingested or accumulated by high order animals
such as fish, cnidarians, arthropods, molluscs, and echinoderms [1, 2]. Several hypotheses about the role of
these compounds in biological systems have been formulated and are still on discussion. Among other func-
tions, osmotic and reproduction regulation, UV transduction in photosynthesis, intracellular nitrogen reser-
voir and ecological connectivity have been suggested [3–5]. The MAAs in particular have been recognized as
molecules of key significance in marine ecosystems [6]. Beyond the controversy about their multifunctionality,
MAAs have mainly attracted interest around their role as UV sunscreens in a wide variety of organisms [4, 7–9].
Mycosporine and MAAs are water soluble compounds; their basic chemical structure contains a cyclohex-
enone or a cyclohexenimine unit (Fig. 1). Fungal mycosporines and only two MAAs from marine sources
(mycosporine-glycine and mycosporine-taurine) consist of a cyclohexenone ring system linked to an amino
acid, with maximal absorption between 310 and 320 nm. The rest of the MAAs generally includes a glycine
moiety attached to a cyclohexenimine ring and a second substituent group (amino acid, amino alcohol or an
Article note: A collection of invited papers based on presentations at the 16
th
International Congress on Photobiology (ICP-16),
Córdoba, Argentina, 7–12 September 2014.
*Corresponding author: María Sandra Churio, Facultad de Ciencias Exactas y Naturales, Departamento de Química, Universidad
Nacional de Mar del Plata, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Dean Funes 3350, B7602AYL,
Mar del Plata, Argentina, e-mail: schurio@mdp.edu.ar; sanchurio@hotmail.com
Raul Losantos and Diego Sampedro: Departamento de Química, Universidad de La Rioja, Centro de Investigación en Síntesis
Química (CISQ), Madre de Dios, 51, 26006 Logroño, Spain
© 2015 IUPAC & De Gruyter
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980
R. Losantos etal.: Photochemistry and photophysics of MAAs and gadusols
enaminone system). This results in a diversity of more than 20 structures with UV-absorption bands at maximum
wavelengths from 320nm to ca. 360nm and molar absorptions coefficients in the range from 28 000 to 60 000
M
−1
cm
−1
[7]. In fact, MAAs have been considered as the strongest UVA absorbing compounds in nature.
On the other hand, related compounds such as gadusol and deoxygadusol (Fig. 1) are natural cyclohex-
enones resembling the mycosporine core. They absorb intensively towards the UVB and UVC spectrum with
pH-dependent distinctive maxima: 269 (pH 2) and 296 (pH 7) [10, 11].
Both compounds are generally involved as intermediates in the proposed schemes for MAAs biosynthesis
[2, 8, 12]. Their role in UV photoprotection is particularly relevant from an evolutionary point of view since
primitive organisms that developed in the absence of atmospheric O
2
may have required UVB/C screening,
hypothetically fulfilled by gadusols and oxo-mycosporines [13].
UV-induced synthesis and accumulation have been broadly reported and generally regarded as central
evidence supporting the sunscreening role of MAAs in living organisms [4, 14–17]. However the chemistry and
molecular basis of the photoprotective potential of MAAs and gadusols have been examined to a lesser extent.
The exploration of the basic photochemical and photophysical features of MAAs is important for the
understanding of natural mechanisms of photoprotection which becomes particularly significant in the
context of the stratospheric ozone depletion phenomenon and the consequent effects of UV radiation on
living beings [18]. This problematic has led to consider MAAs and other natural photoprotective molecules
as valued alternatives to synthetic compounds for the formulation of topical sunscreens [19, 20]. Besides, the
characterization at a molecular level of the light induced behaviour of this family of compounds may help
to designing derivatives for future applications in the cosmeceutical and pharmaceutical industries [21, 22].
In this work we review the in vitro experimental studies and theoretical calculations reported to date on
the photochemistry and photophysics of MAAs and gadusols.
Mycosporine-like amino acids
As seen, MAAs are low-molecular-weight, generally colorless and water-soluble compounds that are also
resistant to thermodegradation and photodegradation under environmental conditions. Most of the photo-
N
O
NH
CO
2
H
HO
HO
CO
2
H
HO
N
O
NH
CO
2
H
HO
HO
CO
2
H
HO
NH
O
NH
CO
2
H
HO
HO
Porphyra-334
Shinorine Palythine
Mycosporine-glycine
Mycosporine-taurine
O
O
NH
CO
2
H
HO
HO
O
O
NH
HO
HO
SO
3
H
N
O
NH
CO
2
H
HO
HO
Palythene
N
O
NH
CO
2
H
HO
HO
Usujirene
N
O
NH
CO
2
H
HO
HO
OH
Palythinol
O
O
OH
HO
HO
HO
O
O
OH
HO
HO
Gadusol
6-deoxygadusol
Fig. 1: Chemical structures of some relevant MAAs and gadusols.
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R. Losantos etal.: Photochemistry and photophysics of MAAs and gadusols
981
physical and photochemical properties of these molecules are quite general and independent of the specific
substituents linked to the core structure. Thus, general aspects will be described in the following sections
with specific examples to illustrate the behavior of these compounds.
Photophysical properties
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 chromo-
phore 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 impor-
tant 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 por-
phyra-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.
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 stabil-
ity of this compound in very acidic water solutions. In contrast, under relatively low basic conditions of pH
NH
O
NH
CO
2
HO
HO
CO
2
H
HO
N
O
NH
CO
2
HO
HO
CO
2
HO
NH
O
NH
CO
2
H
HO
HO
CO
2
H
HO
+H
+
+H
+
–H
+
–H
+
pH>3
pH 2–
3p
H 1
λ=334 nm λ=332 nm
λ=330 nm
Fig. 2: Molecular structures of porphyra-334 at different pH values.
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982
R. Losantos etal.: Photochemistry and photophysics of MAAs and gadusols
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 avail-
able 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 some-
times 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 dissi-
pates 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 tuber-
culosa [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 impu-
rity and not to palythine.
According to these results, the suggested role of MAAs as transducers of the UV light to more useful wave-
lengths 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-naph-
thalene-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 gener-
ate 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 benzophe-
none 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].
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R. Losantos etal.: Photochemistry and photophysics of MAAs and gadusols
983
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 shi-
norine 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.
Photochemical properties
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 pho-
tostability 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 unal-
tered 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 spec-
troscopy [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 pho-
tostability 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 N
2
atmosphere
and Φ
R
= 3.4 × 10
−4
in 1 atm O
2
-equilibrated aqueous solutions, by using valerophenone as actinometer. On
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Frequently asked questions

Q1. What is the main characteristic of an ideal photoprotective compound?

Beyond the strong absorption in the relevant regions of the UV spectrum, the photostability is a key feature of an ideal photoprotective compound. 

Q2. how did the decay of triplet RB follow the Stern-Volmer dependence?

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 kq = 2 × 108 M−1 s−1, thus below the diffusion limit in water. 

Q3. What is the effect of the amino and alcohol groups on the chromophore?

The amino and alcohol groups directly bonded to the chromophore only affect the wavelength of absorption while keeping the photostability almost unchanged. 

Q4. What is the main reason for the high photostability of MAAs?

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. 

Q5. What is the way to avoid the formation of long lived triplets?

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. 

Q6. What are the characteristics that make MAAs a good sunscreen?

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. 

Q7. How does porphyra-334 show a hypsochromic shift?

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. 

Q8. What type of charge stabilization has been previously proposed for other types of compounds?

This type of push–pull charge stabilization has been previously proposed for other types of compounds showing also high proton affinity. 

Q9. What is the role of mycosporine and mycosporine-like amino acids?

Since the late 1970s, hundreds of publications have addressed the isolation, structural elucidation, occurrence and roles of mycosporine and mycosporine-like amino acids (MAAs). 

Q10. What is the key aspect for the photoprotective capability of MAAs?

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. 

Q11. What is the effect of the double bond on the photodecomposition of usuji?

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 ΦR = 2.9 × 10−5 [43]. 

Q12. What is the basic chemical structure of mycosporine and MAAs?

Mycosporine and MAAs are water soluble compounds; their basic chemical structure contains a cyclohexenone or a cyclohexenimine unit (Fig.  1). 

Q13. What are the main effects of the different substituents in the basic core of MAAs?

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. 

Q14. How many kJ mol1 does palythine have?

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.