PAPER www.rsc.org/pps | Photochemical & Photobiological Sciences

Experimental study of the excited-state properties and photostability of themycosporine-like amino acid palythine in aqueous solution

Federico Ruben Conde,a Marıa Sandra Churio*a,b and Carlos Mario Previtalib,c

Received 15th December 2006, Accepted 2nd March 2007First published as an Advance Article on the web 20th March 2007DOI: 10.1039/b618314j

Characterization of the excited states of the mycosporine-like amino acid palythine (kmax = 320 nm) inaqueous solutions was achieved experimentally. The low value for the photodegradation quantumyield, (1.2 ± 0.2) × 10−5, confirms that palythine is highly photostable in air saturated-aqueoussolutions. Laser flash photolysis of acetone in the presence of palythine allowed for the observation of atransient spectrum which is consistent with the triplet–triplet absorption of palythine. Kinetic treatmentof the transient signals yields a lifetime of the triplet state of ca. 9 ls and a triplet energy around330 kJ mol−1. The photoacoustic calorimetry results are consistent with non-radiative decay as themajor fate of excited palythine. A comparison of the photodegradation quantum yields andphotophysical properties of palythine with those previously determined for the other mycosporine-likeamino acids, shinorine and porphyra-334, suggests that geometrical isomerization around the C=Nbond may contribute to the rapid deactivation of this group of molecules.

Introduction

Reduction of effective UV radiation by synthesis of inter- orextracellular screening agents is one of the major types ofstrategies that many organisms use to minimize UV-induceddamage.1 Mycosporine-like amino acids (MAAs) constitute agroup of intracellular substances absorbing intensively in theUV range that seem to play such a role in photoprotection ofdiverse forms of aquatic life.2–6 In fact, MAAs are considered asmultifunction secondary metabolites also exhibiting antioxidantproperties,7–9 activity in osmotic and reproductive regulation10,11

and, according to recent reports, utility as nitrogen reservoirs too.12

Formerly, it was hypothesized that MAAs may act as auxiliarypigments in photosynthesis by transferring the absorbed energythrough fluorescence to chlorophylls.13 However, in vivo spectralfluorescence excitation on colonies of phytoplankton have shownthat chlorophyll a fluorescence does not correlate with MAAsabsorption in the UV region.14 On the other hand, the suppositionis not in line with the reduced fluorescence quantum yieldsmeasured by Conde et al.15,16 and confirmed by Inoue et al.,17 thusadding evidence against a key role of MAAs in photosynthesis.

Studies on MAAs as UV sunscreens in physiological and ecolog-ical contexts are abundant in the literature (for examples see ref. 2–7, 18–20, and references therein) however only a few reports focuson their physicochemical properties and on the molecular basisunderlying possible UV-protective mechanisms.15–17,21–23 Indeed, asinclusion of MAAs in sunscreen formulations becomes commer-cially attractive, evaluation of potential hazards associated withthis use needs the examination of the photochemistry of the com-

aDepartamento de Quımica, FCEyN, Universidad Nacional de Mar delPlata, Funes 3350, B7602AYL, Mar del Plata, Argentina. E-mail: schurio@mdp.edu.ar; Fax: 54 223 4753150; Tel: 54 223 4756167bConsejo Nacional de Investigaciones Cientıficas y Tecnicas (CONICET),ArgentinacDepartamento de Quımica, Universidad Nacional de Rıo Cuarto, 5800, RıoCuarto, Argentina

pounds.24,25 Within this frame, an assessment of the mechanismresponsible for the photoprotective role and antioxidant activity isan issue that deserves special attention. In particular, it is impor-tant to establish whether these functions are exerted as passiveradiation filters or through molecular energy transfer, for instanceby avoiding the formation of thymine photodimers, or both.26

A deeper knowledge about the influence of the molecular struc-ture on the photochemical behavior of this kind of compoundsmay contribute as well to the field of medical- and cosmetic-drugsdesign. In addition, evaluation of the photostability of each MAAis a primary requirement for the complete interpretation of theradiation-induced in vivo transformations of biosynthetized oracquired MAAs that may explain their high diversity in marineorganisms.20,27,28

With this perspective, our group has been interested in thephotophysical and photochemical characterization of MAAs andrelated compounds in vitro.15,16,27 Our results on shinorine andporphyra-334 confirm that the electronic-excited states of themolecules relax mainly by non-radiative pathways,15,16 which isconsistent with the assigned photoprotective role.

Palythine, a MAA frequently found in alga, cnidarians andplanktonic organisms,20,28–30 represents the structurally-simplestpattern bearing the basic cyclohexenimine unit common to most ofthe members of this family of compounds (see Scheme 1). Photo-degradation rate constants for palythine under polychromatic

Scheme 1

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irradiation in the presence of riboflavin or Rose Bengal, and alsoin sea water medium, have been recently determined.21 Neverthe-less, a direct comparison of photostabilities of structure-relatedcompounds can be achieved by considering photodecompositionquantum yields.

In this work, we report on the experimental results concerningthe characterization of the excited states of palythine in aqueoussolution. The study has been aimed to the determination ofthe photodegradation quantum yield, the exploration of tripletstate production and the direct quantification of the non-radiativerelaxation pathways.

Experimental

Materials

Palythine was isolated from the red algae Palmaria decipienscollected from Antarctica.27 Methanolic extracts of the algaewere treated and finally purified by HPLC according to reportedprocedures for the isolation of the MAAs shinorine and porphyra-334.15,16 The chromatographic peak ascribed to palythine showedmaximal absorption at 320 nm. The retention time was calibratedwith a standard solution.

Indigo carmine (IC) from Sigma-Aldrich was used withoutfurther purification. Sodium chloride was analytical grade fromMerck. Acetone and acetonitrile, PA reactants from Sintorgan,were used as received. Phenylglyoxylic acid (PGA) from Sigmawas recrystallized from carbon tetrachloride (PA, Dorwill) andstored in a desiccator in the dark. Water was Millipore Milli-Qgrade.

Methods

Steady-state photolysis experiments. Air-saturated aqueoussolutions of palythine ca. 1.5 × 10−5 M were continuouslyirradiated in a 1 cm path length quartz cuvette under stirringat room temperature. The output of a 1000 W Hanovia Hg–Xehigh-pressure lamp, dispersed by a high-intensity grating mono-chromator (Schoeffel-Kratos), was tuned in 320 ± 10 nm anddirected perpendicularly to the cuvette wall. Changes in the com-position of the solutions were monitored by UV-vis absorptionspectrophotometry and/or HPLC analysis.

Actinometry. The total intensity of irradiation at 320 ± 10 nm,measured as the volumetric photon flux, was determined bychemical actinometry with PGA. Bleaching of 25 mM solutionsof PGA in acetonitrile : water (3 : 1) under N2 atmosphere wasmonitored by optical absorption at 380 nm.31

Spectrophotometric measurements. Spectral changes duringthe photolysis and actinometry experiments were recorded on aShimadzu UV-2001 PC scanning double beam UV-vis absorptionspectrophotometer. Corrected steady-state emission and excita-tion spectra were obtained on a Spex Fluoromax spectrofluorime-ter at room temperature.

Transient absorption measurements for the characterizationof the triplet states were carried out by laser flash photolysis(LFP) with a N2 laser (337 nm, Laseroptics, Argentina) or aNd:YAG laser (266 nm, Spectron Laser SL40, UK) as excitationsources. Further details concerning the LFP system are described

elsewhere.32 Solutions analyzed by this technique were kept free ofO2 by previous bubbling with Ar during 20 min.

Photoacoustic calorimetry (PAC). The photoacoustic signalswere induced by the N2-laser beam with 1 mm width collimationon palythine in 0.1 M NaCl aqueous solutions with absorbanceca. 0.26 at 337 nm. The experimental set-up was analogous to theone used in a previous work.16 IC in 0.1M NaCl aqueous solutionwas employed as the calorimetric reference. Measurements wereconducted at 296.5 and 283.5 ± 0.1 K.

Results

Aqueous palythine probed to be stable in the dark at pH between6 and 7 and below 45 ◦C. The solutions show one wide absorptionband with maximum at 320 nm (Fig. 1).

Fig. 1 Absorption spectra of aqueous palythine, initial concentration1.5 × 10−5 M, after different irradiation times at 320 ± 10 nm, as indicatedon the left side of the curves. The inset shows time evolution of theabsorbance at 320 nm. The initial photolysis rate was estimated fromthe initial slope of the data (dashed line).

A weak emission around 390 nm was observed upon excitationat 320 nm of a sample of aqueous palythine with maximal ab-sorbance A320 = 0.19. However, the excitation spectrum registeredwhen monitoring the emission at 390 nm did not agree with theabsorption spectrum of the sample indicating that the emissionmay arise from some impurity.

Continuous photolysis experiments on palythine aqueous so-lutions were carried out at 320 ± 10 nm. The pH of thesamples was ca. 6.8 and did not change upon irradiation. Thephotodecomposition rate was determined by following the spectralchanges as a function of time. Fig. 1 shows typical results.Quantitative analysis of palythine by HPLC on the photolyzedsolutions were in agreement with absorbance measurements. Nonew absorbing species in the spectral range between 290 and360 nm was detected by HPLC of the irradiated samples. The initialphotolysis rate, vi was determined from plots of the absorbance at320 nm vs. photolysis time (Fig. 1, inset) and the molar absorptionof palythine (e320 = 36200 M−1 cm−1).29 The incident light intensityI 0 was assessed by photolysis of PGA. The quantum yield forpalythine photodecomposition UR was calculated from the ratio

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of vi and the initial absorbed intensity I a = I 0(1 − 10−〈A〉), where<A> stands for the absorbance at 320 nm averaged over thephotolysis period. Finally it results UR = (1.2 ± 0.2) × 10−5.

In order to attain the triplet-excited state characterization ofthe MAA, 6 × 10−5 M palythine was analyzed by LFP withexcitation at 337 nm. No transient absorption was observedin the microsecond range within the detection limit of theexperimental set-up. However, a net absorption signal between 350and 600 nm appeared in sensitization experiments with acetone.These measurements were carried out by excitation at 266 nm of3 × 10−2 M acetone in the presence of 4.5 × 10−4 M palythine. Fig. 2shows the transient absorption spectrum taken 3 ls after the laserpulse. Fig. 3 includes signal profiles observed at 300 nm (whereonly triplet acetone absorbs) in the absence or in the presenceof palythine, traces a and b, respectively. The third trace c wasrecorded at 400 nm and reflects the growing and decay of thesensitized transient.

Fig. 2 Triplet–triplet absorption spectra obtained via sensitization of4.5 × 10−4 M palythine with 3 × 10−2 M acetone by LFP at 266 nm.The spectrum was taken 3 ls after the laser pulse.

Fig. 3 Time evolution of the absorbance at a single wavelength obtainedby LFP at 266 nm. Trace a was recorded at 300 nm for 3 × 10−2 M acetonein the absence of palythine. Traces b and c were obtained for a mixture of3 × 10−2 M acetone and 4.5 × 10−4 M palythine at 300 nm and 400 nm,respectively.

The non-radiative decay of excited-state palythine was assessedby PAC. With the aim of preventing from usual fluctuations oflaser intensity, the amplitudes H of the photoacoustic signals forpalythine and for IC at a given temperature were normalized bythe absorbed laser energy Ea = E0(1 − 10−A), where E0 standsfor the incident laser fluence and A is the absorbance at thelaser wavelength.33 Fig. 4 shows the plots of H/(1 − 10−A) vs.E0 and the curves that represent the linear regression of thedata up to ca. 80% of full laser fluence, thereby avoiding therange where non-linearities due to multiphotonic processes mayarise. Hence, energy-normalized amplitudes Hn were estimatedas the slope of the linear plots of H/(1 − 10−A) vs. E0. Theratio of normalized amplitudes, i.e. Hn(palythine)/Hn(IC), wasevaluated at two different temperatures in order to discriminatepossible contributions to the photothermal signal arising fromphotoinduced structural volume changes.33 The results at 296.5and 283.5 K did not differ significantly within the experimentalerror, affording respectively 0.91 ± 0.05 and 0.95 ± 0.05.

Fig. 4 Plots of the photocalorimetric signal amplitude normalized by thefraction of the absorbed energy H/(1 − 10−A) vs. the incident laser energy,expressed as percentage of full laser fluence, E0. Full symbols refer to IC,the calorimetric reference, and empty symbols correspond to palythine ca.2.6 × 10−5 M at 283.5 K (circles) and 296.5 K (squares). Dotted lines showthe linear regression of the data up to ca. 80% E0.

Discussion

The lack of correspondence between the excitation and absorptionspectra of the aqueous solution of palythine suggests that theweak emission should not be ascribed to the fluorescence of thisMAA but to some impurity in the sample. This result is in linewith the low fluorescence quantum yields previously reportedfor porphyra-334 and shinorine, two closely structure-relatedMAAs.15,16

The high photostability of palythine is corroborated by the lowvalue for the photolysis quantum yield in water UR = (1.2 ±0.2) × 10−5. In fact, the elevated resistance to photodegradationof palythine was addressed in the work by Whitehead andHedges.21 However, the study was carried out under polychromaticirradiation in the presence of assorted sensitizers, thus makingthe normalization of the kinetic data difficult. Our result instead

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allows for the direct analysis of the structure-dependence of thephotochemical behaviour of different MAAs. In this context,the comparatively larger photodecomposition quantum yieldsfor aqueous porphyra-334 and shinorine determined under thesame conditions (2.4 × 10−4 and 3.4 × 10−4, respectively)16

seem to indicate that substitution in the nitrogen atom of thecyclohexenimine unit contributes to reduce the photochemicalrobustness of the molecule in more that one order of magnitude.Involvement of competitive relaxation pathways of the excitedstates through geometrical isomerization around the C=N bondcan be relevant to this point.34 In fact, the energy barriers to C=Nrotation and to nitrogen inversion may be substantially influencedby structural effects. Particularly, nitrogen inversion rates dependmarkedly on the substituents on nitrogen.35 On the other hand,a more efficient geometrical relaxation from the excited singletstate of palythine, in comparison with the cases of shinorine andporphyra-334, may explain the absence of fluorescence within thedetection limits for the first MAA. Regarding the production ofpalythine in triplet state, Fig. 2 shows an absorption spectrumwhich is consistent with the energy-transfer process from excited-state acetone. Triplet acetone absorbs below 360 nm,36 thus theabsorption transient increasing towards 400 nm is ascribed to thetriplet state of palythine. The bleaching around 320 nm is explainedby the absorption of ground-state palythine. The absorption-timeprofiles in Fig. 3 reinforce the above interpretation and are in linewith the following mechanism:

Ahm−−−→

3

A (1)

3AkA

D−−−→A (2)

3A + PkA-P

q−−−→3P + A (3)

3PkP

D−−−→P (4)

where A and P stand for acetone and palythine respectively,and the superscripts indicate the excited-triplet states of thespecies.

This simple mechanism assumes that absorption of the laserpulse by acetone molecules (eqn (1)) promotes them to the excited-triplet state with maximal efficiency (UT = 1).37 Eqn (2) accountsfor every first-order deactivation step of triplet acetone takingplace with a global constant kD

A. Alternatively, excited-stateacetone is quenched by palythine with a second order constantkq

A-P (eqn (3)). This last step generates palythine in an excited-triplet state which decays with an overall first order constant kD

P

(eqn (4)).The outcome of eqn (2)–(4) can be considered on the basis of

a two-steps consecutive kinetics involving 3P as an intermediate.The growth and decay of the transient signal at 400 nm in trace c(Fig. 3) is naturally expected from such a scheme, the absorptionbeing assigned to 3P. First, the increasing portion of the traceresponds to the formation of 3P by energy transfer from 3A whichdecays with an apparent kinetic constant k1 = kD

A + kqA-P[P].

In turn, the decrease of the signal is determined by the decayof 3P to the ground state and is described in terms of the firstorder constant k2 = kD

P. According to the consecutive kinetics,

the analytical expression for the concentration of 3P as a functionof time is given by eqn (5).

[3P

] = [3A

] k1

(k2 − k1)[exp (−k1t) − exp (−k2t)] (5)

The fit of trace c in Fig. 3 to this expression affords k1 = 1.04 ×106 s−1 and k2 = 1.16 × 105 s−1.

Besides, 3A lifetime is experimentally determined from themonoexponential fit of trace a in Fig. 3 as s0

A = (kDA)−1 = 1.81 ls.

From the result of k1, and the concentration of palythine 4.5 ×10−4 M, the value of kq

A-P yields 1.08 × 109 M−1 s−1. Finally, k2

leads to the lifetime of triplet palythine, yielding sTP = 8.6 ls.

The consistency of the above assignations can be checked bydetermination of the quenching rate constant from the lifetime ofacetone in the presence of palythine. Thus, the fit of trace b in Fig. 3gives sA = 0.99 ls. According to the Stern–Volmer relationshipfor the quenching process, the quenching rate constant can becalculated using the lifetime values in eqn (6).

kA-Pq = sA−1 − sA−1

0

[P](6)

In this way, it results kqA-P = 1.02 × 109 M−1 s−1 in reasonable

concordance with the determination from the fit of eqn (5).The magnitude of this quenching rate constant is indicative of

a diffusion-controlled process although its value is rather smallerthan that corresponding to neutral species in water. This in turnmay suggest that the triplet energy of palythine lays not far awayfrom the triplet energy of the donor, i.e. acetone (332 kJ mol−1).15,37

The result is relevant for the analysis of the issue raised by Misonouet al.26 who proposed that the MAAs mixture present in thered alga Porphyra yezoensis (mainly composed by porphyra-334,shinorine and palythine) may protect against UV-induced damagenot only through a filtering effect but also by quenching the excitedstate of thymine. In fact, the triplet energy of thymine in DNA hasbeen estimated in 270 kJ mol−1,38 thereby preventing from energytransfer to palythine with a triplet energy level larger than thisvalue.

Finally, the direct quantification of the non-radiative decayof the excited palythine was achieved by PAC. The ratio of theenergy-normalized signal-amplitudes for the MAA and for IC,the calorimetric reference, shows no dependence on temperaturewithin the error of the determinations. However, in the rangeexplored (13 ◦C below room temperature) the thermoelasticparameters of the solvent vary considerably. On this basis, it canbe assumed that the photocalorimetric signal is dominated bya thermal component, and consequently the molecular volumechanges induced by absorption of photons are negligible.33 Hence,the ratio of normalized amplitudes for palythine and IC representsdirectly the fraction of the absorbed energy delivered as heat afterthe photon absorption. Therefore our averaged result of 0.93 ±0.05 sets a lower limit of 88% for the portion of the excitationenergy of palythine that relaxes as heat to the surroundings.

Conclusions

The results obtained here confirm that palythine is highly pho-tostable in aqueous solution. The absorption assigned to tripletpalythine could be characterized by sensitization experiments with

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acetone. The kinetic treatment of the transient signals accountsfor a lifetime of the triplet state of ca. 9 ls, and a triplet energyof ca. 332 kJ mol−1 at the most. Non-radiative decay is themajor fate of excited palythine according to the results derivedfrom PAC. A comparison of the photodegradation quantumyields and photophysical properties of this MAA with thosepreviously determined for shinorine and for porphyra-334, pointsto an apparent influence of the substitution in the nitrogen atomof the cyclohexenimine unit. It is suggested that geometricalisomerization around the C=N bond may contribute to the rapiddeactivation of these MAAs.

Acknowledgements

The authors are grateful to Dr J. I. Carreto for providing thestandard of palythine and to Dr G. Ferreyra who facilitated thecollection of red algae samples. This research was supported byFundacion Antorchas (project 14022-36) and CONICET (PEI6125/01).

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