J. Photochem. Photobiol. A: Chem., 67 (1992) 245-254 245

Solubilization and photophysical and photochemical behaviour of depsides and depsidones in water and Brij-35 solutions at different pH values

M. E. Hidalgo, E. Fernandez and W. Quilhot Chem&y and Pharmacy School, Faculty of Medicine, University of Valparaiso, Valparaiso

(Chile)

E. A. Lissi

Chemist Department, Faculty of Science, University of Santiago of Chile, Santiago (Chile)

(Received October 21, 1991; accepted March 5, 1992)

Abstract

Atranorine, pannarine and 1-chloropannarine are lichenic metabolites bearing ortho- hydroxybenzaldehyde chromophoric units. The photochemical and photophysical behaviour in aqueous solutions, as well as in Brij-35 solutions, is strongly pH dependent_ At pH 2, the behaviour is similar to that observed in aprotic solvents. The singlet lifetimes are extremely short (less than 1 ns), fluorescence yields are low, and the compounds show high photostability. This behaviour is compatible with a fast intramolecular enolization. At pH 10, the absorption and emission spectra are red-shifted, the singlets are longer lived and fluorescence yields are higher. The compounds readily photodecompose. The possible role of these compounds in the lichen is discussed.

1. Introduction

The present state of knowledge of the physicochemical properties and photophysical behaviour of lichenic metabolites is very limited. This general statement applies in particular to depsides and depsidones, two chemically related compounds that are specific to Lichens and that can be present in large quantities (up to 5% of the lichen dry weight) in different lichenic species [I]_ This lack of information precludes an understanding of the possible roles that they can play in the lichen. Rao and Le Blanc [2] have proposed that the depside atranorine I could contribute to photosynthesis by absorbing short wavelength radiation which is re-emitted as fluorescence at longer wavelengths which can be absorbed by chlorophyll. Lichenic metabolites could also play a role as radiation filters avoiding photodamage induced by short wavelength radiation [3. 41. However, these considerations are highly speculative owing to a lack of information regarding the state of these compounds in lichen (i.e. how much is incorporated in the cellular membranes or .whether their phenolic groups are protonated or deprotonated) as well as about their spectroscopic (absorption spectra under different conditions), photophysical (i.e. fluorescence yields) andphotoehemical (i.e. photo&ability) characteristics.

Rao and Le Blanc [2] have reported that atranorine presetits a fluorescence band at 425 nm, but the study was carried out in toluene and the fluorescence quantum

lOlO-6030/92/$.5.00 Q 1992 - Elsevier Sequoia. All rights reserved

246

yields were not reported. With regard to the photochemistry, Takani and Takahashi [53 have studied the photolysis of usnic acid II in tetrahydrofuran-methanol and have proposed that photoaddition to the solvent is the dominant pathway, the process taking place from an excited triplet of nm* character localized in the carbonyl group. In the present work we report the solubility and spectroscopic and photochemical behaviour of three closely related lichenic metabolites: atranorine (I), pannarine (III) and l- chloropannarine (IV), characterized by having a carbonyl group (CHO) bound to a benzene ring bearing at least an ortho-hydroxyl group. These data have been obtained in water and in Brij-35 micellar solutions (to mimic a lipidic pseudophase) at different pH values. The behaviour of these compounds was compared with that of the simplest coumpound having the same chromophoric unit, ortho-hydroxybenzaldehyde (V).

“*coo@ocn~ *TRANOR,HE

CHO OH H CH3

I- CLOROPANNARINE

ORTO-HYDROXYBENZALDEHYM

OH

Scheme 1.

Atranorine, pannarine and 1-chloropannarine were isolated and characterized by the standard procedure [6, 73. o-Hydroxybenzaldehyde (Merck), Brij-35 (Aldrich) and acetonitrile (Merck) were employed as received. Bi-distilled water, whose pH was adjusted by acid (HCl) or base (NaOH) addition, was used. Micellar solutions were prepared at 3% Brij-35 concentrations.

2; Experimental details

Absorption spectra were recorded with a Beckman-35 spectrophotometer. Fluor- escence spectra and fluorescence yields were measured in a Shimadzu RF-540 and/ or a Perkin Elmer LS-5 spectrofluorimeter. Fluorescence quantum yields were evaluated by comparison of the fluorescence spectra of the compounds considered with that of anthracene in ethanol (@+1 = 0.27 [S]). Excited state lifetimes were measured by the phase modulation technique employing a Gregg 200 equipment. All measurements were carried out at room temperature (21* 1 “C).

Water or micellar solutions were prepared by addition of aliquots of a concentrated solution of the compounds in chloroform with subsequent evaporation of the solvent. The solubility of the compounds was estimated from the absorbance of the saturated

247

solution (after evaluation of the extinction coefficient) and/or from the break in an absorbance vs. added concentration pkot.

Photolyses were carried out employing light of 366 nm wavelength from a medium pressure mercury lamp in nitrogen or oxygen standard solutions. Photoconsumption was evaluated from changes in the absorption spectra. Most irradiations were carried out at pH 10 in mice&r solutions. Photoconsumption yields were evaluated by comparing the bleaching rates with the bleaching rates of anthracene irradiated, under matched conditions, in ethanolic solutions containing 10% carbon tetrachloride [9].

3. Experimental results

3.1 Absovtion spectra The spectroscopic properties and the photochemistry and photophysics of the

excited molecules are extremely solvent and pH dependent. The absorption spectra of V in water at pH 2 and pH 10 are shown in Fig. 1 (where a strong red-shift at basic pH is observed). Similar behaviour was observed for all the compounds considered, both in water and in micellar solutions of Brij-35 (Fig. 2). The absorption spectra obtained at pH 2 are similar to those obtained in acetonitrile (Table 1).

The absorption bands observed at pH 10 (Figs. 1 and 2) are more intense and considerably red-shifted, and can be ascribed‘ to the phenolate anion. As solids, compounds I, III and IV show only a very broad, structureless band with maxima clearly below 400 nm (Fig. 3).

3.2 pKiz deternina tions The difference between the absorption spectra of the protonated and deprotonated

forms can be employed to determine the pKa of the compounds. The protonation status can be characterized by the changes in absorbance at 375 or 310 nm. A typical plot of the absorbance change at 375 nm as a function of pH is shown in Fig. 4. In this plot the pI(a can be equated to the pII at which

(1)

400 500 300 340 380 420

Xlnml x [nml

Fig. 1. Ortho-hydroxybenzaldehyde absorption spectra in Brij-35 solution at pH 2 (-----) and pH 10 (-).

Fig. 2. Pannarine absorption spectra in Brij-35 solutions at pH 2 (- - -) and pH 10 {-).

248

TABLE 1

Extinction coefficients and wavelengths of maxima absorbance

Compound Medium pH 10

max E

pH 2”

max E

I Water Brij-35 Acetonitrile

III Water Brij-35 Acetonitrile

IV Water Brij-35 Acetonitrile

V Water Brij-35 Acetonitrile

‘Values in acetonitrile included.

380 5500 344 2700 386 14500 346 4000

332 7900

370 5500 340 2400 370 10600 340 4000

335 2900

370 6700 340 2900 370 14700 340 4200

336 4750

370 7600 326 3500 380 19000 340 6700

335 2800

Abs

04 .;__ -- ’ :

02 k ‘: j,’

j:

_____-;7’

,I’

____- -.--

500 300 8 12

Xlnml FH

Fig. 3. Absorption spectra of pannarine (-), 1-chloropannarine (- - -) and atranorine (- . -) in the solid state.

Fig. 4. Pannarine (0.1 mM) absorbance at 375 nm in Brij-35 solution as a function of the solution

PH.

The values obtained applying eqn. (1) are collected in Table 2. The pKa determined for V in water agrees with that reported in the literature [lo), rendering support to the proposed procedure.

3.3 Determination of solubilities and extinction coefficients A typical plot of absorbance against the analytica concentration of the added

compound is shown in Fig. 5. In order to avoid contribution to the absorbance from solid particles after saturation, all the solutions were centrifuged prior to their absorbance

249

TABLE 2

pKu values determined according to eqn. (1)

Compound Pa

Water Micellar solution

I 8.8 5.7 II 8.0 5.5 IV 5.1 V ::: (8.37)a 8.6

“Data from ref. 10.

A

01 0.2 0.3 400 600

Concentration (mM1 x “In

Fig. 5. Ortho-hydroqbenzaldehyde absorbance at 325 nm as a function of its concentration pH 2 in Brij-35 solutions.

at

Fig. 6. Fluorescence spectra of pannarine in Brij-35 (pH 10) (---), water (pH 2) (---) and water at pH 10 (-). Intensities are given in arbitrary units.

determination. From plots like that shown in Fig. 5 it is possible to obtain the extinction coefficient (E) from the slope and the solubility (S) from the break point and/or from

s =A, l(EI) (2)

where A _, is the absorbance after saturation and I is the path of the cell. The values of E are given in Table 1 and the solubilities are collected in Table 3.

3.4 Fluorescence spectra, fluorescence yields and excited lifeiimes Fluorescence spectra and fluorescence quantum yields are extremely sensitive to

the solvent and, in water, to the solution pH (Fig. 6). The values of the wavelengths of maxima fluorescence (CD& and the fluorescence quantum yields (&&I are given in Table 4. Compounds I, IKI and IV in the solid state, when excited with light of wavelengths between 310 and 340 nm, present a broad fluorescence band with a peak at about 475 mu.

Lifetimes obtained in water and Brij-35 solutions at pH 10 are given in Table 4. In all cases but I-chloropannarine in water, the decays can be represented by a monoexponential function. However, the lifetimes reported contain rather large errors owing to the low absorbances and rather short lifetimes of the samples. At low pH

250

TABLE 3

Solubilities in water and in Brij-35 (3% v/v) micellar solutions

Compound Medium SoIubili ty K”

PH 2 pH 10 PH 2 pH 10

I

III

IV

V

IIb

Water 0.50 190 z 1 Brij-35 1.0 Water 0.23 14 5 0.7 Brij-35 12.5 130 Water 2.4 60 50 8 Brij-35 15 100 Water 100 430 0.7 0.4 Brij-35 330 600 Water 6x 1O-4 4.5

‘Partition constant evaluated according to eqn. (3). bData from ref. 1.

TABLE 4

Wavelengths of maxima fluorescence, fluorescence quantum yields and excited singlet lifetimes

Compound Medium PH Max 103+F1 r (ns)

I Water 10 410 Acetonitrile 455

III Water 2 = 450 10 470

Brij-35 10 490 Acetonitrile 470

IV Water 2 = 435 10 485

Brij-35 10 490 Acetonitrile 480

V Water 10 490 2 400

Brij-35 10 490 Acetonitrile

“A shorter lived (0.44 ns) component was also present.

=0.16 2.1 zzo.15

1 3.3 8 1.4 0.4

0.8 3.2” 9 2.0 0.2 3 0.3

3.5 0.7 0.1

values, fluorescence intensities were too small to allow lifetime determinations with our available instrumentation.

3.5 Photosta bility determinations Irradiation of the compounds at pH 2, with light of 366 and 310 nm, did not

produce any significant change in the samples’ absorbances. The same photostability was observed when the irradiation was performed in acetonitrile. Irradiation of the aqueous solutions at pII 10 lead to ready photobleaching. The decrease in absorbance takes place without significant changes in the shape of the longest wavelength band, allowing a direct estimation of the photoconsumption from the absorbance decrease.

251

8000

Time 1 s 1

Fig. 7. Plot of ln@l’%l) ~1s. time for 1-chloropannarine irradiated at 366 nm in Brij-35 at pH 10 in nitrogen purged solution.

TABLE 5

Photoconsumption quantum yields”

Compound @air @*itrogen

I 0.144 0.036 III 0.033 0.33 IV 0.048 0.144 V 0.034 0.017

“Micelkar solutions (pH lo), irradiated at 366 nm during 180 min. Matched at absorbance 0.307.

The plots of [ln(&/!)] were nearly linear (Fig. 7), showing that the process follows a pseudo first-order kinetics. From these plots and the absorbed intensity (determined employing matched solutions of anthracene) the photoconsumption yields given in Table 5 were evaluated.

4. Discussion

The carbonyl group in a-hydroxy aromatic aldehydes is strongly influenced by the solvent and the protonation status of the neighbouring hydroxyl groups. For the protonated compounds, the structures shown in Scheme 2 can be envisaged, with the

CLOSE STRUCTURES

OPEN STRUCTURE

Scheme 2.

252

“Open” structure being present only in protic solvents. On the other hand, the deprotonated compound can be considered to have an open structure like that shown in Scheme 3.

Scheme 3.

The PIG values of monophenols III and IV are very close to that of compound V, as expected from the similarities of their structures, the small differences observed being attributable to the electron donor character of the substituents. The p&z determined for atranorine is higher than those of the other compounds. Atranorine presents three phenolic groups, two of them adjacent to the carbonyl group. The deprotonation of these phenols is the only one that can be detected by the present procedure. The larger pKa value found would imply that changes in the chromophore take place only after deprotonation of both phenols vecinals to the carbonyl. In this regard it is interesting to note that, while the pKa of phenol is 9.97, the deprotonation of m- dihydroxy benzene takes place at pH values of 9.3 and 11.06 [lo].

Evaluation of the pKa in Brij-35 solutions is subject to rather large uncertainties, since the relative absorbances change gradually over several pH units, particularly for the lichenic metabolites. These pKa values are difficult to interpret since they are conditioned by the proton concentration at the micellar interphase and by the solubilities of the protonated and deprotonated compounds in the micelles, but the data show that at pH <4 all the compounds can be considered as totally protonated both in water and in micellar solutions. At pH > 10, almost quantitative deprotonation takes place for all the compounds but atranorine in Brij-35 solutions.

Solubilities of the compounds considered are considerably larger than that reported previously for usnic acid, the only other related lichenic compound whose solubility in aqueous solution has been reported [l J. As expected from their charge, the solubilities of all the compounds are considerably higher in the basic solutions. The larger solubility of atranorine at pH 10, and also the very large ratio between its solubilities at pH 10 and pH 2, can be related to its polyphenol character, which can lead to higher negative charges by successive deprotonation.

The solubilities in the micellar solutions are considerably larger than in water (Table 3), pointing to significant incorporation to the Brij-35 micelles. This would indicate that, in the lichens, these compounds could be partially incorporated in the lipidic regions of membranes.

The incorporation of solutes to lipidic michrophases are usually expressed in terms of a partition constant [ll]:

K= [~ol~telpseu~op~ase/([so~~teJ~~~~~ x [pseudophase]) (3) The values of K, at a given pH, can be broadIy related to the probes’ hydrophobicities, determined by the balance of hydrophobic and hydrophylic contributions. Atranorine is a larger molecule but with three polar hydroxyl groups that reduce its incorporation to the micelles. The larger size of the hydrophobic part of the molecules explains the higher Kvalues measured for compounds III and IV than for V. For all the compounds, K values are larger at pH 2 than at pH 10. This implies that the negative charges, although favouring incorporation to the micelles from the solid, decrease the partitioning between the solvent and the micelles. Similar results have been reported for the

2.53

partition of alkyl phenols and alkyl phenolates in neutral micelles [12]. These results can be explained either by considering that the charge of the probes associated to the micelles is located in a microenvironment of smaller dielectric constant than water, and/or that the charge remains in a “water-like” environment but pushes part of the hydrophobic groups to which it is attached out of the “oil-like” micellar interior.

Nagaoka and co-workers have reported the absorption spectra of o-hydroxyben- zaldehyde and its derivatives [13, 141. Both in non-polar and polar (ethanol) solvents, the absorption spectra show a broad, structureless band centred at about 340 nm that, given its rather high absorption coefficient (E,,,,, =4000), was assigned to a -rr-rr* transition, as previously pointed out by Seliskar [15]. The n# absorption band of the aromatic carbonyls commonly observed in the 370-340 nm range was considered to be blue-shifted because of hydrogen bonding and presumably hidden by the stronger absorption of the d band. Similar considerations can appIy to the absorption bands observed for the present compounds in water and the micellar solutions at low pH values (Table 1).

The spectrum of the phenolates appears to be considerably red-shifted and, from the intensity of the band, could also be considered to correspond to a 7.r-‘lr* type transition. The solids present an absorption band at wavelengths close to those of the compounds in solutions_ The position of these bands is then compatible with a structure of the solid involving significant contribution of intra- and/or intermolecular hydrogen- bridging stabilization of the carbonyl group.

Fluorescence of compound V in non-polar solvents show a significant Stokes shift which has been considered to be a result of fluorescence from the enol tautomers [13]_ Emission in polar, protic, solvents appears to be considerably more complex, since the fluorescence excitation spectrum is considerably red-shifted in comparison with the absorption spectrum, suggesting the presence of several species in the ground state [13, 143. The data given in Table 4 also show that the fluorescence spectrum measured in aqueous solutions at low pH present a significant Stokes shift. This shift is also evidenced in the solid state, where enol tautomers could also participate. In agreement with these considerations, smaller Stokes shifts are present when the anions are considered. However, in spite of the fact that for these compounds, and particularly compound IV, the participation of enolic tautomers can be disregarded, the rather large shift observed still suggests large changes in the geometry and/or solvation of the open structure between the ground and excited states.

Efficient enol formation from the excited singlet ctin also explain the very low fluorescence yields (less than 10m3) measured in water at low pH and in non-aqueous solvents. The readiness of this process leads to extremely short (below 100 ps) singlet lifetimes [13] and explains the high photostability found in the present work both in acetonitrile or in aqueous solutions at low PH. These considerations imply that, although the fluorescence of the protonated compounds in solution or as solids could be reabsorbed by chlorophyl, the very low quantum yields makes the process questionable as a procedure for increasing the photosynthetic efficiency of the lichens. The very short lifetime of the excited singlet renders long-range energy transfer to the chlorophyl a very unlikely process. The efficient enol formation is a waste process that could protect lichenic compounds from photodecomposition. In this regard, these depsides and depsidones with carbonyl groups neighbouring to one or more hydroxyl groups appear as nearly ideal compounds for filtering, without irreversible photochemistry, short wavelength radiation.

At high pH values (about 10) we observe a stronger fluorescence band without structure, which can be ascribed to the open anion. The observed fluorescence yields

254

range from 0.16 X 10M3 (atranorine) to roughly 8 X low3 (pannarine and l-chloropan- narine in Brij). These higher fluorescence yields are possibly a result of the longer lifetime of the excited singlet. Table 4 shows that, under these conditions, singlet lifetimes are in the few nanosecond range. Furthermore, the impossibility of going back to the ground state via enolization renders these compounds more prone to be involved in photochemical processes. The data given in Table 5 show that all the compounds photodecompose when irradiated in micellar solutions at high pH values. However, the photoconsumption behaviour shows noticeabIe differences for chlorinated (compounds III and IV) and unchlorinated compounds. For the former compounds, oxygen considerably reduces the photoconsumption. while for o-hydroxybenzaIdehyde and atranorine, the presence of oxygen increases the photoconsumption rate. Preliminary results obtained by quenching the photoconsumption by naphthalene addition suggest that most of the decomposition arises from the excited triplet. Further results elucidating the mechanism of this process will be published elsewhere.

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